Allogeneic and xenogeneic response in mixed leukocyte cultures.

The mixed lymphocyte culture (MLC) test has been regarded as an in vitro model of the recognition or sensitization phase of the homograft or graft-versus-host reaction. It has been suggested that the graft-versus-host response in vivo is less in xenogeneic combinations than in allogeneic ones and that there is a similar quantitative relationship in MLC responses. Given the above interpretation of the MLC test, this could suggest that the lesser reactivity in xenogeneic combinations may be due to a lesser recognition of the stimulus. We have done nine experiments testing allogeneic and xenogeneic combinations in MLC, largely in combinatorial fashion. The results indicate that the response in xenogeneic MLC may be as great as that in allogeneic MLC and that, as in different allogeneic mixtures, there is great variation in the extent to which xenogeneic mixtures may respond.

Responsiveness of germfree mice in mixed leukocyte culture. Reexamining the nature of the responding unit.

Conventional (cv) and germfree (gf) mice are able to give a good proliferative response to allogeneic cells in the mixed leukocyte culture (MLC) test, while the response to xenogeneic stimulating cells has been in question. Previous studies by others have suggested only a low MLC response in cv animals and none in gf ones. We have found that both cv and gf animals can give a good MLC response to xenogeneic as well as allogeneic cells. These findings are of importance for our understanding of both MLC stimulation and response.

Serologically defined and lymphocyte-defined components of the major histocompatibility complex in the mouse.

The mixed leukocyte culture (MLC) test is an in vitro model of the recognition phase of the homograft response. For the most part, activation in MLC is dependent on differences of the major histocompatibility complex (MHC). Our present studies in the mouse suggest that activation is primarily associated with differences of genetic regions of the MHC other than those which control the serologically defined (H-2) antigens. These differences do not lead to cytotoxic or agglutinating antibody formation despite extensive immunization; we have called these differences lymphocyte-defined (LD) differences. The strongest stimulation in MLC is associated with differences of the Ir region. It is possible that the Ir product is the T cell receptor and that it is this same molecule which can act as the stimulatory agent in MLC. Other possibilities are discussed.

Interleukin 7 enhances cytolytic T lymphocyte generation and induces lymphokine-activated killer cells from human peripheral blood.

The effects of purified recombinant interleukin 7 (IL-7) on the generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte culture (MLC) and on the induction of lymphokine-activated killer (LAK) cells in autologous cultures of human peripheral blood mononuclear cells were investigated. IL-7 was found to induce the generation of both CTL and LAK cells in bulk cultures. The appearance of peak CTL activity in MLC established with exogenous IL-7 was delayed in comparison with replicate cultures containing exogenous IL-2, but both cytokines stimulated quantitatively similar levels of antigen-specific lytic activity. An IL-2-neutralizing antiserum inhibited substantially, but not completely, the effect of IL-7 on CTL generation, implying the existence of both an indirect component of IL-7 activity via IL-2 utilization, as well as an IL-2-independent component. Cell surface phenotypic analysis of IL-2- or IL-7-generated CTL effector cells revealed that CD8+ cells were responsible for the vast majority of lytic activity. Limiting dilution analysis (LDA) revealed that essentially identical frequencies of CTL precursors (CTL-P) were capable of clonal expansion and/or differentiation in the presence of exogenous IL-2, IL-4, or IL-7, supporting the concept that all three of these cytokines are capable of exerting a major influence on T cell growth and differentiation. Approximately half of the CTL-P that responded in IL-7-supplemented LDA cultures did so in an IL-2-independent manner. IL-7 stimulated the development of LAK cells in autologous bulk cultures, but only weakly in comparison with IL-2. In contrast to its effects on CTL generation, the induction of LAK cells by IL-7 was virtually independent of IL-2. LAK cells induced by IL-7, like those induced by IL-2, were phenotypically heterogeneous and included CD8+, CD56+, and gamma/delta+ cells. Limiting dilution analysis indicated that IL-2 stimulated fivefold more LAK-P than IL-7 and 220-fold more than IL-4. Collectively, these data suggest that IL-7 has potent regulatory effects on human cytolytic cell populations and, either alone or in combination with other cytokines, could be important for the in vitro expansion of cells for adoptive immunotherapy.

Regulation of cytolytic cell populations from human peripheral blood by B cell stimulatory factor 1 (interleukin 4).

