Anti-LFA-1 therapy induces long-term islet allograft acceptance in the absence of IFN-gamma or IL-4

mAb therapy directed against a variety of cell surface accessory molecules has been effectively utilized to prolong allograft acceptance in various models of tissue and organ transplantation. The purpose of this study was to determine whether transient therapy directed against the adhesion molecule LFA-1 (CD11a) was sufficient to induce donor-specific tolerance to pancreatic islet allografts. Anti-LFA-1 monotherapy was found to be efficacious in inducing long-term islet allograft acceptance in multiple donor-recipient strain combinations. Graft acceptance following anti-LFA-1 therapy was not simply due to clonal ignorance of donor Ags in that the majority of recipients bearing established islet allografts resisted rejection induced by immunization with donor-type APCs. Furthermore, donor-specific tolerance from anti-LFA-1-treated animals could be transferred to secondary immune-deficient animals. Taken together, these results indicated that transient anti-LFA-1 monotherapy resulted in donor-specific tolerance. In vitro, functionally tolerant animals retained normal anti-donor reactivity as assessed by proliferative, cytotoxic, and cytokine release assays that demonstrated that tolerance was not secondary to general clonal deletion or anergy of donor-reactive T cells. Finally, anti-LFA-1 treatment was effective in both IL-4-deficient and IFN-gamma-deficient recipients, indicating that neither of these cytokines are universally required for allograft acceptance. These results suggest that anti-adhesion-based therapy can induce a nondeletional form of tolerance that is not overtly dependent on the prototypic Th1 and Th2 cytokines, IFN-gamma and IL-4, respectively, in contrast to results in other transplantation models.     

Suppressor T cells, distinct from "veto cells," are induced by alloantigen priming and mediate transferable suppression of cytotoxic T lymphocyte responses in vivo

Primary and secondary cytotoxic T lymphocyte responses to minor alloantigens can be suppressed by priming host mice with a high dose (10(8) cells) of alloantigenic donor spleen cells (SC). Such suppression is antigen specific and transferable into secondary hosts with T cells. One interpretation of this is that antigen-specific host suppressor T cells (Ts) are activated. Alternatively, donor Lyt-2+ T cells, introduced in the priming inoculum, may inactivate host CTL precursors (CTLp) that recognize the priming (donor) alloantigens. Donor cells that act in this way are termed veto T cells. The experiments described here exclude veto T cell participation in transferable alloantigen-specific suppression, and demonstrate the operation of an alloantigen-specific host-derived T suppressor (Ts) cell. The origin of the Ts has been studied directly by using Thy-1-disparate BALB/c mice. The cell responsible for the transfer of suppression of a secondary CTL response to B10 minors was of the host Thy-1 allotype, and so originated in the host spleen and was not introduced in the priming inoculum. Secondly, antigen-specific Ts generated in CBA female mice against B10 minors could act on CTL responses to an unequivocally non-cross-reactive-third party antigen (H-Y), provided the two antigens were expressed on the same cell membrane. Such third-party suppression is incompatible with the operation of veto T cells. Depletion of Thy-1.2+ or Lyt-2+ cells from the suppression-inducing donor SC inoculum did not abrogate suppression induction in BALB/c mice; instead, suppression was enhanced. The demonstration of veto cell activity in similarly primed mice by other groups of investigators indicates that both types of suppression may operate. However, our results show that only antigen-specific Ts can mediate the transferable suppression of CTL responses to alloantigens.     

