A critical role for donor‐derived IL‐22 in cutaneous chronic GVHD

Graft‐versus‐host disease (GVHD) is the major cause of nonrelapse morbidity and mortality after allogeneic stem cell transplantation (allo‐SCT). Prevention and treatment of GVHD remain inadequate and commonly lead to end‐organ dysfunction and opportunistic infection. The role of interleukin (IL)‐17 and IL‐22 in GVHD remains uncertain, due to an apparent lack of lineage fidelity and variable and contextually determined protective and pathogenic effects. We demonstrate that donor T cell–derived IL‐22 significantly exacerbates cutaneous chronic GVHD and that IL‐22 is produced by highly inflammatory donor CD4⁺ T cells posttransplantation. IL‐22 and IL‐17A derive from both independent and overlapping lineages, defined as T helper (Th)22 and IL‐22⁺ Th17 cells. Donor Th22 and IL‐22⁺ Th17 cells share a similar IL‐6–dependent developmental pathway, and while Th22 cells arise independently of the IL‐22⁺Th17 lineage, IL‐17 signaling to donor Th22 directly promotes their development in allo‐SCT. Importantly, while both IL‐22 and IL‐17 mediate skin GVHD, Th17‐induced chronic GVHD can be attenuated by IL‐22 inhibition in preclinical systems. In the clinic, high levels of both IL‐17A and IL‐22 expression are present in the skin of patients with GVHD after allo‐SCT. Together, these data demonstrate a key role for donor‐derived IL‐22 in patients with chronic skin GVHD and confirm parallel but symbiotic developmental pathways of Th22 and Th17 differentiation.     

Imaging Tolerance Induction in the Classic Medawar Neonatal Mouse Model: Active Roles of Multiple F1‐Donor Cell Types

The immature immune system is uniquely susceptible to tolerance induction and thus an attractive target for immunomodulation strategies for organ transplantation. Newborn mice injected with adult semi‐allogeneic lymphohematopoietic cells accept transplants without immunosuppressive drugs. Early in vivo/in situ events leading to neonatal tolerance remain poorly understood. Here, we show by whole body/organ imaging that injected cells home to lymphoid organs and liver where various F1‐donor cell types selectively alter neonatal immunity. In host thymus, F1‐donor dendritic cells (DC) interact with developing thymocytes and regulatory T cells suggesting a role in negative selection. In spleen and lymph nodes, F1‐donor regulatory T/B cells associate with host alloreactive cells and by themselves prolong cardiac allograft survival. In liver, F1‐donor cells give rise to albumin‐containing hepatocyte‐like cells. The neonatal immune system is lymphopenic, Th‐2 immunodeviated and contains immature DC, suggesting susceptibility to regulation by adult F1‐donor cells. CD8a T cell inactivation greatly enhances chimerism, suggesting that variable emerging neonatal alloreactivity becomes a barrier to tolerance induction. This comprehensive qualitative imaging study systematically shows contribution of multiple in vivo processes leading simultaneously to robust tolerance. These insights into robust tolerance induction have important implications for development of strategies for clinical application.     

Interleukin-23 receptor signaling by interleukin-39 potentiates T cell pathogenicity in acute graft-versus-host disease

