Treg Signature Research Articles

1142-P: Update on BCG Clinical Trial Programs in Patients with Advanced Type 1 Diabetes

The bacillus Calmette-Guerin (BCG) vaccine was first introduced a century ago for tuberculosis prevention and is today being tested in clinical trials for treatment of diverse forms of autoimmunity, including type 1 diabetes (T1D). Long-term follow up of a Phase I study of the BCG vaccine in longstanding T1D revealed potential disease-modulating effects after repeated BCG vaccination, including long-term reductions of HbA1c, death of autoreactive cells, modest restoration of insulin secretion and induction of beneficial regulatory T cells (Tregs). Here we provide an update on the BCG clinical trial program current underway at Massachusetts General Hospital (MGH): enrollment numbers, study timelines and interim outcomes for key T1D biomarkers. To date, 236 patients have been enrolled in the randomized and open-label MGH trials and 143 patients have received at least two doses of BCG, 34 of whom are being followed in open-label, unblinded trials. Enrollment of 150 participants in a pediatric study is planned for 2021. Data from ongoing analysis of the open-label BCG trials shows lowered insulin needs and decreases in HbA1c as compared to reference populations. Patients are also being monitored by RNAseq, epigenetics, Treg cell numbers and Treg signature gene expression to better define the mechanism of beneficial effects. We show a gradual and stable 3-year time course of demethylation of signature Treg genes known to be associated with Treg potency, including Foxp3, which was observed to be over-methylated at baseline in T1D and, over 3 years after BCG vaccination, demethylated to a level close to controls without T1D. The clinical time course of BCG-related improvements (i.e., improvement in HbA1C) appears to correlate with the time course of Treg induction through epigenetic modifications. Disclosure W. Kuhtreiber: None. H. Takahashi: None. R. Keefe: None. K. Nelson: None. N. Ng: None. J. Braley: None. H. Zheng: None. D. L. Faustman: None.

The role of tyrosine phosphatase SHP-1 in regulatory T cell function and immune tolerance

Abstract Our immune system relies on multiple levels of controls to balance its response to danger while maintaining self-tolerance. The functional interactions between conventional T cells (Tcon) and regulatory T cells (Treg) are crucial in this process. Previous studies from our lab revealed that the tyrosine phosphatase SHP-1 plays a critical role in immune tolerance, and while its regulatory role in Tcon cells is widely studied, the role of SHP-1 in Treg is much less understood. Here, we investigated the role of SHP-1 in Treg cells by using a genetic mouse model carrying a Treg-specific deletion of SHP-1 to address two questions: (I) how SHP-1 affects the activity of Treg cells; and (II) how SHP-1-deficient Treg cells shape the overall immune response. (I) A characterization of SHP-1-deficient Treg cells demonstrated an increase in the expression of a subset of Treg signature markers compared to SHP-1-sufficient Treg cells. Upon stimulation, SHP-1 deficiency in Tregs increases IL-17 cytokine expression. Moreover, we observed that SHP-1 decreases AKT phosphorylation in Treg cells, consistent with previous studies in Tcon cells from us and others. Finally, we found that SHP-limits Treg suppressive activity in vitro. (II) Next, we asked whether SHP-1 deficiency in Treg cells affects the overall immune system and found a significant increase in CD44hi CD4+ and CD8+ T cells, indicating a secondary in vivo effect of SHP-1-deficient Treg cells on SHP-1-sufficient Tcon cells. Using murine models of autoimmune diseases, we are currently addressing the underlying mechanism of this phenotype. In summary, our studies suggest an important regulatory role of SHP-1 in Treg activity and function thereby additionally contributing to the overall immune balance.

Single-cell chromatin accessibility landscape identifies tissue repair program in human regulatory T cells

SummaryMurine regulatory T (Treg) cells in tissues promote tissue homeostasis and regeneration. We sought to identify features that characterize human Treg cells with these functions in healthy tissues. Single-cell chromatin accessibility profiles of murine and human tissue Treg cells defined a conserved, microbiota-independent tissue-repair Treg signature with a prevailing footprint of the transcription factor BATF. This signature, combined with gene expression profiling and TCR fate mapping, identified a population of tissue-like Treg cells in human peripheral blood that expressed BATF, chemokine receptor CCR8 and HLA-DR. Human BATF+CCR8+ Treg cells from normal skin and adipose tissue shared features with nonlymphoid T follicular helper-like (Tfh-like) cells, and induction of a Tfh-like differentiation program in naive human Treg cells partially recapitulated tissue Treg regenerative characteristics, including wound healing potential. Human BATF+CCR8+ Treg cells from healthy tissue share features with tumor-resident Treg cells, highlighting the importance of understanding the context-specific functions of these cells.

