Adoptive immunity in immune-deficient scid/scid mice. I. Differential requirements of naive and primed lymphocytes for CD4+ T cells during rejection of minor histocompatibility antigen-disparate skin grafts

Using a model system in which mature lymphocytes were adoptively transferred into immunodeficient C.B17-scid/scid recipients, the requirement for CD4+ T cells in rejection of previously healed-in multiple minor-H-antigen-disparate skin grafts was investigated. Depletion of functional CD4+ cells was accomplished by anti-CD4 antibody + complement treatment prior to adoptive transfer followed by chronic anti-CD4 serotherapy in vivo. Homozygous scid mice harboring nondepleted naive donor cells effectively rejected minor-H-antigen-disparate grafts, whereas scid/scid mice harboring CD4+ T-cell-depleted naive cells did not. Homozygous scid mice harboring minor-H-antigen-primed cells rejected the minor-H-antigen-disparate allografts regardless of whether or not the animals had received anti-CD4 treatment, although rejection by the mice receiving anti-CD4 treatment was retarded compared to mice not receiving anti-CD4 treatment. All the mice that received viable donor cells rejected minor + H-2 disparate allografts, demonstrating effective immunity in this case was not dependent upon the activity of CD4+ T cells. These data suggest that in responses against multiple minor-H-antigen-disparate tissue, primary allograft rejection is absolutely dependent upon CD4+ cells, and secondary allograft rejection is not. The implications of these findings in understanding interactions of T cell subsets in vivo and of the utility of using homozygous scid mice as recipients for exploring the function of transferred lymphoid cell populations are discussed.     

Immunosuppression by anti-CD4 treatment in vivo. Cellular and humoral responses to alloantigens

Antibody-mediated elimination of CD4+ lymphocytes in vivo has been successfully used to suppress the humoral response to foreign antigens and to induce long-term tolerance. However, secondary humoral responses, as well as secondary cytolytic responses specific for viral antigens, could not be prevented, providing evidence for functional heterogeneity within the helper cell compartment. Data presented here support the notion that helper cell requirements for cellular responses to alloantigens are unique and do not involve CD4+ T lymphocytes. While the administration of anti-CD4 mcAb failed to suppress allospecific CTL responses, the formation of alloantibodies was initially inhibited in parallel to the deficiency in CD4+ helper cells. After regeneration of CD4+ T cells, the animals regained the ability to produce specific IgG alloantibodies. The dichotomy of helper pathways in humoral and cellular alloreactive responses challenges the concept of a single CD4+ helper cell population. Insights into the functional heterogeneity of helper cells for primary, secondary, and allospecific responses might open new avenues for selective manipulation of helper subpopulations.     

Deletion of naïve T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers inhibits cognate CTL responses and alters immunodominance

Alloreactive T-cell responses directed against minor histocompatibility (H) antigens, which arise from diverse genetic disparities between donor and recipient outside the MHC, are an important cause of rejection of MHC-matched grafts. Because clinically significant responses appear to be directed at only a few antigens, the selective deletion of naïve T cells recognizing donor-specific, immunodominant minor H antigens in recipients before transplantation may be a useful tolerogenic strategy. We have previously demonstrated that peptide-MHC class I tetramers coupled to a toxin can efficiently eliminate specific TCR-transgenic T cells in vivo. Here, using the minor histocompatibility antigen HY as a model, we investigated whether toxic tetramers could inhibit the subsequent priming of the two H2-D^b-restricted, immunodominant T-cell responses by deleting precursor CTL. Immunization of female mice with male bone marrow elicited robust CTL activity against the Uty and Smcy epitopes, with Uty constituting the major response. As hypothesized, toxic tetramer administration prior to immunization increased survival of cognate peptide-pulsed cells in an in vivo CTL assay, and reduced the frequency of corresponding T cells. However, tetramer-mediated decreases in either T-cell population magnified CTL responses against the non-targeted epitope, suggesting that D^b-Uty⁺ and D^b-Smcy⁺ T cells compete for a limited common resource during priming. Toxic tetramers conceivably could be used in combination to dissect manipulate CD8⁺ T-cell immunodominance hierarchies, and to prevent the induction of donor-specific, minor H antigen CTL responses in allotransplantation.     

