WO1997030590A1 - Cellular immunotherapy - Google Patents

Abstract

Broadly, disclosed is a novel approach to the adoptive cellular therapy of HIV infection that exploits the potentially effective cellular immune response that is initially generated in HIV-infected individuals. One aspect is a method for preparing cells for treating patients afflicted with human immunodeficiency virus (HIV), which includes subjecting cytokine-producing cells derived from lymph nodes excised from patients infected with HIV to mitogenic stimulation in serum-free media for their expansion. The resulting therapeutic agent for treating patients afflicted with human immunodeficiency virus (HIV) includes in a pharmaceutically-acceptable carrier cytokine-producing cells having been produced by the step of subjecting cytokine-producing cells derived from lymph nodes excised from patients infected with HIV to mitogenic stimulation in serum-free media for their expansion. As another aspect of the present invention, disclosed is a method for treating patients afflicted with human immunodeficiency virus (HIV) which includes administering to the patient an effective amount of the therapeutic agent disclosed herein. The invention also is capable of inhibiting replication of HIV as measured by the viral load reductions exhibited by patients that receive the inventive therapeutic and in inducing an immunorestorative effect in HIV patients.

Description

CELLULAR IMMUNOTHERAPY

BACKGROUND OF THE INVENΗON

The present invention relates to immune mediated diseases as typified by immunosuppressive virus infections exemplified by immunosuppressive virus infection

(both human, e.g., HIV, and animal, e.g., FIV) and more particularly to the adoptive cellular therapy of such virus infections. Much of the discussion will focus on HIV, although such discussion is by way of illustration and not by way of limitation.

Antiviral therapy in persons with AIDS or advanced HIV infection has failed to cure the infection or arrest the deterioration of the immune system. One possible approach to treating the infection and reconstituting or restoring the immune system is to enhance the patient's cellular antiviral capacity. Certain cytokines are capable of inducing the replication of HTV; therefore, selective blocking of cytokines is an important strategy in the treatment of HTV disease. However, cytokines also are important for the expression and regulation of normal immune function. Defects in the production of, e.g., interleukin-2 (IL-2) and interferon (IFN) have been reported in HIV infection, and the administration of cytokines in order to partially reconstitute or stimulate the defective cellular immune system in HTV disease is a strategy that is being pursued. Fauci, "Multifactorial nature of human immunodeficiency virus disease: implications for therapy", Science, 262:1011-1018, 1993. A number of chemical immune enhancers have also been proposed for the treatment of HTV-infected individuals, with clinical trials generally showing variable results. Turowsky, et. al., "Clinical application of chemical immunomodulators in cancer and AIDS", Cancer Invest, 1994, vol. 12, 620-643 (1994).

The most direct and potentially most effective form of immunologic reconstitution is the replacement of cellular elements of the immune system, i.e., adoptive cellular therapy. Based on studies showing that CD8⁺ cytolytic T cells (CTL) from HTV-infected patients have anti-HTV activities, most efforts have focused on CD8⁺ cells. Ho et al. conducted a phase I study to determine the safety and feasibility of infusing in vitro purified, activated, and expanded CD8⁺ cells in seven patients with AIDS-related complex or AIDS. Ho, et al., "A phase I study of adoptive transfer of autologous CD8⁺ T lymphocytes in patients with acquired immunodeficiency syndrome (ATDS)-related complex or AIDS", Blood, 81:2093-2101 , 1993. Autologous CD8⁺ cells were first selectively isolated in monoclonal antibody-coated flasks from peripheral blood mononuclear cells (PBMC) recovered by leukopheresis. They were then cultured and expanded with phytohemagglutinin and IL-2 before infusion. Five cycles of isolations and infusions of increasing numbers of CD8⁺ T cells were achieved in five of seven subjects. Five cycles could not be completed in two subjects with AIDS whose CD4⁺ cell counts were < 48/μL. Infusions of CD8⁺ cells alone were well tolerated. Four patients received IL-2 by continuous infusion for 5 days with their final cycle of CD8⁺ cells. All developed reversible adverse effects attributable to IL-2. After infusion, ^In-labeled CD8⁺ cells quickly accumulated in the lungs, with less than 10% of the labeled cells remaining in the circulation. After 24 hours, labeled CD8⁺ cells were reduced in the lungs, but increased and persisted in liver, spleen, and bone marrow. Four of five patients who were treated with multiple infusions as CD8⁺ cells remained clinically stable, but a fifth developed Pneumocystis carinii pneumonia. Other attempts to reconstitute cellular elements under investigation include the transfusion of ex vivo IL-2 expanded HTV-specific CD8⁺ T cell clones, bone marrow transplantation, and thymic transplantation. Riddell, et al., "CD8⁺ cytotoxic T cell therapy of cytomegalovirus and HTV infection", Curr Opin Immunol, 5:484-491, 1993; and Fauci, et al, supra. Results are too preliminary for conclusions to be reached concerning the feasibility and long-term benefit of adoptive cellular therapy programs focusing on CD8⁺ CTL. However, there are a number of potential limitations: (i) Normally CD8⁺ cells do not make enough IL-2 to support their own expansion and are dependent on IL-2 and possibly other cytokines from CD4+ cells for "help". In the absence of T_H activity, HTV-specific CTLs would not be expected to expand and eliminate HIV in vivo, (ii) Virus-specific CD8⁺ CTLs have been demonstrated to cause immunopathology if a significant reservoir of actively infected cells is established in a vital organ as the response is elicited. HTV-specific CTLs have been implicated in the pathogenesis of AIDS dementia and lymphocytic alveolitis (Riddell, supra), (iii) Peripheral blood-derived CD8⁺ do not appear to traffick to major sites of HIV activity, e.g., lymph nodes (Ho, et al., supra), (iv) There is evidence that HIV reactive CD8⁺ cells may contribute to the immunodeficiency by lysing HTV-infected CD4 cells. Grant, et. al., "Lysis of CD4+ lymphocytes by non-HLA-restricted cytotoxic T lymphocytes from HTV-infected individuals", Clin Exp Immunol, 93:356-362, 1993. v) A major role in ώe progression of HIV infection is played by the HTV-mediated destruction of the microenvironment of the lymphoid organs. It is unclear whether the severely damaged immune systems o^r patients with advanced HIV disease would be capable of spontaneous regeneraπoi even if viru replication could be completely inhibited by totally effective CD8⁺ CTLs. In fact, there is suggestive evidence that spontaneous regeneration would not occur under these circumstances (Fauci, supra).

The fundamental distinguishing feature between immunotherapy of HTV infection and that for other persistent viruses is that HIV undermines the ability of the host to appropriately respond to efforts to augment of elicit potentially protective host immune responses by inducing dysfunction and depletion of CD4+ TJ_J cells. The presentation of an antigen (Ag) to CD4⁺ TJJ cells and the generation of T_H activity are the central processes in any specific cellular immune response, such as the specific response to a HIV, upon which all the other elements depend. Murine CD4⁺ Tu cells comprise at least three subsets (Powrie, et al., "Cytokine regulation of T-cell function: potential for therapeutic intervention", Immunol Today, 14:270-274, 1993): The T_H1 subset, which secretes TL-2 and TFN, but not TL-4, appears to be responsible for delayed type hypersensitivity (DTH) responses. Tγ{2 cells, which secrete TL-4 and TL- 5, but not IL-2, provide B-cell help. T cells produce TL-2, TFN, TL-4, and IL-5. Several groups have now isolated human clones similar to the mouse Tul, Tu2, and TjrO phenotype. The factors influencing TJJ differentiation are not completely understood; current evidence suggests the following: Tjjl differentiation occurs in response to intracellular viruses and bacteria and is promoted by IFN, IFNa, and TL- 12. Tu2 differentiation occurs in response to soluble allergens and some helminth components and is promoted by TL-4 and, in some studies, transforming growth factor (TGF) β. In addition to the depletion of the T_j-_j arm of the immune response of

HTV-infected individuals, recent reports suggest that there is a progressive imbalance immune systems with a selective defect in Tjjl responses and a predominance of Tu2 responses. Correcting this imbalance by the administration of Tul-type cytokines such as TL-2 or TL- 12 is under investigation. It is now evident that the initial host cellular immune response to HIV reduces the level of HIV in the peripheral blood. In the early, "immune activation" stage of infection, HTV is harbored in lymphatic tissues. Lymph nodes are characterized by germinal center hyperplasia and a CD4⁺ cell pleocytosis that includes a substantial number of TL-2 and TFN secreting cells suggesting that a TJJI differentiation has occurred. Emille, et. al., "Production of interleukins in human immunodeficiency virus- 1 -replicating lymph nodes", J Clin Invest, 86:148-159, 1990. Recently, the use of sensitive molecular techniques combined with careful histopathologic examination has indicated that in the early stages HIV exists primarily as extracellular virus trapped in the follicular dendritic cell network of the germinal center. Intracellular virus also is detectable but usually in a latent form. It is postulated that the activation of CD4⁺ T cells by antigen presenting cells (APC), which normally occurs in the follicular center, renders uninfected cells susceptible to infection by the locally retained virus and results in the reactivation of HIV in latently infected cell, thus contributing to the gradual depletion of CD4⁺ Tfj cells observed during the course of HIV infection. Nonetheless, it does appear that early in the course of HTV infection an appropriate and potentially effective immune response is generated in lymph nodes. We are proposing to examine the possibility that cells that can provide appropriate TJJ function can be isolated and expanded ex vivo from the lymph nodes infected with HIV and that adoptive cellular therapy using the expanded lymph node lymphocytes (LNL) will generate antiviral immunity in vivo. It may appear counter-intuitive to utilize a major reservoir of HTV, i.e., lymph nodes, and the central target of HTV infection, i.e., activated CD4⁺ cells, in the adoptive cellular therapy of HTV infection. Based on the central role of lymph nodes and CD4⁺ cells in immune response, the possibility does exist that under certain circumstances, and perhaps in association with the adrninistration of effective antiretroviral agents, stimulation of the immune system by CD4+. LNL secreting appropriate cytokines might have a beneficial effect in reconstituting immune competence in HTV infected individuals. It should be noted that although CD8+ CTL suppress the replication of HTV in PBMC of patients with AIDS, Walker et al. discovered that the virus infected cells were not killed during the process, but rather that HTV replication was inhibited by a diffusible cytokine elaborated by CD8⁺ cells. Walker et al, "A diffusible lymphokine produced by CD8+ T lymphocytes suppresses HTV replication", Immunology, 66:628, 1989. Furthermore, it has been demonstrated that collaboration between CD4+ T cells and B cells and the factors that these cells produce are essential for the normal development of the lymphoid microenvironment (Fauci, supra). Lymph nodes are also attractive as a source of cells not only because they contain all the critical elements to develop an immune response, e.g., APCs, but also because the yield of immunologically active cells is orders of magnitude greater than that from the peripheral blood. The locomotor capability of LNL, and, thus, the ability to traffick to lymph nodes, also may be greater than that of PBL. Triozzi, "Identification and activation of tumor-reactive cells for adoptive immunotherapy", Stem Cells, 11:204-211, 1993.

