Failure of TGF β1 and IL-12 to regulate human FasL and mTNF alloreactive cytotoxic T-cell pathways

Abstract: The effect of TGFβ1 and IL-12 on calcium-independent cytotoxic pathways was investigated. We have previously demonstrated that the regulatory effect of TGFβ1 and IL-12 on human alloreative CTL activity was associated with regulation of perform and granzyme B gene expression. To determine the effect of both cytokines on the alternative cytotoxic pathway involving FasL and mTNF, we first investigated the expression of both molecules on human primary alloactivated T cells. Our results show that human allostimulated T lymphocytes express FasL. Cell lysis experiments demonstrate that the FasL cytotoxic pathway is involved in the killing of specific target cells mediated by human alloreactive CTL. In addition, allogeneic stimulation induced significant mTNF expression on both CD4+ and CD8+ responder T cells. Using TNF-sensitive target cells, we also demonstrate that the mTNF-mediated cytotoxic pathway is involved in the cytotoxic activity of human primary allostimulated T lymphocytes. Neither TGFβ1 nor IL-12 had an effect on FasL or mTNF expression. Furthermore, addition of TGFβ1 or IL-12 at the initiation of the MLR had no significant effect on Fas- and mTNF-mediated cytotoxicity.
Taken together, our results provide a novel insight into the differences between regulation by cytokines of perforin-dependent and -independent cytotoxic mechanisms. Unlike their role in the perforin/granzyme B pathway, TGFβ1 and IL-12 do not appear to mediate any regulatory effect on FasL and mTNF cytotoxic pathways used by human alloreactive primary CTL.     

Generation of cytotoxic lymphocytes in lympho-epidermal mixed cultures in man

The ability of human normal skin epidermal cells (EC) to induce the generation of alloreactive cytotoxic T lymphocytes (CTL) was investigated in vitro using the Mixed Skin Cell lymphocyte Reaction (MSLR) model. In human MSLR, EC stimulated the proliferation of allogeneic peripheral blood lymphocytes (L) as measured, after 6 days, by 3H-thymidine uptake. In parallel, the generation of alloreactive CTL was tested in 18 hr 51CR release assays against L targets (targets autologous to EC that stimulated in MSLR). Allogeneic, not autologous MSLR, lead to the generation of CTL; alloreactive CTL were not generated against targets allogeneic to stimulating EC; no CTL activity occurred without previous stimulation by EC. These data indicate that in vitro MSLR may provide an useful tool for the investigation of lympho-epidermal interactions in man and our understanding of lymphocytotoxicity mechanisms that occur in vivo in response and/or directed to epidermal constituents .     

Interleukin-4 Bypass of the Immunosuppressive Effect Mediated by Interleukin-2 Receptor Antibodies

Numerous studies have demonstrated that the generation of alloreactive effector cells depends on cytokines. Conversely, there is evidence that cytokine metabolism is altered at the clonal level in tolerant chimaeras. This has led to preclinical and clinical studies using antibodies that antagonize interleukin-2 (IL-2), with the hope of achieving immunosuppression and inducing tolerance. Monoclonal antibodies against the α-chain (p55) of the human IL-2 receptor are being applied to prevent transplant rejection and graft-versus-host disease in several clinical trials. The antibodies that have been applied clinically so far antagonize the binding of IL-2 to the IL-2 receptor α-chain which is part of the high affinity IL-2 receptor, but they do not deplete the receptor-bearing cells. Our study investigates the immunosuppressive effect of monoclonal antibodies against the α-chain (p55) and β-chain (p75). In mixed lymphocyte cultures the p55 antibody causes a reduction in T-cell proliferation to about 50%. The generation of cytotoxic T cells is reduced more effectively (up to 80%). By additional blocking of the IL-2 receptor β-chain we achieved an additional but still incomplete immunosuppressive effect. Moreover we show that IL-2 receptor-blocked alloreactive T cells escape suppression by using IL-4 as an alternative stimulating signal. To prevent T lymphocytes benefiting from this alternative and thwarting the immunosuppressive effect, cytotoxic IL-2 receptor antibodies that deplete the high affinity receptor-bearing cells are needed.     

