Anti‐LFA‐1 or rapamycin overcome costimulation blockade‐resistant rejection in sensitized bone marrow recipients

While costimulation blockade‐based mixed chimerism protocols work well for inducing tolerance in rodents, translation to preclinical large animal/nonhuman primate models has been less successful. One recognized cause for these difficulties is the high frequency of alloreactive memory T cells (Tmem) found in the (pre)clinical setting as opposed to laboratory mice. In the present study, we therefore developed a murine bone marrow transplantation (BMT) model employing recipients harboring polyclonal donor‐reactive Tmem without concomitant humoral sensitization. This model was then used to identify strategies to overcome this additional immune barrier. We found that B6 recipients that were enriched with 3 × 10⁷ T cells isolated from B6 mice that had been previously grafted with Balb/c skin, rejected Balb/c BM despite costimulation blockade with anti‐CD40L and CTLA4Ig (while recipients not enriched developed chimerism). Adjunctive short‐term treatment of sensitized BMT recipients with rapamycin or anti‐LFA‐1 mAb was demonstrated to be effective in controlling Tmem in this model, leading to long‐term mixed chimerism and donor‐specific tolerance. Thus, rapamycin and anti‐LFA‐1 mAb are effective in overcoming the potent barrier that donor‐reactive Tmem pose to the induction of mixed chimerism and tolerance despite costimulation blockade.     

Rapamycin and CTLA4Ig Synergize to Induce Stable Mixed Chimerism Without the Need for CD40 Blockade

The mixed chimerism approach achieves donor‐specific tolerance in organ transplantation, but clinical use is inhibited by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti‐CD154 monoclonal antibodies (mAbs) is exceptionally potent in inducing mixed chimerism, but these mAbs are clinically not available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that CD4 or CD8 activation through an intact direct or CD4 T cell activation through the indirect pathway is sufficient to trigger BM rejection despite CTLA4Ig treatment. In the absence of CD4 T cells, CD8 T cell activation via the direct pathway, in contrast, leads to a state of split tolerance. Interruption of the CD40 signals in both the direct and indirect pathway of allorecognition or lack of recipient CD154 is required for the induction of chimerism and tolerance. We developed a novel BMT protocol that induces mixed chimerism and donor‐specific tolerance to fully mismatched cardiac allografts relying on CD28 costimulation blockade and mTOR inhibition without targeting the CD40 pathway. Notably, MHC‐mismatched/minor antigen‐matched skin grafts survive indefinitely whereas fully mismatched grafts are rejected, suggesting that non‐MHC antigens cause graft rejection and split tolerance.     

The Spleen Is the Major Source of Antidonor Antibody‐Secreting Cells in Murine Heart Allograft Recipients

Antibody‐mediated allograft rejection is an increasingly recognized problem in clinical transplantation. However, the primary location of donor‐specific alloantibody (DSA)‐producing cells after transplantation have not been identified. The purpose of this study was to test the contribution of allospecific antibody‐secreting cells (ASCs) from different anatomical compartments in a mouse transplantation model. Fully MHC‐mismatched heart allografts were transplanted into three groups of recipients: nonsensitized wild type, alloantigen‐sensitized wild‐type and CCR5^?/? mice that have exaggerated alloantibody responses. We found that previous sensitization to donor alloantigens resulted in the development of antidonor alloantibody (alloAb) with accelerated kinetics. Nevertheless, the numbers of alloantibody‐secreting cells and the serum titers of antidonor IgG alloantibody were equivalent in sensitized and nonsensitized recipients 6 weeks after transplantation. Regardless of recipient sensitization status, the spleen contained higher numbers of donor‐reactive ASCs than bone marrow at days 7 – 21 after transplantation. Furthermore, individual spleen ASCs produced more antidonor IgG alloantibody than bone marrow ASCs. Taken together, our results indicate that the spleen rather than bone marrow is the major source of donor‐reactive alloAb early after transplantation in both sensitized and nonsensitized recipients.     

