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.     

Down-Regulation of Surface CD28 under Belatacept Treatment: An Escape Mechanism for Antigen-Reactive T-Cells

Background

The co-stimulatory inhibitor of the CD28-CD80/86-pathway, belatacept, allows calcineurin-inhibitor-free immunosuppression in kidney transplantation. However, aggressive T-cell mediated allogeneic responses have been observed in belatacept-treated patients, which could be explained by effector-memory T-cells that lack membrane expression of CD28, i.e. CD28-negative (CD28^NULL) T-cells. CD28-positive (CD28^POS) T-cells that down regulate their surface CD28 after allogeneic stimulation could also pose a threat against the renal graft. The aim of this study was to investigate this potential escape mechanism for CD28^POS T-cells under belatacept treatment.

Materials & Methods

PBMCs, isolated T-cell memory subsets and isolated CD28^POS T-cells were obtained from end-stage renal disease (ESRD) patients and co-cultured with allo-antigen in the presence of belatacept to mimic allogeneic reactions in kidney-transplant patients under belatacept treatment. As a control, IgG was used in the absence of belatacept.

Results

Despite high in vitro belatacept concentrations, a residual T-cell growth of ±30% was observed compared to the IgG control after allogeneic stimulation. Of the alloreactive T-cells, the majority expressed an effector-memory phenotype. This predominance for effector-memory T-cells within the proliferated cells was even larger when a higher dose of belatacept was added. Contrary to isolated naïve and central-memory T cells, isolated effector-memory T cells could not be inhibited by belatacept in differentiation or allogeneic IFNγ production. The proportion of CD28-positive T cells was lower within the proliferated T cell population, but was still substantial. A fair number of the isolated initially CD28^POS T-cells differentiated into CD28^NULL T-cells, which made them not targetable by belatacept. These induced CD28^NULL T-cells were not anergic as they produced high amounts of IFNγ upon allogeneic stimulation. The majority of the proliferated isolated originally CD28^POS T-cells, however, still expressed CD28 and also expressed IFNγ.

Conclusion

This study provides evidence that, apart from CD28^NULL T-cells, also CD28^POS, mostly effector-memory T-cells can mediate allogeneic responses despite belatacept treatment.     

Role of donor-specific regulatory T cells in long-term acceptance of rat hind limb allograft

Background

Vascularized bone marrow transplantation (VBMT) is widely accepted as an efficient means of establishing chimerism and inducing tolerance. However, the mechanism underlying is poorly understood. Recently, regulatory T cells (Tregs) have been shown to play an important role in regulating immune responses to allogeneic antigens. In this study, we explored the role of Tregs in the induction of tolerance in an allogeneic hind limb transplantation model.

Methodology/Principal Findings

Forty-eight Lewis rats were divided into 6 groups. They received isografts and allografts from Brown-Norway hind limbs. Recipients in groups 1 and 2 received isografts and those in the other groups received allografts. The bone components of donor limbs were kept intact in groups 1, 3, and 5 but removed before transplantation into groups 2, 4, and 6. Tapered cyclosporin A (CsA) was administered to recipients in groups 5 and 6 after transplantation. During the 100-day observation period, all isografts survived, but the allografts in groups 3 and 4 were rejected within 8 to 12 days. CsA-treated intact allografts survived rejection-free for more than 100 days, and CsA-treated allografts lacking bone elements were rejected within 2 months. Stable peripheral chimerism and myeloid chimerism were observed in group 5. Declining peripheral chimerism and a lack of myeloid chimerism were observed in group 6. Donor-specific Tregs were exclusively detected in both peripheral blood and in the spleens of long-term recipient rats in group 5, with an increased FoxP3 mRNA expression in the allografts. This was further demonstrated to be responsible for donor-specific hyporeactivity by in vitro one-way mixed lymphocyte reaction (MLR).

Conclusion/Significance

Bone components in the allogeneic hind limbs can induce myeloid chimerism and donor-specific Tregs may be essential to tolerance induction. The bone-removal hind limb model may be a suitable counterpart to the induction of tolerance in the study of limb transplantation.     

Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment

Allogeneic bone marrow transplantation (in immunocompetent adults) has always required cytoreductive treatment of recipients with irradiation or cytotoxic drugs to achieve lasting engraftment at levels detectable by non-PCR-based techniques ('macrochimerism' or 'mixed chimerism'). Only syngeneic marrow engraftment at such levels has been achieved in unconditioned hosts. This requirement for potentially toxic myelosuppressive host pre-conditioning has precluded the clinical use of allogeneic bone marrow transplantation for many indications other than malignancies, including tolerance induction. We demonstrate here that treatment of naive mice with a high dose of fully major histocompatibility complex-mismatched allogeneic bone marrow, followed by one injection each of monoclonal antibody against CD154 and cytotoxic T-lymphocyte antigen 4 immunoglobulin, resulted in multi-lineage hematopoietic macrochimerism (of about 15%) that persisted for up to 34 weeks. Long-term chimeras developed donor-specific tolerance (donor skin graft survival of more than 145 days) and demonstrated ongoing intrathymic deletion of donor-reactive T cells. A protocol of high-dose bone marrow transplantation and co-stimulatory blockade can thus achieve allogeneic bone marrow engraftment without cytoreduction or T-cell depletion of the host, and eliminates a principal barrier to the more widespread use of allogeneic bone marrow transplantation. Although efforts have been made to minimize host pre-treatment for allogeneic bone marrow transplantation for tolerance induction, so far none have succeeded in eliminating pre-treatment completely. Our demonstration that this can be achieved provides the rationale for a safe approach for inducing robust transplantation tolerance in large animals and humans.     

Studies on the mechanism of transplantation tolerance: I. Do suppressor cells play a role in the induction and maintenance of neonatal transplantation tolerance?

Neonatal transplantation tolerance was induced in CBA (H-2^k) mice to A (H-2^a) mice by injection of (CBA × A)F₁ spleen cells. Animals carrying an A-skin test allograft for more than 4 months without any visible sign of rejection were considered to be permanently tolerant. Permanently tolerant CBA mice were given normal syngeneic spleen cells to abrogate the state of tolerance. Abrogation of tolerance was greatly facilitated by antithymocyte serum (ATS) treatment of tolerant mice prior to the normal syngeneic cell transfer. Survival of A allografts on normal, adult, ATS-treated CBA mice was significantly prolonged (and in many cases “adult” tolerance was achieved) by transfer of spleen cells of syngeneic mice made permanently tolerant at neonatal age. The possible role of the F₁-cell “contamination” in the tolerance-inducing effect of the transferred “tolerant” spleen cells was excluded. The results indicate that ATS-sensitive suppressor cells play a definite role in the induction, maintenance, and transfer of neonatally induced transplantation tolerance.     

Treatment with immunotoxin

T-cell depletion prior to or beginning at the time of transplantation has been shown to be a valuable adjunct to the induction of immunological unresponsiveness. Both total lymphoid irradiation and anti-lymphocyte globulin have been used for this purpose in experimental models of transplantation as well as in human organ transplant recipients. However, these methods of T-cell depletion are limited in their ability to deplete T cells selectively due to non-specific targeting and limited efficacy. A new anti-CD3 immunotoxin has been developed with a far more potent ability to deplete T cells selectively as measured by flow cytometry analysis of peripheral blood T lymphocytes as well as lymph node lymphocytes. This immunotoxin is well tolerated by rhesus monkeys when administered in vivo. When administered as a single immunosuppressive agent pretransplant, it substantially promotes allograft survival, inducing tolerance in at least one-third of recipients as measured by subsequent acceptance of donor skin grafts and rejection of third-party skin grafts. When administered on the day of transplant in combination with steroid pretreatment and a brief course of deoxyspergualin or mycophenolate mofetil (4 to 14 days), long-term unresponsiveness is also produced and in a more reliable manner than using immunotoxin alone. A new immunotoxin directed at the human CD3epsilon has been developed with excellent potency in T-cell killing and lacking the Fc portion of the CD3 antibody. This construct may be useful for T-cell depletion in humans and has a potential application in tolerance induction in human organ transplantation. Lessons learned from anti-CD3 immunotoxin in the non-human primate model to date include (i) profound (2-3 log) depletion of T-cells can be accomplished safely without inducing lymphoma or infection, (ii) such depletion is a useful adjunct for tolerance induction to allogeneic organ transplants, and (iii) tolerance to both allogeneic renal transplants and xenogeneic islet transplants has been accomplished using such strategies to date in non-human primates and in pigs. Immunotoxin may be useful for the induction of chimerism using strategies that include donor bone marrow infusion. Successful strategies for tolerance induction have also been developed using immunotoxin without the adjunct of donor bone marrow or stem cell infusion. Clinical application of immunotoxin will use a newly engineered construct with the potential for causing cytokine release, less susceptibility to neutralization by anti-diphtheria antibody and not dependent on chemical conjugation of an antibody and toxin. The usefulness of immunotoxin is directly related to its tremendous potency for depleting T cells. Based on results in nonhuman primates, it is anticipated that it will become a useful agent in tolerance induction in humans.     