Evidence that long-term cardiac allograft survival induced by anti-CD4 monoclonal antibody does not require depletion of CD4+ T cells.

Monoclonal antibodies that deplete cells carrying their target antigen are being used increasingly for immunosuppression in clinical and experimental transplantation. We have characterized a panel of rat antimouse CD4 monoclonal antibodies with the aim of establishing, in a vascularized organ transplant model, whether prolonged graft survival can be induced without recipient T cell depletion. The spatial relationship of the epitopes recognized by the anti-CD4 mabs was established. Mabs of the IgG2b isotype were found to profoundly deplete CD4+ T cells in vivo, whereas IgG2a mabs did not. The IgG2b anti-CD4 mab YTS191 and the IgG2a mab KT6 both blocked proliferation of C3H/He leukocytes in mixed leukocyte culture. Potent suppression of rejection and indefinite survival of cardiac allografts, mismatched for both major and multiple minor histocompatibility antigens (C57BL/10, H-2b into C3H/He, H-2k), was achieved with the IgG2b anti-CD4 mab YTS191 that depleted CD4+ T cells, (n = 9, median survival time (MST) greater than 100 days, P less than 0.001). The non-depleting IgG2a anti-CD4 mab, KT6, which had been shown to recognize and epitope on the CD4 molecule closely related to that recognized by YTS191 and to block comparably in MLC, was also shown to be capable of producing long-term cardiac graft survival in this strain combination (n = 6, MST greater than 100 days P less than 0.001). The kinetics of the KT6 therapy on the blocking of the CD4 molecule in vivo were investigated and shown to correlate with the effectiveness of the mab in prolonging graft survival.     

Roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection

An essential role of murine CD4+ T cells in immune reactivity and skin graft rejection in discordant xenogeneic combinations have been reported. Our study was conducted to further clarify the roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection, by using CD4 and CD8 knockout [C57BL/6 Cr Slc (B6; H-2b) background] mice. When human skins were grafted on CD8 knockout mice or B6 mice, both hosts rejected human skin grafts within 12 days after grafting. By contrast, survival of human skin grafts was significantly prolonged in CD4 knockout mice (mean survival times=19.3+/-(SD) 1.6 days; median 19 days). Fully allogeneic C3H/He Slc (H-2k) skin grafts were rejected within 14 days in CD4 knockout mice, suggesting that non-CD4+ T cells in CD4 knockout mice were immunocompetent for allograft rejection. In spleens of these recipient mice, CD8+ T cells seemed to be activated 10 days after human skin grafting. Immunohistological analysis revealed the infiltration of CD8+ T cells at the site of transplanted human skin on CD4 knockout mice. To further examine the role of CD8+ T cells in CD4 knockout mice, human skin grafting was performed on day 0 followed by administration of anti-CD8 monoclonal antibody on days 0, 5, and 14. The administration of anti-CD8 monoclonal antibodies caused the significant prolongation of human skin graft survival. These results indicate the following two conclusions: (1) CD4+ T cells have an essential role in rejecting discordant human skin xenografts rapidly and (2) however, CD8+ T cells also are capable of rejecting discordant human skin xenografts.     

Mediation of skin allograft rejection in scid mice by CD4+ and CD8+ T cells.