BROAD STATEMENT OF THE INVENTION

Broadly, disclosed is a novel approach to the adoptive cellular therapy of immune mediated diseases exemplified by HTV infection which therapy exploits the potentially effective cellular immune response that is initially generated in HTV-infected individuals. One aspect is a method for preparing cells having an immunorestorative effect on patients afflicted with human immunodeficiency virus (HIV), which includes subjecting cytokine-producing cells derived from lymph nodes excised from patients infected with HTV to mitogenic stimulation in serum-free media for their expansion. The resulting therapeutic agent is effe e in inducing an immunorestorative effect on patients afflicted with human immunodeficiency virus (HIV) and includes in a pharmaceutically-acceptable carrier cytokine-producing cells in an amount effective for HTV reduction, the cytokine-producing cells having been produced by the step of subjecting cytokine-producing cells derived from lymph nodes excised from patients infected with HTV to mitogenic stimulation in serum-free media for their expansion. As another aspect of the present invention, disclosed is a method for inducing an immunorestorative effect on patients afflicted with human immunodeficiency virus (HTV) which includes administering to the patient an effective amount of the therapeutic agent disclosed herein.

The invention apparently is capable of inhibiting replication of HIV as determined by the viral load reductions exhibited by patients administered the therapeutic agent of the present invention. Thus, another aspect of the present invention is a therapeutic agent effective in reducing the viral load in patients afflicted with human immunodeficiency virus (HIV) and includes in a pharmaceutically- acceptable carrier cytokine-producing cells in an amount effective for HIV reduction, the cytokine-producing cells having been produced by the step of subjecting cytokine- producing cells derived from lymph nodes excised from patients infected with HIV to mitogenic stimulation in serum-free media for their expansion. As another aspect of the present invention, disclosed is a method for inducing a viral load reduction in patients afflicted with human immunodeficiency virus (HTV) which includes administering to the patient an effective amount of the therapeutic agent disclosed herein.

Advantages of the present invention include a culture procedure that is easy, expedient, and reproducible. Another advantage is a therapeutic agent that is negative for HTV; yet, is effective in inhibiting the replication of HIV. A further advantage is that the capacity of the therapeutic agent to secrete cytokine may obviate the need for exogenous systemic cytokines to maintain their activity. A further advantage is that total lack of adverse side effects when using the novel therapeutic agent. A yet further advantage is the lack of adverse reactions between the novel therapeutic agent and other drugs taken by the patient. These and other advantages will be readily apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 graphically depicts the influence of anti-CD3 MAb concentration on lymph node cell expansion. Cells were cultured for 10 days with a range of concentrations of anti-CD2 MAb (OKT3) and constant concentrations of TL-2. Data represent range and mean values for 5 different donors with peripheral blood CD4 counts from 46 to 210/mm³.

Figs. 2 and 3 graphically depict expansion of lymph node and peripheral blood CD4 and CD8 cells from 2 patients. The peripheral blood CD4 count of the patient depicted in A was 23 and that of the patient depicted in B was 125/mm³. Fig. 4 graphically depicts supernatant cytokine levels (A) and cytokine production in response to immobilized anti-CD3 MAb (B). Data represent mean values cells stimulated with 5 (solid), 20 (cross-hatched), or 100 (dotted) ng/ml anti-CD3 MAb (n = 3).

Fig. 5 graphically depicts cytokine production in response to immobilized anti- CD3 MAb of cells cultured for 4, 8, 10, and 12 days. Data represent mean values (n = 3).

Fig. 6 depicts HTV-1 mRNA expression of lymph node cells during ex vivo expansion with anti-CD3/TL-2, TL-2 alone, or with no additions (NA). Fig. 7 graphically depicts the effect of culture supernatants generated with either

5, 20, or 100 ng/ml anti-CD3 MAb on HIV-1 replication added at an 80% concentration. Data are expressed as % suppression of p24 production.

Fig. 8 graphically depicts proliferative responses of expanded cells to oligopeptides corresponding to HTV-1 envelope sequence. The drawings will be described in more detail below.

DETAILED DESCRIPTION OF THE IN VΕNTION The infusion of immunologically active cells is an attractive approach to AIDS. Cellular irnmunotherapy has been extensively studied in patients with cancer and has resulted in reported sustained complete responses and reported improved survival and quality of life (Rosenberg, S.A., "Adoptive Cellular Therapy: Clinical Applications", DeVita VT, Hellman, S., Rosenberg, S.A. (eds). Biologic Therapy of Cancer, Philadelphia: J.B. Lippincott, 1991, p. 214; Osband, et al, "Effect of Autolymphocyte Therapy on Survival and Quality of Life in Patients with Metastatic Renal-Cell Carcinoma", Lancet 335:994-998,1990). Antiviral responses to, e.g., cytomegalovirus, have also been effected in clinical trials (Riddell, et al, "Restoration of Viral Immunity in Immunodeficient Humans by Adoptive Transfer of T-cell Clones", Science 257:238-240, 1992). A limited number of early phase clinical trials have been conducted in patients with HIV-1 infection. Ho, et al, "A Phase I Study of Adoptive Transfer of Autologous CD8⁺ T Lymphocytes in Patients with Acquired Immunodeficiency Syndrome (ATDS)-Related Complex or AIDS", Blood 81:2093- 2101, 1993, infused purified autologous, peripheral blood CD8+ CTL, expanded ex vivo with TL-2 and phytohemagglutinin (PHA). Four of five patients treated remained clinically stable, but a fifth developed Pneumocystis carinii pneumonia. Increases in circulating HTV- 1 -specific CTL activity were reported Torpey, et al, "Effects of Adoptive Irnmunotherapy with Autologous CD8⁺ T Lymphocytes on Immunologic Parameters: Lymphocyte Subsets and Cytotoxic Activity", Clin. Immunol Immunopath. 68:263-272, 1993. Klimas, et al, "Clinical and Immunological Changes in AIDS Patients Following Adoptive Therapy with Activated Autologous CD8 T Cells and Interleukin-2 Infusion, AIDS 8:1073-1081, 1994 infused six patients with TL- 2/PHA-expanded, peripheral-blood, CD8⁺ cells along with TL-2. Clinical improvement in lymphadenopathy, hairy leukoplakia, and Kaposi's sarcoma was observed. Carter, et al, "Expansion of Syngeneic T Lymphocytes for Adoptive Irnmunotherapy in Twins Discordant for HTV Infection", Blood 82:416a, 1993, treated twelve HIV-1 patients with repeated infusion of peripheral-blood lymphocytes from their HTV-1- twin that had been expanded ex vivo using anti-CD3 MAb and TL-2. Increases in CD4 counts were observed in patients with initial counts less than 200/mm³; after a transient increase, p24 levels were also observed to decrease slightly. Other approaches under investigation include HTV- 1 -specific CTL generated by culturing peripheral blood lymphocytes with HTV-1 peptides, autologous cloned HTV- 1 -specific CTL transduced with a fusion marker/suicide gene, and CD8⁺ cells transduced with genes coding for chimeric T-cell receptors that bind HTV- 1 -infected cells (reviewed by Bridges, et al, "Gene Therapy and Immune Restoration for HTV Disease", Lancet 345:427-432, 1995).

Dysregulation of the cytokine system plays a central role in the progression of HTV-1 infection and related immunosuppression. The inventive approach to the cellular therapy of AIDS relies on lymph-node cell infusions as a means of modulating the cytokine system, rather than as a means of administrating HTV- 1- specific CTL. The results reported herein indicate that significant numbers of cytokine-releasing cells with anti-HIV-1 activity can be expanded in short-term cultures from the lymph nodes of TTTV-1 infected individuals, even from individuals with advanced disease. A variety of cytokines that could inhibit HTV-1 replication are potentially produced by the expanded cells, including: TL-8, IL-10, TNFa, and TGFb (Mackewicz, et al, "CD8⁺ Cell and Anti-HTV Activity: Nonlytic Suppression of Virus Replication" AIDS Res. Hum. Retrovirus 8:629-640, 1992). Supernatants from the expanded cells were capable of suppressing HTV-1 mRNA expression in cells initially cultured in TL-2 alone. Thus, it also is possible that CD8 cell anti-viral factor, as described by Levy and coworkers (Mackewicz, et al, (supra); Mackewicz, et al, "Effect of Cytokines on HTV Replication in CDR⁺ Lymphocytes: Lack of Identity with CD8⁺ Cell Antiviral Factor", Cell Immunol. 153:329-343, 1994; and Mackewicz, et al, "CD8⁺ T Cells Suppress Human Immunodeficiency Virus Replication by Inhibiting Viral Transcription", Proc. Natl Acad. Sci. USA 92:2308-2312, 1995), is present. The cytokines produced would also be predicted to have a variety of immunorestorative effects. The heterogeneity of HTV- 1 makes is difficult to select cells reactive with all strains present at the time of re-infusion. Walker et al, "Long-Term Culture and Fine Specificity of Human Cytotoxic T-Lymphocyte Clones Reactive with Human Immunodeficiency Virus Type 1", Proc. Natl. Acad. Sci. USA 86:9514-9518, 1989, have shown that MHC class I-restricted CTL specific for HIV-1 reverse transcriptase can be expanded from HTV- 1 -infected individuals. These CTL recognized multiple epitopes in conjunction with different host MHC molecules. There was a lack of cross- reactivity when CTL from other subjects were evaluated, suggesting that the immunogenic epitopes were different in different individuals. To generate cells with a wide spectrum of HTV- 1 reactivities, the invention used HTV- 1 -infected lymph nodes as a source of cells, rather than peripheral blood (PB). The relative frequencies of CTL specific for HIV-1 proteins from lymphoid organs differ at least one log from that of peripheral blood (Hadida, et al, "CTLs from Lymphoid Organs Recognize an Optimal HLA-A2-Restricted and HLA-B52-Restricted Nonpeptide and Several Epitopes in the C-Terminal Region of HTV-1", Nef. J. Immunol 154:4174-4186, 1995). The high levels of local viral replication and production of viral antigens together with presence of "professional" APCs, such as macrophages and dendritic cells, should maintain the activation and differentiation of a large array of CTL clones within lymph nodes. It also would seem likely that polyclonal response against HIV-1 are first elicited in lymphoid tissues before being disseminated in the peripheral blood.

