Manipulating regulatory T cells: is it the key to unlocking effective immunotherapy for pancreatic ductal adenocarcinoma?

The five-year survival rates for pancreatic ductal adenocarcinoma (PDAC) have scarcely improved over the last half-century. It is inherently resistant to FDA-approved immunotherapies, which have transformed the outlook for patients with other advanced solid tumours. Accumulating evidence relates this resistance to its hallmark immunosuppressive milieu, which instils progressive dysfunction among tumour-infiltrating effector T cells. This milieu is established at the inception of neoplasia by immunosuppressive cellular populations, including regulatory T cells (Tregs), which accumulate in parallel with the progression to malignant PDAC. Thus, the therapeutic manipulation of Tregs has captured significant scientific and commercial attention, bolstered by the discovery that an abundance of tumour-infiltrating Tregs correlates with a poor prognosis in PDAC patients. Herein, we propose a mechanism for the resistance of PDAC to anti-PD-1 and CTLA-4 immunotherapies and re-assess the rationale for pursuing Treg-targeted therapies in light of recent studies that profiled the immune landscape of patient-derived tumour samples. We evaluate strategies that are emerging to limit Treg-mediated immunosuppression for the treatment of PDAC, and signpost early-stage trials that provide preliminary evidence of clinical activity. In this context, we find a compelling argument for investment in the ongoing development of Treg-targeted immunotherapies for PDAC.

Blood-based epigenome-wide analyses of chronic low-grade inflammation across diverse population cohorts.

Chronic inflammation is a hallmark of age-related disease states. The effectiveness of inflammatory proteins including C-reactive protein (CRP) in assessing long-term inflammation is hindered by their phasic nature. DNA methylation (DNAm) signatures of CRP may act as more reliable markers of chronic inflammation. We show that inter-individual differences in DNAm capture 50% of the variance in circulating CRP (N = 17,936, Generation Scotland). We develop a series of DNAm predictors of CRP using state-of-the-art algorithms. An elastic-net-regression-based predictor outperformed competing methods and explained 18% of phenotypic variance in the Lothian Birth Cohort of 1936 (LBC1936) cohort, doubling that of existing DNAm predictors. DNAm predictors performed comparably in four additional test cohorts (Avon Longitudinal Study of Parents and Children, Health for Life in Singapore, Southall and Brent Revisited, and LBC1921), including for individuals of diverse genetic ancestry and different age groups. The best-performing predictor surpassed assay-measured CRP and a genetic score in its associations with 26 health outcomes. Our findings forge new avenues for assessing chronic low-grade inflammation in diverse populations.

High sensitivity of host Helios+/Neuropilin-1+ Treg to pretransplant conditioning hampers development of OX40bright/integrin-ß7+ regulatory cells in acute gastrointestinal GvHD.

This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4+/CD25hi/Foxp3+ Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-ß7, LAG3, TGFß/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios+/Nrp1+ Treg, shifting the balance toward Helios-/Nrp1- Treg in the host. Upregulation of integrin-ß7 and OX40 occurred in GI-aGvHD-dependent manner in Helios+/Nrp1+ cells but not in Helios-/Nrp1- Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-ß7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1+/Helios+ Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.

Selective targeting or reprogramming of intra-tumoral Tregs.

Regulatory T cells (Tregs) are critical immunosuppressive cells that are frequently present in the tumor microenvironment of solid cancers and enable progression of tumors toward metastasis. The cells expand in response to tumor-associated antigens and are actively involved in bypassing immunotherapy with immune checkpoint inhibitors through integrating numerous environmental signals. A point here is that Tregs are clonally distinct in peripheral blood from tumor area. Currently, an effective and novel task in cancer immunotherapy is to selectively destabilize or deplete intra-tumoral Tregs in order to avoid systemic inflammatory events. Helios is a transcription factor expressed selectively by Tregs and promotes their stabilization, and Trps1 is a master regulator of intra-tumoral Tregs. Anti-CCR8 and the IL-2Rßγ agonist Bempegaldesleukin selectively target intra-tumoral Treg population, with the former approved to not elicit autoimmunity. Disarming Treg-related immunosuppression in tumors through diverting their reprogramming or promoting naïve T cell differentiation into cells with effector immune activating profile is another promising area of research in cancer immunotherapy. Blimp-1 inhibitors and glucocorticoid-induced TNFR-related protein agonists are example approaches that can be used for diverting Treg differentiation into Th1-like CD4+ T cells, thereby powering immunogenicity against cancer. Finally, selective target of intra-tumoral Tregs and their reprogramming into effector T cells is applicable using low-dose chemotherapy, and high-salt and high-tryptophan diet.

