Effects of cyclosporin A, antilymphocyte serum and donor-specific transfusions on murine delayed-type hypersensitivity and skin graft survival

The effect of immunosuppressive reagents cyclosporin A (CsA) and rabbit anti-mouse antilymphocyte serum (ALS) on the response to alloantigens was studied in inbred mouse strains. Alloantigen was given either as a cell suspension which induced a delayed-type hypersensitivity reaction (DTH), or as a full-thickness skin graft. Dose-response studies showed that DTH reactions in CBA mice sensitised to BALB/c cells were reduced to background levels when recipient mice were treated with 100 mg/kg CsA on days 0, 4 and 6 after primary alloantigenic challenge. The response to a second challenge was significantly decreased by CsA treatment during primary or secondary exposure to alloantigen and CsA was as effective as ALS in abrogating both primary and secondary DTH reactions. Survival of full-thickness grafts of BALB/c skin on CBA mice was increased from 9 to 23 days by ALS treatment on days -1 and +2, with grafts given on day 0. Long-term treatment with CsA, from day -14 to +12, also prolonged graft survival from 9 to 18 days but donor-specific transfusions, with or without concomitant ALS or CsA treatment, decreased graft survival and often sensitised the recipients. This occurred with transfusions administered from -63 to -7 days and on the day of grafting. Thus, in H-2 mismatched mice, both CsA and ALS treatments produced a state of tolerance when administered during short-term exposure to alloantigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

In Vivo Responses of Alloreactive Lymphocytes Stimulated In Vitro

Mouse lymphocytes that have been primed in vitro against alloantigens show a specific increase in cells reactive to the priming antigens in mixed lymphocyte response (MLR) and include cells that are specifically cytotoxic in vitro. The primed population also contains cells capable of causing rejection of skin grafts when injected into nude mice. Functional enrichment of cells capable of rejecting skin grafts bearing specific alloantigens and depletion of cells capable of rejecting a third-party graft have been shown. Priming the cells a second time in vitro may result in a moderate enrichment of cells capable of rejecting the specific graft and depletion of cells reactive to third-party skin compared with once-primed cells. These findings support the prediction that the MLR is an in vitro model of allograft responses in vivo.     

Autologous lymphoid cells exposed to recombinant interleukin-2 in vitro in the absence of antigen can induce the rejection of long-term tolerated skin allografts.

The intraperitoneal (i.p.) administration of autologous spleen cells treated with recombinant interleukin-2 (IL-2) in vitro resulted in the rejection of C57BL/10ScSn (B10) skin grafts that had survived for 18 months on CBA/Ca mice tolerant to B10 alloantigens. Cytotoxic lymphocytes specific for the graft alloantigens, assayed by limiting dilution analysis, appeared after the breaking of this immunological tolerance, whereas they were not detectable in tolerant mice. In these mice the frequencies of precursor cytotoxic cells specific for third-party alloantigens were unaffected by the IL-2 treatment.     

Cyclosporin alters the induction of allospecific tolerance in vivo

CBA mice were made hyporesponsive to A/J alloantigens by either neonatal inoculation of (CBA X A)F1 hybrid lymphoid cells or by intravenous injection of adult mice with A/J bone marrow cells. Specific alloreactivity was assessed in vitro by induction of anti-A/J cytotoxic T lymphocytes (CTL) or in vivo by skin graft rejection. Cyclosporin A given at the same time as the tolerance-inducing regimen of F1 (or parental) lymphoid cells abolished the hyporesponsiveness normally induced by these injections.     