Cell-Mediated Immunoreactivity of Poly(2-isopropenyl-2-oxazoline) as Promising Formulation for Immunomodulation.

Poly(2-isopropenyl-2-oxazoline) (PIPOx) represents a functional polymer with high potential for drug delivery, tissue engineering, and immunomodulation. The immunomodulatory efficiency of the PIPOx formulation has been studied in vitro following splenic cells and RAW 264.7 macrophages exposition. The cell-specific immunomodulative effect on production of Th1, Th2, Th17, and Treg signature cytokines has been demonstrated. The impact on the functionality of PIPOx-sensitized RAW 264.7 macrophages was assessed by cell phagocytosis. Time- and concentration-dependent cell internalization and intracellular organelles colocalization of fluorescently labeled PIPOx has been examined. The in vitro results demonstrated the PIPOx bioavailability and the capability of triggering immune cell responses resulting in the induced production of cell-specific signature interleukins, important prerequisite properties for future potential biomedical applications.

Adipose Tissue T Regulatory Cells: Implications for Health and Disease.

Obesity dramatically increases the risk of numerous conditions, including type 2 diabetes mellitus and other components of the metabolic syndrome. Pro-inflammatory changes that occur in adipose tissue are critical to the pathogenesis of these obesity-induced complications. Adipose tissue is one of the body's largest endocrine organs, and the cells that comprise the adipose tissue immunoenvironment secrete multiple factors (including adipokines and cytokines) that impact systemic metabolism. In particular, immunosuppressive regulatory T cells (Tregs) decline in obesity, partly in response to its complex interaction with adipocytes, and this decline contributes to disruption of the typical homeostasis observed in lean adipose tissue. Although the regulation of Treg differentiation, function, and enrichment is incompletely understood, factors including various cell-surface co-stimulatory molecules, certain lipid species, and cytokines such as PPARγ, adiponectin, and leptin are important mediators. It is also clear that there may be depot-specific differences in Tregs, rendering adipose tissue Tregs distinct from lymphoid or circulating Tregs, with implications on maintenance and functionality. While most of these findings are derived from studies in murine models, comparatively little is known about the human adipose tissue Treg signature, which requires further investigation.

