Reversing donor-specific antibody responses and antibody-mediated rejection with bortezomib and belatacept in mice and kidney transplant recipients.

Active antibody-mediated rejection (AMR) is a potentially devastating complication and consistently effective treatment remains elusive. We hypothesized that the reversal of acute AMR requires rapid elimination of antibody-secreting plasma cells (PC) with a proteasome inhibitor, bortezomib, followed by the sustained inhibition of PC generation with CTLA4-Ig or belatacept (B/B). We show in mice that B/B therapy selectively depleted mature PC producing donor-specific antibodies (DSA) and reduced DSA, when administered after primary and secondary DSA responses had been established. A pilot investigation was initiated to treat six consecutive patients with active AMR with B/B. Compassionate use of this regimen was initiated for the first patient who developed early, severe acute AMR that did not respond to steroids, plasmapheresis, and intravenous immunoglobulin after his third kidney transplant. B/B treatment resulted in a rapid reversal of AMR, leading us to treat five additional patients who also resolved their acute AMR episode and had sustained disappearance of circulating DSA for ≤30 months. This study provides a proof-of-principle demonstration that mouse models can identify mechanistically rational therapies for the clinic. Follow-up investigations with a more stringent clinical design are warranted to test whether B/B improves on the standard of care for the treatment of acute AMR.

TGR5 signaling mitigates parenteral nutrition-associated liver disease.

Bile acid receptors regulate the metabolic and immune functions of circulating enterohepatic bile acids. This process is disrupted by administration of parenteral nutrition (PN), which may induce progressive hepatic injury for unclear reasons, especially in the newborn, leading to PN-associated liver disease. To explore the role of bile acid signaling on neonatal hepatic function, we initially observed that Takeda G protein receptor 5 (TGR5)-specific bile acids were negatively correlated with worsening clinical disease markers in the plasma of human newborns with prolonged PN exposure. To test our resulting hypothesis that TGR5 regulates critical liver functions to PN exposure, we used TGR5 receptor deficient mice (TGR5-/-). We observed PN significantly increased liver weight, cholestasis, and serum hepatic stress enzymes in TGR5-/- mice compared with controls. Mechanistically, PN reduced bile acid synthesis genes in TGR5-/-. Serum bile acid composition revealed that PN increased unconjugated primary bile acids and secondary bile acids in TGR5-/- mice, while increasing conjugated primary bile acid levels in TGR5-competent mice. Simultaneously, PN elevated hepatic IL-6 expression and infiltrating macrophages in TGR5-/- mice. However, the gut microbiota of TGR5-/- mice compared with WT mice following PN administration displayed highly elevated levels of Bacteroides and Parabacteroides, and possibly responsible for the elevated levels of secondary bile acids in TGR5-/- animals. Intestinal bile acid transporters expression was unchanged. Collectively, this suggests TGR5 signaling specifically regulates fundamental aspects of liver bile acid homeostasis during exposure to PN. Loss of TGR5 is associated with biochemical evidence of cholestasis in both humans and mice on PN.NEW & NOTEWORTHY Parenteral nutrition is associated with deleterious metabolic outcomes in patients with prolonged exposure. Here, we demonstrate that accelerated cholestasis and parental nutrition-associated liver disease (PNALD) may be associated with deficiency of Takeda G protein receptor 5 (TGR5) signaling. The microbiome is responsible for production of secondary bile acids that signal through TGR5. Therefore, collectively, these data support the hypothesis that a lack of established microbiome in early life or under prolonged parenteral nutrition may underpin disease development and PNALD.

Cutting Edge: CTLA-4Ig Inhibits Memory B Cell Responses and Promotes Allograft Survival in Sensitized Recipients.

Sensitized recipients with pretransplant donor-specific Abs are at higher risk for Ab-mediated rejection than nonsensitized recipients, yet little is known about the properties of memory B cells that are central to the recall alloantibody responses. Using cell enrichment and MHC class I tetramers, C57BL/6 mice sensitized with BALB/c splenocytes were shown to harbor H-2K(d)-specific IgG(+) memory B cells with a post-germinal center phenotype (CD73(+)CD273(+)CD38(hi)CD138(-)GL7(-)). These memory B cells adoptively transferred into naive mice without memory T cells recapitulated class-switched recall alloantibody responses. During recall, memory H-2K(d)-specific B cells preferentially differentiated into Ab-secreting cells, whereas in the primary response, H-2K(d)-specific B cells differentiated into germinal center cells. Finally, our studies revealed that, despite fundamental differences in alloreactive B cell fates in sensitized versus naive recipients, CTLA-4Ig was unexpectedly effective at constraining B cell responses and heart allograft rejection in sensitized recipients.

