Number Of Tregs Research Articles

Costimulatory Molecule CD226 Regulates Atopic Dermatitis in a Mouse Model

This study investigated the role of CD226 in a 2,4-dinitrochlorobenzene (DNCB)-induced mouse model of atopic dermatitis. The results showed that the lack of CD226 (global and CD4+ T-cell specific) significantly increased ear thickness, reddening, swelling, and scaling of the skin as well as inflammatory cell and mast cell infiltration. RT-qPCR results demonstrated that the mRNA expressions of atopic dermatitis-related inflammatory cytokines and chemokines were markedly increased in the draining lymph nodes and lesioned ear skin tissues of global and CD4+ T-cell-specific CD226-deficient mice compared with that in control mice. Invitro assessment revealed that CD226 directly modulates TGFβ-mediated regulatory T (Treg) cell differentiation and proliferation. Notably, Treg cell-specific deletion of CD226 (Cd226fl/flFoxp3cre mice) resulted in more severe dermatitis and epidermal thickening than those observed in littermate mice upon DNCB treatment. Subsequent analysis showed that the infiltration of Treg cells in ear lesions and the number of Tregs in the spleen were significantly reduced in Cd226fl/flFoxp3cre mice after DNCB treatment. In addition, the lack of CD226 induced apoptosis of Treg cells through the activation of caspase 3. Therefore, these results suggest that CD226 has potential efficacy in atopic dermatitis, correlating with Treg cell inhibition.

Phosphorylation of hnRNP A1-Serine 199 Is Not Required for T Cell Differentiation and Function.

hnRNP A1 is an important RNA-binding protein that influences many stages of RNA processing, including transcription, alternative splicing, mRNA nuclear export, and RNA stability. However, the role of hnRNP A1 in immune cells, specifically CD4+ T cells, remains unclear. We previously showed that Akt phosphorylation of hnRNP A1 was dependent on TCR signal strength and was associated with Treg differentiation. To explore the impact of hnRNP A1 phosphorylation by Akt on CD4+ T cell differentiation, our laboratory generated a mutant mouse model, hnRNP A1-S199A (A1-MUT) in which the major Akt phosphorylation site on hnRNP A1 was mutated to alanine using CRISPR Cas9 technology. Immune profiling of A1-MUT mice revealed changes in the numbers of Tregs in the mesenteric lymph node. We found no significant differences in naive CD4+ T cell differentiation into Th1, Th2, Th17, or T regulatory cells (Tregs) invitro. In vivo, Treg differentiation assays using OTII-A1-Mut CD4+ T cells exposed to OVA food revealed migration and homing defects in the A1-MUT but no change in Treg induction. A1-MUT mice were immunized with NP- keyhole limpet hemocyanin, and normal germinal center development, normal numbers of NP-specific B cells, and no change in Tfh numbers were observed. In conclusion, Akt phosphorylation of hnRNP A1 S199 does not play a role in CD4+ T cell fate or function in the models tested. This hnRNP A1-S199A mouse model should be a valuable tool to study the role of Akt phosphorylation of hnRNP A1-S199 in different cell types or other mouse models of human disease.

Low-dose Interleukin-2 Therapy: Fine-tuning Treg in Solid Organ Transplantation?

Regulatory T cells (Treg), a subset of CD4 + T cells, are potent regulators of immune reactions, which have been shown to be a promising therapeutic alternative to toxic immunosuppressive drugs. Data support the utility of Treg in managing immunopathologies, including solid organ transplant rejection, graft-versus-host disease, and autoimmune disorders. Notably, reports suggest that interleukin-2 (IL-2) is critical to survival of Treg, which constitutively express high levels of CD25, that is, the IL-2 receptor α-chain, and are exquisitely sensitive to IL-2, even at very low concentrations in contrast to effector T cells, which only upregulate IL-2 receptor α-chain on activation. This has led to the notion of using low doses of exogenous IL-2 therapeutically to modulate the immune system, specifically Treg numbers and function. Here, we summarize developments of clinical experience with low-dose IL-2 (LD-IL-2) as a therapeutic agent. So far, no clinical data are available to support the therapeutic use of LD-IL-2 therapy in the solid organ transplant setting. For the latter, fine-tuning by biotechnological approaches may be needed because of the narrow therapeutic window and off-target effects of LD-IL-2 therapy and so to realize the therapeutic potential of this molecule.

Identification of a non-canonical chemokine-receptor pathway suppressing regulatory T cells to drive atherosclerosis

CCL17 is produced by conventional dendritic cells, signals through CCR4 on regulatory T (Treg) cells and drives atherosclerosis by suppressing Treg functions through yet undefined mechanisms. Here we show that conventional dendritic cells from CCL17-deficient mice display a pro-tolerogenic phenotype and transcriptome that is not phenocopied in mice lacking its cognate receptor CCR4. In the plasma of CCL17-deficient mice, CCL3 was the only decreased cytokine/chemokine. We found that CCL17 signaled through CCR8 as an alternate high-affinity receptor, which induced CCL3 expression and suppressed Treg functions in the absence of CCR4. Genetic ablation of CCL3 and CCR8 in CD4+ T cells reduced CCL3 secretion, boosted FoxP3+ Treg numbers and limited atherosclerosis. Conversely, CCL3 administration exacerbated atherosclerosis and restrained Treg differentiation. In symptomatic versus asymptomatic human carotid atheroma, CCL3 expression was increased, whereas FoxP3 expression was reduced. Together, we identified a non-canonical chemokine pathway whereby CCL17 interacts with CCR8 to yield a CCL3-dependent suppression of atheroprotective Treg cells.