Analysis of the donor-specific cytotoxic T lymphocyte repertoire in a patient with a long term surviving allograft. Frequency, specificity, and phenotype of donor-reactive T cell receptor (TCR)-alpha beta+ and TCR-gamma delta+ clones

In the present study the transplant specific CTL repertoire of a patient (HLA:A1,3, B8,18, Cw5,7 DR3, DQw2, DPw3) with a long term surviving HLA mismatched kidney graft (HLA: A1,24 B8,27 Cw2,7, DR3, w13 DQw2,6 DPw1,3) has been investigated. This patient was unable to generate specific cytolytic activity against donor-derived PHA-blasts in the MLC in which donor spleen cells or B lymphoblastoid cell line were used as stimulator cells. In addition, the CTL precursor frequencies against donor alloantigens were very low (1/67,000). The patient had otherwise normal immune responses in vivo and in vitro and no signs of transplant rejection. Transplant specific CTL clones were generated in high frequencies (1/195) from T cell bulk cultures activated by PHA in the absence of any sensitization by donor Ag in vitro. The repertoire of 14 donor-reactive CTL clones (12 TCR-alpha beta+ and 2 TCR-gamma delta+) was analyzed. Two TCR-alpha beta+ CD8+ clones were specific for B27. Ten TCR-alpha beta+ CTL clones directed against class II HLA Ag were isolated. Seven of these were CD4+ and recognized DRw13 (3), DQw6 (3), and DPw1 (1), whereas three of these clones were CD4-CD8+ recognizing DRw13 (1) and DQw6 (2). In addition, two donor-specific TCR-gamma delta+ CTL clones were obtained recognizing HLA-A9(23,24) and DQw6. Our data indicate that the precursors of CTL clones specifically directed against donor class I or II HLA Ag are not deleted from the repertoire and that part of this reactivity resides in the TCR-gamma delta+ fraction.     

Rapid deletional peripheral CD8 T cell tolerance induced by allogeneic bone marrow: role of donor class II MHC and B cells

Mixed chimerism and donor-specific tolerance are achieved in mice receiving 3 Gy of total body irradiation and anti-CD154 mAb followed by allogeneic bone marrow (BM) transplantation. In this model, recipient CD4 cells are critically important for CD8 tolerance. To evaluate the role of CD4 cells recognizing donor MHC class II directly, we used class II-deficient donor marrow and were not able to achieve chimerism unless recipient CD8 cells were depleted, indicating that directly alloreactive CD4 cells were necessary for CD8 tolerance. To identify the MHC class II(+) donor cells promoting this tolerance, we used donor BM lacking certain cell populations or used positively selected cell populations. Neither donor CD11c(+) dendritic cells, B cells, T cells, nor donor-derived IL-10 were critical for chimerism induction. Purified donor B cells induced early chimerism and donor-specific cell-mediated lympholysis tolerance in both strain combinations tested. In contrast, positively selected CD11b(+) monocytes/myeloid cells did not induce early chimerism in either strain combination. Donor cell preparations containing B cells were able to induce early deletion of donor-reactive TCR-transgenic 2C CD8 T cells, whereas those devoid of B cells had reduced activity. Thus, induction of stable mixed chimerism depends on the expression of MHC class II on the donor marrow, but no requisite donor cell lineage was identified. Donor BM-derived B cells induced early chimerism, donor-specific cell-mediated lympholysis tolerance, and deletion of donor-reactive CD8 T cells, whereas CD11b(+) cells did not. Thus, BM-derived B cells are potent tolerogenic APCs for alloreactive CD8 cells.     

Suppressor T cell recognition of major and minor histocompatibility alloantigens: selected suppression of MHC-directed responses by minor alloantigen Ts

