Tolerance and withdrawal of immunosuppressive drugs in patients given kidney and hematopoietic cell transplants.

Sixteen patients conditioned with total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) were given kidney transplants and an injection of CD34+ hematopoietic progenitor cells and T cells from HLA-matched donors in a tolerance induction protocol. Blood cell monitoring included changes in chimerism, balance of T-cell subsets and responses to donor alloantigens. Fifteen patients developed multilineage chimerism without graft-versus-host disease (GVHD), and eight with chimerism for at least 6 months were withdrawn from antirejection medications for 1-3 years (mean, 28 months) without subsequent rejection episodes. Four chimeric patients have just completed or are in the midst of drug withdrawal, and four patients were not withdrawn due to return of underlying disease or rejection episodes. Blood cells from all patients showed early high ratios of CD4+CD25+ regulatory T cells and NKT cells versus conventional naive CD4+ T cells, and those off drugs showed specific unresponsiveness to donor alloantigens. In conclusion, TLI and ATG promoted the development of persistent chimerism and tolerance in a cohort of patients given kidney transplants and hematopoietic donor cell infusions. All 16 patients had excellent graft function at the last observation point with or without maintenance drugs.

Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells.

In this pilot study, we investigated the ability of autologous dendritic cells pulsed ex vivo with tumor-specific idiotype protein to stimulate host antitumor immunity when infused as a vaccine. Four patients with follicular B-cell lymphoma received a series of three or four infusions of antigen-pulsed dendritic cells followed, in each instance, by subcutaneous injections of soluble antigen two weeks later. All patients developed measurable antitumor cellular immune responses. In addition, clinical responses have been measured with one patient experiencing complete tumor regression, a second patient having partial tumor regression, and a third patient resolving all evidence of disease as detected by a sensitive tumor-specific molecular analysis.

Antibodies to membrane structures that distinguish suppressor/cytotoxic and helper T lymphocyte subpopulations block the mixed leukocyte reaction in man.

Two major subsets of human T lymphocytes that are functionally analogous to the mouse Lyt-2+ and Lyt-2- subsets have been defined by their expression of two thymus-dependent membrane antigens, Leu-2 and Leu-3. Leu-2+,3- cells have suppressor/cytotoxic functions and Leu-2-,3+ cells have helper functions. These studies were designed to determine the effects of adding IgG1 monoclonal anti-Leu-2 and anti-Leu-3 antibodies to the mixed leukocyte reaction (MLR). At high concentrations, each antibody partially inhibited the proliferative response of unseparated T cells and abolished the response of the isolated subset having the appropriate phenotype. An IgG1 monoclonal antibody to HLA-A2 and an IgG2a antibody to Leu-1, a pan-T antigen, failed to inhibited the MLR. These results suggest that the Leu-2 and Leu-3 antigens may have a direct role in the mechanism whereby T cells recognize and respond to alloantigen.

Ia antigen on peripheral blood mononuclear leukocytes in man. II. Functional studies of HLA-DR-positive T cells activated in mixed lymphocyte reactions.

Blast transformation of T cells in response to allogeneic lymphocytes is followed by expression of HLA-DR antigen on up to 60% of the T cells. Murine monoclonal antibody to HLA-DR antigen was used to separate alloactivated T cells into those T cells that express high quantities of DR antigen (DR+) and those that express little or no DR antigen (DR-), and each population was tested in a variety of assays. DR+, but not DR-, T cells stimulated fresh allogeneic and autologous T cells to proliferate and supported proliferation by fresh autologous T cells to soluble antigens. Alloactivated T cells were suppressive of fresh mixed lymphocyte reactions (MLR) and suppression by irradiated DR+ T cells was specific for the DR antigens of the initial stimulator cell. Suppression of the MLR by DR+ T cells was not a result of altered kinetics or cell-mediated cytotoxicity. DR+ T cells released soluble factors that suppressed fresh allogeneic responses. These data indicate that alloactivated DR+ T cells may provide antigen-specific feedback inhibition of the MLR.