Antilymphocytic antibodies and marrow transplantation. V. Suppression of secondary disease by host-versus-theta-graft reaction.

An approach to block secondary disease was investigated in mice sensitized against the Th-1.1 (theta-AKR) alloantigen on the marrow donor's T cells. To avoid a concomitant sensitization against the donor's histocompatibility antigens, prospective marrow recipients were sensitized against thymocytes of a third-party strain sharing the donor's Th-1 alloantigen but not his histocompatibility antigens. Advantage was taken of the fact that rats carry a Th-1.1-like theta-antigen which induces anti-Th-1.1 antibodies in Th-1.2 mice. CBA/J and (C57BL/6 x CBA)F1 Th-1.2 mice were sensitized against rat thymocytes and tranfused with spleen and bone marrow of AKR/J Th-1.1 after irradiation with 800 to 900 R. Although unsensitized recipients died within 3 weeks of acute secondary disease, sensitized mice survived the observation period of 50 days as chimaeras. Sensitized recipients were killed by the transplantation of spleen cells from congenic AKR/Cum carrying the Th-1.2 antigen. The host-versus-theta-graft approach suppressed secondary disease following H-2-compatible and -incompatible marrow grafts. Its hemopoietic and Tcell chimaeras tolerated skin grafts of the donor strain while rejecting third-party skin grafts.     

Recipient Dendritic Cells,But Not B Cells,Are Required Antigen‐Presenting Cells for Peripheral Alloreactive CD8+ T‐Cell Tolerance

Induction of mixed allogeneic chimerism is a promising approach for achieving donor‐specific tolerance, thereby obviating the need for life‐long immunosuppression for solid organ allograft acceptance. In mice receiving a low dose (3Gy) of total body irradiation, allogeneic bone marrow transplantation combined with anti‐CD154 tolerizes peripheral CD4 and CD8 T cells, allowing achievement of mixed chimerism with specific tolerance to donor. With this approach, peripheral CD8 T‐cell tolerance requires recipient MHC class II, CD4 T cells, B cells and DCs. Recipient‐type B cells from chimeras that were tolerant to donor still promoted CD8 T‐cell tolerance, but their role could not be replaced by donor‐type B cells. Using recipients whose B cells or DCs specifically lack MHC class I and/or class II or lack CD80 and CD86, we demonstrate that dendritic cells (DCs) must express CD80/86 and either MHC class I or class II to promote CD8 tolerance. In contrast, B cells, though required, did not need to express MHC class I or class II or CD80/86 to promote CD8 tolerance. Moreover, recipient IDO and IL‐10 were not required. Thus, antigen presentation by recipient DCs and not by B cells is critical for peripheral alloreactive CD8 T cell tolerance.     

Natural killer T cell facilitated engraftment of rat skin but not islet xenografts in mice

