Optimal CXCR5 Expression during Tfh Maturation Involves the Bhlhe40-Pou2af1 Axis.

The pair of transcription factors Bcl6-Blimp1 is well-known for follicular T helper (Tfh) cell fate determination, however, the mechanism(s) for Bcl6-independent regulation of CXCR5 during Tfh migration into germinal center (GC) is still unclear. In this study, we uncovered another pair of transcription factors, Bhlhe40-Pou2af1, that regulates CXCR5 expression. Pou2af1 was specifically expressed in Tfh cells whereas Bhlhe40 expression was found high in non-Tfh cells. Pou2af1 promoted Tfh formation and migration into GC by upregulating CXCR5 but not Bcl6, while Bhlhe40 repressed this process by inhibiting Pou2af1 expression. RNA-Seq analysis of antigen-specific Tfh cells generated in vivo confirmed the role of Bhlhe40-Pou2af1 axis in regulating optimal CXCR5 expression. Thus, the regulation of CXCR5 expression and migration of Tfh cells into GC involves a transcriptional regulatory circuit consisting of Bhlhe40 and Pou2af1, which operates independent of the Bcl6-Blimp1 circuit that determines the Tfh cell fate.

Long-term control of diabetes by tofacitinib-based immunosuppressive regimen after allo islet transplantation in diabetic rhesus monkeys that rejected previously transplanted porcine islets.

Porcine islet xenotransplantation has been highlighted as an alternative to allo islet transplantation. Despite the remarkable progress that has been made in porcine-islet pre-clinical studies in nonhuman primates, immunological tolerance to porcine islets has not been achieved to date. Therefore, allo islet transplantation could be required after the failure of porcine islet xenotransplantation. Here, we report the long-term control of diabetes by allogeneic pancreatic islet transplantation in diabetic rhesus monkeys that rejected previously transplanted porcine islets. Four diabetic male rhesus monkeys received the porcine islets and then allo islets (5700-19 000 IEQ/kg) were re-transplanted for a short or long period after the first xeno islet rejection. The recipient monkeys were treated with an immunosuppressive regimen consisting of ATG, humira, and anakinra for induction, and sirolimus and tofacitinib for maintenance therapy. The graft survival days of allo islets in these monkeys were >440, 395, >273, and 127, respectively, similar to that in allo islet transplanted cynomolgus monkeys that received the same immunosuppressive regimen without xeno sensitization. Taken together, it is likely that prior islet xenotransplantation does not affect the survival of subsequent allo islets under clinically applicable immunosuppressants.

Transcription factor TCF-1 regulates the functions, but not the development, of lymphoid tissue inducer subsets in different tissues.

Lymphoid tissue inducer (LTi) cells, a subset of innate lymphoid cells (ILCs), play an essential role in the formation of secondary lymphoid tissues. However, the regulation of the development and functions of this ILC subset is still elusive. In this study, we report that the transcription factor T cell factor 1 (TCF-1), just as GATA3, is indispensable for the development of non-LTi ILC subsets. While LTi cells are still present in TCF-1-deficient mice, the organogenesis of Peyer's patches (PPs), but not of lymph nodes, is impaired in these mice. LTi cells from different tissues have distinct gene expression patterns, and TCF-1 regulates the expression of lymphotoxin specifically in PP LTi cells. Mechanistically, TCF-1 may directly and/or indirectly regulate Lta, including through promoting the expression of GATA3. Thus, the TCF-1-GATA3 axis, which plays an important role during T cell development, also critically regulates the development of non-LTi cells and tissue-specific functions of LTi cells.

Mesenchymal stem cell-derived extracellular vesicles subvert Th17 cells by destabilizing RORγt through posttranslational modification.