Ia antigens on alloreactive T cells in man detected by monoclonal antibodies. Evidence for synthesis of HLA-D/DR molecules of the responder type.

A monoclonal antibody (Ab 2.06) directed against a nonpolymorphic determinant of HLA-D/DR molecules was used to study the expression and the biosynthesis of Ia molecules on human T cells before and after allogeneic stimulation. Normal resting peripheral T cells failed to synthesize and/or express Ia antigens. However, at day 7 of a mixed lymphocyte reaction, 40-60% of alloreactive T cells express and synthesize HLA-D/DR molecules of the responder type as assessed by two-dimensional gel electrophoresis genotyping. This Ia+ alloreactive population originates from an Ia - T cell pool and not from an Ia+ T cell population. Moreover, the two-dimensional polyacrylamide gel electrophoresis pattern of Ia on T cells is similar to that obtained with B cells from the same individual.

Ia antigen on peripheral blood mononuclear leukocytes in man. I. Expression, biosynthesis, and function of HLA-DR antigen non-T cells.

Murine monoclonal antibodies to monomorphic components of HLA-DR antigen were used to analyze the distribution and function of DR molecules on non-T mononuclear leukocytes from peripheral blood. On the basis of indirect immunofluorescence and complement-mediated cytolysis. DR antigen was detected on approximately 70% of non-T cells (DR+) and could not be detected on approximately 30% of non-T cells (DR-). A fluorescence-activated cell sorter was used to separate non-T cells into DR+ and DR- populations, and each population was studied. At least one-third of DR- cells were monocytes, and the remainder were surface-immunoglobulin-negative lymphocytes. Analysis of [35S]methionine-labeled proteins by the method of two-dimensional polyacrylamide gel electrophoresis indicated that DR+, but not DR-, cells biosynthesize DR molecules DR+ cells stimulated strongly in the autologous and allogeneic mixed lymphocyte reactions (MLR) and supported T cell proliferation to soluble antigens, whereas DR- cells stimulated in the allogeneic MLR but failed to stimulate in the autologous MLR or to support T cell proliferation to soluble antigens. When present continuously in culture, one monoclonal anti-DR antibody (antibody 2.06) modestly inhibited T cell proliferative responses. Another antibody (antibody 1.35) markedly enhanced the autologous MLR and the proliferative response to soluble antigens, but had no effect on the allogeneic MLR. These data suggest that DR+ and DR- non-T populations are functionally distinct, and that DR antigen may be required for presentation of soluble antigen and stimulation in the autologous MLR. Antigens in addition to DR may stimulate allogeneic T cell proliferation.

Three HLA-D region antigens defined by primed LD typing.

We have recently described a new method, primed LD typing or PLT, for specific identification of HLA-D antigens. Highly discriminatory PLT cells have been developed which clearly differentiate between cells of individuals that restimulate strongly and those that restimulate weakly. Seven such discriminatory PLT cells have been used to define three antigens called PL1, PL2, and PL3; two more PLT cells may define antigen(s) PL4.

Activation of antigen-specific suppressor T cells in the presence of cyclosporin requires interactions between T cells of inducer and suppressor lineage.

To probe the mechanism of suppressor T cell generation in man, we have carried out mixed leukocyte reactions (MLR) in the presence of cyclosporin (CsA), a fungal metabolite which prevents the generation of cytotoxic lymphocytes while permitting activation of suppressor cells. After a 12-d MLR in the presence of 1 microgram/ml CsA, T cells were fractionated into subsets with monoclonal antibodies, and each subset was tested for the ability to inhibit a second fresh MLR that is devoid of CsA. The results show that Leu 2+ T cells derived from the first culture suppress the second MLR in an HLA-DR antigen-specific manner and in the absence of detectable lysis of stimulator cells. However, Leu 2+ cells do not develop into suppressor cells unless acted upon by alloantigen-primed Leu 3+ inducer cells. Furthermore, only those Leu 3+ cells that also express the Leu 8 marker (Leu 3+, 8+) are capable of inducing suppressor cells. Thus, antigen-specific feedback inhibition of an immune response in man results from an ordered series of interactions between T cells of distinct phenotype.