Resistance of neonatal tolerance in mice to abrogation by normal immunocytes

The mechanisms of neonatally induced tolerance in CAF₁ mice to bovine serum albumin were investigated with cell transfer experiments and primary interaction between radiolabeled antigen and antisera for antibody measurements. Adoptive transfer of normal syngeneic splenocytes, of thymocytes, or of thymus-bone marrow cell combinations did not break this tolerance though proved effective for breaking irradiation-caused tolerance. Similar transfer of spleen cells taken from immunized mice did abrogate neonatal tolerance; but spleen cells transferred from immunized mice that subsequently also had been desensitized did not, even though they could provide nontolerant recipients with anamnestic responses. Neither allogeneic nor xenogeneic spleen cells broke the tolerance.Thus, the neonatal tolerance studied appears to suppress primary immunologic response but not an anamnestic response. It appears more likely to be an active suppressive phenomenon than one of clone loss.     

Responsiveness of Thymus Cells to Syngeneic and Allogeneic Lymphoid Cells

THE thymus is necessary for the normal development of cell-mediated immunity in mice as shown by the immunological defects after neonatal thymectomy¹. Thymus cells themselves can be stimulated by allogeneic lymphoid cells in mixed leucocyte reaction (MLR)² and become killer cells or cytotoxic lymphocytes after stimulation with allogeneic spleen cells in vitro (H. Wagner and M. Feldmann, unpublished work) and in vivo^3,4. This suggests that the thymus as well as peripheral lymphoid tissues contain T cells which can be stimulated by foreign histocompatibility antigen to divide and differentiate into the cytotoxic lymphocytes which mediate cellular immunity. There have been suggestions that thymus cells might be stimulated to divide by “self” antigen, as well as foreign cells: incorporation of ³H-thymidine above background levels has been found in cultures with syngeneic spleen and thymus cells of adult rats⁵, although the experiments do not determine whether thymus or spleen cells have been stimulated. In contrast to these experiments, Howe et al. reported that only thymus cells of neonatal CBA mice reacted to allogeneic and syngeneic spleen cells of adult animals in “one way” MLR cultures^6,7. Whether the reaction of neonatal thymus cells to syngeneic adult spleen cells is recognition of “self” antigens is uncertain, since spleens of adult mice could carry antigens which do not occur in neonatal animals and are therefore “unknown” for neonatal thymus cells. We demonstrate here that neonatal thymus cells do not react to 4-day-old CBA spleen cells, but adult thymus cells do react against both allogeneic and syngeneic adult spleen cells.     

Immunomodulation after allogeneic bone marrow transplantation by CD4+CD25+ regulatory T cells

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation (BMT) caused by donor T cells. Immunosuppression mediated by CD4(+)CD25(+) regulatory T cells has been shown to ameliorate such pathogenic immune responses in animal models. Here, we summarize recent findings from experimental and clinical studies and propose a model for peripheral tolerance induction after BMT.     

Allogeneic offspring produced by male germ line stem cell transplantation into infertile mouse testis

The testis is one of several immune-privileged organs and is known for its unique ability to support allogeneic or xenogeneic tissue transplants. We investigated the possibility of deriving offspring from mice that underwent transplantation with allogeneic male germ line stem cells in the testis. Although mature adult mice rejected allogeneic germ cells and were infertile, offspring were obtained by intracytoplasmic germ cell injection using partially differentiated donor cells. In contrast, complete spermatogenesis occurred when allogeneic germ cells were transplanted into immature pup testes. Tolerance induction by monoclonal antibody administration allowed the pup transplant recipients to produce allogeneic offspring by natural mating, whereas no spermatozoa were found in the epididymis of untreated recipients. Thus, these results indicate that a histoincompatible recipient can serve as a "surrogate father" to propagate the genetic information of heterologous male donors.     