When aging meets tregs: It’s all about ROS stress

The E3 ligase DCAF1 is a key regulator of Treg senescence and protects Tregs from ROS stress. Guo, Z, Wang, G, Wu, B, et al. DCAF1 regulates Treg senescence via the ROS axis during immunological aging. J Clin Invest. 2020; 130: 5893- 5908. Aging affects all organs and systems, including the immune system, and a prominent feature of the aging immune system is a progressive decline in protective responses, a phenomenon called immunosenescence. At a cellular level, aging is associated with a marked expansion of effector memory T cells in the periphery, which is believed to contribute to the age-related chronic inflammatory state in the elderly (inflammaging). However, whether and how aging impacts Foxp3+ regulatory T cells (Tregs), a key cell type dedicated to tolerance and immune homeostasis, remains poorly defined. As the elderly nowadays represent the fastest growing transplant population, studies in this area will undoubtedly have significant clinical ramifications. A recent study in the Journal of Clinical Investigation by Guo et al. reported a surprising finding that the Foxp3+ Tregs are more likely to become senescent than T effector cells in aged mice: Tregs tended to more readily lose proliferative and suppressive capacities. The authors identified the E3 ligase DDB1- and CUL4-associated factor 1 (DCAF1) as a key regulator of Treg senescence and, by partnering with glutathione-S-transferase P (GSTP), plays a critical role in protecting Tregs from oxidative stress induced by reactive oxygen species (ROS). They further showed that Treg-specific deletion of DCAF1 led to accelerated aging of Tregs in young mice and that therapeutic blockade of the DCAF1/GSTP/ROS pathway rejuvenated aged Tregs and restored their suppressive activities. The authors initially took multiple approaches to compare Tregs and T effector cells in aged versus young naïve mice and found that aged Tregs proliferated poorly to T cell receptor stimulation and showed reduced suppressive activities compared with their young , counterparts. Unbiased genome-wide RNA-seq analysis revealed selective enrichment of aging-related programs as well as preferential upregulation of a senescence gene signature in aged Tregs (i.e., p16, p19, and p21). Interestingly, this senescence signature is more pronounced in Tregs than in T effector cells, regardless of age. They provide further evidence that aged, compared with young, Tregs showed normal expression of Treg signature genes (Foxp3, Il2ra, and Tnfrsf18) but a clear tendency of enhanced expression of proinflammatory cytokines. In an irradiation-induced aging model, young, but not aged, Tregs effectively prevented the phenotype of inflammaging. Clearly, these data support the notion that Tregs can readily become senescent and contribute to the inflammaging phenotype in aged mice. Guided by their RNA-seq data, the authors honed their efforts on DCAF1 and, by creating a set of genetically modified animal models, they demonstrated that deletion of Dcaf1 in Tregs produced an autoimmune phenotype in young mice, in which the Tregs resembled the phenotype of aged Tregs, i.e., they proliferate poorly, show deteriorated suppressive functions, and die prematurely. They found that in Dcaf1-deleted Tregs, there was marked ROS accumulation and Erk activation. Mechanistically, they showed that DCAF1 binds GSTP to form a complex that is required for intracellular detoxification of ROS by conjugating glutathione to hydrophobic and electrophilic substances (e.g., ROS). Thus, reduced expression of DCAF1 or its genetic deletion results in the accumulation of ROS in Tregs, which is known to activate the Erk pathway. Erk activation then leads to the expression of the senescence gene signature in Tregs and consequently Treg senescence and Treg dysfunction. This notion is further strengthened by the finding that interfering with the DCAF1/GSTP/ROS axis by genetic and pharmacological means rejuvenated aged Tregs and restored their suppressive functions both in vitro and in vivo. Finally, the authors extended their findings to human Tregs in the elderly, showing that similar mechanisms are also operative in the control of human Treg senescence. This new discovery, combined with the demonstration that the senescent Treg phenotype is reversible and can be therapeutically targeted, should both motivate and direct future inquiries and translational efforts. Xian C. Li, MD, PhD, is professor and director at the Immunobiology and Transplant Science Center and Department of Surgery at Houston Methodist Hospital in Texas.

Immuno-priming durvalumab with bevacizumab in HER2-negative advanced breast cancer: a pilot clinical trial

BackgroundPreclinical research suggests that the efficacy of immune checkpoint inhibitors in breast cancer can be enhanced by combining them with antiangiogenics, particularly in a sequential fashion. We sought to explore the efficacy and biomarkers of combining the anti-PD-L1 durvalumab plus the antiangiogenic bevacizumab after bevacizumab monotherapy for advanced HER2-negative breast cancer.MethodsPatients had advanced HER2-negative disease that progressed while receiving single-agent bevacizumab maintenance as a part of a previous chemotherapy plus bevacizumab regimen. Treatment consisted of bi-weekly durvalumab plus bevacizumab (10 mg/kg each i.v.). Peripheral-blood mononuclear cells (PBMCs) were obtained before the first durvalumab dose and every 4 weeks and immunophenotyped by flow-cytometry. A fresh pre-durvalumab tumor biopsy was obtained; gene-expression studies and immunohistochemical staining to assess vascular normalization and characterize the immune infiltrate were conducted. Patients were classified as “non-progressors” if they had clinical benefit (SD/PR/CR) at 4 months. The co-primary endpoints were the changes in the percentage T cell subpopulations in PBMCs in progressors versus non-progressors, and PFS/OS time.ResultsTwenty-six patients were accrued. Median PFS and OS were 3.5 and 11 months; a trend for a longer OS was detected for the hormone-positive subset (19.8 versus 7.4 months in triple-negatives; P = 0.11). Clinical benefit rate at 2 and 4 months was 60% and 44%, respectively, without significant differences between hormone-positive and triple-negative (P = 0.73). Non-progressors’ tumors displayed vascular normalization features as a result of previous bevacizumab, compared with generally abnormal patterns observed in progressors. Non-progressors also showed increased T-effector and T-memory signatures and decreased TREG signatures in gene expression studies in baseline—post-bevacizumab—tumors compared with progressors. Notably, analysis of PBMC populations before durvalumab treatment was concordant with the findings in tumor samples and showed a decreased percentage of circulating TREGs in non-progressors.ConclusionsThis study reporting on sequential bevacizumab+durvalumab in breast cancer showed encouraging activity in a heavily pre-treated cohort. The correlative studies agree with the preclinical rationale supporting an immunopriming effect exerted by antiangiogenic treatment, probably by reducing TREGs cells both systemically and in tumor tissue. The magnitude of this benefit should be addressed in a randomized setting.Trial registration(www.clinicaltrials.gov): NCT02802098. Registered on June 16, 2020.

