The effect of ultraviolet B irradiation on delayed-type hypersensitivity, cytotoxic T lymphocyte activity, and skin graft rejection

The influence of ultraviolet B irradiation on the induction of delayed-type hypersensitivity reactions to alloantigens by epidermal cells (EC), on the generation of cytotoxic T lymphocyte activity to alloantigens, and on skin graft rejection was studied. After the skin was irradiated with UVB in vitro, EC were obtained. The EC were injected subcutaneously, and the DTH reaction was compared with that induced by non-UVB-irradiated EC. A reduction in the DTH reaction was observed (from 62% to 99.1%). CTL activity in these mice was assessed after in vitro stimulation. CTL activity in mice sensitized with UVB-irradiated EC was significantly reduced. Furthermore, mice sensitized with UVB-irradiated EC did not reject a subsequent skin allograft in an accelerated fashion, whereas mice sensitized with non-UVB-irradiated EC did. The mechanism(s) of these reactions and the clinical application of the UVB irradiation prior to grafting are discussed.     

T cell help in cytotoxic T lymphocyte responses. Role of the I region in helper cell induction

We have studied the in vivo induction of T helper (TH) cells that participate in the generation of cytotoxic T (TC) lymphocytes. Helper activity was measured by the ability of the cells to help resting thymic TC cell precursors develop into effector TC cells in vitro. Direct injection of allogeneic spleen cells into the footpads of mice led to the generation of alloantigen-specific helper cells in the draining popliteal lymph nodes within 4 to 6 days. Helper activity was mediated by nylon-wool-nonadherent Lyt-1+ T lymphocytes; some activity was associated with Lyt-1,2+ cells. The genetic requirements for both the induction and restimulation of C3H anti-H-2d TH cells were investigated using cells from H-2k/H-2d recombinant mice as in vivo immunogens and in vitro stimulators. Evidence is presented that shows in a direct assay that TH cells themselves are specific for I region-coded determinants. Thus, disparity at the left side of the H-2 complex (K to I-E) but not at H-2K alone was necessary and sufficient to induce and reactivate TH cells. Proliferation in mixed lymphocyte culture was measured in combinations in which TH cells were not detectable, supporting the idea that proliferation cannot be strictly considered a measurement of helper cells.     

Prolongation of allograft survival by Nippostrongylus brasiliensis is associated with decreased allospecific cytotoxic T lymphocyte activity and development of T cytotoxic cell type 2 cells

BACKGROUND: We have demonstrated that infection with Nippostrongylus brasiliensis (Nb), which induces strong type 2 responses, prolongs kidney allograft survival in rats. Here, we confirm that this effect is not species-specific and address immune modulation in allospecific T-cell responses mediated by nematode infection. METHODS: C57BL/6 mice were injected with Nb or phosphate-buffered saline. Four days later, mice were transplanted with BALB/c hearts and graft survival was assessed. In other experiments, Nb-infected mice were immunized with BALB/c spleen cells and allospecific T-cell responses were determined in vitro. RESULTS: In this study, we show that Nb prolongs cardiac allograft survival in mice. Further, spleen T cells from Nb-infected, allo-immunized mice exhibit reduced allospecific cytotoxic T-lymphocyte activity. In contrast, allospecific proliferation of T cells in the mixed lymphocyte reaction was not reduced by Nb, ruling out immunosuppression as the mechanism of Nb-induced allograft survival. Nb infection induced IL-4 and IL-6 and inhibited IFN-gamma production by T cells in response to allo-antigen. Furthermore, anti-IL-4 treatment reduced allospecific T-cell proliferation from Nb-infected but not control mice, indicating that type 2 allospecific T cells develop in the presence of Nb. We also double-stained T cells for CD8 and IL-4 and showed that Nb induces an 8-fold increase in Tc2 cell numbers. CONCLUSIONS: These results are consistent with a hypothesis that Nb mediates prolongation of allograft survival through induction of type 2 immunity, including the development of regulatory Tc2 cells, and subsequent inhibition of allospecific cytotoxic T-lymphocyte activity.     