Vascularized composite islet-kidney transplantation in a miniature swine model

Previous work from this laboratory has demonstrated that transplantation of allogeneic thymic tissue as part of a composite vascularized graft is far more successful in terms of both engraftment and long-term survival than transplantation of thymic tissue or cells alone. We have subsequently extended this concept to transplantation of allogeneic islets, comparing survival of islet cell suspensions to that of vascularized composite islet-kidneys (IK), prepared by injection of autologous islets underneath the renal capsule 2-3 months prior to allogeneic transplantation of the composite organ. We have utilized partially inbred miniature swine with defined MHC loci as the experimental large animals for this study, permitting reproducible transplantation across specific MHC barriers. Composite IK have been transplanted successfully across minor and full MHC mismatch barriers, using treatment regimens previously demonstrated to induce long-term tolerance of kidney allografts across these barriers. IK allografts containing ≥5000 islet equivalents (IE)/kg recipient body weight were found capable of reversing surgically induced diabetes, while injection of comparable numbers of purified islets via the portal vein or under the renal capsule did not. Studies are also being directed toward preparation of autologous “thymo-islet-kidneys” (TIK), for potential use as xenografts, in which the thymic component is intended to induce tolerance and the islets to reverse diabetic hyperglycemia. The use of both types of composite organ transplants may eventually be applicable to the treatment of type I diabetic patients suffering from end-stage diabetic nephropathy.     

Tolerogenic function of dimeric forms of HLA-G recombinant proteins: a comparative study in vivo

HLA-G is a natural tolerogenic molecule involved in the best example of tolerance to foreign tissues there is: the maternal-fetal tolerance. The further involvement of HLA-G in the tolerance of allogeneic transplants has also been demonstrated and some of its mechanisms of action have been elucidated. For these reasons, therapeutic HLA-G molecules for tolerance induction in transplantation are actively investigated. In the present study, we studied the tolerogenic functions of three different HLA-G recombinant proteins: HLA-G heavy chain fused to β2-microglobulin (B2M), HLA-G heavy chain fused to B2M and to the Fc portion of an immunoglobulin, and HLA-G alpha-1 domain either fused to the Fc part of an immunoglobulin or as a synthetic peptide. Our results demonstrate the tolerogenic function of B2M-HLA-G fusion proteins, and especially of B2M-HLA-G5, which were capable of significantly delaying allogeneic skin graft rejection in a murine in vivo transplantation model. The results from our studies suggest that HLA-G recombinant proteins are relevant candidates for tolerance induction in human transplantation.     

Mixed chimerism

Induction of mixed chimerism has the potential to overcome the current limitations of transplantation, namely chronic rejection, complications of immunosuppressive therapy and the need for xenografts to overcome the current shortage of allogeneic organs. Successful achievement of mixed chimerism had been shown to tolerize T cells, B cells and possibly natural killer cells, the lymphocyte subsets that pose major barriers to allogeneic and xenogeneic transplants. Current understanding of the mechanisms involved in tolerization of each cell type is reviewed. Considerable advances have been made in reducing the potential toxicity of conditioning regimens required for the induction of mixed chimerism in rodent models, and translation of these strategies to large animal models and in a patient are important advances toward more widespread clinical application of the mixed chimerism approach for tolerance induction.     

Development of either split tolerance or robust tolerance along with humoral tolerance to donor and third-party alloantigens in nonmyeloablative mixed chimeras

Hematopoietic chimerism is considered to generate robust allogeneic tolerance; however, tissue rejection by chimeras can occur. This "split tolerance" can result from immunity toward tissue-specific Ags not expressed by hematopoietic cells. Known to occur in chimeric recipients of skin grafts, it has not often been reported for other donor tissues. Because chimerism is viewed as a potential approach to induce islet transplantation tolerance, we generated mixed bone marrow chimerism in the tolerance-resistant NOD mouse and tested for split tolerance. An unusual multilevel split tolerance developed in NOD chimeras, but not chimeric B6 controls. NOD chimeras demonstrated persistent T cell chimerism but rejected other donor hematopoietic cells, including B cells. NOD chimeras also showed partial donor alloreactivity. Furthermore, NOD chimeras were split tolerant to donor skin transplants and even donor islet transplants, unlike control B6 chimeras. Surprisingly, islet rejection was not a result of autoimmunity, since NOD chimeras did not reject syngeneic islets. Split tolerance was linked to non-MHC genes of the NOD genetic background and was manifested recessively in F(1) studies. Also, NOD chimeras but not B6 chimeras could generate serum alloantibodies, although at greatly reduced levels compared with nonchimeric controls. Surprisingly, the alloantibody response was sufficiently cross-reactive that chimerism-induced humoral tolerance extended to third-party cells. These data identify split tolerance, generated by a tolerance-resistant genetic background, as an important new limitation to the chimerism approach. In contrast, the possibility of humoral tolerance to multiple donors is potentially beneficial.     