We analyzed the role of CD4+ and CD8+ T cells in H-2-disparate skin allograft rejection in the mutant mouse strain C.B-17/Icr scid with severe combined immunodeficiency. On the day of skin allografting, scid mice were adoptively transferred with negatively selected CD4+ or CD8+ splenocytes from normal unsensitized C.B-17/Icr mice. These populations were obtained using a double-mAb--plus--complement elimination protocol using anti-CD4 or anti-CD8 mAb that resulted in no detectable CD4+ or CD8+ cells by FACS and negligible numbers of cytolytic T lymphocytes by limiting dilution analysis in anti-CD8 treated populations. Spleen cells were removed from grafted mice at the time of rejection and were tested in vitro for antidonor reactivity in several assays: mixed lymphocyte culture, cell-mediated lympholysis, and LDA for CTL and for IL-2-producing HTL. The presence of Thy 1.2+, CD4+, or CD8+ cells was determined by FACS. All control C.B-17 mice and scid mice adoptively transferred with nondepleted CD4+, and CD8+ cells rejected skin allografts with similar mean survival times (15.6 +/- 1.5, 18.8 +/- 3.4, 18.0 +/- 5.4, respectively), whereas control scid mice retain skin allografts indefinitely (all greater than 100 days). C.B-17 syngeneic grafts survived indefinitely in all groups. At the time of rejection, splenocytes from scid mice receiving CD4+ cells had negligible donor-specific cytotoxicity in CML and negligible numbers of CTL by LDA, but demonstrated a good proliferative response in MLC and IL-2-producing cells by LDA (frequency = 1/1764). There were no detectable CD8+ cells present by FACS analysis. Conversely, splenocytes from scid mice adoptively transferred with CD8+ cells had strong donor-specific cytotoxicity in CML (58.8% +/- 16.1%) and CTL by LDA (frequency = 1/3448), but no significant proliferation was detected in MLC. There were no detectable CD4+ cells by FACS, but there were small numbers of IL-2-producing cells by LDA (frequency = 1/10,204). These data demonstrate that CD4+ cells adoptively transferred into scid mice are capable of mediating skin allograft rejection in the absence of any detectable CD8+ cells or significant functional cytolytic activity. The adoptive transfer of CD8+ cells also results in skin allograft rejection in the absence of detectable CD4+ cells. The detection of small numbers of IL-2 secreting cells in these mice may indicate that CD(8+)-mediated allograft rejection in this model is dependent on IL-2-secreting CD8+ cells.     

Induction of transplantation tolerance in adults using donor antigen and anti-CD4 monoclonal antibody.

The T-cell-mediated immune response usually results in the rapid destruction of organ allografts transplanted between murine strains incompatible for major and minor histocompatibility antigens. This response may be modified by pretreatment with either donor-specific antigen or anti-CD4 monoclonal antibody. Previous work by others has shown that combined treatment of mice with soluble protein antigens and anti-CD4 monoclonal antibody can produce antigen-specific B cell unresponsiveness that continues long after the nonspecific immunosuppressive effect of the mAb treatment has resolved. Following this principle we have shown that adult C3H/He mice can be made specifically unresponsive to vascularized C57BL/10 cardiac allografts by pretreating the recipient with donor alloantigen under the cover of a brief course of mAb against CD4. A full-dose response analysis shows that the dose of mAb is critically important for the successful induction of tolerance. Tolerance induction using this protocol is dependent on treatment with donor major histocompatibility complex antigens and occurs in the presence of marked depletion but not complete elimination of the CD4+ T cell subset. The unresponsiveness to alloantigen is antigen specific, as determined by the ineffectiveness of third-party (C57BL/10) alloantigen when combined with anti-CD4 mAb to induce long-term survival of BALB/c allografts in C3H/He recipients. The tolerant state is specific and effective in the long-term as indicated by the specific acceptance of C57BL/10 skin grafts in recipients with surviving C57BL/10 cardiac allografts. This study provides a simple method for the successful induction of specific transplantation tolerance in the adult across a full H-2 major and minor antigen mismatch strain combination. The results illustrate the important role of the CD4 molecule in the T cell response to alloantigen in vivo and suggest possibilities for the therapeutic manipulation of complex immune reactions.     

Rejection of grafts with no H-2 disparity in TAP1 mutant mice: CD4 T cells are important effector cells and self H-2b class I molecules are target