The present invention uses the capacity of anti-CD3 MAb to mimic the normal pathways of T-cell activation and the capacity of TL-2 to expand multiple T-cell subpopulations from lymph nodes. HTV- 1 -specific T-cell lines can be expanded by nonspecific stimulation with anti-CD3 MAb and TL-2 without the need for re-exposure to viral antigen (Walker, et al, (supra); Johnson, et al, "HIV-1 Gag Specific Cytotoxic T Lymphocyte Recognize Multiple Highly Conserved Epitopes. Fine Specificity of the Gag-Specific Response Defined by Using Unstimulated Peripheral Blood Mononuclear Cells and Cloned Effector Cells", J. Immunol 147:1512-1521, 1991; Littaua, et al, "An HLA C Restricted CD8+ Cytotoxic T Lymphocyte Clone Recognizes a Highly Conserved Epitope on Human Immunodeficiency Virus Type 1 Gag", J. Virol. 65: 4051-4056, 1991; Lieberman, et al, "Cytotoxic T Lymphocytes from HTV-1 Seropositive Individuals Recognize Immunodominant Epitopes in GP160 and Reverse Transcriptase", J. Immunol. 148:2738-2747, 1992; and Buseyne, et al, "Gag-Specific Cytotoxic T Lymphocytes from Human Immunodeficiency Virus Type 1 -Infected Individuals: Gag Epitopes are Clustered in three Regions of the p218^ag Protein", J . Virol. 67:694-702, 1993). It is difficult to assess the spectrum of HIV-1 reactivities in vitro of cells cultured in the presence of polyclonal activators, such as anti-CD3 MAb and TL-2, particularly in short-term bulk cultures. A proliferative response to a variety of HTV-1 antigens was observed. Although the expanded cells did proliferate in response to HIV-1 antigens, specific CTL activity against these targets was not demonstrated. HTV-1 specific CTL and CTL precursors likely were present in culture and may have played a role in the anti-HIV-1 activity. The inventive expansion regimen, however, was not designed to maximize cytolytic activity, and cells were exposed to decreasing concentrations of TL-2 with the intent of reducing their dependency on TL-2. Although the possibility was not examined, cells with reactivities against other pathogens to complicate HTV-1 infection also may have been expanded.

Culture conditions were optimized to maintain the viability of the APCs present in the lymph nodes, so that at least theoretically, any HTV- 1 -associated antigens released in vitro could be presented by the APCs. Cells were cultured in serum-free conditions using a media designed to maintain the viability of macrophages and dendritic cells, and not lymphocytes. Cells expanded in serum-free conditions are potentially less toxic: the only significant toxicity observed in the study of Carter, et al, (supra), was hypersensitivity to the bovine serum proteins that were adherent to the expanded lymphocytes. The concentration of anti-CD3 MAb influenced cell expansion. Moran, et al, "Regulation of HTV Production by Blood Mononuclear Cells from HTV- infected Donors: I. Lack of Correlation between HIV-1 Production and T Cell Activation", AIDS Res. Human Retroviruses 9:455-464, 1993, have shown that although soluble anti-CD3 is a stimulus for HIV-1 production, immobilized anti-CD3 stimulation produces a factor that decreases HIV-1 replication. Both T cells and monocytes were found to be required for soluble anti-CD3 to induce high levels of HTV-1 production. A concentration of anti-CD3 MAb was used to elicit the effect of immobilized anti-CD3, by associating with the APCs present, and also elicit the effect of soluble anti-CD3 MAb and the possibility of in vitro immunization. The concentration of anti-CD3 MAb is 0.5% of that used by Moran, et al (supra) to induce the effects of soluble anti-CD3. The transient increase in HIV-1 mRNA observed would be a predicted consequence of exposure to soluble anti-CD3 (Moran, et al, supra). The presence of TNFa and TL-6 in the culture supernatants would be a predicted effect of immobilized anti-CD3 MAb as would the presence of soluble factors that inhibit HTV-1 (Moran, et al, supra; Mackewicz, et al, 1994, supra). Culture interval also influenced cytokine production; the greatest amounts of cytokines were produced by cells cultured for 8 to 10 days.

The expanded cells secreted both THL e.g., TFNg, and TH2, e.g., IL-5, cytokines, a result that would not be unexpected in light of the mixed population of cells present. Most of the cells expanding were CD8⁺, as one of the unique features of lymph nodes during HTV-1 infection is the accumulation of large numbers of CD8⁺ cells (Emilie, etal, "Production of Interleukins in Human Immunodeficiency Virus- 1- Replicating Lymph Nodes", J. Clin. Invest. 86:148-159, 1990). CD8+ cells are the major producers of TFNg, a THl-type cytokine in HTV- 1 -infected lymph nodes; they also produce TL-4, TL-5, and IL-10, TH2-type cytokines (Emilie, et al, supra; Manetti, et al, "CD30 Expression by CD8⁺ T. Cells Producing Type 2 Helper Cytokines. Evidence for Large Numbers of CD8⁺CD30⁺ T Cell Clones in Human Immunodeficiency Virus Infection", J. Exp. Med. 180:2407-2411, 1994; Graziosi, et al, "Lack of Evidence of the Dichotomy of THI and TH2 Predominance in HTV- Infected Individuals", Science 265:248-252, 1994). The CD8+ cells that expanded were CD1 lb-, which is the predominant phenotype of anti-HIV-1 CTL and CD8⁺ cells that suppress HTV-1 replication non-lytically (Mackewicz, et al, 1992, supra). Although expansion rates varied, the cytokine profiles of cells expanded from individuals with CD4 counts <100 and those with CD4 counts >100 were comparable. These results are consistent with those of Graziosi, et al. (supra), who found no correlation between the clinical stage of HTV- 1 -infected subjects as defined by CD4 counts and either the constitutive expression or that induced with immobilized anti- CD3-MAb-induced of THI and TH2 cytokines from lymph node cells in longitudinal and/or cross-sectional studies.

The expansion rates of CD4+ cells present were comparable to the CD8⁺ cells. In several model systems, the adoptive transfer of a mixed population of CD4+ and CD8⁺ cells has been more effective than purified CD8+ cells, even when CD8⁺ cells are central to desired response (Byrne, et al, "Biology of Cloned Cytotoxic T Lymphocytes Specific for Lymphocyte choriomeningitis Virus: Clearance of Virus in vivo", J. Virol 51:682-686, 1984; Larsen, et al, "Role of T-Lymphocyte Subsets in Recovery from Herpes Simplex Virus Infection", J. Vriol. 50:56-59, 1984; Lukacher, et al, "In Vivo Effector Function of Influenza Virus-Specific T Lymphocyte Clones is Highly Specific", /. Exp. Med. 160:814-823, 1984). Although the infusion of CD4+ cells, activated CDF⁺ in particular, are the principal target for HIV- 1 and critical to the progression of the infection, there are theoretical advantages to infusing CD4+ cells- activated CD4+ in particular-are the principal target for HTV-1 and critical to the progression of the infection. There are theoretical advantages to infusing CD4⁺ cells along with CD8⁺ cells. CD8+ cells normally do not make enough IL-2 to support their own expansion and are dependent on TL-2, and possibly other cytokines from CD4⁺ cells for "help". In the absence of TH activity, an infusion of HIV- 1 -specific CTL would not be expected to expand in vivo. There also is evidence, at least in the case of influenza infections, that ex vivo expanded CD4⁺ cells can mediate antiviral effects directly (Scherle, et al, "Functional Analyses of Influenza-Specific Helper T Cell Clones In Vivo: T Cells Specific for Internal Viral Protein Provide Cognate Help for B Cell Responses to Hemagglutinin", J. Exp. Med. 164:1114-1121, 1986). There may be other advantages of using a mixed population of cells. Antibodies have been able to protect against experimental retroviral infections under some circumstances (Vaslin, et al, "Induction of Humoral and Cellular Immunity to Simian Immunodeficiency Virus: What are the Requirements for Protection", Vaccine 12:1132-1140, 1994); correlative evidence suggests that some antibody may be associated with protection against progress of HTV- 1 infection (Salk, "Prospects for the Control of AIDS by Immunizing Seropositive Individuals", Nature 327:473-476, 1987); long-term survivors of HIV-1 have been characterized by a strong neutralizing-antibody response (Pantaleo, et al, "Studies in Subjects with Long-term Nonprogressive Human Immunodeficiency Virus Infection", N. Eng. J. Med. 332:209-216, 1995, Cao, et al, "Virologic and Immunologic Characterization of Long-Term Survivors of Human Immunodeficiency Virus Type 1 Infection", N. Engl J. Med. 332:201-208, 1995); and the infusion of plasma rich in anti-HIV-1 antibody has been reported to delay the appearance of the first ATDS-defining event (Vittecoq, et al, "Passive Irnmunotherapy in AIDS: A Double-Blind Randomized Study Based on Transfusions of Plasma Rich in Anti- Human Immunodeficiency Virus 1 Antibodies vs. Transfusion of Seronegative Plasma, Proc. Natl Acad. Sci. USA, 92:1195-1199, 1995). CD8+ T_H cells are well- recognized. Thus, there is a potential advantage to the infusion of cells that can provide TH activity to B-cells. Some of the CD8+ cells were also CD45RA+ or CD30⁺, suggesting the possibility of CD8⁺ TH function in vivo, including the induction of anti- HTV-1 antibody (Manetti, et al, supra) The release of THI cytokines, such as IFNg, suggests the possibility that DTH responses can be enhanced. The study of Carter, et al, (supra) has suggested the feasibility and safety of infusing a mixed population of uninfected CD4+ and CD8⁺ cells into HTV- 1 -infected individuals.

