Interleukin-2-induced skin inflammation.

Recombinant human IL-2 has been used to treat inflammatory diseases and cancer; however, side effects like skin rashes limit the use of this therapeutic. To identify key molecules and cells inducing this side effect, we characterized IL-2-induced cutaneous immune reactions and investigated the relevance of CD25 (IL-2 receptor α) in the process. We injected IL-2 intradermally into WT mice and observed increases in immune cell subsets in the skin with preferential increases in frequencies of IL-4- and IL-13-producing group 2 innate lymphoid cells and IL-17-producing dermal γδ T cells. This overall led to a shift toward type 2/type 17 immune responses. In addition, using a novel topical genetic deletion approach, we reduced CD25 on skin, specifically on all cutaneous cells, and found that IL-2-dependent effects were reduced, hinting that CD25 - at least partly - induces this skin inflammation. Reduction of CD25 specifically on skin Tregs further augmented IL-2-induced immune cell infiltration, hinting that CD25 on skin Tregs is crucial to restrain IL-2-induced inflammation. Overall, our data support that innate lymphoid immune cells are key cells inducing side effects during IL-2 therapy and underline the significance of CD25 in this process.

Non-IL-2-blocking anti-CD25 antibody inhibits tumor growth by depleting Tregs and has synergistic effects with anti-CTLA-4 therapy.

CD25, also known as the interleukin-2 receptor α chain (IL-2Rα), is highly expressed on regulatory T cells (Tregs), but relatively lower on effector T cells (Teffs). This makes it a potential target for Treg depletion, which can be used in tumor immunotherapy. However, marketed anti-CD25 antibodies (Basiliximab and Daclizumab) were originally developed as immunosuppressive drugs to prevent graft rejection, because these antibodies can block IL-2 binding to CD25 on Teffs, which in turn destroys the function of Teffs. Recent studies have shown that non-IL-2-blocking anti-CD25 antibodies have displayed exciting antitumor effects. Here, we screened out a non-IL-2-blocking anti-CD25 monoclonal antibody (mAb) 7B7 by hybridoma technology, and confirmed its antitumor activity via depleting Tregs in a CD25 humanized mouse model. Subsequently, we verified that the humanized 7B7, named as h7B7-15S, has comparable activities to 7B7, and that its Treg depletion is further increased when combined with anti-CTLA-4, leading to enhanced remodeling of the tumor immune microenvironment. Moreover, our findings reveal that the Fab form of h7B7-15S has the ability to deplete Tregs, independent of the Fc region. Taken together, our studies expand the application of anti-CD25 in tumor immunotherapy and provide insight into the underlying mechanism.

Celastrol, which targets IL-2/CD25 binding inhibition, induces T cell-mediated antitumor activity in melanoma.

Interleukin-2 (IL-2) induces contrasting immune responses depending on its binding receptor subunit; thus, selective receptor binding is considered a key challenge in cancer therapeutic strategies. In this study, we aimed to investigate the inhibition of IL-2 action and antitumor activity of celastrol (CEL), a compound identified in a screen for IL-2/CD25 binding inhibitors, and to elucidate the underlying role of CEL in immune cells. We found that CEL selectively impairs the binding of IL-2 and CD25 and directly binds to IL-2 but not to CD25. CEL significantly suppressed the proliferation and signaling of IL-2-dependent murine T cells and interfered with IL-2-responsive STAT5 phosphorylation in IL-2 reporter cells and human PBMCs. After confirming the impact of CEL on IL-2, we evaluated its antitumor activity in C57BL/6 mice bearing B16F10 tumors and found that CEL significantly inhibited tumor growth by increasing CD8+ T cells. We also found that CEL did not inhibit tumor growth in T cell-deficient BALB/c nude mice, suggesting that its activity was mediated by the T-cell response. Moreover, combination therapy with low-dose CEL and a TNFR2 antagonist synergistically improved the therapeutic efficacy of the individual monotherapies by increasing the ratio of intratumoral CD8/Treg cells and suppressing Foxp3 expression. These findings suggest that CEL, which inhibits CD25 binding by targeting IL-2, exerts antitumor activity by mediating the T-cell response and could be a promising candidate for combination therapy in cancer immunotherapy against melanoma.

Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4+CD25+FOXP3+ T Cells.

Staphylococcal superantigens induce massive activation of T cells and inflammation, leading to toxic shock syndrome. Paradoxically, increasing evidence indicates that superantigens can also induce immunosuppression by promoting regulatory T cell (Treg) development. In this study, we demonstrate that stimulation strength plays a critical role in superantigen-mediated induction of immunosuppressive human CD4+CD25+FOXP3+ T cells. Suboptimal stimulation by a low dose (1 ng/ml) of staphylococcal enterotoxin C1 (SEC1) led to de novo generation of Treg-like CD4+CD25+FOXP3+ T cells with strong suppressive activity. In contrast, CD4+CD25+ T cells induced by optimal stimulation with high-dose SEC1 (1 µg/ml) were not immunosuppressive, despite high FOXP3 expression. Signal transduction pathway analysis revealed differential activation of the PI3K signaling pathway and expression of PTEN in optimal and suboptimal stimulation with SEC1. Additionally, we identified that FOXP3 isoforms in Treg-like cells from the suboptimal condition were located in the nucleus, whereas FOXP3 in nonsuppressive cells from the optimal condition localized in cytoplasm. Sequencing analysis of FOXP3 isoform transcripts identified five isoforms, including a FOXP3 isoform lacking partial exon 3. Overexpression of FOXP3 isoforms confirmed that both an exon 2-lacking isoform and a partial exon 3-lacking isoform confer suppressive activity. Furthermore, blockade of PI3K in optimal stimulation conditions led to induction of suppressive Treg-like cells with nuclear translocation of FOXP3, suggesting that PI3K signaling impairs induction of Tregs in a SEC1 dose-dependent manner. Taken together, these data demonstrate that the strength of activation signals determined by superantigen dose regulates subcellular localization of FOXP3 isoforms, which confers suppressive functionality.

The effect of gemigliptin treatment on immune parameters including regulatory T cells in patients with type 2 diabetes and moderate to very severe chronic renal impairment.

It is crucial to understand the impact of DPP-4 inhibitors on the immune system, particularly T cell differentiation, maturation, and proliferation, in patients with type 2 diabetes and CKD. This prospective observational study aimed to investigate the distribution of immune cells (particularly regulatory T cells), following the administration of gemigliptin, a DPP-4 inhibitor, in patients with type 2 diabetes mellitus and chronic kidney disease. We enrolled 28 patients with type 2 diabetes, aged 20 to 69, who had been taking a daily dose of 50mg gemigliptin for <3 months and had chronic kidney disease stages 3, 4, or 5, including that undergoing dialysis. T regulatory cells were defined as CD4 + CD25 high CD127 low/- FoxP3 + phenotype, and flow cytometry was used to examine the distribution of T regulatory cells. In the patient group, blood samples were collected at baseline, as well as at 3 and 6 months after initiating medication. Of the 28 patients, 17 (60.7%) were male and the mean age was 61.82 ±â€…8.03 years. Serum Cr ≥ 1.5 mg/dL was 16 (57%), and Cr < 1.5 mg/dL was 12 (43%). The number of CD4(+)/CD25(+) cells did not significantly increase or decrease in baseline, 3 months, and 6 months time changes, and the number of CD127(-/FoxP3(+) cells did not change significantly. Treatment with gemigliptin for 3 and 6 months did not significantly alter the number, percentage, or ratio of circulating Treg cells in patients with type 2 diabetes and CKD. Therefore, the administration of gemigliptin may help maintain regulatory T cells or have no significant impact.

Distinct use of super-enhancer elements controls cell type-specific CD25 transcription and function.

The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (Tregs) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and Tregs, with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type-specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type-specific fashion.

Role of Regulatory T Cells and Their Potential Therapeutic Applications in Celiac Disease.

