The Role of Recombinant Glycodelin in the Differentiation of Regulatory T-Lymphocytes.

The effect of recombinant glycodelin (GdA) on the number of T-regulatory lymphocytes (Treg) in a culture of activated CD4+ lymphocytes was investigated, while simultaneously assessing the proliferative status of the cells. Recombinant GdA from E. coli and from HEK293 cells were used in the study at concentrations of 0.2; 2 and 10 µg/mL. It was found that only a low concentration (0.2 µg/mL) of recombinant GdA of bacterial origin reduced the number of proliferating CD4+ lymphocytes as well as the number of Treg (CD4+CD25highCD127-/low) in the experimental system in vitro.

Interleukin-7 regulates CD127 expression and promotes CD8+ T cell activity in patients with primary cutaneous melanoma.

BACKGROUND: Interleukin (IL)-7 signaling through CD127 is impaired in lymphocytes in cancers and chronic infections, resulting in CD8+ T cell exhaustion. The mechanisms underlying CD8+ T cell responses to IL-7 in melanoma remain not completely elucidated. We previously showed reduced IL-7 level in melanoma patients. Thus, the aim of this study was to investigate the effect of IL-7 regulation on CD127 expression and CD8+ T cell responses in melanoma. METHODS: Healthy controls and primary cutaneous melanoma patients were enrolled. Membrane-bound CD127 (mCD127) expression on CD8+ T cells was determined by flow cytometry. Soluble CD127 (sCD127) protein level was measured by ELISA. Total CD127 and sCD127 mRNA level was measured by real-time PCR. CD8+ T cells were stimulated with recombinant human IL-7, along with signaling pathway inhibitors. CD8+ T cells were co-cultured with melanoma cell line, and the cytotoxicity of CD8+ T cells was assessed by measurement of lactate dehydrogenase expression. RESULTS: Plasma sCD127 was lower in melanoma patients compared with controls. The percentage of CD8+ T cells expressing mCD127 was higher, while sCD127 mRNA level was lower in peripheral and tumor-infiltrating CD8+ T cells from melanoma patients. There was no significant difference of total CD127 mRNA expression in CD8+ T cells between groups. IL-7 stimulation enhanced total CD127 and sCD127 mRNA expression and sCD127 release by CD8+ T cells. However, mCD127 mRNA expression on CD8+ T cells was not affected. This process was mainly mediated by phosphatidylinositol 3-kinase (PI3K) pathway. CD8+ T cells from melanoma patients exhibited decreased cytotoxicity. IL-7 stimulation promoted CD8+ T cell cytotoxicity, while inhibition of PI3K dampened IL-7-induced elevation of CD8+ T cell cytotoxicity. CONCLUSION: The current data suggested that insufficient IL-7 secretion might contribute to CD8+ T cell exhaustion and CD127 dysregulation in patients with primary cutaneous melanoma.

Mutant IL7R collaborates with MYC to induce T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric cancer. Amongst the wide array of driver mutations, 10% of T-ALL patients display gain-of-function mutations in the IL-7 receptor α chain (IL-7Rα, encoded by IL7R), which occur in different molecular subtypes of this disease. However, it is still unclear whether IL-7R mutational activation is sufficient to transform T-cell precursors. Also, which genes cooperate with IL7R to drive leukemogenesis remain poorly defined. Here, we demonstrate that mutant IL7R alone is capable of inducing T-ALL with long-latency in stable transgenic zebrafish and transformation is associated with MYC transcriptional activation. Additionally, we find that mutant IL7R collaborates with Myc to induce early onset T-ALL in transgenic zebrafish, supporting a model where these pathways collaborate to drive leukemogenesis. T-ALLs co-expressing mutant IL7R and Myc activate STAT5 and AKT pathways, harbor reduced numbers of apoptotic cells and remake tumors in transplanted zebrafish faster than T-ALLs expressing Myc alone. Moreover, limiting-dilution cell transplantation experiments reveal that activated IL-7R signaling increases the overall frequency of leukemia propagating cells. Our work highlights a synergy between mutant IL7R and Myc in inducing T-ALL and demonstrates that mutant IL7R enriches for leukemia propagating potential.

