The expanding universe of type I regulatory T cell biology: a new role in cancer immunotherapy.

In this article, we discuss new findings which suggest that type I regulatory T (Tr1) cells can interfere with cancer vaccine efficacy in mice by exerting strong regulatory control over antitumor immune responses.

Tolerogenic IL-10-engineered dendritic cell-based therapy to restore antigen-specific tolerance in T cell mediated diseases.

Tolerogenic dendritic cells play a critical role in promoting antigen-specific tolerance via dampening of T cell responses, induction of pathogenic T cell exhaustion and antigen-specific regulatory T cells. Here we efficiently generate tolerogenic dendritic cells by genetic engineering of monocytes with lentiviral vectors co-encoding for immunodominant antigen-derived peptides and IL-10. These transduced dendritic cells (designated DCIL-10/Ag) secrete IL-10 and efficiently downregulate antigen-specific CD4+ and CD8+ T cell responses from healthy subjects and celiac disease patients in vitro. In addition, DCIL-10/Ag induce antigen-specific CD49b+LAG-3+ T cells, which display the T regulatory type 1 (Tr1) cell gene signature. Administration of DCIL-10/Ag resulted in the induction of antigen-specific Tr1 cells in chimeric transplanted mice and the prevention of type 1 diabetes in pre-clinical disease models. Subsequent transfer of these antigen-specific T cells completely prevented type 1 diabetes development. Collectively these data indicate that DCIL-10/Ag represent a platform to induce stable antigen-specific tolerance to control T-cell mediated diseases.

Immune tolerance in multiple sclerosis and neuromyelitis optica with peptide-loaded tolerogenic dendritic cells in a phase 1b trial.

There are adaptive T-cell and antibody autoimmune responses to myelin-derived peptides in multiple sclerosis (MS) and to aquaporin-4 (AQP4) in neuromyelitis optica spectrum disorders (NMOSDs). Strategies aimed at antigen-specific tolerance to these autoantigens are thus indicated for these diseases. One approach involves induction of tolerance with engineered dendritic cells (tolDCs) loaded with specific antigens. We conducted an in-human phase 1b clinical trial testing increasing concentrations of autologous tolDCs loaded with peptides from various myelin proteins and from AQP4. We tested this approach in 12 patients, 8 with MS and 4 with NMOSD. The primary end point was the safety and tolerability, while secondary end points were clinical outcomes (relapses and disability), imaging (MRI and optical coherence tomography), and immunological responses. Therapy with tolDCs was well tolerated, without serious adverse events and with no therapy-related reactions. Patients remained stable clinically in terms of relapse, disability, and in various measurements using imaging. We observed a significant increase in the production of IL-10 levels in PBMCs stimulated with the peptides as well as an increase in the frequency of a regulatory T cell, known as Tr1, by week 12 of follow-up. In this phase 1b trial, we concluded that the i.v. administration of peptide-loaded dendritic cells is safe and feasible. Elicitation of specific IL-10 production by peptide-specific T cells in MS and NMOSD patients indicates that a key element in antigen specific tolerance is activated with this approach. The results warrant further clinical testing in larger trials.

Effect of IL-27, Teriflunomide and Retinoic Acid and Their Combinations on CD4+ T Regulatory T Cells-An In Vitro Study.