A decreased functional capacity of CD4+ T cells underlies the impaired DTH reactivity in old mice

The data presented in this paper show that the in vivo delayed-type-hypersensitivity (DTH) reaction to both H-2 and non-H-2 alloantigens declines with increasing age. It is also shown that cells generated in vitro are capable to transfer DTH to young naive syngeneic recipients. Using this in vitro system it could be demonstrated that cells from old CBA/Rij mice induced lower DTH responses than cells from young CBA/Rij mice. Depletion experiments with the effector T cell population showed that the DTH effector phase is mediated by CD4+ T cells. Lower responses in old mice were not due to increased CD8+ suppressor T cell activity, since after removal of the CD8+ T cells old CD4+ cells were still less effective in the generation of DTH effector T cells than young CD4+ cells. Addition of IL-2 containing supernatant to in vitro cultures did not improve the subsequent DTH response. From these data it can be concluded that the reduced DTH responses in old mice are not solely due to CD8+ suppressor cell activity and/or lack of IL-2, but that rather intrinsic defects of the CD4+ T cell population appear to play a major role in the impaired DTH reactivity during ageing.     

Differences in the Rejection of an Allogeneic Tumor Graft in two Strains of Inbred Mice and Their F1 Hybrids1

The rejection of a Sarcoma tumor (Sal) was compared in two histoin-compatible strains of mice (C57BL/6, CBA) and in their F₁ hybrids. Based on the tumor growth curves, allograft rejection appeared much stronger in C57BL/6 than in CBA mice, the former rejecting the tumor graft rapidly, the latter showing delayed regression with occasional tumor enhancement. Differences in the response to the tumor allograft between male and female recipients were more pronounced in CBA than in C57BL/6 mice. Male and female C57BL/6 recipients exhibited the same rate of rejection, whereas the mortality in male CBA mice was higher than in females. This difference was even more striking for the (C57BL/6 x CBA) F₁ hybrids: the female hybrids rejected tumor grafts as rapidly as C57BL./6 recipients, whereas the male hybrids failed to reject the tumor allograft in most of the cases (80%) and died from progressive tumor growth. The humoral and cellular immune responses tested in vitro were much weaker and somewhat delayed in CBA mice as compared to those of C57BL/6 recipients. These findings would suggest that the immunogenicity of the different H-2 specificities is not identical in these two strains for the different immune responses and that histoincompatibility at the H-2 locus is not necessarily the major factor responsible for allograft rejection.     

Ly-6A is critical for the function of double negative regulatory T cells

We have recently demonstrated that CD3+CD4-CD8- double negative (DN) T cells can down-regulate allogeneic immune responses both in vitro and in vivo by killing activated syngeneic CD8+ T cells. The goal of this study was to identify molecules that are crucial for DN T cell-mediated suppression. We demonstrate that Ly-6A (Sca-1) is highly expressed on DN T cells. Incubation with IL-10 significantly reduced Ly-6A expression and the function of DN T cells. DN T cell-mediated killing was significantly reduced when Ly-6A was blocked.Ly-6A-deficient mice showed an accelerated allograft rejection when compared to wild-type controls. Furthermore we demonstrate that pretransplantation donor lymphocyte infusion (DLI) led to activation and proliferation of recipient DN T cells and prolongation of bm1-->B6 skin allograft survival. However, when the recipients were deficient in Ly-6A, the beneficial effect of DLI on allograft survival was abolished. Moreover, deficiency in Ly-6A did not affect the activation and proliferation of DN T cells. Rather, it impaired the ability of DN T cells to kill activated anti-donor CD8+ T cells. Taken together, our data indicate that Ly-6A plays a crucial role in DN T cell-mediated regulation in vitro and in vivo, perhaps by enhancing DN-CD8+ T cell signaling.     