The effects of B cell stimulatory factor 1 (BSF-1) on the generation of human CTL and lymphokine-activated killer (LAK) cells in vitro were investigated. Both L-2 and BSF-1 were potent helper factors for the generation of antigen-specific CTL in MLC; detection of optimal BSF-1-induced CTL activity in this system occurred when BSF-1 was added to cultures after an initial period of activation during which exogenous BSF-1 was not present. In contrast to IL-2, BSF-1 failed to induce an LAK cell population, as detected with Daudi tumor targets, in cultures that had not been allosensitized. Furthermore, when both lymphokines were added together at culture initiation, BSF-1 inhibited the IL-2-driven generation of cytolytic cells. The differential ability of BSF-1 to promote the generation of CTL but not LAK could have important implications for lymphokine-mediated immunotherapy.

Human interleukin 4 receptor confers biological responsiveness and defines a novel receptor superfamily.

IL-4, a pleiotropic cytokine produced by T lymphocytes, plays an important role in immune responsiveness by regulating proliferation and differentiation of a variety of lymphoid and myeloid cells via binding to high affinity receptors. In this report we describe the isolation and functional expression of a human IL-4-R cDNA. When transfected into COS-7 cells, the cDNA encodes a 140-kD cell-surface protein. After transfection into a murine T cell line, the cDNA encodes a protein that binds human IL-4 with high affinity and can confer responsiveness to human IL-4. The predicted extracellular domain of the IL-4-R exhibits significant amino acid sequence homology with the beta subunit of the IL-2-R (p75), and the receptors for IL-6, erythropoietin, and prolactin. These receptors comprise a novel superfamily with extracellular domains characterized by four conserved cysteine residues and a double tryptophan-serine (WSXWS) motif located proximal to the transmembrane region.

Genetic and immunological complexity of major histocompatibility regions.

There are genetic differences within the major histocompatibility complex of the mouse which lead to skin graft rejection but which cannot be detected serologically. When confronted with these differences on allogeneic cells, lymphocytes proliferate in vitro. In other cases, in vitro lymphocyte proliferation but no skin graft rejection is associated with loci that are linked to but genetically separable from the loci controlling the serologically defined antigens.

Regulation of alloreactivity in vivo by a soluble form of the interleukin-1 receptor.

In vitro studies have shown that cytokines are involved in the regulation of the immune response, but their role in vivo is less well defined. Specific cytokine antagonists enable the identification of particular cytokines involved in the response and offer a means for modifying it. Systemic administration of a soluble, extracellular portion of the receptor for interleukin-1 (sIL-1R) had profound inhibitory effects on the development of in vivo alloreactivity. Survival of heterotopic heart allografts was prolonged from 12 days in controls to 17 days in mice treated with sIL-1R. Lymph node hyperplasia in response to a localized injection of allogeneic cells was completely blocked by sIL-1R treatment. The inhibition was overcome by simultaneous administration of interleukin-1 (IL-1); thus, sIL-1R acts by neutralizing IL-1. These results implicate IL-1 as a regulator of allograft rejection and demonstrate the in vivo biological efficacy of a soluble cytokine receptor.

cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily.

Interleukin-1 alpha and -1 beta (IL-1 alpha and IL-1 beta) are cytokines that participate in the regulation of immune responses, inflammatory reactions, and hematopoiesis. A direct expression strategy was used to clone the receptor for IL-1 from mouse T cells. The product of the cloned complementary DNA binds both IL-1 alpha and IL-1 beta in a manner indistinguishable from that of the native T cell IL-1 receptor. The extracellular, IL-1 binding portion of the receptor is 319 amino acids in length and is composed of three immunoglobulin-like domains. The cytoplasmic portion of the receptor is 217 amino acids long.

Nature and specificity of lymphokine independence induced by a selectable retroviral vector expressing v-src.

