Frequency Of CD4 Research Articles

Prognostic significance of the frequencies of bone marrow lymphocyte subsets in adult acute myeloid leukemia at diagnosis.

Immune microenvironment plays an important role in the occurrence and development of acute myeloid leukemia (AML). Studies assessing the prognostic significance of bone marrow (BM) lymphocyte subsets' frequencies at diagnosis in patients with AML were limited. Fresh BM samples collected from 97 adult AML patients at diagnosis were tested for lymphocyte, T, CD4+ T, CD8+ T, γδT, NK, and B cell frequencies using multi-parameter flow cytometry. Low frequencies of lymphocytes, T, CD4+ T, and CD8+ T cells were associated with significantly lower rates of one-course complete remission (CR) (all p < 0.05). Moreover, the frequency of CD4+ T cells independently predicted one-course CR achievement (p = 0.021). Low frequencies of T and CD8+ T cells were significantly associated with lower relapse-free survival (RFS) rates (p = 0.032; 0.034), respectively, and a low frequency of CD8+ T cells was associated with a significantly lower overall survival (OS) rate (p = 0.028). Combination of frequency of CD8+ T cells and ELN risk stratification showed that patients with ELN-intermediate/adverse risk + high CD8+ T cell frequency had a similar RFS rate to those with ELN-favorable risk + high CD8+ T cell frequency and those with ELN-favorable risk + low CD8+ T cell frequency (p = 0.88; 0.76), respectively. The RFS rate of patients with ELN intermediate/adverse risk + low CD8+ T cell frequency was significantly lower than that of all aforementioned patients (p = 0.021; 0.0007; 0.028), respectively. The frequencies of BM lymphocyte subsets at diagnosis predicted clinical outcomes and could help improve risk stratification in AML.

Too Much of a Good Thing: Extended Duration of Gut Microbiota Depletion Reverses Protection From Experimental Autoimmune Uveitis.

Using the model of experimental autoimmune uveitis (EAU) induced by immunization with a retinal antigen, two studies have reported contradictory results on disease development following oral antibiotic treatment (ABX). We showed that long-term ABX did not affect EAU, but another study showed that short-term ABX was protective. We therefore studied the effects of ABX on EAU, gut microbiota, and host immune responses as a function of treatment duration. EAU-susceptible mice were treated orally with broad-spectrum antibiotics starting at least 10 weeks (long-term) or 1 week (short-term) before immunization until termination of the experiment. Gut microbiota were characterized by 16S amplicon sequencing, and host gut immune elements were studied phenotypically and functionally. Long-term ABX had no effect, whereas short-term ABX delayed EAU, as previously reported by us and others, respectively. Microbial sequencing revealed progressive reduction of gut microbiota that showed some differences in the two ABX groups. Interestingly, duration of ABX was associated with a gradual disappearance of the CD4+ and CD4+CD8+ subset of gut intraepithelial lymphocytes (IELs). This IEL subset is microbiota dependent and is absent in germ-free mice. Relative abundance of Lactobacillus reuteri correlated with the frequencies of CD4+CD8+ IELs. IELs suppressed antigen-specific activation of autoreactive T cells in culture. Gut microbiota may play dual roles in uveitis development: They promote EAU development but also help maintain IEL populations that have regulatory function against autoreactive T cells. We propose that the progressive loss of this population during long-term ABX reverses the EAU-ameliorating effects of microbiota depletion.

Phenotypic and Molecular Landscape of Bone Marrow CD8+ T Cells in Myeloid Neoplasms Predicts Response to Treatment with Hypomethylating Agents

Background and aims Myeloid malignancies encompass a diverse group of conditions, including myelodysplastic neoplasms (MDS) and chronic myelomonocytic leukemia (CMML). There is evidence suggesting the role of bone marrow (BM) environment and immune cells in the development and progression of MDS and CMML. Azacytidine (AZA) remains the backbone therapy for higher-risk MDS and non-proliferative CMML. To date, molecular predictors of response to AZA are not yet well-defined. Herein, we sought to investigate the immune landscape of BM samples from patients with lower-risk MDS (LR-MDS; i.e., IPSS-R &amp;lt;3.5), CMML, higher-risk MDS (HR-MDS; i.e., IPSS-R ≥3.5), and acute myeloid leukemia (AML). Methods Samples were collected before treatment initiation. Mass cytometry (CyTOF) was performed on isolated BM mononuclear cells from 48 patients, using the Maxpar® Direct™ Immune Profiling Assay. Flow cytometry was performed on a secondary cohort of 56 patients. scRNA-sequencing was performed on sorted BM CD8+ T cells from patients with HR-MDS (n=4) and AML (n=5), using a 10x Genomics pipeline. Mann-Whitney U test and the Kruskal-Wallis test were used as appropriate. Kaplan-Meier analysis was utilized for survival analysis, and the log-rank test was employed to assess the significance. The level of significance was established at P &amp;lt; 0.05. Results The immunophenotypic analysis using CyTOF demonstrated increased expression of CXCR3 on cytotoxic CD8+ T-cells in CMML and AML samples compared to samples from patients with MDS. Increased frequency of the CD57 +CXCR3 +CD8 + T-cell subset was further observed in patients with HR-MDS and AML that were non-responders to AZA, compared to responders, as observed by both CyTOF and flow cytometry. After determining the optimal cut-off for the frequency of CD8 +CD57 +CXCR3 + T cells at 29%, we observed that HR-MDS and AML patients (Figure 1A) with a frequency of CD57 +CXCR3 +CD8 + of &amp;lt;29% had better overall survival (OS).Multivariate analysisshowed that a frequency of CD57 +CXCR3 +CD8 + of &amp;gt;29% was independently associated with decreased survival in patients with TET2 mutations (HR=2.603; 1.275-5.317). scRNA-seq analysis of isolated BM CD8 + T cells was further performed to characterize the molecular signature that favors response to AZA, which resulted in the identification of several cell clusters based on gene expression. The cluster characterized by the expression of genes associated with cytotoxicity showed significant differences between responders and non-responders. Specifically, within the cytotoxic cluster, genes associated with IFN-related pathways were upregulated in cells from responders to AZA. In contrast, genes upregulated in non-responders displayed an enrichment for the TGF-β signaling pathway, which is known to be associated with the suppression of the killing capacity of T cells (Figure 1B). The aforementioned molecular signatures were further associated with a higher cytotoxic molecular signature in the cytotoxic cell cluster from the group of responders. Conclusion In this study, we focused on BM CD8 + T cells in samples from patients with myeloid neoplasias and performed deep phenotypic analysis using CyTOF coupled with scRNA, in order to characterize the BM immune environment. We demonstrate for the first time that BM CD8 + T cells show different phenotypic and transcriptomic characteristics in responders to AZA. Specifically, we show that the frequency of the effector CD57 +CXCR3 +CD8 + subset is decreased in responders before treatment initiation, and this is associated with better survival. Despite the decreased proportion of this cytotoxic cell population in responders, there are specific signatures associated with effector T cell functionality that can explain the improved outcomes in these patients. These findings underscore the significance of the crosstalk between leukemic and immune cells in the progression of the disease and the effect of treatment. Figure legend A. Increased (%) CD57 +CXCR3 + correlates significantly with worse survival of HR-MDS and AML patients. Kaplan Meier curves for Overall Survival in HR-MDS and AML patients respectively. B. Gene signature derived from the scRNAseq analysis. Interferon-stimulated gene (ISG) signature is increased in the cytotoxic cell cluster in responders and and TGF-β signature in non-responders.