Autologous mixed lymphocyte reaction in patients with Hodgkin's disease. Evidence for a T cell defect.

The proliferative response of T lymphocytes cultured with autologous non-T lymphocytes is known as the autologous mixed lymphocyte reaction (MLR). This reaction can be demonstrated reproducibly in healthy individuals and has been shown to generate specific cytotoxic T cells, as well as T cells that regulate antibody synthesis and cell-mediated immunity. In this study, we demonstrate that the autologous MLR is impaired or absent in most patients with Hodgkin's disease regardless of age, sex, pathologic stage, or histologic classification. In 64 patients, the mean autologous MLR was 3,084+/-1,878 cpm compared to 16,552+/-6,532 in 29 healthy donors. A defect in autologous MLR was observed in newly diagnosed patients before the initiation of therapy, but was also found in patients without evidence of recurrent disease up to 15 yr after treatment. These findings could not be explained by abnormal kinetics or poor viability of stimulator or responder cells. The possibility that suppressor cells are responsible for the reduction of T cell autoreactivity was examined by comparing the autologous MLR of a healthy HLA-identical sibling in the presence and absence of T or non-T cells of an affected sibling. No inhibitory effects were observed. Similarly, substitution of patient plasma for pooled AB serum failed to inhibit the autologous responses of normal donors. Increasing the number of responder T cells in the culture or removing adherent cells from the stimulator population enhanced autoreactivity in some patients, indicating that the defect is not absolute. In two families, T cells of healthy HLA-A, B, and DR-identical siblings of patients responded normally to the non-T cells of their affected siblings, whereas patients' T cells failed to respond both to their own stimulator cells and those of their healthy HLA-identical siblings. These data indicate that the impairment of autologous MLR in some patients is due to a reduction or dysfunction of responder T cell activity and not to a defect of autologous stimulator cells.

Inhibition of cell mediated immune responses by 8-methoxypsoralen and long-wave ultraviolet light: a possible explanation for the clinical effects of photoactivated psoralen.

Human thymus-derived lymphocytes proliferate when cultured with lymphocyes or epidermal cells from unrelated individuals because such cells express HLA-D antigens, which are recognized as foreign by thymus-derived lymphocytes. The current study demonstrates that these responses are inhibited if either the stimulator cells or responder cells are pretreated with the combination of 8-methoxypsoralen and long-wave ultraviolet light. Additional studies revealed that photoactivated 8-methoxypsoralen has a lethal effect on lymphocytes and monocytes but not on the majority of epidermal cells. These obervations suggest that the dramatic beneficial effect of PUVA on patients with psoriasis and other skin disorders may be due to a toxic effect on immunocompetent cells in the epidermis, which results in inhibition of cell mediated immune responses.

Cultured human epidermal cells do not synthesize HLA-DR.

All nucleated cells express HLA-A, B, and C antigens. However, only a few cells, including epidermal cells, demonstrate HLA-DR antigens which are potent transplantation immunogens in man. The current study was undertaken to determine if epidermal cell continue to synthesize and/or express HLA-DR antigens after prolonged in vitro culture. Epidermal cells cultured for 7 days or more no longer stimulated allogeneic lymphocytes in the epidermal cell-lymphocyte reaction. Indirect immunofluorescence light microscopy of cultured cells using mouse monoclonal antibody to HLA-DR antigen confirmed that these cells do not express HLA-DR antigens whereas they retain beta 2-microglobulin. Detergent extracts of 12-day cultured epidermal cells biosynthetically labeled with 35 S-methionine were immunoprecipitated with monoclonal anti-DR antibody and analyzed by the method of two-dimensional polyacrylamide gel electrophoresis. No radiolabeled proteins were found on these gels in the regions where HLA-DR molecules are known to migrate. These data indicate that HLA-DR antigen is absent from cultured epidermal cells. Finally, we describe a technique for growing epidermal cells on a gelatin membrane which allows subsequent removal of intact cell monolayers from the culture dish. Such monolayers may be useful for purposes of transplantation.