Mesenchymal stem cell (MSC)-derived small extracellular vesicles (MSC-sEVs) are known to exert immunosuppressive functions. This study showed that MSC-sEVs specifically convert T helper 17 (Th17) cells into IL-17 low-producer (ex-Th17) cells by degrading RAR-related orphan receptor γt (RORγt) at the protein level. In experimental autoimmune encephalomyelitis (EAE)-induced mice, treatment with MSC-sEVs was found to not only ameliorate clinical symptoms but also to reduce the number of Th17 cells in draining lymph nodes and the central nervous system. MSC-sEVs were found to destabilize RORγt by K63 deubiquitination and deacetylation, which was attributed to the EP300-interacting inhibitor of differentiation 3 (Eid3) contained in the MSC-sEVs. Small extracellular vesicles isolated from the Eid3 knockdown MSCs by Eid3-shRNA failed to downregulate RORγt. Moreover, forced expression of Eid3 by gene transfection was found to significantly decrease the protein level of RORγt in Th17 cells. Altogether, this study reveals the novel immunosuppressive mechanisms of MSC-sEVs, which suggests the feasibility of MSC-sEVs as an attractive therapeutic tool for curing Th17-mediated inflammatory diseases.

Galectin-4 increases the ability of M2 macrophages to enhance antiviral CD4+ T-cell responses.

Galectin-4 (Gal-4) is a ß-galactoside-binding protein belonging to the galectin family. Although Gal-4 is known to be involved in several physiologic processes of the gastrointestinal tract, its immunomodulatory roles remain unclear. In this study, we investigated whether Gal-4 influences the function of M1 and M2 macrophages. Gal-4 treatment drove more robust changes in the gene expression of M2 macrophages compared to M1 macrophages. Antiviral immune response-related genes were significantly upregulated in Gal-4-treated M2 macrophages. Gal-4 significantly enhanced the immunostimulatory activity of M2 macrophages upon Toll-like receptor 7 stimulation or infection with lymphocytic choriomeningitis virus (LCMV). Moreover, the antibody production against LCMV infection and the antiviral CD4+ T-cell responses, but not the antiviral CD8+ T-cell responses, were greatly increased by Gal-4-treated M2 macrophages in vivo. The present results indicate that Gal-4 enhances the ability of M2 macrophages to promote antiviral CD4+ T-cell responses. Thus, Gal-4 could be used to boost antiviral immune responses.

Gata3 ZsG and Gata3 ZsG-fl: Novel murine Gata3 reporter alleles for identifying and studying Th2 cells and ILC2s in vivo.

T helper-2 (Th2) cells and type 2 innate lymphoid cells (ILC2s) play crucial roles during type 2 immune responses; the transcription factor GATA3 is essential for the differentiation and functions of these cell types. It has been demonstrated that GATA3 is critical for maintaining Th2 and ILC2 phenotype in vitro; GATA3 not only positively regulates type 2 lymphocyte-associated genes, it also negatively regulates many genes associated with other lineages. However, such functions cannot be easily verified in vivo because the expression of the markers for identifying Th2 and ILC2s depends on GATA3. Thus, whether Th2 cells and ILC2s disappear after Gata3 deletion or these Gata3-deleted "Th2 cells" or "ILC2s" acquire an alternative lineage fate is unknown. In this study, we generated novel GATA3 reporter mouse strains carrying the Gata3 ZsG or Gata3 ZsG-fl allele. This was achieved by inserting a ZsGreen-T2A cassette at the translation initiation site of either the wild type Gata3 allele or the modified Gata3 allele which carries two loxP sites flanking the exon 4. ZsGreen faithfully reflected the endogenous GATA3 protein expression in Th2 cells and ILC2s both in vitro and in vivo. These reporter mice also allowed us to visualize Th2 cells and ILC2s in vivo. An inducible Gata3 deletion system was created by crossing Gata3 ZsG-fl/fl mice with a tamoxifen-inducible Cre. Continuous expression of ZsGreen even after the Gata3 exon 4 deletion was noted, which allows us to isolate and monitor GATA3-deficient "Th2" cells and "ILC2s" during in vivo immune responses. Our results not only indicated that functional GATA3 is dispensable for regulating its own expression in mature type 2 lymphocytes, but also revealed that GATA3-deficient "ILC2s" might be much more stable in vivo than in vitro. Overall, the generation of these novel GATA3 reporters will provide valuable research tools to the scientific community in investigating type 2 immune responses in vivo.

Six-month sustained delivery of anti-VEGF from in-situ forming hydrogel in the suprachoroidal space.