A murine retroviral vector, LSNLsrc, has been constructed and examined for its ability to induce growth factor independence in cells normally dependent on interleukin 2 (IL-2) or interleukin 3 (IL-3) for growth. The LSNLsrc vector coexpressed the v-src gene of Rous sarcoma virus and the neo gene from transposon Tn5, allowing infected cells to be selected on the basis of G418 resistance. The murine cell lines CTLL-2 and FD.C/1, which are dependent for growth on IL-2 and IL-3, respectively, were both readily infected with the LSNLsrc virus. LSNLsrc-infected, G418-resistant cultures of FD.C/1 cells were able to give rise to IL-3-independent progeny, but all G418-resistant CTLL-2 cells retained normal IL-2 dependence. The induction of IL-3 independence by v-src was not a direct event, since limiting dilution analysis of the LSNLsrc-infected FD.C/1 cells showed that most of them were IL-3 dependent, despite expression of v-src mRNA and active pp60v-src kinase. However, clones selected from this population in the presence of IL-3 were able to undergo a subsequent progression event and generate IL-3-independent progeny. The generation of factor-independent variants in the clonal cultures was a rare event, as witnessed by the death of most of the cells in each clone when IL-3 was withdrawn. Together, these data indicate that a secondary event, in addition to v-src expression, was required to generate IL-3-independent growth. No evidence was found for an autocrine mechanism of transformation involving IL-2, IL-3, interleukin 4, or granulocyte-macrophage colony-stimulating factor.

Induction of lymphokine-activated killer activity by interleukin 4 in human lymphocytes preactivated by interleukin 2 in vivo or in vitro.

In an attempt to augment the generation of human cytotoxic effector cells for potential cancer therapy with interleukin 2 (IL2) and lymphokine-activated killer (LAK) cells, the effect of interleukin 4 (IL4) on LAK cell induction was studied. In normal human peripheral blood lymphocytes (PBL), IL4 does not induce LAK activity and inhibits LAK induction by IL2. However, since lymphocyte activation, such as with antigen or mitogen, can render them responsive to IL4, the ability of IL4 to induce LAK activity in lymphocytes preactivated in vivo or in vitro with IL2 was investigated. PBL obtained from 12 patients with advanced cancer 1 to 3 days after IL2 therapy and from eight healthy control subjects were cultured 4 to 5 days with or without IL4 and/or IL2 and then tested for LAK activity as assessed by lysis of Daudi in a 4-h 51Cr release assay. In normal PBL, IL4 failed to induce LAK activity and consistently inhibited LAK induction by a suboptimal concentration of IL2 (10 units/ml). By contrast, IL4 induced LAK activity in PBL from seven of twelve IL2-treated patients and augmented LAK induction by the suboptimal IL2 in PBL from five of twelve IL2-treated patients. With an optimal LAK-inducing concentration of IL2 (1000 units/ml), IL4 less consistently inhibited LAK induction in normal PBL and had a variable effect upon LAK induction in PBL from IL2-treated patients. IL4 induced LAK activity in PBL obtained from a cancer patient after, but not before, systemic IL2 therapy. Similarly, IL4 induced LAK activity in normal PBL only after they had been preincubated with IL2. Thus, IL4 induces LAK activity in lymphocytes preactivated by IL2 in vivo or in vitro. Fluorescence-activated cell sorting revealed that the LAK activity, whether induced by IL4 or by IL2, was mediated largely by non-T (CD5-) natural killer-like (CD56+) cells. The results suggest a regulatory relationship between IL2 and IL4 in the induction and/or maintenance of LAK activity, which might be exploited to augment the generation of cytotoxic cells for lymphokine-mediated immunotherapy of human cancer.

Inhibition of cytokine function: potential in autoimmune disease.

Multiple lines of evidence implicate cytokines in the pathogenesis of autoimmune and inflammatory diseases. Neutralization of the biological activities of cytokines by interfering with ligand binding to specific cell-surface cytokine receptors thus represents an approach to controlling such diseases. Several agents, including novel, receptor-based antagonists, are being developed and tested in disease models.

Antigen specificity and helper characteristics of antigen-driven helper-cell-independent cytolytic T lymphocytes.

Antigen-driven, helper-cell-independent cytolytic T lymphocytes, which proliferate in response to antigenic stimulation independently of exogenous lymphokines, were characterized with regard to antigen specificity and helper factor production. Mapping data indicate that the determinants recognized by such cells for cytotoxicity and proliferation induction are identical. In addition, the cells were shown to function like helper cells in that they secrete soluble IL-2-like factor upon stimulation with the sensitizing antigens. The potential importance of this cell type in the immune response is discussed.

Recognition of major histocompatibility complex antigens on murine glial cells.