Lnc-IL7R alleviates PM2.5-mediated cellular senescence and apoptosis through EZH2 recruitment in chronic obstructive pulmonary disease.

BACKGROUND: Long-term exposure to PM2.5 (particulate matter with an aerodynamic diameter of ≤ 2.5 µm) is associated with pulmonary injury and emphysema in patients with chronic obstructive pulmonary disease (COPD). We investigated mechanisms through which the long noncoding RNA lnc-IL7R contributes to cellular damage by inducing oxidative stress in COPD patients exposed to PM2.5. METHODS: Associations of serum lnc-IL7R levels with lung function, emphysema, and previous PM2.5 exposure in COPD patients were analyzed. Reactive oxygen species and lnc-IL7R levels were measured in PM2.5-treated cells. The levels of lnc-IL7R and cellular senescence-associated genes, namely p16INK4a and p21CIP1/WAF1, were determined through lung tissue section staining. The effects of p16INK4a or p21CIP1/WAF1 regulation were examined by performing lnc-IL7R overexpression and knockdown assays. The functions of lnc-IL7R-mediated cell proliferation, cell cycle, senescence, colony formation, and apoptosis were examined in cells treated with PM2.5. Chromatin immunoprecipitation assays were conducted to investigate the epigenetic regulation of p21CIP1/WAF1. RESULTS: Lnc-IL7R levels decreased in COPD patients and were negatively correlated with emphysema or PM2.5 exposure. Lnc-IL7R levels were upregulated in normal lung epithelial cells but not in COPD cells exposed to PM2.5. Lower lnc-IL7R expression in PM2.5-treated cells induced p16INK4a and p21CIP1/WAF1 expression by increasing oxidative stress. Higher lnc-IL7R expression protected against cellular senescence and apoptosis, whereas lower lnc-IL7R expression augmented injury in PM2.5-treated cells. Lnc-IL7R and the enhancer of zeste homolog 2 (EZH2) synergistically suppressed p21CIP1/WAF1 expression through epigenetic modulation. CONCLUSION: Lnc-IL7R attenuates PM2.5-mediated p21CIP1/WAF1 expression through EZH2 recruitment, and its dysfunction may augment cellular injury in COPD.

Cytokine-induced transient monocyte IL-7Ra expression and the serum milieu in tuberculosis.

Bacterial components and cytokines induce IL-7 receptor (IL-7Rα) expression in monocytes. Aberrant low IL-7Rα expression of monocytes has been identified as a feature of tuberculosis immunopathology. Here, we investigated the mechanisms underlying IL-7Rα regulation of monocytes and tuberculosis serum effects on IL-7Rα expression. Serum samples from tuberculosis patients and healthy controls, cytokine candidates, and mycobacterial components were analyzed for in vitro effects on IL-7Rα expression of primary monocytes, monocyte-derived macrophages (MDM), and monocyte cell lines. IL-7Rα regulation during culture and the role of FoxO1 were characterized. In vitro activation-induced IL-7Rα expression in human monocytes and serum samples from tuberculosis patients boosted IL-7Rα expression. Although pathognomonic tuberculosis cytokines were not associated with serum effects, we identified cytokines (i.e., GM-CSF, IL-1ß, TNF-α, IFN-γ) that induced IL-7Rα expression in monocytes and/or MDM comparable to mycobacterial components. Blocking of cytokine subsets (i.e., IL-1ß/TNF-α in monocytes, GM-CSF in MDM) largely diminished IL-7Rα expression induced by mycobacterial components. Finally, we showed that in vitro-induced IL-7Rα expression was transient and dependent on constitutive FoxO1 expression in primary monocytes and monocyte cell lines. This study demonstrated the crucial roles of cytokines and constitutive FoxO1 expression for transient IL-7Rα expression in monocytes.