Neonatal immunology and cord blood transplantation

The increased susceptibility of human newborns to infections is usually ascribed to the immaturity of the neonatal immune system. The neonatal immune system has never met microbial antigens, and thus the repertoire of its adaptative arm (T and B cells) is entirely pre-immune, or "na?ve". However this neonatal pre-immune repertoire is similar to the adult pre-immune repertoire, and cord blood natural killer cells studies show that the innate immunity cells harbor the full killing machinery that characterize mature cells. Moreover, human neonates are able to show an adult-like allogeneic response. Taken together, several lines of evidence suggest that the neonatal immune system, although na?ve, is fully mature. However, newborns display phenotypic and functional differences with adults in both adaptative and innate arms. Specific properties may explain these differences, as high number of regulatory T cells, low plasmacytoid dendritic cell response to stimuli and high IL-10 production. These properties are in line with the high susceptibility of newborns to infections and the low incidence of graft-versus-host-disease after cord blood transplantation. To explain these differences, we introduce a new model. Although naive, the neonatal immune system is mature, and these functional differences are due to a message originating from the placenta and aimed at inducing the foetus tolerance to its mother. Full understanding of the involved mechanisms will help to protect the newborn against infections and to improve cord blood transplantation outcome.     

Replacement of T-Cell Function by a T-Cell Product

WE have recently shown that the immune response to sheep red blood cells (SRBC) in vitro is T-cell dependent, that is, it can be abrogated by pretreatment of the spleen cells with anti-θ serum and complement¹. We further reported that reconstitution of the system can be achieved by, among other things, the addition of allogeneic thymocytes², while syngeneic thymocytes failed to function as helper cells. Positive allogeneic effects were only obtained if the added thymocytes could recognize the remaining B-cells as foreign. One of the explanations evoked for this allogeneic effect was the participation of a potentiating factor^2,3. It seemed possible that on the heavy antigenic stimulation provided by the histocompatibility antigens carried by the B-cells, added T-cells produce a soluble factor which stimulates the immune response of B-cells to unrelated antigens. We now want to report the existence of two distinct factors, one of which can completely replace T-cells.     

Partial tolerance and immunity after adoptive abrogation of transplantation tolerance in the rat

Lewis rats were rendered hematopoietic and lymphoid cell chimeras by injection of (LBN)F₁ hybrid cells at birth or following treatment with cyclophosphamide in adult life. The establishment of transplantation tolerance was indicated by acceptance of (LBN)F₁ skin grafts and specific unresponsiveness in graft vs. host reaction (GvHR) and mixed lymphocyte interaction (MLI) in vitro. Tolerance was abolished by adoptively transferred Lewis lymphocytes, and the loss of chimerism and recovery of specific reactivity by blood lymphocytes were monitored independently by mixed lymphocyte cultures. Recovery of competence to initiate GvHR by splenic and lymph node cells was monitored by the local renal graft vs. host technique. Both techniques measure essentially the proliferative response of certain lymphocytes to foreign cellular AgB antigens, and both detected a prolonged, but gradually weakening, state of partial tolerance to the AgB factors to which tolerance had originally been induced. During this phase of partial tolerance the former chimera rejects skin and lymph node cell grafts from (LBN)F₁ donors with alacrity, but in some cases accepts (LBN)F₁ kidney grafts. Cytotoxic antibodies appear in the serum soon after allogeneic chimerism is terminated. These results are interpreted to indicate that a state of partial tolerance exists among the cells which proliferate in response to certain AgB antigens in GvHR and MLI in the formerly tolerant chimera, and that a state of transplantation immunity (possibly to other determinants) coexists with this partial tolerance.     

Hematopoietic stem cell transplantation for the treatment of autoimmunity in type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease that leads to the destruction of the insulin-producing pancreatic b cells. While there is no current cure, recent work in the field of allogeneic hematopoietic stem cell transplantation (HSCT) and the induction of mixed chimerism, a state in which multilineage hematopoietic populations of both recipient and donor co-exist, has demonstrated that it is possible to provide protection from disease onset, as well as reverse the autoimmune state in spontaneously diabetic mice. Furthermore, the establishment of mixed chimerism induces donor-specific tolerance, providing the potential to normalize glucose regulation via pancreatic islet transplantation without the requirement of life-long immunosuppression. Current studies are aimed at understanding the mechanisms involved in both the reversal of autoimmunity and the induction of tolerance, with the aim of moving this promising approach to curing T1D into the clinic.