Blood-transfusion-induced suppression of cytotoxic T lymphocyte responses in mice

B6AF1 (H-2KbkDbd) mice were transfused weekly with 0.1 ml of whole blood from DBA/2 (H-2d) mice. One week after each transfusion, spleen and serum samples were collected. Blood transfusions did not induce blood donor alloantigen-specific cytotoxic T lymphocytes (CTL) in spleens of B6AF1 mice. When spleen cells from transfused mice were sensitized to alloantigens in mixed lymphocyte culture in vitro, it was observed that 1-3 transfusions induced suppression of blood donor-specific CTL activity. No suppression of CTL activity was found after 4 transfusions. The cell-mixing experiments demonstrated that the suppression of CTL activity following initial 2 blood transfusions was due to the presence of suppressor cells. The presence of antibodies in sera of transfused B6AF1 mice capable of inhibiting CTL was investigated using the CTL-inhibition test. In these experiments, cytotoxic T lymphoblasts generated in MLC in vitro by culturing normal B6AF1 spleen cells with x-irradiated DBA/2 cells were treated with serum before testing them for cytotoxicity. The antibodies capable of inhibiting CTL responses were demonstrable in sera from transfused mice. Three and four BT sera caused significant inhibition of CTL responses. The CTL-inhibitory antibodies were specific for effector cells of the B6AF1 mice and for target cells of the blood donor DBA/2 mice. These results suggest that the inhibition of CTL responses is caused by antibodies directed against the recognition sites on effector T lymphocytes. The data from this study, therefore, demonstrate that BT cause suppression of the recipient's CTL responses against alloantigens present on the blood donor, and that this suppression is mediated by suppressor cells after the initial 1 to 2 transfusions and by antibodies directed against the CTL antigen-specific receptors after subsequent transfusions.     

Apoptosis and expression of cytotoxic T lymphocyte effector molecules in renal allografts.

Cytotoxic T lymphocyte (CTL) mediated apoptosis is thought to play a major role in the rejection of renal allografts following transplantation, however, the CTL effector mechanism that is primarily responsible for immunological rejection is unknown. The two major effector pathways of CTL killing which lead to apoptosis involve the Fas/Fas ligand (Fas L) lytic pathway, and the perforin/granzyme degranulation pathway. The expression of CTL effector molecules which influence these pathways include Fas, Fas L and TiA-1 (cytotoxic granule protein). This study has investigated apoptosis by in situ terminal deoxytransferase-catalysed DNA nick end labelling (TUNEL), and the expression of CTL effector molecules by immunohistochemistry, in renal allograft biopsies obtained from patients following kidney transplantation. Renal biopsies were classified into three histological groups; acute cellular rejection, chronic rejection, or no rejection. The extent of T-cell infiltration of renal tissues was assessed by immunohistochemical staining with an anti-CD3 monoclonal antibody. Numerous TUNEL positive cells were detected in all transplant biopsies examined; these consisted mainly of renal tubular cells and infiltrating cells, with some TUNEL positive cells also detected in the glomeruli. In the case of normal kidney tissue, renal cells also stained positive for TUNEL but there was no lymphocytic infiltration. There was significantly more T-cell infiltration observed in acute rejection biopsies compared to the no rejection biopsies. In the case of Fas L expression, there was little expression in all three biopsy groups, apart from one case of chronic rejection. Conversely, although there were no significant differences in TiA-1 expression between the three biopsy groups, TiA-1 expression was more prominent in acute rejection biopsies. Furthermore, Fas expression was significantly decreased in acute rejection biopsies when compared to those of chronic and no rejection in which Fas was predominantly localized in the renal tubular cells. These results indicate that the mechanism of CTL killing leading to the rejection of renal allografts may be different in acute and chronic rejection. Moreover, our data indicate the potential for cytotoxic granule-based CTL killing in acute renal allograft rejection but not in chronic rejection.     

Effect of cyclosporine on secondary cytotoxic T lymphocyte responses

Cyclosporine, a potent immunosuppressive agent, inhibits the development of the cytotoxic response in a secondary mixed lymphocyte reaction (MLR) in a dose-dependent manner. Exogenous lymphokines can partially overcome this inhibition. We present evidence that the CsA-resistant cells detected in these responses represent an activated population of memory CTL precursors that require only lymphokines for clonal growth and that kill targets of the original stimulator type only. Recombinant IL-4, when used at high concentrations, can support the generation of CTL in the presence of CsA during a secondary MLR response. The magnitude of the cytotoxic response though is far below the maximal levels achieved either by saturating quantities of rIL-2 or a combination of subsaturable quantities of rIL-2 and rIL-4.     