IRF4 affects the protective effect of regulatory T cells on the pulmonary vasculature of a bronchopulmonary dysplasia mouse model by regulating FOXP3

BackgroundBronchopulmonary dysplasia (BPD) is a common chronic lung disease in preterm infants, characterised by compromised alveolar development and pulmonary vascular abnormalities. Emerging evidence suggests that regulatory T cells (Tregs) may confer protective effects on the vasculature. Knockdown of their transcription factor, interferon regulatory factor 4 (IRF4), has been shown to promote vascular endothelial hyperplasia. However, the involvement of Tregs and IRF4 in the BPD pathogenesis remains unclear. This study aimed to investigate the regulation of Tregs by IRF4 and elucidate its potential role in pulmonary vasculature development in a BPD mouse model.MethodsThe BPD model was established using 85% hyperoxia exposure, with air exposure as the normal control. Lung tissues were collected after 7 or 14 days of air or hyperoxia exposure, respectively. Haematoxylin–eosin staining was performed to assess lung tissue pathology. Immunohistochemistry was used to measure platelet endothelial cell adhesion molecule-1 (PECAM-1) level, flow cytometry to quantify Treg numbers, and Western blot to assess vascular endothelial growth factor (VEGFA), angiopoietin-1 (Ang-1), forkhead box protein P3 (FOXP3), and IRF4 protein levels. We also examined the co-expression of IRF4 and FOXP3 proteins using immunoprecipitation and immunofluorescence double staining. Furthermore, we employed CRISPR/Cas9 technology to knock down the IRF4 gene and observed changes in the aforementioned indicators to validate its effect on pulmonary vasculature development in mice.ResultsElevated IRF4 levels in BPD model mice led to FOXP3 downregulation, reduced Treg numbers, and impaired pulmonary vascular development. Knockdown of IRF4 resulted in improved pulmonary vascular development and upregulated FOXP3 level.ConclusionIRF4 may affect the protective role of Tregs in the proliferation of pulmonary vascular endothelial cells and pulmonary vascular development in BPD model mice by inhibiting the FOXP3 level.

Plexin B1 controls Treg numbers, limits allergic airway inflammation, and regulates mucins.

We investigated the effect of global Plexin B1 deficiency on allergic airway responses to house dust mite (HDM) or ovalbumin (OVA). In the HDM model, there were higher Th2 cytokine levels in the BALF of Plexin B1 knock-out (KO) mice compared to wild type (WT), and tissue inflammation and mucus production were modestly enhanced. In the OVA model, Plexin B1 deficiency led to increases in lung inflammation, mucus production, and lung Th2 cytokines accompanied by dysregulated mucin gene expression without affecting anti-OVA IgE/IgG1 levels. Spleen cells from Plexin B1 KO mice proliferated more robustly than WT cells in vitro to a variety of stimuli. Plexin B1 KO CD4+ T cells from spleens expressed higher levels of Ki-67 and CD69 compared to WT cells. Spleen cells from naïve Plexin B1 KO mice secreted increased amounts of IL-4 and IL-6 when pulsed in vitro with OVA whereas in vivo OVA-primed spleen cells produced IL-4/IL-5 when subjected to in vitro OVA restimulation. The upregulated allergic inflammatory response in Plexin B1 KO mice was associated with a lower number of Tregs in the lung tissues. Moreover, these mice displayed lower numbers of Treg cells in the lymphoid tissues at the baseline. These results demonstrate a previously unrecognized link between Plexin B1, Treg cells, and mucus in allergic lung inflammation.

T-regulatory cells require Sin3a for stable expression of Foxp3.

Histone deacetylases 1 and 2 play a major role in the transcriptional regulation of T-regulatory (Treg) cells via interactions with a myriad of coregulatory factors. Sin3a has been well established as a Hdac1/2 cofactor, while its role within Tregs has not been established. In this study, the effects of conditional deletion of Sin3a within Foxp3+ Tregs were evaluated. Developmental deletion of Sin3a from Foxp3+ Tregs resulted in the rapid onset of fatal autoimmunity. Treg numbers were greatly reduced, while residual Tregs had impaired suppressive function. Mice also showed effector T-cell activation, autoantibody production, and widespread tissue injury. Mechanistically, Sin3a deletion resulted in decreased transcription of Foxp3 with a complete lack of CNS2 CpG demethylation. In addition, Foxp3 protein stability was impaired with an increased ex-Treg population. Thus, Sin3a plays a critical role in the maintenance of Treg identity and function and is essential for the expression and stability of Foxp3.

Blocking the CTLA-4 and PD-1 pathways during pulmonary paracoccidioidomycosis improves immunity, reduces disease severity, and increases the survival of infected mice.