Abstract: Background: We have studied cellular components required for xenograft survival mediated by anti‐CD154 monoclonal antibody (mAb) and a transfusion of donor spleen cells and found that the elimination of CD4⁺ but not CD8⁺ cells significantly improves graft survival. A contribution of other cellular components, such as natural killer (NK) cells and natural killer T (NKT) cells, for costimulation blockade‐induced xenograft survival has not been clearly defined. We therefore tested the hypothesis that NK or NKT cells would promote rat islet and skin xenograft acceptance in mice. Methods: Lewis rat islets or skin was transplanted into wild type B6 mice or into B6 mice that were Jα18^null, CD1^null, or beta2 microglobulin (β2M)^null NK 1.1 depleted, or perforin^null. Graft recipients were pretreated with an infusion of donor derived spleen cells and a brief course of anti‐CD154 mAb treatments. Additional groups received mAb or cells only. Results: We first observed that the depletion of NK1.1 cells does not significantly interfere with graft survival in C57BL/6 (B6) mice. We used NKT cell deficient B6 mice to test the hypothesis that NKT cells are involved in islet and skin xenograft survival in our model. These mice bear a null mutation in the gene for the Jα18 component of the T‐cell receptor. The component is uniquely associated with NKT cells. We found no difference in islet xenograft survival between Jα18^null and wild type B6 mice. In contrast, median skin graft survival appeared shorter in Jα18^null recipients. These data imply a role for Jα18⁺ NKT cells in skin xenograft survival in treated mice. In order to confirm this inference, we tested skin xenograft survival in B6 CD1^null mice because NKT cells are CD1 restricted. Results of these trials demonstrate that the absence of CD1⁺ cells adversely affects rat skin graft survival. An additional assay in β2M^null mice demonstrated a requirement for major histocompatibility complex (MHC) class I expression in the graft host, and we demonstrate that CD1 is the requisite MHC component. We further demonstrated that, unlike reports for allograft survival, skin xenograft survival does not require perforin‐secreting NK cells. Conclusions: We conclude that MHC class I⁺ CD1⁺ Jα18⁺ NKT cells promote the survival of rat skin but not rat islet xenografts. These studies implicate different mechanisms for inducing and maintaining islet vs. skin xenograft survival in mice treated with donor antigen and anti‐CD154 mAb, and further indicate a role for NKT cells but not NK cells in skin xenograft survival.     

Blockade of the CD40/CD154 pathway enhances T-cell-depleted allogeneic bone marrow engraftment under nonmyeloablative and irradiation-free conditioning therapy

BACKGROUND: T-cell-depleted bone marrow transplantation (TDBMT) can prevent graft-versus-host disease (GvHD). However, depleting T cells from allogeneic bone marrow often results in failure of bone marrow engraftment under irradiation conditioning. It is not know whether donor T cells are essential for bone marrow engraftment and whether blocking the CD40/CD154 pathway promotes allogeneic TDBM engraftment under nonmyeloablative and irradiation-free fludarabine phosphate and cyclophosphamide conditioning therapy. METHODS: Using fully major histocompatibility complex (MHC)-matched mouse models, we investigated whether donor T cells are essential for bone marrow engraftment under fludarabine phosphate and cyclophosphamide conditioning therapy. We also determined whether the barrier of allogeneic TDBM could be overcome by blocking the CD40/CD154 pathway. Donor chimerism was detected by flow cytometric analysis. Donor-specific tolerance through establishing mixed chimerism was tested in vivo by skin transplantation and in vitro by mixed leukocyte reaction and enzyme-linked immunospot (ELISPOT) assay. RESULTS: Compared with unmodified bone marrow, TDBM resulted in poor engraftment when fully MHC-mismatched donors were used. However, anti-CD154 monoclonal antibody (mAb) treatment significantly enhanced donor TDBM engraftment. TDBM engraftment was also seen in CD154 knockout mice. A stable and high level of multilinage donor chimerism was achieved. Recovery of host CD3 T cells was suppressed, and recovery of donor CD3 T cells was promoted, after TDBMT and anti-CD154 mAb treatment. Donor chimerism was established by TDBMT induced donor-specific tolerance in vivo and in vitro. CONCLUSION: Donor T cells facilitate bone marrow engraftment under nonmyeloablative and irradiation-free conditioning therapy, and the blocking the CD40/CD154 pathway can replace donor T cells to promote TDBM engraftment.     

Sharing of Class I MHC Molecules Between Donor and Host Promotes the Infiltration of Allografts by mHAg‐Reactive CD8+ T cells