The induction of suppression by i.v. administered alloantigens in the murine host was analyzed as a model of the possible effects of blood transfusion on transplant survival. The results indicated that suppressor T cells (Ts) specific for minor histocompatibility alloantigens could be readily induced by the i.v. presentation of minor alloantigen-disparate spleen cells. In contrast, similar priming with cells differing solely at the H-2 major histocompatibility complex stimulated only positive T cell immunity, with no evidence of suppression. The induction of H-2 directed Ts activity could be accomplished only by i.v. priming with major plus minor incompatible donor cells, suggesting that suppressor cell recognition of minor alloantigens may have facilitated the generation of Ts against H-2-encoded major transplantation antigens. A role for minor histocompatibility antigens in the regulation of H-2-specific immunity at the effector level was also indicated. Ts induced by i.v. pretreatment with minor antigen-disparate donor cells not only suppressed the delayed-type hypersensitivity (DTH) response to the relevant minor alloantigens, but also inhibited DTH against unrelated H-2 alloantigens introduced during subsequent intradermal immunization. Suppression of H-2-directed T cell reactivity was specific in that the presence of the Ts-inducing minor alloantigens was also required and occurred only when the minor and unrelated major alloantigens were presented within the same inoculum, if not on the same cell surface. The capacity of Lyt-2+Ts or Ts-derived suppressive factors specific for one set of cell surface molecules to modulate responses to an unrelated group of surface antigens does not appear to represent a general phenomenon, because similar suppression of immunity to unrelated tumor-specific transplantation antigens by minor-specific Ts was not observed. These results are discussed with respect to the possible mechanism of H-2-directed suppression and the role of the I region in Ts recognition of antigen.     

IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo

We present evidence that donor-reactive CD4(+) T cells present in mice tolerant to donor alloantigens are phenotypically and functionally heterogeneous. CD4(+) T cells contained within the CD45RB(high) fraction remained capable of mediating graft rejection when transferred to donor alloantigen-grafted T cell-depleted mice. In contrast, the CD45RB(low) CD4(+) and CD25(+)CD4(+) populations failed to induce rejection, but rather, were able to inhibit rejection initiated by naive CD45RB(high) CD4(+) T cells. Analysis of the mechanism of immunoregulation transferred by CD45RB(low) CD4(+) T cells in vivo revealed that it was donor Ag specific and could be inhibited by neutralizing Abs reactive with IL-10, but not IL-4. CD45RB(low) CD4(+) T cells from tolerant mice were also immune suppressive in vitro, as coculture of these cells with naive CD45RB(high) CD4(+) T cells inhibited proliferation and Th1 cytokine production in response to donor alloantigens presented via the indirect pathway. These results demonstrate that alloantigen-specific regulatory T cells contained within the CD45RB(low) CD4(+) T cell population are responsible for the maintenance of tolerance to donor alloantigens in vivo and require IL-10 for functional activity.     

Tolerance to antigen-presenting cell-depleted islet allografts is CD4 T cell dependent

Pretreatment of pancreatic islets in 95% oxygen culture depletes graft-associated APCs and leads to indefinite allograft acceptance in immunocompetent recipients. As such, the APC-depleted allograft represents a model of peripheral alloantigen presentation in the absence of donor-derived costimulation. Over time, a state of donor-specific tolerance develops in which recipients are resistant to donor APC-induced graft rejection. Thus, persistence of the graft is sufficient to induce tolerance independent of other immune interventions. Donor-specific tolerance could be adoptively transferred to immune-deficient SCID recipient mice transplanted with fresh immunogenic islet allografts, indicating that the original recipient was not simply "ignorant" of donor antigens. Interestingly, despite the fact that the original islet allograft presented only MHC class I alloantigens, CD8+ T cells obtained from tolerant animals readily collaborated with naive CD4+ T cells to reject donor-type islet grafts. Conversely, tolerant CD4+ T cells failed to collaborate effectively with naive CD8+ T cells for the rejection of donor-type grafts. In conclusion, the MHC class I+, II- islet allograft paradoxically leads to a change in the donor-reactive CD4 T cell subset and not in the CD8 subset. We hypothesize that the tolerant state is not due to direct class I alloantigen presentation to CD8 T cells but, rather, occurs via the indirect pathway of donor Ag presentation to CD4 T cells in the context of host MHC class II molecules.     