Immune checkpoint pathways, i.e., coinhibitory pathways expressed as feedback following immune activation, are crucial for controlling an excessive immune response. Cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death protein-1 (PD-1) are the central classical checkpoint inhibitory (CPI) molecules used for the control of neoplasms and some infectious diseases, including some fungal infections. As the immunosuppression of severe paracoccidioidomycosis (PCM), a chronic granulomatous fungal disease, was shown to be associated with the expression of coinhibitory molecules, we hypothesized that the inhibition of CTLA-4 and PD-1 could have a beneficial effect on pulmonary PCM. To this end, C57BL/6 mice were infected with Paracoccidioides brasiliensis yeasts and treated with monoclonal antibodies (mAbs) α-CTLA-4, α-PD-1, control IgG, or PBS. We verified that blockade of CTLA-4 and PD-1 reduced the fungal load in the lungs and fungal dissemination to the liver and spleen and decreased the size of pulmonary lesions, resulting in increased survival of mice. Compared with PBS-treated infected mice, significantly increased levels of many pro- and anti-inflammatory cytokines were observed in the lungs of α-CTLA-4-treated mice, but a drastic reduction in the liver was observed following PD-1 blockade. In the lungs of α-CPI and IgG-treated mice, there were no changes in the frequency of inflammatory leukocytes, but a significant reduction in the total number of these cells was observed. Compared with PBS-treated controls, α-CPI- and IgG-treated mice exhibited reduced pulmonary infiltration of several myeloid cell subpopulations and decreased expression of costimulatory molecules. In addition, a decreased number of CD4+ and CD8+ T cells but sustained numbers of Th1, Th2, and Th17 T cells were detected. An expressive reduction in several Treg subpopulations and their maturation and suppressive molecules, in addition to reduced numbers of Treg, TCD4+, and TCD8+ cells expressing costimulatory and coinhibitory molecules of immunity, were also detected. The novel cellular and humoral profiles established in the lungs of α-CTLA-4 and α-PD-1-treated mice but not in control IgG-treated mice were more efficient at controlling fungal growth and dissemination without causing increased tissue pathology due to excessive inflammation. This is the first study demonstrating the efficacy of CPI blockade in the treatment of pulmonary PCM, and further studies combining the use of immunotherapy with antifungal drugs are encouraged.

FOXP3+ regulatory T cells are associated with the severity and prognosis of sarcoidosis.

Sarcoidosis is an inflammatory granulomatous disease of unknown etiology with predominant lung involvement. Organ involvement and disease severity, as well as the nature of immune alterations, vary among patients leading to a range of clinical phenotypes and outcomes. Our objective was to evaluate the association of disease course and immune responses in pulmonary sarcoidosis. In this prospective cohort study of 30 subjects, most of whom were followed for one year, we evaluated 14 inflammatory markers in plasma, 13 Treg/T cell flow cytometry markers and 8 parameters of FOXP3+ Treg biology, including suppressive function, epigenetic features and stability. We identified a set of 13 immunological parameters that differ in sarcoidosis subjects in comparison with healthy donors. Five of those were inversely correlated with suppressive function of Tregs in sarcoidosis, and six (TNFα, TNFR I and II, sCD25, Ki-67 and number of Tregs) were particularly upregulated or increased in subjects with thoracic lymphadenopathy. Treg suppressive function was significantly lower in patients with thoracic lymphadenopathy, and in patients with higher burdens of pulmonary and systemic symptoms. A combination of five inflammatory markers, Ki-67 expression, Treg function, and lung diffusion capacity evaluated at study entry predicted need for therapy at one year follow-up in 90% of cases. Tregs may suppress ongoing inflammation at local and systemic levels, and TNFα, TNFR I and II, sCD25 and Ki-67 emerge as attractive biomarkers for in vivo sarcoid inflammatory activity.

The effect of post-covid syndrome on the cytokine cascade in patients with type 2 diabetes mellitus

With the increasing prevalence of type 2 diabetes, it is of interest to study the characteristics of the immunological status in this category of patients. According to modern data, in type 2 diabetes, a pro-inflammatory type of immune response is formed with an imbalance between T-helper 1 (Th1), T-helper 2 (Th2), T-helper 17 (Th17) types and T-regulatory cells (Treg). The COVID-19 pandemic makes a certain contribution to changes in the immune response in patients with type 2 diabetes mellitus, since in patients who have had coronavirus infection, significant changes in the composition of circulating immune cells are observed: the proportion of Th17 increases and the number of Tregs decreases. This study is devoted to identifying the features of the cytokine cascade in patients with type 2 diabetes mellitus, depending on the presence of a confirmed history of SARS-CoV-2 infection. The study included 75 patients with type 2 diabetes, in 36 of them the diagnosis was verified in the post-Covid period. The study was conducted at least 6 months after pneumonia caused by SARS-CoV-2 infection. All study participants signed informed consent. The study was approved by the Independent Local Ethics Committee at the State Autonomous Institution of Regional Clinical Hospital “City Clinical Hospital No. 1” of Chelyabinsk, protocol No. 8 dated 04/11/2022, on the basis of which these studies were conducted. Using the multiplex assessment method, the concentrations of 15 cytokines in the blood serum were assessed in patients: GM-CSF, IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17A, MIP-1β, TNFα. The results obtained during the study showed not only a change in the number of cytokines, but also a change in the balance of pro- and anti-inflammatory cytokines, cytokines that regulate forms of development of acquired immunity, in the group of patients with type 2 diabetes in the post-Covid period. In the post-Covid period, in patients with type 2 diabetes mellitus, the concentrations of cytokines secreted by Th1, Th2, Th17 cells, and Tregs sharply decreased. It has been determined that patients with type 2 diabetes mellitus after SARS-CoV-2 infection develop a functionally inferior virus-specific T-cell response, characterized by reduced or suppressed secretion of the cytokines GM-CSF, IFNγ, IL-1β, IL-2, IL -4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17A, MIP-1β, TNFα. A weak T-cell response may increase the likelihood of developing pathological processes associated with an inadequate response to viral antigens (including repeated infections with the SARSCoV-2 virus, exacerbation of chronic infections).