The principal goal of the study was to verify the concept of pharmacological induction of Foxp3+CD25+CD4+ T regulatory (Treg) cells which will additionally be characterized by a highly suppressive phenotype, i.e., by extensive CD25 and CD39 expression and IL-10 and TGF-ß production. Stimulated and unstimulated murine lymphocytes were exposed to IL-27, teriflunomide (TER), and all trans retinoic acid (ATRA) alone and to their combinations. The study demonstrated that: (a) IL-27 alone induced CD39 expression on Treg cells and the generation of Tr1 cells; (b) TER alone induced Foxp3-expressing CD4+ T cells and up-regulated density of CD25 on these cells; TER also induced the ability of Treg cells to TGF-ß production; (c) ATRA alone induced CD39 expression on Treg cells. The experiments revealed a strong superadditive effect between IL-27 and ATRA with respect to increasing CD39 expression on Treg cells. Moreover, IL-27 and ATRA in combination, but not alone, induced the ability of Treg cells to IL-10 production. However, the combination of IL-27, TER, and ATRA did not induce the generation of Treg cell subset with all described above features. This was due to the fact that TER abolished all listed above desired effects induced by IL-27 alone, ATRA alone, and their combination. IL-27 alone, ATRA alone, and their combination affected TER-induced effects to a lesser extent. Therefore, it can be concluded that in the aspect of pharmacological induction of Treg cells with a highly suppressive phenotype, the triple combination treatment with TER, IL-27, and ATRA does not provide any benefits over TER alone or dual combination including IL-27 and ATRA.

TCR/ITK Signaling in Type 1 Regulatory T cells.

Type 1 regulatory T (Tr1) cells can modulate inflammation through multiple direct and indirect molecular and cellular mechanisms and have demonstrated potential for anti-inflammatory therapies. Tr1 cells do not express the master transcription factor of conventional regulatory T cells, Foxp3, but express high levels of the immunomodulatory cytokine, IL-10. IL-2-inducible T-cell kinase (ITK) is conserved between mouse and human and is highly expressed in T cells. ITK signaling downstream of the T-cell receptor (TCR) is critical for T-cell subset differentiation and function. Upon activation by TCR, ITK is critical for Ras activation, leading to downstream activation of MAPKs and upregulation of IRF4, which further enable Tr1 cell differentiation and suppressive function. We summarize here the structure, signaling pathway, and function of ITK in T-cell lineage designation, with an emphasis on Tr1 cell development and function.

Association between IL-27 and Tr1 cells in severe form of paracoccidioidomycosis.

Interleukin-27, a cytokine of the IL-12 family, is secreted by antigen-presenting cells such as macrophages and dendritic cells (DCs). Recent studies suggest an anti-inflammatory role for IL-27 by inducing IL-10 producing Tr1 cells capable of inhibiting Th1 and Th17 type responses. Our study aimed to investigate the involvement of IL-27 and Tr1 cells in the immunomodulation of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Brazil. The presence of IL-27 was evaluated in serum and biopsies of patients with PCM by ELISA, immunohistochemistry, and immunofluorescence. The presence of Tr1 in peripheral blood was analyzed by flow cytometry. In vitro assays were performed to verify the ability of P. brasiliensis yeast to induce IL-27 production by DCs and macrophages, as well as the polarization of lymphocytes to the Tr1 phenotype. Patients with the acute form and severe chronic form, the most severe and disseminated forms of PCM, presented higher serum concentrations of IL-27 and higher percentage of Tr1 cells compared to patients with mild chronic form. IL-27 was also detected in lesions of patients with PCM and associated with DCs and macrophages. P. brasiliensis Pb18 yeasts were able to induce IL-27 production by both DCs and macrophages. We found that DCs pulsed with Pb18 were able to induce Tr1 lymphocytes in vitro. Our data suggest that IL-27 and Tr1 cells could contribute to the deficient immune response to P. brasiliensis that leads to severe and disseminated forms of the disease.

Granulysin- and granzyme-dependent elimination of myeloid cells by therapeutic ova-specific type 1 regulatory T cells.

