Alpharetroviral Vector-Mediated Gene Therapy for IL7RA-Deficient Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) encompasses rare primary immunodeficiency disorders characterized by deficient T-cell development, which leads to a severely compromised immune system and susceptibility to life-threatening infections. Among SCID subtypes, IL7RA-SCID is caused by mutations in the interleukin 7 receptor alpha chain (IL7RA) and represents a significant subset of patients with limited treatment options. This study investigated the efficacy of a self-inactivating (SIN) alpharetroviral vector (ARV) engineered to deliver a codon-optimized IL7RA cDNA to restore T-cell development in Il7r-knockout mice. We compared the elongation factor 1 alpha short (EFS) promoter and the lymphoid-restricted Lck promoter for their ability to drive IL7RA expression and found that the EFS promoter enabled robust and sustained IL7RA expression that led to the functional rescue of T-lymphopoiesis in vitro and in vivo. Conversely, though effective in vitro, the Lck promoter failed to produce viable T-cell populations in vivo. Our results highlight the potential of using SIN-ARVs as a gene therapy (GT) strategy for treating IL7RA-SCID. Importantly, sustained production of T-lymphocytes was found in both primary and secondary transplant recipient animals with no adverse effects, supporting the safety and feasibility of this approach. Overall, this study provides valuable insights into the development of GT for IL7RA-SCID and underscores the clinical potential of an EFS-driven SIN-ARV to restore IL7RA-deficient immune function.

Activation of Cell-Intrinsic Signaling in CAR-T Cells via a Chimeric IL7R Domain.

Chimeric antigen receptor (CAR) T cells can effectively treat leukemias, but sustained antitumor responses can be hindered by a lack of CAR T-cell persistence. Cytotoxic effector T cells are short-lived, and establishment of CAR-T cells with memory to ensure immune surveillance is important. Memory T cells depend on cytokine support, with IL7 activation of the IL7 receptor (IL7R) being critical. However, IL7R surface expression is negatively regulated by exposure to IL7. We aimed to support CAR T-cell persistence by equipping CAR-T cells with a sustained IL7Rα signal. We engineered T cells to constitutively secrete IL7 or to express an anti-acute myeloid leukemia-targeted IL7Rα-chimeric cytokine receptor (CCR) and characterized the phenotype of these cell types. Canonical downstream signaling was activated in CCR-T cells with IL7R activation. When coexpressed with a cytotoxic CAR, functionality of both the CCR and CAR was maintained. We designed hybrid CAR-CCR and noted membrane proximity of the intracellular domains as vital for signaling. These data show cell-intrinsic cytokine support with canonical signaling, and functionality can be provided via expression of an IL7Rα domain whether independently expressed or incorporated into a cytotoxic CAR for use in anticancer therapy. SIGNIFICANCE: To improve the phenotype of tumor-directed T-cell therapy, we show that provision of cell-intrinsic IL7R-mediated signaling is preferable to activation of cells with exogenous IL7. We engineer this signaling via independent receptor engineering and incorporation into a CAR and validate maintained antigen-specific cytotoxic activity.

CD161+CD127+CD8+ T cell subsets can predict the efficacy of anti-PD-1 immunotherapy in non-small cell lung cancer with diabetes mellitus.