An instructive role for Interleukin-7 receptor α in the development of human B-cell precursor leukemia.

Kinase signaling fuels growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Yet its role in leukemia initiation is unclear and has not been shown in primary human hematopoietic cells. We previously described activating mutations in interleukin-7 receptor alpha (IL7RA) in poor-prognosis "ph-like" BCP-ALL. Here we show that expression of activated mutant IL7RA in human CD34+ hematopoietic stem and progenitor cells induces a preleukemic state in transplanted immunodeficient NOD/LtSz-scid IL2Rγnull mice, characterized by persistence of self-renewing Pro-B cells with non-productive V(D)J gene rearrangements. Preleukemic CD34+CD10highCD19+ cells evolve into BCP-ALL with spontaneously acquired Cyclin Dependent Kinase Inhibitor 2 A (CDKN2A) deletions, as commonly observed in primary human BCP-ALL. CRISPR mediated gene silencing of CDKN2A in primary human CD34+ cells transduced with activated IL7RA results in robust development of BCP-ALLs in-vivo. Thus, we demonstrate that constitutive activation of IL7RA can initiate preleukemia in primary human hematopoietic progenitors and cooperates with CDKN2A silencing in progression into BCP-ALL.

Selective dependence on IL-7 for antigen-specific CD8 T cell responses during airway influenza infection.

Interleukin-7 (IL-7) is a cytokine known for its importance in T cell development and survival. How IL-7 shapes CD8 T cell responses during an acute viral infection is less understood. We had previously shown that IL-7 signaling deficient mice have reduced accumulation of influenza-specific CD8 T cells following influenza infection. We sought to determine whether IL-7 affects early CD8 T cell expansion in the mediastinal lymph node and effector function in the lungs. Using IL-7Rα signaling deficient mice, we show that IL-7 is required for a normal sized mediastinal lymph node and the early clonal expansion of influenza-specific CD8 T cells therein. We show that IL-7 plays a cell-intrinsic role in the accumulation of NP366-374 and PA224-233-specific CD8 T cells in the lymph node. We also found that IL-7 shapes terminal differentiation, degranulation and cytokine production to a greater extent in PA224-233-specific than NP366-374-specific CD8 T cells. We further demonstrate that IL-7 is induced in the lung tissue by viral infection and we characterize multiple cellular sources that contribute to IL-7 production. Our findings on IL-7 and its effects on lower respiratory diseases will be important for expanding the utility of therapeutics that are currently available.

Signature of long-lived memory CD8+ T cells in acute SARS-CoV-2 infection.

Immunological memory is a hallmark of adaptive immunity and facilitates an accelerated and enhanced immune response upon re-infection with the same pathogen1,2. Since the outbreak of the ongoing COVID-19 pandemic, a key question has focused on which SARS-CoV-2-specific T cells stimulated during acute infection give rise to long-lived memory T cells3. Here, using spectral flow cytometry combined with cellular indexing of transcriptomes and T cell receptor sequencing, we longitudinally characterized individual SARS-CoV-2-specific CD8+ T cells of patients with COVID-19 from acute infection to 1 year into recovery and found a distinct signature identifying long-lived memory CD8+ T cells. SARS-CoV-2-specific memory CD8+ T cells persisting 1 year after acute infection express CD45RA, IL-7 receptor-α and T cell factor 1, but they maintain low expression of CCR7, thus resembling CD45RA+ effector memory T cells. Tracking individual clones of SARS-CoV-2-specific CD8+ T cells, we reveal that an interferon signature marks clones that give rise to long-lived cells, whereas prolonged proliferation and mechanistic target of rapamycin signalling are associated with clonal disappearance from the blood. Collectively, we describe a transcriptional signature that marks long-lived, circulating human memory CD8+ T cells following an acute viral infection.

Interleukin-7 receptor α mutational activation can initiate precursor B-cell acute lymphoblastic leukemia.