MD-1 expression regulates direct and indirect allorecognition

Expression of the molecule MD-1 was previously described to regulate allogeneic and xenogeneic skin graft survival, as documented by the decrease in rejection seen following functional blockade of MD-1 expression in vivo, using antisense oligodeoxynucleotides (ODNs) or anti-MD-1 antibodies. It was unclear from these data whether blockade of expression of MD-1 on donor or recipient cells was crucial. We have investigated the effect on allorecognition of treating skin graft donors, and/or recipients, of either fully major histocompatibility complex (MHC)-mismatched allogeneic skin grafts (C3H with C57BL/6 grafts and vice versa) or grafts differing at only multiple minor alloantigens (C3H with B10.BR grafts; C57BL/6 with C3H.SW), with antisense ODNs to MD-1, or in some cases, following transplantation of class II-deficient cells into class I-deficient mice. Graft-specific cytotoxic T lymphocytes (CTLs) were measured in spleen cells recovered at sacrifice of recipients and following donor-specific restimulation in vitro. In the latter case, we also measured cell proliferation and (by enzyme-linked immunosorbent assay) production of interleukin-2 (IL-2)/interferon-gamma (IFN-gamma) or IL-4/IL-10 in vitro (nominal type-1 vs type-2 cytokines). CTL responses to minor-incompatible grafts were diminished, only if graft recipients were treated with ODNs. However, treatment of graft donor and/or recipient of MHC-incompatible grafts produced inhibition of CTL production. Optimal inhibition came from treating both. Specific suppression of CTL production coincided with inhibition of proliferation and preferential production of IL-4 and IL-10 at the expense of IL-2 and IFN-gamma. Our data are consistent with the hypothesis that MD-1 expression regulates both the direct and indirect pathways of allorecognition and that regulation of MD-1 expression may thus help regulate clinical graft rejection.     

Prolongation of allogeneic skin graft survival by injection of anti-Ly49A monoclonal antibody YE1/48

Ly49A receptors expressed on NK, NKT, and T cells play inhibitory roles in regulating the immune responses in vivo and in vitro. Whether or not injection of anti-Ly49A monoclonal antibody (mAb) YE1/48 can block allograft rejection has not been evaluated. Balb/c mouse recipients received intraperitoneal injections of YE1/48 mAb (0.5 mg) or control mAb or phosphate-buffered saline on days -1 and 10. On day 0, fully MHC-mismatched allogeneic C57BL/6 (B6) skin grafts were implanted. The skin graft survival and anti-donor humoral responses were observed. Whereas allogeneic B6 skin grafts survived 14 days in isotopy control antibody-treated or nontreated Balb/c mice, injection of YE1/48 mAb significantly prolonged B6 skin graft survival to 19 days (P < 0.0005). Injection of YE1/48 mAb into presensitized Balb/c recipients did not significantly delay B6 skin graft rejection. On the other hand, after depleting recipient NK, NKT, and some cytotoxic T cells by injection of anti-asialo GM1, YE1/48 failed to prolong B6 skin graft survival in Balb/c recipients. The present studies indicate that injection of YE1/48 mAb significantly delays allogeneic skin graft rejection in nonsensitized recipients but not in sensitized recipients. The presence of NK, NKT cells, and some cytotoxic T cells may be essential for YE1/48-mediated immunosuppression in vivo.     

Specific manipulation in vivo of immunity to skin grafts bearing multiple minor histocompatibility differences.

Naive CBA mice injected with AKR spleen cells via the portal vein (p.v.) subsequently showed decreased stimulation in vitro in a primary MLR or cell-mediated lympholysis assay with irradiated AKR stimulator cells. No inhibition of stimulation by B10.BR cells is seen. These mice also show specific prolongation of survival of AKR skin grafts in vivo and diminished capacity for in vivo priming for (secondary) anti-AKR responses in vitro. These effects are not seen if initial challenge is with AKR cells injected subcutaneously (s.c.) or via the lateral tail vein (i.v.). Moreover, if immune CBA anti-AKR mice are similarly challenged with AKR cells via the portal vein, no suppression of anti-AKR immunity is elicited, as determined by subsequent in vitro assays or in vivo graft rejection. However, spleen cells from CBA anti-AKR immune mice can be used to induce, in further naive CBA mice, a specific suppression of subsequent anti-AKR graft reactivity (assayed in vitro or in vivo). Active T cell-mediated suppression can be documented using both these protocols though the additional involvement of specific serum-mediated suppression cannot be eliminated.     