No Discernable Dysfunction of Peripheral T-Cells and NK-Cells Despite Distinct Phenotypes in MYC Rearranged Versus Non Rearranged B-Cell Lymphoma Patients

Background: Patients with diffuse large B-cell lymphoma/high-grade B-cell lymphoma with MYC and BCL2 rearrangements (DLBCL/HGBL- MYC/BCL2, hereafter HGBL- MYC/BCL2) respond poorly to standard immunochemotherapy treatment compared with DLBCL not otherwise specified (LBCL-NOS) patients without a MYC rearrangement. This suggests a negative interaction between MYC and the immune system. As novel T-cell and NK-cell based immunotherapeutic approaches emerge as potential treatment options for aggressive lymphomas, we conducted comprehensive immune profiling to compare peripheral blood T-cells and NK-cells of HGBL- MYC/BCL2 with LBCL NOS patients. Methods: Peripheral blood mononuclear cells (PBMCs) were prospectively collected from HGBL- MYC/BCL2 patients (registered in the HOVON-152 trial, NCT03620578), LBCL-NOS (registered in the HOVON-902 cohort, NCT04139252) and age-matched healthy donors (HDs). Flow-cytometry-based immune profiling of T-cells and NK-cells was performed on baseline samples available from n=66 HGBL- MYC/BCL2 patients (median age 62 years), n=67 LBCL NOS patients (median age 69 years) and n=17 HDs (median age 64 years). The flow cytometry data were computationally analyzed using FlowSOM and UMAP. T-cells were also functionally assessed for intracellular cytokine secretion (ICS) after stimulation with PMA/ionomycin (4h) and proliferation after stimulation with CD3/CD28 beads (5 days) using CellTrace Violet. NK-cells were assessed for cytotoxicity against K562 and degranulation (CD107a/b upregulation) after 4h. Mann-Whitney's U tests were used to compare HGBL-MYC/BCL2 patients with LBCL-NOS patients (primary comparison), and for pairwise comparisons of the lymphoma groups with HDs. To correct for baseline characteristics (age), linear regression analysis was performed. Results: CD4 + helper T-cells did not significantly between the lymphoma subgroups, but were decreased in LBCL-NOS patients as compared to HDs. Frequencies of cytotoxic CD8 + and CD4 +CD8 + T-cells did not significantly between the lymphoma subgroups and were similar as in HDs. Regulatory T-cell (Treg) frequencies of HGBL- MYC/BCL2 patients were similar to that of HDs, but were decreased in LBCL NOS patients. The frequency of CD4 -CD8 - T-cells was increased in LBCL NOS patients. Previously activated (HLA-DR +) CD4 + and CD8 + T-cells, as well as recently activated CD25 +, CD38 + or CD127 + CD4 + T-cells (Figure 1A) or activated/exhausted (PD-1 +) CD4 + T-cells and PD1 + Tregs were increased in both lymphoma subtypes (Figure 1B), but only HGBL- MYC/BCL2 displayed an increase in PD1 + CD8 + T-cells (Figure 1B). Conversely, recently activated CD8 + T-cells were decreased exclusively in LBCL NOS. HGBL- MYC/BCL2 patients and HDs had similar percentages of T-cells with a senescent phenotype (CD28 -CD57 +KLRG1 +), but the frequency of T-cells with this senescent phenotype was increased in LBCL NOS patients (Figure 1A), also after adjustment for age. Despite all these phenotypic differences, functional in vitro assays detected no differences in proliferation or cytokine secretion of peripheral T-cells from HGBL- MYC/BCL2 patients, LBCL NOS patients and HDs. In the NK-cell compartment, both lymphoma subtypes contained lower frequencies of CD56 brightTim3 +, CD56 bright/dimDNAM-1 + NK-cells, but LBCL NOS patients expressed a higher amount of NK-cells expressing inhibitory receptor NKG2A compared with HDs. NK-cells of HGBL- MYC/BCL2 patients, LBCL NOS patients and HDs displayed similar cytotoxic activity, suggesting no apparent functional impairment. Conclusions: HGBL- MYC/BCL2 patients have a distinct peripheral T-cell and NK-cell phenotype from LBCL NOS patients without a MYC rearrangement, but show no apparent functional deficiencies in terms of proliferation, cytokine secretion or cytotoxic activity. These results are not contradicting the application of T-cell and NK-cell based immunotherapeutic approaches for patients with aggressive B-cell lymphoma. Simultaneously, it emphasize the need to investigate the potential immunomodulatory impact of lymphoma intrinsic MYC in the tumor microenvironment.