Interleukin-7 receptor α (encoded by IL7R) is essential for lymphoid development. Whether acute lymphoblastic leukemia (ALL)-related IL7R gain-of-function mutations can trigger leukemogenesis remains unclear. Here, we demonstrate that lymphoid-restricted mutant IL7R, expressed at physiological levels in conditional knock-in mice, establishes a pre-leukemic stage in which B-cell precursors display self-renewal ability, initiating leukemia resembling PAX5 P80R or Ph-like human B-ALL. Full transformation associates with transcriptional upregulation of oncogenes such as Myc or Bcl2, downregulation of tumor suppressors such as Ikzf1 or Arid2, and major IL-7R signaling upregulation (involving JAK/STAT5 and PI3K/mTOR), required for leukemia cell viability. Accordingly, maximal signaling drives full penetrance and early leukemia onset in homozygous IL7R mutant animals. Notably, we identify 2 transcriptional subgroups in mouse and human Ph-like ALL, and show that dactolisib and sphingosine-kinase inhibitors are potential treatment avenues for IL-7R-related cases. Our model, a resource to explore the pathophysiology and therapeutic vulnerabilities of B-ALL, demonstrates that IL7R can initiate this malignancy.

iNKT subsets differ in their developmental and functional requirements on Foxo1.

Invariant natural killer T (iNKT) cells play important roles in regulating immune responses. Based on cytokine profiling and key transcriptional factors, iNKT cells are classified into iNKT1, iNKT2, and iNKT17 subsets. However, whether the development and functions of these subsets are controlled by distinct mechanisms remains unclear. Here, we show that forkhead box protein O1 (Foxo1) promotes differentiation of iNKT1 and iNKT2 cells but not iNKT17 cells because of its distinct contributions to IL7R expression in these subsets. Nuclear Foxo1 is essential for Il7r expression in iNKT1 and iNKT2 cells at early stages of differentiation but is dispensable in iNKT17 cells. RORγt, instead of Foxo1, promotes IL7R expression in iNKT17 cells. Additionally, Foxo1 is required for the effector function of iNKT1 and iNKT2 cells but not iNKT17 cells. Cytoplasmic Foxo1 promotes activation of mTORC1 in iNKT1 and iNKT2 cells through inhibiting TSC1-TSC2 interaction, whereas it is dispensable for mTORC1 activation in iNKT17 cells. iNKT17 cells display distinct metabolic gene expression patterns from iNKT1 and iNKT2 cells that match their different functional requirements on Foxo1. Together, our results demonstrate that iNKT cell subsets differ in their developmental and functional requirements on Foxo1.

CD127+ CD94+ innate lymphoid cells expressing granulysin and perforin are expanded in patients with Crohn's disease.

Phenotypic definition of helper ILC1 and NK cells is problematic due to overlapping markers. Recently we showed the identification of cytotoxic ILC3s characterized by expression of CD94. Here we analyse CD127+ ILCs and NK cells in intestinal lamina propria from healthy donors and Crohn's disease patients and identify two populations of CD127+CD94+ ILCs, designated population A and B, that can be distinguished on the expression of CD117, CD18 and cytotoxic molecules. Population B expresses granulysin, a cytotoxic molecule linked to bacterial lysis and/or chemotaxis of monocytes. Granulysin protein is secreted by population B cells upon stimulation with IL-15. Activation of population B in the presence of TGF-ß strongly reduces the expression of cytotoxic effector molecules of population B. Strikingly, samples from individuals that suffer from active Crohn's disease display enhanced frequencies of granulysin-expressing effector CD127+CD94+ ILCs in comparison to controls. Thus this study identifies group 1 ILC populations which accumulate in inflamed intestinal tissue of Crohn's disease patients and may play a role in the pathology of the disease.

Expression and Polymorphism of TSLP/TSLP Receptors as Potential Diagnostic Markers of Colorectal Cancer Progression.