The role of CD161+CD127+CD8+ T cells in non-small cell lung cancer (NSCLC) patients with diabetes remains unexplored. This study determined the prevalence, phenotype, and function of CD8+ T cell subsets in NSCLC with diabetes. We recruited NSCLC patients (n = 436) treated with anti-PD-1 immunotherapy as first-line treatment. The progression-free survival (PFS), overall survival (OS), T cells infiltration, and peripheral blood immunological characteristics were analyzed in NSCLC patients with or without diabetes. NSCLC patients with diabetes exhibited shorter PFS and OS (p = 0.0069 and p = 0.012, respectively) and significantly lower CD8+ T cells infiltration. Mass cytometry by time-of-flight (CyTOF) showed a higher percentage of CD161+CD127+CD8+ T cells among CD8+T cells in NSCLC with diabetes before anti-PD-1 treatment (p = 0.0071) than that in NSCLC without diabetes and this trend continued after anti-PD-1 treatment (p = 0.0393). Flow cytometry and multiple-immunofluorescence confirmed that NSCLC with diabetes had significantly higher CD161+CD127+CD8+ T cells to CD8+T cells ratios than NSCLC patients without diabetes. The RNA-sequencing analysis revealed immune-cytotoxic genes were reduced in the CD161+CD127+CD8+ T cell subset compared to CD161+CD127-CD8+ T cells in NSCLC with diabetes. CD161+CD127+CD8+ T cells exhibited more T cell-exhausted phenotypes in NSCLC with diabetes. NSCLC patients with diabetes with ≥ 6.3% CD161+CD127+CD8+ T cells to CD8+T cells ratios showed worse PFS. These findings indicate that diabetes is a risk factor for NSCLC patients who undergo anti-PD-1 immunotherapy.CD161+CD127+CD8+ T cells could be a key indicator of a poor prognosis in NSCLC with diabetes. Our findings would help in advancing anti-PD-1 therapy in NSCLC patients with diabetes.

Human CD127 negative ILC2s show immunological memory.

ILC2s are key players in type 2 immunity and contribute to maintaining homeostasis. ILC2s are also implicated in the development of type 2 inflammation-mediated chronic disorders like asthma. While memory ILC2s have been identified in mouse, it is unknown whether human ILC2s can acquire immunological memory. Here, we demonstrate the persistence of CD45RO, a marker previously linked to inflammatory ILC2s, in resting ILC2s that have undergone prior activation. A high proportion of these cells concurrently reduce the expression of the canonical ILC marker CD127 in a tissue-specific manner. Upon isolation and in vitro stimulation of CD127-CD45RO+ ILC2s, we observed an augmented ability to proliferate and produce cytokines. CD127-CD45RO+ ILC2s are found in both healthy and inflamed tissues and display a gene signature of cell activation. Similarly, mouse memory ILC2s show reduced expression of CD127. Our findings suggest that human ILC2s can acquire innate immune memory and warrant a revision of the current strategies to identify human ILC2s.

Identification of amino acids in transmembrane domains of mutated cytokine receptor-like factor 2 and interleukin-7 receptor α required for constitutive signal transduction.

Cytokine receptor-like factor 2 (CRLF2) and interleukin-7 receptor α (IL-7Rα) form a receptor for thymic stromal lymphopoietin (TSLP). A somatic mutation consisting of the substitution of five amino acids (SLLLL) in the transmembrane domain of CRLF2 with three amino acids, including glutamic acid, isoleucine, and methionine (insEIM), which has been identified in acute lymphocytic leukemia, causes the TSLP-independent dimerization with IL-7Rα and activation. However, the dimerization mechanism remains unclear. In this study, we examined the involvement of the amino acids in the transmembrane domains of EIM CRLF2 and IL-7Rα in TSLP-independent activation. HEK293 cells were transfected with vectors encoding CRLF2 and IL-7Rα, or their mutants, in which the amino acid of the transmembrane domain was replaced with alanine. STAT5 phosphorylation was detected using western blotting, and receptor dimerization was analyzed using the NanoBiT assay. The substitution of glutamic acid within the insEIM mutation for alanine failed to cause the STAT5 phosphorylation in the absence of TSLP. Moreover, the alanine substation of the specific leucine residues in the transmembrane domains of both CRLF2 and IL-7Rα abrogated the TSLP-independent signal transduction and dimerization. The mutation of IL-7Rα W264 partially reduced the phosphorylation of STAT5 without affecting receptor dimerization. These results suggest that the amino acids in the transmembrane domains of EIM CRLF2 and IL-7Rα play at least three possible functions: interaction through hydrogen bonds, hydrophobic interaction, and signal transduction. Our findings contribute to a better understanding of the function of the transmembrane domains of cytokine receptors in their dimerization and signal transduction.

Phase I Trial of GD2.CART Cells Augmented With Constitutive Interleukin-7 Receptor for Treatment of High-Grade Pediatric CNS Tumors.