Our previous results showed that TAP1 mutant mice rejected heart and skin grafts from donors with no H-2 disparity that express normal density of MHC class I molecules at the cell surface. During rejection, CD4 cells were predominant and essentially, no CD8 cells were found infiltrating the grafts. We hypothesized that TAP1 mutant mice, which developed and matured in an MHC class I-deficient environment, may have selected a repertoire of T cells with distinct reactivity to self class I molecules. The rejection of grafts with no H-2 disparity could be mediated by CD4+ T cells reactive to wild type H-2b class I molecules, or derived peptides, in the context of self-APC. Accordingly, we observed that transplanted TAP1 mutant mice presented a significant amplification of the proliferative T cell response to H-2Kb peptides, indicating that the stimulus with the graft was sufficient to induce peripheral expansion of these T cell repertoires. Therefore, the response to H-2Kb molecules could be a relevant pathway of activating T cells and triggering rejection of grafts expressing normal levels of these class I molecules. To test our hypothesis, we investigate the effect of pre-transplantation H-2Kb peptide-immunization on TAP1 mutant, which were then transplanted with C57BL/6 skin grafts (H-2b). Mice were immunized with a pool of five peptides derived from the polymorphic region of Kb alpha chain, before tail skin grafting. To study the role of CD4+ T cells in the rejection of C57BL/6 skin grafts, mice were in vivo depleted with an anti-CD4 monoclonal antibody GK1.5, and transplant evolution was observed. Sensitization of TAP1 mutant mice with H-2Kb peptides accelerated the rejection of skin grafts. Immunized mice rejected grafts with a MST of 13 days, compared to 16 days for the non-immunized mice (P=0.0089). The significant acceleration of graft rejection, induced by immunization with H-2Kb peptides, indicates that these peptides are capable of mobilizing effector T-cells that participate in rejection. These results support our hypothesis that class I molecules may be a target in the rejection of grafts with no MHC disparity. Depletion of CD4 T-cells resulted in a significant delay in rejection compared with the untreated control group. The MST of skin grafts in the controls was 16 days, whereas CD4-depleted recipients rejected skin grafts with a MST of 41 days (P=0.025). Moreover, some animals did not show macroscopic signs of rejection up to > 100 days posttransplantation. The contribution of CD4+ T cells to skin graft rejection, in our model, may reflect the occurrence of the presentation of H-2b peptides during graft rejection, in the context of self-APC. In conclusion, our results demonstrate an important role for H-2b molecules and CD4 T cells in the rejection of C57BL/6 grafts by TAP1 mutant mice. The low expression of MHC-I molecules on TAP1-/- mice may be determinant in the selection of a T cell repertoire strongly reactive to self MHC class I molecules which probably escapes the control of peripheral regulatory mechanisms.     

Prolonged minor allograft survival in intravenously primed mice--a test of the veto hypothesis

Experiments were performed to test the hypothesis that veto cells are responsible for the prolonged survival of minor allografts of skin that is observed in recipients primed intravenously with spleen cells from mice syngeneic with the skin donors. This prolonged survival was observed for each of several minor histocompatibility (H) antigens and is antigen-specific. Gamma radiation (3300 rads) abolished the ability of male spleen cells infused i.v. to delay the rejection of male skin grafts (H-Y antigen) on female recipients. However, depletion of Thy-1+ cells from the i.v. infusion failed to abolish the ability to prolong male skin graft survival. Furthermore, the prolonged survival accorded to B6 (H-2b) male skin grafts on CB6F1 (H-2b/H-2d) female recipients given i.v. infusions of B6 male spleen cells extended to BALB/c (H-2d) male skin grafts as well, indicating a lack of MHC restriction. Thus, prolongation of minor allograft survival by i.v. infusion of minor H antigen-bearing spleen cells appears not to depend on veto T cells that others have found to be responsible for the suppression of CTL generation (2, 3, 5).     

Contribution of CD40-CD154-mediated costimulation to an alloresponse in vivo.

BACKGROUND: Costimulation through CD40-CD154 plays an important role in T-cell activation. Although systemic administration of anti-CD154 antibody prevents or delays rejection of organ allografts in animal models, the molecular mechanisms responsible for this effect are not well defined. METHODS: We have previously demonstrated that priming of mice (H2d) with CD40-/- but not with wildtype naive B cells (H2b) leads to alloantigen-specific T-cell hyporesponsiveness in vitro. In the present study, we investigated whether such priming modifies allograft rejection in a major histocompatibility complex-mismatched murine cardiac transplantation model. RESULTS: Priming of hosts with donor-specific CD40-/- B cells delayed rejection of subsequently transplanted wild-type cardiac allografts by 8.0 days (P<0.001). The lack of CD40 on the cardiac graft delayed rejection in unprimed or primed hosts by 3-5 days. Prolongation of graft survival correlated with the failure of infused CD40-/- B cells to express B7.2 and ICAM-1 in vivo. CONCLUSIONS: Our data suggest that CD40-CD154 costimulation contributes to T cell priming to alloantigens in vivo and to a second set rejection phase in which donor antigens are presented to primed T cells.     