Pre-Existing T Cell Subsets Determine Anti-PD1 Blockade Response in Richter's Transformation

Pre-Existing T Cell Subsets Determine Anti-PD1 Blockade Response in Richter's Transformation

Deep Phenotyping Reveals Distinct Immune Signatures Correlating with Prognostication, Treatment Responses, and MRD Status in Multiple Myeloma.

Simple SummaryIn Multiple Myeloma (MM) malignant cells accumulate in the bone marrow (BM), where they interact with various cell populations. These complex interactions impose mechanisms of tumor growth and proliferation, immune surveillance and immune evasion. The aim of the present study was a detailed immune characterization of MM during the course of the disease, in order to highlight signatures which are clinically relevant. Analyses of both BM and peripheral blood (PB) in matched patients’ samples, we showed that PB cannot representatively reflect the BM microenvironment. Particular immune signatures in BM and PB significantly correlated with established prognostic features and could independently associate with distinct responses to the same induction therapy. Moreover, our data provide evidence of a diverse immune profile according to patients’ MRD status post treatment. Finally, we provide insights that unique PB immune profiles may be used for the prediction of MRD status through a simple non-invasive approach.Despite recent advances, Multiple Myeloma (MM) remains an incurable disease with apparent heterogeneity that may explain patients’ variable clinical outcomes. While the phenotypic, (epi)genetic, and molecular characteristics of myeloma cells have been thoroughly examined, there is limited information regarding the role of the bone marrow (BM) microenvironment in the natural history of the disease. In the present study, we performed deep phenotyping of 32 distinct immune cell subsets in a cohort of 94 MM patients to reveal unique immune profiles in both BM and peripheral blood (PB) that characterize distinct prognostic groups, responses to induction treatment, and minimal residual disease (MRD) status. Our data show that PB cells do not reflect the BM microenvironment and that the two sites should be studied independently. Adverse ISS stage and high-risk cytogenetics were correlated with distinct immune profiles; most importantly, BM signatures comprised decreased tumor-associated macrophages (TAMs) and erythroblasts, whereas the unique Treg signatures in PB could discriminate those patients achieving complete remission after VRd induction therapy. Moreover, MRD negative status was correlated with a more experienced CD4- and CD8-mediated immunity phenotype in both BM and PB, thus highlighting a critical role of by-stander cells linked to MRD biology.

Prognostic significance and immune correlates of CD73 expression in renal cell carcinoma

BackgroundCD73–adenosine signaling in the tumor microenvironment is immunosuppressive and may be associated with aggressive renal cell carcinoma (RCC). We investigated the prognostic significance of CD73 protein expression in RCC leveraging...

Human Tregs at the materno-fetal interface show site-specific adaptation reminiscent of tumor Tregs.