Regulation of delayed-type hypersensitivity. VI. Antigen-specific suppressor T cells and suppressor factor for delayed-type hypersensitivity to histocompatibility antigens

Mice develop highly significant levels of delayed-type hypersensitivity (DTH) to major and minor histocompatibility antigens when injected s.c. with lymphoid cells from X-irradiated allogeneic donors. However, when mice are inoculated i.v. with a high dose of X-irradiated allogeneic lymphoid cells, they not only fail to develop DTH to the allogeneic cells, but their ability to respond to an immunogenic challenge of the alloantigens is also significantly depressed. This suppression is adoptively transferable by antigen-specific suppressor T cells and not by immune serum. Cell surface phenotypic analysis shows that the primary suppressor cells for alloantigens are Thy-1+, Lyt-1+2-, and Ia-, whereas the secondary suppressor cells appearing after boosting injection are Thy-1+, Lyt-1+2+, and Ia-. These suppressor T (Ts) cells localize in the lymphoid organs shortly after their induction and are largely absent from the spleen or lymph node 1 month later. However, "suppressor memory" can be recalled by an immunogenic dose of alloantigens which would normally induce DTH effector cells rather than suppressor cells in naive mice. When the suppressor cells were cultured in vitro for 48 hr, the supernatant contained suppressive activity. It appears likely that the manifestation of the suppressor cells is via soluble, antigen-specific suppressor factor(s), the production of which is dependent on viable T cells.     

Tolerance induction of alloreactivity by portal venous inoculation with allogeneic cells followed by the injection of cyclophosphamide. I. Specific suppression of alloreactive cytotoxic and delayed-type hypersensitivity responses as well as allograft rejection

BALB/c mice receiving allogeneic C3H/He spleen cells via portal venous (p.v.) route or a single administration of cyclophosphamide (Cy) were capable of rejecting allogeneic X5563 tumor cells (C3H/He origin). In contrast, the combined treatment of p.v. inoculation with C3H/He cells and Cy administration abrogated the capability of rejecting allogeneic X5563 tumor cells. The dose of Cy and interval between p.v. presensitization and Cy injection needed to be more than 1.5 mg Cy/mouse and less than 2 days, respectively. Such abrogation of alloreactivity was alloantigen-specific, since p.v. inoculation of C3H/He cells followed by Cy injection resulted in selective inhibition of rejecting allogeneic C3H/He tumor cells (X5563 plasmacytoma) without suppressing the capability of rejecting allogeneic C57BL/6 tumor cells (EL4 leukemia). Most important, the induction of alloantigen-specific unreactivity by p.v. presensitization plus Cy injection contrasted with the failure of intravenous or subcutaneous presensitization plus Cy injection to induce tolerance that permits the growth of allogeneic tumor cells inoculated. This was substantiated by the finding that p.v. presensitization with C3H/He cells prior to Cy injection eliminated anti-C3H/He cytotoxic T lymphocyte (CTL) and delayed-type hypersensitivity (DTH) potentials under conditions in which appreciable CTL and DTH responses are induced in mice inoculated via the i.v. route before Cy injection. These results demonstrate that p.v. inoculation of allogeneic cells followed by a single administration of Cy results in more efficient elimination of antialloantigen CTL and DTH reactivities, leading to the abrogation of potential to reject the allogenic tumor graft.     

Immune mechanisms in organ allograft rejection. II. T helper cells, delayed-type hypersensitivity, and rejection of renal allografts

The cellular requirements for renal allograft rejection have been assessed by adoptive transfer studies in a rat model. Sublethally irradiated (780 rads) LEW (RT1l) recipients of WF (RT1u) renal allografts were selectively reconstituted with spleen cells from specifically sensitized donors. In some experiments the reconstituting inocula were depleted of SIg+ cells by passage over anti-Ig columns or subjected to additional depletion of cytotoxic T cells (Tc) and their precursors reactive with monoclonal antibody OX8. WF renal allografts underwent acute rejection in the unmodified LEW recipient with day 7 serum creatinines of 6.8 +/- 0.9 mg/dL (mean +/- SD; n = 7), graft histology characterized by marked mononuclear cell infiltration and evidence of a brisk humoral response (complement-dependent cytotoxicity (CDC) titer greater than 2(6] and generation of Tc demonstrable by in vitro monitoring. Sublethally irradiated recipients mounted no detectable immune response, and day 7 serum creatinine and graft histological findings were not significantly different from those obtained in isograft controls. Renal allografts were, however, rejected in sublethally irradiated recipients reconstituted with unfractionated immune spleen cells, as evidenced by functional and histologic criteria (day 7 serum creatinine of 5.5 +/- 1.2 mg/dL; histology characterized by extensive interstitial hemorrhage, fibrinoid necrosis of blood vessels, and polymorphic infiltration). Neither antibody nor Tc appear, in this model, to be required to effect rejection, because recipients reconstituted with inocula depleted of SIg+ cells (day 7 CDC titer less than 2l) or subjected to additional depletion of Tc and their precursors (day 7 lymphocyte-mediated cytotoxicity assay: % specific chromium release at 100/1 E/T ratios less than 5%) underwent acute rejection with a day 7 serum creatinine of 5.0 +/- 1 and 4.3 +/- 1.5 mg/dL, respectively, and histological findings were characterized by marked mononuclear cell infiltration and a paucity of hemorrhage.     