PURPOSE: T cells modified with chimeric antigen receptors (CARTs) have demonstrated efficacy for hematologic malignancies; however, benefit for patients with CNS tumors has been limited. To enhance T cell activity against GD2+ CNS malignancies, we modified GD2-directed CART cells (GD2.CARTs) with a constitutively active interleukin (IL)-7 receptor (C7R-GD2.CARTs). METHODS: Patients age 1-21 years with H3K27-altered diffuse midline glioma (DMG) or other recurrent GD2-expressing CNS tumors were eligible for this phase I trial (ClinicalTrials.gov identifier: NCT04099797). All subjects received standard-of-care adjuvant radiation therapy or chemotherapy before study enrollment. The first treatment cohort received GD2.CARTs alone (1 × 107 cells/m2), and subsequent cohorts received C7R-GD2.CARTs at two dose levels (1 × 107 cells/m2; 3 × 107 cells/m2). Standard lymphodepletion with cyclophosphamide and fludarabine was included at all dose levels. RESULTS: Eleven patients (age 4-18 years) received therapy without dose-limiting toxicity. The GD2.CART cohort did not experience toxicity, but had disease progression after brief improvement of residual neurologic deficits (≤3 weeks). The C7R-GD2.CART cohort developed grade 1 tumor inflammation-associated neurotoxicity in seven of eight (88%) cases, controllable with anakinra. Cytokine release syndrome was observed in six of eight (75%, grade 1 in all but one patient) and associated with increased circulating IL-6 and IP-10 (P < .05). Patients receiving C7R-GD2.CARTs experienced temporary improvement from baseline neurologic deficits (range, 2 to >12 months), and seven of eight (88%) remained eligible for additional treatment cycles (range 2-4 cycles). Partial responses by iRANO criteria were observed in two of seven (29%) patients with DMG treated by C7R-GD2.CARTs. CONCLUSION: Intravenous GD2.CARTs with and without C7R were well tolerated. Patients treated with C7R-GD2.CARTs exhibited transient improvement of neurologic deficits and increased circulating cytokines/chemokines. Treatment with C7R-GD2.CARTs represents a novel approach warranting further investigation for children with these incurable CNS cancers.

Memory CD8 T cells are vulnerable to chronic IFN-γ signals but not to CD4 T cell deficiency in MHCII-deficient mice.

The mechanisms by which the number of memory CD8 T cells is stably maintained remains incompletely understood. It has been postulated that maintaining them requires help from CD4 T cells, because adoptively transferred memory CD8 T cells persist poorly in MHC class II (MHCII)-deficient mice. Here we show that chronic interferon-γ signals, not CD4 T cell-deficiency, are responsible for their attrition in MHCII-deficient environments. Excess IFN-γ is produced primarily by endogenous colonic CD8 T cells in MHCII-deficient mice. IFN-γ neutralization restores the number of memory CD8 T cells in MHCII-deficient mice, whereas repeated IFN-γ administration or transduction of a gain-of-function STAT1 mutant reduces their number in wild-type mice. CD127high memory cells proliferate actively in response to IFN-γ signals, but are more susceptible to attrition than CD127low terminally differentiated effector memory cells. Furthermore, single-cell RNA-sequencing of memory CD8 T cells reveals proliferating cells that resemble short-lived, terminal effector cells and documents global downregulation of gene signatures of long-lived memory cells in MHCII-deficient environments. We propose that chronic IFN-γ signals deplete memory CD8 T cells by compromising their long-term survival and by diverting self-renewing CD127high cells toward terminal differentiation.

Novel Synonymous Variant in IL7R Causes Preferential Expression of the Soluble Isoform.