Cyclosporine-induced tolerance in experimental organ transplantation. Evidence of diminished donor-specific cytotoxicity relative to donor-specific proliferative response

Alloreactivity of intragraft and peripheral blood lymphocytes from tolerant canine lung allograft recipients was examined. Tolerance was induced by variable periods of treatment with cyclosporine. Analysis of effector cells from lung allografts (obtained by bronchoalveolar lavage) revealed the absence of specific cytolytic T lymphocyte (CTL) activity and the presence of a low level of cytolytic activity detected in a lectin-dependent cell-mediated cytotoxicity assay. In contrast, high levels of specific CTL activity and lectin-dependent activity were detected in cell preparations from lung allografts undergoing rejection. Tolerant recipients retained normal ability to generate specific CTL activity to third party alloantigens in mixed lymphocyte cultures (MLC) but had diminished ability to generate CTL to donor alloantigens in recipient X donor MLC. Addition of exogenous interleukin 2 to these MLC was unable to restore donor-specific CTL activity. Lymphocytes from tolerant recipients were, however, capable of generating proliferative responses and lectin-dependent cytotoxicity on exposure to donor alloantigens in MLC. Evidence presented in this report suggests that the lectin-dependent cytolytic activity generated in these MLC is mediated by lymphokine-activated killer cells. Such cells are likely to be activated by interleukin 2 released in the proliferative response. The results support the proposal that the cyclosporine-induced tolerant state is characterized by the relative inability to respond against major histocompatibility complex class I antigens in contrast to class II antigens and/or minor histocompatibility antigens since MLC-induced CTL are directed, for the most part, against class I molecules.     

Neonatal tolerance of H-2 alloantigens. II. I region dependence of tolerance expressed to K and D antigens

Third-party skin allografts were employed to test the specificity of transplantation tolerance achieved by neonatal inoculation of cells bearing H-2 alloantigens. Tolerant animals rejected with normal vigour third-party grafts expressing strong Class I alloantigens foreign to the host and to the donor of the tolerance-conferring inoculum. However, these animals rejected with exceptional vigour third-party grafts expressing weak Class II alloantigens plus the tolerated Class I alloantigen; even third-party grafts comprised of the host's own Class II antigens in conjunction with the tolerated Class I alloantigen were acutely rejected. It is proposed, but there is no direct evidence to prove, that rejection of these third-party grafts is mediated by killer T cells directed at the tolerated Class I alloantigens and that these cells are activated by the presentation of the putative tolerogen in an inappropriate I region context. Inconsistency of these data with a clonal deletion mechanism is discussed.     

Suppression of cytotoxic response to histoincompatible cells. I. Evidence for two types of T lymphocyte-derived suppressors acting at different stages in the induction of a cytotoxic response.

Spleen and thymus cell populations from normal or allograft tolerant mice have been cultured for 5 days with specific alloantigens and examined for their reactivity in three assay systems. No consistent correlation was observed between the production of cytotoxic T cells (CTL) in these cultures and the ability of such cultured cells to inhibit specifically a CML response from fresh normal spleen cells directed to the priming alloantigens. Furthermore, suppressor cells measured in this latter assay were apparently distinct from those able to inhibit the production of cytotoxic lymphocyte precursors (CTLp) from bone marrow stem cells in lethally irradiated bone marrow protected mice. Velocity sedimentation experiments confirmed that both the precursor and effector cells for the two suppressor systems were physically separable, and were distinct from CTLp or CTL, respectively. Precursor cells for the two suppressor systems investigated belong to the short-lived cortical thymus cell population.     