IL-12 (p35/p40) and IL-23 (p19/p40) signal through IL-12R (IL-12Rβ2/β1) and IL-23R (IL-23Rα/IL-12Rβ1), respectively, which can promote pathogenic T lymphocyte activation, differentiation, and function in graft-versus-host disease (GVHD). With the use of murine models of allogeneic hematopoietic cell transplantation (HCT), we found that IL-12Rβ1 on donor T cells was dispensable to induce acute GVHD development in certain circumstances, while IL-23Rα was commonly required. This observation challenges the current paradigm regarding IL-12Rβ1 as a prerequisite to transmit IL-23 signaling. We hypothesized that p19/EBI3 (IL-39) may have an important role during acute GVHD. With the use of gene transfection and immunoprecipitation approaches, we verified that p19 and EBI3 can form biological heterodimers. We found that IL-39 levels in recipient serum positively correlated with development of acute GVHD in experimental models and in clinical settings, thereby implicating IL-39 in the pathogenesis of acute GVHD. Furthermore, we observed that human T cells can signal in response to IL-39. In chronic GVHD, IL-23Rα and IL-12Rβ1 were similarly required for donor T cell pathogenicity, and IL-39 levels were not significantly different from controls without GVHD. Collectively, we identify a novel cytokine, IL-39, as a pathogenic factor in acute GVHD, which represents a novel potential therapeutic target to control GVHD and other inflammatory disorders.     

Donor plasmacytoid dendritic cells modulate effector and regulatory T cell responses in mouse spontaneous liver transplant tolerance

We assessed the role of donor liver non-conventional plasmacytoid dendritic cells (pDCs) in spontaneous liver transplant tolerance in a fully MHC-mismatched (C57BL/6 (H2^b) to C3H (H2^k)) mouse model. Compared with spleen pDCs, liver pDCs expressed higher levels of DNAX-activating protein of 12 kDa and its co-receptor, triggering receptor expressed by myeloid cells 2, and higher ratios of programed death ligand-1 (PD-L1):costimulatory CD80/CD86 in the steady state and after Toll-like receptor 9 ligation. Moreover, liver pDCs potently suppressed allogeneic CD4⁺ and CD8⁺ T cell proliferative responses. Survival of pDC-depleted livers was much poorer (median survival time: 25 days) than that of either untreated donor livers or pDC-depleted syngeneic donor livers that survived indefinitely. Numbers of forkhead box p3 (FoxP3)⁺ regulatory T cells in grafts and mesenteric lymph nodes of mice given pDC-depleted allogeneic livers were reduced significantly compared with those in recipients of untreated livers. Graft-infiltrating CD8⁺ T cells with an exhausted phenotype (programed cell death protein 1⁺, T cell immunoglobulin and mucin domain-containing protein 3⁺) were also reduced in recipients of pDC-depleted livers. PD1-PD-L1 pathway blockade reversed the reduction in exhausted T cells. These novel observations link immunoregulatory functions of liver interstitial pDCs, alloreactive T cell exhaustion, and spontaneous liver transplant tolerance.     

R707, a fully human antibody directed against CC‐chemokine receptor 7, attenuates xenogeneic acute graft‐versus‐host disease

Acute graft‐versus‐host disease (aGVHD) remains a barrier to the success of allogeneic hematopoietic stem cell transplantation (HSCT). Previously, we demonstrated that CC‐chemokine receptor 7 (CCR7) is critical for aGVHD pathogenesis but dispensable for beneficial graft‐versus‐leukemia responses. As a result, we evaluated a fully human anti‐CCR7‐blocking antibody as a new approach to prevent aGVHD in preclinical models. Here we report that antibody R707 is able to block human CCR7 signaling and function in vitro in response to its 2 natural ligands. The antibody was less active against the murine orthologue, however, and failed to substantially limit aGVHD in a standard murine allogeneic HSCT model. Nevertheless, R707 significantly reduced xenogeneic aGVHD induced by human peripheral blood mononuclear cells (PBMCs). R707 limited CD4⁺ and in particular CD8⁺ T cell expansion during the period of antibody administration. These effects were transient, however, and T cell numbers recovered after antibody cessation. R707 did not substantially impair the antitumor potential of the PBMC inoculum as antibody‐treated mice retained their capacity to reject a human acute myeloid leukemia cell line. Collectively, these data indicate for the first time that an antibody directed against CCR7 might represent a viable new approach for aGVHD prevention.     