Base Edited HSPCs Are Shielded from Targeted CD33 Therapy but Preserve CD33 Expression

Targeted therapy for AML remains challenging as there is broad overlap between targetable surface proteins on transformed cells and healthy hematopoietic stem and progenitor cells (HSPC). CD33 is expressed in about 90% of patients with AML but also on healthy myeloid cells with strong expression on progenitor cells and only weak expression on mature neutrophils. Given the higher expression on leukemic blasts compared to their healthy counterparts, CD33 is an attractive target in AML. However, targeting CD33 e.g. with the antibody-drug conjugate (ADC) gemtuzumab ozogamicin often leads to prolonged cytopenia due to suppression of normal myelopoiesis. Transplantation of genetically engineered CD33 knock-out allogenic HSPCs protects reconstituting hematopoietic cells from targeted immunotherapy administered for disease control after transplantation. Early clinical trials explore this option but the long-term effect of loss-of-function of CD33 remains unknown. We recently demonstrated that single amino acid substitutions engineered into CD45, CD117 or CD123 protect HSPCs from targeted therapies against these antigens while preserving their function. Here, we describe the identification of CD33 shielding amino acid substitutions that can be engineered by base editing and preserve CD33 expression on HSPCs. We identified binding sites of two CD33 antibodies by alanine scanning of the entire extracellular domain. The most favorable amino acid substitutions at key residues were analyzed computationally and binding reduction was subsequently validated by flow cytometry. 23 individual amino acid substitutions at 3 positions were tested. Eleven retained similar expression, purity and biophysical properties as wildtype CD33. Those variants are shielded from antibody 1 even at high concentrations when assessed by biolayer interferometry. In addition, three variants are shielded from antibody 2 binding. We used the ABE8e_SpRY base editor to engineer HSPCs and tiled 21 sgRNAs centered around the intended edit. Five days after electroporation, HSPCs were evaluated for editing efficiency (Sanger sequencing) and binding to antibody 1 (flow cytometry). sgRNA4 showed high editing efficiency of about 80% as well as loss of binding to antibody 1 (Figure A) while binding to the control antibody that binds a different epitope was maintained. Importantly, Sanger sequencing did not show off-target editing of the closely related SIGLEC22P pseudogene. Edited (sgRNA4) and non-edited control (NTC) HSPCs formed comparable numbers of myeloid and erythroid colonies (Figure B). Furthermore, in vitro differentiation showed no difference in frequency of CD14, CD15, CD19, CD33, CD41 and GlyA positive cells indicating that the edited HSPCs did not display major functional alterations. In summary, CD33 variants that can be engineered into HSPCs with high editing efficiency by base editing protected from binding to antibody 1 but preserved CD33 expression. These edited HSPCs could be therapeutically useful, in particular for continued immunotherapy after allogenic HSC transplantation. Similar to HSPCs devoid of CD33 these engineered cells could enable tumor-selective immunotherapy using ADCs or CAR T cells but with preserved CD33 expression and function. Furthermore, since base editing is suitable for multiplexing, they may in the future be multiplexed to protect CD33 and other targets for combination immunotherapy.

Targeting of CD25+ Activated T Cells in Gvhd-Associated Marrow Suppression and Cytopenias

Introduction: Secondary cytopenias occurring after hematopoietic cell transplant (HCT) are an observed complication of graft-versus-host disease (GVHD). Causes include peripheral immune mediated destruction (hemolytic anemia or thrombocytopenia), usually associated with autoantibody production, and peripheral destruction associated with microangiopathy (TMA). However, cases have been observed where marrow production of the affected lineage is abnormal. Immune-mediated cases are treated with increased immunosuppression, e.g., steroids or calcineurin inhibitors (CNI), and TMA with complement inhibition. To better understand the mechanisms and treatment of hypoproductive cytopenias in GVHD, we combined analyses of hematopoietic stem and progenitor cells (HSPC) and peripheral blood and marrow activated T cells. We describe 3 cases of GVHD-associated cytopenias marked by HSPC depletion and presence of CD25 (IL-2R alpha subunit)-positive activated T cells in the marrow that responded to combinations of immunosuppression containing basiliximab (basil) anti-CD25 monoclonal antibody blockade. Methods/Results: We describe combined HSPC and T cell analyses in 3 patients (P) with severe GVHD-associated cytopenias. P1 was at 5 years post-HCT for acute lymphoblastic leukemia, P2 at 2 years post-HCT for severe aplastic anemia (SAA), and P3 at 1.5 months post-liver transplant for alcohol use disorder-associated cirrhosis and hepatocellular carcinoma ( Table 1). Because of refractory cytopenias, all were being considered for HCT. However, marrow analyses revealed that each had normal frequencies of phenotypic HSC, with variable decreases in cellularity and frequencies of committed progenitors ( Table 2). Chimerism analyses showed that 100% of CD34+ cells in P1 and P2 were of donor origin, and in P3 of host origin. All 3 marrows had activated CD3+ T cells per expression of CD69 and/or CD38, and frequency of CD25 on &amp;gt;10% of T cells. In addition, P1 had evidence of IgG bound to committed progenitor populations. The treatment strategy was to block IL-2 signaling with basil (induction of 20 mg on day 1, 4, 7, 14, 21, 28, followed by alternate week and then monthly dosing for 2-9 months), combined with suppression of IL-2 production with steroid and/or CNI. After induction, repeat analyses of the blood and marrow T-cells confirmed complete blockade of CD25. Therapy was also individualized for specific clinical conditions or as noted on marrow evaluation. P1 received prednisone, CNI, and rituximab for a presumed anti-HSPC autoantibody. At 9 months he was weaned off all therapy and remains on no immunosuppressive treatment 2 years later. P2 did not receive CNI due to chronic renal failure but received steroids and later sirolimus. At ~18 months, his cytopenias recurred, but subsequently responded to basil re-induction and steroids. P3 has received prednisone, CNI, and sirolimus indefinitely for prevention of liver rejection and/or hepatocellular carcinoma relapse. P1 had no viral reactivation; P2 had CMV pneumonia with his second course responsive to antiviral therapy, completion of basil, and tapering of steroids; and P3 had CMV reactivation responsive to antiviral therapy and EBV viremia. P3 has a recent diagnosis of EBV+ intrahepatic B cell lymphoma. In all cases, blood counts normalized except P2 who has anemia due to CKD. Discussion: HSPC analyses in these patients with GVHD and cytopenias demonstrate that peripheral changes are not due to loss of HSC, but instead to quantitative and/or qualitative defects in committed progenitor populations, likely mediated by activated CD25+ T cells. In addition, the presence of IgG on HSPCs in P1 demonstrates a novel role for antibody-mediated humoral suppression in some cases. The presence of HSC indicates that there may be no need for re-transplant. Previous studies of SAA have shown activated T cells to be drivers of aplasia and is the basis for immune suppressive therapy (anti-thymocyte globulin and CNI). Our analyses of patients with GVHD-associated cytopenias demonstrates that targeting of IL-2 signaling via combined CD25 receptor blockade and pharmacologic inhibition of IL-2 production can correct the marrow defects and cytopenias, albeit with need for attention to viral infections. Targeting CD25 in patients with evidence of CD25+ activated T cells is a rational approach to the difficult problem of GVHD-associated marrow suppression and cytopenias.