Suppression of delayed-type hypersensitivity to third party "bystander" alloantigens by antigen-specific suppressor T cells

Delayed-type hypersensitivity (DTH) against alloantigens can be induced by sc immunization with allogeneic cells. The induction of DTH can be suppressed by iv preimmunization of the mice with similar allogeneic spleen cells, provided the cells are irradiated before injection. This suppression is mediated by T cells. The suppressor activity can be induced not only by H-2-and non-H-2-coded antigens, but also by H-2 subregion-coded antigens. Suppression induced by K, I, or D subregion-coded antigens is specific for that particular subregion as well as for its haplotype. I-J-coded alloantigens were found to not be necessary for the induction of antigen-specific suppressor T cells. After restimulation of suppressor T cells by the "specific" alloantigens, the DTH to simultaneously administered third-party alloantigens becomes suppressed as well. This nonspecific suppression of DTH to third party "bystander" alloantigens also occurs when the specific and the third-party antigens are presented on separate cells, provided that both cell types are administered together at the same site. The simultaneous presentation of both sets of alloantigens during the induction phase of DTH only is sufficient to prevent the normal development of DTH to the third-party antigens.     

Cellular events during radiation-induced thymic leukemogenesis in mice: abnormal T cell differentiation in the thymus and defect of thymocyte precursors in the bone marrow after split-dose irradiation

Cellular events during the development of thymic lymphomas in young B10.BR mice given leukemogenic split-dose irradiation were studied by examining the differentiation of functional T lymphocyte precursors in the regenerating thymus. It was found that leukemogenic radiation treatment resulted in a sustained depression of the level of thymic cytotoxic T lymphocyte precursors (CTLp) and of mixed lymphocyte reactivity of thymus cells when assessed between 1 and 4 mo after irradiation, in spite of the fact that the total number of thymocytes was restored to the normal level within 2 mo and continued to increase thereafter. In vitro mixing studies of normal thymocytes with thymus cells from split-dose irradiated mice provided no evidence for active suppression as a mechanism for this depressed activity. The ability of bone marrow cells from split-dose irradiated mice to regenerate the thymus and to differentiate into functional CTLp was examined by use of supralethally irradiated Thy-1 congenic recipients. Reconstitution of supralethally irradiated B10.BR Thy-1.2 mice with normal bone marrow from B10.BR Thy-1.1 mice resulted in the complete repopulation of host-thymus with donor-derived cells when assessed at 4 wk after reconstitution. Lymphocytes from the regenerating thymus of these animals were shown to contain high levels of CTLp which were donor-derived. On the other hand, when the recipient mice were reconstituted with bone marrow cells from donor mice which had been split-dose irradiated 1 mo earlier, regeneration of the recipient thymus was severely depressed when assessed at 4 wk to 3 mo after reconstitution. Although variable but small numbers of donor-derived Thy-1+ cells were detected, CTL activity for alloantigen could not be induced in these donor-derived cells. The results suggest that T cell precursors derived from split-dose irradiated donor mice were unable to undergo active proliferation and differentiation into functional CTLp. The significance of these findings on radiation-induced thymic leukemogenesis is discussed.     

CD25+CD4+ regulatory T cells prevent graft rejection: CTLA-4- and IL-10-dependent immunoregulation of alloresponses.

Specific and selective immunological unresponsiveness to donor alloantigens can be induced in vivo. We have shown previously that CD25+CD4+ T cells from mice exhibiting long-term operational tolerance to donor alloantigens can regulate rejection of allogeneic skin grafts mediated by CD45RB(high)CD4+ T cells. In this study, we wished to determine whether donor-specific regulatory cells can be generated during the induction phase of unresponsiveness, i.e., before transplantation. We provide evidence that pretreatment with anti-CD4 Ab plus a donor-specific transfusion generates donor-specific regulatory CD25+CD4+ T cells that can suppress rejection of skin grafts mediated by naive CD45RB(high)CD4+ T cells. Regulatory cells were contained only in the CD25+ fraction, as equivalent numbers of CD25-CD4+ T cells were unable to regulate rejection. This pretreatment strategy led to increased expression of CD122 by the CD25+CD4+ T cells. Blockade of both the IL-10 and CTLA-4 pathways abrogated immunoregulation mediated by CD25+ T cells, suggesting that IL-10 and CTLA-4 are required for the functional activity of this population of immunoregulatory T cells. In clinical transplantation, the generation of regulatory T cells that could provide dynamic control of rejection responses is a possible route to permanent graft survival without the need for long-term immunosuppression.     