The intrinsic immunosuppressive properties of regulatory T (Treg) cells can be harnessed for therapeutic approaches aiming at down-modulating harmful immune reactions. In this context, expanded type 1 Treg cells (Tr1 cells) specific for ovalbumin (ova-Tr1 cells) have been tested for clinical efficacy in the treatment of autoimmune disorders such as refractory Crohn's disease (CD). The clinical use of these therapeutic products warrants exploration of their mechanism of action. Here, we identified a relationship between the CD activity index and the expression of lytic molecules by the ova-Tr1 cells administered in the previously reported First-in-Man study [Crohn's And Treg cells Study 1 (CATS1) study]. Accordingly, ova-Tr1 cells were found to carry granules containing high levels of lytic molecules, including multiple granzymes and granulysin. These cells displayed a T-cell receptor (TCR)-independent cytotoxic activity, which was preferentially directed toward myeloid cell lines and monocyte-derived dendritic cells. Upon contact with myeloid cells, ova-Tr1 cells induced their apoptosis via a perforin-independent and a granulysin/granzyme-dependent mechanism. As compared to CD8+ cytotoxic T cells, ova-Tr1 cells required more time to lyse target cells and displayed a more gradual lytic activity over time. Notably, this activity was sustained over days resulting in the control of myeloid cell populations at a relatively low ratio. Our study reveals that ova-Tr1 cells are endowed with a sustained cytotoxic activity that relies on a unique combination of granulysin and granzymes and that preferentially eliminates myeloid target cells in a TCR-independent manner.

Treg-specific IL-27Rα deletion uncovers a key role for IL-27 in Treg function to control autoimmunity.

Dysregulated Foxp3+ Treg functions result in uncontrolled immune activation and autoimmunity. Therefore, identifying cellular factors modulating Treg functions is an area of great importance. Here, using Treg-specific Il27ra-/- mice, we report that IL-27 signaling in Foxp3+ Tregs is essential for Tregs to control autoimmune inflammation in the central nervous system (CNS). Following experimental autoimmune encephalomyelitis (EAE) induction, Treg-specific Il27ra-/- mice develop more severe EAE. Consistent with the severe disease, the numbers of IFNγ- and IL-17-producing CD4 T cells infiltrating the CNS tissues are greater in these mice. Treg accumulation in the inflamed CNS tissues is not affected by the lack of IL-27 signaling in Tregs, suggesting a functional defect of Il27ra-/- Tregs. IL-10 production by conventional CD4 T cells and their CNS accumulation are rather elevated in Treg-specific Il27ra-/- mice. Analysis with Treg fate-mapping reporter mice further demonstrates that IL-27 signaling in Tregs may control stability of Foxp3 expression. Finally, systemic administration of recombinant IL-27 in Treg-specific Il27ra-/- mice fails to ameliorate the disease even in the presence of IL-27-responsive conventional CD4 T cells. These findings uncover a previously unknown role of IL-27 in regulating Treg function to control autoimmune inflammation.

Intestinal type 1 regulatory T cells migrate to periphery to suppress diabetogenic T cells and prevent diabetes development.

Growing insight into the pathogenesis of autoimmune diseases and numerous studies in preclinical models highlights the potential of regulatory T cells to restore tolerance. By using non-obese diabetic (NOD) BDC2.5 TCR-transgenic (Tg), and IL-10 and Foxp3 double-reporter mice, we demonstrate that alteration of gut microbiota during cohousing experiments or treatment with anti-CD3 mAb significantly increase intestinal IL-10-producing type 1 regulatory T (Tr1) cells and decrease diabetes incidence. These intestinal antigen-specific Tr1 cells have the ability to migrate to the periphery via a variety of chemokine receptors such as CCR4, CCR5, and CCR7 and to suppress proliferation of Th1 cells in the pancreas. The ability of Tr1 cells to cure diabetes in NOD mice required IL-10 signaling, as Tr1 cells could not suppress CD4+ T cells with a dominant-negative IL-10R. Taken together, our data show a key role of intestinal Tr1 cells in the control of effector T cells and development of diabetes. Therefore, modulating gut-associated lymphoid tissue to boost Tr1 cells may be important in type 1 diabetes management.

Tolerising cellular therapies: what is their promise for autoimmune disease?