Donor‐targeted serotherapy as a rescue therapy for steroid‐resistant acute GVHD after HLA‐mismatched kidney transplantation

Acute graft‐versus‐host disease (GVHD) is a rare but frequently lethal complication after solid organ transplantation. GVHD occurs in unduly immunocompromised hosts but requires the escalation of immunosuppression, which does not discriminate between host and donor cells. In contrast, donor‐targeted therapy would ideally mitigate graft‐versus‐host reactivity while sparing recipient immune functions. We report two children with end‐stage renal disease and severe primary immune deficiency (Schimke syndrome) who developed severe steroid‐resistant acute GVHD along with full and sustained donor T cell chimerism after isolated kidney transplantation. Facing a therapeutic dead end, we used a novel strategy based on the adoptive transfer of anti‐HLA donor‐specific antibodies (DSAs) through the transfusion of highly selected plasma. After approval by the appropriate regulatory authority, an urgent nationwide search was launched among more than 3800 registered blood donors with known anti‐HLA sensitization. Adoptively transferred DSAs bound to and selectively depleted circulating donor T cells. The administration of DSA‐rich plasma was well tolerated and notably did not induce antibody‐mediated rejection of the renal allografts. Acute GVHD symptoms promptly resolved in one child. This report provides a proof of concept for a highly targeted novel therapeutic approach for solid organ transplantation–associated GVHD.     

Murine Mobilized Peripheral Blood Stem Cells Have a Lower Capacity than Bone Marrow to Induce Mixed Chimerism and Tolerance

Allogeneic bone marrow transplantation (BMT) under costimulation blockade allows induction of mixed chimerism and tolerance without global T‐cell depletion (TCD). The mildest such protocols without recipient cytoreduction, however, require clinically impracticable bone marrow (BM) doses. The successful use of mobilized peripheral blood stem cells (PBSC) instead of BM in such regimens would provide a substantial advance, allowing transplantation of higher doses of hematopoietic donor cells. We thus transplanted fully allogeneic murine granulocyte colony‐stimulating factor (G‐CSF) mobilized PBSC under costimulation blockade (anti‐CD40L and CTLA4Ig). Unexpectedly, PBSC did not engraft, even when very high cell doses together with nonmyeloablative total body irradiation (TBI) were used. We show that, paradoxically, T cells contained in the donor PBSC triggered rejection of the transplanted donor cells. Rejection of donor BM was also triggered by the cotransplantation of unmanipulated donor T cells isolated from naïve (nonmobilized) donors. Donor‐specific transfusion and transient immunosuppression prevented PBSC‐triggered rejection and mixed chimerism and tolerance were achieved, but graft‐versus‐host disease (GVHD) occurred. The combination of in vivo TCD with costimulation blockade prevented rejection and GVHD. Thus, if allogeneic PBSC are transplanted instead of BM, costimulation blockade alone does not induce chimerism and tolerance without unacceptable GVHD‐toxicity, and the addition of TCD is required for success.     

Migration of host and donor T cells in small bowel transplantation

In this study migration of host and donor CD4⁺ and CD8⁺ T cells in a fully allogeneic model was described and compared with the migration pattern in a graft-versushost reaction (GVHR) model, where the T-cell traffic in the graft served as a physiological control. Heterotopic small bowel transplantations were performed in a rat model, with animals being sacrificed on postoperative days (POD) 2, 3, 4, 5, and 7. Graft and host mesenteric lymph nodes were harvested, homogenized, and stained with monoclonal antibodies against MHC class 1, CD4⁺, and CD8⁺ antigens. The host and donor T cell migration patterns were studied using a doublestaining flow cytometric technique. We found that during the development of rejection, the normal physiological circulation of graft and host T cells was disrupted. In the graft of the allogeneic model, a shift from host cell to graft cell dominance occurred on POD 3–4. This change in migration pattern coincided in the host with a 6 % peak in graft cell infiltration, which disappeared on POD 7. These patterns of T-cell migration may be further explored for diagnostic purposes.     