Colorectal cancer (CRC) is the third most common malignancy and the fourth leading cause of cancer-related mortality worldwide. Inflammation is considered as a critical driver for CRC development and growth. We investigated the association between polymorphisms/expression levels of thymic stromal lymphopoietin (TSLP) /TSLP receptors and CRC risk in Saudi population. DNA samples were isolated from blood samples from 220 participants. Case subjects were 112 patients diagnosed with CRC, while control subjects were 108 healthy individuals, who were not diagnosed with any type of malignancy. We selected two single nucleotide polymorphisms (SNPs) located in the thymic stromal lymphopoietin gene (rs10043985 and rs2289276), three SNPs in TSLP receptor gene (TSLPR; rs36139698, rs36177645, and rs36133495), and two other SNPs in interleukin-7 receptor gene (IL-7R; rs12516866 and rs1053496), and designated these SNPs for a case-control genotyping study. The gene expression was analyzed using quantitative RT-PCR and immunohistochemistry assays array on 20 matching colorectal cancer/normal tissues. mRNA expressions and protein levels of TSLP, TSLPR-α subunit, and IL-7R-α subunit showed a 4-fold increase in colon cancer tissues when compared to normal colon tissues. Furthermore, two SNPs (rs10043985 of TSLP and rs1053496 of IL-7R) showed statistically significant correlations with CRC susceptibility. Interestingly, only rs10043985 showed a statistically significant association (p < 0.0001) in the genotypic and phenotypic levels with CRC for all clinical parameters (age, gender, and tumor location) tested. However, IL-7R rs1053496 genotyping results presented a significant correlation (p < 0.05) in male CRC patients and in individuals under 57 years of age. TSLP rs2289276, IL-7R rs12516866, and all TSLPR variants did not display any significant genotypic or phenotypic correlations in all tested clinical parameters. This study identified that TSLP rs10043985 and IL-7R rs1053496 SNPs, and the expression levels of TSLP and TSLPR-α subunit, can be used as markers for CRC development and treatment. However, additional investigations are required on larger group of patients from diverse ethnicities to confirm the genetic association of these variants to CRC.

Expression and clinical significance of IL7R, NFATc2, and RNF213 in familial and sporadic multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory and autoimmune disorder of the central nervous system characterized by myelin loss and axonal dysfunction. Increased production of inflammatory factors such as cytokines has been implicated in axon destruction. In the present study, we compared the expression level of IL7R, NFATc2, and RNF213 genes in the peripheral blood of 72 MS patients (37 familial MS, 35 sporadic MS) and 74 healthy controls (34 individuals with a family history of the disease, 40 healthy controls without a family history) via Real-time PCR. Our results showed that the expression level of IL7R was decreased in the sporadic patients in comparison with other groups. Additionally, there was an increased NFATc2 expression level in MS patients versus healthy controls. Increased expression of NFATc2 in sporadic and familial groups compared to the controls, and familial group versus FDR was also seen. Our results also represented an increased expression level of RNF213 in familial patients as compared to the control group. The similar RNF213 expression between sporadic and control group, as well as FDR and familial group was also seen. Diagnostic evaluation was performed by receiver operating characteristic (ROC) curve analysis and area under the curve (AUC) calculation. The correlation of clinical parameters including onset age and Expanded Disability Status Scale (EDSS) with our gene expression levels were also assessed. Overall, decreased expression level of IL7R in the sporadic cases and increased expression level of NFATc2 may be associated with the pathogenesis of MS disease. Confirmation of the effects of differential expression of RNF213 gene requires further studies in the wider statistical populations.

Standard Peripheral Blood Mononuclear Cell Cryopreservation Selectively Decreases Detection of Nine Clinically Relevant T Cell Markers.

Biobanking is an operational component of various epidemiological studies and clinical trials. Although peripheral blood is routinely acquired and stored in biobanks, the effects of specimen processing on cell composition and clinically relevant functional markers of T cells still require a systematic evaluation. In this study, we assessed 25 relevant T cell markers in human PBMCs and showed that the detection of nine membrane markers (e.g., PD-1, CTLA4, KLRG1, CD25, CD122, CD127, CCR7, and others reflecting exhaustion, senescence, and other functions) was reduced among at least one T cell subset following standard processing, although the frequency of CD4, CD8, and regulatory T cells was unaffected. Nevertheless, a 6-mo-long cryopreservation did not impair the percentages of cells expressing many other membrane and all the eight tested intracellular lineage or functional T cell markers. Our findings uncover that several clinically relevant markers are particularly affected by processing and the interpretation of those results in clinical trials and translational research should be done with caution.