Helios characterized circulating follicular helper T cells with enhanced functional phenotypes and was increased in patients with systemic lupus erythematosus.

Helios was related to the immunosuppressive capacity and stability of regulatory T cells. However, the significance of Helios in follicular help T (TFH) and follicular regulatory T (TFR) cells is unclear. This research aimed to clarify the significance of Helios (IKZF2) in TFH and TFR cells and its clinical value in systemic lupus erythematosus (SLE). IKZF2 mRNA in different cell subsets was analyzed. Helios+ percentages in TFH and TFR cells were identified in the peripheral blood of 75 SLE patients and 62 HCs (healthy controls). PD-1 and ICOS expression were compared between Helios+ and Helios- cells. The capacity of TFH cells to secrete IL-21 and TFR cells to secrete IL-10 was measured. Correlation analysis and receiver operating characteristic (ROC) curve analysis were conducted to assess the clinical significance of Helios-related TFH and TFR cell subsets in SLE. There was Helios expression in TFH and TFR cells. PD-1 and ICOS were lower in Helios+ TFR than in Helios- TFR. ICOS was increased in Helios+ TFH cells compared with Helios- TFH cells, and ICOS in Helios+ TFH cells was downregulated in SLE. Helios+ TFH cells secreted more IL-21 than Helios- TFH cells, and Helios+ TFH cells from SLE patients had a stronger IL-21 secretion than HCs. Helios+ TFH percentages were negatively correlated with C3 and C4 and positively related to CRP and SLEDAI, and the AUC of Helios+ TFH to distinguish SLE from HC was 0.7959. Helios characterizes circulating TFH cells with enhanced function. Increased Helios+ TFH cells could reflect the autoimmune status of SLE.

Temporal changes in regulatory T cell subsets defined by the transcription factor Helios in stroke and their potential role in stroke-associated infection: a prospective case-control study.

BACKGROUND: Regulatory T cells (Tregs) are involved in the systemic immune response after ischemic stroke. However, their role remains unclear, and the effect appears to be both neuroprotective and detrimental. Treg suppressor function may result in immunodepression and promote stroke-associated infection (SAI). Thus we assume that the bidirectional effects of Tregs may be in part attributed to the intracellular transcription factor Helios. Tregs with Helios expression (H+ Tregs) constitute 70-90% of all Treg cells and more frequently than Helios-negative Tregs (H- Tregs) express molecules recognized as markers of Tregs with suppressor abilities. METHODS AND RESULTS: We prospectively assessed the circulating Treg population with flow cytometry in 52 subjects on days 1, 3, 10 and 90 after ischemic stroke and we compared the results with those obtained in concurrent age-, sex- and vascular risk factor-matched controls. At all studied time points the percentage of H+ Tregs decreased in stroke subjects-D1: 69.1% p < 0.0001; D3: 62.5% (49.6-76.6), p < 0.0001; D10: 60.9% (56.5-72.9), p < 0.0001; D90: 79.2% (50.2-91.7), p = 0.014 vs. controls: 92.7% (81.9-97.0) and the percentage of H- Tregs increased accordingly. In patients with SAI the percentage of pro-suppressor H+ Tregs on post-stroke day 3 was higher than in those without infection (p = 0.03). After adjustment for confounders, the percentage of H+ Tregs on day 3 independently correlated with SAI [OR 1.29; CI 95%: 1.08-1.27); p = 0.02]. Although the percentage of H+ Tregs on day 3 correlated positively with NIHSS score on day 90 (rS = 0.62; p < 0.01) and the infarct volume at day 90 (rS = 0.58; p < 0.05), in regression analysis it was not an independent risk factor. CONCLUSIONS: On the first day after stroke the proportion of H+ vs. H- Tregs changes in favor of pro-inflammatory H- Tregs, and this shift continues toward normalization when assessed on day 90. A higher percentage of pro-suppressive H+ Tregs on day 3 independently correlates with SAI and is associated positively with NIHSS score, but it does not independently affect the outcome and stroke area in the convalescent phase of stroke.