A Novel Xenogeneic Graft‐Versus‐Host Disease Model for Investigating the Pathological Role of Human CD4+ or CD8+ T Cells Using Immunodeficient NOG Mice

Graft‐versus‐host disease (GVHD) is a major complication of allogenic bone marrow transplantation and involves the infiltration of donor CD4⁺ and/or CD8⁺ T cells into various organs of the recipient. The pathological role of human CD4⁺ and CD8⁺ T cells in GVHD remains controversial. In this study, we established two novel xenogeneic (xeno)‐GVHD models. Human CD4⁺ or CD8⁺ T cells were purified from peripheral blood and were transplanted into immunodeficient NOD/Shi‐scid IL2rg^null (NOG) mice. Human CD8⁺ T cells did not induce major GVHD symptoms in conventional NOG mice. However, CD8⁺ T cells immediately proliferated and induced severe GVHD when transferred into NOG mice together with at least 0.5 × 10⁶ CD4⁺ T cells or into NOG human interleukin (IL)‐2 transgenic mice. Human CD4⁺ T cell–transplanted NOG mice developed skin inflammations including alopecia, epidermal hyperplasia, and neutrophilia. Pathogenic T helper (Th)17 cells accumulated in the skin of CD4⁺ T cell–transplanted NOG mice. Further, an anti‐human IL‐17 antibody (secukinumab) significantly suppressed these skin pathologies. These results indicate that pathogenic human Th17 cells induce cutaneous GVHD via IL‐17–dependent pathways. This study provides fundamental insights into the pathogenesis of xeno‐GVHD, and these humanized mouse models may be useful as preclinical tools for the prevention of GVHD.     

Lactobacillus rhamnosus GG probiotic enteric regimen does not appreciably alter the gut microbiome or provide protection against GVHD after allogeneic hematopoietic stem cell transplantation

Graft‐versus‐host disease (GVHD) is a major adverse effect associated with allogeneic stem cell transplant. Previous studies in mice indicated that administration of the probiotic Lactobacillus rhamnosus GG can reduce the incidence of GVHD after hematopoietic stem cell transplant. Here we report results from the first randomized probiotic enteric regimen trial in which allogenic hematopoietic stem cell patients were supplemented with Lactobacillus rhamnosus GG. Gut microbiome analysis confirmed a previously reported gut microbiome association with GVHD. However, the clinical trial was terminated when interim analysis did not detect an appreciable probiotic‐related change in the gut microbiome or incidence of GVHD. Additional studies are necessary to determine whether probiotics can alter the incidence of GVHD after allogeneic stem cell transplant.     

Graft-versus-host disease in the absence of the spleen after allogeneic bone marrow transplantation

BACKGROUND: The spleen is considered to be an important secondary lymphoid organ where acute graft-versus-host disease (GVHD) is initiated by donor T cells that recognize host alloantigens after allogeneic bone-marrow transplantation (BMT). The influence of splenectomy on the development of GVHD prior to BMT has yet to be determined. METHODS: The mortality and severity of murine GVHD of unsplenectomized, splenectomized, and sham-operated recipients of allogeneic BMT were compared in a blinded fashion. Serum levels of interferon (IFN)-gamma were measured 7 days after BMT, as an index of systemic donor T-cell responses. RESULTS: Mortality and morbidity of acute GVHD were not significantly affected by splenectomy in a major histocompatibility complex (MHC)-mismatched, CD4-driven murine GVHD model and a minor histocompatibility antigen (MiHA)-mismatched, CD8-driven GVHD model. Serum levels of IFN-gamma also were not different between the groups. CONCLUSION: GVHD can readily develop after allogeneic BMT, even in the absence of the spleen, in these mouse models.     