The current management of autoimmunity involves the administration of immunosuppressive drugs coupled to symptomatic and functional interventions such as anti-inflammatory therapies and hormone replacement. Given the chronic nature of autoimmunity, however, the ideal therapeutic strategy would be to reinduce self-tolerance before significant tissue damage has accrued. Defects in, or defective regulation of, key immune cells such as regulatory T cells have been documented in several types of human autoimmunity. Consequently, it has been suggested that the administration of ex vivo generated, tolerogenic immune cell populations could provide a tractable therapeutic strategy. Several potentially tolerogenic cellular therapies have been developed in recent years; concurrent advances in cell manufacturing technologies promise scalable, affordable interventions if safety and efficacy can be demonstrated. These therapies include mesenchymal stromal cells, tolerogenic dendritic cells and regulatory T cells. Each has advantages and disadvantages, particularly in terms of the requirement for a bespoke versus an 'off-the-shelf' treatment but also their suitability in particular clinical scenarios. In this review, we examine the current evidence for these three types of cellular therapy, in the context of a broader discussion around potential development pathway(s) and their likely future role. A brief overview of preclinical data is followed by a comprehensive discussion of human data.

The IL-10/STAT3 axis: Contributions to immune tolerance by thymus and peripherally derived regulatory T-cells.

The signal transducer and activator of transcription (STAT) proteins are important mediators for the integration of extrinsic signals provided by cytokines and hormones and thereby adapt cellular processes to their surroundings. In the past decade, the involvement of STAT3 in the regulation of T-cell responses has become a topic of increasing interest. STAT3 is activated in response to multiple cytokines, many of which have been shown to influence T-cell responses. Interestingly, many of these factors have been described with apparent opposing roles, such as the highly pro-inflammatory potency of IL-6 and the anti-inflammatory properties of IL-10, thus raising the possibility that STAT3 signaling may fulfill diverse roles in CD4+ T-cells. Here, we review the contribution of STAT3 to the induction and function of both peripherally induced as well as thymus-derived regulatory T-cells. Indeed, experimental approaches as well as studies of human patients suffering from e.g. Job's (hyper IgE) syndrome or inflammatory bowel disease (IBD) have now established a clear-cut role for the IL-10/STAT3 axis in immune tolerance; further understanding of these processes could lead to novel therapeutic approaches for autoimmune diseases.

AhR activation increases IL-2 production by alloreactive CD4+ T cells initiating the differentiation of mucosal-homing Tim3+ Lag3+ Tr1 cells.

Activation of the aryl hydrocarbon receptor (AhR) by immunosuppressive ligands promotes the development of regulatory T (Treg) cells. Although AhR-induced Foxp3+ Treg cells have been well studied, much less is known about the development and fate of AhR-induced Type 1 Treg (AhR-Tr1) cells. In the current study, we identified the unique transcriptional and functional changes in murine CD4+ T cells that accompany the differentiation of AhR-Tr1 cells during the CD4+ T-cell-dependent phase of an allospecific cytotoxic T lymphocyte (allo-CTL) response. AhR activation increased the expression of genes involved in T-cell activation, immune regulation and chemotaxis, as well as a global downregulation of genes involved in cell cycling.  Increased IL-2 production was responsible for the early AhR-Tr1 activation phenotype previously characterized as CD25+ CTLA4+ GITR+ on day 2. The AhR-Tr1 phenotype was further defined by the coexpression of the immunoregulatory receptors Lag3 and Tim3 and non-overlapping expression of CCR4 and CCR9. Consistent with the increased expression of CCR9, real-time imaging showed enhanced migration of AhR-Tr1 cells to the lamina propria of the small intestine and colon. The discovery of mucosal imprinting of AhR-Tr1 cells provides an additional mechanism by which therapeutic AhR ligands can control immunopathology.

Interleukin-10 Gene-Modified Dendritic Cell-Induced Type 1 Regulatory T Cells Induce Transplant-Tolerance and Impede Graft Versus Host Disease After Allogeneic Stem Cell Transplantation.