Patients with wet age-related macular degeneration (AMD) require intravitreal injections of bevacizumab (Bev) or other drugs, often on a monthly basis, which is a burden on the healthcare system. Here, we developed an in-situ forming hydrogel comprised of Bev and hyaluronic acid (HA) crosslinked with poly(ethylene glycol) diacrylate for slow release of Bev after injection into the suprachoroidal space (SCS) of the eye using a microneedle. Liquid Bev formulations were cleared from SCS within 5 days, even when formulated with high viscosity, unless Bev was conjugated to a high molecular-weight HA (2.6 MDa), which delayed clearance until 1 month. To extend release to 6 months, we synthesized in-situ forming Bev-HA hydrogel initially as a low-viscosity mixture suitable for injection and flow in the SCS to cover a large area extending to the posterior pole of the eye where the macula is located in humans. Within 1 h after injection, Bev and HA were crosslinked, which retained Bev for slow release as the hydrogel biodegraded. In vivo studies in the rabbit eye reported Bev release for >6 months, depending on gel formulation and Bev assay. The in-situ forming Bev-HA hydrogel was well tolerated, as assessed by clinical exam, fundus imaging, histological analysis, and intraocular pressure measurement. We conclude that Bev released from an in-situ forming hydrogel may enable long-acting treatments of AMD and other posterior ocular indications.

Acetyl-CoA carboxylase-1/2 blockade locks dendritic cells in the semimature state associated with FA deprivation by favoring FAO.

Immunometabolism is rising as an intriguing topic that reveals the connection between immune cell function and metabolic processes. Especially, fatty acid metabolism plays an essential role in the dendritic cells (DCs) during the differentiation and maturation period. We questioned whether regulation of acetyl-CoA carboxylases 1 and 2-(ACC1/2), the core enzymes of fatty acid synthesis (FAS), would control DC function. Here, we report that blocking ACC1/2 to prevent FAS during DC maturation switched their cellular metabolism into fatty acid oxidation to fuel oxidative phosphorylation. This action turned DCs to utilize exogenous fatty acids to sustain their basal energy demand and maintain a stable cellular respiration rate. Coincidentally, under the ACC1/2 inhibitor treatment, LPS-treated DCs exhibited a semimaturation phenotype with a maturation-resistance feature, with decreased expression of costimulatory molecules including CD86 and CD40, along with the reduction of IL-12 and IL-6. The migratory capability of DCs has been known to relate to the glycolysis pathway, and here we showed that the ACC1/2 blockade did not affect the expression of CCR7 and DC migration. Furthermore, we found that under the ACC1/2 blocking condition, DCs pulsed with OVA failed to activate OVA-specific CD4+ T cell proliferation even though their antigen uptake capacity was intact. Together, our data suggest ACC1/2 as a promising target to control DC fate.

Long-term control of diabetes in a nonhuman primate by two separate transplantations of porcine adult islets under immunosuppression.

Porcine islet transplantation is an alternative to allo-islet transplantation. Retransplantation of islets is a routine clinical practice in islet allotransplantation in immunosuppressed recipients and will most likely be required in islet xenotransplantation in immunosuppressed recipients. We examined whether a second infusion of porcine islets could restore normoglycemia and further evaluated the efficacy of a clinically available immunosuppression regimen including anti-thymocyte globulin for induction; belimumab, sirolimus, and tofacitinib for maintenance and adalimumab, anakinra, IVIg, and tocilizumab for inflammation control in a pig to nonhuman primate transplantation setting. Of note, all nonhuman primates were normoglycemic after the retransplantation of porcine islets without induction therapy. Graft survival was >100 days for all 3 recipients, and 1 of the 3 monkeys showed insulin independence for >237 days. Serious lymphodepletion was not observed, and rhesus cytomegalovirus reactivation was controlled without any serious adverse effects throughout the observation period in all recipients. These results support the clinical applicability of additional infusions of porcine islets. The maintenance immunosuppression regimen we used could protect the reinfused islets from acute rejection.

Long-term porcine islet graft survival in diabetic non-human primates treated with clinically available immunosuppressants.