Immune Profiling of Newly Diagnosed Multiple Myeloma (NDMM) Treated with Quadruplet Induction and Autologous Stem Cell Transplantation (ASCT) and Comparison with Achievement of Minimal Residual Disease (MRD) Negativity

Background Incorporation of anti-CD38 monoclonal antibodies (mAb) to the triplet backbone of proteasome inhibitor, immunomodulatory agent and corticosteroid has resulted in unprecedented depth of response including MRD negativity in transplant eligible NDMM. While numerous immunological mechanisms of modern quadruplet combinations are proposed, the modulation of the immunoprofiles bone marrow (BM) and peripheral blood (PB) with quadruplet therapy and ASCT are unknown. The baseline as well as longitudinal study of the composition and functionality of the immune microenvironment with treatment may have implications on disease response. Methods We prospectively collected clinically annotated patient (pt) samples from a phase II single institution clinical trial in transplant eligible NDMM (NCT04991103). Pts received quadruplet combination with daratumumab, bortezomib, lenalidomide and dexamethasone for six cycles followed by MRD adapted deferral of ASCT for pts who achieved MRD&amp;lt;10 -5. Pts deferring ASCT received three additional cycles of consolidation followed by maintenance. Pts who did not meet threshold for ASCT deferral received ASCT followed by maintenance therapy per protocol. We collected paired PB and BM samples at the time of screening (SCN), post-induction (I), and post-consolidation/ASCT (C). We purified and biobanked viable PB mononuclear cells (PBMCs). We thawed PBMCs, treated with FcR blocking Ab, stained with fluorochrome-labeled antibodies and a live/dead fixable stain, and then analyzed using the FACSymphony A5 SE flow cytometer. We identified immune cell subsets in PBMCs using FlowJo v10.2. We report data as percentage of each population within total live PBMCs. Results We collected 48 samples from 8 pts at three timepoints (SCN, I and C) and performed initial immunophenotyping analysis on the PBMCs. We observed no significant changes over time in the frequencies of peripheral CD45 +, CD3 +, NK T cells, monocytes or dendritic cells (not shown). We noted decreased frequencies of multiple B cell subsets, including naïve, transitional, memory and double negative cells (not shown), as well as CD56 +CD16 + NK cells (Fig. panel A) post-induction with no recovery following consolidation . While the frequencies of CD3 + T cells did not change with time, we observed a shift in the CD4:CD8 ratio, with an increase in the proportion of CD8 + T cells post-consolidation (Fig. panel B). Within the post-consolidation CD8 + T cell compartment, we noted increased frequencies of cells that presented with senescent (PD1 negCD57 +), exhausted (PD1 +CD57 neg) or anergic (PD1 +CD57 +) profiles. Consistent with this, the frequencies of terminally differentiated effector memory cells re-expressing CD45RA (TEMRA) CD8 + T cells increased at consolidation (Fig. panel C) and many of these cells exhibited an anergic profile (CD57 +PD1 +, Fig. panel D). Comparison of the baseline immune profiles and MRD levels post-induction revealed that the pt attaining MRD&amp;lt;10 -5 had the highest frequency of-NK cells as well as proportionally higher T CM/T EM and lower TEMRA/T Naive cells at diagnosis. This subject was able to defer ASCT per protocol and subsequently exhibited the lowest frequency of CD8 + TEMRA with an anergic phenotype (PD1 +CD57 +) post quadruplet consolidation. The subject with the least response to quadruplet induction and highest MRD post-induction had a high proportion of CD8 + T cells with an exhausted phenotype (PD1 +CD57 neg) at screening. Conclusion This is the first longitudinal analysis comparing MRD levels to the immune profiles of PB and BM cells derived from a cohort of NDMM patients prospectively treated with quadruplet induction and ASCT. We conclude that quadruplet induction negatively affects the NK cell and B cell compartment during induction and these compartments do not rapidly recover in the immediate post-transplant period. ASCT post quadruplet therapy is associated with preferential expansion of the effector CD8 + T cell compartments, including terminally differentiated TEMRA cells, which express markers associated with T cell exhaustion and anergy. Additional studies evaluating BM immune profile and T cell exhaustion are ongoing and shown at the meeting.