Aggressive skin allograft rejection in CD28-/- mice independent of the CD40/CD40L costimulatory pathway.

CD28-/- mice have been utilized to study the role of B7/CD28 and B7-CTLA4 interactions. There is evidence that CTLA4 ligation may be critical for tolerance induction. The aim of the current study is to further investigate rejection responses of CD28-/- mice and to define the role of B7-CTLA4 interactions in the absence of the CD40 and CD28 pathways. Balb/c skin allografts were transplanted onto C57BL/6 (B6) wild type or CD28-/- mice treated with anti-CD40L, CTLA4-Ig, or combination blockade. To investigate the cellular mechanism of rejection in CD28-/- recipients, mice were treated with anti-CD4 or anti-CD8 antibodies prior to treatment with costimulation blockade. The fluoroscein dye CFSE was utilized to study T cell expansion in vivo. Surprisingly, treatment of B6 CD28-/- mice with CTLA4-Ig alone (MST 12d), anti-CD40L alone (MST 13d), or combined blockade (MST 13d) had no effect on allograft survival compared to untreated B6 CD28 mice (MST 11d). CD28-/- recipients depleted of CD4+ cells and treated with CTLA4-Ig, anti-CD40L, or combination blockade also did not have prolonged survival compared with untreated mice (MST 10d). In contrast, CD28-/- recipients depleted of CD8+ cells had markedly prolonged allograft survival when treated with either anti-CD40L alone (MST 49d) or with combination blockade (MST 57d). Studies utilizing CFSE demonstrated that CD28-/- CD8+ T cells are not defective in in vivo proliferation responses compared with wild type CD8 cells. Thus, CD28-/- CD8+ T cells are responsible for aggressive rejection responses of CD28-/- mice independent of the CD40 pathway. In addition, CD40L blockade does not result in CD4+ T cell tolerance in CD28 recipients, despite an intact B7-CTLA4 pathway.     

Dominant tolerance and linked suppression induced by therapeutic antibodies do not depend on Fas-FasL interactions

BACKGROUND: Nonlytic anti-CD4 monoclonal antibody therapy can be used to induce transplantation tolerance in rodent models. Such tolerance is often associated with dominant regulation, mediated by CD4+ cells, and characterized by infectious tolerance and linked suppression. Understanding the mechanisms by which CD4+ regulatory cells function may improve the manner in which current immunosuppressants are applied and may lead to the development of new tolerance-inducing therapeutics. Fas-mediated apoptosis has been characterized as an important mechanism of peripheral self-tolerance and we here examine whether it has any role in anti-CD4 monoclonal antibody-induced dominant tolerance. METHODS: Tolerance to transplanted skin and bone marrow, mismatched for multiple minor histocompatibility antigens, was induced in Fas mutant and control mice using anti-CD4 and anti-CD8 monoclonal antibodies. To test for linked suppression, animals were transplanted with a second graft-bearing tolerated and third party antigens. The ability of splenocytes from tolerant animals to suppress graft rejection was assessed by transfer into partially immunocompromised recipients. RESULTS: Monoclonal antibody therapy rendered Fas mutant mice tolerant of minor disparate skin and bone marrow. Splenocytes from these and control tolerant animals when transferred into partially immunocompromised Fas mutant or control recipients, induced antigen-specific suppression of graft rejection. Additionally, tolerant Fas mutant mice accepted grafts bearing tolerated and third party antigens. CONCLUSIONS: Signal transduction through the Fas receptor plays no essential role in the induction of tolerance using anti-CD4 and anti-CD8 monoclonal antibodies or its maintenance by active regulation.     