Regulation of the IL-10 production by human T cells

Interleukin (IL)-10, an immunomodulatory cytokine predominantly produced by monocytes/macrophages and T cells, inhibits several functions of dendritic cells (DC), monocytes and T cells including their cytokine production, but it stimulates B cell immunoglobulin (Ig) production and cytotoxic T lymphocyte (CTL) generation. A precise knowledge of the mechanisms that control the IL-10 production is therefore highly important for understanding the immunoregulation. The IL-10 production was studied in cultures of freshly isolated human T cells. A rise in intracellular calcium as well as the common gamma-chain containing cytokine receptor triggering or CD28 triggering were found to be important signals for IL-10 induction. CD80, CD58, rIL-12 and rIFN-alpha all had efficacious and independent costimulatory activities on the IL-10 production, while PGE2 was inhibitory. Dependence on autocrine IL-2 signalling was shown by the effects of anti-IL-2 and anti-IL-2R monoclonal antibodies (MoAb), but the IL-10 production proceeded partly IL-2-independent when CD80 provided costimulation. Sensitivity to inhibition by CsA was not removed by CD80 or CD58 costimulation and/or by addition of rIL-12 or rIFN-alpha, pointing to the absolute requirement for calcineurin activity. These data reveal important differences in the regulatory pathways between IL-10 (a cytokine-inhibitory interleukin) and IL-2 (a cytokine-inducing interleukin), which can potentially be exploited therapeutically. The fact that CsA blocks the production of IL-10, which itself has important immunosuppressive properties, should be taken into account in defining immunosuppressive treatment schedules which include the use of CsA.     

Trophoblast Does Not Cause the Cytotoxic T Lymphocyte Generation Due to the Lack of Ability to Stimulate lnterleukin-2 Production

ABSTRACT: Trophoblast was demonstrated to be unable to cause the production of interleukin-2 (IL-2) by allogeneic splenocytes in vitro in two ways: 1) The addition of lymphocyte-trophoblast culture-supernatant (LTC-SN) did not stimulate the proliferation of IL-2 dependent cytotoxic T lymphocyte (CTL-L cells; 2) When responder cells were cultured with heat-treated splenocytes (usually no CTL generation) an increase of CTL formation could be seen in the presence of mixed lymphocyte culture-supernatant (MLC-SN) but not of SN from the cultures in which trophoblast cells served as stimulators. In parallel, the trophoblast cells were found to be very poor stimulators of alloreactive CTL. The addition of interleukin-2-enriched media resulted in a significant amplification of trophoblast-induced CTL generation. The resulting killing lymphocytes were capable of destroying the only specific targets, did not lyse syngeneic target cells, and could not be generated in the absence of allogeneic trophoblast. The incubation of these lymphocytes with anti-Thy 1.2 monoclonal antibody in the presence of complement eliminated their killing effect. Lack of class II antigenic determinants on the surface of trophoblast and/or local immunosuppression of IL-2 production by trophoblast cells seem to be responsible for nondelivery of helper factor, which is essential for CTL production.     

Suppression of T-helper cell function in mice following exposure to the carcinogen 7,12-dimethylbenz[a]anthracene and its restoration by interleukin-2

Previous studies in this laboratory have demonstrated that exposure of mice to the carcinogenic polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthrance (DMBA) results in suppression of cell-mediated immunity (CMI), specifically the ability to generate cytotoxic T cells. This is accompanied by an increased susceptibility to challenge with transplantable tumors. Our previous studies have demonstrated no appreciable change in the composition of splenic lymphocyte populations following exposure to DMBA, suggesting a modulation of lymphocyte function, probably at the level of the T lymphocyte. The purpose of this study was to examine the mechanism of DMBA-induced T lymphocyte dysfunction following DMBA exposure, and to determine whether CTL function could be reconstituted by the addition of untreated lymphocytes or lymphokines. Exposure to DMBA in vivo at doses of 50 and 100 micrograms/g and in vitro at doses of 20 and 40 microM suppressed the ability of splenic lymphocytes to generate cytotoxic T-lymphocytes (CTL). CTL-mediated lysis of allogeneic tumor target cells could be restored by the addition of 20% T cell-enriched naive lymphocytes and by 10% T-helper cell-enriched lymphocytes. DMBA suppressed splenocyte production of the lymphokine interleukin-2 (IL-2) in response to mitogenic or allogeneic stimulation by greater than 70% following in vitro exposure and greater than 45% following in vivo exposure. Although DMBA-exposed lymphocytes were impaired in their ability to produce IL-2, CTL responsiveness could be reconstituted by the addition of exogenous IL-2 (purified or recombinant DNA-produced). Complete restoration of CTL responsiveness by the addition of exogenous IL-2 suggests that the T-helper cell, rather than the T-cytotoxic cell, is the target for the DMBA-induced CMI lesion.     