同种异系抗原门静脉接种诱导特异性免疫抑制的研究

目的 探讨同种异系抗原经门静脉接种后机体对该抗原免疫反应状态的改变。方法 以ＮＩＨ／ｑ小鼠为供体,ＢＡＬＢ／Ｃ小鼠为受体预先用ＮＩＨ／ｑ小鼠脾细胞或与供受体无关Ｃ５７／ＢＬ小鼠脾细胞注射至ＢＡＬＢ／Ｃ小鼠门静脉或下腔静脉,１周后用ＮＩＨ／ｑ小鼠脾细胞注射至ＢＡＬＢ／Ｃ小鼠背部皮下免疫,２击后第２次皮下免疫。第２次皮下接种后１周,测定供受试体之间的混合淋巴细胞反应（ＭＬＢ）和受体对供体抗原的迟发性超     

Cytokine regulation of the balance between alloindifferent and allospecific suppressor induction in mixed lymphocyte cultures.

The effects of exogenous cytokines on the generation of alloindifferent, MHC-unrestricted suppressive activity early on in mixed lymphocyte culture interactions have been investigated. Interleukin 4 strongly blocked the generation of suppression, whereas IL-1, IL-2, and IL-6 enhanced it to some extent. Tumor necrosis factor-alpha, interferons-alpha and -gamma, granulocyte/macrophage colony-stimulating factor, granulocyte CSF, IL-3 and IL-5, and a number of combinations of these factors were without effect in this system. Insofar as the alloindifferent suppression studied here also inhibited the development of allospecific, MHC restricted suppressive activity later in MLC, reduction by IL-4 of its development may have relevance for the use of this cytokine to facilitate the induction of specific suppressor cell-mediated transplantation tolerance in vivo.     

In vitro reactivity of allospecific cytotoxic T lymphocytes does not explain the taboo phenomenon

Matching for human leucocyte antigens (HLA) is important for graft survival in kidney transplantation. Nevertheless, most patients receive a kidney graft with multiple HLA mismatches. Some of these mismatches seem to be more harmful than others. By studying the effect of single HLA mismatches in the context of the patients' own HLA, we have previously identified donor/recipient combinations with a significantly higher incidence of early graft failure, the so-called taboo combinations. In the present study we investigated whether a higher cytotoxic T lymphocyte (CTL) response towards taboo mismatches may be involved in this phenomenon. CTL reactivity was determined both in taboo and control combinations by in vitro CTL precursor assays, using peripheral blood mononuclear cells and proximal tubular epithelial cells as target cells. Inhibition studies with CD8-antibody as well as Cyclosporin A were performed to identify high avidity and primed CTLs. Furthermore, in committed CTLp assays indirect recognition of the taboo mismatch was tested using synthetic peptides. The CTL precursor frequencies in taboo combinations were always lower than the CTL precursor frequencies in control combinations. No difference in avidity and activation status of the CTLs could be detected when taboo combinations were compared with the controls. In the committed CTLp assays no reactivity towards any of the synthetic peptides was observed. The significantly poorer graft survival of taboo combinations cannot be explained by a higher number of donor-specific CTLs. Furthermore, the avidity or activation status of these CTLs does not provide a clue to the taboo phenomenon.     

转染肿瘤mRNA的树突状细胞疫苗诱导抗肝癌免疫研究

目的探讨转染原发性肝癌(HCC)mRNA的树突状细胞(DC)能否诱导抗肿瘤特异性细胞毒性T淋巴细胞(CTL)。方法采用HCC患者外周血单核细胞(PBMC)体外刺激分化为DC细胞；从人肝癌HepG-2细胞和3例HCC患者的肝癌组织中体外扩增mRNA。以mRNA转染DC细胞，并与PBMC混合培养诱导扩增CTL。流式细胞计数仪检测培养细胞中CD3^ 、CD4^ 、CD8^ 细胞的比例。^51Cr释放法测定CTL的杀瘤活性。结果经扩增人肝癌HepG-2mRNA和2例AFP( )患者的AFP( )HCCmRNA诱导3周后，CD3^ 、CD8^ 细胞占淋巴细胞总数由诱导前的27．8％、26．5％、29．6％升高至89．3％、73．6％、86．8％；而经扩增AFP(-)HCCmRNA诱导3周后，CD3^ 、CD8^ 细胞占淋巴细胞总数由诱导前的25．4％升高至53．6％。转染HepG-2细胞和AFP( )的患者HCCmRNA的DC诱导的CTL对HepG-2细胞杀瘤活性明显高于AFP(-)的患者，其杀瘤特性由MHC-I限制的CD8^ T细胞所介导。结论HCCmRNA体外转染DC能诱导肿瘤特异性CTL，可为肝癌的免疫治疗提供新的有效手段。     