Celiac disease (CeD) is a T-cell-mediated immune disease, in which gluten-derived peptides activate lamina propria effector CD4+ T cells. While this effector T cell subset produces proinflammatory cytokines, which cause substantial tissue injury in vivo, additional subsets of T cells exist with regulatory functions (Treg). These subsets include CD4+ type 1 regulatory T cells (Tr1) and CD4+ CD25+ T cells expressing the master transcription factor forkhead box P3 (Foxp3) that may have important implications in disease pathogenesis. In this review, we provide an overview of the current knowledge about the effects of immunomodulating cytokines on CeD inflammatory status. Moreover, we outline the main Treg cell populations found in CeD and how their regulatory activity could be influenced by the intestinal microenvironment. Finally, we discuss the Treg therapeutic potential for the development of alternative strategies to the gluten-free diet (GFD).

IL-2 can signal via chemokine receptors to promote regulatory T cells' suppressive function.

Canonical interleukin-2 (IL-2) signaling via the high-affinity CD25-containing IL-2 receptor-Janus kinase (JAK)1,3-signal transducer and activator of transcription 5 (STAT5) pathway is essential for development and maintenance of CD4+CD25HiFoxp3+ regulatory T cells (Tregs) that support immune homeostasis. Here, we report that IL-2 signaling via an alternative CD25-chemokine receptor pathway promotes the suppressive function of Tregs. Using an antibody against CD25 that biases IL-2 signaling toward this alternative pathway, we establish that this pathway increases the suppressive activity of Tregs and ameliorates murine experimental autoimmune encephalomyelitis (EAE). Furthermore, heparan sulfate, an IL-2-binding element of cell surfaces and extracellular matrix, or an engineered IL-2 immunocytokine can also direct IL-2 signaling toward this alternative pathway. Overall, these data reveal a non-canonical mechanism for IL-2 signaling that promotes suppressive functions of Tregs, further elucidates how IL-2 supports immune homeostasis, and suggests approaches to promote or suppress Treg functions.

Effects of ruxolitinib on murine regulatory T cells are immune-context dependent.

Aplastic anemia is a bone marrow failure (BMF) disorder characterized by pancytopenia and hypocellular marrow from an immune-mediated etiology. Regulatory T cells (Tregs) prevent autoimmunity by suppressing autoreactive T cells. We recently demonstrated the efficacy of ruxolitinib (RUX), a JAK 1/2 inhibitor, in attenuating murine BMF. Herein, we investigated the changes of Tregs in the context of RUX treatment for murine BMF. Tregs are conventionally identified by surface expression of CD4 and CD25, in addition to intracellular transcription factor FoxP3. RUX promoted the expansion of Tregs in BMF mice defined by increased expression of FoxP3 in CD4 T cells but suppressed expression of activation marker CD25 in CD4 and CD8 T cells. In this context, CD25 is no longer a reliable surface marker for Tregs. We observed strong co-expression of FoxP3 with surface marker GITR instead of CD25 in RUX-treated BMF mice. Fluorescence-activated cell sorting (FACS)-sorted CD4+GITRhi cells showed high FoxP3 expression and intact suppressive function in vitro, suggesting GITR to be a surrogate marker for Tregs. In contrast to its expansive effect on Tregs in BMF, RUX suppressed Tregs in normal and sublethal irradiation conditions, indicating that the effects of RUX on Tregs are immune-context dependent.

Unsolved mystery of Fas: mononuclear cells may have trouble dying in patients with Sjögren's syndrome.

BACKGROUND: Patients with Sjögren's syndrome, like other patients with autoimmune disorders, display dysregulation in the function of their immune system. Fas and Fas Ligand (FasL) are among the dysregulated proteins. METHODS: We studied Fas and FasL on IL-2Rα+ cells and in serum of patients with Sjögren's syndrome (n = 16) and healthy individuals (n = 16); both from same ethnic and geographical background. We used flow cytometry and enzyme-linked immunosorbent for this purpose. We also measured the expression of Bcl-2 and Bax by reverse transcription quantitative real-time PCR (RT-qPCR) and percentage of apoptotic and dead cells using Annexin V and 7-AAD staining in lymphocytes. RESULTS: FasL was increased in patients' T and B cells while Fas was increased in patients' monocytes, T and B cells. No signs of increased apoptosis were found. sFas and sFasL in patients' serum were increased, although the increase in sFasL was not significant. We suspect an effect of non-steroidal anti-inflammatory therapy on B cells, explaining the decrease of the percentage Fas+ B cells found within our samples. In healthy individuals, there was a noticeable pattern in the expression of FasL which mutually correlated to populations of mononuclear cells; this correlation was absent in the patients with Sjögren's syndrome. CONCLUSIONS: Mononuclear cells expressing IL-2Rα+ had upregulated Fas in Sjögren's syndrome. However, the rate of apoptosis based on Annexin V staining and the Bcl-2/Bax expression was not observed in mononuclear cells. We suspect a functional role of abnormal levels of Fas and FasL which has not been cleared yet.