Pregnancy dedifferentiates memory CD8+ T cells into hypofunctional cells with exhaustion-enriched programs.

Alloreactive memory, unlike naive, CD8+ T cells resist transplantation tolerance protocols and are a critical barrier to long-term graft acceptance in the clinic. We here show that semiallogeneic pregnancy successfully reprogrammed memory fetus/graft-specific CD8+ T cells (TFGS) toward hypofunction. Female C57BL/6 mice harboring memory CD8+ T cells generated by the rejection of BALB/c skin grafts and then mated with BALB/c males achieved rates of pregnancy comparable with naive controls. Postpartum CD8+ TFGS from skin-sensitized dams upregulated expression of T cell exhaustion (TEX) markers (Tox, Eomes, PD-1, TIGIT, and Lag3). Transcriptional analysis corroborated an enrichment of canonical TEX genes in postpartum memory TFGS and revealed a downregulation of a subset of memory-associated transcripts. Strikingly, pregnancy induced extensive epigenetic modifications of exhaustion- and memory-associated genes in memory TFGS, whereas minimal epigenetic modifications were observed in naive TFGS. Finally, postpartum memory TFGS durably expressed the exhaustion-enriched phenotype, and their susceptibility to transplantation tolerance was significantly restored compared with memory TFGS. These findings advance the concept of pregnancy as an epigenetic modulator inducing hypofunction in memory CD8+ T cells that has relevance not only for pregnancy and transplantation tolerance, but also for tumor immunity and chronic infections.

Pregnancy programs epigenetic and transcriptional exhaustion in memory CD8+ T cells.

Alloreactive memory T cells, unlike naive T cells, fail to be restrained by transplantation tolerance protocols or regulatory T cells, and therefore represent a critical barrier to long-term graft acceptance. Using female mice sensitized by rejection of fully-mismatched paternal skin allografts, we show that subsequent semi-allogeneic pregnancy successfully reprograms memory fetus/graft-specific CD8+ T cells (TFGS) towards hypofunction in a manner that is mechanistically distinct from naive TFGS. Post-partum memory TFGS were durably hypofunctional, exhibiting enhanced susceptibility to transplantation tolerance induction. Furthermore, multi-omics studies revealed that pregnancy induced extensive phenotypic and transcriptional modifications in memory TFGS reminiscent of T cell exhaustion. Strikingly, at loci transcriptionally modified in both naive and memory TFGS during pregnancy, chromatin remodeling was observed exclusively in memory and not naive TFGS. These data reveal a novel link between T cell memory and hypofunction via exhaustion circuits and pregnancy-mediated epigenetic imprinting. This conceptual advance has immediate clinical relevance to pregnancy and transplantation tolerance.

A novel triptolide analog downregulates NF-κB and induces mitochondrial apoptosis pathways in human pancreatic cancer.

Pancreatic cancer is the seventh leading cause of cancer-related death worldwide, and despite advancements in disease management, the 5 -year survival rate stands at only 12%. Triptolides have potent anti-tumor activity against different types of cancers, including pancreatic cancer, however poor solubility and toxicity limit their translation into clinical use. We synthesized a novel pro-drug of triptolide, (E)-19-[(1'-benzoyloxy-1'-phenyl)-methylidene]-Triptolide (CK21), which was formulated into an emulsion for in vitro and in vivo testing in rats and mice, and used human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids. A time-course transcriptomic profiling of tumor organoids treated with CK21 in vitro was conducted to define its mechanism of action, as well as transcriptomic profiling at a single time point post-CK21 administration in vivo. Intravenous administration of emulsified CK21 resulted in the stable release of triptolide, and potent anti-proliferative effects on human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids in vitro, and with minimal toxicity in vivo. Time course transcriptomic profiling of tumor organoids treated with CK21 in vitro revealed <10 differentially expressed genes (DEGs) at 3 hr and ~8,000 DEGs at 12 hr. Overall inhibition of general RNA transcription was observed, and Ingenuity pathway analysis together with functional cellular assays confirmed inhibition of the NF-κB pathway, increased oxidative phosphorylation and mitochondrial dysfunction, leading ultimately to increased reactive oxygen species (ROS) production, reduced B-cell-lymphoma protein 2 (BCL2) expression, and mitochondrial-mediated tumor cell apoptosis. Thus, CK21 is a novel pro-drug of triptolide that exerts potent anti-proliferative effects on human pancreatic tumors by inhibiting the NF-κB pathway, leading ultimately to mitochondrial-mediated tumor cell apoptosis.