The complexity of the cytokine system is an obstacle to effective systemic therapy. Cytokines can mediate both beneficial and detrimental effects depending on a variety of factors, including the nature of the responding cell, the stage of infection, and the concentration of the cytokine. For example, certain cytokines, including TL-4 and IL-10, enhance the replication of HTV- 1 in lymphocytes but inhibit HIV-1 replication in macrophages (Akridge, et al, "TL- 10 is Induced During HTV-1 Infection and is Capable of Decreasing Viral Replication in Human Macrophage", J. Immunol 153:5782-5789, 1994; Montaner et al, "T_H2 Downregulation of Macrophage HTV-1 Replication", Science 267:538-539, 1995). Low concentrations of TL-4 enhance HIV- 1 replication in lymphocytes, while high concentrations inhibit replication (Mackewicz, et al, 1994, supra). Systemic therapy with intermittent courses of TL-2 has been reported to increase the number of CD4⁺ cells; however, perhaps because CD4 cells also are activated, plasma HIV-1 RNA levels increase (Kovacs, et al, "Increases in CD4 T Lymphocytes with Intermittent Courses of Interleukin-2 in Patients with Human Immunodeficiency Virus Infection~A Prekminary Study", N. Engl. J. Med. 332:567- 575, 1995). Cytokines also are unlikely to be secreted or act individually, but rather as a "cascade" involving other cytokines, and regulated, local, i.e., paracrine, release appears to be critical to optimal activity. Finally, the experience to date, including that with TL-2 therapy, suggests that systemic cytokine therapy will unlikely be of benefit in patients with advanced TflV-1 infection.

The infusion of cells as a vehicle for modulating the cytokine system addresses many of the limitations inherent to the systemic cytokine therapy. Theoretically, cells could traffic to reservoirs of HTV-1 activity and specifically release a variety of cytokines in a regulated fashion that could mediate both antiviral and immunorestorative effects. Several lines of evidence suggest a role for CD8⁺ CTL in the control of HIV-1 infection; and, thus, the focus of most cellular irnmunotherapy programs has been on expanding CD8⁺ CTL. Longitudinal studies have shown, however, that HIV- 1 can escape specific CTL recognition (Phillips, et al, "Human Immunodeficiency Virus Genetic Variation that Can Escape Cytotoxic T Cell Recognition", Nature 354:453-459, 1991). HTV- 1 -specific CD8+ CTL have been demonstrated to cause immunopathology, including dementia and lymphocytic alveolitis, and may contribute to the immunodeficiency by lysing HTV- 1 -infected CDC4 cells (Riddell, et al, supra; Grant, et al, "Lysis of CDr+ Lymphocytes by Non-HLA- Restricted Cytotoxid T Lymphocytes from HTV-infected Individuals", Clin. Exp. Immunol. 93:356-362, 1993). It also is unclear whether the severely damaged immune systems of patients with advanced disease would be capable of spontaneous regeneration even if virus replication could be completely inhibited by totally effective CD8+ CTL (Fauci, "Multifactorial Nature of Human Immunodeficiency Virus Disease: Implications for Therapy", Science 262:1011-1018, 1993). Other cytolytic cells, including NK cells, can mediate antiviral activity; but, whether they can reverse the immunosuppression is unknown and unlikely. It is of interest that in a number of preclinical and clinical studies targeting cancer, the effectiveness of cellular immunotherapy programs has correlated better with the ability of the infused cells to specifically secrete cytokines than with cytotoxicity in vitro (Barth, et al, "Interferon g and Tumor Necrosis Factor have a Role in Tumor Regression Mediated by Murine CD8⁺ Tumor-Infiltrating Lymphocytes", J. Exp. Med. 173: 647-658, 1991; Kim, et al, "Effects of the Adoptive Transfer of Cells Secreting Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) in Patients with Colon Cancer", J. Immunother 16-239, 1994; Schwartzentruber, et al, "In Vitro Predictors of Therapeutic Response in Melanoma Patients Receiving Tumor-Infiltrating Lymphocytes and Interleukin-2' J. Clin. Oncol 12: 1475-1483, 1994). Another unexpected, yet fortuitous, benefit of the present invention is the reduction in viral load that the patients experience when presented with the inventive therapeutic. Recent evidence has shown patient benefit in reducing viral loads in AIDS patients (Deeks, et al, "HTV-1 Protease Inhibitors - A Review for Clinicians, JAMA, Vol. 277, No. 2, 145-153, January 8, 1997; Mellors, et al, "Quantitation of HTV-1 RNA in Plasma Predicts Outcome after Seroconversion", Ann. Intern. Med., 1995; 122: 573-579; and Coombs, et. al, "Association of Plasma Human Immunodeficiency Virus Type 1 RNA Level with Risk of Clinical Progression in Patients with Advanced Infection", J. Inf. Dis., 1996; 174: 704-712). The data below will expand on this benefit of the present invention. In broader terms and based on the data presented below, it is believed that the inventive cellular therapy regimen has applicability in the treatment of immune mediated diseases (of which HIV is an example) in both animals and humans. While some of these diseases are bacterial (e.g., tuberculosis) and some are of unknown cause (autoimmune diseases,e.g., rheumatoid arthritis), most such immune mediated diseases are viral induced and result from persistent and acute infections, including latent infection (e.g., human herpes virus), chronic infections (e.g., "old dog encephalitis" following canine distemper virus (CDV) infection or lymphocyteic choriomeningitis in mice), and slow infections (both lentiviruses including HIV, feline immunodeficiency virus (FIV), and simian immunodeficiency virus (STV); and a group of unclassified agents which cause subacute spongioform encephalopathies including Cruetzfeld- Jakob disease, Kuru, and Mad Cow Disease). Such immunosuppressive or chronic diseases that lead to an immunosuppressed state in the host (both human and animal) should be treatable in accordance with the precepts of the present invention including, for example, HTV, tuberculosis, measles, dengue fever, malaria, hepatitis (chronic), leprosy, rheumatoid arthritis, multiple sclerosis, canine distemper virus, and the like.

Chronic and acute viruses are classified as being DNA viruses or RNA viruses, enveloped and non-enveloped. RNA viruses are exemplified by, for example, picornaviruses, togaviruses, paramyxoviruses, orthomyxoviruses, rhandoviruses, reoviruses, retroviruses, bunyaviruses, coronaviruses, and arenaviruses. DNA viruses are typified by panoviruses, papoviruses, adenoviruses, herpesviruses, and poxviruses. For more information on viruses, reference is made to the following texts: Fenner, et al, Veterinarian Virology, 2nd Edition, Academic Press, New York, New York (1993); Mims, et al, Viral Pathogenesis and Immunology", Blackwell Scientific Publications, London, England (1984); Virology, B.N. Field, Editor, Raven Press, 3rd edition; Shulman, et al, The Biological and Clinical Basis of Infectious Diseases, 5th edition, W.B. Saunders Co. (1997), the disclosures of which are expressly incorporated herein by reference.

The following examples illustrate the present invention, but they should not be construed as limiting. All citations referred to herein are expressly incorporated herein by reference.

EXAMPLES

EXAMPLE I

IN VITRO DATA

MATERIALS AND METHODS

Cells. Samples of lymph nodes not needed for diagnosis were obtained from resected specimens. The major part of each lymph node was used for histopathology; approximately 20% to 50% was used for the immunologic studies. Lymph nodes with histologic evidence of malignancy or mycobacterial infection were excluded. Dissociation of tissue was carried out under sterile conditions in a laminar flow hood. Tissue was rinsed in a centrifuge tube with a serum-free medium, Macrophage SFM (Gibco/BRL, Grand Island, NY), and was transferred to a Petri dish. Extraneous tissue was excised with a scalpel, and the tissue was minced into pieces approximately 2 to 3 mm in diameter. Tissue fragments were placed in centrifugation tubes with collagenase pre-warmed to 37^*C. Tubes were vortexed at a speed that caused tumbling, but not foaming. Free cells were decanted through three layers of sterile medium-wet nylon mesh into centrifuge tubes. The cells were centrifuged (250 X g) in a refrigerated centrifuge for ten minutes. The supernatant fluid was poured off, and the procedure was repeated until sufficient cells were obtained. Cells from the different digests then were pooled and counted, and the cell viability was assessed by the Trypan blue exclusion test. Cells not needed immediately were cryopreserved in dimethyl- sulfoxide with 5% autologous serum.

Cell Expansion. Lymph node lymphocytes (lOfyml) were suspended in Macrophage SFM, 600 IU/ml human recombinant TL-2 (Proleukin, Cetus Oncology, Emeryville, CA), and MAb anti-CD3 (Orthoclone, OKT3, Ortho Pharmaceutical Corporation, Raritan, NY) at 37^*C for four days. Cells were counted and resuspended every three to four days depending on growth in fresh media with additions.

Flow Cvtometrv. Cell surface marker analysis was performed on an EPICS ELITE cytofluorgraph using fluoresceinated or phycoerythrinated MAb as previously described (Triozzi, et al, 1994). The following MAbs were used: anti-CD3 (Leu4), - CD4 (Leu3a), -CD8 (Leu2a), -l ib (Leul5), -CD14 (LeuM3), and -CD56 (Leul9) (all from Becton-Dickinson); anti-CD30 (DAKO Corporation, Carpinteria, CA); anti- CDw60 (Pharmingen, San Diego, CA); anti-CD45RA (2H4) and -CD45RO (UCHL1; both from AMAC, Westbrook, ME); and anti-HLA-DR (HB103; ATCC, Rockville, MD).