A novel interleukin-2-based fusion molecule, HCW9302, differentially promotes regulatory T cell expansion to treat atherosclerosis in mice.

Atherosclerosis is a chronic inflammatory disease caused by deposition of oxidative low-density lipoprotein (LDL) in the arterial intima which triggers the innate immune response through myeloid cells such as macrophages. Regulatory T cells (Tregs) play an important role in controlling the progression or regression of atherosclerosis by resolving macrophage-mediated inflammatory functions. Interleukin-2 (IL-2) signaling is essential for homeostasis of Tregs. Since recombinant IL-2 has an unfavorable pharmacokinetic profile limiting its therapeutic use, we constructed a fusion protein, designated HCW9302, containing two IL-2 domains linked by an extracellular tissue factor domain. We found that HCW9302 exhibited a longer serum half-life with an approximately 1000-fold higher affinity for the IL-2Rα than IL-2. HCW9302 could be administered to mice at a dosing range that expanded and activated Tregs but not CD4+ effector T cells. In an ApoE-/- mouse model, HCW9302 treatment curtailed the progression of atherosclerosis through Treg activation and expansion, M2 macrophage polarization and myeloid-derived suppressor cell induction. HCW9302 treatment also lessened inflammatory responses in the aorta. Thus, HCW9302 is a potential therapeutic agent to expand and activate Tregs for treatment of inflammatory and autoimmune diseases.

Insulin Receptor Substrate 1 Signaling Inhibits Foxp3 Expression and Suppressive Functions in Treg Cells through the mTORC1 Pathway.

Regulatory T (Treg) cells play an important role in immune homeostasis by inhibiting cells within the innate and adaptive immune systems; therefore, the stability and immunosuppressive function of Treg cells need to be maintained. In this study, we found that the expression of insulin receptor substrate 1 (IRS1) by Treg cells was lower than that by conventional CD4 T cells. IRS1-overexpressing Treg cells showed the downregulated expression of FOXP3, as well as Treg signature markers CD25 and CTLA4. IRS1-overexpressing Treg cells also showed diminished immunosuppressive functions in an in vitro suppression assay. Moreover, IRS1-overexpressing Treg cells were unable to suppress the pathogenic effects of conventional T cells in a transfer-induced colitis model. IRS1 activated the mTORC1 signaling pathway, a negative regulator of Treg cells. Moreover, IRS1 destabilized Treg cells by upregulating the expression of IFN-γ and Glut1. Thus, IRS1 acts as a negative regulator of Treg cells by downregulating the expression of FOXP3 and disrupting stability.

Soluble CD25 imposes a low-zone IL-2 signaling environment that favors competitive outgrowth of antigen-experienced CD25high regulatory and memory T cells.

This study focused on soluble (s)CD25-mediated regulation of IL-2 signaling in murine and human CD4+ T cells. Recombinant sCD25 reversibly sequestered IL-2 to limit acute maximal proliferative responses while preserving IL-2 bioavailability to subsequently maintain low-zone IL-2 signaling during prolonged culture. By inhibiting IL-2 signaling during acute activation, sCD25 suppressed T-cell growth and inhibited IL-2-evoked transmembrane CD25 expression, thereby resulting in lower prevalence of CD25high T cells. By inhibiting IL-2 signaling during quiescent IL-2-mediated growth, sCD25 competed with transmembrane CD25, IL2Rßγ, and IL2Rαßγ receptors for limited pools of IL-2 such that sCD25 exhibited strong or weak inhibitory efficacy in IL-2-stimulated cultures of CD25low or CD25high T cells, respectively. Preferential blocking of IL-2 signaling in CD25low but not CD25high T cells caused competitive enrichment of CD25high memory/effector and regulatory FOXP3+ subsets. In conclusion, sCD25 modulates IL-2 bioavailability to limit CD25 expression during acute activation while enhancing CD25highT-cell dominance during low-zone homeostatic IL-2-mediated expansion, thereby 'flattening' the inflammatory curve over time.