Durable natural killer cell response after three doses of SARS-CoV-2 inactivated vaccine in HIV-infected individuals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine can induce a potent cellular and humoral immune response to protect against SARS-CoV-2 infection. However, it was unknown whether SARS-CoV-2 vaccination can induce effective natural killer (NK) cell response in people living with human immunodeficiency virus (PLWH) and healthy individuals. Forty-seven PLWH and thirty healthy controls (HCs) inoculated with SARS-CoV-2 inactivated vaccine were enrolled from Beijing Youan Hospital in this study. The effect of SARS-CoV-2 vaccine on NK cell frequency, phenotype, and function in PLWH and HCs was evaluated by flow cytometry, and the response of NK cells to SARS-CoV-2 Omicron Spike (SARS-2-OS) protein stimulation was also evaluated. SARS-CoV-2 vaccine inoculation elicited activation and degranulation of NK cells in PLWH, which peaked at 2 weeks and then decreased to a minimum at 12 weeks after the third dose of vaccine. However, in vitro stimulation of the corresponding peripheral blood monocular cells from PLWH with SARS-2-OS protein did not upregulate the expression of the aforementioned markers. Additionally, the frequencies of NK cells expressing the activation markers CD25 and CD69 in PLWH were significantly lower than those in HCs at 0, 4 and 12weeks, but the percentage of CD16 + NK cells in PLWH was significantly higher than that in HCs at 2, 4 and 12weeks after the third dose of vaccine. Interestingly, the frequency of CD16 + NK cells was significantly negatively correlated with the proportion of CD107a + NK cells in PLWH at each time point after the third dose. Similarly, this phenomenon was also observed in HCs at 0, 2, and 4 weeks after the third dose. Finally, regardless of whether NK cells were stimulated with SARS-2-OS or not, we did not observe any differences in the expression of NK cell degranulation markers between PLWH and HCs. s:SARS-CoV-2 vaccine elicited activation and degranulation of NK cells, indicating that the inoculation of SARS-CoV-2 vaccine enhances NK cell immune response.

Nicotinamide mononucleotide impacts HIV-1 infection by modulating immune activation in T lymphocytes and humanized mice

HIV-1-associated immune activation drives CD4+ T cell depletion and the development of acquired immunodeficiency syndrome. We aimed to determine the role of nicotinamide mononucleotide (NMN), the direct precursor of nicotinamide adenine dinucleotide (NAD) co-enzyme, in CD4+ T cell modulation during HIV-1 infection. We examined HIV-1 integrated DNA or transcribed RNA, intracellular p24 protein, and T cell activation markers in CD4+ T cells including invitro HIV-1-infected cells, reactivated patient-derived cells, and in HIV-1-infected humanized mice, under NMN treatment. RNA-seq and CyTOF analyses were used for investigating the effect of NMN on CD4+ T cells. We found that NMN increased the intracellular NAD amount, resulting in suppressed HIV-1 p24 production and proliferation in infected CD4+ T cells, especially in activated CD25+CD4+ T cells. NMN also inhibited CD25 expression on reactivated resting CD4+ T cells derived from cART-treated people living with HIV-1 (PLWH). In HIV-1-infected humanized mice, the frequency of CD4+ T cells was reconstituted significantly by combined cART and NMN treatment as compared with cART or NMN alone, which correlated with suppressed hyperactivation of CD4+ T cells. Our results highlight the suppressive role of NMN in CD4+ T cell activation during HIV-1 infection. It warrants future clinical investigation of NMN as a potential treatment in combination with cART in PLWH. This work was supported by the Hong Kong Research Grants Council Theme-Based Research Scheme (T11-706/18-N), University Research Committee of The University of Hong Kong, the Collaborative Research with GeneHarbor (Hong Kong) Biotechnologies Limited and National Key R&D Program of China (Grant2021YFC2301900).

T-regulatory Cells in Acute Leukemia and their Relation with Induction Outcome

Abstract Background: The leukemic microenvironment has become an area of interest for researchers in recent times. Nonmalignant T and B lymphocytes form a significant part of leukemic microenvironment and are postulated to have an effect on its progression and behavior. These lymphocytes are constantly interacting with blasts and are actively involved in innate and acquired immune responses against these blasts. We decided to look at the role of T-regulatory lymphocytes in tumor surveillance and treatment outcome. Aim and Objectives: The aims and objectives of the study are to evaluate the role of T-regulatory lymphocytes in acute leukemia cases and its correlation with treatment outcome. Materials and Methods: We evaluated consecutive 55 acute leukemia cases to investigate the prognostic effect of T-regulatory cells (T-regs) in acute leukemia. The study period was between January 2021 and December 2021. All patients whose complete records were available till the completion of induction were included in the study. The immunophenotype used to characterize and evaluate T-regs was CD4+CD25+ T lymphocytes. For comparison, a total of 10 healthy controls were included in the study. Clinicohematological parameters including fever, lymphadenopathy, hepatosplenomegaly, extramedullary involvement, day 1 total leukocyte count, and blast percentage in peripheral blood and bone marrow were studied and documented to establish any possible correlation with T-regs percentage and treatment outcome. The treatment outcome evaluated in the study was complete remission postfirst induction. Results: As compared to those of healthy participants, the frequencies of CD4+CD25+ T-regs in the peripheral blood of acute leukemia patients were significantly increased (1.5% [range: 0.50%–4.3%] vs. 0.58% [range: 0.40%–1.05%], P = 0.0001). Furthermore, the frequencies of CD4+CD25+ T-regs in the peripheral blood of acute leukemia patients who did not achieve remission were significantly increased as compared to those who achieved remission (2.9% [range: 0.20%–4.3%] vs. 1.3% [range: 0.30%–2.9%], P = 0.002). No significant correlation was noted in the T-reg cells and parameters such as fever, lymphadenopathy, hepatosplenomegaly, extramedullary involvement, day 1 total leukocyte count, and blast percentage. Conclusion: This study suggests the possibility of using T-regs percentages as a prognostic indicator of acute leukemia and possible role of anti-T-regulatory immunomodulators in improving the outcome of disease.

Resident Memory-like CD8+ T Cells Are Involved in Chronic Inflammatory and Neurodegenerative Diseases in the CNS.