Cvtolvtic Activity. Target cells were labeled with Na chromate (⁵¹Cr) using 100 μCi/5 X 10⁶ cells/0.5 ml for one hour at 37X. Target cells (lOVlOO μl) were added to triplicate 6-mm round-bottomed plates. Effector cells generated, as described above, were added in the same media (in 100 μl) to achieve effector-to-target (E:T) ratios of 40:1, 20:1, 5:1, and 1.5:1. Also included were three maximum release wells containing only labeled target cells plus TRITON X-100 surface active agent and three spontaneous release wells. The plate was centrifuged (200 X g for 5 minutes) and incubated at 37^*C for four hours. The plate was centrifuged again and 100 μl of supernatant was removed. The percent of lysis was determined by the formula: (experimental cpm - spontaneous cpm) / (total cpm - spontaneous cpm). Each variable was tested in triplicate. Cytokine Production. Standard semi-quantitative polymerase chain reaction

(PCR) was employed to assess cytokine mRNA expression. Total RNA was extracted by quanidium thiocyanate phenol chloroform using RNA-zol (Cinna/Biotec, Friendswood, TX). One μl of cDNA was mixed with 10 μl of 10 X PCR buffer (500 mM KCl, 200 mM Tris HCl, pH 8.3, MgCtø concentration optimized for each oligo set), 2 μl of 20 pM of the relevant oligonucleotide, 0.5 μl of 10 mM dNTP, and 1.2 U Taq polymerase (Ampli Taq, Perkin-Elmer Cetus) to a final volume of 100 μl. Amplification was performed with a Perkin-Elmer Cetus thermocycler for 30 cycles (1 min. 94°C melting, 2 min. 55°C annealing, and 2 min. 72^*C extension). To ensure that equal amounts of starting material was used, cDNA product were amplified with G3PDH primers. Amplified products were separated on a 2% agarose gel and blotted onto nylon filters. The filters were incubated in a pre-hybridization solution for four hours at 65^* C and then hybridized to a digoxigen-labeled internal Cb probe (Boehringer Mannheim, Indianapolis, IN). The filters were washed twice with 2 X SSC 0.5 SDS, then in 2 X SSC 0.1 SDS before being developed with dioxigenin-specific alkaline phosphatase-coupled antibody. mRNA expression was scored as: - (negative); + (positive); and ++ (strongly positive).

Commercially available enzyme-linked immunoabsorbent assay (ELISA) kits were used to quantify IL-4, TL-5, TL-6, GM-CSF (R&D Systems, Inc., Minneapolis, MN) and TFNg, and TNFa (Genzyme Corporation, Cambridge, MA) levels. Assays were conducted according to the recommendations of the manufacturers.

HTV-1 Co-Culture. Quantitative HIV-1 co-cultures were performed per the ACTG Virology Manual.

HΓV-1 mRNA. PCR was also used to quantify HTV-1 mRNA expression. mRNA was obtained and PCR performed as described above. To verify the presence of HTV- 1 in a sample, oligomer SK38 and SK39 were used (Varas, et al, "Influence of PCR Parameters on Amplifications of HTV-1 DNA: Establishment of Limiting Sensitivity", Biotechniques 11: 384-391, 1991). These oligomers ampkfy a 115-bp fragment of the HIV-1 gag gene. PCR was optimized using the SK38/SK39 primer set at the 0.8 mM dNTPS and 2.8 mM Mg+-

Proliferation Assay. Proliferation assays were performed by incubation 10⁵ lymphocytes, in both the presence and absence of IL-2, with synthetic oligopeptides corresponding to HTV-1 envelope sequence (The Ohio State University Comprehensive Cancer Center Peptide Laboratory). Cells were pulse-labeled with 1 μCi [³H] thymidine for one hour at 37"C and harvested using a Ska ron cell harvester.

HTV-1 Suppression Assay. The effects of culture supernatants on HIV-1 production were assessed using previously described methods (Mackewicz, et al, 1992, supra). Peripheral blood CD4+ T cells were isolated from a normal volunteer using negative selection (Human T cell CD4 Subset Column Kit, R&D Systems, Inc., Minneapolis, MN). These purified CD4⁺ cells were activated with 10 ng/ml OKT3 and grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco/BRL, Grand Island, NY) and 100 TU/ml IL-2. Cells were maintained between 0.5 and 2 x lOfyml by adding fresh complete medium weekly. CD4⁺ T cells (5 X lOfyml/well) were added to a 24-well plate containing either 20% or 80% of supernatants from lymph node cell expansion cultures from HIV- 1 -infected donors in which 5, 20, or 100 ng/ml OKT3 was used. Control wells were estabkshed from cells similarly expanded from the lymph node of a cancer patient. AD wells, except the "no virus" control wells, were infected with HIV-1 culture supernatant known to contain sufficient HTV-1 to infect lymphocyte cultures at the proportions used. Supernatants were collected from the 24-well plate at twice weekly intervals. At each collection, the same proportion of supernatants from the original expansion cultures was added. Day- 4 and day- 12 supernatants from the 24-well plate were analyzed by quantitative ELISA for HIV-1 p24 antigen (Coulter, Hiyalea, FL) as were the supernatants from the original expansion cultures. The amount of p24 produced was calculated by subtracting the p24 present in the 20% or 80% of the original expansion culture supernatant from the p24 detected in the 24-well plate. The p24 produced in control wells with fresh media alone was compared to the p24 produced in well with 20% or 80% supernatant from the original HTV- 1 lymph node expansion culture or from the control cancer patient expansion culture.

RESULTS Cell expansion. Lymph nodes were obtained from 12 HIV- 1 -infected donors. The number of cells obtained per lymph node specimen for immunologic studies varied from 0.3 x 10⁸ to 30 x 10⁸. In general, the CD4/CD8 ratio of lymph nodes paralleled that of the peripheral blood. Expansion of lymph node cells was examined in the presence of 600 TU/ml of TL-2 and a range of concentrations of anti-CD3 MAb. Initial exposure to lower concentrations of anti-CD3 MAb, namely 5 ng/ml, resulted in higher cell numbers and also favored CD4⁺ cell expansion at 10 days (Fig. 1). After 14 days of culture, the concentration of anti-CD3 MAb did not influence overall cell yields and cells were predominantly CD8⁺. It was determined that the concentration of IL-2 could be reduced after four days to 20 TU/ml without substantially reducing the expansion rates. Expansion was examined in a serum-free medium formulated to maintain the viability of APCs. Large cells having the morphologic appearance of macrophages were visuakzed in the cultures throughout. These adherent and semi-adherent cells were associated with lymphocyte colonies. Fold-expansions in serum-free conditions were equivalent to that of serum-supplemented media. There was a tendency for CD4+ ceU expansion to be greater in serum-free conditions. However, these differences were not statistically significant. Culturing lymph node cells initially at lOfyml in serum- free media with 5 ng ml of anti-CD3 MAb and 600 TU/ml IL-2 for the initial four days, resuspending cells at 0.25 x 10⁶ ceks/ml in media with 20 TU/ml TL-2 for three days, and then resuspending cells at 0.35 x 10⁶ cells/ml with media and 20 TU/ml 1-2 for three more days (total of 10 days) appeared to be optimal. Using this regimen, lymph node cells expanded 6.0-fold to 48.5-fold in 10-days; expansion rates declining after 10 days. The rate of cell expansion appeared to be positively related to the number of CD4+ cells present initially. Log-fold expansion, however, was achievable even from patients with peripheral blood CD4 counts of less than 50/mm³. The expansion rates of lymph nodes were greater than that of the peripheral blood cells. The percentage of CD4 and CD8 cells remained constant throughout the expansion process. Fig. 2 displays expansion of lymph node cells to peripheral blood for two representative patients.

Detailed phenotypic analysis was performed on cells from five patients using the optimal expansion regimen described above.

TABLE 1 Phenotype (% positive) of Cells Pre and Post Expansion

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5

Phenotype Pre Post Pre Post Pre Post Pre Post Pre Post

CD4+ 5 6 21 31 15 44 19 41 32 41

CD8+ 80 90 71 75 50 66 29 47 29 73

CD14+ 1 0 2 0 6 0 -- — -- —

CD56+ 3 6 5 7 ~ 15 5 12 — —

CD4+CD25+ 8 43 — — — -- — -- — —

CD4+CD45RA+ — 3 — 16 — 17 6 10 -- —

00

I CD4+CD45RO+ 11 64 24 57 13 — — 40 29 38

CD4+HLA-DR+ 23 84 29 90 34 91 28 95 — —

CD8+11B+ 3 2 5 2 1 2 1 0 2 3

CD8+CD30+ 21 33 24 65 1 44 2 30 10 37

CD8+CD45RA+ 88 85 73 69 — -- -- — — —

CD8+CD45RO+ 20 12 16 17 ~ ~ — -. -- —

CD8+CDw60+ 15 78 24 56 17 49 22 42 22 61

The majority of expanded ceks were CD8⁺. Importantly, >90% of ak of the CD8⁺ ceks were CDl lb", the phenotype of CTL and also CD8⁺ cells which inhibit HTV-1 repkcation non-lyticaky (Mackewicz, et al, 1992, supra). The expression of CDllb, the CR3 receptor, by CD8⁺ ceks has been associated with T- suppressor activity (Landay, et al, "Characterization of a Phenotypicaky Distinct Subpopulation of Leu-2⁺ Ceks which Suppress T Cell Prokferative Responses", J. Immunol. 131:2757- 2761, 1983). Approximately 3.0% - 16.9% of the CD8⁺ cells were CD45RA⁺, 34.0% - 65% were CD30⁺, and 42.0% - 78.7% were CDw60⁺, the phenotype of CD8⁺ T_H ceks (Manetti, et al, supra; Rieber, et al, "CDw60: A Marker for Human CD8⁺ T Helper Cells", /. Exp. Med. 170:1385-1390, 1994). Most of the CD4+ ceks that resulted expressed the activation and "memory" marker CD45RO. Very few CD56⁺ NK ceks (<5%) and no B cells (<1%) expanded. Less than 6% of the expanded expressed the monocyte/macrophage marker, CD 14.

Cytokine Production. Cytokine production was evaluated by assessing cytokine mRNA expression using standard semi-quantitative PCR, measuring cytokine levels of the culture supernatants using commercially avakable ELISA kits, and by measuring cytokine production (ELISA) of the expanded ceks exposed to immobilized anti-CD3 MAb. Expression of cytokine mRNA over the 10-day culture period is displayed in Table 2 below.