Recognition of autologous major histocompatibility complex (MHC) antigens by T cells is an essential step in the induction of an immunologic reaction to either endogenous or exogenous antigens. We investigated the ability of murine glial cells of different ages to stimulate clones of allospecific T lymphocytes. We also investigated the effects of supernatants from cultures of activated T cells on the immunologic recognition of MHC antigens on murine glial cells. Lymphocyte clones specific for Class I, Class II and non-MHC, background antigens were obtained from C57B1/6J-anti-DBA/2 mixed lymphocyte cultures. Glial cell cultures were prepared from newborn syngeneic (C57B1/6J) and allogeneic (DBA/2) mouse brains. Glial cultures 1-4 weeks of age were able to stimulate alpha-Class I-specific clones. No stimulation of alpha-Class II or alpha-background clones was noted. Incubation of glial cells with supernatants from cultures of alloantigen-activated spleen cells (C57B1/6J-anti-DBA/2) resulted in a decreased ability of glial cells to stimulate alpha-Class I responses. In contrast supernatant-treated cultures acquired the capacity to stimulate alpha-Class II-specific clones. No responses were noted in clones responsive to non-MHC antigens. The ability to stimulate alpha-Class II-specific clones was most prominent with one-week-old glial cultures and was lost by four weeks of culture. The increased susceptibility of younger glial cultures to the modulatory effects of lymphokines from activated T cells may be a factor in the increased susceptibility of the immature central nervous system to persistent viral infections and the development of autoimmune phenomena.

Mediation of cellular recruitment in vivo by functionally distinct T cell clones.

The ability of functionally distinct alloreactive T cell clones to mediate cellular recruitment in vivo was examined in a modified sponge matrix allograft model. Changes in cellular recruitment to paired healed s.c. urethane sponge grafts injected with cytolytic T lymphocyte (CTL), helper, or helper-independent CTL clones, or bulk resting mixed leukocyte culture (MLC) cells, and allogeneic or syngeneic blasts were studied. Injection of indium-111-labeled unsensitized cells i.v. was used to assess cellular recruitment to the graft site. All three alloreactive T cell clones and bulk MLC cells mediated preferential recruitment of circulating labeled cells when injected with allogeneic cells. The helper clone mediated significantly greater recruitment than the CTL clone. These results confirm at the clonal level our previous observations that populations of allosensitized cells enriched for either cytolytic or noncytolytic T lymphocytes can mediate cellular recruitment in vivo and extends them to include helper-cell-independent cytolytic T lymphocytes.

Interleukin-1 alpha-mediated promotion of long-term alloengraftment and short-term neutrophil expansion does not require the presence of either donor or host T cells.

In earlier studies, we showed that a 14-day continuous subcutaneous infusion of recombinant human interleukin (IL)-1 accelerated neutrophil recovery and enhanced long-term chimerism in a bone marrow (BM) transplant model in which T-cell-depleted BALB/c donor BM was given to irradiated C57BL/6 fully allogeneic recipients. We have extended these studies to a model entirely devoid of donor and host T cells. In the model, donor BALB/c congenic severe combined immunodeficient (C.B-17-scid/scid) BM cells are T cell depleted. The cells are then transplanted into adult irradiated C57BL/6 hosts that have been thymectomized and treated with anti-CD4 and CD8. When IL-1 alpha was delivered subcutaneously using a mini-osmotic pump, it enhanced short-term neutrophil recovery and longer term alloengraftment despite the absence of T cells in the donors and the hosts. Therefore, T cells were not required for the promotional effects of IL-1 alpha on neutrophil recovery and alloengraftment. Studies also showed that the potency of the IL-1 alpha effects was related to the degree of donor cell engraftment, which was related to the irradiation dose and the presence of T cells. We conclude that IL-1 alpha can augment post-BM transplantation hematopoietic recovery and alloengraftment via a T-cell-independent mechanism by favoring donor allogeneic hematopoietic progenitor cell competition over limited numbers of host progenitor cells.

IL-15 administration following syngeneic bone marrow transplantation prolongs survival of lymphoma bearing mice.

The toxicity and antitumor efficacy of simian IL-15 was compared with human IL-2 in the context of syngeneic BMT. Groups of mice receiving or not receiving anti-CD3 activated splenocytes, termed "T-activated killer" (T-AK) cells, were treated between days 7 and 12 with escalating doses of IL-2 or IL-15 given twice daily. Recipients of IL-2+T-AK or IL-15+T-AK had significantly higher survival rates than saline+T-AK. Tissues from IL-2+T-AK, but not IL-15+Y-AK, treated mice revealed the presence of perivascular infiltrates in the lung and liver consisting of CD8+ T cells and Mac-1+ cells. Our findings demonstrate that IL-15 can be used effectively to stimulate antitumor responses post-BMT and may be associated with less toxicity than IL-2.

Analysis of cloned T cell function. II. Differential blockade of various cloned T cell functions by cyclosporine.