Tregs are crucial for maintaining maternal immunotolerance against the semiallogeneic fetus. We investigated the elusive transcriptional profile and functional adaptation of human uterine Tregs (uTregs) during pregnancy. Uterine biopsies, from placental bed (materno-fetal interface) and incision site (control) and blood were obtained from women with uncomplicated pregnancies undergoing cesarean section. Tregs and CD4+ non-Tregs were isolated for transcriptomic profiling by Cel-Seq2. Results were validated on protein and single cell levels by flow cytometry. Placental bed uTregs showed elevated expression of Treg signature markers, including FOXP3, CTLA-4, and TIGIT. Their transcriptional profile was indicative of late-stage effector Treg differentiation and chronic activation, with increased expression of immune checkpoints GITR, TNFR2, OX-40, and 4-1BB; genes associated with suppressive capacity (HAVCR2, IL10, LAYN, and PDCD1); and transcription factors MAF, PRDM1, BATF, and VDR. uTregs mirrored non-Treg Th1 polarization and tissue residency. The particular transcriptional signature of placental bed uTregs overlapped strongly with that of tumor-infiltrating Tregs and was remarkably pronounced at the placental bed compared with uterine control site. In conclusion, human uTregs acquire a differentiated effector Treg profile similar to tumor-infiltrating Tregs, specifically at the materno-fetal interface. This introduces the concept of site-specific transcriptional adaptation of Tregs within 1 organ.

New Treg cell-based therapies of autoimmune diseases: towards antigen-specific immune suppression.

Naturally occurring FoxP3+CD4+ regulatory T (Treg) cells indispensable for the maintenance of immunological self-tolerance and homeostasis are instrumental in treating autoimmune and other immunological disorders. Stable function of natural Treg cells requires not only the expression of Foxp3 and other Treg signature genes such as CD25 and CTLA-4 but also the generation of Treg-specific epigenetic changes, especially Treg-specific DNA hypomethylation, at these gene loci. Recent studies have shown that the Treg-specific transcriptional and epigenetic changes can be induced in antigen-specific conventional T cells in vivo and in vitro, converting them to functionally stable Treg cells. Such natural or induced Treg cells bear the potential to achieve stable antigen-specific immune suppression and reestablish immunological self-tolerance in treating and preventing autoimmune diseases.

Abstract CT044: Genomic correlates of clinical benefits from atezolizumab combined with bevacizumab vs. atezolizumab alone in patients with advanced hepatocellular carcinoma (HCC)

Abstract Background: Atezolizumab (atezo) and bevacizumab (bev) combination therapy has demonstrated robust clinical activity in patients with unresectable HCC who have not received prior systemic therapy (Lee et al., APPLE 2019; Cheng et al., ESMO Asia 2019). In this exploratory analysis, we aimed to identify tumor-based molecular biomarkers that may be associated with clinical response or resistance to atezo + bev. We also investigated how VEGF blockade with bev could potentiate PD-L1 checkpoint inhibition with atezo in pts with advanced HCC. Methods: Archival tumor tissues or fresh biopsies taken prior to treatment were collected from HCC pts enrolled in the Phase 1b trial GO30140 (NCT02715531). Arm A (n = 104) was a single-arm evaluation of atezo + bev; Arm F (n = 119) was a randomized arm comparing atezo + bev with atezo. Whole-exome sequencing was carried out on these tumor tissues to determine tumor mutation burden (TMB). Gene expression in tumors was profiled by RNAseq analysis. The association between biomarker expression and clinical response (responders [R] vs. non-responders [NR]) or PFS was assessed by t-tests or Cox regression models, respectively. All p-values are descriptive. Results: In Arm A, 90/104 pts were biomarker evaluable. TMB was not associated with response to atezo + bev or PFS. In contrast, analysis of baseline tumor gene expression showed that pre-existing immunity appeared to be associated with clinical response and longer PFS, which included high expression of CD274 (PD-L1) (R vs. NR, p < 2.1 × 10−5; PFS: HR = 0.42 [0.25-0.72]), and T effector signature (GZMB, PRF1, CXCL9) (R vs. NR, p < 0.0004; PFS: HR = 0.46 [0.27-0.78]). Gene expression related to Notch pathway activation (i.e. high expression of HES1) appeared to be associated with lack of response to atezo + bev (p < 0.039) and shorter PFS (HR = 2.1 [1.3-3.6]). In Arm F, 91/119 pts were biomarker evaluable. High expression of VEGF receptor 2 (VEGFR2; HR = 0.36 [0.16-0.81]), Treg (HR = 0.35 [0.15-0.82]), myeloid inflammation (HR = 0.43 [0.19-0.95]), and TREM1/MDSC signatures (HR = 0.43 [0.19-0.94]) was associated with longer PFS in patients treated with atezo + bev than in those treated with atezo alone. Analysis of 12 serial biopsy pairs confirmed reduced levels of VEGFR2 and Treg signatures after atezo + bev treatment. Conclusion: We identified candidate biomarkers for predicting response to atezo + bev in HCC. Furthermore, the findings in Arm F are consistent with previous preclinical studies supporting a multi-faceted role of VEGF/VEGFR signaling in promoting immune suppression in addition to angiogenesis. Overall, the data presented here further support the mechanistic hypotheses on how anti-VEGF may combine with immune checkpoint blockade to increase its clinical benefit. As these results are exploratory, future study is needed to confirm these findings in a larger population. Citation Format: Andrew X. Zhu, Yinghui Guan, Alexander R. Abbas, Hartmut Koeppen, Shan Lu, Chih-Hung Hsu, Kyung-Hun Lee, Michael S. Lee, Aiwu Ruth He, Amit Mahipal, Beiying Ding, Jessica Spahn, Wendy Verret, Baek-Yeol Ryoo, Yulei Wang. Genomic correlates of clinical benefits from atezolizumab combined with bevacizumab vs. atezolizumab alone in patients with advanced hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT044.