Indirect recognition of minor histocompatibility antigens (mHAg) and/or MHC‐encoded allopeptides is an important barrier to long‐term allograft acceptance following solid organ transplantation. Efficient priming of CD8⁺ T cells can occur after allotransplantation as a consequence of cross‐presentation of donor‐derived proteins by the graft recipient's APC. Consistent with this, draining lymph node clonal expansion of OVA‐reactive OT‐I CD8⁺ T cells following placement of OVA‐transgenic skin grafts did not depend on graft expression of K^b. However, OT‐I T cells did accumulate in OVA‐transgenic skin grafts most efficiently only when both the donor and host expressed K^b. OT‐I infiltration of (B6‐OVA × BALB/c)F₁ grafts in B6 recipients was not suppressed by graft expression of H‐2^d. Furthermore, B6 animals transplanted with both B6‐OVA and BALB/c‐OVA skin had more OT‐I T cells infiltrating their B6‐OVA MHC‐matched graft. Therefore, class I MHC matching between donor and host may not always favor an avoidance of alloreactivity within the graft tissue.     

Donor apoptotic cell–based therapy for effective inhibition of donor‐specific memory T and B cells to promote long‐term allograft survival in allosensitized recipients

Allosensitization constitutes a major barrier in transplantation. Preexisting donor‐reactive memory T and B cells and preformed donor‐specific antibodies (DSAs) have all been implicated in accelerated allograft rejection in sensitized recipients. Here, we employ a sensitized murine model of islet transplantation to test strategies that promote long‐term immunosuppression‐free allograft survival. We demonstrate that donor‐specific memory T and B cells can be effectively inhibited by peritransplant infusions of donor apoptotic cells in combination with anti‐CD40L and rapamycin, and this treatment leads to significant prolongation of islet allograft survival in allosensitized recipients. We further demonstrate that late graft rejection in recipients treated with this regimen is associated with a breakthrough of B cells and their aggressive graft infiltration. Consequently, additional posttransplant B cell depletion effectively prevents late rejection and promotes permanent acceptance of islet allografts. In contrast, persistent low levels of DSAs do not seem to impair graft outcome in these recipients. We propose that B cells contribute to late rejection as antigen‐presenting cells for intragraft memory T cell expansion but not to alloantibody production and that a therapeutic strategy combining donor apoptotic cells, anti‐CD40L, and rapamycin effectively inhibits proinflammatory B cells and promotes long‐term islet allograft survival in such recipients.     

Flt3L‐mobilized dendritic cells bearing H2‐Kbm1 apoptotic cells do not induce cross‐tolerance to CD8+ T cells across a class I MHC mismatched barrier

Tolerization of allogeneic CD8⁺ T cells is still a pending issue in the field of transplantation research to achieve long‐term survival. To test whether dendritic cells (DC) bearing allogeneic major histocompatibility complex (MHC) class I mismatched apoptotic cells could induce cross‐tolerance to alloreactive CD8⁺ T cells, the following experimental strategy was devised. Rag2/γ_c KO B6 mice were treated with Fms‐like tyrosine kinase 3 ligand (Flt3L)‐transduced B16 melanoma cells to drive a rapid expansion and mobilization of DC in vivo. Of all DC populations expanded, splenic CD11c⁺CD103⁺CD8α⁺ DC were selectively involved in the process of antigen clearance of X‐ray irradiated apoptotic thymocytes in vivo. Considering that CD11c⁺CD103⁺CD8α⁺ DC selectively take up apoptotic cells and that they are highly specialized in cross‐presenting antigen to CD8⁺ T cells, we investigated whether B6 mice adoptively transferred with Flt3L‐derived DC loaded with donor‐derived apoptotic thymocytes could induce tolerance to bm1 skin allografts. Our findings on host anti‐donor alloresponse, as revealed by skin allograft survival and cytotoxic T lymphocyte assays, indicated that the administration of syngeneic DC presenting K^bm1 donor‐derived allopeptides through the indirect pathway of antigen presentation was not sufficient to induce cross‐tolerance to alloreactive CD8⁺ T cells responding to bm1 alloantigens in a murine model of skin allograft transplantation across an MHC class I mismatched barrier.     