PURPOSE: The interleukin-7 receptor (IL-7R) is primarily expressed on lymphoid cells and plays a crucial role in the development, proliferation, and survival of T cells. Autosomal recessive mutations that disrupt IL-7Rα chain expression give rise to a severe combined immunodeficiency (SCID), which is characterized by lymphopenia and a T-B+NK+ phenotype. The objective here was to diagnose two siblings displaying the T-B+NK+ SCID phenotype as initial clinical genetic testing did not detect any variants in known SCID genes. METHODS: Whole genome sequencing (WGS) was utilized to identify potential variants causing the SCID phenotype. Splicing prediction tools were employed to assess the deleterious impact of the mutation. Polymerase Chain Reaction (PCR), Sanger sequencing, flow cytometry, and ELISA were then used to validate the pathogenicity of the detected mutation. RESULTS: We discovered a novel homozygous synonymous mutation in the IL7R gene. Our functional studies indicate that this variant is pathogenic, causing exon 6, which encodes the transmembrane domain, to be preferentially spliced out. CONCLUSION: In this study, we identified a novel rare synonymous mutation causing a loss of IL-7Rα expression at the cellular membrane. This case demonstrates the value of reanalyzing genetic data based on the clinical phenotype and highlights the significance of functional studies in determining the pathogenicity of genetic variants.

[Circulating populations of CD4+ CD25+ CD127- regulatory t lymphocytes in peripheral blood of allergic asthmatic children]. / Determinación de poblaciones circulantes en sangre periférica de linfocitos T, reguladores CD4+ CD25+ CD127-, en niños asmáticos alérgicos.

OBJECTIVE: To carry out a preliminary analysis on the Treg lymphocyte counts present in the peripheral blood of allergic asthmatic children from the city of Cartagena, Colombia, compared to healthy controls. METHODS: We compared cytometry counts of ten asthmatic patients (age 7-16 years) and seven healthy controls (6-12 years), recruited in the city of Cartagena. Peripheral blood samples were stained using Cytek's 14-color cFluor Immunoprofiling kit (Cytek® cFluor® Immunoprofiling Kit 14 Color RUO kit), and analyzed on a Northern Lights™ spectral cytometer (Cytek® Biosciences, Fremont, CA, USA), to read 50.000 events per sample. The data obtained were analyzed in SpectroFlo® and FlowJo. The study was approved by the ethics committee of the University of Cartagena (SGR, Grant BPIN2020000100405). RESULTS: The frequency of CD3+, CD4+, CD25+, CD127- Tregs was 11% of all CD4+ T cells, with a range of minimum 8,1% and maximum 17,7%. There was no significant difference in the proportion of Tregs between allergic asthmatic patients and healthy controls (P = 0,2). CONCLUSIONS: With this preliminary sample size, no significant differences were found in the Treg lymphocyte population between allergic asthmatic patients and healthy controls. The 14-color multiplexed panel is a useful tool not only to count CD3+ and CD4+ populations, but also to obtain the percentage of regulatory T cells using cell surface markers.

OBJETIVO: Realizar un análisis preliminar sobre los conteos de linfocitos Tregs presentes en sangre periférica de niños asmáticos alérgicos de la ciudad de Cartagena, comparado con controles sanos. MÉTODOS: Se compararon los conteos de citometría de diez pacientes asmáticos (entre 7 y16 años) y siete controles sanos (entre 6 y12 años), reclutados en la ciudad de Cartagena. La muestra de sangre periférica fue teñida empleando el kit de inmunofenotipo multiplexado de 14 colores de Cytek (Cytek® cFluor® Immunoprofiling Kit 14 Color), y analizada en un citómetro espectral Northern Lights™ (Cytek® Biosciences, Fremont, CA, USA), a lectura de 50.000 eventos por muestra. Los datos obtenidos fueron analizados en SpectroFlo® y FlowJo. El estudio fue aprobado por el Comité de Ética de la Universidad de Cartagena. RESULTADOS: El panel de tinción funcionó apropiadamente y dentro de los parámetros apropiados. Se obtuvo un promedio de células Tregs CD3+, CD4+, CD25+ y CD127- del 11% de todos los CD4+ en las muestras estudiadas, con un rango de mínimo de 8,1% y un máximo de 17,7%. No hubo diferencias significativas en la proporción de linfocitos Tregs entre los pacientes asmáticos alérgicos y los controles sanos (P = 0.2). CONCLUSIONES: Con este tamaño de muestra preliminar, no se encontraron diferencias significativas en la población de linfocitos Tregs entre los pacientes asmáticos alérgicos y los controles sanos. El panel multiplexado de 14 colores es una herramienta útil no solo para derivar las poblaciones CD3+ y CD4+, sino también para obtener el porcentaje de células T reguladoras empleando marcadores de superficie celular.