Pancreatic cancer is a major cause of cancer-related deaths worldwide, with only 12% of patients surviving for five years after diagnosis. Individuals generally experience few symptoms of the disease in the early stages and are often diagnosed once the cancer has already spread to other parts of the body. By this point, options for treatment are limited. A molecule known as triptolide has been shown to kill breast, lung, pancreatic and other types of cancer cells. However, triptolide is toxic to humans and other animals, making it unsuitable for use in patients. One way to make drugs safer without compromising their beneficial effects is to modify their molecular structure. By formulating triptolide into an emulsion ­ a mixture of liquids allowing it to dissolve ­ Tian, Zhang et al. synthesized a new analogue called CK21. Experiments showed that CK21 inhibited the growth of human pancreatic cancer cells grown in a laboratory including cells grown in artificial organs similar to the pancreas, known as pancreatic tumor organoids. Furthermore, CK21 killed large tumors in mice pancreases with very few side effects, suggesting the structural modification of triptolide increased safety of the drug. To better understand how CK21 works, Tian, Zhang et al. examined the genes that were induced in the pancreatic tumor organoids at various time points after treatment with the drug. This revealed that CK21 switched off genes involved in the NF-κB cell signaling pathway, which regulates how cells grow and respond to stress. In turn, it triggered programmed cell death, killing the tumor cells in a controlled manner. The findings suggest that CK21 could be a promising candidate for treating pancreatic cancer. In the future, clinical trials will be required to establish whether CK21 is a safe and effective therapy for humans.

MHC Tetramers Specifically Identify High- and Low-avidity Donor-specific B Cells in Transplantation Tolerance and Rejection.

BACKGROUND: Although donor-specific antibody pre- and posttransplantation is routinely assessed, accurate quantification of memory alloreactive B cells that mediate recall antibody response remains challenging. Major histocompatibility complex (MHC) tetramers have been used to identify alloreactive B cells in mice and humans, but the specificity of this approach has not been rigorously assessed. METHODS: B-cell receptors from MHC tetramer-binding single B cells were expressed as mouse recombinant immunoglobulin G1 (rIgG1) monoclonal antibodies, and the specificity was assessed with a multiplex bead assay. Relative binding avidity of rIgG1 was measured by modified dilution series technique and surface plasmon resonance. Additionally, immunoglobulin heavy chain variable regions of 50 individual B-cell receptors were sequenced to analyze the rate of somatic hypermutation. RESULTS: The multiplex bead assay confirmed that expressed rIgG1 monoclonal antibodies were preferentially bound to bait MHC class II I-E d over control I-A d and I-A b tetramers. Furthermore, the dissociation constant 50 binding avidities of the rIgG1 ranged from 10 mM to 7 nM. The majority of tetramer-binding B cells were low avidity, and ~12.8% to 15.2% from naive and tolerant mice and 30.9% from acute rejecting mice were higher avidity (dissociation constant 50 <1 mM). CONCLUSIONS: Collectively, these studies demonstrate that donor MHC tetramers, under stringent binding conditions with decoy self-MHC tetramers, can specifically identify a broad repertoire of donor-specific B cells under conditions of rejection and tolerance.

Heterogeneity in allospecific T cell function in transplant-tolerant hosts determines susceptibility to rejection following infection.