Deciphering the immune landscape of head and neck squamous cell carcinoma: A single-cell transcriptomic analysis of regulatory T cell responses to PD-1 blockade therapy

Immunotherapy is changing the Head and Neck Squamous Cell Carcinoma (HNSCC) landscape and improving outcomes for patients with recurrent or metastatic HNSCC. A deeper understanding of the tumor microenvironment (TME) is required in light of the limitations of patients’ responses to immunotherapy. Here, we aimed to examine how Nivolumab affects infiltrating Tregs in the HNSCC TME. We used single-cell RNA sequencing data from eight tissues isolated from four HNSCC donors before and after Nivolumab treatment. Interestingly, the study found that Treg counts and suppressive activity increased following Nivolumab therapy. We also discovered that changes in the CD44-SSP1 axis, NKG2C/D-HLA-E axis, and KRAS signaling may have contributed to the increase in Treg numbers. Furthermore, our study suggests that decreasing the activity of the KRAS and Notch signaling pathways, and increasing FOXP3, CTLA-4, LAG-3, and GZMA expression, may be mechanisms that enhance the killing and suppressive capacity of Tregs. Additionally, the result of pseudo-temporal analysis of the HNSCC TME indicated that after Nivolumab therapy, the expression of certain inhibitory immune checkpoints including TIGIT, ENTPD1, and CD276 and LY9, were decreased in Tregs, while LAG-3 showed an increased expression level. The study also found that Tregs had a dense communication network with cluster two, and that certain ligand-receptor pairs, including SPP1/CD44, HLA-E/KLRC2, HLA-E/KLRK1, ANXA1/FPR3, and CXCL9/FCGR2A, had notable changes after the therapy. These changes in gene expression and cell interactions may have implications for the role of Tregs in the TME and in response to Nivolumab therapy.

Regulatory T cells in the peripheral blood of women with gestational diabetes: a systematic review and meta-analysis.

Gestational diabetes (GDM) affects approximately 14% of pregnancies globally and is associated with short- and long-term complications for both the mother and child. In addition, GDM has been linked to chronic low-grade inflammation with recent research indicating a potential immune dysregulation in pathophysiology and a disparity in regulatory T cells. This systematic review and meta-analysis aimed to determine whether there is an association between GDM and the level of Tregs in the peripheral blood. Literature searches were conducted in PubMed, Embase, and Ovid between the 7th and 14th of February 2022. The inclusion criteria were any original studies published in the English language, measuring differentiated Tregs in women with GDM compared with glucose-tolerant pregnant women. Meta-analysis was performed between comparable Treg markers. Statistical tests were used to quantify heterogeneity: τ 2, χ 2, and I 2. Study quality was assessed using a modified version of the Newcastle-Ottawa scale. The search yielded 223 results: eight studies were included in the review and seven in the meta-analysis (GDM = 228, control = 286). Analysis of Tregs across all trimesters showed significantly lower Treg numbers in women with GDM (SMD, -0.76; 95% CI, -1.37, -0.15; I 2=90%). This was reflected in the analysis by specific Treg markers (SMD -0.55; 95% CI, -1.04, -0.07; I 2=83%; third trimester, five studies). Non-significant differences were found within subgroups (differentiated by CD4+FoxP3+, CD4+CD127-, and CD4+CD127-FoxP3) of both analyses. GDM is associated with lower Treg numbers in the peripheral maternal blood. In early pregnancy, there is clinical potential to use Treg levels as a predictive tool for the subsequent development of GDM. There is also a potential therapeutic intervention to prevent the development of GDM by increasing Treg populations. However, the precise mechanism by which Tregs mediate GDM remains unclear. https://www.crd.york.ac.uk/prospero, identifier CRD42022309796.

Abstract C077: STC-15, a small molecule inhibitor of the RNA methyltransferase METTL3, activates anti-tumor immunity and reshapes the tumor microenvironment

Abstract Background METTL3 is the RNA methyltransferase responsible for the deposition of N-6-methyladenosine modification (m6A) on mRNA, to regulate its stability, splicing and protein translation. Small molecule inhibitors of METTL3 catalytic activity have previously demonstrated anti-tumor efficacy in pre-clinical models of acute myeloid leukemia (AML) and solid tumors. Inhibition of METTL3 was shown to induce a cell-intrinsic innate immune response through activation of the double strand RNA (dsRNA) sensing machinery and interferon (IFN) signalling. Here we present new pre-clinical data, investigating the combination of METTL3 inhibitor (METTL3i) with DNA damaging therapies. We show that METTL3 inhibition profoundly changes the tumor microenvironment (TME), and we further demonstrate the generation of durable immune memory in mice treated with METTL3i, with or without anti-PD1 therapy. Materials & Methods The orally bioavailable small molecule clinical-stage METTL3i STC-15, or the equipotent tool compound STM3675 were used in these studies. To characterise transcriptomic changes following METTL3 inhibition, RNA sequencing was performed. Induction of specific genes was validated by Western Blot, and cytokine secretion assessed by ELISA. In-vitro cell viability was assessed by Cell TiterGlo (Promega). In vivo efficacy studies used subcutaneous MC38 and GL261 syngeneic tumor models, and characterisation of the TME was performed by flow cytometry. Results Treatment with METTL3i sensitized cells to DNA damaging agents such as doxorubicin. On the molecular level, further enhancement of the IFN pathway by the combination treatment was observed. Moreover, combination of METTL3i with radiation treatment (RT) in vivo in the MC38 colorectal syngeneic mouse model demonstrated a clear benefit of the combination over each of the single treatments. METTL3i alone or in combination with RT led to a major TME remodulation, reducing the number of Tregs, M2 macrophages and neutrophils, and increasing the number of M1 macrophages and CD8+ T-cells in tumors. The GL261 syngeneic glioma model is exquisitely sensitive to anti-PD1 treatment. Similarly, METTL3i treatment led to multiple complete regressions, and the combination of METTL3i and anti-PD1 further accelerated the occurrence of regressions. Following a 3-weeks’ observation period, tumor free mice were re-inoculated with GL261 cells. All tumor-free mice remained tumor free after the rechallenge. Conclusions In pre-clinical cancer models, treatment with METTL3i such as STC-15 resulted in activation of innate immune pathways and subsequent enhancement of cytotoxic T-cell activity. In-vivo, STC-15 had a profound effect on the TME, shifting it from an immunosuppressive, pro-tumorigenic state to an immunostimulatory, anti-tumor state. In combination with anti-PD1, METTL3 inhibitors generate a durable anti-tumor response and lasting immune memory. A First-in-Human dose escalating study evaluating STC-15 monotherapy in patients with solid tumors is ongoing (NCT05584111). Citation Format: Yaara Ofir-Rosenfeld, Lina Vasiliauskaitė, Joanna Obacz, Georgia Tsagkogeorga, Josefin-Beate Holz, Jerry McMahon, Oliver Rausch. STC-15, a small molecule inhibitor of the RNA methyltransferase METTL3, activates anti-tumor immunity and reshapes the tumor microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C077.