Inhibition of the production of a soluble helper mediator by cyclosporin A results in the failure to generate alloreactive cytolytic cells in mixed-lymphocyte culture

The mechanism of action of cyclosporin A (CsA) in inhibiting the induction of alloreactive cytolytic T lymphocytes (CTL) in mixed-lymphocyte culture (MLC) was investigated. CsA at concentrations of 10(-3) to 10(-1) micrograms/ml completely prevented the generation of CTL. However, the addition of culture supernatants from mitogen-activated lymphocytes to MLC not only significantly reversed the suppressive effect of CsA but also fully restored the reactivity of lymphocytes already treated with CsA. By measuring the presence of a soluble helper mediator (SHF) in MLC supernatants, we found that CsA-treated lymphocytes produced no SHF, possibly interleukin 2 (IL-2). The effect of CsA on receptors for IL-2 was subsequently studied and it was found that the binding capacity of 125I-labeled IL-2 to lymphocytes was not altered by the presence of CsA. These findings suggest that the prevention of helper cells from producing SHF, rather than the inhibition of the response of effector cells to SHF, is a possible explanation for the immunosuppression mediated by CsA.     

Alloantigenic stimulation bypasses CD28-B7 costimulatory blockade by an interleukin-2-dependent mechanism

Allogeneic leukocytes have been used as biological adjuvants for T cell-specific responses to tumor and recall antigens, but the mechanisms underlying this effect have not been fully understood. The present study investigates whether alloantigen stimulation of human T cells would bypass an in vitro T cell costimulatory dysfunction induced by CTLA4Ig blockage of CD28-B7 interaction. Here, we demonstrate that costimulation with intact allogeneic leukocytes plus viral antigen circumvented the inhibition of this costimulatory pathway via interleukin-2 (IL-2) production, resulting in the generation of influenza-specific cytotoxic T lymphocytes (CTL). The alloantigen-induced help for influenza-specific CTL generation did not require cell-to-cell contact between responding and allogeneic stimulator cells. These results suggest that alloantigens can be used to bypass defects in the CD28-B7 costimulatory pathway and, therefore, may contribute to understanding the mechanisms of alloantigen-induced restoration of T cell-mediated immunity.     

Regulation of human cytotoxic lymphocyte responses. I. Non-cytolytic suppression mediated by alloantigen-activated cells.

Alloantigen sensitized human lymphocytes obtained from a 2-3 day mixed lymphocyte culture (MLC) suppressed the in vitro generation of alloreactive cytotoxic lymphocytes (CTL). The inhibition of CTL responses was demonstrated in MLC after both 1 day and 14 days' allosensitization. The suppressor cells were nylon wool non-adherent, lacked Fc receptors and adhered to histamine columns. The MLC-activated suppressor cell population had an associated very low and transient cytotoxic response directed against the allogeneic sensitizing cell. Several procedures were used to dissociate this activity from suppressor cell function: (1) donors were preselected which showed minimal cross-killing between allogeneic stimulating cells, (2) suppressor cultures were added to the test cultures prior to the development of maximal CTL activity, (3) suppressor cultures were irradiated preventing cell proliferation associated with differentiating CTL. Lastly, by increasing stimulating antigen concentration suppressor cell activity was increased rather than being competitively diminished, which would be predicted if suppression was occurring through a cytolytic or inactivation of stimulating antigen. It was therefore concluded that alloantigen stimulation in MLC activates a non-cytolytic regulatory cell population which is capable of inhibiting CTL responses to third-party allogeneic lymphocytes.     