Even when successfully induced, immunological tolerance to solid organs remains vulnerable to inflammatory insults, which can trigger rejection. In a mouse model of cardiac allograft tolerance in which infection with Listeria monocytogenes (Lm) precipitates rejection of previously accepted grafts, we showed that recipient CD4+ TCR75 cells reactive to a donor MHC class I-derived peptide become hypofunctional if the allograft is accepted for more than 3 weeks. Paradoxically, infection-induced transplant rejection was not associated with transcriptional or functional reinvigoration of TCR75 cells. We hypothesized that there is heterogeneity in the level of dysfunction of different allospecific T cells, depending on duration of their cognate antigen expression. Unlike CD4+ TCR75 cells, CD4+ TEa cells specific for a peptide derived from donor MHC class II, an alloantigen whose expression declines after transplantation but remains inducible in settings of inflammation, retained function in tolerant mice and expanded during Lm-induced rejection. Repeated injections of alloantigens drove hypofunction in TEa cells and rendered grafts resistant to Lm-dependent rejection. Our results uncover a functional heterogeneity in allospecific T cells of distinct specificities after tolerance induction and reveal a strategy to defunctionalize a greater repertoire of allospecific T cells, thereby mitigating a critical vulnerability of tolerance.

p73 protein regulates DNA damage repair.

Although the p53 tumor suppressor is relatively well characterized, much less is known about the functions of other members of the p53 family, p73 and p63. Here, we present evidence that in specific pathological conditions caused by exposure of normal cells to bile acids in acidic conditions, p73 protein plays the predominant role in the DNA damage response. These pathological conditions frequently occur during gastric reflux in the human esophagus and are associated with progression to esophageal adenocarcinoma. We found that despite strong DNA damage induced by bile acid exposure, only p73 (but not p53 and p63) is selectively activated in a c-Abl kinase-dependent manner. The activated p73 protein induces DNA damage repair. Using a human DNA repair PCR array, we identified multiple DNA repair genes affected by p73. Two glycosylases involved in base excision repair, SMUG1 and MUTYH, were characterized and found to be transcriptionally regulated by p73 in DNA damage conditions. Using a surgical procedure in mice, which recapitulates bile acid exposure, we found that p73 deficiency is associated with increased DNA damage. These findings were further investigated with organotypic and traditional cell cultures. Collectively our studies demonstrate that p73 plays an important role in the regulation of DNA damage repair.

Linked sensitization by memory CD4+ T cells prevents costimulation blockade-induced transplantation tolerance.

Dominant infectious tolerance explains how brief tolerance-inducing therapies result in lifelong tolerance to donor antigens and "linked" third-party antigens, while recipient sensitization and ensuing immunological memory prevent the successful induction of transplant tolerance. In this study, we juxtapose these 2 concepts to test whether mechanisms of dominant infectious tolerance can control a limited repertoire of memory T and B cells. We show that sensitization to a single donor antigen is sufficient to prevent stable transplant tolerance, rendering it unstable. Mechanistic studies revealed that recall antibody responses and memory CD8+ T cell expansion were initially controlled, but memory CD4+Foxp3- T cell (Tconv) responses were not. Remarkably, naive donor-specific Tconvs at tolerance induction also acquired a resistance to tolerance, proliferating and acquiring a phenotype similar to memory Tconvs. This phenomenon of "linked sensitization" underscores the challenges of reprogramming a primed immune response toward tolerance and identifies a potential therapeutic checkpoint for synergizing with costimulation blockade to achieve transplant tolerance in the clinic.

Glutathione peroxidase-3 produced by the kidney binds to a population of basement membranes in the gastrointestinal tract and in other tissues.

Glutathione peroxidase-3 (Gpx3), the extracellular glutathione peroxidase synthesized largely in the kidney, binds to basement membranes of renal cortical epithelial cells. The present study assessed extrarenal expression of Gpx3 using RT-PCR and presence of Gpx3 protein using immunocytochemistry. Gpx3 expression was higher in kidney and epididymis than in other tissues. Gpx3 bound to basement membranes of epithelial cells in the gastrointestinal tract, the efferent ducts connecting the seminiferous tubules with the epididymis, the bronchi, and type II pneumocytes. It was not detected on the basement membrane of type I pneumocytes. Gpx3 was also present in the lumen of the epididymis. Transplantation of Gpx3(+/+) kidneys into Gpx3(-/-) mice led to Gpx3 binding to the same basement membranes to which it bound in Gpx3(+/+) mice but not to its presence in the epididymal lumen. These results show that Gpx3 from the blood binds to basement membranes of specific epithelial cells and indicate that the cells modify their basement membranes to cause the binding. They further indicate that at least two Gpx3 compartments exist in the organism. In one compartment, kidney supplies Gpx3 through the blood to specific basement membranes in a number of tissues. In the other compartment, the epididymis provides Gpx3 to its own lumen. Tissues other than kidney and epididymis express Gpx3 at lower levels and may supply Gpx3 to other compartments.