Resident memory T (Trm) cells are a unique population that can survive and function in a compartmentalized tissue with inflammatory potential. We aim to investigate the alteration of Trm population in acute/chronic inflammatory and neurodegenerative diseases in the CNS. The frequencies of CD4+ and CD8+ T cells expressing both CD69 and CD103, the markers for Trm cells, were quantified in the peripheral blood and CSF (n = 80 and 44, respectively) in a cross-sectional manner. The transcriptional profile of Trm-like population in the CSF was further analyzed using a public single-cell dataset. The frequency of CD69+CD103+CD8+ T cells was strikingly higher in the CSF than in the peripheral blood (among memory fraction, 13.5% vs 0.11%, difference (mean [SE]): 13.4% [2.9]). This CD69+CD103+CD8+ T-cell population was increased in the CSF from patients with chronic inflammatory diseases including multiple sclerosis and with neurodegenerative diseases such as Parkinson disease and Alzheimer disease compared with controls (11.5%, 13.0%, 8.1% vs 2.9%, respectively). By contrast, the frequency was not altered in acute inflammatory conditions in the CNS (4.0%). Single-cell RNAseq analysis confirmed Trm signature in CD69+CD103+CD8+ T cells in the CSF, supporting their Trm-like phenotype, which was not clear in controls. Collectively, an increase in CD69+CD103+CD8+ Trm-like population in the CSF is related with both chronic neuroinflammatory and some neurodegenerative diseases in the CNS, suggesting a partially shared pathology in these diseases.

A CD38/CD28xCD3 Trispecific T-Cell Engager (TCE) As a Potentially Active Agent in Multiple Myeloma Patients Relapsed and/or Refractory (RRMM) to Anti-CD38 Monoclonal Antibodies (mAbs)

Background: Despite great advances in the treatment of RRMM, including anti-CD38 mAbs, patients become resistant and therapeutic options are limited. Some factors involved in resistance to anti-CD38 mAbs have been described. On the one hand, their efficacy is partially dependent on baseline CD38 expression in MM plasma cells (MMPC), but the treatment induces a rapid and sustained CD38 downregulation. On the other hand, their tumor-killing activity relies on NK cells, but its frequency rapidly decreases following initiation of treatment, and persistent NK cells exhibit a dysfunctional phenotype. Given the reduction of CD38 levels in MMPC and the impaired NK-cell activity in resistance to anti-CD38 mAbs, we hypothesized that RRMM patients previously treated with these agents could benefit from T-cell-based immunotherapeutic strategies that depend less on CD38 antigen density and NK cell function. Aim: Evaluate a CD38/CD28xCD3 trispecific TCE (SAR442257) as a potential therapeutic agent in the RRMM setting. Results: CD38 expression in MMPC and NK-cell frequency in bone marrow were significantly reduced in 29 RRMM patients previously exposed to anti-CD38 mAbs vs 45 newly-diagnosed MM (NDMM) patients ( P &amp;lt;.001 and P =.013, respectively). Moreover, the percentage of CD57+ NK cells was significantly lower in RRMM than NDMM patients ( P =.006), suggesting that both CD38 downregulation and a contraction of cytotoxic NK cells may be associated with resistance to anti-CD38 mAbs. By contrast, T cells did not change significantly between stages, which ignited further interest in the preclinical efficacy of the CD38/CD28xCD3 TCE. First, we observed that, contrary to isatuximab and daratumumab, CD38/CD28xCD3 TCE did not reduce surface CD38 levels in the MOLP 8, RPMI 8226 and KMS 12 BM cell lines. Fluorescence microscopy imaging revealed that CD38 was present in cell membrane up to one week of treatment with CD38/CD28xCD3 TCE, while isatuximab induced an internalization of CD38 at 24h. Next, in a co-culture of the OPM-2 cell line with purified T or NK cells from healthy individuals (E:T ratio 2:1), we confirmed that the activity of CD38/CD28xCD3 TCE relied on T but not NK cells. Namely, no direct NK-cell stimulation was found whereas a significant T-cell activation was observed upon CD38/CD28xCD3 TCE treatment. Surprisingly, when OPM-2 were co-cultured with freshly-isolated peripheral blood mononuclear cells (PBMCs, E:T ratio 10:1), CD38/CD28xCD3 TCE induced activation of T- and NK-cells, suggesting a bystander NK-cell activation. This dual stimulation resulted in a significant reduction of OPM-2 cell viability at 48h. Interestingly, we found a comparable tumor-killing activity and T-cell activation in the presence of the CD38 null trispecific isotype (CD3xCD28 ΔCD38) compared with CD38/CD28xCD3 TCE, suggesting that the latter targets CD28 not only in T-cells, but also in OPM-2 cells, which express high CD28 basal levels. Altogether, these data demonstrate the potential dual CD38/CD28 targeting in RRMM patients previously exposed to anti-CD38 mAbs, particularly in those with double positive MMPC. Additionally, CD38 regulation was analyzed in the co-culture system (measured with a non-competing anti-CD38 antibody), and an unaltered and increased CD38 expression was observed in OPM-2 and T-cells, respectively, indicating again T-cell stimulation. We tested the ex vivo efficacy of CD38/CD28xCD3 TCE (48h, 700 pM) in primary bone marrow samples from eight RRMM patients, which were collected after a median of 10 months since the last anti-CD38 treatment (range, 0.5 - 37). T-cell activation and MMPC lysis were found in six and four samples, respectively, which was accompanied by a reduction in the frequency of CD4 T cells (from 100% to 76%; P =.014). A positive and borderline-significant correlation (r = 0.764, P =.057) was found between the response to CD38/CD28xCD3 TCE and the time since last anti-CD38 treatment, and no differences in CD38 expression were observed between CD38/CD28xCD3 TCE responders and non-responders. Conclusions: We observed a reduction in CD38 levels in MMPC and an impaired cytotoxic activity of NK cells in RRMM patients previously exposed to anti-CD38 mAbs. We propose a second targeting of CD38 using T-cell-based immunotherapeutic agents whose efficacy depends less on CD38 antigen density, especially after longer washout periods, as a potential therapeutic strategy in the RRMM setting.