Human Lung-Resident Macrophages Express and Are Targets of Thymic Stromal Lymphopoietin in the Tumor Microenvironment.

Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine highly expressed by epithelial cells and several innate and adaptive immune cells. TSLP exerts its biological effects by binding to a heterodimeric complex composed of TSLP receptor (TSLPR) and IL-7Rα. In humans, there are two TSLP isoforms: the short form (sfTSLP), constitutively expressed, and the long form (lfTSLP), which is upregulated in inflammation. TSLP has been implicated in the induction and progression of several experimental and human cancers. Primary human lung macrophages (HLMs), monocyte-derived macrophages (MDMs), and peripheral blood monocytes consitutively expressed sfTSLP mRNA. Incubation of HLMs, MDMs, and monocytes with lipopolysaccharide (LPS) or IL-4, but not with IL-13, induced TSLP release from HLMs. LPS, but not IL-4 or IL-13, induced CXCL8 release from HLMs. LPS, IL-4 alone or in combination with IL-13, induced the expression of lfTSLP, but not of sfTSLP from HLMs. Preincubation of HLMs with IL-4, alone or in combination with IL-13, but not IL-13 alone, synergistically enhanced TSLP release from LPS-activated macrophages. By contrast, IL-4, alone or in combination with IL-13, inhibited LPS-induced CXCL8 release from HLMs. Immunoreactive TSLP was detected in lysates of HLMs, MDMs, and monocytes. Incubation of HLMs with TSLP induced the release of proinflammatory (TNF-α), angiogenic (VEGF-A, angiopoietin 2), and lymphangiogenic (VEGF-C) factors. TSLP, TSLPR, and IL-7Rα were expressed in intratumoral and peritumoral areas of human lung cancer. sfTSLP and lfTSLP mRNAs were differentially expressed in peritumoral and intratumoral lung cancer tissues. The TSLP system, expressed in HLMs, MDMs, and monocytes, could play a role in chronic inflammatory disorders including lung cancer.

Expression of KLRG1 and CD127 defines distinct CD8+ subsets that differentially impact patient outcome in follicular lymphoma.

BACKGROUND: CD8+ T-lymphocyte subsets defined by killer lectin-like receptor G1 (KLRG1) and CD127 expression have been reported to have an important role in infection, but their role in the setting of lymphoid malignancies, specifically follicular lymphoma (FL), has not been studied. METHODS: To characterize the phenotype of KLRG1/CD127-defined CD8+ subsets, surface and intracellular markers were measured by flow cytometry and Cytometry by time of flight (CyTOF), and the transcriptional profile of these cells was determined by CITE-Seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing). The functional capacity of each subset was determined, as was their impact on overall survival (OS) and event-free survival (EFS) of patients with FL. RESULTS: We found that intratumoral CD8+ cells in FL are skewed toward effector cell subsets, particularly KLRG+CD127- and KLRG1-CD127- cells over memory cell subsets, such as KLRG1-CD127+ and KLRG1+CD127+ cells. While effector subsets exhibited increased capacity to produce cytokines/granules when compared with memory subsets, their proliferative capacity and viability were found to be substantially inferior. Clinically, a skewed distribution of intratumoral CD8+ T cells favoring effector subtypes was associated with an inferior outcome in patients with FL. Increased numbers of CD127+KLRG1- and CD127+KLRG1+ were significantly associated with a favorable OS and EFS, while CD127-KLRG1- correlated with a poor EFS and OS in patients with FL. Furthermore, we demonstrated that interleukin (IL)-15 promotes CD127-KLRG1+ cell development in the presence of dendritic cells via a phosphoinositide 3-kinase (PI3K)-dependent mechanism, and treatment of CD8+ T cells with a PI3K inhibitor downregulated the transcription factors responsible for CD127-KLRG1+ differentiation. CONCLUSIONS: Taken together, these results reveal not only a biological and prognostic role for KLRG1/CD127-defined CD8+ subsets in FL but also a potential role for PI3K inhibitors to manipulate the differentiation of CD8+ T cells, thereby promoting a more effective antitumor immune response.