BACKGROUND/AIMS: Tr1 cells can induce peripheral tolerance to self- and foreign antigens, and have been developed as a therapeutic tool for the induction of tolerance to transplanted tissue. We explored the feasibility of generating Tr1 cells by using IL-10 gene-modified recipient DCs (DCLV-IL-10) to stimulate donor naive CD4+ T cells. We also investigated some biological properties of Tr1 cells. METHODS: DCLV-IL-10 were generated through DCs transduced with a lentivirus vector carrying the IL-10 gene, and Tr1 cells were produced by using DCLV-IL-10 to stimulate naive CD4+ T cells. The effects of Tr1 cells on T-cell proliferation and the occurrence of graft versus host disease (GVHD) following allogeneic stem-cell transplantation (allo-HSCT) were investigated. RESULTS: The DCLV-IL-10-induced Tr1 cells co-expressed LAG-3 and CD49b. Moreover, they also expressed CD4, CD25, and IL-10, but not Foxp3, and secreted significantly higher levels of IL-10 (1,729.36 ± 185.79 pg/mL; P < 0.001) and INF-γ (1,524.48 ± 168.65 pg/mL; P < 0.01) than the control T cells upon the stimulation by allogeneic DCs. Tr1 cells markedly suppressed T-lymphocyte proliferation and the mixed lymphocytic response (MLR) in vitro. The mice used in the allo-HSCT model had longer survival times and lower clinical and pathological GVHD scores than the control mice. CONCLUSION: IL-10 gene-modified DC-induced Tr1 cells may be used as a potent cellular therapy for the prevention of GVHD after allo-HSCT.

The aryl hydrocarbon receptor/microRNA-212/132 axis in T cells regulates IL-10 production to maintain intestinal homeostasis.

Aryl hydrocarbon receptor (Ahr), a transcription factor, plays a critical role in autoimmune inflammation of the intestine. In addition, microRNAs (miRNAs), small non-coding oligonucleotides, mediate pathogenesis of inflammatory bowel diseases (IBD). However, the precise mechanism and interactions of these molecules in IBD pathogenesis have not yet been investigated. We analyzed the role of Ahr and Ahr-regulated miRNAs in colonic inflammation. Our results show that deficiency of Ahr in intestinal epithelial cells in mice exacerbated inflammation in dextran sodium sulfate-induced colitis. Deletion of Ahr in T cells attenuated colitis, which was manifested by suppressed Th17 cell infiltration into the lamina propria. Candidate miRNA analysis showed that induction of colitis elevated expression of the miR-212/132 cluster in the colon of wild-type mice, whereas in Ahr (-/-) mice, expression was clearly lower. Furthermore, miR-212/132(-/-) mice were highly resistant to colitis and had reduced levels of Th17 cells and elevated levels of IL-10-producing CD4(+) cells. In vitro analyses revealed that induction of type 1 regulatory T (Tr1) cells was significantly elevated in miR-212/132(-/-) T cells with increased c-Maf expression. Our findings emphasize the vital role of Ahr in intestinal homeostasis and suggest that inhibition of miR-212/132 represents a viable therapeutic strategy for treating colitis.

STAT3 governs hyporesponsiveness and granzyme B-dependent suppressive capacity in human CD4+ T cells.