Memory alloreactive B cells and alloantibodies prevent anti-CD154-mediated allograft acceptance.

The impact of memory B cells and alloantibodies on the ability to induce transplantation tolerance has not been elucidated. We have developed a murine heart transplant model that isolates the contributions of functional memory B cells from memory T cells in allograft rejection. Memory 3-83 B cells with dual specificity for H-2K(k) and H-2K(b) were generated in 3-83 Igi BCR knockin (BALB/c background) mice by the transplantation of C3H (H-2K(k)) hearts in the absence of immunosuppression. To test the effect of functional memory 3-83 B cells, C3H-primed 3-83 Igi recipients were challenged with C57BL/6 hearts (H-2K(b)) at 60-90 days post-C3H heart transplant and treated with anti-CD154 mAbs. Despite immunosuppression, the C57BL/6 hearts were acutely rejected within 10-13 days and graft rejection was associated with increased frequencies of C57BL/6-specific IFN-gamma-producing T cells. Histology revealed significant numbers of infiltrating T cells, consistent with acute T cell-mediated rejection. The resistance to tolerance induction was dependent on the synergistic effects of memory 3-83 B cells and alloantibodies, whereas memory T cells are not necessary. We conclude that the combined effects of functional memory B cells and alloantibodies prevent anti-CD154-mediated graft acceptance by facilitating the CD40-CD154-independent activation of alloreactive T cells. This study provides insight into the potential ability of memory B cells and alloantibodies to prevent anti-CD154-mediated graft acceptance.

Pregnancy-induced humoral sensitization overrides T cell tolerance to fetus-matched allografts in mice.

Immunological tolerance to semiallogeneic fetuses is necessary to achieving successful first pregnancy and permitting subsequent pregnancies with the same father. Paradoxically, pregnancy is an important cause of sensitization, resulting in the accelerated rejection of offspring-matched allografts. The underlying basis for divergent outcomes following reencounter of the same alloantigens on transplanted organs versus fetuses in postpartum females is incompletely understood. Using a mouse model that allows concurrent tracking of endogenous fetus-specific T and B cell responses in a single recipient, we show that semiallogeneic pregnancies simultaneously induce fetus-specific T cell tolerance and humoral sensitization. Pregnancy-induced antibodies, but not B cells, impeded transplantation tolerance elicited by costimulation blockade to offspring-matched cardiac grafts. Remarkably, in B cell-deficient mice, allogeneic pregnancy enabled the spontaneous acceptance of fetus-matched allografts. The presence of pregnancy-sensitized B cells that cannot secrete antibodies at the time of heart transplantation was sufficient to precipitate rejection and override pregnancy-established T cell tolerance. Thus, while induction of memory B cells and alloantibodies by pregnancies establishes formidable barriers to transplant success for multigravid women, our observations raise the possibility that humoral desensitization will not only improve transplantation outcomes, but also reveal an unexpected propensity of multiparous recipients to achieve tolerance to offspring-matched allografts.

Transplantation tolerance modifies donor-specific B cell fate to suppress de novo alloreactive B cells.

The absence of alloantibodies is a feature of transplantation tolerance. Although the lack of T cell help has been evoked to explain this absence, herein we provide evidence for B cell-intrinsic tolerance mechanisms. Using a murine model of heart tolerance, we showed that alloreactive B cells were not deleted but rapidly lost their ability to differentiate into germinal center B cells and secrete donor-specific antibodies. We inferred that tolerant alloreactive B cells retained their ability to sense alloantigen because they continued to drive T cell maturation into CXCR5+PD-1+ T follicular helper cells. Unexpectedly, dysfunctional alloreactive B cells acquired the ability to inhibit antibody production by new naive B cells in an antigen-specific manner. Thus, tolerant alloreactive B cells contribute to transplantation tolerance by foregoing germinal center responses while retaining their ability to function as antigen-presenting cells and by actively suppressing de novo alloreactive B cell responses.

CpG oligodeoxynucleotide triggers the liver inflammatory reaction and abrogates spontaneous tolerance.