BACKGROUND: Although pancreatic islet transplantation is becoming an effective therapeutic option for patients with type 1 diabetes (T1D) who suffer from a substantially impaired awareness of hypoglycemia, its application is limited due to the lack of donors. Thus, pig-to-human islet xenotransplantation has been regarded as a promising alternative due to the unlimited number of "donor organs." Long-term xenogeneic islet graft survival in pig-to-non-human primate (NHP) models has mainly been achieved by administering the anti-CD154 mAb-based immunosuppressant regimen. Since the anti-CD154 mAb treatment has been associated with unexpected fatal thromboembolic complications in clinical trials, the establishment of a new immunosuppressant regimen that is able to be directly applied in clinical trials is an urgent need. METHODS: We assessed an immunosuppressant regimen composed of clinically available agents at porcine islet transplantation in consecutive diabetic NHPs. RESULTS: Porcine islet graft survival in consecutive diabetic NHPs (n = 7; >222, >200, 181, 89, 62, 55, and 34 days) without severe adverse events. CONCLUSION: We believe that our study could contribute greatly to the initiation of islet xenotransplantation clinical trials.

The blockade of cytoplasmic HMGB1 modulates the autophagy/apoptosis checkpoint in stressed islet beta cells.

High mobility group (HMGB1) is an alarmin known to be harmful to pancreatic beta cells and associated with diabetes mellitus pathogenesis and pancreatic islet graft failure. It has been long thought that the suppression of HMGB1 molecule is beneficial to the beta cells. However, recent studies have indicated that cytoplasmic HMGB1 (cHMGB1) could function as a modulator to relieve cells from apoptotic stress by autophagy induction. Particularly, pancreatic beta cells have been known to utilize the autophagy-to-apoptosis switch when exposed to hypoxia or lipotoxicity. This study aimed to investigate the beta cells under hypoxic and lipotoxic stress while utilizing a small molecule inhibitor of HMGB1, inflachromene (ICM) which can suppress cHMGB1 accumulation. It was revealed that under cellular stress, blockade of cHMGB1 accumulation decreased the viability of islet grafts, primary islets and MIN6 cells. MIN6 cells under cHMGB1 blockade along with lipotoxic stress showed decreased autophagic flux and increased apoptosis. Moreover, cHMGB1 blockade in HFD-fed mice produced unfavorable outcomes on their glucose tolerance. In sum, these results suggested the role of cHMGB1 within beta cell autophagy/apoptosis checkpoint. Given the importance of autophagy in beta cells under apoptotic stresses, this study might provide further insights regarding HMGB1 and diabetes.

Mesenchymal stem cell-derived exosomes suppress proliferation of T cells by inducing cell cycle arrest through p27kip1/Cdk2 signaling.

Mouse mesenchymal stem cells (MSCs) have been shown to suppress T cells. Especially, MSC-cultured media have shown suppressive functions against various immune cells including the T cells. However, the underlying immunosuppressive mechanisms of the MSC-cultured medium are not yet fully understood. In this study, we confirmed the T cell-suppression capacity of MSC culture supernatant (MSC-CS) through both apoptosis and cell cycle arrest, and hypothesized that the exosomes were the major immunosuppressive agents in the MSC-CS. MSC-derived exosomes (MSC-exo) exhibited potent suppressive effects on T cell proliferation while the rest of the supernatant fraction did not. Interestingly, the exosomes derived from MSC only induced the cell cycle arrest, and it was through the upregulation of p27kip1 protein and downregulation of Cdk2 protein. In conclusion, the exosomes secreted from MSCs could suppress the activated T cell proliferation through the induction of cell cycle arrest.

The effect of preexisting HMGB1 within fetal bovine serum on murine pancreatic beta cell biology.

High-mobility group box 1 (HMGB1) can act as a structural protein of the chromatin and at the same time as a mediator of the immune system. Its high correlation with the graft acceptance in pancreatic islet recipients makes it a biomarker in islet transplantation. With the suspicion that preexisting HMGB1 in the fetal bovine serum (FBS) would be detrimental to the viability and function of murine beta cells, HMGB1 was removed from FBS and its impact was investigated. Interestingly, the elimination of HMGB1 from FBS seemed unfavorable to the viability and function of cultured murine beta cells, suggesting that the preexisting HMGB1 in the FBS may be an indispensable component of islet cell culture.

CD4+ /CD8+ T-cell ratio correlates with the graft fate in pig-to-non-human primate islet xenotransplantation.