Allograft Rejection in a New Allospecific CD4+ TCR Transgenic Mouse

The application of TCR transgenic mice to transplantation immunology is hampered by the limited lines available. Recently, we reported CD4+ T cell receptor (TCR) transgenic mice specific for I-Abm12 expressed on B6.C.H-2bm12 mice. Here, we characterized rejection of skin and vascularized cardiac allografts in these mice, which we term ABM (for anti-bm12). In vivo proliferative experiments reveal that all CD4 T cells in ABM mice react to bm12 antigens. Surprisingly, while ABM mice have accelerated (compared to B6 recipients) rejection of bm12 skin allografts, they, like B6 recipients, fail to acutely reject bm12 cardiac allografts. This is not due to lack of immunogenicity of bm12 hearts, as these grafts are acutely rejected by primed ABM recipients, although not by primed B6 recipients. Lastly, long-term surviving bm12 grafts in ABM recipients are relatively free from chronic rejection (compared with B6 recipients), which may be due to skewing of the CD4 repertoire towards direct alloreactivity, and consequent lack of CD4 mediated indirect allorecognition as evidenced by the lack of IgG deposition in those grafts. The results indicate that a complex interplay between responder frequency, priming and repertoire dictates the occurrence, or lack thereof, of acute and chronic rejection.     

Influence of direct and indirect allorecognition pathways on CD4+CD25+ regulatory T-cell function in transplantation

While both direct and indirect allorecognition are involved in allograft rejection, evidence to date suggests that tolerance is primarily dependent on indirect pathway-triggered CD4+CD25+ T cell-mediated immunoregulation. However, the precise influence of these two pathways on CD4+CD25+ T-cell function has not been addressed. In the current study, we have utilized an adoptive transfer model to assess selectively how the absence of either direct or indirect allorecognition affects CD4+CD25+ T-cell function. The effects of the loss of the direct pathway were assessed by transplanting skin grafts from minor histocompatibility mismatched B10.D2 (H-2d) donors onto Balb/c (H-2d) recipients, or by placing bone marrow chimeric DBA/2 (H-2d/H-2b) allografts onto C57BL/6 (H-2b) hosts. The requirement for indirect allorecognition was tested by grafting DBA/2 skin allografts onto either C57BL/6- or MHC-II-deficient C57BL/6 recipients. We report here that although CD4+CD25+ regulatory T cells can suppress both directly and indirectly generated alloresponses, immunoregulation is favored when indirect presentation is the sole mechanism of allorecognition. Hence, in the absence of indirect presentation, net CD4+CD25+ T cell-dependent immunoregulation is weak, and high ratios of CD4+CD25+ to CD4+CD25 T cells are required to ensure graft survival.     

Anti-CD2 monoclonal antibodies alter cell-mediated immunity in vivo.

The antimurine CD2 mAb 12-15 was administered intravenously to investigate the role of CD2 in cell-mediated immunity in vivo. The anti-CD2 mAb was able to diminish the contact sensitivity response to the hapten trinitrophenyl and was most effective when administered during the efferent or elicitative phase of immunity. Antibody treatment was also able to inhibit in vivo priming for subsequent generation of secondary, TNP-specific CTL in vitro. This inhibitory effect was most effective in the afferent or early phase of immunity. Indeed antibody could be injected at least 3 weeks prior to in vivo antigen priming, and the subsequent CTL response was still suppressed. Additional experiments showed a well-defined dose-response relationship between the amount of anti-CD2 administered and subsequent immunosuppression. Control experiments showed that other isotype-matched antibodies were not suppressive and that the anti-CD2 was not merely shifting the kinetics of the CTL response. Further experiments revealed that in vivo mAb treatment could also inhibit the subsequent development of primary, alloantigen-specific CTL in vitro while the mixed lymphocyte reaction (MLR) remained unchanged. FACS analysis revealed a marked downmodulation of CD2 in vivo, a small and variable decrease in CD8, and essentially no change in CD3 or CD4 after treatment with anti-CD2. The F(ab')2 fragment was not able to downmodulate CD2 or to suppress CTL activity at the doses tested. These results support a major role for CD2 in diverse aspects of cell-mediated immunity affecting both CD4+ and CD8+ effector T cells. The anti-CD2 mAb functions not by deleting or depleting relevant cell populations but rather by altering the array of cell surface receptors and subsequent responses to antigenic challenge.     