The IL-7R antagonist lusvertikimab reduces leukemic burden in xenograft ALL via antibody-dependent cellular phagocytosis.

ABSTRACT: Acute lymphoblastic leukemia (ALL) arises from the uncontrolled proliferation of B-cell precursors (BCP-ALL) or T cells (T-ALL). Current treatment protocols obtain high cure rates in children but are based on toxic polychemotherapy. Novel therapies are urgently needed, especially in relapsed/refractory (R/R) disease, high-risk (HR) leukemias and T-ALL, in which immunotherapy approaches remain scarce. Although the interleukin-7 receptor (IL-7R) plays a pivotal role in ALL development, no IL-7R-targeting immunotherapy has yet reached clinical application in ALL. The IL-7Rα chain (CD127)-targeting IgG4 antibody lusvertikimab (LUSV; formerly OSE-127) is a full antagonist of the IL-7R pathway, showing a good safety profile in healthy volunteers. Here, we show that ∼85% of ALL cases express surface CD127. We demonstrate significant in vivo efficacy of LUSV immunotherapy in a heterogeneous cohort of BCP- and T-ALL patient-derived xenografts (PDX) in minimal residual disease (MRD) and overt leukemia models, including R/R and HR leukemias. Importantly, LUSV was particularly effective when combined with polychemotherapy in a phase 2-like PDX study with CD127high samples leading to MRD-negativity in >50% of mice treated with combination therapy. Mechanistically, LUSV targeted ALL cells via a dual mode of action comprising direct IL-7R antagonistic activity and induction of macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). LUSV-mediated in vitro ADCP levels significantly correlated with CD127 expression levels and the reduction of leukemia burden upon treatment of PDX animals in vivo. Altogether, through its dual mode of action and good safety profile, LUSV may represent a novel immunotherapy option for any CD127+ ALL, particularly in combination with standard-of-care polychemotherapy.

Intragraft memory-like CD127hiCD4+Foxp3+ Tregs maintain transplant tolerance.

CD4+Foxp3+ regulatory T cells (Tregs) play an essential role in suppressing transplant rejection, but their role within the graft and heterogeneity in tolerance are poorly understood. Here, we compared phenotypic and transcriptomic characteristics of Treg populations within lymphoid organs and grafts in an islet xenotransplant model of tolerance. We showed Tregs were essential for tolerance induction and maintenance. Tregs demonstrated heterogeneity within the graft and lymphoid organs of tolerant mice. A subpopulation of CD127hi Tregs with memory features were found in lymphoid organs, presented in high proportions within long-surviving islet grafts, and had a transcriptomic and phenotypic profile similar to tissue Tregs. Importantly, these memory-like CD127hi Tregs were better able to prevent rejection by effector T cells, after adoptive transfer into secondary Rag-/- hosts, than naive Tregs or unselected Tregs from tolerant mice. Administration of IL-7 to the CD127hi Treg subset was associated with a strong activation of phosphorylation of STAT5. We proposed that memory-like CD127hi Tregs developed within the draining lymph node and underwent further genetic reprogramming within the graft toward a phenotype that had shared characteristics with other tissue or tumor Tregs. These findings suggested that engineering Tregs with these characteristics either in vivo or for adoptive transfer could enhance transplant tolerance.

A novel 268 kb deletion combined with a splicing variant in IL7R causes of severe combined immunodeficiency in a Chinese family: a case report.