Use of the fluorescence-activated cell sorter to quantitate and enrich for subpopulations of human skin cells.

A variety of immunologic staining techniques were compared in a quantitative study of antigen expression by human epidermal cells. Virtually all nucleated epidermal cells express beta 2-microglobulin, which is associated with HLA-A, -B, and -C antigens, whereas only about 4% expressed T6, an antigen expressed by Langerhans cells but not other cells in the skin. With the fluorescence-activated cell sorter (FACS), epidermal cell suspensions were selectively enriched 10- to 15-fold for T6-positive Langerhans cells. An average of 6.5% of cells were specifically stained by anti-HLA-DR antibody. When dispersed cells stained with anti-DR plus peroxidase were examined with the technique of immunoelectron microscopy, only mononuclear leukocytes (probably Langerhans cells) were stained. After separating HLA-DR positive skin cells with the FACS, the DR-positive population but not the DR-negative population stimulated proliferation of allogeneic responder lymphocytes, indicating that sorted cells are metabolically active. We conclude that HLA-DR antigen is not expressed by keratinocytes in normal human skin cell suspensions and that the FACS can be used to selectively enrich or deplete skin cell suspensions of antigenically distinct subpopulations such as Langerhans cells.

Clinical transplantation tolerance twelve years after prospective withdrawal of immunosuppressive drugs: studies of chimerism and anti-donor reactivity.

BACKGROUND: Previous studies showed the feasibility of inducing transplantation tolerance to cadaveric renal allografts in patients given pretransplant total lymphoid irradiation (TLI). Microchimerism has been theorized to be an important or necessary factor in long-term graft acceptance and tolerance in humans. METHODS: A cadaveric renal transplant recipient given pretransplant total lymphoid irradiation and withdrawn from immunosuppressive drugs more than 12 years ago was tested for microchimerism using a sensitive nested polymerase chain reaction technique, and for anti-donor reactivity using the mixed leukocyte reaction and an ELISA screen for anti-HLA antibodies. Donor and recipient were mismatched for all HLA-A, B, and DR antigens. RESULTS: The "tolerant" recipient had good graft function, no detectable donor-type cells in the blood by polymerase chain reaction analysis, vigorous reactivity to donor stimulator cells in the mixed leukocyte reaction, and no detectable serum anti-HLA antibodies. CONCLUSIONS: Operational tolerance to HLA-A, B, and DR mismatched organ allografts can be induced prospectively in humans for at least 12 years after withdrawal of immunosuppressive drugs. The allograft can be maintained in the absence of detectable donor microchimerism and in the presence of anti-donor reactivity in the mixed leukocyte reaction, suggesting that neither chimerism nor clonal deletion or anergy of recipient T cells to alloantigens presented by donor Class II HLA molecules is required for persistence of the tolerant state using this total lymphoid irradiation protocol.

A pilot clinical trial of HIV antigen-pulsed allogeneic and autologous dendritic cell therapy in HIV-infected patients.