The Role of CABIN1 in the Pathogenesis of Idiopathic Multicentric Castleman Disease

Introduction: Idiopathic Multicentric Castleman Disease (iMCD) is a rare disease characterized by flares of hypercytokinemia, leading to life threatening complications. Although several hypotheses have emerged including autoimmune, immune dysregulation, and/or infectious etiology, to date the exact pathogenesis of iMCD is unknown. IL-6 is thought to be the key cytokine that drives symptoms in a proportion of patients and current standard of care targets IL-6; however, it is only successful in one third of patients. Therefore, there is a dire need to identify additional driver cytokines/pathways to address treatment-refractory cases. Lately, the pathogenic role of CXCL13 in iMCD has emerged, as proteomic studies have shown that serum CXCL13 is elevated in iMCD and a biomarker of response to therapy. Given the heterogeneity of the disease, research into iMCD has been limited by the lack of an animal model. Through whole exome sequencing of an iMCD patient, we recently discovered a potential disease-causing mutation in a protein known as CABIN1. CABIN1 in known to play a role in T cell activation as a negative regulator of calcineurin and MEF2. Using Crispr/Cas9 technology, we generated a mouse model that harbors this gene mutation (CABIN1 V2185M). Methods: Following LCMV infection or ovalbumin (OVA)-mRNA-LNP immunization serum cytokine levels were measured using Luminex assay or CXCL13 specific ELISA kit. Different immune cell populations and Nur77 levels were assessed by flow cytometry. MEF2D protein levels were determined by Western blot analysis. Results: Although CABIN1 V2185M mice do not demonstrate spontaneous pathology at baseline, we found that upon LCMV CL13 infection, mice harboring the CABIN1 V2185M mutation displayed significantly higher morbidity and mortality compared to control mice. LCMV-infected CABIN1 V2185M mice displayed elevated serum levels of cytokines/chemokines including IL-6 and CXCL13, which are associated with human iMCD flares. Concurrently, we also measured a robust increase in the frequency of CD4 + T cells expressing CXCR5, which is the receptor for CXCL13. Similar to LCMV CL13, infection with LCMV Armstrong or immunization with OVA-mRNA-LNP also led to an increase in serum CXCL13 levels and CXCR5 +CD4 + T cells in the associated secondary lymphoid organ in CABIN1 V2185M compared to control mice. In terms of the underlying mechanism, we found that T cells of CABIN1 V2185M mice exhibit increased MEF2D protein and expression of a MEF2D-regulated gene, Nur77 following CD3/CD28 stimulation, suggesting that CABIN1 V2185M mutation acts through MEF2 dysregulation in T cells. Conclusion: Our preliminary data shows that LCMV-infected CABIN1 mutant mice recapitulate the cytokine milieu of human iMCD. Therefore, CABIN1 could be the first ever described mutation that could drive iMCD disease. Furthermore, this mouse model could serve as a model platform to study the underlying pathogenic pathway(s) in iMCD.

Th2 cell clonal expansion at diagnosis in human type 1 diabetes

Soon after diagnosis with type 1 diabetes (T1D), many patients experience a period of partial remission. A longer partial remission is associated with a better response to treatment, but the mechanism is not known. The frequency of CD4+CD25+CD127hi (127-hi) cells, a cell subset with an anti-inflammatory Th2 bias, correlates positively with length of partial remission. The purpose of this study was to further characterize the nature of the Th2 bias in 127-hi cells. Single cell RNA sequencing paired with TCR sequencing of sorted 127-hi memory cells identifies clonally expanded Th2 clusters in 127-hi cells from T1D, but not from healthy donors. The Th2 clusters express GATA3, GATA3-AS1, PTGDR2, IL17RB, IL4R and IL9R. The existence of 127-hi Th2 cell clonal expansion in T1D suggests that disease factors may induce clonal expansion of 127-hi Th2 cells that prolong partial remission and delay disease progression.

Impacts of Combining Pulsed Low Dose Rate Radiotherapy (PLDR) and Anti-PD-1 Antibody on Tumor Growth and Its Mechanism for Lung Cancer in Mice

As an emerging anti-tumor strategy, Immunotherapies has been approved for the treatment of a variety of tumors. Recent data suggest that the efficacy of radiotherapy in various cancers can be augmented when combined with immune checkpoint blockade. Then, pulsed low dose rate radiotherapy (PLDR) is a new radiotherapy segmentation method. Therefore, this study investigated the inhibitory effect of PLDR combined with anti-PD-1 antibody on lung cancer in mice and its impact on tumor immune microenvironment. By transplanting murine LLC cells into the right leg of C57BL/6 mice with immune activity, a transplanted subcutaneous tumor model was established. The mice were randomly divided into five groups: control, conventional radiotherapy (RT)±anti-PD-1 antibody (mAb),PLDR±anti-PD-1 antibody (mAb). RT was delivered as a dose of 5 × 2 Gy whereas PLDR involved delivering a dose of 2 Gy as 10 pulses of 0.2 Gy, each 3 minutes apart and lasting for 5 days. Anti-PD-1 antibody and isotype control were administered intraperitoneally once every three days at a dose of 5mg/kg, three times in total. The tumor, blood and tumor-draining lymph nodes (TDLNs) were harvested after treatment, and a single cell suspension was prepared for flow cytometry to analyze the changes in the immune microenvironment of the tumor tissue, the expression of PD-L1. PD-1 and the activated systemic immune response; Finally, this study explored a mechanism able to explain the observed synergy of combined therapy. PLDR combined with anti-PD-1 antibody can better inhibit the growth of tumor than RT,PLDR and RT combined group. Survival analysis demonstrated a statistically significant advantage for PLDR+anti-PD-1 than other groups. Median survival with PLDR+anti-PD-1mAb was 63d compared with 54d with RT+anti-PD-1mAb,41d with RT and 40d with PLDR and 33 with control. Meanwhile both RT and PLDR induced up-regulation of PD-L1 expression on tumor surface and PD-1 expression on lymphocytes. Then, the frequency of CD4+,CD8+T cells were higher in the PLDR combined treatment group in tumor blood and draining lymph nodes, and synergistically reduce the local accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs) than others. Finally, the main reason for the better tumor inhibition effect of PLDR combined group is that it upgrades the number and activity of CD8+T cells in tumor. PLDR combined with anti-PD-1 antibody can result in better tumor growth inhibition and significantly delay the survival time of mice, which was mainly through the cytotoxic T cell-dependent mechanism, meanwhile increasing the infiltration of CD4 + and CD8 +T-cells in tissues.