Promotion of skin graft tolerance across MHC barriers by mobilization of dendritic cells in donor hemopoietic cell infusions

Flt3 ligand (FL) dramatically increases the number of immunostimulatory dendritic cells (DC) and their precursors in bone marrow (BM) and secondary lymphoid tissues. Herein we tested the ability of FL-mobilized donor hemopoietic cells to promote induction of skin graft tolerance across full MHC barriers. C57BL/10 (B10; H2(b), IE(-)) mice were given 10(8) spleen cells (SC) from normal or FL-treated, H-2-mismatched B10.D2 (H2(d), IE(+)) donors i.v. on day 0, 200 mg/kg i.p. cyclophosphamide on day 2, and 10(7) T cell-depleted BM cells from B10.D2 mice on day 3. B10.D2 skin grafting was performed on day 14. Indefinite allograft survival (100 days) was induced in recipients of FL-SC, but not in mice given normal SC. Tolerance was associated with blood macrochimerism and was confirmed by second-set skin grafting with donor skin 100 days after the first graft. In tolerant mice, peripheral donor-reactive T cells expressing TCR Vbeta11 were deleted selectively. Immunocompetence of tolerant FL-SC-treated mice was proven by rapid rejection of third-party skin grafts. To our knowledge this is the first report that mobilization of DC in donor cell infusions can be used to induce skin graft tolerance across MHC barriers, accompanied by specific deletion of donor-reactive T cells.     

Role of double-negative regulatory T cells in long-term cardiac xenograft survival

A novel subset of CD3(+)CD4(-)CD8(-) (double negative; DN) regulatory T cells has recently been shown to induce donor-specific skin allograft acceptance following donor lymphocyte infusion (DLI). In this study, we investigated the effect of DLI on rat to mouse cardiac xenotransplant survival and the ability of DN T cells to regulate xenoreactive T cells. B6 mice were given either DLI from Lewis rats, a short course of depleting anti-CD4 mAb, both DLI and anti-CD4 treatment together, or left untreated. DLI alone did not prolong graft survival when compared with untreated controls. Although anti-CD4-depleting mAb alone significantly prolonged graft survival, grafts were eventually rejected by all recipients. However, the combination of DLI and anti-CD4 treatment induced permanent cardiac xenograft survival. We demonstrate that recipients given both DLI and anti-CD4 treatment had a significant increase in the total number of DN T cells in their spleens when compared with all other treatment groups. Furthermore, DN T cells harvested from the spleens of DLI plus anti-CD4-treated mice could dose-dependently inhibit the proliferation of syngeneic antidonor T cells. Suppression mediated by these DN T cells was specific for antidonor T cells as T cells stimulated by third-party Ags were not suppressed. These results demonstrate for the first time that a combination of pretransplant DLI and anti-CD4-depleting mAb can induce permanent survival of rat to mouse cardiac xenografts and that DN T regulatory cells play an important role in preventing long-term concordant xenograft rejection through the specific suppression of antidonor T cells.     

Differential susceptibility of cytotoxic and helper T cell precursors to neonatal tolerization to histocompatibility antigens

