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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.

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.

Transplantation is a cure for end-stage organ failure but, in the absence of pharmacological immunosuppression, allogeneic organs are acutely rejected. Such rejection invariably results in allosensitization and accelerated rejection of secondary donor-matched grafts. Transplantation tolerance can be induced in animals and a subset of humans, and enables long-term acceptance of allografts without maintenance immunosuppression. However, graft rejection can occur long after a state of transplantation tolerance has been acquired. When such an allograft is rejected, it has been assumed that the same rules of allosensitization apply as to non-tolerant hosts and that immunological tolerance is permanently lost. Using a mouse model of cardiac transplantation, we show that when Listeria monocytogenes infection precipitates acute rejection, thus abrogating transplantation tolerance, the donor-specific tolerant state re-emerges, allowing spontaneous acceptance of a donor-matched second transplant. These data demonstrate a setting in which the memory of allograft tolerance dominates over the memory of transplant rejection. An infection can break the immune tolerance of a transplanted organ, resulting in its rejection. Here the authors show that the immunological memory of transplantation tolerance dominates over the memory of allograft rejection, so that another organ transplanted later can be spontaneously accepted.

Successful allograft-specific tolerance induction would eliminate the need for daily immunosuppression and improve posttransplant quality of life. Adoptive cell therapy with regulatory T cells expressing donor-specific chimeric antigen receptors (CAR Tregs) is a promising strategy but, as monotherapy, cannot prolong survival with allografts with multiple MHC mismatches. Using an HLA-A2–transgenic haplo-mismatched heart transplantation model in immunocompetent C57BL/6 recipients, we showed that HLA-A2–specific CAR (A2.CAR) Tregs were able to synergize with a low dose of anti-CD154 to enhance graft survival. Using haplo-mismatched grafts expressing the 2W-OVA transgene and tetramer-based tracking of 2W- and OVA-specific T cells, we showed that in mice with accepted grafts, A2.CAR Tregs inhibited donor-specific T cell, B cell, and antibody responses and promoted a substantial increase in endogenous FOXP3 + Tregs with indirect donor specificity. By contrast, in mice where A2.CAR Tregs failed to prolong graft survival, FOXP3 – A2.CAR T cells preferentially accumulated in rejecting allografts, and endogenous donor-specific responses were not controlled. This study therefore provides evidence for synergy between A2.CAR Tregs and CD154 blockade to promote infectious tolerance in immunocompetent recipients of haplo-mismatched heart grafts and defines features of A2.CAR Tregs when they fail to reshape host immunity toward allograft 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-A 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-A 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-A 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-A 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.

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 (T FGS ) 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 + T FGS from skin-sensitized dams upregulated expression of T cell exhaustion (T EX ) markers (Tox, Eomes, PD-1, TIGIT, and Lag3). Transcriptional analysis corroborated an enrichment of canonical T EX genes in postpartum memory T FGS 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 T FGS , whereas minimal epigenetic modifications were observed in naive T FGS . Finally, postpartum memory T FGS durably expressed the exhaustion-enriched phenotype, and their susceptibility to transplantation tolerance was significantly restored compared with memory T FGS . 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.

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 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.

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.

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.

Autoimmune destruction of β cells is the predominant cause of type 1 diabetes mellitus (T1DM) in humans and is modeled in non-obese diabetic (NOD) mice. Many therapeutic interventions prevent the development of T1DM in NOD mice, but few can induce its reversal once established. Intervention with Freund's complete adjuvant, semi-allogeneic splenocytes, and temporary islet transplantation has been reported to cure NOD mice of established T1DM. Using the same approach, we report here that this treatment cured 32% of NOD mice of established diabetes (>340 milligrams per deciliter blood glucose), although β cells in these mice were not derived from donor splenocytes.