Alterations in Helios+ T cell subsets in peripheral blood of early-stage lung adenocarcinoma patients: Implications for early diagnosis.

OBJECTIVE: This study aimed to investigate the changes and significance of circulating Helios-associated T cell subsets in patients with early-stage lung adenocarcinoma (LUAD). METHODS: Blood samples were collected from 35 healthy controls and 34 patients with early-stage LUAD. Flow cytometry was used to analyze various CD4+ T cell subsets, including regulatory T(Treg) cells, follicular regulatory T(Tfr) cells, follicular helper T (Tfh) cells, and conventional T (con-T) cells. Correlation analysis was conducted to investigate the association of Helios-related subsets with clinical indicators. The ROC curve was used to explore the potential clinical value of Helios+ T cell subsets in the screening of patients with early LUAD. Fifteen of these patients were tracked after lung cancer resection and changes in Helios+ T cell subsets before and after treatment were analyzed. RESULTS: The percentage and absolute number of Tregs were up-regulated in LUAD patients while Tfh and con-T cells expressing Helios were down-regulated. Absolute counts of Tfr and con-T cells and Helios expression in Tfr and Treg decreased significantly after resection. Helios+ Tfh and con-T were negatively correlated with certain tumor markers. Areas under the curve (AUCs) of percentages and absolute counts of Helios+ Tfh, Treg, Tfr and con-T cells to distinguish early LUAD from healthy individuals were 0.7277, 0.5697, 0.5718, 0.7210 (percentages), 0.7336, 0.7378, 0.5908 and 0.7445(absolute numbers), respectively. CONCLUSION: Helios+ T cell subsets in peripheral blood of early-stage LUAD patients has changed significantly, which may be related to the pathogenesis of LUAD and could help for early diagnosis of LUAD.

CD4+ regulatory T cells lacking Helios and Eos.

The transcriptional regulators that drive regulatory T (Treg) cell development and function remain partially understood. Helios (Ikzf2) and Eos (Ikzf4) are closely-related members of the Ikaros family of transcription factors. They are highly expressed in CD4+ Treg cells and functionally important for Treg cell biology, as mice deficient for either Helios or Eos are susceptible to autoimmune diseases. However, it remains unknown if these factors exhibit specific or partially redundant functions in Treg cells. Here we show that mice with germline deletions of both Ikzf2 and Ikzf4 are not very different from animals with single Ikzf2 or Ikzf4 deletions. Double knockout Treg cells differentiate normally, and efficiently suppress effector T cell proliferation in vitro. Both Helios and Eos are required for optimal Foxp3 protein expression. Surprisingly, Helios and Eos regulate different, largely non-overlapping, sets of genes. Only Helios is required for proper Treg cell aging, as Helios deficiency results in reduced Treg cell frequencies in the spleen of older animals. These results indicate that Helios and Eos are required for distinct aspects of Treg cell function.

Co-expression of Foxp3 and Helios facilitates the identification of human T regulatory cells in health and disease.

Foxp3 is regarded as the major transcription factor for T regulatory (Treg) cells and expression of Foxp3 is used to identify and quantitate Treg cells in mouse models. However, several studies have demonstrated that human CD4+ T conventional (Tconv) cells activated in vitro by T cell receptor (TCR) stimulation can express Foxp3. This observation has raised doubt as to the suitability of Foxp3 as a Treg marker in man. Helios, a member of the Ikaros gene family, has been shown to be expressed by 80-90% of human Foxp3+ Treg cells and can potentially serve as a marker of human Treg. Here, we confirm that Foxp3 expression is readily upregulated by Tconv upon TCR stimulation in vitro, while Helios expression is not altered. More importantly, we show that Foxp3 expression is not elevated by stimulation of hTconv in a humanized mouse model of graft versus host disease (GVHD) and in patients with a wide variety of acute and chronic inflammatory diseases including sickle cell disease, acute and chronic GVHD, systemic lupus erythematosus, as well as critical COVID-19. In all patients studied, an excellent correlation was observed between the percentage of CD4+ T cells expressing Foxp3 and the percentage expressing Helios. Taken together, these studies demonstrate that Foxp3 is not induced upon Tconv cell activation in vivo and that Foxp3 expression alone can be used to quantitate Treg cells in humans. Nevertheless, the combined use of Foxp3 and Helios expression provides a more reliable approach for the characterization of Treg in humans.