BACKGROUND: Severe combined immunodeficiency (SCID) is a group of fatal primary immunodeficiencies characterized by the severe impairment of T-cell differentiation. IL7R deficiency is a rare form of SCID that usually presents in the first months of life with severe and opportunistic infections, failure to thrive, and a high risk of mortality unless treated. Although recent improvements in early diagnosis have been achieved through newborn screening, few IL7R-related SCID patients had been reported in the Chinese population. CASE PRESENTATION: Here, we retrospectively analyzed a case of SCID in a 5-month-old girl with symptoms, including severe T-cell depletion, recurrent fever, oral ulcers, pneumonia, hepatosplenomegaly, bone marrow hemophagocytosis, and bacterial and viral infections. Whole-exome sequencing (WES), quantitative PCR (qPCR), and chromosome microarray analysis (CMA) were performed to identify the patient's genetic etiology. We identified a 268 kb deletion and a splicing variant, c.221 + 1G > A, in the proband. These two variants of IL7R were inherited from the father and mother. CONCLUSIONS: To our knowledge, this is the first report of whole IL7R gene deletion in combination with a pathogenic splicing variant in a patient with SCID. This deletion also expands the pathogenic variation spectrum of SCID caused by IL7R. The incorporation of exome-based copy number variant analysis makes WES a powerful molecular diagnostic technique for the clinical diagnosis of pediatric patients.

Investigation of the IL7Rα Gene Polymorphism rs6897932 and the Expression Levels of the CDH1, TTPAL, and FHIT Genes in Patients with Breast Cancer.

OBJECTIVE: The aim of this study was to determine the expression levels of CDH1, FHIT, and TTPAL genes and to determine the genotype and allele frequencies of the IL7Rα gene polymorphism rs6897932 in patients with breast cancer. METHODS: The expression levels of genes and the distribution of the IL7Rα gene polymorphism rs6897932 were analyzed by real-time polymerase chain reaction. RESULTS: No differences in genotype ratios or allele frequencies were observed between the 2 groups for the IL7Rα gene polymorphism rs6897932. The frequency of the IL7Rα rs6897932 T risk allele was found to be similar between breast cancer patients and controls. CDH1 messenger RNA (mRNA) levels decreased (0.714-fold and 0.834-fold, respectively), and TTPAL mRNA levels increased (2.675-fold [P < .05] and 1.169-fold, respectively) in tumor tissues and peripheral blood samples. FHIT mRNA levels decreased (0.559-fold) in tumor tissue samples and increased (2.21-fold) in peripheral blood samples. CONCLUSION: Our results are compatible with those reported in the literature. It can be suggested that the upregulation observed in the TTPAL gene might be a marker for breast cancer. The downregulation of CDH1 and FHIT gene expression has been validated in our study. An increase in the copy numbers of FHIT mRNA in blood samples and a decrease in the tumor samples can also be considered an abnormal condition.

IL7RA rs10491434 polymorphism is related to spontaneous HIV infection control in naïve HIV-infected patients: A retrospective study.

Interleukin 7 receptor (IL7R) is vital in the adaptive immune response against human immunodeficiency viruses (HIV). We assessed IL7RA polymorphisms (SNPs) in antiretroviral therapy (ART)-naïve HIV patients for their association with spontaneous HIV infection control. We conducted a retrospective cohort study involving 667 ART-naïve patients categorized by HIV progression (ordinal variable): 150 rapid progressors, 334 moderate/typical progressors, 86 long-term nonprogressors elite controllers (LTNPs-EC), and 97 LTNPs-non-EC. We genotyped three IL7RA SNPs using Agena Bioscience's MassARRAY platform. The association between IL7RA SNPs and spontaneous HIV infection control was evaluated using ordinal logistic regression. Individuals carrying the rs10491434 G allele have a higher likelihood of spontaneous HIV infection control (adjusted odds ratio [aOR] = 1.33; p = 0.023). Moreover, the IL7RA GCT haplotype, consisting of three specific SNPs (rs6897932, rs987106, and rs10491434), demonstrated an association with the control of untreated HIV infection (aOR = 1.34; p = 0.050). Remarkably, the rs10491434 SNP and the IL7RA GCT haplotype exhibited similar aOR values, suggesting that rs10491434 may be primarily responsible for the observed effect of the haplotype. IL7RA rs10491434 G allele is associated with a higher likelihood of spontaneous HIV infection control, indicating its significant role in the pathogenesis of HIV, possibly influencing infection course and viral replication control.