TABLE 2 Cytokine mRNA Expression

The addition of MAb anti-CD3 to TL-2 resulted in increased expression of TL-5 mRNA. TL-2/anti-CD3-expanded ceks consistently expressed mRNA for interferon (TFN) g, TL-1, TL-4, TL-8, tumor necrosis factor (TNF) a, and transforming growth factor (TGF) b-cytokines that have been shown to suppress HIV-1. High concentrations of GM-CSF were present in the culture supernatants (> 500 pg/ml). TL- 5, TL-6, IL-10, and TNF were also present in the cek culture supernatants; secretion of these cytokines increased in response to immobilized anti-CD MAb (Fig. 3). In general, maximal secretion of cytokines in response to immobkized anti-CD3 MAb was observed from ceks expanded for 8 to 10 days (Fig. 4). Note, also that MTP-la also was expressed. The importance of this expression recently has been reported by Cocci, et al, Sci., vol. 270, pp 1811 et seq. (1995).

HΓV-1 Production. Freshly collected lymph nodes grew out HIV-1; cultures of the expanded ceks were negative (n = 4). HTV-1 mRNA, determined by PCR, increased after 1 to 3 days of culture, by day 6 HTV- 1 mRNA it was undetectable. In contrast, HIV-1 mRNA remained positive in lymph node cells expanded in TL-2 alone (Fig. 2).

Effect of Culture Supernatants on HTV-1 Production. Culturing HTV- 1 -infected peripheral blood lymphocytes with immobkized anti-CD3 MAb and TL-2 has been shown previously by Levy and coworkers to induce the release of a soluble factor or factors that have been shown to inhibit HIV-1 rephcation in a non-major histocompatibikty complex (MHC)-restricted manner (Mackewicz, et al, 1994, supra). Whether culture conditions resulted in the production of soluble factors capable of inhibiting HTV-1 replication was evaluated by assaying the capacity of the supernatants to suppress HIV-1 replication in previously uninfected, normal CD4⁺ cells (Mackewicz, et al, 1994, supra). Wells with normal CD4+ cells infected with HTV-1 were highly positive for the p24 antigen at both day 4 and day 12; wells not infected with virus had no p24 antigen. Supernatant from lymph node cultures from HIV-1- infected donors inhibited replication of HIV-1 in the normal CD4⁺ cells infected with HTV-1 in vitro. Inhibition of HIV-1 rephcation was not observed at day 4 using 20% supernatant from HTV- 1 -infected lymph node cultures; the use of 80% supernatant, however, did result in inhibition (Fig. 6). Both 20% and 80% supernatant inhibited

HTV-1 rephcation at day 12. HIV-1 inhibition was greater when using the supernatant form lymph node cell expanded with the highest amount, namely 100 ng/ml, of OKT3.

Proliferative Responses. To examine the spectrum of HTV-reactivity, the proliferative response of expanded cell to oligopeptides corresponding to HIV-1 envelope sequence was assessed in the presence of autologous EBV-transformed B- cells as APCs. Significant proliferative response to all epitopes was observed (Fig. 7).

Cvtolvtic Activity. The cytotoxic activity of the expanded cells was assessed using ⁵¹Cr-release assays with a variety of target cells including autologous EBV- transformed B-cells pulsed with HTV-associated oligopeptides. Four-day expansion demonstrated significant cytotoxicity (% specific lysis greater than 40% at effector to target ratios of 10:1) versus NK-resistant Daudi cells, i.e., lymphokine activated kiker (LAK) cek activity. Ten-day expanded ceks were weakly cytolytic versus NK-cek sensitive Raji and NK-resistant Daudi cells (% specific lysis < 10% at effector to target ratio of 10:1). They did not specificaky lyse autologous EBV-transformed B-ceks that were pulsed with oligopeptides corresponding to the HIV-1 envelope sequence (% specific lysis < 10% at effector to target ratio of 10:1).

EXAMPLE II

IN VΓVO DATA

1. A pilot study was conducted at The Ohio State University (Columbus, Ohio) under an IND granted by the FDA in accordance with the teachings herein. 2. Ekgibility criteria in the study included: HIV-1 infection documented by ELISA and confirmed by either a clinical history compatible with HIV-1 disease, Western blot, positive HIV-1 culture, positive HIV-1 antigen, or plasma viremia; CD4 count of less then 200 but greater than 100/mm³ within 4 weeks of the time of lymph node excision; palpable peripheral lymph nodes that were considered to be easky accessible/resectable under local anesthesia; Karnofsky performance status greater than 70; adequate organ function defined by an absolute neutrophil count greater than 1000 mm³, hemoglobin greater than 9.5 g/dL, platelet count greater than 75,000/ mm³, creatinine less than 1.5 mg/dl, bilirubin less than 1.5 mg/dl, and hepatic transaminases less than 3 times the upper limit of normal; and signed informed consent.

3. Exclusion criteria included: any active ATDS-related opportunistic infection, encephalopathy, or makgnancy except mild Kaposi's sarcoma; significant autoimmune disease or non- AIDS -associated makgnancy; prior splenectomy; prior immunotherapy; or concurrent experimental HIV-1 therapy. Patients had to be receiving approved antiretroviral treatment and Pneumocystis carinii pneumonia

(PCP) prophylaxis, but there could not have been any change in therapy for the 4 weeks prior to enrollment.

4. Consenting, ekgible patients underwent excision of a clinically palpable lymph node. The lymph node was examined for malignancy and for opportunistic infection. If histologic and microbiologic examinations were negative, lymph node cells were processed and underwent an ex vivo expansion. Patients received a single infusion of autologous expanded cells into a peripheral vein. They were premedicated only with 650 mg of acetaminophen. Patients were monitored frequently for clinical and laboratory evidence of toxicity. Toxicity was graded using World Health Organization Common Toxicity Criteria. Virologic and immunologic response determinations 1 day, 2 days, 1 week, 2 weeks, and 5 weeks after cek infusion and thereafter every 4 weeks. Peripheral blood immunophenotyping was performed by The OSU Hospital Aids Cknical Trial Unit Cekular Immunology Laboratory. Viral loads were assessed by NASBA

(Advanced Bioscience Laboratory, Kensington, MD) and by quantitative polymerase chain reaction (PCR; Coming Clinical Laboratories, Pittsburgh, PA; and Lab Corp., Dubkn, OH). The sensitivity of these assays are 400 copies/sample (viz., 100 μl or 1 ml). 5. Adequate portions of each lymph node was used for histopathology; the remainder was used for therapy. Lymph nodes with histologic evidence of makgnancy or opportunistic infection were excluded. After extraneous fatty and connective tissue was removed, lymph nodes were minced into pieces approximately 2 to 3 mm in diameter under sterile conditions in a laminar flow hood. The minced lymph nodes were transferred to a 50 ml conical centrifuge tube and allowed to stand at 1 x g for one to two minutes in a serum-free medium (Macrophage SFM, Gibco BRL, Grand Island, NY) to allow dense capsular material to settle. Cells and supernatant were pipetted into a fresh 50 ml tube. The ceks were then centrifuged (250 x g) at room temperature for six minutes. The supernatant and any fatty accumulation on the surface were carefully aspirated and discarded. The cek peket was resuspended in fresh serum-free medium. An aliquot of cells was counted and viabikty assessed by Trypan blue exclusion.

6. Lymph node cells were cultured in serum-free medium at a density of 10⁶/ml with

10 ng/ml of anti-CD-3 monoclonal antibody (MAb) (Orthoclone, OKT3, Ort o Pharmaceutical Corporation, Raritan, NY) and recombinant human IL-2

(Proleukin, Cetus Oncology Corporation, Emeryville, CA) at 600 TU/ml. Cultures were maintained in air-porous plastic bags (Ethox, Buffalo, NY) in 5% CO₂ in humidified air at 37^*C. Ceks were resuspended after 3 to 4 days, depending on growth, at 0.25 x 10⁶/ml in fresh medium containing TL-2 at 600 TU/ml, and then resuspended after 3 to 4 more days at a density of 0.35 x 10⁶/ml in fresh medium containing TL-2 at 120 TU/ml. Ceks were harvested and washed in 1 L of 0.9% NaCl and 1.25% human serum albumin (American Red Cross) using a SteriCell harvester (E.I. DuPont de Nemours and Co., Glenolden, PA) and suspended in 300 ml of 0.9% NaCl, 1.25% normal human serum albumin in a transfer bag. The fokowing were performed to characterize the ceks prior to infusion: cek number and viabUity, endotoxin assay of final product supernatant, morphology of cytocentrifuge preparations stained with Diff Quik (Baxter Healthcare Corp., Miami, FL); gram stain; cultures for bacterial contamination; and HTV-1 mRNA. 7. RT-PCR was used to assess an HTV-1 expression. PCR primers were designed using primer analysis software (Oligo, National Biosciences, Inc., Plymouth, MN) and obtained from Stratagene (La Jolla, CA). The HTV-1 primer corresponded to the gag gene (5 '-primer = ATAATCCACCTATCCCAGTAGGAGAAAT; 3 -primer

TTTGGTCCTTGTCTTATGTCCAGAATGC; PCR product = 873 bp). Total RNA was extracted via guanidine isothiocyanate phenol using TRIzol (Gibco/BRL, Gaithersburg, MD). Equal starting concentrations of total RNA (0.5 μg) were used as a template for the RT reaction. RT synthesized cDNA (2 μL) was mixed with 1.8 μL of 10X PCT buffer (500 mM KCl, 200 mM Tris HCl, pH

8.3), 1.625 mM MgCl₂, 1 μL of the relevant 10 μM okgonucleotide primer set, and 0.5 U Taq Polymerase (Gibco/BRL) to a final volume of 20 μL. RNA from HTV- 1 -infected and uninfected cells were used as controls. GADPH cDNA was amplified for each sample to serve as an internal control (5'-primer = CTACTGGCGCTGCCAAGGCTGT; 3'-primer

GCCATGAGGTCCACCACCCTGT; PCR product = 358). Amplification was performed with a Perkin-Elmer Cetus thermocycler for 40 cycles of template denaturing (30 seconds at 94°C), annealing and extension (2 minute at 60^*C). Amplified products were separated on a 1% agarose gel and visualized using a UV kluminator.