Human small intestine contains 2 functionally distinct regulatory T-cell subsets.

BACKGROUND: Regulatory T (Treg) CD4 cells in mouse gut are mainly specific for intestinal antigens and play an important role in the suppression of immune responses against harmless dietary antigens and members of the microbiota. However, information about the phenotype and function of Treg cells in the human gut is limited. OBJECTIVE: We performed a detailed characterization of Foxp3+ CD4 Treg cells in human normal small intestine (SI) as well as from transplanted duodenum and celiac disease lesions. METHODS: Treg cells and conventional CD4 T cells derived from SI were subjected to extensive immunophenotyping and their suppressive activity and ability to produce cytokines assessed. RESULTS: SI Foxp3+ CD4 T cells were CD45RA-CD127-CTLA-4+ and suppressed proliferation of autologous T cells. Approximately 60% of Treg cells expressed the transcription factor Helios. When stimulated, Helios-negative Treg cells produced IL-17, IFN-γ, and IL-10, whereas Helios-positive Treg cells produced very low levels of these cytokines. By sampling mucosal tissue from transplanted human duodenum, we demonstrated that donor Helios-negative Treg cells persisted for at least 1 year after transplantation. In normal SI, Foxp3+ Treg cells constituted only 2% of all CD4 T cells, while in active celiac disease, both Helios-negative and Helios-positive subsets expanded 5- to 10-fold. CONCLUSION: The SI contains 2 subsets of Treg cells with different phenotypes and functional capacities. Both subsets are scarce in healthy gut but increase dramatically in active celiac disease.

Decreased frequency of regulatory T cells and level of helios gene expression in secondary progressive multiple sclerosis patients: Evidence about the development of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an aggressive disease characterized by central nervous system (CNS) inflammatory and demyelinating lesions. Tolerance failure is implicated in the development of several autoimmune disorders, including MS. Due to their involvement in maintaining environmental tolerance, regulatory T cells (Tregs) are regarded as efficient immune cells. We examined the frequency of Tregs in this study using CD4/CD25/forkhead box protein P3 (FOXP3)/Helios markers. METHODS: Fifty participants, including 25 patients with secondary progressive MS (SPMS) and 25 healthy controls (HCs), were enrolled in this study, and their demographic characteristics were recorded. Peripheral blood samples ranging from 5 to 6 mL were obtained, and the Ficoll technique was used to extract peripheral blood mononuclear cells (PBMCs). Then, the percentage of CD4+CD25+FOXP3+Helios+ regulatory T lymphocytes was examined by flow cytometry in the study groups. Real-time polymerase chain reaction (PCR) was also used to assess the Helios gene expression level. RESULTS: This study showed that the percentage of Tregs with CD4 and CD25 markers did not reveal a significant difference compared with HCs despite the decrease in SPMS patients (P = 0.6). However, lymphocytes with CD4/CD25/FOXP3/Helios markers were significantly reduced in the patients (P = 0.01). Additionally, SPMS patients had statistically significantly lower Helios gene expression levels (P = 0.002). CONCLUSION: In SPMS patients, a decrease in the frequency of the CD4+CD25+FOXP3+Helios+ Treg population can result in an imbalanced immune system. In other words, one of the immunological mechanisms involved in this disease may be a deficiency in Tregs. Helios gene expression was also decreased in these patients, which may exacerbate functional defects in Tregs.

Helios as a Potential Biomarker in Systemic Lupus Erythematosus and New Therapies Based on Immunosuppressive Cells.