Sodium Selenite Diminished the Regulatory T Cell Differentiation In Vitro.

Sodium selenite modulates the activity of lymphocytes. It negatively regulates the suppressive activity of cells and increases the immune response. In this study, we evaluated whether the regulatory T cell differentiation was modulated by sodium selenite. The percentages of CD4+CD25+Foxp3+, CD4+CD25+, and CD4+CTLA-4+ cells in CD4+ T cells cultures stimulated with IL-2 and TGF-ß in the presence or absence of selenium, in the form of sodium selenite (2.0×10-6M), were evaluated by flow cytometry. The mRNA expression of TET2/3 enzymes and IL-10 was analyzed by RT-qPCR and the levels of IL-10 were measured by an ELISA. We observed a decrease in CD4+CD25+Foxp3+ and CD4+CTLA-4+ cells in presence of selenium. However, normal percentages were reached again after selenium removal. An increase in CD4+CTL4-4+ cells was detected in selenium-primed cell cultures in absence of IL-2 and TGF-ß. In addition, we observed a decrease in TET3 in presence of selenium. Finally, we observed an augment in IL-10 transcription and protein levels and relative expression of TET2 in cultures exposed to selenium. We suggest that selenium reversibly affects the regulatory T cell differentiation in vitro. Likewise, selenium may modulate Treg percentages promoting optimal immune responses and, at the same time, the expression of specific suppressor molecules.

Whole exome sequencing identified a novel splice donor site variant in interleukin 2 receptor alpha chain.

Interleukin 2 receptor alpha chain (IL-2Rα or CD25) deficiency (OMIM #606367) is an immune dysregulation disorder segregating in autosomal recessive form. The disease is caused by biallelic variants in the IL-2Rα gene encoding IL-2Rα also known as CD25 protein. IL-2Rα combines with γ and ß chains of interleukin 2 receptor to form a functional interleukin 2 receptor (IL-2R). In the present study, we identified a Pakistani family presenting a unique presentation of IL-2Rα deficiency. Clinical whole exome sequencing revealed a novel splice donor site variant (NM_001378789.1 (NP_001365718); c.64 + 1G > A) in the IL-2Rα gene. American College of Medical Genetics (ACMG) guidelines interpreted the identified variant as likely pathogenic. The IL-2Rα gene mutation usually presents with autoimmunity and immunodeficiency but in our patient, it presents with congenital diarrhea, metabolic crisis, and strong family history of death in infancy due to the similar complications. Her congenital diarrhea is attributed to autoimmunity in the form of autoimmune enteropathy and eczema. The laboratory findings revealed severe metabolic acidosis hypokalemia and elevated lactate and ammonia levels. This is a new presentation of IL-2Rα gene mutation. The present study highlights the importance of clinical whole exome sequencing in the correct diagnosis of congenital disorders. The study will also help clinical geneticists for genetic counseling and prevention of the disease in the affected family.

Increased sIL-2Rα leads to obstruction of IL-2 biological function and Treg cells differentiation in SLE patients via binding to IL-2.