Allospecific Rejection of MHC Class I‐Deficient Bone Marrow by CD8 T Cells

Avoidance of long‐term immunosuppression is a desired goal in organ transplantation. Mixed chimerism offers a promising approach to tolerance induction, and we have aimed to develop low‐toxicity, nonimmunodepleting approaches to achieve this outcome. In a mouse model achieving fully MHC‐mismatched allogeneic bone marrow engraftment with minimal conditioning (3 Gy total body irradiation followed by anti‐CD154 and T cell–depleted allogeneic bone marrow cells), CD4 T cells in the recipient are required to promote tolerance of preexisting alloreactive recipient CD8 T cells and thereby permit chimerism induction. We now demonstrate that mice devoid of CD4 T cells and NK cells reject MHC Class I‐deficient and Class I/Class II‐deficient marrow in a CD8 T cell–dependent manner. This rejection is specific for donor alloantigens, since recipient hematopoiesis is not affected by donor marrow rejection and MHC Class I‐deficient bone marrow that is syngeneic to the recipient is not rejected. Recipient CD8 T cells are activated and develop cytotoxicity against MHC Class I‐deficient donor cells in association with rejection. These data implicate a novel CD8 T cell–dependent bone marrow rejection pathway, wherein recipient CD8 T cells indirectly activated by donor alloantigens promote direct killing, in a T cell receptor–independent manner, of Class I‐deficient donor cells.     

CD25+CD4+ regulatory T cells develop in mice not only during spontaneous acceptance of liver allografts but also after acute allograft rejection

BACKGROUND: Liver grafts transplanted across a major histocompatibility barrier are accepted spontaneously and induce donor specific tolerance in some species. Here, we investigated whether liver allograft acceptance is characterized by, and depends upon, the presence of donor reactive CD25CD4 regulatory T cells. METHODS: CD25 and CD25CD4 T cells, isolated from CBA. Ca (H2) recipients of C57BL/10 (B10; H2) liver and heart allografts 10 days after transplantation, were transferred into CBA. Rag1 mice to investigate their influence on skin allograft rejection mediated by CD45RBCD4 effector T Cells. RESULTS: Fully allogeneic B10 liver allografts were spontaneously accepted by naive CBA.Ca recipient mice, whereas B10 cardiac allografts were acutely rejected (mean survival time=7 days). Strikingly, however, CD25CD4 T cells isolated from both liver and cardiac allograft recipients were able to prevent skin allograft rejection in this adoptive transfer model. Interestingly, CD25CD4 T cells isolated from liver graft recipients also showed suppressive potency upon adoptive transfer. Furthermore, depletion of CD25CD4 T cells in primary liver allograft recipients did not prevent the acceptance of a secondary donor-specific skin graft. CONCLUSIONS: Our data provide evidence that the presence of CD25CD4 regulatory T cells is not a unique feature of allograft acceptance and is more likely the result of sustained exposure to donor alloantigens in vivo.     

Contrasting effects of B cell depletion on CD4+ and CD8+ memory T cell responses generated after transplantation

Alloreactive memory T cells play a key role in transplantation by accelerating allograft rejection and preventing tolerance induction. Some studies using µMT mice, which are constitutionally devoid of B cells, showed that B cells were required for the generation of memory T cells after allotransplantation. However, whether B cell depletion in normal adult mice has the same effect on memory responses by CD4⁺ and CD8⁺ T cells activated after transplantation has not been thoroughly investigated. In this study, we tested the effect of anti‐CD20 antibody‐mediated B cell depletion on CD4⁺ and CD8⁺ memory T cell alloresponses after skin transplantation in wild‐type mice. We found that B cell depletion prevented the development of memory alloresponses by CD4⁺ T cells but enhanced that of CD8⁺ memory T cells. Next, we tested the influence of B cell depletion on hematopoietic chimerism. In OT‐II CD4⁺ anti‐OVA TCR transgenic mice sensitized to ovalbumin antigen, B cell depletion also impaired allospecific memory T cell responses and thereby enhanced donor hematopoietic chimerism and T cell deletion after bone marrow transplantation. This study underscores the complexity of the relationships between B and T cells in the generation and reactivation of different memory T cell subsets after transplantation.     