The Helios protein (encoded by the IKZF2 gene) is a member of the Ikaros transcription family and it has recently been proposed as a promising biomarker for systemic lupus erythematosus (SLE) disease progression in both mouse models and patients. Helios is beginning to be studied extensively for its influence on the T regulatory (Treg) compartment, both CD4+ Tregs and KIR+/Ly49+ CD8+ Tregs, with alterations to the number and function of these cells correlated to the autoimmune phenomenon. This review analyzes the most recent research on Helios expression in relation to the main immune cell populations and its role in SLE immune homeostasis, specifically focusing on the interaction between T cells and tolerogenic dendritic cells (tolDCs). This information could be potentially useful in the design of new therapies, with a particular focus on transfer therapies using immunosuppressive cells. Finally, we will discuss the possibility of using nanotechnology for magnetic targeting to overcome some of the obstacles related to these therapeutic approaches.

Treg in inborn errors of immunity: gaps, knowns and future perspectives.

Regulatory T cells (Treg) are essential for immune balance, preventing overreactive responses and autoimmunity. Although traditionally characterized as CD4+CD25+CD127lowFoxP3hi, recent research has revealed diverse Treg subsets such as Tr1, Tr1-like, and CD8 Treg. Treg dysfunction leads to severe autoimmune diseases and immune-mediated inflammatory disorders. Inborn errors of immunity (IEI) are a group of disorders that affect correct functioning of the immune system. IEI include Tregopathies caused by genetic mutations affecting Treg development or function. In addition, Treg dysfunction is also observed in other IEIs, whose underlying mechanisms are largely unknown, thus requiring further research. This review provides a comprehensive overview and discussion of Treg in IEI focused on: A) advances and controversies in the evaluation of Treg extended subphenotypes and function; B) current knowledge and gaps in Treg disturbances in Tregopathies and other IEI including Treg subpopulation changes, genotype-phenotype correlation, Treg changes with disease activity, and available therapies, and C) the potential of Treg cell-based therapies for IEI with immune dysregulation. The aim is to improve both the diagnostic and the therapeutic approaches to IEI when there is involvement of Treg. We performed a non-systematic targeted literature review with a knowledgeable selection of current, high-quality original and review articles on Treg and IEI available since 2003 (with 58% of the articles within the last 6 years) in the PubMed database.

HELIOS-expressing human CD8 T cells exhibit limited effector functions.

Introduction: The transcription factor HELIOS is primarily known for its expression in CD4 regulatory T cells, both in humans and mice. In mice, HELIOS is found in exhausted CD8 T cells. However, information on human HELIOS+ CD8 T cells is limited and conflicting. Methods: In this study, we characterized by flow cytometry and transcriptomic analyses human HELIOS+ CD8 T cells. Results: These T cells primarily consist of memory cells and constitute approximately 21% of blood CD8 T cells. In comparison with memory HELIOS- T-BEThigh CD8 T cells that displayed robust effector functions, the memory HELIOS+ T-BEThigh CD8 T cells produce lower amounts of IFN-γ and TNF-α and have a lower cytotoxic potential. We wondered if these cells participate in the immune response against viral antigens, but did not find HELIOS+ cells among CD8 T cells recognizing CMV peptides presented by HLA-A2 and HLA-B7. However, we found HELIOS+ CD8 T cells that recognize a CMV peptide presented by MHC class Ib molecule HLA-E. Additionally, a portion of HELIOS+ CD8 T cells is characterized by the expression of CD161, often used as a surface marker for identifying TC17 cells. These CD8 T cells express TH17/TC17-related genes encoding RORgt, RORa, PLZF, and CCL20. Discussion: Our findings emphasize that HELIOS is expressed across various CD8 T cell populations, highlighting its significance beyond its role as a transcription factor for Treg or exhausted murine CD8 T cells. The significance of the connection between HELIOS and HLA-E restriction is yet to be understood.

Correlations between Circulating and Tumor-Infiltrating CD4+ Treg Subsets with Immune Checkpoints in Colorectal Cancer Patients with Early and Advanced Stages.