Background: The decrease of IL-2 level is believed to play an important role in the disease occurrence and development of SLE, but the relevant mechanisms have not been fully clarified. Many studies have found that the level of soluble interleukin 2 receptor α (sIL-2Rα) in SLE patients is significantly increased. Considering the fact that sIL-2Rα has the ability to bind IL-2, we want to know whether the increased sIL-2Rα has some impact on the level and function of IL-2 in SLE patients. Methods: New onset SLE patients, treated SLE patients and healthy volunteers were recruited. The levels of serum IL-2, IL-2 mRNA in CD3+ T cells and serum sIL-2Rα were detected and compared in these subjects. Two mixed solid-phase sandwich ELISA system were designed to measure exclusively the heterodimers complex of sIL-2Rα/IL-2. The sera from SLE patients were pretreated with or without immune complex dissociation solution and detected for IL-2 levels. IL-2 standard or serum from HCs were used to co-incubate with recombinant sIL-2Rα or serum samples with high levels of sIL-2Rα and detected for IL-2 levels by ELISA. The inhibitory effect of sIL-2Rα on IL-2 biological activity was investigated by CTLL-2 cell proliferation assay. The frequencies and absolute counts of Treg cells were detected by flow cytometry before and after the addition of recombinant sIL-2Rα. Results: The levels of serum IL-2 in SLE patients were significantly decreased and negatively correlated with SLEDAI. However, there was no significant difference in IL-2 mRNA levels in CD3+ T cells between SLE patients and healthy controls. The levels of serum sIL-2Rα in SLE patients were significantly increased, positively correlated with the SLEDAI and negatively correlated with the levels of serum IL-2. sIL-2Rα was shown to bind to IL-2 to form immune complex, resulting in false reduction in the detection level of serum IL-2 and significant decrease in biological activity of IL-2. The increase of sIL-2Rα was demonstrated to be one of the important mechanisms for the obstruction of Treg cells differentiation in SLE patients. Conclusion: Increased serum sIL-2Rα can bind to IL-2, leading to obstruction of IL-2 activity and Treg cells differentiation.

A novel membrane-bound interleukin-2 promotes NK-92 cell persistence and anti-tumor activity.

A major challenge in natural killer (NK) cell immunotherapy is the limited persistence of NK cells in vivo. However, the proliferation of NK cells is dependent on cytokines such as interleukin-2 (IL-2). Although IL-2 is a critical cytokine for NK cell activation and survival, IL-2 administration in adoptive NK cell therapy can induce adverse toxicities. To improve the persistence of NK cells and attenuate the systemic toxicity of IL-2, we constructed a cell-restricted artificial IL-2, named membrane-bound IL-2 (mbIL-2), comprising human IL-2 and human IL-2Rα joined by a classic linker. We found that mbIL-2-activated NK-92 cells can survive and proliferate in vitro and in vivo, independent of exogenous IL-2, while mbIL-2-expressing NK-92 cells do not support bystander cell survival or proliferation. Additionally, mbIL-2 enhanced NK-92 cell-mediated antitumor activity by tuning the IL-2 receptor downstream signals and NK cell receptor repertoire expression. To conclude, our novel mbIL-2 improves NK-92 cell persistence and enhances NK-92 cell-mediated antitumor activity. NK-92 cells genetically modified to express the novel mbIL-2 with potential significance for clinical development.

[The decreased proportion of CD4+CD25+FOXP3+ regulatory T cells in peripheral blood of children with juvenile idiopathic arthritis and its negative correlation with cytokine level].

Objective To investigate the expression of CD4+CD25+FOXP3+regulatory T cells(Tregs), IL-6 and TNF-α in patients with juvenile idiopathic arthritis (JIA) and its clinical significance. Methods The percentages of CD4+CD25+FOXP3+ Tregs in 58 children with JIA and 40 healthy controls were detected by flow cytometry. Serum levels of IL-6 and TNF-α in each patient was detected by chemiluminescence. The correlations between the expression of CD4+CD25+FOXP3+ Tregs and IL-6, TNF-α were analyzed by pearson correlation analysis. We measured the expression level of CD4+CD25+FOXP3+regulatory T cells, IL-6 and TNF-α of 18 cases six weeks after the treatment of tocilizumab (TCZ) in order to figure out the dynamic changes using methods above. Results The percentages of CD4+CD25+FOXP3+Tregs in juvenile idiopathic arthritis were significantly lower than those in 40 healthy volunteers, while levels of IL-6 and TNF-α were significantly higher. However, no obvious difference in the levels of CD4+CD25+FOXP3+Tregs, IL-6 or TNF-α was observed between patients with systemic and poly-articular JIA. Pearson correlation analysis showed that the percentages of CD4+CD25+FOXP3+Tregs negatively correlated with the levels of IL-6 and TNF-α, while levels of IL-6 positively correlated with the levels of TNF-α. Compared with pre-treatment of TCZ, levels of CD4+CD25+FOXP3+Tregs in post-treatment markedly increased, which however were still lower than control group while the levels of IL-6 significantly decreased, yet remained higher than control group. There was no statistical difference between post and pre-treatment in the levels of TNF-α. Conclusion The percentages of CD4+CD25+FOXP3+ Tregs in peripheral blood of JIA children decreases, and it has a negative correlation with IL-6 and TNF-α. Furthermore, the levels of CD4+CD25+FOXP3+ Tregs and IL-6 are partially restored after treatment with TCZ, which may be helpful to assess the activity of systemic JIA and the efficacy of therapy.