The ability of various (C57BL/6J X CBA/HT6T6)F1 spleen cell subpopulations to induce tolerance to allogeneic histocompatibility antigens after injection into neonatal CBA/HT6T6 mice was examined. The requirements for tolerization of cytotoxic T lymphocyte precursors (CTLp) and IL 2-producing helper T cell precursors (IL 2Tp) appear to be coordinated but not identical. CTLp frequencies measured in limiting dilution analysis (LDA) were found to be decreased by 90 to 99% in mice injected neonatally with unseparated or a variety of semiallogeneic spleen cell fractions, including T cells, T cell-depleted spleen, the Ig+ and Ig- fractions of nylon-adherent, T-depleted spleen cells, Sephadex-G10 (G10)-nonadherent spleen cells, and T-depleted allogeneic C57BL/6J spleen cells. In contrast, IL 2Tp showed tolerization only after neonatal injection of unseparated or T cell-depleted F1 spleen cells, and not after injection of T or B cells or of G10-nonadherent or T-depleted allogeneic spleen cells. These studies show that the CTLp and IL 2Tp compartments have different requirements for neonatal tolerization, which appear to correlate with the presence of cells expressing class I or class II alloantigens in the inoculum: all spleen cell types tested were capable of tolerizing the CTLp compartment, whereas only whole spleen and T-depleted spleen cells could tolerize IL 2Tp; donor T cells, although capable of inducing CTLp tolerance, are not necessary for either CTLp or IL 2Tp tolerance induction; Ig+ B cells alone are marginally effective in tolerization of IL 2Tp, and G10-nonadherent cells are ineffective, suggesting that macrophages or another type of G10-adherent accessory cell may be required for tolerization of IL 2Tp, although it is not clear whether they are sufficient; and tolerization of CTLp can occur in the presence of a normal IL 2Tp compartment when certain inocula, such as T cells, are used for tolerance induction at birth.     

Antibody-mediated rejection of cardiac allografts in CCR5-deficient recipients

Rejected MHC-mismatched cardiac allografts in CCR5(-/-) recipients have low T cell infiltration, but intense deposition of C3d in the large vessels and capillaries of the graft, characteristics of Ab-mediated rejection. The roles of donor-specific Ab and CD4 and CD8 T cell responses in the rejection of complete MHC-mismatched heart grafts by CCR5(-/-) recipients were directly investigated. Wild-type C57BL/6 and B6.CCR5(-/-) (H-2(b)) recipients of A/J (H-2(a)) cardiac allografts had equivalent numbers of donor-reactive CD4 T cells producing IFN-gamma, whereas CD4 T cells producing IL-4 were increased in CCR5(-/-) recipients. Numbers of donor-reactive CD8 T cells producing IFN-gamma were reduced 60% in CCR5(-/-) recipients. Day 8 posttransplant serum titers of donor-specific Ab were 15- to 25-fold higher in CCR5(-/-) allograft recipients, and transfer of this serum provoked cardiac allograft rejection in RAG-1(-/-) recipients within 14 days, whereas transfer of either serum from wild-type recipients or immune serum from CCR5-deficient recipients diluted to titers observed in wild-type recipients did not mediate this rejection. Wild-type C57BL/6 and B6.CCR5(-/-) recipients rejected A/J cardiac grafts by day 11, whereas rejection was delayed (day 12-60, mean 21 days) in muMT(-/-)/CCR5(-/-) recipients. These results indicate that the donor-specific Ab produced in CCR5(-/-) heart allograft recipients is sufficient to directly mediate graft rejection, and the absence of recipient CCR5 expression has differential effects on the priming of alloreactive CD4 and CD8 T cells.     

Loss of direct and maintenance of indirect alloresponses in renal allograft recipients: implications for the pathogenesis of chronic allograft nephropathy.

Chronic allograft nephropathy (CAN) is the principal cause of late renal allograft failure. This complex process is multifactorial in origin, and there is good evidence for immune-mediated effects. The immune contribution to this process is directed by CD4(+) T cells, which can be activated by either direct or indirect pathways of allorecognition. For the first time, these pathways have been simultaneously compared in a cohort of 22 longstanding renal allograft recipients (13 with good function and nine with CAN). CD4(+) T cells from all patients reveal donor-specific hyporesponsiveness by the direct pathway according to proliferation or the secretion of the cytokines IL-2, IL-5, and IFN-gamma. Donor-specific cytotoxic T cell responses were also attenuated. In contrast, the frequencies of indirectly alloreactive cells were maintained, patients with CAN having significantly higher frequencies of CD4(+) T cells indirectly activated by allogeneic peptides when compared with controls with good allograft function. An extensive search for alloantibodies has revealed significant titers in only a minority of patients, both with and without CAN. In summary, this study demonstrates widespread donor-specific hyporesponsiveness in directly activated CD4(+) T cells derived from longstanding recipients of renal allografts, whether they have CAN or not. However, patients with CAN have significantly higher frequencies of CD4(+) T cells activated by donor Ags in an indirect manner, a phenomenon resembling split tolerance. These findings provide an insight into the pathogenesis of CAN and also have implications for the development of a clinical tolerance assay.     

