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Regulatory T (Treg) cells are classically known for their critical immunosuppressive functions that support peripheral tolerance. More recent work has demonstrated that Treg cells produce pro-repair mediators independent of their immunosuppressive function, a process that is critical to repair and regeneration in response to numerous tissue insults. These factors act on resident parenchymal and structural cells to initiate repair in a tissue-specific context. This review examines interactions between Treg cells and tissue-resident non-immune cells-in the context of tissue repair, fibrosis, and cancer-and discusses areas for future exploration., (© 2024 Loffredo et al.)

Production of amphiregulin (Areg) by regulatory T (Treg) cells promotes repair after acute tissue injury. Here, we examined the function of Treg cells in non-alcoholic steatohepatitis (NASH), a setting of chronic liver injury. Areg-producing Treg cells were enriched in the livers of mice and humans with NASH. Deletion of Areg in Treg cells, but not in myeloid cells, reduced NASH-induced liver fibrosis. Chronic liver damage induced transcriptional changes associated with Treg cell activation. Mechanistically, Treg cell-derived Areg activated pro-fibrotic transcriptional programs in hepatic stellate cells via epidermal growth factor receptor (EGFR) signaling. Deletion of Areg in Treg cells protected mice from NASH-dependent glucose intolerance, which also was dependent on EGFR signaling on hepatic stellate cells. Areg from Treg cells promoted hepatocyte gluconeogenesis through hepatocyte detection of hepatic stellate cell-derived interleukin-6. Our findings reveal a maladaptive role for Treg cell-mediated tissue repair functions in chronic liver disease and link liver damage to NASH-dependent glucose intolerance., Competing Interests: Declaration of interests T.M.S. and N.A. have filed a provisional patent related to this work (U.S. Provisional Patent Application No. 63/440,641)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tumors use multiple mechanisms to actively exclude immune cells involved in antitumor immunity. Strategies to overcome these exclusion signals remain limited due to an inability to target therapeutics specifically to the tumor. Synthetic biology enables engineering of cells and microbes for tumor-localized delivery of therapeutic candidates previously unavailable using conventional systemic administration techniques. Here, we engineer bacteria to intratumorally release chemokines to attract adaptive immune cells into the tumor environment. Bacteria expressing an activating mutant of the human chemokine CXCL16 (hCXCL16 K42A ) offer therapeutic benefit in multiple mouse tumor models, an effect mediated via recruitment of CD8 + T cells. Furthermore, we target the presentation of tumor-derived antigens by dendritic cells, using a second engineered bacterial strain expressing CCL20. This led to type 1 conventional dendritic cell recruitment and synergized with hCXCL16 K42A -induced T cell recruitment to provide additional therapeutic benefit. In summary, we engineer bacteria to recruit and activate innate and adaptive antitumor immune responses, offering a new cancer immunotherapy strategy.

Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality worldwide, develops almost exclusively in patients with chronic liver disease and advanced fibrosis 1,2 . Here we interrogated functions of hepatic stellate cells (HSCs), the main source of liver fibroblasts 3 , during hepatocarcinogenesis. Genetic depletion, activation or inhibition of HSCs in mouse models of HCC revealed their overall tumour-promoting role. HSCs were enriched in the preneoplastic environment, where they closely interacted with hepatocytes and modulated hepatocarcinogenesis by regulating hepatocyte proliferation and death. Analyses of mouse and human HSC subpopulations by single-cell RNA sequencing together with genetic ablation of subpopulation-enriched mediators revealed dual functions of HSCs in hepatocarcinogenesis. Hepatocyte growth factor, enriched in quiescent and cytokine-producing HSCs, protected against hepatocyte death and HCC development. By contrast, type I collagen, enriched in activated myofibroblastic HSCs, promoted proliferation and tumour development through increased stiffness and TAZ activation in pretumoural hepatocytes and through activation of discoidin domain receptor 1 in established tumours. An increased HSC imbalance between cytokine-producing HSCs and myofibroblastic HSCs during liver disease progression was associated with increased HCC risk in patients. In summary, the dynamic shift in HSC subpopulations and their mediators during chronic liver disease is associated with a switch from HCC protection to HCC promotion., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Cancer-associated fibroblasts (CAF) may exert tumor-promoting and tumor-suppressive functions, but the mechanisms underlying these opposing effects remain elusive. Here, we sought to understand these potentially opposing functions by interrogating functional relationships among CAF subtypes, their mediators, desmoplasia, and tumor growth in a wide range of tumor types metastasizing to the liver, the most common organ site for metastasis. Depletion of hepatic stellate cells (HSC), which represented the main source of CAF in mice and patients in our study, or depletion of all CAF decreased tumor growth and mortality in desmoplastic colorectal and pancreatic metastasis but not in nondesmoplastic metastatic tumors. Single-cell RNA-Seq in conjunction with CellPhoneDB ligand-receptor analysis, as well as studies in immune cell-depleted and HSC-selective knockout mice, uncovered direct CAF-tumor interactions as a tumor-promoting mechanism, mediated by myofibroblastic CAF-secreted (myCAF-secreted) hyaluronan and inflammatory CAF-secreted (iCAF-secreted) HGF. These effects were opposed by myCAF-expressed type I collagen, which suppressed tumor growth by mechanically restraining tumor spread, overriding its own stiffness-induced mechanosignals. In summary, mechanical restriction by type I collagen opposes the overall tumor-promoting effects of CAF, thus providing a mechanistic explanation for their dual functions in cancer. Therapeutic targeting of tumor-promoting CAF mediators while preserving type I collagen may convert CAF from tumor promoting to tumor restricting.

In humans receiving intestinal transplantation (ITx), long-term multilineage blood chimerism often develops. Donor T cell macrochimerism (≥4%) frequently occurs without graft-versus-host disease (GVHD) and is associated with reduced rejection. Here we demonstrate that patients with macrochimerism had high graft-versus-host (GvH) to host-versus-graft (HvG) T cell clonal ratios in their allografts. These GvH clones entered the circulation, where their peak levels were associated with declines in HvG clones early after transplant, suggesting that GvH reactions may contribute to chimerism and control HvG responses without causing GVHD. Consistently, donor-derived T cells, including GvH clones, and CD34+ hematopoietic stem and progenitor cells (HSPCs) were simultaneously detected in the recipients' BM more than 100 days after transplant. Individual GvH clones appeared in ileal mucosa or PBMCs before detection in recipient BM, consistent with an intestinal mucosal origin, where donor GvH-reactive T cells expanded early upon entry of recipient APCs into the graft. These results, combined with cytotoxic single-cell transcriptional profiles of donor T cells in recipient BM, suggest that tissue-resident GvH-reactive donor T cells migrated into the recipient circulation and BM, where they destroyed recipient hematopoietic cells through cytolytic effector functions and promoted engraftment of graft-derived HSPCs that maintain chimerism. These mechanisms suggest an approach to achieving intestinal allograft tolerance.