The Sphingosine-1-Phosphate Receptor Agonist FTY720 Modulates Dendritic Cell Trafficking In Vivo

The pro‐drug FTY720 is undergoing phase III clinical trials for prevention of allograft rejection. After phosphorylation, FTY720 targets the G protein‐coupled –sphingosine‐1‐phosphate receptor 1 (S1PR1) on lymphocytes, thereby inhibiting their egress from lymphoid organs and their recirculation to inflammatory sites. Potential effects on dendritic cell (DC) trafficking have not been evaluated. Here, we demonstrate the expression of all five S1PR subtypes (S1PR1–5) by murine DCs. Administration of FTY720 to C57BL/10 mice markedly reduced circulating T and B lymphocytes within 24 h, but not blood‐borne DCs, which were enhanced significantly for up to 96 h, while DCs in lymph nodes and spleen were reduced. Numbers of adoptively transferred, fluorochrome‐labeled syngeneic or allogeneic DCs in blood were increased significantly in FTY720‐treated animals, while donor‐derived DCs and allostimulatory activity for host naïve T cells within the spleen were reduced. Administration of the selective S1PR1 agonist SEW2871 significantly enhanced circulating DC numbers. Flow analysis revealed that CD11b, CD31/PECAM‐1, CD54/ICAM‐1 and CCR7 expression on blood‐borne DCs was downregulated following FTY720 administration. Transendothelial migration of FTY720‐P‐treated immature DCs to the CCR7 ligand CCL19 was reduced. These novel data suggest that modulation of DC trafficking by FTY720 may contribute to its immunosuppressive effects.     

The role of donor lymphoid cells in the transfer of allograft tolerance

Tolerance to murine skin allografts across a MHC disparity was induced by conditioning primary hosts with sublethal fractionated total-body irradiation (FTBI) and transfusion of allogeneic bone marrow (BM). Tolerance could be adoptively transferred to secondary hosts conditioned by FTBI with infusion of spleen cells from hosts bearing intact skin allografts greater than 60 days. Tolerance could not be transferred by tolerant host spleen (THS) preparations from which cells of the donor genotype had been deleted by cytotoxic alloantisera. Deletion of host genotype cells, however, did not diminish the capability of THS to transfer tolerance. All of the tolerizing activity of THS appeared to reside within cells of the donor genotype. Small numbers of normal donor spleen cells could induce tolerance in FTBI hosts but only at the expense of very high mortality, in contrast to the low mortality observed with tolerizing injections of allogeneic donor cells from THS or injections of normal semiallogeneic F1 hybrid spleen cells. If an active immune response is responsible for tolerance induction/transfer in this model, allogeneic donor lymphoid cells derived from BM, in contrast to donor spleen cells, must be capable of mounting this response without concomitant severe GVHD. In future experiments, cells of donor genotype can be isolated from THS and purified in sufficient numbers to compare their tolerizing efficiency vs. that of normal donor cells, detect possible suppression of normal host cell alloreactivity in vitro and identify the donor cell phenotypes involved.     

Study of murine T cell migration using the Thy-1 allotypic marker. Demonstration of antigen-specific homing to lymph node germinal centers

We describe the use of Thy-1 alloantigen as a marker for in vivo T lymphocyte homing studies. Following transfer of 5 x 10(7) peripheral node T cells i.v., 32% of the transferred cells could be recovered in the host lymphoid organs (spleen, lymph nodes, Peyer's patches, and thymus); 11% of the T cells in the lymph nodes were donor derived. The transferred T cells assume the same microenvironmental and immunophenotypic distribution as the host T cells. The transferred T cells are identifiable in peripheral lymph nodes up to 170 days posttransfer, gradually declining in number during this time without evidence of rejection. This Thy-1 transfer technique permits T lymphocyte homing studies to be performed under physiologic conditions without problems of loss of lymphocyte subsets, selective labeling of lymphocyte populations, or long-term marker loss or dilution. We then employ this technique to demonstrate the antigen-directed homing of peripheral T cells to lymph node germinal centers.     