Structural basis of γ chain family receptor sharing at the membrane level.

Common γ chain (γc) cytokine receptors, including interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, are activated upon engagement with a common γc receptor (CD132) by concomitant binding of their ectodomains to an interleukin. In this work, we find that direct interactions between the transmembrane domains (TMDs) of both the γc and the interleukin receptors (ILRs) are also required for receptor activation. Moreover, the same γc TMD can specifically recognize multiple ILR TMDs of diverse sequences within the family. Heterodimer structures of γc TMD bound to IL-7 and IL-9 receptor TMDs-determined in a lipid bilayer-like environment by nuclear magnetic resonance spectroscopy-reveal a conserved knob-into-hole mechanism of recognition that mediates receptor sharing within the membrane. Thus, signaling in the γc receptor family requires specific heterotypic interactions of the TMDs.

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.

Research Note: Development and characterization of monoclonal antibodies specific for chicken interleukin-7 receptor α (CD127).

CD127, also named interleukin-7 receptor (IL-7R), is expressed on various cell types including naive and memory T cells, and plays a critical role in the differentiation and activation of T lymphocytes. The availability of poultry-specific immune reagents to identify and measure chicken CD127 response will enhance fundamental and applied research in poultry immunology. Mouse monoclonal antibodies (MAbs) against chicken CD127 (chCD127) were developed and characterized. More specifically, a 678 bp ectodomain of chCD127 gene was cloned in the pET28a (+) vector and expressed in BL21-AI E. coli competent cells. The recombinant chCD127 protein with a size of 30 KDa which was also recognized by a mouse anti-human CD127 MAb (Clone G-11) was used to immunize mice, and 6 new mouse MAbs which specifically detected chicken CD127 were developed and characterized. Availability of these new sets of chCD127-specific MAbs will facilitate the immunological studies on CD127 in poultry, especially in understanding effector and memory T immune cell responses in normal and diseased states.

Low IL7R Expression at Diagnosis Predicted Relapse in Adult Acute Myeloid Leukemia Patients With t(8;21).

Background: Acute myeloid leukemia (AML) with t(8;21) needs to be further stratified. In addition to leukemia cells, immune cells in tumor microenvironment participate in tumor initiation, growth and progression. Interleukins (ILs)/interleukin receptors (ILRs) interaction plays important roles in the antitumor immune response. IL7R is reported to be relevant to prognosis in solid tumor and acute lymphoblastic leukemia. However, the prognostic significance of IL7R in t(8;21) AML remains to be clarified. Methods: Bone marrows collected from 156 newly diagnosed t(8;21) AML patients were used for testing IL7R transcript level by TaqMan-based real-time quantitative PCR (RQ-PCR), and RNAseq were performed in 15 of them. Moreover, IL7R expression at diagnosis were measured by RQ-PCR and flow cytometry (FCM) simultaneously in other 13 t(8;21) AML patients. Results: t(8;21) AML patients had varied IL7R transcript levels and were categorized into low-expression (IL7R-L) and high-expression (IL7R-H) groups; IL7R-L was significantly associated with a lower relapse-free survival (RFS) rate (P=0.0027) and KITD816/D820 mutation (P=0.0010). Furthermore, IL7R-L was associated with a lower RFS rate in KITD816/D820 group (P=0.013) and IL7R-H/KITD816/D820 patients had similar RFS to KITN822/e8/WT patients (P=0.35). GO analysis enrichment showed that down-regulated genes were predominantly involved in the regulation of T cell and leukocyte activation, proliferation and differentiation in IL7R-L group. IL7R-L had significantly lower levels of Granzymes A/B, CCR7, CD28 and CD27 than IL7R-H group (all P<0.05). FCM analysis showed IL7R protein was primarily expressed in CD4+ T and CD8+ T cell subset. A significant association was found between the transcript level of IL7R and the percentage of CD8+ T cells in nucleated cells (P=0.015) but not CD4+ T cells (P=0.47). Conclusion: Low IL7R transcript level of bone marrow at diagnosis predicted relapse in t(8;21) AML, which might be caused by the difference in the amount, status and function of T cells.

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.