Epigenetic conversion of conventional T cells into regulatory T cells by CD28 signal deprivation

Foxp3-expressing regulatory T cells (Tregs) can be generated in vitro by antigenic stimulation of conventional T cells (Tconvs) in the presence of TGF-β and IL-2. However, unlike Foxp3+ naturally occurring Tregs, such in vitro induced Tregs (iTregs) are functionally unstable mainly because of incomplete Treg-type epigenetic changes at Treg signature genes such as Foxp3 Here we show that deprivation of CD28 costimulatory signal at an early stage of iTreg generation is able to establish Treg-specific DNA hypomethylation at Treg signature genes. It was achieved, for example, by TCR/TGF-β/IL-2 stimulation of CD28-deficient Tconvs or CD28-intact Tconvs without anti-CD28 agonistic mAb or with CD80/CD86-blocked or -deficient antigen-presenting cells. The signal abrogation could induce Treg-type hypomethylation in memory/effector as well as naive Tconvs, while hindering Tconv differentiation into effector T cells. Among various cytokines and signal activators/inhibitors, TNF-α and PKC agonists inhibited the hypomethylation. Furthermore, CD28 signal deprivation significantly reduced c-Rel expression in iTregs; and the specific genomic perturbation of a NF-κB binding motif at the Foxp3 CNS2 locus enhanced the locus-specific DNA hypomethylation even in CD28 signaling-intact iTregs. In addition, in vitro maintenance of such epigenome-installed iTregs with IL-2 alone, without additional TGF-β or antigenic stimulation, enabled their expansion and stabilization of Treg-specific DNA hypomethylation. These iTregs indeed stably expressed Foxp3 after in vivo transfer and effectively suppressed antigen-specific immune responses. Taken together, inhibition of the CD28-PKC-NF-κB signaling pathway in iTreg generation enables de novo acquisition of Treg-specific DNA hypomethylation at Treg signature genes and abundant production of functionally stable antigen-specific iTregs for therapeutic purposes.

A paradoxical role for classical dendritic cells and Flt3 ligand in tumor immune response

Abstract Flt3 ligand (FL) induces differentiation and proliferation of classical dendritic cells (cDCs) and is a promising molecule in therapies of cancer and vaccination, but has mixed effects in clinical trials. The role of cDCs in antitumor response is ambiguous as they may induce priming of anti-tumor lymphocytes, but also may favor recruitment of tumor-protecting regulatory T cells (Tregs). To better understand the role of FL/DCs axis in cancer, we monitored tumor growth in a mouse model of melanoma in the context of low, normal or high levels of FL. Paradoxically, both genetic FL depletion and overexpression stalled tumor growth and increased animal survival as compared to control. We show that FL-deficient mice tumor growth is predominantly controlled by the adaptive immune system unleashed by a diminution of Tregs, while high FL levels favor the innate anti-tumor response through the recruitment of NK cells. Analysis of human gene expression data confirmed the paradoxical effect of FL as well as cDCs on survival in several types of cancer. Tumor samples from patients in these groups expressed Treg and NK cells signatures in correlation with the levels of cDCs, FL, IL15 and IL15RA expression. As a proof-of-principle, we showed that the combination of FL overexpression and Tregs depletion resulted in regression of established tumors in mice. We propose that FL is a master regulator of immune control of tumor growth owing to its key role in DCs function.