Differential effects of IL-12 on the generation of alloreactive CTL mediated by murine and human dendritic cells: a critical role for nitric oxide

We examined the mechanisms involved in interleukin (IL)-12-mediated suppression of cellular immunity in mice using allogeneic mixed leukocyte reaction (MLR) stimulated by dendritic cells (DCs) in vitro and compared the effect of IL-12 on MLR in mice and humans. Although IL-12 stimulated human MLR, the addition of IL-12 or interferon-gamma (IFN-gamma) resulted in a dose-dependent suppression of MLR in mice. The treatment with N(G)-monomethyl-L-arginine (L-NMMA) completely abrogated IL-12- and IFN-gamma-mediated suppression of MLR in mice. Furthermore, IL-12 enhanced the alloreactive cytolytic T lymphocyte (CTL) induction in human MLR, whereas the addition of L-NMMA was required to generate alloreactive CTLs in the presence of IL-12 in mice. Nitric oxide (NO) was detected only in mouse MLR. Murine DCs could produce NO, but neither human CD34(+) cell- nor monocyte-derived DCs produced a detectable amount of NO. These results suggest that NO produced by DCs might play an important role in IL-12-mediated immune suppression in mice but not in humans.     

In vitro co-stimulation with anti-CD28 synergizes with IL-12 in the generation of T cell immune responses to leukaemic cells; a strategy for ex-vivo generation of CTL for immunotherapy

The existence of an immune based graft-versus-leukaemia (GvL) effect highlighted the prospect of managing relapsed leukaemias with T cell-based adoptive immunotherapy. Thus, various strategies have been explored for the in vitro expansion of acute myeloid leukaemia (AML)-specific T cells. In a popular approach, AML blasts have been genetically modified to express co-stimulatory molecules essential for effective T cell priming. One such tactic has been the modification of AML cells to express the B7/CD80 co-stimulatory molecule that binds to CD28 on T cells initiating events that culminate in enhanced cytokine production, proliferation and development of effector functions by T cells. The success of these strategies has been limited by difficulties in attaining sufficient transduction efficiencies and associated high levels of CD80 expression. We demonstrate that these problems can be circumvented by using anti-CD28 monoclonal antibody. Furthermore, we show that the synergistic relationship between CD80/CD28 pathway and interleukin 12 cytokine (IL-12), documented in the generation of cytotoxic T lymphocytes (CTL) for solid tumours, also applies to AML. CD28/IL-12 synergy facilitated the proliferation of allogeneic T cells in response to stimulation with primary AML blasts. The synergy also favoured generation of a Th1-type immune response, evidenced by gamma interferon (IFN-gamma) secretion and facilitated naive and memory T cell proliferation. Unlike some methods of in vitro T cell expansion, use of CD28/IL-12 synergy left T cells in the physiologically appropriate CD45RA-/CCR7- subsets known to be associated with immediate cytotoxic functions.     

Human cytotoxic T lymphocytes. II. Frequency analysis of cyclosporin A-sensitive alloreactive cytotoxic T-lymphocyte precursors.

A limiting dilution (LD) culture system was used to investigate the effect of cyclosporin A (CsA) on the activation and differentiation of human alloreactive cytotoxic T-lymphocyte precursors (CTL-p). CsA reduced in a dose-dependent fashion the frequency of alloantigen-inducible CTL-p. With most normal individuals tested there was a 20- to 50-fold reduction of alloreactive CTL-p frequencies in the presence of 500-1000 ng/ml CsA. Both unseparated T cells and CD8+ T cells were CsA-sensitive under LD culture conditions. Importantly, however, alloreactive CTL-p from two out of 21 normal individuals were found to be largely CsA-resistant. CsA did not affect the growth of MLR-primed CTL in secondary LD culture. Furthermore, CsA slightly inhibited the cytolytic activity of some alloantigen-specific CTL clones. These results are discussed with respect to the clinical use of CsA in transplantation medicine.     