The existence of various T regulatory cell (Treg) subsets in colorectal cancer (CRC) could play a variety of functions in the regulation of anti-cancer immunity. We studied correlations between CD4+ Treg subsets with the expression of immunological checkpoints on CD4+ T cells, including PD-1, TIM-3, LAG-3, and CTLA-4 in CRC patients with early and advanced TNM staging. Strong positive correlations were found between frequencies of FoxP3+ Tregs and FoxP3+Helios+ Tregs with frequencies of various immune checkpoint-expressing CD4+ T cells in the tumor microenvironment (TME). However, there were strong negative correlations between frequencies of FoxP3-Helios- T cells and these immune checkpoint-expressing CD4+ T cells. Specifically, in the TME, we found that the correlations between FoxP3+ Tregs, FoxP3+Helios+ Tregs, FoxP3+Helios- Tregs, and FoxP3-Helios- T cells with CD4+LAG-3+ T cells and CD4+CTLA-4+ T cells were higher in patients with early stages, suggesting the potential of these highly immunosuppressive cells in inhibiting inflammatory responses in the TME. However, the correlations between FoxP3+ Tregs, FoxP3+Helios+ Tregs, and FoxP3-Helios- T cells with CD4+TIM-3+ T cells were higher in patients with advanced stages. This is the first study to explore correlations of Treg subpopulations with immune checkpoint-expressing CD4+ T cells in CRC based on clinicopathological features of CRC patients. The findings of our study provide a justification for focusing on these cells that possess highly immunosuppressive features. Understanding the correlations between different immune checkpoints and Treg subsets in CRC patients has the potential to enhance our understanding of core mechanisms of Treg-mediated immunosuppression in cancer.

Helios Expression Is Downregulated on CD8+ Treg in Two Mouse Models of Lupus During Disease Progression.

T-cell-mediated autoimmunity reflects an imbalance in this compartment that is not restored by tolerogenic immune cells, e.g., regulatory T cells or tolerogenic dendritic cells (tolDCs). Although studies into T-cell equilibrium have mainly focused on regulatory CD4+FoxP3+ T cells (CD4+ Tregs), recent findings on the lesser known CD8+ Tregs (CD44+CD122+Ly49+) have highlighted their non-redundant role in regulating lupus-like disease and their regulatory phenotype facilitated by the transcription factor Helios in mice and humans. However, there are still remaining questions about Helios regulation and dynamics in different autoimmune contexts. Here, we show the absence of CD8+ Tregs in two lupus-prone murine models: MRL/MPJ and MRL/lpr, in comparison with a non-prone mouse strain like C57BL/6. We observed that all MRL animals showed a dramatically reduced population of CD8+ Tregs and a greater Helios downregulation on diseased mice. Helios induction was detected preferentially on CD8+ T cells from OT-I mice co-cultured with tolDCs from C57BL/6 but not in MRL animals. Furthermore, the Helios profile was also altered in other relevant T-cell populations implicated in lupus, such as CD4+ Tregs, conventional CD4+, and double-negative T cells. Together, these findings could make Helios a versatile maker across the T-cell repertoire that is capable of differentiating lupus disease states.

Natural and Induced Tolerance to Hymenoptera Venom: A Single Mechanism?

Inducing tolerance in Hymenoptera-allergic patients, bee venom immunotherapy (BVIT) is a widely accepted method to treat severe allergy to bee stings. In order to increase the existing knowledge on the underlying immunological mechanisms and look for possible biomarkers predictive of efficacy, a group of 20 bee-venom-allergic patients (AG) were thoroughly examined during their first year of BVIT. In addition, the results of treated patients with those of an untreated group of 20 tolerant beekeepers (TG) who had previously shown a firm suppressor-regulatory profile were compared. Tolerance in AG patients was invariably associated with a significant regulatory response characterised by the expansion of Helios- subpopulation and increased IL-10, specific IgG4 (sIgG4), and kynurenine levels. Although specific IgE (sIgE) levels increased transiently, surprisingly, the T helper type 2 (Th2) population and IL-4 levels rose significantly after one year of immunotherapy. Thus, the picture of two parallel phenomena emerges: a tolerogenic response and an allergenic one. Comparing these results with those obtained from the TG, different immunological mechanisms appear to govern natural and acquired tolerance to immunotherapy. Of particular interest, the kynurenine levels and T regulatory (Treg) Helios- population could be proposed as new biomarkers of response to BVIT.

Regulatory T Cells from Patients with Rheumatoid Arthritis Are Characterized by Reduced Expression of Ikaros Zinc Finger Transcription Factors.