Cyclosporine has profound suppressive effects on selected in vitro functions of cloned T lymphocytes. Cyclosporine inhibits the antigen-induced proliferation of the helper T cell clone 12-11. The effective dose required to reduce this response by 50% (ED50) is 28 ng/ml. In contrast, the proliferation of clone 12-11 induced by exogenous growth factors in secondary mixed lymphocyte culture supernatant (2 degrees MLC SN), is relatively insensitive to cyclosporine (ED50 = 4600 ng/ml). Furthermore, cyclosporine abrogates both antigen-induced and mitogen-induced secretion of lymphokines by clone 12-11, indicating that cloned helper T cell function is sensitive to cyclosporine even when interactions between specific alloantigens and their cell surface receptors are bypassed with mitogen. The suppressive effect of cyclosporine is not limited to helper T cell clones. The cytolytic T lymphocyte (CTL) clone 5MD2-2 is also sensitive to cyclosporine. Again, cyclosporine (100 ng/ml) blocks the antigen-driven, but not the exogenous lymphokine-driven, component of clone 5MD2-2 proliferation. This suppression does not result from the occlusion of antigen receptors or from antigen deformation by cyclosporine, because clone 5MD2-2 remains capable of antigen-specific cytolysis in the presence of cyclosporine concentrations that can suppress its proliferation. Finally, the ability of clone 5MD2-2 to remove IL-2 activity from culture media, a function that is significantly enhanced by contact with specific alloantigen, is not influenced by suppressive cyclosporine concentrations.

Precursor frequency and lytic specificity of interleukin 2 and interleukin 4-responsive murine lymphocytes.

The response to IL-2 and IL-4 of several functionally defined lymphoid cell types was assessed at the individual responding cell level in limiting dilution experiments in which exogenous IL-2 or IL-4 was used to promote cellular activation and expansion. Splenic precursors that give rise to alloreactive CTL in LDA supplemented with IL-2 were approximately 3-fold more frequent than those detected in IL-4. Of special interest was the observation that cytolytic cultures arising in primary allogeneic LDA supplemented with IL-4 exhibited a greater degree of lytic specificity than those arising in IL-2. This difference may be due to an inherently broader specificity of CTL cultured in IL-2 or to a concomitant activation of CTL and lymphokine-activated killer(s) (LAK) in individual IL-2 supplemented microcultures. In this regard, estimation of LAK precursor(s) (LAK-P) frequencies in LDA cultures established without an overt antigenic stimulus revealed that IL-2 activates a 20-fold higher frequency of LAK-P than does IL-4. Finally, an examination of the proliferative response to IL-2 and IL-4 of several CTL and PTL clones demonstrated that IL-4 responsive cells comprise a subset of cells that respond to IL-2, irrespective of their functional phenotype.

Inhibition of the effects of TNF in renal allograft recipients using recombinant human dimeric tumor necrosis factor receptors.

Tumor necrosis factor alpha (TNFa) and lymphotoxin (LT) or TNF beta are closely linked cytokines produced by macrophages and activated T lymphocytes, which play important regulatory roles in the immune response to allografts. They have also been implicated as mediators of the adverse reactions observed during OKT3 therapy. Therefore, anti-TNF agents could be useful both for immunosuppression and for limiting the systemic response observed in patients receiving OKT3. Recombinant TNFR:Fc is a fusion protein that binds TNFa and LT, thereby neutralizing their effects in vitro. The present study investigates the potential clinical application of TNFR:Fc in a nonhuman primate renal allograft model. Cynomolgus renal allograft recipients were treated with TNFR:Fc induction therapy alone or in combination with subtherapeutic doses of cyclosporine. Control animals received no immunosuppression or subtherapeutic cyclosporine. TNFR:Fc, administered as the only immunosuppressive agent, successfully prolonged renal allograft survival in the majority of treated animals. The prolongation of allograft survival was even more impressive when TNFR:Fc was combined with subtherapeutic doses of cyclosporine. Onset of rejection was significantly delayed as well in the TNFR:Fc treated groups. No adverse side effects were observed in any of the TNFR:Fc treated animals. Precursor cytotoxic T cells were detected in peripheral blood samples of treated recipients but the level of effector CTLs in vivo was below the threshold of detection. These results demonstrate that TNFR:Fc can be safely administered and is effective in prolonging renal allograft survival and in delaying the onset of rejection when administered alone or in combination with cyclosporine.