A pilot study was carried out to assess the safety and antigen-presenting properties of allogeneic or autologous dendritic cells (DCs) in six HLA-A2+, HIV-infected patients. Allogeneic DCs obtained from the peripheral blood of HLA-identical, HIV-seronegative siblings were pulsed with recombinant HIV-1 MN gp160 or synthetic peptides corresponding to HLA-A2-restricted cytotoxic epitopes of envelope, Gag, and Pol proteins. The antigen-pulsed cells were infused intravenously six to nine times at monthly intervals and HIV-specific immune responses were monitored. One allogeneic DC recipient with a CD4+ T cell count of 460/mm3 showed increases in envelope-specific CTL- and lymphocyte-proliferative responses, as well as in IFN-gamma and IL-2 production. Another allogeneic DC recipient with a CD4+ T cell count of 434/mm3 also showed an increase in HIV envelope-specific lymphocyte-proliferative responses. A recipient of autologous DCs with a CD4+ T cell count of 730/mm3 showed an increase in peptide-specific lymphocyte-proliferative responses after three infusions. Three other allogeneic DC recipients with CD4+ T cell counts <410/mm3 did not show increases in their HIV-specific immune responses. No clinically significant adverse effects were noted in this study and CD4+ T cell numbers and plasma HIV-1 RNA detected by RT-PCR of all six patients were stable during the study period. Thus, both allogeneic and autologous DC infusions were well tolerated and in patients with normal or near normal CD4+ T cell counts administration of these antigen-pulsed cells enhanced the immune response to HIV. However, since no effect on viral load was observed there was no evidence that this approach provided clinical benefit.

Allogeneic dendritic cell induction of HIV-specific cytotoxic T lymphocyte responses from T cells of HIV type 1-infected and uninfected individuals.

The potential benefit of T cell-based vaccination for HIV-1 infection remains to be determined. Cytotoxic T lymphocytes (CTLs) appear to clear substantial populations of HIV-1 virus in vivo, although CTL activity may contribute to the decline in CD4+ T cell count observed in the course of the disease. To investigate further the role of specific CTL responses in the control of HIV-1 replication, we raised primary CTL lines against a panel of conserved HIV-1 epitopes using blood-derived dendritic cells as antigen-presenting cells (APCs). Specific primary human CTL responses were induced against HLA-A*0201-restricted peptides with dendritic cells from HIV-1-seronegative donors. This method of immunization elicited cytotoxic activities capable of recognizing endogenously processed antigen. The CTL induction protocol was extended in order to explore the capacity of HLA-matched allogeneic dendritic cells to evoke novel CTL responses in T cells from an HIV-seropositive asymptomatic individual. Allogeneic peptide-pulsed dendritic cells from a healthy sibling were capable of eliciting a CTL response directed against an HIV epitope (env814: SLLNATDIAV) that was initially not detected in the CTL effector population of the HIV-1-infected patient. The possibility of manipulating CTL specificity directed against multiple conserved HIV-1 epitopes represents a significant step in the evaluation of T cell-based vaccination for treatment of disease.

Generation of primary peptide-specific CD8+ cytotoxic T-lymphocytes in vitro using allogeneic dendritic cells.

Dendritic cells (DC) are potent antigen-presenting cells (APC) capable of inducing strong T-cell-mediated immunity. Infusion of lymphoma-specific antigen-loaded autologous DC has been demonstrated to result in the generation of antigen-specific immunity and reduction in tumor burden in B-cell lymphoma patients. Cellular immunotherapy employing antigen-loaded DC could have a potential therapeutic impact in tumors and viral infections, including HIV infection. However, DC in HIV-infected individuals and breast cancer patients are believed to be functionally defective. Therefore, the potential of using allogeneic DC offers significant implications for DC immunotherapy in AIDS and immunocompromised cancer patients. To explore the potential of allogeneic DC therapy in vivo, we tested the ability of allogeneic DC to generate primary peptide-specific CD8+ cytotoxic T-lymphocyte (CTL) responses in vitro. Our results indicate that DC from HLA class I-matched individuals elicit primary immune responses in vitro using viral peptides as naive antigens. A primary peptide-specific immune response could also be detected even when only one HLA allele (HLA-A*0201) was matched between the allogeneic DC and T-lymphocytes. The ability to generate primary peptide-specific responses in vitro is strongly indicative of the in vivo therapeutic potential of allogeneic DC.