8. Cek surface marker analysis was performed on an EPICS ELITE cytofluorograph using fluoresceinated and phycoerythrinated MAb as previously described (Triozzi, et al, "Identification of tumor-reacting lymph node lymphocytes in vivo using radiolabeled monoclonal antibody", Cancer, 73:580-589, 1994). The fokowing MAbs were used: anti-CD3 (LEU4), -CD4 (LEU3a), -CD8 (Leu2a), - l ib (Leul5), -CD14 (LeuM3), and -CD56 (Leul9) (all from Becton-Dickinson); anti-CD30 (DAKO Corporation, Carpinteria, CA); anti-CDwόO (PharMingen, San Diego, CA); anti-CD45RA (2H4) and -CD45RO (UCHL1; both from AMAC, Westbrook, ME); and anti-HLA-DR (HB103; ATCC, Rockvike, MD). 9. Expanded ceks were stimulated with irradiated (75 Gy), autologous, Epstein-Barr virus (EBV)-transformed B-cell lines transfected with either empty Vaccinia control vector or the Vaccinia vPE16 vector, which contains the HIV-1 env gene. The EBV-transformed cek lines were generated from peripheral blood lymphocytes using B95-8 marmoset cek line supernatant by standard methods (Blumberg, et al, "Effects of human immunodeficiency virus on the cekular immune response to Epstein-Barr virus in homosexual men: characterization of the cytotoxic response and lymphokine production, J. Infect. Dis., 155:877-890 1987). The vectors were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Health (Vaccinia control, Dr. Bernard Moss; vPE16, from Drs. Patricia Earl and Bernard Moss). Expanded ceks were cultured for four additional days with the stimulator ceks at a ratio of 10:1, and then supernatants were cokected. ELISA kits were used to quantify MTP-la and RANTES levels (R&D Systems Inc., Minneapolis, MN). Assays were conducted in dupkcate according to the recommendations of the manufacturer, and data are presented as mean values of these determinations.

10. Cells were cultured at 10:1 with irradiated, autologous EBV-transformed B-cek knes transfected with either empty Vaccinia control vector or Vaccinia vPE16 vector as described above. After four additional days, the cells were pulsed with Η-thymidine for four hours, harvested, and counted. Data are presented as mean cpm ± SD for quadrupkcate determinations.

11. Cytolytic activity versus the autologous B-ceks expressing env protein, natural kiker (NK) sensitive Raji cells, and NK-resistant Daudi cells was assessed at an effector-to-target ratio of 10:1 for four hours, as previously described (Triozzi, et al, supra). Also included were three maximum release wells containing only labeled target cells plus TRITON X-100 surfactant and three spontaneous release weks (no effector cells). The percent of lysis was determined by the formula: (experimental cpm - spontaneous cpm)/(total cpm - spontaneous cpm). Each variable was tested in triplicate; data are presented as mean ± SD. Expanded/activated lymph node lymphocytes from HIV-1 infected donors did not exhibit any specific cytolytic activity against autologous B cek targets expressing the HTV-1 envelope protein gpl20 (% specific lysis was less than 10% at an effector to target ratio of 10: 1.

12. Skin testing for a delayed type hypersensitivity (DTH) response to common recak antigens was performed prior to and four weeks after cek infusion using the

Multitest CMI skin test reaction file (Connaught Laboratories, Swiftwater, PA). Forty-eight hours after application, the largest perpendicular diameters were measured according to the recommendation of the manufacturers. Data are presented as the sum of the means of these determinations. A re-establishment of sensitivity to such antigens would establish immunorestoration of the patients.

Lymph nodes were resected from five patients. Characteristics of these patients are displayed in Table 3 below. TABLE 3 Patient Characteristics

Substantiaky fewer cells were obtained from two resections (Patients Nos. 3 and 4). On pathologic examination, these two lymph nodes were characterized by lymphocyte depletion and dense plasma cell and eosinophil infiltration replacing most of the normal lymph-node architecture. The three lymph nodes that yield more than 10⁸ ceks were characterized histologically by follicular and interfokicular hyperplasia. All lymph nodes were negative for malignancy and evidence of microorganisms, including acid fast bacilk and fungus.

Cells were expanded ex vivo for 10 to 14 days. Characteristics of the cells, which expanded 2.3 to 200-fold, are displayed in Table 4 below.

TABLE 4 Cell Characteristics

* Percent CD4 cek also positive for CD45RO. ^b Percent CD8 cell also positive for CD 1 lb or CDw60.

The expanded cells were predominantly CD8⁺ (Table 4). Less than 3% of the expanded ceks were CD14, CD19, or CD16 positive. When challenged and transfected autologous B-cell targets expressing TIIV-1 env protein, the expanded ceks proliferated and secreted MTP-la and RANTES. Expanded ceks were weakly cytolytic versus NK- cek sensitive Raji (data not shown), NK-resistant Daudi cells, and the autologous EBV- transformed B-cells expressing the env gene (Table 4). Expression of HIV-1 mRNA was negative by cells expanded from Patients Nos. 1, 2, and 5; it was positive by ceks expanded from Patents Nos. 3 and 4. Because of the persistent positivity, Patents Nos. 3 and 4 did not receive cell infusions and were excluded from the study. Patients Nos. 1, 2, and 5 received a single cell infusion. The cek infusion was wek tolerated with only mkd (grade 1) arthralgia reported by one patient. No acute or chronic toxicities were observed. Importantly, the increases in viral load that occurred the day after cek infusion (see below) were not associated with any clinical symptoms. Infectious complications, opportunistic or otherwise, have not been observed for up to 25 weeks after infusion.

Viral loads were performed by certified clinical laboratories using NASBA (copies/100 μL) or quantitative PCR methodologies (copies/mL, where indicated). Tables 5A-C displays the changes in viral load after infusion. As expected, viral load was observed to increase the day after cek infusion. Viral load was observed to decrease to undetectable levels (<400 copies/sample being the detection limit) four weeks after cell infusion.

Tables 5A-C display the changes in peripheral blood CD4 and CD8 lymphocyte counts. Significant changes attributable to cell infusion were/were not observed. Patient No. 2 manifested sustained increases in CD4 count that may have been due, in part, to better compliance with anti-retroviral therapy. As an assessment of immunocompetence, DTH skin test reactivity to common recak microbial antigens was evaluated. All patients were poorly reactive prior to cell infusion; skin test reactivity increased after cek infusion (see Tables 5 and 6, below).

TABLE 5A Patient 1 Results

* CD4, etc. counts are enrollment numbers 3 days prior to surgery.

TABLE 5B Patient 2 Results

* 2,000 copies/ml by PCR 4 days after surgery. ** copies/ml by PCR.

TABLE 5C

Patient 5 Results

* Copies/ml by PCR about 1 month prior to surgery.

TABLE 6 Skin Test Reaction

* Patients 3 and 4 were not treated.

I

OJ I

The results of this study indicate that a single infusion of autologous, noncytolytic, chemokine-producing, lymph node lymphocytes is safe and can mediate beneficial virologic and immunologic activity, even in patients with advanced disease. There were no adverse effects associated with the cek infusion. After a transient increase, HTV- 1 viral load was observed to decrease to undetectable levels.

Importantly, there was evidence of an improvement in immunologic function, as skin test reactivity to recak microbial antigens improved. Without the treatment described herein, negative skin test results would remain negative. "Immunorestoration" or "immunorestorative effect", as described herein, relates to the restoration of immune function (as measured by skin tests to antigens) which results by dint of the therapeutic administration of expanded lymph node lymphocytes as disclosed herein.

Technicaky, the procedure is easy, expedient, and reproducible. The technique employed as disclosed herein was not designed to maximize cytolytic activity, as ceks were exposed to decreasing concentrations of TL-2 in serum- free media.

EXAMPLE III MITOGENIC STIMULATION DATA In order to demonstrate the use of different mitogens in the stimulation of the lymph node lymphocytes, lymph node lymphocytes from an HIV+ donor were stimulated with three different mitogenic stimuli: (1) anti-CD3 and IL-2 (control); (2) anti-CD3, anti-CD28, and TL-2; and (3) phytohemagglutinin (PHA) and IL-2. Anti-CD3 was used at 10 ng/ml, anti-CD28 was used at 10 ng/ml, and PHA was used at 0.5 μg/ml. Instead of the usual 100 Cetus units/ml, only 20 Cetus units/ml was used throughout the culture period. Ak cultures were split 1:5 on day 5 of the culture (approximately 0.25 x 10⁶ cells/ml after split), then split to 0.35 x 10⁶ cells/ml on day 8 of culture, and then were harvested on day 11 of culture.

Ak 3 cultures proliferated in response to the mitogenic stimuli used, and all 3 cultures showed functional activity against HIV in that they cleared HTV from the cultures as determined by RT-PCR performed on day 11, as demonstrated in Table 7 below. TABLE 7

Fold Expansion

Mitogenic Stimulus After 8 Days After 11 Days HIV RT-PCT

Anti-CD3, TL-2 3.6 9.05 Negative

Anti-CD3, Anti-CD28, TL-2 10.0 25.14 Negative

PHA, TL-2 8.2 17.34 Negative

Phenotypicaky, all 3 cultures were similar. Also, all 3 cultures were 98%-99% CD3+ (T ceks) and displayed other surface markers in comparable quantities as demonstrated in Table 8, below.

TABLE 8

(1) Numbers represent the percentage positive cells for the designated marker(s).

(2) Brief explanation of phenotypic markers:

CD3 T ceks

CD44 HERMES antigen, lymphocyte homing receptor

CD4 Class Il-restricted T cells (helper cells)

CD45RO activation marker, "memory" T cells

CD8 Class I-restricted T cells (cytotoxic/suppressor cells)

CDllb Mac-1 adhesion molecule

CD8+CDl lb = cytotoxic T cell

CD56 Natural Killer cell (LAK, Lymphokine activated killer cell)

Taken together, these data indicate that equivalent stimulation results from using any of these mitogenic regimens. Equivalent results, thus, would be expected when using any of these stimulated cells in HfV+ patients as described in Example II, above. EXAMPLE IV

Culture Supernatants from Human Lymph Nodes Inhibit the in vitro Replication of Human Rhinoviruses A. Experimental design:

Cultured supernatants from human lymph nodes were tested for antiviral activity against a DNA virus (herpes simplex virus type 1, HSV-1) and an RNA virus (human rhinovirus type 13, HRV-13). Host ceks for antiviral studies were grown in 12- well tissue culture plates for 3 days before being used. After 3 days of cekular growth, mature monolayers were treated for 1-1/2 hours with either experimental material (lymph node supernatants) or control material (cell culture medium). The experimental material was used full strength (neat). After treatment of cek monolayers for 1-1/2 hours, viruses (HSV-1 orHRV-13) were added to monolayers and each virus was allowed to adsorb for 2 hours. Each virus was used at a low input of infection so individual viral plaques could be detected for quantitation in regards to plaque number and size. Following viral adsorption, all viral inoculums were removed from the monolayers and a 0.6% agarose overlay was added to each monolayer. Incorporated into the agarose overlay was either experimental material at a concentration of 80% (V/V) or control material at a concentration of 80% (V/V). After adding the agarose overlay medium, cultures were incubated for 5 days at the appropriate temperature (36.5° C for HSV-1 and 32° C for HRV-13). After 5 days of incubation, cultures were fixed and stained for the quantitation of plaque numbers and plaque size.