Signal transducer and activator of transcription 3 (STAT3) integrates key signals of cell surface immune receptors, yet its precise role in cluster of differentiation (CD)4(+) T cells is not well-established. Current research has indicated T-helper cell 17-inducing roles but also tolerogenic roles. To address this issue, human T cells were transduced with the constitutively active STAT3 mutant STAT3C. Following stimulation, STAT3C(+) T cells up-regulated IL-10 (4.1 ± 0.5-fold; P < 0.001) and granzyme B (2.5 ± 1.2, P < 0.05) secretion, combined with significantly reduced IFN-γ (35 ± 5%), IL-2 (57 ± 4%), TNF-α (64 ± 8%), and IL-13 (89 ± 3%) secretion (P < 0.001). CD3/CD2- or CD3/CD28-activated STAT3C(+) T cells revealed reduced proliferation (53.4 ± 23.5% and 70.5 ± 10.4%, respectively), which was independent of IL-10 production and significantly suppressed effector T cell proliferation by 68.7 ± 10.6% and 65.9 ± 2.6%, respectively (P < 0.001). Phenotypically, STAT3C-transgenic CD4(+) T cells resembled effector T cells regarding expression of T regulatory cell markers, but up-regulated granzyme B expression levels by 2.4-fold (P < 0.05). Suppression was cell contact dependent and mediated by granzyme B-induced cell death, but was independent of IL-10 and TGF-ß. Notably, peripheral blood CD4(+)CD45RA(-)lymphocyte activation gene-3(+)CD49(+) type 1 regulatory T cells revealed activation-induced hyperphosphorylation of STAT3. In agreement, pharmacological inhibition of STAT3 activation partially reverted hyporesponsiveness of peripheral type 1 regulatory T cells (increasing their division index from 0.46 ± 0.11 to 0.89 ± 0.04; P < 0.01). These observations indicate a clear-cut relation between activation of STAT3 and the acquisition of a tolerogenic program, which is also used by peripheral blood type 1 regulatory T cells.

Tr-1-like CD4+CD25-CD127-/lowFOXP3- cells are the main source of interleukin 10 in patients with cutaneous leishmaniasis due to Leishmania braziliensis.

CD4(+)CD25(+)FOXP3(+) regulatory T cells have long been shown to mediate susceptibility to Leishmania infection, mainly via interleukin 10 production. In this work, we showed that the main sources of interleukin 10 in peripheral blood mononuclear cells (PBMCs) from patients with cutaneous leishmaniasis due to Leishmania braziliensis are CD4(+)CD25(-)CD127(-/low)FOXP3(-) cells. Compared with uninfected controls, patients with CL had increased frequencies of circulating interleukin 10-producing CD4(+)CD25(-)CD127(-/low) cells, which efficiently suppressed tumor necrosis factor α production by the total PBMC population. Also, in CL lesions, interleukin 10 was mainly produced by CD4(+)CD25(-) cells, and interleukin 10 messenger RNA expression was associated with interleukin 27, interleukin 21, and interferon γ expression, rather than with FOXP3 or transforming growth factor ß expressions. Active production of both interleukin 27 and interleukin 21, together with production of interferon γ and interleukin 10, was also detected in the lesions. Since these cytokines are associated with the differentiation and activity of Tr-1 cells, our results suggest that this cell population may play an important role in the immunomodulation of CL. Therefore, development of treatments that interfere with this pathway may lead to faster parasite elimination.

Adult-onset type 1 diabetes patients display decreased IGRP-specific Tr1 cells in blood.

The breakdown of immune tolerance against islet antigens causes type 1 diabetes (T1D). The antigens associated with adult-onset T1D (AT1D) remain largely undefined. It is possible that AT1D patients display a unique type of CD4(+) T cells specific for a certain islet antigen. Here we analyzed the cytokine production profiles of CD4(+) helper T (Th) cells that are specific for three islet antigens; GAD65, preproinsulin, and IGRP in patients with AT1D, juvenile-onset T1D (JT1D), and age-, gender- and human leukocyte antigen (HLA)-matched control adults. While IGRP-specific Th cells in AT1D patients were dominantly Th1 cells, IGRP-specific Th cells in control adults and JT1D patients were dominantly Th2 and T regulatory type 1 (Tr1) cells. Notably, the frequency of IGRP-specific Tr1 cells was significantly lower in AT1D patients than in control adults and JT1D patients. In conclusion, our study suggests that IGRP-specific Th cells play a unique pathogenic role in AT1D.

Type I interferon potentiates T-cell receptor mediated induction of IL-10-producing CD4⁺ T cells.