LFA‐1 Antagonism Inhibits Early Infiltration of Endogenous Memory CD8 T Cells into Cardiac Allografts and Donor‐Reactive T Cell Priming

Alloreactive memory T cells are present in virtually all transplant recipients due to prior sensitization or heterologous immunity and mediate injury undermining graft outcome. In mouse models, endogenous memory CD8 T cells infiltrate MHC‐mismatched cardiac allografts and produce IFN‐γ in response to donor class I MHC within 24 h posttransplant. The current studies analyzed the efficacy of anti‐LFA‐1 mAb to inhibit early CD8 T cell cardiac allograft infiltration and activation. Anti‐LFA‐1 mAb given to C57BL/6 6 (H‐2^b) recipients of A/J (H‐2^a) heart grafts on days –1 and 0 completely inhibited CD8 T cell allograft infiltration, markedly decreased neutrophil infiltration and significantly reduced intragraft expression levels of IFN‐γ‐induced genes. Donor‐specific T cells producing IFN‐γ were at low/undetectable numbers in spleens of anti‐LFA‐1 mAb treated recipients until day 21. These effects combined to promote substantial prolongation (from day 8 to 27) in allograft survival. Delaying anti‐LFA‐1 mAb treatment until days 3 and 4 posttransplant did not inhibit early memory CD8 T cell infiltration and proliferation within the allograft. These data indicate that peritransplant anti‐LFA‐1 mAb inhibits early donor‐reactive memory CD8 T cell allograft infiltration and inflammation suggesting an effective strategy to attenuate the negative effects of heterologous immunity in transplant recipients.     

Mechanism of allorecognition and skin graft rejection in CD28 and CD40 ligand double-deficient mice

BACKGROUND: It has been shown that simultaneous blockade of CD28- and CD40-mediated costimulatory signals significantly prolongs allograft survival. Although these results led to an expectation of the establishment of specific immunotolerant therapy for organ transplantation, it became evident that these treatments rarely resulted in indefinite allograft survival. To uncover the mechanisms underlying these costimulation blockade-resistant allograft rejections, we studied the process of allogenic skin graft rejection in CD28 and CD40 ligand (L) double-deficient (double-knockout [dKO]) mice. METHODS: Skin grafts from BALB/c or BALB.B mice were transplanted to C57BL/6 background dKO mice. The frequency of CD4+ and CD8+ T cells responding to alloantigens presented by direct or indirect pathways were defined by the use of a cytostaining assay. RESULTS: BALB/c skin grafts were rapidly rejected by dKO mice. This CD28 and CD40L independent allograft rejection was inhibited by the depletion of CD8+ T cells. In vitro studies indicated that CD8+ T cells from BALB/c skin-grafted dKO mice responded to donor antigen presented only by the direct pathway. Unlike major histocompatibility complex (MHC)-mismatched donors, allogenic skin grafts from MHC-matched donors were accepted by dKO mice. CONCLUSION: In the absence of CD28 and CD40 costimulatory signals, CD8+ T cells recognize MHC antigens by the direct pathway, resulting in the rejection of skin grafts from MHC-mismatched donors. In contrast, MHC-matched and non-MHC-mismatched donor skin grafts indefinitely survive in dKO mice. These results indicated that donor-host MHC matching may still be critical to costimulation blockade therapy for organ transplantation.     