Accelerated Skin Graft Rejection Following Transfer of Cells Sensitized to a “Weak” Histocompatibility Antigen(s)

C3H/HeJ mice were immunized with either CBA/J or C57L/J skin grafts and then tested for secondary responses against both strains. Secondary responses were measured by six-day graft rejection, ability of syngeneic spleen cells to transfer skin-graft imnunity to sublethally irradiated mice, and graft-versus-host disease (GVHD) in lethally irradiated CBA/J mice receiving C3H/HeJ spleen cells. Although failure to transfer a primary response against “Creak” histocompatibility antigens with syngeneic spleen cells was noted, the secondary responses demonstrated a cross-reacting antigen(s) which seems to be of greater imnunologic strength than the postulated H-13 difference. Possible explanations for this finding include the existence of multiple undescribed loci with cross-reacting alleles, or an increase in immunogenicity of “weak” transplantation antigens when acting in concert with “stronger” antigens.     

Evidence for a limited contribution of immune regulation to cardiac allograft acceptance

We have used donor-reactive DTH responses to study the immune regulation that is displayed by C57BL/6 mice after they accept DBA/2 cardiac or renal allografts. This regulation is expressed by splenocytes from the allograft acceptors, and involves their alloantigen-induced production of the anti-inflammatory cytokines transforming growth factor beta (TGFbeta) and/or interleukin-10 (IL-10) at DTH challenge sites, and presumably within accepted allografts. In cardiac allograft acceptors the production of these cytokines depends on a population of CD25(+) splenocytes. During these studies, we have encountered several situations in which allograft acceptance does not correlate with DTH-detectable immune regulation: (1) splenocytes from cardiac or kidney allograft acceptors lose TGFbeta-mediated inhibition of donor-reactive DTH responses by 150 days post-transplant, although they retain ongoing allograft function; (2) cardiac allograft acceptors rapidly reject donor-matched skin allografts, retain good cardiac allograft function, but lose DTH-detectable immune regulation; (3) Balb/c mice accept C57BL/6 cardiac allografts when treated with anti-CD40L mAb (MR1), but fail to express DTH-detectable immune regulation; and (4) infusion of C57BL/6 mice with peritoneal exudate cells (PEC) that were educated ex vivo to DBA/2 alloantigens in the presence of IL-10 and TGFbeta, causes them to exhibit DTH-detectable immune regulation mediated by both TGFbeta and IL-10, but they fail to accept DBA/2 cardiac allografts. These observations suggest that the process of allograft acceptance, as it is studied in murine transplant models, is metastable, and does not necessarily reflect the achievement of allograft tolerance. Further, the development of allograft tolerance probably requires more than regulatory T cells, representing a coordinated evolution of multiple immune processes over a prolonged period of time.     

Characterisation of cyclosporine-induced suppressor cells in murine delayed-type hypersensitivity responses

The effect of Cyclosporine A (CsA) on T cell-mediated delayed-type hypersensitivity (DTH) reactions to murine alloantigens was analysed by transferring spleen cells from sensitised and suppressed mice into irradiated naive recipients. Selective removal of Thy1+, Ly1+ or Ly2+ cells prior to intravenous transfer of cells from alloantigen-sensitised mice showed that Ly1+ Thy1+ cells transferred sensitivity. Concomitant treatment of mice with alloantigen and CsA suppressed the DTH response to alloantigens, and spleen cells transferred from these suppressed mice abrogated the response of sensitised cells transferred at the same time. The suppressor cells were strain-restricted and antigen-specific with the surface phenotype Thy1+, Ly1-Ly2+.     

Accelerated Skin Graft Rejection Following Transfer of Cells Sensitized to a “Weak” Histocompatibility Antigen(s)

C3H/HeJ mice were immunized with either CBA/J or C57L/J skin grafts and then tested for secondary responses against both strains. Secondary responses were measured by six-day graft rejection, ability of syngeneic spleen cells to transfer skin-graft imnunity to sublethally irradiated mice, and graft-versus-host disease (GVHD) in lethally irradiated CBA/J mice receiving C3H/HeJ spleen cells. Although failure to transfer a primary response against “Creak” histocompatibility antigens with syngeneic spleen cells was noted, the secondary responses demonstrated a cross-reacting antigen(s) which seems to be of greater imnunologic strength than the postulated H-13 difference. Possible explanations for this finding include the existence of multiple undescribed loci with cross-reacting alleles, or an increase in immunogenicity of “weak” transplantation antigens when acting in concert with “stronger” antigens.     