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti-inflammatory activity. We analyzed the integrated effect of IFN-α, TCR signal strength, and CD28 costimulation on human CD4⁺ T-cell differentiation into cell subsets producing the anti- and proinflammatory cytokines IL-10 and IFN-γ. We show that IFN-α boosted TCR-induced IL-10 expression in activated peripheral CD45RA⁺CD4⁺ T cells and in whole blood cultures. The functional cooperation between TCR and IFN-α efficiently occurred at low engagement of receptors. Moreover, IFN-α rapidly cooperated with anti-CD3 stimulation alone. IFN-α, but not IL-10, drove the early development of type I regulatory T cells that were mostly IL-10⁺ Foxp3⁻ IFN-γ⁻ and favored IL-10 expression in a fraction of Foxp3⁺ T cells. Our data support a model in which IFN-α costimulates TCR toward the production of IL-10 whose level can be amplified via an autocrine feedback loop.

Phosphatidylserine promotes immunotherapy for airway allergy.

Type 1 regulatory T cells (Tr1 cells) play an important role in the maintenance of the immune homeostasis in the body. The induction of Tr1 cell is to be further investigated. The interaction of phosphatidylserine (PS) with TIM3 has immune regulation functions. The objective of this study is to elucidate the role of PS-TIM3 signals in inducing Tr1 cells. In this study, mice were treated using PS or specific immunotherapy by nasal instillation. A murine model of allergic rhinitis was developed using ovalbumin as a specific antigen. We found that PS-containing nasal instillation induced Tr1 cells in the airway tissues. PS promoted gene activities associated with IL-10 through activation of TIM3 in CD4+ T cells. TIM3 mediated the effects of PS on inducing Tr1 cells, in which the TIM3- PI3K-AKT pathway played a critical role. PS boosted allergen-specific immunotherapy by inducing specific antigen Tr1 cell generation. Concomitant administration of PS and SIT resulted in better therapeutic effects on AR. In conclusion, the data demonstrate that PS can promote the specific immunotherapy for AR through inducing antigen specific Tr1 cells in the airway tissues.

Leptin favors imbalance of antigen-specific CD4+ T-cells associated with severity of cat allergy.

Introduction: Obesity can complicate IgE-mediated allergic diseases. In the present study, we aimed to investigate the ability of obesity-related concentrations of leptin to modulate the in vitro effector and regulatory Fel d1-specific CD4+ T-cell subsets in patients allergic to cat, considered the third most common cause of respiratory allergy in humans. Methods: For this study, plasma and peripheral blood mononuclear cells (PBMC) from 30 cat-allergic patients with mild, moderate and severe respiratory symptoms were obtained. The PBMC cultures were stimulated with Fel d1 antigen (10 µg/mL) in the presence or absence of obesity-related leptin dose (50 ηg/mL). After 6 days, the levels of cytokines and IgE in the supernatants were evaluated by multiplex and ELISA, respectively. The frequency of different non-follicular (CXCR5-) and follicular (CXCR5+) Fel d1-specific CD4+ T cell subsets was determined by flow cytometry. The plasma levels of leptin and IgE anti-cat titers were evaluated by ELISA and ImmunoCAP, respectively. Results and conclusions: Fel d1 induced both IgE production and release of cytokines related to Th2, Th9 and Th17 cell phenotypes. Feld1 was more efficient in increasing the frequency of TFHIL-21- cells positive for IL-4, IL-5 and IL-13 than TFHIL-21+ cell subsets. Leptin favored the expansion Th2-like and Th9-like cells and TFHIL-21- cells positive for IL-4, IL-5 and IL-13, but reduced the proportion of conventional (Treg/Tr-1) and follicular (TFR) regulatory CD4+ T-cell subsets expressing or not CD39 marker. Finally, many of the imbalances between Fel d1-specific CD4+ T-cells were also correlated with plasma leptin and anti-Fel d1 IgE titers. In summary, hyperleptinemia should negatively impact on the severity of cat allergies by favoring the expansion of pathogenic Fel d1-specific CD4+ T-cell phenotypes and damaging the functional status of regulatory CD4+ T-cell subsets.