Inhibition of T cell homing by down-regulation of CD62L and the induction of a Th-2 response as a method to prevent acute allograft rejection in mice.

OBJECTIVE: For a successful immune response, migration of lymphocytes to lymphoid organs and other tissues is a key step, as the initial recognition of foreign antigens and activation of lymphocytes takes place in these organs. CD62L is a homing receptor that mediates entry of na?ve T cells to peripheral lymph nodes. Maybe the preventing of T cell homing will change the immune response against allogeneic tissue and suppress rejection. METHODS: We treated different mouse strains with pertussis toxin to manipulate T cell homing and measured the rejection of allografts in terms of allogeneic tumor cells. We transferred pertussis toxin treated or nontreated transgenic T cells into BALB/c wild type mice. The transgenic T cells could be followed ex vivo by specific antibodies. Cytokine production from purified (1x10(5)/ml) T cells after different stimulations in vitro and expression of surface markers on T cells following pertussis toxin treatment by FACS analysis were performed. RESULTS: Pertussis toxin-treated C57BL/6 mice with the MHC class I molecule H-2K(b) could not reject allogeneic tumor cells R1.1, which expressed the MHC class I molecule H-2K(k) and were killed by these cells. This allograft survival could be demonstrated for various allogeneic cells in different mouse strains with different MHC class I expression and emphasizes the general mechanism in these studies. In vivo CD62L expression on T cells was down-regulated by pertussis toxin in normal mice and transgenic mice that produce only one specific T cell, and after the pertussis toxin treatment the mice showed 4-5 times larger spleens compared to untreated mice. In transfer experiments, we demonstrated that CD62L low transgenic T cells could not home to lymph nodes. Furthermore, spleen cells from pertussis toxin-treated mice produced high amounts of the Th-2 cytokine interleukin 4 after stimulation in primary culture. CONCLUSIONS: Our data suggest that the inhibition of T cell homing changes the immune response. Prevention of homing of T cells in combination with the induction of a Th-2 response is a mechanism to prevent specific acute rejection of allogeneic tissue.     

Apparent difference in the roles of spleen and lymph nodes in the pathogenesis of host-versus-graft disease in murine parent/F1 chimeras

Host-versus-graft (HVG) disease is the often fatal immunodeficiency syndrome that can be induced in susceptible strains of mice by the perinatal inoculation of semiallogenic spleen cells. To determine the distribution and engraftment of the donor cells in the spleens and lymph nodes of RFM hosts, sequential tests were done for the presence of (T6 X RFM)F1 cells marked by their ability to form donor-specific antibodies to horseradish peroxidase (HRP) and by the T6 chromosome. Quantitation of cells with intracytoplasmic antibody that bound HRP (HRPBC) and of metaphases with the T6 marker showed that greater than 90% of donor cells were located in the hyperplastic lymph nodes. The pattern of sequential changes in numbers of HRPBC corresponded with the rise and fall in titers of antibodies to HRP. The marked differences in localization of donor cells suggested that host nodes and spleens played different roles. The lymph nodes became the main sources of donor antibodies and the principal repositories of (T6 X RFM)F1 cells capable of replication. The spleen evidently served as a major site of host resistance to engraftment. This was attributed to the ability of host cells there to inhibit selectively the proliferation of the semiallogenic donor cells. It is also proposed that counts of HRPBC measured the vigor of the HVG reaction in host spleen and lymph nodes, because the appearance of virtually all these antibody-forming cells was the result of the maturational effect of the allogenic reaction on F1 donor B memory cells.     