Presence of regulatory T-cells in endometrial cancer predicts poorer overall survival and promotes progression of tumor cells.

PURPOSE: Endometrial cancer (EC) is one of the most common gynaecologic malignancies. Tumor infiltrating regulatory T-cells (Treg) have been reported to have a prognostic impact in many malignancies. Immunotherapeutic strategies are gaining interest for advanced and recurrent EC cases, where treatment options are rare. Our study was aimed at determining the value of Treg in EC progression. METHODS: EC specimens from 275 patients and 28 controls were screened immunohistochemically for the presence of Treg represented by FoxP3. Correlations with clinicopathological and survival parameters were performed. Functional assays were performed using EC cell lines Ishikawa + and RL95-2 after co-culturing with isolated CD4 + CD25 + CD127dim Treg. To assess the influence of EC on the composition of peripheral blood mononuclear cells (PBMC), flow cytometric analyses were performed. RESULTS: We found that an increased infiltration of Treg was associated with high grades and a reduced overall survival. Treg were almost absent in endometrium tissues from healthy control patients. Co-culture of tumor cells with CD4 + CD25 + CD127dim Treg led to functional changes: enhanced invasion, migration and viability indicated that increased levels of Treg in the tumor microenvironment may promote tumor growth. Furthermore, we found that the presence of EC cells led to phenotypic changes in PBMC, showing significantly increased levels of CD25 and FoxP3. CONCLUSION: Our results indicate that the presence of Treg in the EC tumor environment is associated with a poorer outcome. A remarkable impact of Treg on tumor cell behaviour and vice versa of tumor cells on PBMC subpopulations support this notion mechanistically. Our findings provide a basis for focusing on Treg as potential future therapeutic targets in EC.

Regulatory T Cell Depletion Using a CRISPR Fc-Optimized CD25 Antibody.

Regulatory T cells (Tregs) are major drivers behind immunosuppressive mechanisms and present a major hurdle for cancer therapy. Tregs are characterized by a high expression of CD25, which is a potentially valuable target for Treg depletion to alleviate immune suppression. The preclinical anti-CD25 (αCD25) antibody, clone PC-61, has met with modest anti-tumor activity due to its capacity to clear Tregs from the circulation and lymph nodes, but not those that reside in the tumor. The optimization of the Fc domain of this antibody clone has been shown to enhance the intratumoral Treg depletion capacity. Here, we generated a stable cell line that produced optimized recombinant Treg-depleting antibodies. A genome engineering strategy in which CRISPR-Cas9 was combined with homology-directed repair (CRISPR-HDR) was utilized to optimize the Fc domain of the hybridoma PC-61 for effector functions by switching it from its original rat IgG1 to a mouse IgG2a isotype. In a syngeneic tumor mouse model, the resulting αCD25-m2a (mouse IgG2a isotype) antibody mediated the effective depletion of tumor-resident Tregs, leading to a high effector T cell (Teff) to Treg ratio. Moreover, a combination of αCD25-m2a and an αPD-L1 treatment augmented tumor eradication in mice, demonstrating the potential for αCD25 as a cancer immunotherapy.