Rejection responses to allogeneic hepatocytes by reconstituted SCID mice, CD4, KO, and CD8 KO mice

INTRODUCTION: The purpose of the current study was to investigate the capacity of CD4+, CD8+, or non-T cells to independently initiate acute rejection of allogeneic hepatocytes using reconstituted SCID, CD4 or CD8 knockout (KO) recipient mice. METHODS: Allogeneic hepatocytes (FVB/N, H-2q) were transplanted into C57BL/6.SCID (H-2b), CD4 KO (H-2b), CD8 KO (H-2b), or beige/beige (H-2b) mice. SCID mice with functioning hepatocellular allografts subsequently received purified non-T cells (NTC), CD4+, or CD8+ splenocytes. Some mice were treated with anti-CD4, anti-CD8, and/or anti-nkl.1 mAb. Recipient mice were also assessed for donor-reactive delayed-type hypersensitivity (DTH) responses and donor-reactive alloantibody production. RESULTS: Median hepatocellular allograft survival time (MST) was 28 days in CD4+ reconstituted SCID mice and 14 days in CD8+ reconstituted SCID mice. SCID hosts reconstituted with NTC demonstrated indefinite hepatocellular allograft survival (>120 days). MST was 10 days in untreated beige/beige (NK cell deficient) mice. MST was 14 days in untreated, 35 days in anti-CD4 mAb treated, and 10 days in anti-nkl.1 mAb treated CD8 KO mice. MST was 10 days in untreated, 35 days in anti-CD8 mAb treated, and 7 days in anti-nk1.1 mAb treated CD4 KO mice. Donor-reactive DTH responses were not detected in reconstituted SCID mice, were minimal in CD4 KO mice, and were prominent in CD8 KO mice after rejection of allogeneic hepatocytes. Similarly, donor-reactive alloantibody, was not detected in CD4 KO hosts, but was readily detected in CD8 KO hosts. CONCLUSIONS: These studies show that both CD4+ and CD8+ T cells (but not host NTC) can independently initiate the rejection of allogeneic hepatocytes. While hepatocyte rejection by isolated CD4+ T cells is not surprising, rejection by CD8+ T cells (in the absence of CD4+ T cells) was unusual, and may explain the failure of "standard" immunosuppressive regimens to suppress acute rejection of allogeneic hepatocytes, as noted in prior studies. Furthermore, NK cells do not appear to be required for either CD4+ T cell or CD8+ T cell initiated hepatocyte rejection.     

Identification and characterization of the antigen-specific subpopulation of alloreactive CD4+ T cells in vitro and in vivo

We report the identification and characterization of the small subpopulation of alloantigen-specific T cells in vitro and in vivo. This subpopulation of T cells was distinguished by up-regulation of cell surface CD4 expression. These CD4high T cells were alloantigen specific in proliferation assays in vitro, and they expressed memory/activation markers, including CD44high and CD69high. Further studies demonstrated that these allospecific CD4high cells were also present (< or = 1% of CD4+ T cells) in vivo in BALB/c (H-2d) recipients of C57BL/6 (H-2b) skin allografts. CD4high T cells isolated from regional draining lymph nodes in these skin graft recipients reacted in a donor-specific fashion to C57BL/6 splenocyte stimulator cells in mixed lymphocyte culture. Adoptive transfer of CD4high, but not CD4normal T cells, just before skin engraftment in CD4 knockout mice, reconstituted rejection. The discovery that a small subpopulation of CD4high lymph node cells contained all of the alloantigen-specific T cells may allow study of tissue-specificity and subsequent alloantigen identification in transplantation.     

Allograft tolerance induction in adult mice associated with functional deletion of specific CTL precursors

Rejection of H-2 class I bm 1 mutant skin allografts by B6 recipient mice is mediated by a population of CD8+ anti-bm1 cytotoxic T-lymphocytes that produces and consumes its own T helper factor in response to bm1 skin allografts (dual function CTL). Previously we have demonstrated that transfusion of allogeneic lymphocytes across an H-2 class I disparity induces specific long-lasting skin allograft survival. We now show that intravenous injection of allogeneic spleen cells across the bm1 mutant disparity results in a temporary decrease of donor-reactive CTLp in the spleen of recipient mice, lasting for approximately five weeks. The sponge matrix allograft model was used to show that allograft tolerance is caused by a specific functional clonal deletion of CTLp within the allograft. We propose that dual function CTL are vetoed by donor T cells, resulting in skin allograft tolerance.     