Establishment of experimental malignant glioma-specific cytotoxic T lymphocyte clone by T cell growth factor

In an attempt to facilitate the long-term proliferative growth and subsequent cloning of cytotoxic T lymphocytes (CTL's) against syngeneic murine 203-glioma (20-methylcholanthrene-induced ependymoblastoma of C57BL/6 mouse origin), sensitized T lymphocytes from tumor-bearing mice were cultured in the presence of T cell growth factor (TCGF). Of five clones established by a limiting dilution technique, two clones (G-CTLL 1 and 2) exhibited tumor-specific cytotoxicity. G-CTLL 1 cells, which possessed much higher cytotoxic activity than G-CTLL 2 cells, were further analyzed. G-CTLL 1 cells were maintained in a TCGF-dependent exponential proliferative culture for over 18 months and continued to mediate an extremely high cytotoxic activity with the target specificity (50- to 100-fold increases over the peak cytotoxic activity of sensitized T lymphocytes in tumor-bearing mice). Their phenotypes of surface antigens were Thy-1+ (weak positive), Lyt-1.-2.+3+, and asialo-GM1-, and their cytotoxicity was blocked by adding only anti-Lyt-2 monoclonal antibodies. These results indicated that the cloned cells originated from CTL's. The cloned cells were characterized by the production of immune interferon with the glioma antigen-stimulation, suggesting that the immune interferon could enhance the cytotoxic activity of the CTL clone at the site of a clone-target cell recognition event.     

Evidence of intragraft interleukin-2-activated killer cells and allospecific cytolytic T lymphocytes in rejecting lung allografts

In vivo cell-mediated effector mechanisms of allograft destruction were investigated in a canine single-lung transplantation model. This large animal model permits direct longitudinal studies of immune effector cells from the grafts of individual recipients by bronchoalveolar lavage (BAL). Evidence was obtained that two types of cytolytic lymphocytes act as effectors of allograft destruction. Typical allospecific cytolytic T lymphocytes, were detected late in the course of rejection in nonimmunosuppressed recipients and in cyclosporine-treated recipients during the latter stage of drug tapering. The other type of intragraft cytolytic lymphocyte was observed in the early stages of CsA dose tapering and was characterized by ability to lyse xenogeneic targets in a lectin-dependent cytotoxicity assay but inability to kill allogeneic target cells from the lung donor. These cytolytic cells were also detected in the initial stage of lung rejection in non immunosuppressed recipients and in the early period (3 days) of mixed lymphocyte culture. Current interpretation of these data is that these latter effector cells have the characteristics of IL-2-activated killer cells (IAK). Substantial delays in the detection of intragraft donor-specific CTL relative to IAK activity were observed in recipients undergoing CsA dose tapering compared with nonimmunosuppressed recipients. This finding suggests that appropriate CsA treatment may lead to prolonged inhibition of the generation of donor-specific CTL compared with induction of IAK activity. Delayed detection of intragraft donor-specific CTL paralleled the absence of such activity in donor-specific MLC of tolerant lung allografter recipients. The result of CsA therapy may, therefore, be characterized as a state of "partial unresponsiveness," since certain pathways of immune effector activity remain intact after termination of treatment. The differential effect of CsA on various pathways of allograft destruction may have important implications regarding concepts of alloreactivity and T cell-mediated immune responses.     

Blocking by cyclosporine of antigen-induced maturation and lymphokine secretion by cytotoxic T lymphocyte hybridomas

We have investigated the effect of cyclosporin (Cys) on the maturation of cytotoxic T lymphocytes (CTL), and on the induction of interleukin-2 (IL-2) secretion using two bifunctional CTL hybridomas. Both hybridomas can be stimulated with allogeneic cells to secrete IL-2 and to specifically kill target cells. Cys, at 10-50 ng/ml, eliminates the induction of both functions (secretion and lysis). Although maturation of both specific and lectin-mediated killing by the hybrid cells exhibits high sensitivity to Cys, the actual killing of target cells by previously activated cells is less affected. Our results suggest that pharmacological levels of Cys directly interfere with the antigen-responsiveness of helper-independent cytotoxic T cells, as represented by these hybridomas, and prevent their maturation.