Abstract B004: Neoantigen adenoviral cancer vaccine generates improved CD8+ T-cell responses compared to adjuvanted peptide vaccine

Abstract Development of large, functional CD8+ T cell responses is crucial for successful cancer vaccine therapeutics. Neoantigens have demonstrated their potential as cancer vaccine immunogens, but improvements in delivery method are still necessary. In this work, we characterized five candidate Adenovirus Serotype 26 (Ad26) cancer vaccines for prophylactic efficacy in the MC-38 cancer model. All five single shot vaccines generated cellular immune responses at a higher level than prime boost peptide vaccination. The vaccines generated varying degrees of protection against tumor growth. The best Ad26 vaccine candidates showed improved protection compared to long synthetic peptide vaccines containing the same immunogens, the current standard in the field. Ad26 expressing seven neoantigens conjugated to the Herpes Viral Protein 22 (VP22) (Ad26.VP22.7Epi) was found to reduce tumor growth the most. Ad26.VP227Epi was compared to peptide vaccination in immune recall post challenge studies, in both the spleen and the tumor. Both Ad26.VP22.7Epi and Peptide showed a clear recall response after challenge in the spleen. This response was primarily focused on the Adpgk neoantigen but Ad26.VP22.7Epi additionally induced responses to the Irgq neoantigen while the peptide vaccine had stronger responses against the Reps1 neoantigen. The Ad26.VP227Epi vaccine generated increased numbers of infiltrating CD8+ T-cells, IFN+ CD8+ T-cells, and CD107a+ CD8+ T-cells, which all correlated with reduced tumor growth. We used single cell RNA-seq (scRNA-seq) to characterize tumor infiltrating lymphocyte populations (defined as CD45+) generated by the two platforms. We found that both Ad26.VP22.7Epi and Peptide induced higher numbers of infiltrating CD8+ T-cells within the tumor as compared to Sham. Single-cell analysis also showed higher numbers of tumor infiltrating Tregs in peptide vaccinated mice. Higher numbers of CD8+ T-cells correlated with protection as expected but interestingly increased numbers of Tregs positively correlated with tumor volume only among vaccinated mice, which could further explain differences between Ad vector and peptide vaccination. Differential gene expression analysis of CD8+ T-cells showed an upregulation of Th1 pathways and general T cell activation when comparing Ad vector vaccinated mice to peptide vaccinated. Peptide vaccinated CD8+ T-cells showed upregulation in the IL-10 pathway, potentially caused by an increased Tregs. Further investigation of these differences among CD8+ T cell and Treg populations could further elucidate potential mechanisms explaining the differences in platform efficacy. Citation Format: Gabriel Dagotto, Alessandro Colarusso, Robert Patio, David Li, Tochi Anioke, Victoria Giffin, Malika Aid, Dan Barouch. Neoantigen adenoviral cancer vaccine generates improved CD8+ T-cell responses compared to adjuvanted peptide vaccine [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B004.