Ionizing radiation modulates the phenotype and function of human CD4+ induced regulatory T cells

BackgroundThe use of immunotherapy strategies for the treatment of advanced cancer is rapidly increasing. Most immunotherapies rely on induction of CD8+ tumor-specific cytotoxic T cells that are capable of directly killing cancer cells. Tumors, however, utilize a variety of mechanisms that can suppress anti-tumor immunity. CD4+ regulatory T cells can directly inhibit cytotoxic T cell activity and these cells can be recruited, or induced, by cancer cells allowing escape from immune attack. The use of ionizing radiation as a treatment for cancer has been shown to enhance anti-tumor immunity by several mechanisms including immunogenic tumor cell death and phenotypic modulation of tumor cells. Less is known about the impact of radiation directly on suppressive regulatory T cells. In this study we investigate the direct effect of radiation on human TREG viability, phenotype, and suppressive activity.ResultsBoth natural and TGF-β1-induced CD4+ TREG cells exhibited increased resistance to radiation (10 Gy) as compared to CD4+ conventional T cells. Treatment, however, decreased Foxp3 expression in natural and induced TREG cells and the reduction was more robust in induced TREGS. Radiation also modulated the expression of signature iTREG molecules, inducing increased expression of LAG-3 and decreased expression of CD25 and CTLA-4. Despite the disconcordant modulation of suppressive molecules, irradiated iTREGS exhibited a reduced capacity to suppress the proliferation of CD8+ T cells.ConclusionsOur findings demonstrate that while human TREG cells are more resistant to radiation-induced death, treatment causes downregulation of Foxp3 expression, as well as modulation in the expression of TREG signature molecules associated with suppressive activity. Functionally, irradiated TGF-β1-induced TREGS were less effective at inhibiting CD8+ T cell proliferation. These data suggest that doses of radiotherapy in the hypofractionated range could be utilized to effectively target and reduce TREG activity, particularly when used in combination with cancer immunotherapies.

Coexpression of FOXP3 and a Helios isoform enhances the effectiveness of human engineered regulatory T cells

Regulatory T cells (Tregs) are a subset of immune cells that suppress the immune response. Treg therapy for inflammatory diseases is being tested in the clinic, with moderate success. However, it is difficult to isolate and expand Tregs to sufficient numbers. Engineered Tregs (eTregs) can be generated in larger quantities by genetically manipulating conventional T cells to express FOXP3. These eTregs can suppress in vitro and in vivo but not as effectively as endogenous Tregs. We hypothesized that ectopic expression of the transcription factor Helios along with FOXP3 is required for optimal eTreg immunosuppression. To test this theory, we generated eTregs by retrovirally transducing total human T cells (CD4+ and CD8+) with FOXP3 alone or with each of the 2 predominant isoforms of Helios. Expression of both FOXP3 and the full-length isoform of Helios was required for eTreg-mediated disease delay in a xenogeneic graft-versus-host disease model. In vitro, this corresponded with superior suppressive function of FOXP3 and full-length Helios-expressing CD4+ and CD8+ eTregs. RNA sequencing showed that the addition of full-length Helios changed gene expression in cellular pathways and the Treg signature compared with FOXP3 alone or the other major Helios isoform. Together, these results show that functional human CD4+ and CD8+ eTregs can be generated from total human T cells by coexpressing FOXP3 and full-length Helios.

Dysregulation of Glutamate Transport Enhances Treg Function That Promotes VEGF Blockade Resistance in Glioblastoma.