CD4+CD25+CD127low regulatory T cells associated with the effect of CD19 CAR-T therapy for relapsed/refractory B-cell acute lymphoblastic leukemia.

BACKGROUND: CD19-specific chimeric antigen receptor T-cell (CAR-T) therapy has shown promising clinical outcomes in relapsed/refractory acute B-cell lymphoblastic leukemia (R/R B-ALL) patients. However, some patients did not respond to this therapy or relapsed after remission. Regulatory T cells (Tregs) have shown great importance in promoting tumor escape, but little is known about their role in R/R B-ALL patients with CAR-T therapy. Our previous study has proved that higher Tregs before infusion was an independent high-risk factor for relapse-free survival (RFS). To further clarify the relationship between Tregs and the efficacy of CAR-T therapy, the present study tested the levels of CD4+CD25+CD127low Tregs in peripheral blood (PB) of R/R B-ALL patients at different stages of CD19 CAR-T therapy, and evaluate their impact on the efficacy and prognosis of CAR-T therapy. METHODS: From November 2015 to May 2019, 47 R/R B-ALL patients successfully received CD19 CAR-T therapy at our institution and followed up for at least 1 month. Among them, one patient did not tested for Tregs, so 46 patients enrolled in this study. We collected clinical information of them and dynamically detected the frequency of CD4+CD25+CD127low Tregs within CD4 + T cells at different time points (before infusion and at 1 week after infusion) by flow cytometry, and validated the relationship of circulating Tregs with clinical efficacy, OS, and recurrence of CAR-T therapy. RESULTS: Circulating Tregs of R/R B-ALL patients in pre-infusion group (median 6.67%) and in 1 week after infusion group (median 6.80%) were all higher than that of the healthy control group (median 5.04%), with statistical significance (P < 0.05). The frequencies of Tregs in not remission (NR) group at baseline (pre-infusion) and at 1 week after infusion were all significantly higher than those in remission group. With cut-off values of 11.54% (before infusion) and 13.56% (1 week after infusion), the specificity for Tregs were 94.6% and 100% , respectively. In remission group, 11 patients underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) after achieving remission by Sino 19 cell therapy. No significant differences of Tregs expression were found between transplantation and non-transplantation groups. Time-dependent Cox model showed that transplantation group had lower risk of relapse and death when compared with non-transplantation group (HR = 0.664 for RFS and HR = 0.364 for OS), however, no statistical significances were found (P = 0.403 and 0.106, respectively). Higher Tregs before infusion and at 1 week after infusion were significant associated with shorter RFS and OS by Kaplan-Meier analysis. Multivariate analysis showed that higher Tregs at 1 week after infusion was the independently factor for poor RFS (P = 0.032) and shorter OS (P = 0.025) in R/R B-ALL patients with CD19 CAR-T therapy. Besides, Tregs levels before and at 1 week after infusion were negatively correlated with the persistence time of Sino 19 cell. CONCLUSION: Higher circulating Tregs, especially 1 week after CD19 CAR-T cell infusion, was a poor predict indicator for CD19 CAR-T therapy in R/R B-ALL patients.

Multiplexed histology analyses for the phenotypic and spatial characterization of human innate lymphoid cells.