Iguratimod Regulates CD4+ T-Cell Homeostasis and Function By Restoring PINK1/Parkin-Mediated Mitophagy in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP), an acquired autoimmune disease, is characterized by immune-mediated platelet destruction and suppressed platelet production, resulting in isolated thrombocytopenia. Subpopulation imbalance and dysfunction of CD4+ T cells are involved in the pathogenesis of ITP. However, the exact mechanism of CD4+ T-cell abnormalities is not fully understood. Mitophagy, a process that eliminates damaged and dysfunctional mitochondria, has conventionally been considered the primary mechanism responsible for mitochondrial quality control. Mitophagy deficiency has been implicated in a series of inflammatory and autoimmune diseases, such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD) and rheumatoid arthritis (RA). However, whether it plays a role in ITP immune abnormalities and its specific mechanism remain unclear. Iguratimod is a small molecule compound that is widely used as a novel antirheumatic drug in the treatment of rheumatoid arthritis. Iguratimod inhibits the production of several inflammatory cytokines, including IL-1, IL-6, IL-8, and tumor necrosis factor (TNF), by enhancing mitophagy. Our previous clinical study demonstrated the efficacy of iguratimod in the treatment of ITP. This study aimed to explore whether iguratimod regulates CD4+ T-cell homeostasis and function by enhancing mitophagy in ITP. Methods BM CD4+ T cells were isolated from ITP patients and healthy donors. The subgroup of CD4+ T cells was analyzed by flow cytometry. ELISA was used to evaluate cytokine and chemokine levels in bone marrow plasma. Mitochondrial mass, morphology, quality and mitophagy were evaluated by flow cytometry, confocal microscopy, electron microscopy and western blotting. RNA sequencing and metabolomics were performed. Epigenetic state alteration was assessed by a transposase-accessible chromatin assay with high throughput sequencing (ATAC-seq). Iguratimod-treated BM-derived ITP-CD4+ T cells and an ITP active mouse model were used to explore its regulation of mitophagy and effects on T-cell homeostasis and function. Results Th1/Th2 imbalance and Treg reduction were observed in the bone marrow of ITP patients, consistent with a previous report. Flow cytometry and confocal microscopy revealed an increase in mitochondrial content and mtROS in CD4+ T cells but a decrease in mitochondrial membrane potential. Furthermore, electron microscopy revealed structural damage to mitochondria in ITP-CD4+ T cells. These data confirmed an accumulation of damaged mitochondria in ITP-CD4+ T cells. The expression levels of LC3II/LC3I, PINK1 and parkin decreased in CD4+ T cells, and the colocalization of mitochondria and lysosomes decreased, which suggested compromised PINK1/parkin-mediated mitophagy in ITP-CD4+ T cells. RNA-seq revealed transcriptional changes associated with mitochondrial function, mitophagy and metabolic pathways in ITP-CD4+ T cells. Moreover, metabolomics showed alterations in TCA cycle metabolites. Since epigenetic programs are regulated by metabolites, ATAC-seq was performed to obtain the epigenetic state. We found that ITP-CD4+ T cells altered chromatin accessibility in regions with T cell differentiation, transcription factors and T-cell function. After in vitro treatment with iguratimod, the Th1/Th2 imbalance was corrected, and the number of Tregs was restored. The content, morphology and membrane potential of mitochondria in ITP-CD4+ T cells were restored after iguratimod intervention. The expression level of PINK1 and parkin and the colocalization of mitochondria and lysosomes were increased, suggesting an improvement in mitophagy. In vivo, iguratimod accelerated platelet recovery in an ITP active mouse model. After treatment, the Th1/Th2 imbalance and the Treg number were restored, and the mitophagy of CD4+ T cells was corrected in the bone marrow of the ITP mouse model. Conclusion PINK1/Parkin-mediated mitophagy deficiency contributed to mitochondrial dysfunction and altered the metabolism of ITP-CD4+ T cells, which affected their differentiation and function by regulating the epigenetic state of immune response genes. Iguratimod could restore CD4+ T-cell subpopulations and treat ITP by enhancing mitophagy.

CXCR4 Enriched T Regulatory Cells Preferentially Home to Bone Marrow and Decrease Inflammation

Background and aim Adoptive therapy with T regulatory cells (Tregs) is emerging as an important therapeutic strategy for several autoimmune and inflammatory diseases. Optimal homing and payload delivery of these anti-inflammatory cells remain a cornerstone of optimizing their therapeutic efficacy. CXCR4 cell surface expression has been shown to be critical for homing of hematopoietic cells to the bone marrow (BM). Also, CXCR4/CXCL12 axis plays a major role in the pathogenesis of BM inflammatory disorders including myelofibrosis. We hypothesize that enrichment of CXCR4 on the Tregs cell surface will enable their trafficking to BM and ensure a homogenous delivery. Methods/results/conclusion Magnetic enrichment of CD25 + cells was performed on umbilical cord blood on day 0, followed by dual enrichment on CXCR4 using CRANE TM (cord blood Tregs, activated and enriched) process on day 3 or 4 (Fig 1A). The enrichment process led to increased number of Tregs expressing CXCR4 on their cell surface (Fig 1B; Treg CXCR4) as well as a CXCR4 hi Treg cells when compared to Treg control (Fig 1C, p<0.0001; n=16). In a Transwell TM system, a higher number of Treg CXCR4 migrated to the bottom well in chemotactic response to SDF1α at 15 minutes as compared to the Treg control cells. Such migration was inhibited by CXCR4 antagonist, AMD3100 (Fig 1D). The migration superiority of Treg CXCR4 was maintained at 30 and 60 min (Fig 1E). In order to understand in vivo transit kinetics, Treg cells were injected into NSG mice and organs were harvested at 12 and 24 hours (Fig 1F). Treg CXCR4 showed preferential homing to BM (Fig 1G), where a higher expression of CXCR4 and CXCL12 than control, was observed (Fig 1H). Other markers including CD62L (homing and suppression), CD39 & CD73 (conversion of ATP to immunosuppressive adenosine), and CXCR5 (Follicular Treg) were also increased in Treg CXCR4 recipients. A decrease in biomarkers including TGFα and TNFβ (inflammation), IL-13 (fibrosis) (Fig 1I) with an increase in GM-CSF (increase Treg potency) (Fig 1J), was observed in Treg CXCR4 recipients with a corresponding decrease in TGFβ family (hematopoietic suppressor) (Fig 1K). We conclude that Treg CXCR4 shows superior homing to BM and is currently being studied in a clinical setting to treat myelofibrosis with suboptimal response to ruxolitinib (NCT05423691).

Metabolic Reprograming of Exhausted Intratumoral CD8+ T-Cell Underlies Anti-Tumor Activity of Sumoylation Inhibitors in Large B Cell Lymphoma