Anti-VEGF therapy prolongs recurrence-free survival in patients with glioblastoma but does not improve overall survival. To address this discrepancy, we investigated immunologic resistance mechanisms to anti-VEGF therapy in glioma models. A screening of immune-associated alterations in tumors after anti-VEGF treatment revealed a dose-dependent upregulation of regulatory T-cell (Treg) signature genes. Enhanced numbers of Tregs were observed in spleens of tumor-bearing mice and later in tumors after anti-VEGF treatment. Elimination of Tregs with CD25 blockade before anti-VEGF treatment restored IFNγ production from CD8+ T cells and improved antitumor response from anti-VEGF therapy. The treated tumors overexpressed the glutamate/cystine antiporter SLC7A11/xCT that led to elevated extracellular glutamate in these tumors. Glutamate promoted Treg proliferation, activation, suppressive function, and metabotropic glutamate receptor 1 (mGlutR1) expression. We propose that VEGF blockade coupled with glioma-derived glutamate induces systemic and intratumoral immunosuppression by promoting Treg overrepresentation and function, which can be pre-emptively overcome through Treg depletion for enhanced antitumor effects. SIGNIFICANCE: Resistance to VEGF therapy in glioblastoma is driven by upregulation of Tregs, combined blockade of VEGF, and Tregs may provide an additive antitumor effect for treating glioblastoma.

Rapamycin and abundant TCR stimulation are required for the generation of stable human induced regulatory T cells.

ObjectivesRegulatory T cells (Tregs) are a vital sub‐population of CD4+ T cells with major roles in immune tolerance and homeostasis. Given such properties, the use of regulatory T cells for immunotherapies has been extensively investigated, with a focus on adoptive transfer of ex vivo expanded natural Tregs (nTregs). For immunotherapies, induced Tregs (iTregs), generated in vitro from naïve CD4+ T cells, provide an attractive alternative, given the ease of generating cell numbers required for clinical dosage. While the combination of TGF‐β, ATRA and rapamycin has been shown to generate highly suppressive iTregs, the challenge for therapeutic iTreg generation has been their instability. Here, we investigate the impact of rapamycin concentrations and α‐CD3/CD28 bead ratios on human iTreg stability.MethodsWe assess iTregs generated with various concentrations of rapamycin and differing ratios of α‐CD3/CD28 beads for their differentiation, stability, expression of Treg signature molecules and T helper effector cytokines, and Treg‐specific demethylation region (TSDR) status.ResultsiTregs generated in the presence of TGF‐β, ATRA, rapamycin and a higher ratio of α‐CD3/CD28 beads were highly suppressive and stable upon in vitro re‐stimulation. These iTregs exhibited a similar expression profile of Treg signature molecules and T helper effector cytokines to nTregs, in the absence of TSDR demethylation.ConclusionThis work establishes a method to generate human iTregs which maintain stable phenotype and function upon in vitro re‐stimulation. Further validation in pre‐clinical models will be needed to ensure its suitability for applications in adoptive transfer.

OX40L-JAG1–Induced Expansion of Lineage-Stable Regulatory T Cells Involves Noncanonical NF-κB Signaling

Foxp3+T regulatory cells (Tregs) control autoimmune response by suppressing proliferation and effector functions of self-reactive Foxp3-CD4+/CD8+ T cells and thereby maintain the critical balance between self-tolerance and autoimmunity. Earlier, we had shown that OX40L-JAG1 cosignaling mediated through their cognate receptors OX40 and Notch3 preferentially expressed on murine Tregs can selectively induce their proliferation in the absence of TCR stimulation. However, the differential molecular mechanisms regulating TCR-independent versus TCR-dependent Treg proliferation and lineage stability of the expanded Tregs remained unknown. In this study, we show that OX40L-JAG1 treatment induced TCR-independent proliferation of Tregs in the thymus and periphery. The use of Src kinase inhibitor permitted us to demonstrate selective inhibition of TCR-dependent T cell proliferation with little to no effect on OX40L-JAG1-induced TCR-independent Treg expansion in vitro, which was critically dependent on noncanonical NF-κB signaling. OX40L-JAG1-expanded Tregs showed sustained lineage stability as indicated by stable demethylation marks in Treg signature genes such as Foxp3, Il2ra, Ctla4, Ikzf2, and Ikzf4. Furthermore, OX40L-JAG1 treatment significantly increased CTLA4+ and TIGIT+ Tregs and alleviated experimental autoimmune thyroiditis in mice. Relevance of our findings to humans became apparent when human OX40L and JAG1 induced TCR-independent selective expansion of human Tregs in thymocyte cultures and increased human Tregs in the liver tissue of humanized NSG mice. Our findings suggest that OX40L-JAG1-induced TCR-independent Treg proliferation is a conserved mechanism that can be used to expand lineage-stable Tregs to treat autoimmune diseases.