Liver allografts are spontaneously accepted in the liver transplantation mouse model; however, the basis for this tolerance and the conditions that abrogate spontaneous tolerance to liver allografts are incompletely understood. We examined the role of CpG oligodeoxynucleotide (ODN) in triggering the liver inflammatory reaction and allograft rejection. Bioluminescence imaging quantified the activation of nuclear transcriptional factor kappaB (NF-kappaB) at different time points post-transplantation. Intrahepatic lymphocyte subsets were analyzed by immunofluorescence assay and flow cytometry. The results showed that liver allografts survived for more than 100 days without a requirement for any immunosuppressive therapy. Donor-matched cardiac allografts were permanently accepted, whereas third-party cardiac grafts were rejected with delayed kinetics; this confirmed donor-specific tolerance. NF-kappaB activation in the liver allografts was transiently increased on day 1 and diminished by day 4; in comparison, it was elevated up to 10 days post-transplantation in the cardiac allografts. When CpG ODN was administered at a high dose (50 microg per mouse x 1) to the recipients on day 7 post-transplantation, it induced an acute liver inflammatory reaction with elevated NF-kappaB activation in both allogeneic and syngeneic liver grafts. Multiple doses of CpG ODN (10 microg per mouse x 3) elicited acute rejection of the liver allografts with significant T cell infiltration in the liver allografts, reduced T regulatory cells, and enhanced interferon gamma-producing cells in the intrahepatic infiltrating lymphocytes. These data demonstrate that CpG ODN initiates an inflammatory reaction and abrogates spontaneous tolerance in the liver transplantation mouse model. Liver Transpl 15:915-923, 2009. (c) 2009 AASLD.

CD4+ T cells are sufficient to elicit allograft rejection and major histocompatibility complex class I molecule is required to induce recurrent autoimmune diabetes after pancreas transplantation in mice.

BACKGROUND: We characterized the role of T cell subsets and major histocompatibility complex molecules in allograft rejection and recurrence of autoimmune diabetes. METHODS: Adoptive cell transfer and vascularized segmental pancreas transplantation were performed in mice. RESULTS: In an alloimmune response model, transfer of nondiabetic CD4, but not CD8 T cells, elicited pancreas allograft rejection in streptozotocin (STZ)-induced diabetic NOD/scid mice. Pancreas allografts were acutely rejected in STZ-induced diabetic NOD/beta2m mice (confirmed the absence of major histocompatibility complex [MHC] class I and CD8 T cells) and permanently accepted in NOD/CIIT mice (confirmed the absence of MHC class II and CD4 T cells). The results suggest that rejection of pancreas allograft is CD4-dependent and MHC class I-independent. In the autoimmune diabetes model, whole spleen cells obtained from diabetic NOD mice induced autoimmune diabetes in NOD/scid and NOD/CIIT mice, but the onset of diabetes was delayed in NOD/beta2m mice. However, the purified diabetic T cells failed to elicit autoimmune diabetes in NOD/beta2m mice. NOD/scid and NOD/CIIT pancreas grafts were acutely destroyed whereas four of six NOD/beta2m pancreas grafts were permanently accepted in autoimmune diabetic NOD mice. CONCLUSION: CD4 T cells are sufficient for the induction of allograft rejection, and MHC class I molecule is required to induce recurrent autoimmune diabetes after pancreas transplantation in mice.

Equal Expansion of Endogenous Transplant-Specific Regulatory T Cell and Recruitment Into the Allograft During Rejection and Tolerance.