B. Results: Human rhinovirus type 13 was inhibited 50% by the experimental material when compared to the control material. In addition, plaque size was also reduced by more than 50% by the experimental material. Human HSV-1 was not inhibited by the test material (plaque numbers and plaque sizes were equivalent for control and experimental samples).

C. Conclusions:

Human rhinovirus type 13 was dramaticaky inhibited by human lymph node supernatant fluids. Inhibition occurred in regard to a reduction in plaque numbers and plaque size. Because of the experimental design and results (reduced plaque size, etc.), the data suggest that the antiviral activity in the experimental material was not due to a direct virocidal effect on the virus. Instead the antiviral activity appears to be targeted at some key viral function (s) involved with rephcation events within host cells. D. Specificity and controls for antiviral activity:

The experimental material was tested against 2 different viruses, an enveloped virus (HSV-1) and a nonenveloped virus (HRV-13). Both viruses were tested at the same time. In addition, the same test conditions were used for each virus. Only one virus was inhibited, the nonenveloped RNA virus. These results argue in support of an antiviral effect against a particular type of RNA virus. Appropriate controls for cek growth, pipetting, inoculum volumes, incubation conditions, and temperatures were included and should not be a factor in the results obtained. These data result from initial testing of the invention with other viruses than HIV. It is believed that these results would improve upon further testing and evaluation of the present invention and, thus, represent the potential that the present invention opens up in the treatment of immune mediated diseases in general and specificaky for chronic viruses.

EXAMPLE V Culture Supernatants from Human Lymph Nodes Inhibit the in vitro Replication of Herpes Simplex Virus (HSV-1) and Human Adenovirus Type 2 (Ad-2)

HSV-1 Virus Herpes Simplex Virus Type 1 (HS V- 1 )

Strain: F(l), ATCC VR-733 (American Type Culture Collection) Negative Control: EMEM with 2% fetal bovine serum (FBS) Test System: Vero monkey kidney cells Ad-2 Virus Human Adenovirus Type 2 (Ad-2)

Strain: Adenoid 6 (American Type Culture Collection) Negative Control: EMEM with 2% fetal bovine serum (FBS) Test System: A549 cells

The appropriate cells were seeded at 150 μl per well, prior to the day of inoculation. On the day of inoculation, the media was aspirated from each well. The stock virus was serially diluted in ten-fold steps in EMEM with 2% FBS. Each dilution of the virus was inoculated into 32 repkcate wells, at 50 μl/well. The viral inoculum was adsorbed for 70 ± 10 minutes at 36 ± 2^*C. After adsorption, the four replicates of each virus dkution were re-fed with an individual cek supernatant or medium control dkution. The cultures were incubated at 36 ± 2^*C until the final CPE observation on day 14 for HSV-1 and on day 17 for Ad-2. TABLE 9

HSV-1 Virus

Supernatant D (80%)

* Theoretical l ter. Note: Cytotoxicity was observed; however, CPE was evaluated

TABLE 10

HSV-1 Virus

Medium Control (80%)

Supernatants and controls titers were within 1 log of the certified titer. The lowest dkution of virus tested in 80% Supernatant D tested had no CPE present for the HSV-1 samples. CPE was observed in 2 of the 4 wells at the lowest dilution of virus tested in control medium. Thus, the noted Supernatant D was able to suppress or reduce virus infectivity as exemplified by HSV-1. While these tests were limited by avakable material, they do show the potential for the lymphocyte proliferation technique disclosed herein to be useful in treating chronic viral infections. Note, that these tests reked on the cek supernatants to achieve virus suppression as a means of screening viruses.

TABLE 11

Ad-2 Virus

Supernatant C (20%)

TABLE 12

Ad-2 Virus

Medium Control (20%)

Supernatants and controls titers were within 1 log of the certified titer. The lowest dilution of virus tested in 20% Supernatant C tested had CPE present in 3 of the 4 wells at the lowest dilution for the Ad-2 samples. CPE was observed in 4 of the 4 weks at the lowest dilution of virus tested in control medium. Thus, the noted Supernatant C was able to suppress or reduce virus infectivity as exemplified by Ad-2. While these tests were limited by available material, they do show the potential for the lymphocyte proliferation technique disclosed herein to be useful in acute viral infections. Note, that these tests relied on the cell supernatants to achieve virus suppression as a means of screening viruses.

Claims

We claim:

1. A method for preparing ceks for treating patients afflicted with human immunodeficiency virus (HIV), which comprises: subjecting ceks derived from lymph nodes excised from patients infected with

HTV to mitogenic stimulation in serum-free media for their expansion.

2. The method of claim 1, wherein said mitogenic stimulation includes the presence of Interleukin-2 (TL-2) and anti-CD3 monoclonal antibody.

3. The method of claim 2, wherein said anti-CD3 monoclonal antibody is present at between about 1 and 100 ng/ml and said TL-2 is present at about 600 TU/ml.

4. The method of claim 3, wherein the amount of TL-2 is lowered to about 120 IU/ml after 7 days of expansion.

5. The method of claim 4, wherein said expansion extends to at least about 10 days.

6. The method of claim 1, wherein said serum-free media comprises serum-free macrophage media.

7. The method of claim 2, wherein said serum-free media comprises serum-free macrophage media.

8. A therapeutic agent for treating patients afflicted with human immunodeficiency virus (HIV), which comprises: in a pharmaceuticaky-acceptable carrier, cytokine-producing cells having been produced by the step of subjecting cells derived from lymph nodes excised from patients infected with HIV to mitogenic stimulation in serum-free media for their expansion.

9. The therapeutic agent of claim 8, wherein said mitogenic stimulation includes the presence of Interleukin-2 (TL-2) and anti-CD3 monoclonal antibody.

10. The therapeutic agent of claim 9, wherein said anti-CD3 monoclonal antibody is present in an amount of between about 1 and 100 ng/ml and said IL-2 is present in an amount of about 600 TU/ml.

11. The therapeutic agent of claim 10, wherein the amount of IL-2 is lowered to about 120 TU/ml after 7 days of expansion.

12. The therapeutic agent of claim 11, wherein said expansion extends to at least about 10 days.

13. The therapeutic agent of claim 8, wherein said serum-free media comprises serum-free macrophage media.

14. The therapeutic agent of claim 9, wherein said serum-free media comprises serum-free macrophage media.

15. A method for treating patients afflicted with human immunodeficiency virus (HTV), which comprises: administering to said patient the therapeutic agent of claim 8.

16. A method for treating patients afflicted with human immunodeficiency virus (HTV), which comprises: administering to said patient the therapeutic agent of claim 9.

17. A method for treating patients afflicted with human immunodeficiency virus (HTV), which comprises: administering to said patient the therapeutic agent of claim 10.

18. A method for treating patients afflicted with human immunodeficiency virus (HTV), which comprises: administering to said patient the therapeutic agent of claim 11.

19. A method for treating patients afflicted with human immunodeficiency virus (HTV), which comprises: administering to said patient the therapeutic agent of claim 12.

20. A method for treating patients afflicted with human immunodeficiency virus (HTV), which comprises: administering to said patient the therapeutic agent of claim 13.

21. A method for treating patients afflicted with human immunodeficiency virus (HTV), which comprises: administering to said patient the therapeutic agent of claim 14.

22. A method for preparing an enriched population of helper cells from lymph nodes excised from patients afflicted with human immunodeficiency virus (HIV), which comprises: subjecting ceks derived from lymph nodes excised from patients infected with HTV to mitogenic stimulation in serum-free media for their expansion.

23. The method of claim 22, wherein said mitogenic stimulation includes the presence of Interleukin-2 (TL-2) and anti-CD3 monoclonal antibody.

24. The method of claim 23, wherein said anti-CD3 monoclonal antibody is present at between about 1 and 100 ng/ml and said TL-2 is present at about 600 TU/ml.

25. The method of claim 24, wherein the amount of IL-2 is lowered to about 120 TU/ml after 7 days of expansion.

26. The method of claim 25, wherein said expansion extends to at least about 10 days.

27. The method of claim 22, wherein said serum-free media comprises serum- free macrophage media.

28. The method of claim 23, wherein said serum-free media comprises serum-free macrophage media.

29. An enriched helper cell population expanded by the method of claim 22.

30. An enriched helper cell population expanded by the method of claim 23.

31. An enriched helper cell population expanded by the method of claim 24.

32. An enriched helper cell population expanded by the method of claim 25.

33. An enriched helper cell population expanded by the method of claim 26.

34. An enriched helper cell population expanded by the method of claim 27.

35. An enriched helper cell population expanded by the method of claim 28.

36. A method for preparing cells for treating patients afflicted with a disease that leads to an immunosuppressed state in the patient, which comprises: subjecting ceks derived from lymph nodes excised from patients infected with said disease to mitogenic stimulation in serum-free media for their expansion.

37. The method of claim 36, wherein said disease results from a persistent or acute virus, a bacterial infection, or an autoimmune disease.

38. The method of claim 37, wherein said persistent or acute virus in an enveloped or non-enveloped RNA or DNA virus.

39. The method of claim 38, wherein said persistent or acute RNA virus is selected from one or more of picornaviruses, togaviruses, paramyxoviruses, oπhomyxoviruses, rhandoviruses, reoviruses, retroviruses, bunyaviruses, coronaviruses, and arenaviruses.

40. The method of claim 38, wherein said persistent or acute DNA virus is selected from one or more of panoviruses, papoviruses, adenoviruses, herpesviruses, and poxviruses.

41. The method of claim 36, wherein said disease is selected from the group consisting essentiaky of HIV, tuberculosis, measles, dinghy fever, malaria, hepatitis (chronic), leprosy, rheumatoid arthritis, multiple sclerosis, and canine distemper virus.