BACKGROUND: Xenogeneic islet transplantation using porcine pancreata has been a promising option for substituting human islet transplantation. Moreover, recent advances in pre-clinical results have put islet xenotransplantation closer to the possibility of clinical application. While preparing for the era of clinical xenotransplantation, developing non-invasive immune monitoring method which could predict the graft fate could benefit the patient. However, there are few reports showing predictive immune parameters associated with the fate of the graft in islet xenotransplantation. METHODS: The absolute number and ratio of T-cell subsets have been measured via flow cytometry from the peripheral blood of 16 rhesus monkeys before and after porcine islet xenotransplantation. The correlation between the graft survival and the absolute number or ratio of T cells was retrospectively analyzed. RESULTS: The ratio of CD4+ versus CD8+ T cells was significantly reduced due to CD8+ effector memory cells' increase. Correlation analyses revealed that CD4+ /CD8+ , CD4+ /CD8+ naïve, CD4+ naïve/CD8+ naïve, and CD4+ central memory/CD8+ naïve cell ratios negatively correlated with the duration of graft survival. Conversely, further analyses discovered strong, positive correlation of CD4+ /CD8+ cell ratios within the early graft-rejected monkeys (≤60 days). CONCLUSIONS: This retrospective study demonstrated that CD4+ /CD8+ ratios correlated with graft survival, especially in recipients which rejected the graft in early post-transplantation periods. CD4+ /CD8+ ratios could be used as a surrogate marker to predict the graft fate in pig-to-NHP islet xenotransplantation.

Bioinformatic analysis of peripheral blood RNA-sequencing sensitively detects the cause of late graft loss following overt hyperglycemia in pig-to-nonhuman primate islet xenotransplantation.

Clinical islet transplantation has recently been a promising treatment option for intractable type 1 diabetes patients. Although early graft loss has been well studied and controlled, the mechanisms of late graft loss largely remains obscure. Since long-term islet graft survival had not been achieved in islet xenotransplantation, it has been impossible to explore the mechanism of late islet graft loss. Fortunately, recent advances where consistent long-term survival (≥6 months) of adult porcine islet grafts was achieved in five independent, diabetic nonhuman primates (NHPs) enabled us to investigate on the late graft loss. Regardless of the conventional immune monitoring methods applied in the post-transplant period, the initiation of late graft loss could rarely be detected before the overt graft loss observed via uncontrolled blood glucose level. Thus, we retrospectively analyzed the gene expression profiles in 2 rhesus monkey recipients using peripheral blood RNA-sequencing (RNA-seq) data to find out the potential cause(s) of late graft loss. Bioinformatic analyses showed that highly relevant immunological pathways were activated in the animal which experienced late graft failure. Further connectivity analyses revealed that the activation of T cell signaling pathways was the most prominent, suggesting that T cell-mediated graft rejection could be the cause of the late-phase islet loss. Indeed, the porcine islets in the biopsied monkey liver samples were heavily infiltrated with CD3+ T cells. Furthermore, hypothesis test using a computational experiment reinforced our conclusion. Taken together, we suggest that bioinformatics analyses with peripheral blood RNA-seq could unveil the cause of insidious late islet graft loss.

A combination regimen of low-dose bortezomib and rapamycin prolonged the graft survival in a murine allogeneic islet transplantation model.

As the first FDA-approved proteasome inhibitor drug, bortezomib has been used for the treatment of multiple myeloma and lymphoma. However, its effects alone or in combination with other immunosuppressants on allogeneic islet transplantation have not been reported so far. In this study, we showed that the short-term combination treatment of low-dose bortezomib and rapamycin significantly prolonged the survival of islet allografts. Short-term treatment of low-dose (0.05 mg/kg or 0.1 mg/kg) bortezomib reduced the MHC class II expression in dendritic cells (DCs) of alloantigen-sensitized mice, and prolonged the islet allograft survival for up to 50 days in diabetic mice. Notably, when bortezomib was combined with rapamycin, it induced islet-specific immunological tolerance which allowed the acceptance of a second graft without additional immunosuppression. This regimen dramatically reduced the alloantigen-specific IFN-γ-producing T cells in the spleen, and increased regulatory T cells both at the graft site and in the spleen. Therefore, we propose that short-term treatment of low-dose bortezomib and rapamycin could be a new tolerance-promoting immunosuppressive regimen for allogeneic islet transplantation.

JAK3 inhibitor-based immunosuppression in allogeneic islet transplantation in cynomolgus monkeys.