Innate lymphoid cells (ILCs) emerge in the last few years as important regulators of immune responses and biological processes. Although ILCs are mainly known as tissue-resident cells, their precise localization and interactions with the microenvironment are still unclear. Here we combine a multiplexed immunofluorescence technique and a customized computational, open-source analysis pipeline to unambiguously identify CD127+ ILCs in situ and characterize these cells and their microenvironments. Moreover, we reveal the transcription factor IRF4 as a marker for tonsillar ILC3, and identify conserved stromal landmarks characteristic for ILC localization. We also show that CD127+ ILCs share tissue niches with plasma cells in the tonsil. Our works thus provide a platform for multiparametric histological analysis of ILCs to improve our understanding of ILC biology.

CHBP induces stronger immunosuppressive CD127+ M-MDSC via erythropoietin receptor.

Erythropoietin (EPO) is not only an erythropoiesis hormone but also an immune-regulatory cytokine. The receptors of EPO (EPOR)2 and tissue-protective receptor (TPR), mediate EPO's immune regulation. Our group firstly reported a non-erythropoietic peptide derivant of EPO, cyclic helix B peptide (CHBP), which could inhibit macrophages inflammation and dendritic cells (DCs) maturation. As a kind of innate immune regulatory cell, myeloid-derived suppressor cells (MDSCs) share a common myeloid progenitor with macrophages and DCs. In this study, we investigated the effects on MDSCs differentiation and immunosuppressive function via CHBP induction. CHBP promoted MDSCs differentiate toward M-MDSCs with enhanced immunosuppressive capability. Infusion of CHBP-induced M-MDSCs significantly prolonged murine skin allograft survival compared to its counterpart without CHBP stimulation. In addition, we found CHBP increased the proportion of CD11b+Ly6G-Ly6Chigh CD127+ M-MDSCs, which exerted a stronger immunosuppressive function compared to CD11b+Ly6G-Ly6Chigh CD127- M-MDSCs. In CHBP induced M-MDSCs, we found that EPOR downstream signal proteins Jak2 and STAT3 were upregulated, which had a strong relationship with MDSC function. In addition, CHBP upregulated GATA-binding protein 3 (GATA-3) protein translation level, which was an upstream signal of CD127 and regulator of STAT3. These effects of CHBP could be reversed if Epor was deficient. Our novel findings identified a new subset of M-MDSCs with better immunosuppressive capability, which was induced by the EPOR-mediated Jak2/GATA3/STAT3 pathway. These results are beneficial for CHBP clinical translation and MDSC cell therapy in the future.

Level of Foxp3, DNMTs, methylation of Foxp3 promoter region, and CD4 + CD25 + CD127low regulatory T cells in vulvar lichen sclerosus.

This study is to investigate the pathogenesis of vulvar lichen sclerosus (VLS) by analyzing the level of Foxp3, DNMTs, methylation of Foxp3 promoter region, and CD4 + CD25 + CD127low Regulatory T cells (Tregs). This study enrolled 15 VLS patients and 25 controls. Lesional and extralesional vulvar skin tissues, normal vulvar skin tissues and peripheral blood were collected. Compared with the control group, Foxp3 protein in the lesional and extralesional skin of VLS group was significantly reduced. The levels of DNMT1 and DNMT3b proteins in lesional skin of VLS group were significantly increased. There was no difference in the total methylation rates of the promoter region of the Foxp3 gene. The methylation rates of CpG1, CpG4, CpG9, and CpG10 were significantly higher in lesional skin of VLS group than in control group. There was no correlation between the total methylation rates of 10 CpG sites and the level of Foxp3 and DNMT1 proteins; there was a positive correlation between Foxp3 and DNMT1 protein in lesional skin of VLS group (r = 0.675, p < 0.05), and a negative correlation (r = -0.665, p < 0.05) in extralesional skin of VLS group. However, there was no correlation of Foxp3 with DNMT3b. The number of CD4 + CD25 + CD127low Tregs VLS decreased significantly. The expression of Foxp3 protein and the quantity of CD4 + CD25 + CD127low Tregs in patients with VLS decreased, which may cause local or systemic abnormal immunosuppression of Tregs, leading to the occurrence of VLS. This may be related with methylation or DNMT1, which needs further verification.