Graft rejection mediated by CD4+ T cells via indirect recognition of alloantigen is associated with a dominant Th2 response

CD4(+) T cells that respond to indirectly presented alloantigen have been shown to mediate chronic rejection, however, the role of the indirect pathway in acute rejection has yet to be completely elucidated. To this end, BALB/c or C57BL/6 mice were depleted of CD8(+) T cells and transplanted with class II transactivator (CIITA)-deficient cardiac allografts, which cannot directly present class II alloantigens to CD4(+) T cells. In this manner, the rejection response by CD4(+) cells was forced to rely upon the indirect recognition pathway. When not depleted of CD8(+) cells, both BALB/c and C57BL/6 mice rejected CIITA-/- allografts and a polarized Th1 response was observed. In contrast, when BALB/c recipients of CIITA-/- allografts were depleted of CD8(+) T cells, the grafts were acutely rejected and a strong Th2 response characterized by eosinophil influx into the graft was observed. Interestingly, CD8-depleted C57BL/6 recipients of CIITA-/- allografts did not acutely reject their transplants and a Th2 response was not mounted. These findings indicate that CD4(+) T cells responding to indirectly presented alloantigens mediate graft rejection in a Th2-dominant manner, and provide further evidence for the role of Th2 responses in acute graft rejection.     

The surface phenotype of a suppressor cell of delayed-type hypersensitivity in the mouse.

Delayed-type hypersensitivity (DTH) to horse red blood cells was induced in cyclophosphamide-treated CBA/H mice. The DTH reaction, represented by an increase (0 x 8--1 x 0 mm) in footpad thickness 24 h after secondary challenge, could be suppressed by the adoptive transfer of 10(7) splenic lymphocytes from syngeneic mice primed with 10(9) HRBC. The surface antigenic phenotype of the suppressor cell was determined by the formation of EA, EAC, or Ig rosettes followed by depleting the rosetted populations on Isopaque-Ficoll. The suppressor cell was found to be Ig-, FcR- and CR-, although some suppression was observed with FcR+ cells. Cell depletions with cytotoxic alloantisera and rabbit complement further characterized the suppressor cell as being Thy-1+, Ly-1+, 2-, 3-, 4-, 5+, 6+, 7-, Ia- and IJ-. This cell surface phenotype if unique and differs from the Ly-1-, 2+, 3+, I-J+ suppressor cell of antibody formation and from the recently described Ly-+, 2+, 3+ feedback suppressor T cell.     

Expression and function of Qa-2 major histocompatibility complex class I molecules in transgenic mice

Qa-2 molecules are weak transplantation antigens encoded by class I genes of the major histocompatibility complex. When expressed in transgenic CBA mice, Qa-2 molecules provoke rapid rejection of skin grafts and strong, Qa-2 specific, cytotoxic T-cell responses. Efficient rejection of skin grafts from Qa-2 transgenic mice takes place when Qa-2 molecules are attached to the cell membrane with a glycophosphatidyl anchor or by a transmembrane protein domain, except that rejection times are slightly longer in the former case. These results demonstrate that Qa-2 molecules can behave as major transplantation antigens, as do closely related H-2 molecules. Failure of Qa-2 molecules to provoke strong T-cell responses in non-transgenic mice is probably due to the very low level of expression of Qa-2 molecules in skin keratinocytes from such mice since these cells express increased levels of Qa-2 molecules in all Qa-2 transgenic mice.     