Suppression extends to major histocompatibility antigens linked to tolerizing minor histocompatibility antigens, but not the other way round

'Active suppression', a mechanism of transplantation tolerance, can spread to newly introduced minor antigens once these antigens are linked to tolerizing antigens. We explored whether this suppression can extend to major histocompatibility (MHC) antigens and whether this phenomenon can be demonstrated once tolerance is induced to a MHC antigen. Mice were tolerized using donor bone marrow plus CD4 and CD8 monoclonal antibodies. The following strain combinations were used: AKR (H-2k) into CBA (H-2k), a multiple minor difference and B6 (H-2b) into B6(bm12) (H-2b), a MHC class II difference. Tolerance was tested by a donorskingraft. CBA mice tolerant to AKR received a second skin carrying either AKR antigens plus additional multiple minor antigens [F1(AKRxBalb.K)] or carrying additional minors and a MHC class I antigen (B10.AKM-H2M). B6(bm12) (H-2b) tolerant to B6 (H-2b) were grafted with skin from a Balb.B donor (Balb minors linked to the tolerizing class II antigen) or from a B10.A(3R) strain (a MHC class I antigen linked to the tolerizing class II antigen). CBA mice tolerant to AKR accepted F1(AKRxBalb.K) skin, whereas F1(CBAxBalb.K) were rejected. Rejection of B10.AKM/H2M skin by tolerant mice was delayed as compared with nontolerant mice. Tolerant and nontolerant B6(bm12) mice rejected Balb.B skin and B10.A(3R) skin within the same time. Thus, in this model, suppression was linked to minors. Alloreactivity against minors and majors could be suppressed. Suppression linked to a class II antigen could not be demonstrated.     

Addition of cyclophosphamide to T-cell depletion-based nonmyeloablative conditioning allows donor T-cell engraftment and clonal deletion of alloreactive host T-cells after bone marrow transplantation

BACKGROUND: Bone marrow (BM) chimerism has been shown to have a beneficial effect on allograft survival. We recently found that production of donor T-cells was highly correlated with induction of tolerance in minimally conditioned chimeras. In the present studies, we demonstrate that nonmyeloablative conditioning and BM cell infusion modulate innate and adaptive host immune responses. METHODS: Chimeras were generated by bone marrow transplantation (B10.BR to B10). Recipients were preconditioned with T-cell depleting antibodies and total body irradiation with or without cyclophosphamide. Donor-specific tolerance was tested by skin grafting. RESULTS: Transfer of tolerant splenocytes to immunocompetent secondary recipients did not transfer tolerance, nor did infusion of tolerant CD4+/CD25+ T-cells into chimeras without donor T-cell production, demonstrating that linked suppression is an unlikely mechanism in tolerance induction in the context of BM cell infusion. The addition of a single dose of cyclophosphamide to the conditioning enhanced engraftment and tolerance. This was associated with production of donor T-cells and effective clonal deletion, and a significant reduction in activated recipient plasmacytoid dendritic cells (pDC) and natural killer (NK) cells. Chimeras without donor T-cell production that eventually lost their chimerism did not generate an antidonor humoral response, whereas unconditioned controls infused with similar numbers of BM cells did, indicating that infusion of donor BM cells into conditioned recipients induced immune deviation for adaptive B-cell immunity, preventing sensitization to major histocompatibility complex (MHC) alloantigens. CONCLUSIONS: These results demonstrate that recipient T-cells, pDC, and NK cells contribute to the host barrier for establishing chimerism, implicate deletional tolerance as the mechanism for total body irradiation-based nonmyeloablative conditioning for BM transplantation, and show a beneficial effect of BM cells in preventing sensitization to MHC alloantigens.     

Analysis of tolerance induction using triple chimeric mice: major histocompatibility complex-disparate thymus, hemopoietic cells, and microenvironment