The critical role of mouse CD4+ cells in the rejection of highly disparate xenogeneic pig thymus grafts

Long-term survival of fetal pig thymus (FP THY) grafts and efficient repopulation of mouse CD4+ T cells is achieved in thymectomized (ATX) B6 mice that receive T and NK cell depletion by injection of a cocktail of mAbs (GK1.5, 2.43, 30-H12, and PK136) and fetal pig thymus/liver (FP THY/LIV) grafts. The requirement for each mAb in this conditioning regimen in order to avoid the rejection of FP THY grafts has not yet been defined. In our present studies, CD4 cell-depleted ATX B6 mice and euthymic MHC class II-deficient (IIKO) mice were employed to investigate the role of mouse CD4+ cells in the rejection of FP THY grafts in vivo. After grafting FP THY/LIV to CD4+ cell-depleted ATX B6 mice, efficient repopulation of mouse CD4+ T cells was observed in the periphery. However, only two of four mice had remaining FP THY grafts by 17 weeks post-implantation, and these were of poor quality, whereas four of four T and NK cell-depleted ATX B6 mice had well-developed FP THY grafts. Furthermore, three of four FP THY/LIV-grafted, CD4+ cell-depleted ATX B6 mice rejected donor MHC-matched pig skin grafts. In contrast, three of three FP THY/LIV grafted, T and NK cell-depleted, ATX B6 mice accepted donor MHC-matched pig skin grafts, suggesting that optimal tolerance to xenogeneic pig antigens was not achieved in mice conditioned only with anti-CD4 mAb. ATX B6 mice treated with only anti-CD8 mAb rejected FP THY completely by 6 weeks post-grafting, a time when CD4+ cell-depleted ATX B6 mice had well-vascularized FP THY grafts. In addition, when euthymic IIKO mice were pre-treated with the standard conditioning regimen that includes four different mAbs, FP THY grafts survived and supported the repopulation of mouse CD4+ T cells in the periphery, while high levels of mouse CD8+ T cells developed in host thymi. These studies suggest that mouse CD4+ T cells play a critical role in the acute rejection of xenogeneic FP THY grafts. Without help from CD4+ cells, mouse CD8+ cells, NK, NK/T, and TCR(gamma/delta)+ T cells do not mediate acute rejection of FP THY grafts. Furthermore, our results suggest that other cell subsets besides CD4+ T cells play a role in the delayed rejection of highly disparate xenogeneic FP THY grafts.     

Allograft rejection in CD4+ T cell-reconstituted athymic nude rats--the nonessential role of host-derived CD8+ cells.

PVG-rnu/rnu nude rats reject fully allogenic renal (DA) and skin (BN, AO) allografts after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many nude-derived CD8+ leukocytes within the graft. In addition, mononuclear cells infiltrating the rejecting renal grafts in these animals display cytotoxic activity in vitro against specific and third-party alloantigens. In this investigation we have treated CD4+ T cell-restored nude rats bearing renal or skin allografts with the mAb MRC OX8 to deplete the host of CD8+ cells. In vivo treatment with OX8 completely eliminated CD8+ cells from rejecting grafts of both kidney and skin, but it did not prevent graft rejection, nor did OX8 treatment abolish the cytotoxic effector cells found in nude rat spleen or in graft-infiltrating cells (GIC) of rejecting renal allografts. The nature of the cytotoxic activity was examined with anti-CD3 mAb 1F4, which was shown to block conventional CD8+ Tc killing in vitro but did not inhibit allogeneic target cell lysis by spleen cells from nude rats. The cytotoxic activity found in GIC of rejecting allografts was not inhibited by anti-CD3 mAb, suggesting that these cytotoxic effector cells were CD3-CD8- and were of extrathymic origin. We conclude that non-thymus-derived CD8+ GIC are not essential for allograft rejection in CD4+ T cell-restored nude rats.