We recently developed a high throughput T cell receptor β chain (TCRβ) sequencing-based approach to identifying and tracking donor-reactive T cells. To address the role of clonal deletion in liver allograft tolerance, we applied this method in samples from a recent randomized study, ITN030ST, in which immunosuppression withdrawal was attempted within 2 years of liver transplantation. We identified donor-reactive T cell clones via TCRβ sequencing following a pre-transplant mixed lymphocyte reaction and tracked these clones in the circulation following transplantation in 3 tolerant and 5 non-tolerant subjects. All subjects showed a downward trend and significant reductions in donor-reactive TCRβ sequences were detected post-transplant in 6 of 8 subjects, including 2 tolerant and 4 non-tolerant recipients. Reductions in donor-reactive TCRβ sequences were greater than those of all other TCRβ sequences, including 3rd party-reactive sequences, in all 8 subjects, demonstrating an impact of the liver allograft after accounting for repertoire turnover. Although limited by patient number and heterogeneity, our results suggest that partial deletion of donor-reactive T cell clones may be a consequence of liver transplantation and does not correlate with success or failure of early immunosuppression withdrawal. These observations underscore the organ- and/or protocol-specific nature of tolerance mechanisms in humans., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Human intestinal transplantation often results in long-term mixed chimerism of donor and recipient blood in transplant patients. We followed the phenotypes of chimeric peripheral blood cells in 21 patients receiving intestinal allografts over 5 years. Donor lymphocyte phenotypes suggested a contribution of hematopoietic stem and progenitor cells (HSPCs) from the graft. Surprisingly, we detected donor-derived HSPCs in intestinal mucosa, Peyer's patches, mesenteric lymph nodes, and liver. Human gut HSPCs are phenotypically similar to bone marrow HSPCs and have multilineage differentiation potential in vitro and in vivo. Analysis of circulating post-transplant donor T cells suggests that they undergo selection in recipient lymphoid organs to acquire immune tolerance. Our longitudinal study of human HSPCs carried in intestinal allografts demonstrates their turnover kinetics and gradual replacement of donor-derived HSPCs from a circulating pool. Thus, we have demonstrated the existence of functioning HSPCs in human intestines with implications for promoting tolerance in transplant recipients., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Allograft tolerance, in which a graft is accepted without long-term immunosuppression, could overcome numerous obstacles in transplantation. Human allograft tolerance has been intentionally induced across HLA barriers via combined kidney and bone marrow transplantation (CKBMT) with a regimen that induces only transient chimerism. Tregs are enriched early after CKBMT. While deletional tolerance contributes to long-term tolerance, the role of Tregs remains unclear. We have optimized a method for identifying the donor-specific Treg repertoire and used it to interrogate the fate of donor-specific Tregs after CKBMT. We expanded Tregs with several different protocols. Using functional analyses and T cell receptor sequencing, we found that expanding sorted Tregs with activated donor B cells identified the broadest Treg repertoire with the greatest potency and donor specificity of suppression. This method outperformed both alloantigen stimulation with CTLA4Ig and sequencing of CFSElo cells from the primary mixed lymphocyte reaction. In 3 tolerant and 1 nontolerant CKBMT recipients, we sequenced donor-specific Tregs before transplant and tracked them after transplant. Preexisting donor-specific Tregs were expanded at 6 months after CKBMT in tolerant patients and were reduced in the nontolerant patient. These results suggest that early expansion of donor-specific Tregs is involved in tolerance induction following CKBMT.

We examined tolerance mechanisms in patients receiving HLA-mismatched combined kidney-bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high-throughput sequencing of T cell receptor-β hypervariable regions of DNA from peripheral blood regulatory T cells (Tregs) and CD4 non-Tregs revealed marked early enrichment of Tregs (CD3 + CD4 + CD25 high CD127 low Foxp3 + ) in blood that resulted from peripheral proliferation (Ki67 + ), possibly new thymic emigration (CD31 + ), and, in one tolerant subject, conversion from non-Tregs. Among recovering conventional T cells, central memory CD4 + and CD8 + cells predominated. A large proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptor sequencing were detected in the peripheral blood and were not donor-reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia-driven peripheral proliferation in the early posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Blood chimerism has been reported sporadically among visceral transplant recipients, mostly in association with graft-vs-host disease (GVHD). We hypothesized that a higher degree of mixed chimerism would be observed in multivisceral (MVTx) than in isolated intestinal (iITx) and isolated liver transplant (iLTx) recipients, regardless of GVHD. We performed a longitudinal prospective study investigating multilineage blood chimerism with flow cytometry in 5 iITx and 4 MVTx recipients up to one year posttransplant. Although only one iITx patient experienced GVHD, T cell mixed chimerism was detected in 8 out of 9 iITx/MVTx recipients. Chimerism was significantly lower in the four subjects who displayed early moderate to severe rejection. Pre-formed high-titer donor-specific antibodies, bound in vivo to the circulating donor cells, were associated with an accelerated decline in chimerism. Blood chimerism was also studied in 10 iLTx controls. Among nonsensitized patients, MVTx recipients exhibited greater T and B cell chimerism than either iITx or iLTx recipients. Myeloid lineage chimerism was present exclusively among iLTx and MVTx (6/13) recipients, suggesting that its presence required the hepatic allograft. Our study demonstrates, for the first time, frequent T cell chimerism without GVHD following visceral transplantation and a possible relationship with reduced rejection rate in MVTx recipients., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)