BACKGROUND: Although bone marrow transplantation (BMT) has become a valuable strategy for the treatment of various intractable diseases in recent years, success rates remain low in elderly patients because of low thymic function. We have previously shown that fetal thymus transplantation (TT) with BMT is effective for elderly recipients in mice. METHODS: We performed fully major histocompatibility complex (MHC)-mismatched fetal TT from B6 (H-2) mice plus allogeneic BMT from C3H/HeN (H-2) mice by intra-bone marrow-BMT (IBM-BMT) using congenitally athymic nude (nu/nu) BALB/c (H-2), or BALB/c adult-thymectomized recipients to obtain triple chimeras. We next carried out the IBM-BMT+TT using senescence-accelerated mouse P1 strain (SAMP1) to examine whether this method would be applicable to aging mice. RESULTS: Triple chimeric mice survived for a long period with sufficient T-cell functions comparable to the mice treated with BMT plus MHC-matched TT, whereas those without TT survived for a short period with insufficient T-cell reconstitution. Almost all the hematolymphoid cells were derived from donor bone marrow cells. Interestingly, they showed tolerance to all three types of MHC determinants with donor-derived thymic dendritic cells in TT. Triple chimeric SAMP1 also survived for long periods with T-cell functions restored in contrast to non-TT SAMP1 recipients. CONCLUSION: These findings suggest that third party combined TT with allogeneic IBM-BMT may be more advantageous for elderly recipients with low thymic function, than IBM-BMT alone (without TT).     

Tolerance induction through megadose bone marrow transplantation with two-signal blockade

BACKGROUND: Induction of mixed chimerism is currently the most promising concept for clinical tolerance induction; however, the toxicity of the required host conditioning for allogeneic bone marrow transplantation (BMT) should be overcome. Therefore, we explored tolerogenic effectiveness of megadose BMT with anti-CD45RB and anti-CD154 mAb (two-signal blockade) in murine recipients without conditioning. MATERIALS AND METHODS: Recipient B6 mice of BALB/c skin allograft received conditioning and an optimal dose (2x10(7) cells) of BMT. For a megadose BMT model, the conditioning was not performed; instead, megadose (2x10(8) cells) of BM was transplanted. The recipients were then treated with anti-CD45RB mAb and anti-CD154 mAb alone or their combination. Flow cytometry was performed to analyze the degree and distribution of donor-derived cells, peripheral deletion of Vbeta5 or Vbeta11 T cells and intrathymic presence of donor MHC class II+ cells. Induction of chimerism-based tolerance to skin allograft was further determined. RESULTS: High levels ( approximately 23.7%) of mixed and multi-lineage chimerism-based tolerance to skin allograft were induced in the recipients (91%) treated with the optimal-dose BMT and the two-signal blockade. The megadose BMT could replace the recipient conditioning and establish low (approximately 10%) and stable multilineage chimerism. Donor-specific tolerance to skin allograft was induced in these chimeras through clonal deletion of donor-reactive cells. CONCLUSIONS: The megadose BMT with the two-signal blockade could effectively establish mixed and multi-lineage chimerism and induce donor-specific tolerance, suggesting its potential for clinical application.     

B Cell Depletion With an Anti‐CD20 Antibody Enhances Alloreactive Memory T Cell Responses After Transplantation

Alloreactive memory T cells mediate accelerated allograft rejection and transplant tolerance resistance. Recent studies have shown that B cell deficient–μMT mice fail to mount donor‐specific memory T cell responses after transplantation. At the same time, other studies showed that pretransplant B cell depletion using rituximab (IgG1 anti‐CD20 mAb) combined with cyclosporine A promoted the survival of islet allografts in monkeys. In this study, we investigated the effect of anti‐CD20 antibody‐mediated B cell depletion on the memory T cell alloresponse in mice. Wild‐type and anti‐OVA TCR transgenic mice were treated with an IgG2a anti‐CD20 monoclonal antibody, which depleted nearly all B cells in the peripheral blood and secondary lymphoid organs but spared some B cells in the bone marrow. B cell depletion did not affect the direct alloresponse but resulted in a marked increase of indirect alloresponse after skin transplantation of naïve mice. Furthermore, in allosensitized mice, anti‐CD20 mAb treatment enhanced the reactivation of allospecific memory T cells and accelerated second set rejection of skin allografts. This suggests that the effect of anti‐CD20 antibodies on alloimmunity and allograft rejection might vary upon the nature of the antibodies as well as the circumstances under which they are delivered.