Despite numerous advances in the definition of a role for regulatory T cells (Tregs) in facilitating experimental transplantation tolerance, and ongoing clinical trials for Treg-based therapies, critical issues related to the optimum dosage, antigen-specificity, and Treg-friendly adjunct immunosuppressants remain incompletely resolved. In this study, we used a tractable approach of MHC tetramers and flow cytometry to define the fate of conventional (Tconvs) and Tregs CD4+ T cells that recognize donor 2W antigens presented by I-Ab on donor and recipient antigen-presenting cells (APCs) in a mouse cardiac allograft transplant model. Our study shows that these endogenous, donor-reactive Tregs comparably accumulate in the spleens of recipients undergoing acute rejection or exhibiting costimulation blockade-induced tolerance. Importantly, this expansion was not detected when analyzing bulk splenic Tregs. Systemically, the distinguishing feature between tolerance and rejection was the inhibition of donor-reactive conventional T cell (Tconv) expansion in tolerance, translating into increased percentages of splenic FoxP3+ Tregs within the 2W:I-Ab CD4+ T cell subset compared to rejection (~35 vs. <5% in tolerance vs. rejection). We further observed that continuous administration of rapamycin, cyclosporine A, or CTLA4-Ig did not facilitate donor-specific Treg expansion, while all three drugs inhibited Tconv expansion. Finally, donor-specific Tregs accumulated comparably in rejecting tolerant allografts, whereas tolerant grafts harbored <10% of the donor-specific Tconv numbers observed in rejecting allografts. Thus, ~80% of 2W:I-Ab CD4+ T cells in tolerant allografts expressed FoxP3+ compared to ≤10% in rejecting allografts. A similar, albeit lesser, enrichment was observed with bulk graft-infiltrating CD4+ cells, where ~30% were FoxP3+ in tolerant allografts, compared to ≤10% in rejecting allografts. Finally, we assessed that the phenotype of 2W:I-Ab Tregs and observed that the percentages of cells expressing neuropilin-1 and CD73 were significantly higher in tolerance compared to rejection, suggesting that these Tregs may be functionally distinct. Collectively, the analysis of donor-reactive, but not of bulk, Tconvs and Tregs reveal a systemic signature of tolerance that is stable and congruent with the signature within tolerant allografts. Our data also underscore the importance of limiting Tconv expansion for high donor-specific Tregs:Tconv ratios to be successfully attained in transplantation tolerance.

Recovery of islet beta-cell function in streptozotocin- induced diabetic mice: an indirect role for the spleen.

Limitations in islet beta-cell transplantation as a therapeutic option for type 1 diabetes have prompted renewed interest in islet regeneration as a source of new islets. In this study we tested whether severely diabetic adult C57BL/6 mice can regenerate beta-cells. Diabetes was induced in C57BL/6 mice with high-dose streptozotocin (160-170 mg/kg). In the absence of islet transplantation, all diabetic mice remained diabetic (blood glucose >400 mg/dl), and no spontaneous reversal of diabetes was observed. When syngeneic islets (200/mouse) were transplanted into these diabetic mice under a single kidney capsule, stable restoration of euglycemia for >/=120 days was achieved. Removal of the kidney bearing the transplanted islets at 120 days posttransplantation revealed significant restoration of endogenous beta-cell function. This restoration of islet function was associated with increased beta-cell mass, as well as beta-cell hypertrophy and proliferation. The restoration of islet cell function was facilitated by the presence of a spleen; however, the facilitation was not due to the direct differentiation of spleen-derived cells into beta-cells. This study supports the possibility of restoring beta-cell function in diabetic individuals and points to a role for the spleen in facilitating this process.

Successful Treatment of T Cell-Mediated Acute Rejection with Delayed CTLA4-Ig in Mice.

Clinical observations that kidney transplant recipients receiving belatacept who experienced T cell-mediated acute rejection can be successfully treated and subsequently maintained on belatacept-based immunosuppression suggest that belatacept is able to control memory T cells. We recently reported that treatment with CTLA4-Ig from day 6 posttransplantation successfully rescues allografts from acute rejection in a BALB/c to C57BL/6 heart transplant model, in part, by abolishing B cell germinal centers and reducing alloantibody titers. Here, we show that CTLA4-Ig is additionally able to inhibit established T cell responses independently of B cells. CTLA4-Ig inhibited the in vivo cytolytic activity of donor-specific CD8+ T cells, and the production of IFNγ by graft-infiltrating T cells. Delayed CTLA4-Ig treatment did not reduce the numbers of graft-infiltrating T cells nor prevented the accumulation of antigen-experienced donor-specific memory T cells in the spleen. Nevertheless, delayed CTLA4-Ig treatment successfully maintained long-term graft acceptance in the majority of recipients that had experienced a rejection crisis, and enabled the acceptance of secondary BALB/c heart grafts transplanted 30 days after the first transplantation. In summary, we conclude that delayed CTLA4-Ig treatment is able to partially halt ongoing T cell-mediated acute rejection. These findings extend the functional efficacy of CTLA4-Ig therapy to effector T cells and provide an explanation for why CTLA4-Ig-based immunosuppression in the clinic successfully maintains long-term graft survival after T cell-mediated rejection.