Islet transplantation is efficacious to prevent severe hypoglycemia and glycemic liability of selected patients of type 1 diabetes. However, since calcineurin inhibitor (CNI) causes ß-cell and nephrotoxicity, alternative drug(s) with similar potency and safety profile to CNI will be highly desirable. Here we tested whether JAK3 inhibitor, tofacitinib could be used instead of tacrolimus in CIT07 immunosuppression regimen in cynomolgus nonhuman primate (NHP) model. Five independent streptozotocin (STZ)-induced diabetic monkeys were transplanted with MHC-mismatched allogeneic islets and three animals were further re-transplanted upon insufficient glycemic control or early islet graft rejection. After islet transplantation, blood glucose levels were quickly stabilized and maximal islet graft survival as measured by serum C-peptide concentration was >330, 98, >134, 31, or 22 days, respectively, after transplantation (median survival day; 98 days). Cellular and humoral immune responses were efficiently suppressed by JAK3 inhibitor-based immunosuppression during the follow-up periods. Although intermittent increases of the genome copy number of cynomolgus cytomegalovirus (CMV) were detected by quantitative real-time PCR analyses, serious infections or posttransplant lymphoproliferative disease (PTLD) was not found in all animals. Taken together, we have shown that JAK3 inhibitor could be used in replacement of tacrolimus in a highly translatable NHP islet transplantation model and these results suggest that JAK3 inhibitor will be potentially incorporated in human allogeneic islet transplantation.

Galectin-4 Interaction with CD14 Triggers the Differentiation of Monocytes into Macrophage-like Cells via the MAPK Signaling Pathway.

Galectin-4 (Gal-4) is a ß-galactoside-binding protein mostly expressed in the gastrointestinal tract of animals. Although intensive functional studies have been done for other galectin isoforms, the immunoregulatory function of Gal-4 still remains ambiguous. Here, we demonstrated that Gal-4 could bind to CD14 on monocytes and induce their differentiation into macrophage-like cells through the MAPK signaling pathway. Gal-4 induced the phenotypic changes on monocytes by altering the expression of various surface molecules, and induced functional changes such as increased cytokine production and matrix metalloproteinase expression and reduced phagocytic capacity. Concomitant with these changes, Gal-4-treated monocytes became adherent and showed elongated morphology with higher expression of macrophage markers. Notably, we found that Gal-4 interacted with CD14 and activated the MAPK signaling cascade. Therefore, these findings suggest that Gal-4 may exert the immunoregulatory functions through the activation and differentiation of monocytes.

Peri-graft porcine-specific CD4+ FoxP3+ regulatory T cells by CD40-CD154 blockade prevented the rejection of porcine islet graft in diabetic mice.

BACKGROUND: Anti-CD154 monoclonal antibody (mAb) treatment has been known to have potential to induce immune tolerance in organ transplantation. Several studies have suggested the involvement of CD4+ regulatory T cells (Treg s) in xeno-immune tolerance. However, the characteristics of Treg s and the mechanisms of their regulatory functions in islet xenotransplantation have not been clearly defined. METHOD: Adult porcine islet cells were isolated and purified, and were transplanted under the kidney capsule of diabetic C57BL/6J mice with the administration of 0.5 mg/mouse of anti-CD154 mAb on 0, 1, 3, 5, and 7 days post-transplantation (DPT). The graft survival was monitored by blood glucose level. The islet graft and recipients' cells were analyzed by immunohistochemistry (IHC), flow cytometry, enzyme-linked immunosorbent spot (ELISPOT) assay, and mixed-lymphocyte reaction. RESULTS: Short-term anti-CD154 mAb monotherapy enabled the porcine islet graft to survive indefinitely in diabetic mice (n = 18). Immunohistochemical staining showed significantly higher ratio of CD4+ Foxp3+ Treg s in the peri-graft site, but not in the spleen and kidney-draining lymph node of the recipient mice. Depletion of Treg s evoked graft rejection, and adoptive transfer of Treg s from anti-CD154 mAb-treated recipients provided protection to the graft from rejection. These Treg s showed more potent suppressive capacity than those from the untreated control and were found to be porcine antigen-specific. CONCLUSIONS: In this study, we showed that anti-CD154 mAb monotherapy resulted in indefinite porcine islet graft survival in mice. The porcine-specific CD4+ Foxp3+ Treg s in the peri-graft site played the critical role in the protection of islet graft from rejection, which suggests a prospective immunosuppressive strategy for islet xenotransplantation.