Biased activation of human T lymphocytes due to low extracellular pH is antagonized by B7/CD28 costimulation

As T cell response to tumor-associated antigens may be impaired by the acidic microenvironment typical of solid tumors, we assessed the effect of extracellular pH (pH(e)) on the activation and proliferation of human T lymphocytes and generation of the cytotoxic response. T lymphocytes stimulated with anti-CD3 mAb or PHA at low pH(e) were unable to secrete IL-2 and IFN-gamma and their ability to progress through the cell cycle was impaired. T lymphocytes also displayed up-regulation of IFN-gammaR2 chain and CTLA-4 expression, rendering them sensitive to negative regulatory signals. Agonistic mAb against CD28, but not against CD2, completely restored cytokine production and cell cycle progression, but down-regulated IFN-gammaR2 and CTLA-4 expression. The anti-CD28mAb rescued the CTL response of allogeneic anti-tumor cultures generated at low pH(e). Following anti-CD28 mAb treatment, T cells synthesized cyclooxygenase-2 (Cox-2) protein, which is involved in the early phases of T cell activation. This rescue of T cell activation was independent of the inducible 6-phosphofructo-2-kinase (iPFK-2) pathway, which stimulates proliferation in hypoxic and acidic conditions. The restoration of proliferative and cytotoxic T cell responses by CD28-triggering provides insight into the mechanisms by which B7 enhances the T cell anti-tumor response in vivo.     

Complement inhibits immune responses: C3 preparations inhibit the generation of human cytotoxic T lymphocytes

C3 fragments have been shown to inhibit mitogen- and antigen-induced human lymphocyte blastogenesis. In this study, C3 preparations and a small fragment of C3 (contained in a preparation called Fraction 2) were examined for their capacity to regulate cytotoxic T lymphocytes (CTL) and proliferative mixed lymphocyte responses (MLR). We found that both preparations inhibited the generation of allogeneic human CTL as well as MLR in a dose-related manner. In contrast, Fraction 1, which contained native C3 and C3b, did not inhibit the generation of CTL nor did it inhibit the MLR. The kinetics of inhibition of proliferation were divergent from the kinetics of inhibition of CTL generation; the active preparations inhibited proliferation significantly when added as late as day 5 of a 6-day culture, whereas no inhibition of CTL generation was seen when these preparations were added after day 3 of culture. Cultures in which C3 preparations caused complete inhibition of CTL generation had normal levels of the nonspecific, anomalous killer cell activity, as assayed on K 562 target cells. Furthermore, C3 preparations and Fraction 2 had no effect on the lytic function of differentiated CTL, on “spontaneous” natural killer cell activity or on interferon-induced augmentation of natural killer cell activity. These findings indicate that C3 fragments may play a negative role in the regulation of CTL responses.     

Corrective effects of interleukin-12 on age-related deficiencies in IFN-gamma production and IL-12Rbeta2 expression in virus-specific CD8+ T cells.

Interleukin-12 receptor beta2 (IL-12Rbeta2) has been shown to be selectively expressed on Th1 T cell subsets, and we have previously shown that influenza-specific CD8+ cytotoxic T lymphocyte (CTL) deficiency in old mice was associated with deficient Th1 (interferon-gamma [IFN-gamma]) cytokine production. This study tested whether IL-12Rbeta2 expression was also deficient in CD8+ CTL from old mice and the effect of IL-12 treatment on these responses. Splenic lymphocytes from influenza-primed old and young BALB/c mice were stimulated with influenza virus in vitro with and without IL-12 and then enriched for CD8+ T cells. IFN-gamma was significantly reduced, whereas IL-4 and IL-12p40 (an antagonist of IL-12 function) were evaluated in old when compared with young mice. This was true for secreted protein measured by ELISA and for mRNA levels quantitated by RT-PCR. IL-12Rbeta2 mRNA expression in CD8+ CTL was also significantly reduced in old mice. IL-12 treatment in vitro caused significant upregulation of IFN-gamma and IL-12Rbeta2 and downregulation of IL-4 in CD8+ T cells from old mice and young mice. The present demonstration of an age-related downregulation in IL-12Rbeta2 expression and our previous data showing reduced IFN-gamma and elevated IL-4 production provide strong evidence that CD8+ CTL deficiency in aging results from a Th1/Th2 cytokine production switch. Agents that increase IL-12Rbeta2 expression and redirect Th2 to Thl immune responses are likely to enhance CD8+ CTL-mediated control of viral infections in aging.     