Induction of transplantation tolerance in rats by spleen allografts. III. The role of T suppressor cells in the induction of specific unresponsiveness

Heterotopic (WAG x AGUS)F1 spleen allografts survive indefinitely when transplanted to normal AGUS recipients and induce long-term donor-specific unresponsiveness. In this report, we have examined the immune reactivity of spleen graft recipients soon after transplantation, in an attempt to define the immunological mechanisms responsible for the induction of donor-specific unresponsiveness. Unresponsiveness develops as early as one week after splenic transplantation. T cells obtained from the recipient lymph node and spleen exhibit reduced mixed lymphocyte reaction responses to donor (WAG) but respond normally to third-party (PVG) stimulators. In contrast, T cells obtained from the spleen graft are unresponsive to both donor and third-party stimulators. Donor specific T suppressor cells (Ts) appear in the recipient's lymph node and spleen by one week posttransplantation--however, at this time antigen nonspecific suppressor cells predominate in the spleen graft. Only minimal cytotoxic T cell activity could be detected in the spleen graft, with the host spleen and lymph nodes being devoid of cytotoxic T lymphocytes. Sera obtained one or two weeks following splenic transplantation did not contain cytotoxic alloantibodies, and only a very weak response could be detected at one month. These data demonstrate that the unresponsiveness associated with the spontaneous acceptance of spleen allografts is correlated with the early induction of antigen specific Ts in recipient lymphoid tissue and the presence of nonspecific suppressor cells at the graft site.     

Graft‐Versus‐Host Disease Following Liver Transplantation: Development of a High‐Incidence Rat Model and a Selective Prevention Method

Graft‐versus‐host disease (GvHD) following liver transplantation (LT) is a rare but serious complication with no presently available animal model and no preventive measures. To develop a rat model of GvHD after LT (LT‐GvHD), we preconditioned hosts with sublethal irradiation plus reduction of natural killer (NK) cells with anti‐CD8α mAb treatment, which invariably resulted in acute LT‐GvHD. Compared with those in the peripheral counterpart, graft CD4⁺CD25^? passenger T cells showed lower alloreactivities in mixed leukocyte culture. Immunohistology revealed that donor CD4⁺ T cells migrated and formed clusters with host dendritic cells in secondary lymphoid organs, with early expansion and subsequent accumulation in target organs. For selectively preventing GvHD, donor livers were perfused ex vivo with organ preservation media containing anti‐TCRαβ mAb. T cell–depleted livers almost completely suppressed clinical GvHD such that host rats survived for >100 days. Our results showed that passenger T cells could develop typical LT‐GvHD if resistant cells such as host radiosensitive cells and host radioresistant NK cells were suppressed. Selective ex vivo T cell depletion prevented LT‐GvHD without affecting host immunity or graft function. This method might be applicable to clinical LT in prediagnosed high‐risk donor–recipient combinations and for analyzing immunoregulatory mechanisms of the liver.     

Direct versus Indirect Allorecognition: Visualization of Dendritic Cell Distribution and Interactions During Rejection and Tolerization

Interactions of donor and recipient dendritic cells (DCs) with CD4+ T cells determine the alloantigenic response in organ transplantation, where recipient T cells respond either directly to donor MHC, or indirectly to processed donor MHC allopeptides in the context of recipient MHC molecules. The present study evaluates donor and recipient alloantigen-presenting DC trafficking and their interactions with CD4+ T cells in the lymph nodes (LNs) and the spleen under tolerogenic treatment with anti-CD2 plus anti-CD3 mAb compared with untreated rejecting conditions. CX3CR1(GFP) BALB/c (I-A(d)) donor hearts were transplanted into C57BL/6 (I-A(b)) mice and quantification of donor DC direct (GFP+ or I-A(d+)) and recipient DC indirect (YAe+) trafficking and interactions with host CD4+ T cells was performed by fluorescent microscopy. Our data indicate that although both direct and indirect interactions between CD4+ T cells and donor and recipient DCs occur shortly after engraftment, only indirect presentation persists in the LN, but not the spleen, of tolerized recipients. These data suggest that distinct anatomic lymphoid compartments play a critical role in peripheral tolerance induction and maintenance, and persistent indirect presentation to CD4+ T cells within the LNs is an important process during tolerization.