Approach to withdrawal from tacrolimus in a fully allogeneic murine skin graft model

With few exceptions, transplant patients must take immunosuppressants throughout their lives. In this study, we used anti-T-cell receptor (TCR/CD3) monoclonal antibodies (mAbs) to induce immunological tolerance to alloantigens after withdrawal from tacrolimus in a fully allogeneic murine skin graft model. Skin grafts from AKR donor mice were maintained in C57BL/6 recipients by administering tacrolimus for one month. Anti-T-cell receptor (TCR) alphabeta mAb was administered to recipient mice on the day of withdrawal from tacrolimus administration. Seven days after mAb administration, the recipient mice were treated with various combinations of the following treatments: low-dose whole body irradiation, AKR bone marrow transfer (BMT), and anti-CD3 mAb administration. The control recipient mice did not receive treatment with either mAb, nor any other treatment. All the control recipient mice showed rejection of AKR skin grafts 42 days after tacrolimus withdrawal (mean skin graft survival: 77 days). Mice treated with a combination of anti-TCR alphabeta antibody, low-dose irradiation and AKR BMT showed stable chimerism in their peripheral blood lymphocytes and significantly prolonged skin graft survival (mean skin graft survival: >151.2+/-15.3 days). Mice given the combination of anti-TCR alphabeta mAb, anti-CD3 mAb, low-dose irradiation, and AKR BMT exhibited more stable chimerism but had earlier skin graft rejection (mean skin graft survival: 116.7+/-17.6 days) than the mice that did not receive anti-CD3 mAb. These results suggest that anti-TCR alphabeta mAb, but not anti-CD3 mAb, in combination with low-dose irradiation and BMT, is useful for long-lasting allograft survival after withdrawal from tacrolimus in mice with fully allogeneic skin grafts.     

Analysis of peripheral immune tolerance uncovers a mouse strain-dependent in situ type of graft tolerance

We screened various mouse strains [C57BL / 6, BALB / c, DBA / 2, CBA / Ca, (CBAxC57L / 6)F1, SJL, C3H] for induction of peripheral immune tolerance. Only CBA / Ca mice treated with anti-CD4 + CD8 monoclonal antibodies and grafted with allogeneic skin showed long-term graft survival (150 to > 200 days). Interestingly, T cells from the tolerant CBA / Ca mice rejected bone marrow / spleen cells of the skin graft donor strain and caused lethal graft-versus-host disease when transplanted to the donor strain. Furthermore, peripheral tolerance was easily broken: CBA / Ca mice could be reactivated to reject their tolerated grafts via immunization with (graft donor x recipient strain)F1 bone marrow cells. Thus, in contrast to the generalized nature of central tolerance, our experiments show that peripheral immune tolerance is strain dependent and locally restricted to graft tissue.     

A direct comparison of rejection by CD8 and CD4 T cells in a transgenic model of allotransplantation

INTRODUCTION: The relative contributions of CD4+ and CD8+ T cells to transplant rejection remain unknown. The authors integrated a previous model of CD4-mediated graft rejection with a complementary model of CD8-mediated rejection to directly compare the function of graft-reactive CD4+ and CD8+ lymphocytes in vivo in a model where rejection requires transgenic T cells. These studies allow direct comparison of CD4 and CD8 T cell responses to the same antigen without the confounding effects of T cell depletion or homeostatic proliferation. MATERIALS AND METHODS: Clone 4 and TS1 mice possess MHC class I- and II-restricted CD8+ and CD4+ T cells, respectively, which express transgenic T cell receptors that recognize the influenza hemagglutinin antigen (HA). We compared the in vivo response of CFSE-labeled, HA-specific transgenic CD8+ and CD4+ T cells after adoptive transfer into syngeneic BALB/c mice grafted with HA-expressing skin. RESULTS: As in the authors' CD4+ model, HA104 skin was consistently rejected by both Clone 4 mice (n=9, MST: 14.2) and by 5 x 10(5) Clone 4 lymphocytes transferred to naive BALB/c hosts that do not otherwise reject HA+ grafts. Rejection correlated with extensive proliferation of either graft-reactive T cell subset in the draining lymph nodes, and antigen-specific CD4+ and CD8+ cells acquired effector function and proliferated with similar kinetics. CONCLUSIONS: These data extend the authors' unique transgenic transplantation model to the investigation of CD8 T cell function. The initial results confirm fundamental functional similarity between the CD4 and CD8 T cell subsets and provide insight into the considerable redundancy underlying T cell mechanisms mediating allograft rejection.