Immune checkpoint inhibitors (ICI) targeting PD1 have revolutionized our approach to solid tumors and Hodgkin lymphoma, but they have not been effective in treating Large B Cell Lymphoma (LBCL). In prior work, we used single cell spatial profiling of human tumors to show that exhausted CD8 tumor and TREG populations were associated with PD1 resistance in LBCL (Colombo et al. Blood Adv 2022). Here we show, using a syngeneic model of LBCL, that PD1 resistance is associated with lower CD8+ T-cell basal metabolic rate (BMR) in tumor infiltrating lymphocytes (TIL), and that treatment with the covalent SUMOylation inhibitor (SB-4826) increases the metabolic rate of TIL by metabolic reprograming. Single cell metabolic profiling of human LBCL is on-going and will be presented as well. In summary these studies demonstrate that metabolic dysfunction of T cells in LBCL underlie resistance to PD1 checkpoint inhibitors and that this can be overcome by treatment with SUMOlyation inhibitors. Direct measurement of intra-tumoral metabolic state is difficult to perform on clinical samples, however large tumor size is hypothesized to be a surrogate for increased metabolic derangement. As tumors expand and become more metabolically active themselves, infiltrating immune cells experience decreased O2 tension as well as nutrient deprivation, ultimately leading to decreased anti-tumor immune responses. In support of this idea, it has been observed that high metabolic tumor volume at baseline predicts survival in LBCL patients, independent of their initial response to therapy. We have shown that mice with established (>125mm 3) A20 tumors are resistant to anti-PD1 therapy. However, A20 bearing mice treated ~5 days post implantation are responsive to anti-PD1 therapy. To understand these findings, we examined sensitive (small) and resistant (large) A20 tumors by flow cytometry analysis and found that resistant tumors had lower relative numbers of T cells, lower absolute numbers of CD8+ T cells, increased absolute numbers of TREG and a strikingly decreased CD8/TREG ratio. Additionally, the resistant tumors had increased absolute numbers of pre-exhausted and exhausted CD8+ T cells as well as decreased BMR. Small ubiquitin-like modifiers (SUMO) proteins regulate a variety of cellular processes in tumor and immune cells and inhibitors of SUMOylation have shown anti-tumor activity in a variety of preclinical tumor models with potential synergy with checkpoint inhibitors targeting PD-1 (Cannon et al. Cancer Res, 2023). We demonstrate that a dose of 100mg/kg of SB-4826 led to a complete response in all mice treated (Fig. 1A) and we also show that this effect is dependent on CD8+ T-cells and elicits a long term memory response, with 0 of 15 mice in remission developing tumor upon rechallenge. A low dose (25mg/kg) of SB-4826 failed to elicit a response in any of the mice treated while anti-PD1 therapy elicited a response in 1/6 mice (Fig. 1A). Combination of 25mg/kg SB-4826 with anti-PD1 therapy demonstrated synergism with a response in 3/6 mice (Fig. 1A). We also demonstrate that global SUMOylation levels are higher in PD1+ TIM3+ CD8+ TILs compared to PD1- TIM3- CD8+ T-cells, suggesting that SUMOylation may be acting as a negative regulator of T-cell function. Using SCENITH (Arguello et al. Cell Metabolism 2020) for single cell metabolic studies, we demonstrate that SB-4826 increases the BMR (Fig. 1B), mitochondrial dependency and proliferative capacity of tumor infiltrating PD1+ TIM3+ CD8+ T-cells (TILs). While both SB-4826 and PD1 inhibition led to increased CD8+ TIL numbers, SB-4826 led to increased BMR in CD8+ TILs while PD1 inhibition did not affect CD8+ TILs BMR. Additionally, PD1 inhibition significantly decreased CD8+ TILs mitochondrial dependency for metabolism, while SB-4826 increased mitochondrial dependency (Fig. 1B). Additionally, we recapitulate these metabolic enhancements in an in vitro system of T-cell activation, further confirming a T-cell intrinsic effect following SB-4826. Mechanistically, we found that SB-4826 led to increased HIF-1a levels in both CD4+ and CD8+ cells following TCR activation, likely through stabilization of HIF-1a due to decreased SUMOylation-dependent turnover (Cheng J et al. Cell 2007). We hypothesize that metabolic reprograming of CD8 T-cells through increased HIF-1a is the basis for synergy between SUMOylation inhibition and immune checkpoint inhibitors in LBCL.

Recirculating Regulatory T Cells Mediate Thymic Regeneration in a ZFP36L1 and Amphiregulin Dependent Manner