Lymphocyte subpopulations in man: induction of cytotoxic T lymphocytes in vitro by allogeneic B and T cells

Testing B and T cells as allogeneic stimulators of cytotoxic T lymphocytes in primary as well as secondary in vitro cultures, reveals that fresh, nonactivated B cells isolated from peripheral blood have an enhanced cytotoxic T cell stimulating capacity compared to T cells, although target determinants are present both on B and T cell blasts. Similarly, the capacity of T and B cells to stimulate proliferation in MLC is also quantitatively different. These results are in accordance with the hypothesis concerning the in vitro generation of alloreactive cytotoxic T lymphocytes, which postulates concurrent stimulation by strong lymphocyte activating determinants and target determinants for the generation of cytotoxic effector T lymphocytes, as both determinants are simultaneously found on B lymphocytes. Three cell experiments performed by coculturing allogeneic stimulating B and T cells with responding T cells show that strong lymphocyte activating determinants found on B cells enhance the cytotoxicity against target determinants on cocultured B cells but not on cocultured T cells, indicating qualitative differences between target determinants on B and T cells with respect to specific CTL stimulating capacity. Furthermore, primed resting CTLs in secondary cultures could unspecifically be restimulated by third party B cells or pokeweed mitogen. These results are the basis for a hypothesis concerning activation of CTLs, postulating nonspecific triggering of cytotoxic precursor cells by lymphocyte activating properties intrinsic to target determinants (TD) on B cells, preferentially activating clones of cytotoxic cells. The clonal proliferation is further unspecifically amplified by products of the T cell recognition of strong lymphocyte activating determinants (LAD).     

Interleukin-12 is necessary for the priming of CD4+ T cells required during the elicitation of HIV-1 gp120-specific cytotoxic T-lymphocyte function

The mechanism of the T-cell response and cytokine induction to restrict human immunodeficiency virus 1 (HIV-1) infection is not clear. During early infection, HIV-infected individuals have a high frequency of virus-specific cytotoxic T lymphocytes (CTLs) that effectively reduces the viral load. However, the CTLs are unable to clear the virus at later stages of infection, leading to disease progression. Dysregulation of cytokines like interleukin-12 (IL-12) and interferon-gamma (IFN-gamma) as a result of the interaction of HIV-1-specific T cells with antigen-presenting cells is one of the possible causes of CTL dysfunction. Secretion of IL-12 is reduced with the progression of HIV infection, correlating with impaired CTL function; however, the role of IL-12 in CTL regulation awaits elucidation. Here, we have studied the role of IL-12 in CTL dysfunction by using DNA immunization of wild-type (WT) and IL-12-deficient mice with HIV-1 gp120 complementary DNA. It was observed that the CTL response in IL-12-deficient mice was significantly less than that in WT mice. Our results further demonstrated that coimmunization with IL-12 vector restored the impaired CTL response in IL-12-deficient mice. However, immunization with IL-12 vector failed to rescue the CTL response in IFN-gamma deficient mice, suggesting that the CTL-promoting function of IL-12 is IFN-gamma-mediated. Our data suggest a phase-specific role of IL-12 in the CTL response, specifically in the priming of CD4+ T cells that provide help to CD8+ T cells. Our results also suggest that IL-12 is vital for the priming of antigen-specific T cells and plays an essential role in IFN-gamma induction in T cells.     

Cyclosporine inhibits soluble antigen and alloantigen presentation by human monocytes in vitro

We examined whether cyclosporine (CsA) interferes with human monocyte processing and presentation of soluble and particulate antigens. Human monocytes were pulsed in the presence of CsA with one of three antigens: tetanus toxoid (TT), diphtheria toxoid (DIP) or cytomegalovirus (CMVx). When these monocytes were co-cultured with immunocompetent T-lymphocytes they were found to be impaired in the induction of proliferative and cytotoxic T-cell responses. At CsA concentrations higher that 0.5 micrograms/ml, proliferation induced by the particulate CMVx antigen was markedly more sensitive than that induced by the soluble TT antigen. In the allogeneic mixed lymphocyte reaction (MLR), pretreatment of responder haplotype monocytes alone with CsA led to reduced reactivity as determined in both proliferative (P less than 0.01) and cell-mediated lympholysis (P less than 0.001) assays. These effects of CsA were not due to direct CsA action of residual CsA carried over with treated monocytes, and were not apparently due to inhibition of interleukin-1 (IL-1) production since exogenous IL-1 could not restore normal responses.