Regulatory T (Treg) cells play an important role in immune tolerance and contribute to the prevention of autoimmune diseases, including rheumatoid arthritis (RA). The differentiation, function and stability of Treg cells is controlled by members of the Ikaros zinc finger transcription factor family. In this study, we aimed to reveal how the expression of Ikaros transcription factors is affected by disease activity in RA. Therefore, we analyzed the ex vivo expression of Ikaros, Helios, Aiolos and Eos in Treg cells, Th17 cells and Th1 cells from RA patients by flow cytometry. We found significantly reduced expression of Helios, Aiolos and Eos in Treg cells from RA patients as compared to healthy controls. Moreover, Helios and Aiolos levels correlated with disease activity, as assessed by DAS28-CRP. In addition, Ikaros, Helios and Aiolos were significantly downregulated in Th1 cells from RA patients, while no difference between healthy individuals and RA was observed in Th17 cells. In summary, Helios and Aiolos expression in Treg cells correlates with disease activity and the expression levels of Ikaros transcription factors are diminished in Treg cells from RA patients. This observation could explain the reduced stability of Treg cells in RA.

Associations of different immune checkpoints-expressing CD4+ Treg/ T cell subsets with disease-free survival in colorectal cancer patients.

There are different subsets of T regulatory cells (Tregs), orchestrating critical roles in the regulation of anti-tumor immunity in colorectal cancer (CRC). In this study, we report that a high frequency of circulating CD4+FoxP3+ Tregs was associated with poorer disease-free survival (DFS), while their higher frequencies in tumor-infiltrating CD4+ Tregs was associated with better DFS. We further investigated such associations with four Tregs/T cells expressing or lacking FoxP3 and Helios (FoxP3±Helios±). For the first time, we report that a high frequency of circulating CD4+FoxP3+Helios+ Tregs was associated with poorer DFS, while a high frequency of tumor-infiltrating CD4+FoxP3-Helios- T cells was associated with poorer DFS. In the four FoxP3±Helios± T cell subsets expressing any of the immune checkpoints (ICs) investigated, we found that a high frequency of CD4+FoxP3+Helios-PD-1+ Tregs in circulation was associated with worse DFS. We also found that high frequencies of FoxP3+Helios+CTLA-4+ Tregs, FoxP3+Helios-CTLA-4+ Tregs, and FoxP3-Helios+CTLA-4+ CD4+ T cells in circulation were associated with worse DFS. In contrast, high frequencies of CD4+TIM-3+ T cells, FoxP3+Helios+TIM-3+ Tregs, and FoxP3-Helios+TIM-3+ CD4+ T cells in circulation were associated with longer DFS. Our data show that certain CD4+ Treg/T cell subsets could serve as independent predictive biomarkers in CRC patients. Identification of the exact subpopulations contributing to clinical outcomes is critical for prognoses and therapeutic targeting.

Co-Expression of TIGIT and Helios Marks Immunosenescent CD8+ T Cells During Aging.

Aging leads to alterations in the immune system that result in ineffective responsiveness against pathogens. Features of this process, collectively known as immunosenescence, accumulate in CD8+ T cells with age and have been ascribed to differentiation of these cells during the course of life. Here we aimed to identify novel markers in CD8+ T cells associated with immunosenescence. Furthermore, we assessed how these markers relate to the aging-related accumulation of highly differentiated CD27-CD28- cells. We found that co-expression of the transcription factor Helios and the aging-related marker TIGIT identifies CD8+ T cells that fail to proliferate and show impaired induction of activation markers CD69 and CD25 in response to stimulation in vitro. Despite this, in blood of older adults we found TIGIT+Helios+ T cells to become highly activated during an influenza-A virus infection, but these higher frequencies of activated TIGIT+Helios+ T cells associate with longer duration of coughing. Moreover, in healthy individuals, we found that TIGIT+Helios+ CD8+ T cells accumulate with age in the highly differentiated CD27-CD28- population. Interestingly, TIGIT+Helios+ CD8+ T cells also accumulate with age among the less differentiated CD27+CD28- T cells before their transit into the highly differentiated CD27-CD28- stage. This finding suggests that T cells with immunosenescent features become prominent at old age also within the earlier differentiation states of these cells. Our findings show that co-expression of TIGIT and Helios refines the definition of immunosenescent CD8+ T cells and challenge the current dogma of late differentiation stage as proxy for T-cell immunosenescence.