T cell reconstitution correlates with outcomes in patients undergoing stem cell transplantation (HCT). A functional thymus is needed for the maturation of T cells, but unfortunately, the thymus is highly sensitive to acute injury, such as cytoreductive conditioning given to HCT patients. To enhance T cell reconstitution, it is therefore imperative to explore therapies that can boost thymic regeneration and T cell output. Regulatory T cells (Tregs) are known to mediate homeostasis and tissue regeneration in a variety of tissues, but their role in thymic injury is not known. This study aims to shed light on the role of regulatory T cells (Tregs) in thymic regeneration. Using a model of thymic injury and regeneration in mice receiving sublethal total body irradiation (SL-TBI, 550cGy), we observed a significant increase in Tregs ( Fig. 1A), located in both the medullary and cortical areas of the thymus on multiparameter immunohistochemistry. Employing a targeted ablation of Tregs at the time of injury using the FOXP3DTR mouse model, we could observe significant delay in thymic regeneration in mice depleted of Tregs (day 7 post SL-TBI, p=<0.0001, n=10, Fig. 1B). RAG2GFP mice, where GFP is expressed under the RAG2 promoter, have been used to discern recirculating and de-novo produced Tregs. To characterize the nature of the Treg expansion, we treated RAG2GFP mice with SL-TBI and analyzed their thymus at multiple time points after injury. Interestingly, the increase in Tregs was mainly due to recirculating RAG2-GFP- Tregs but not de-novo produced RAG2+GFP+ Tregs after injury. Brdu administration after injury indicated proliferation of these RAG2-GFP- Tregs. Parabiotic experiments linking RAG2GFP mice expressing either the congenic marker CD45.1 or CD45.2 confirmed the recirculating nature of these cells, both at steady state and after injury. Furthermore, when adoptively transferring sorted Thymic Tregs from FOXP3RFP mice to mice receiving SL-TBI, we significantly enhanced thymic cellularity after injury and homing of these Tregs to the thymus. To discern the transcriptional profile of these cells, we used single-cell sequencing of CD45+ RAG2- cells in 2- and 18-month-old RAG2GFP mice before and at day 1, 4, and 7 after sublethal irradiation. This enabled us to discover the age- and injury-associated changes in recirculating T cells. While the aged thymus had a similar surge of Treg numbers after injury, this response was blunted compared to their young counterparts. Factor normalization methods enabled us to find two distinct gene regulatory networks highly specific to the RAG2-GFP-Tregs. One gene network was highly enriched for Zfp36l1, a poised RNA known to mediate cell stability. Selectively ablating Zfp36l1 in Tregs using the Foxp3Cre ZFP36L1 flox/flox model impaired regeneration significantly after SL-TBI. The other distinct RAG2-GFP- Treg specific gene network was enriched for the regenerative factor Amphiregulin ( Areg). Amphiregulin was seen to be increased in thymic supernatants, and its receptor Egfr was seen to be transcriptionally upregulated in thymic epithelial cells after injury. In parallel, the most significantly upregulated pathway using cell-to-cell interaction prediction models on single-cell data indicated a strong likelihood of Treg to thymic epithelial cell interactions through amphiregulin to Egfr. Functional inhibition of amphiregulin with an antibody enabled us to observe delayed thymic regeneration, indicating amphiregulin as a factor in the regenerative capacity of thymic recirculating Tregs. Mapping human gene orthologs of the gene regulatory program provided by factor normalization in murine RAG2GFP- Tregs on single cell data of human pediatric thymic samples from children undergoing open heart surgeries enabled us to distinguish a putatively analogous human population of recirculating thymic Tregs. This population was found to be amphiregulin positive. And human thymic epithelial cell cultures with recombinant amphiregulin enhanced the growth of human thymic epithelial cells, as measured by life imaging, and promoted signalling through MAPK ERK1/2. Taken together, our findings reveal a novel role for injury-resistant recirculating thymic regulatory T cells existing in both the human and murine thymus and may enable the development of new therapeutic targets for thymic regeneration for patients undergoing cytoreductive therapies.

A tumor-associated heparan sulfate-related glycosaminoglycan promotes the generation of functional regulatory T cells.

Functional Tregs play a key role in tumor development and progression, representing a major barrier to anticancer immunity. The mechanisms by which Tregs are generated in cancer and the influence of the tumor microenvironment on these processes remain incompletely understood. Herein, by using NMR, chemoenzymatic structural assays and a plethora of in vitro and in vivo functional analyses, we demonstrate that the tumoral carbohydrate A10 (Ca10), a cell-surface carbohydrate derived from Ehrlich's tumor (ET) cells, is a heparan sulfate-related proteoglycan that enhances glycolysis and promotes the development of tolerogenic features in human DCs. Ca10-stimulated human DCs generate highly suppressive Tregs by mechanisms partially dependent on metabolic reprogramming, PD-L1, IL-10, and IDO. Ca10 also reprograms the differentiation of human monocytes into DCs with tolerogenic features. In solid ET-bearing mice, we found positive correlations between Ca10 serum levels, tumor size and splenic Treg numbers. Administration of isolated Ca10 also increases the proportion of splenic Tregs in tumor-free mice. Remarkably, we provide evidence supporting the presence of a circulating human Ca10 counterpart (Ca10H) and show, for the first time, that serum levels of Ca10H are increased in patients suffering from different cancer types compared to healthy individuals. Of note, these levels are higher in prostate cancer patients with bone metastases than in prostate cancer patients without metastases. Collectively, we reveal novel molecular mechanisms by which heparan sulfate-related structures associated with tumor cells promote the generation of functional Tregs in cancer. The discovery of this novel structural-functional relationship may open new avenues of research with important clinical implications in cancer treatment.

Changes in Treg and Breg cells in a healthy pediatric population.

The interpretation of clinical diagnostic results in suspected inborn errors of immunity, including Tregopathies, is hampered by the lack of age-stratified reference values for regulatory T cells (Treg) in the pediatric population and a consensus on which Treg immunophenotype to use. Regulatory B cells (Breg) are an important component of the regulatory system that have been poorly studied in the pediatric population. We analyzed (1) the correlation between the three immunophenotypic definitions of Treg (CD4+CD25hiCD127low, CD4+CD25hiCD127lowFoxP3+, CD4+CD25hiFoxP3+), and with CD4+CD25hi and (2) the changes in Treg and Breg frequencies and their maturation status with age. We performed peripheral blood immunophenotyping of Treg and Breg (CD19+CD24hiCD38hi) by flow cytometry in 55 healthy pediatric controls. We observed that Treg numbers varied depending on the definition used, and the frequency ranged between 3.3-9.7% for CD4+CD25hiCD127low, 0.07-1.6% for CD4+CD25hiCD127lowFoxP3+, and 0.24-2.83% for CD4+CD25hiFoxP3+. The correlation between the three definitions of Treg was positive for most age ranges, especially between the two intracellular panels and with CD4+CD25hi vs CD4+CD25hiCD127low. Treg and Breg frequencies tended to decline after 7 and 3 years onwards, respectively. Treg's maturation status increased with age, with a decline of naïve Treg and an increase in memory/effector Treg from age 7 onwards. Memory Breg increased progressively from age 3 onwards. In conclusion, the number of Treg frequencies spans a wide range depending on the immunophenotypic definition used despite a good level of correlation exists between them. The decline in numbers and maturation process with age occurs earlier in Breg than in Treg.