Human mesenchymal stem cells suppress induction of cytotoxic response to alloantigens

Mesenchymal stem cells (MSC) fail to induce allogeneic responses in mixed lymphocyte reaction assays. Because MSC express HLA class I molecules, here we investigated whether they could be recognized as allogeneic targets by cytolytic T lymphocytes (CTL). With this aim, CTL precursor (CTLp) frequencies were measured following stimulation of T cells with either allogeneic mononuclear cells (MNC) or MSC originated from the same human bone marrow donor. Lysis of MSC was measured at day 10 of culture in standard chromium release assays. In addition, allogeneic PHA blast T cells or B-EBV lymphoblastoid cell lines (LCLs) generated from the same donor were used as positive controls of lysis. Our results showed that when allogeneic MNC were used to stimulate T cells, a high CTLp frequency was detected towards MSC targets. However, when MSC were used as stimulators, CTLp frequencies were markedly altered whatever the targets used, i.e.: MSC, PHA blast T cells or EBV-B LCLs. Moreover, when graded concentrations of MSC were added together with MNC upon stimulation of alloreactive T cells, we observed a dose-dependent decrease in CTLp frequencies towards MSC targets. This inhibition of MSC lysis was partially overcome by adding exogenous rh-IL-2 from the beginning of cultures. In addition, this suppressive effect was totally reproduced when, instead of MSC, supernatant harvested from MSC cultures was added to allogeneic MNC, upon stimulation of alloreactive T cells. In conclusion, our results demonstrate that MSC which can be recognized as targets by pre-activated alloreactive CTLs, may be able to suppress differentiation of CTL precursors into CTL effectors through secretion of suppressive factors.     

Early loss of precursors of CTL and IL 2-producing cells in the development of neonatal tolerance to alloantigens

Bulk culture and limiting dilution analysis (LDA) were used to follow the ontogeny of the tolerant state in CBA/ HT6T6 mice neonatally tolerized to allogeneic histocompatibility antigens. Advantage was taken of the fact that the lymph nodes (LN) of young mice show immunocompetence before spleen cells do, allowing analysis of actual reactivity as early as 1 wk of age. At 1 wk, the LN cells of mice tolerized i.v. showed a loss of CTL reactivity in bulk culture specific for the tolerizing antigens; a corresponding specific decrease was seen in the frequency of CTL precursors (CTLp). At the same age, however, proliferative responses and interleukin 2 (IL 2) production in MLC were nonspecifically depressed in the tolerized animals. LDA of IL 2 producer precursor frequency (IL- 2Tp ) showed that there was a nonspecific loss of 50% of functional alloreactive IL- 2Tp , accompanied by a larger specific decrease of 90% in the frequency of IL- 2Tp responding to the injected alloantigens. These characteristics of the tolerant state persisted through at least 4 wk of age. Neither the proliferative nor CTL response deficiencies could be overcome by the addition of Con A supernatant containing IL 2. Mixing experiments failed to show evidence of suppressor cell involvement in the loss of the proliferative response. Our results indicate that the specific loss of alloreactivity after tolerization is due to clonal inactivation or deletion of both CTLp and IL- 2Tp , which is obvious as early as 7 days of age. In addition, the differences in the specificity of the clonal inactivation between CTLp and IL- 2Tp suggest the existence of independent mechanisms for tolerization.