Differential expression patterns of purinergic ectoenzymes and the antioxidative role of IL-6 in hospitalized COVID-19 patient recovery.

We have acquired significant knowledge regarding the pathogenesis of severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2). However, the underlying mechanisms responsible for disease recovery still need to be fully understood. To gain insights into critical immune markers involved in COVID-19 etiopathogenesis, we studied the evolution of the immune profile of peripheral blood samples from patients who had recovered from COVID-19 and compared them to subjects with severe acute respiratory illness but negative for SARS-CoV-2 detection (controls). In addition, linear and clustered correlations between different parameters were determined. The data obtained revealed a significant reduction in the frequency of inflammatory monocytes (CD14+CD16+) at hospital discharge vs. admission. Remarkably, nitric oxide (NO) production by the monocyte compartment was significantly reduced at discharge. Furthermore, interleukin (IL)-6 plasma levels were negatively correlated with the frequency of NO+CD14+CD16+ monocytes at hospital admission. However, at the time of hospital release, circulating IL-6 directly correlated with the NO production rate by monocytes. In line with these observations, we found that concomitant with NO diminution, the level of nitrotyrosine (NT) on CD8 T-cells significantly diminished at the time of hospital release. Considering that purinergic signaling constitutes another regulatory system, we analyzed the kinetics of CD39 and CD73 ectoenzyme expression in CD8 T-cells. We found that the frequency of CD39+CD8+ T-cells significantly diminished while the percentage of CD73+ cells increased at hospital discharge. In vitro, IL-6 stimulation of PBMCs from COVID-19 patients diminished the NT levels on CD8 T-cells. A clear differential expression pattern of CD39 and CD73 was observed in the NT+ vs. NT-CD8+ T-cell populations. The results suggest that early after infection, IL-6 controls the production of NO, which regulates the levels of NT on CD8 T-cells modifying their effector functions. Intriguingly, in this cytotoxic cell population, the expression of purinergic ectoenzymes is tightly associated with the presence of nitrated surface molecules. Overall, the data obtained contribute to a better understanding of pathogenic mechanisms associated with COVID-19 outcomes.

COVID-19 patients display changes in lymphocyte subsets with a higher frequency of dysfunctional CD8lo T cells associated with disease severity.

This work examines cellular immunity against SARS-CoV-2 in patients from Córdoba, Argentina, during two major waves characterized by different circulating viral variants and different social behavior. Using flow cytometry, we evaluated the main lymphocyte populations of peripheral blood from hospitalized patients with moderate and severe COVID-19 disease. Our results show disturbances in the cellular immune compartment, as previously reported in different cohorts worldwide. We observed an increased frequency of B cells and a significant decrease in the frequency of CD3+ T cells in COVID-19 patients compared to healthy donors (HD). We also found a reduction in Tregs, which was more pronounced in severe patients. During the first wave, the frequency of GZMB, CD107a, CD39, and PD-1-expressing conventional CD4+ T (T conv) cells was significantly higher in moderate and severe patients than in HD. During the second wave, only the GZMB+ T conv cells of moderate and severe patients increased significantly. In addition, these patients showed a decreased frequency in IL-2-producing T conv cells. Interestingly, we identified two subsets of circulating CD8+ T cells with low and high CD8 surface expression in both HD and COVID-19 patients. While the percentages of CD8hi and CD8lo T cells within the CD8+ population in HD are similar, a significant increase was observed in CD8lo T cell frequency in COVID-19 patients. CD8lo T cell populations from HD as well as from SARS-CoV-2 infected patients exhibited lower frequencies of the effector cytokine-producing cells, TNF, IL-2, and IFN-γ, than CD8hi T cells. Interestingly, the frequency of CD8lo T cells increased with disease severity, suggesting that this parameter could be a potential marker for disease progression. Indeed, the CD8hi/CD8lo index helped to significantly improve the patient's clinical stratification and disease outcome prediction. Our data support the addition of, at least, a CD8hi/CD8lo index into the panel of biomarkers commonly used in clinical labs, since its determination may be a useful tool with impact on the therapeutic management of the patients.

Arsenic trioxide demonstrates efficacy in a mouse model of preclinical systemic sclerosis

BackgroundUncontrolled T-cell activation plays a key role in systemic sclerosis (SSc). Arsenic trioxide (ATO) has immunological effects and has demonstrated potential in preclinical SSc models. In this study, we assessed the efficacy of ATO in Fra2 transgenic (Fra2TG) mice, which develop severe vascular remodeling of pulmonary arterioles and nonspecific interstitial pneumonia-like lung disease, closely resembling human SSc-associated pulmonary hypertension, therefore partially resembling to the SSc human disease.MethodsThe efficacy of ATO in Fra2TG mice was evaluated through histological scoring and determination of cell infiltration. Fibrotic changes in the lungs were assessed by measuring collagen content biochemically, using second harmonic generation to measure fibrillar collagen, and imaging via computed tomography. Cardiovascular effects were determined by measuring right ventricular systolic pressure and vessel remodeling. The mechanism of action of ATO was then investigated by analyzing lung cell infiltrates using flow cytometry and bulk RNA with sequencing techniques.ResultsAfter ATO treatment, the Ashcroft histological score was substantially decreased by 33% in ATO-treated mice compared to control mice. Other investigations of fibrotic markers showed a trend of reduction in various measurements of fibrosis, but the differences did not reach significance. Further cardiovascular investigations revealed convergent findings supporting a beneficial effect of ATO, with reduced right ventricular systolic pressure and medial wall thickness, and a significant decrease in the number of muscularized distal pulmonary arteries in ATO-treated Fra2TG mice compared to untreated Fra2TG mice. Additionally, inflammatory cell infiltration was also markedly reduced in lesioned lungs. A reduction in the frequency of CD4 + and T effector memory cells, and an increase in the percentage of CD4 + T naive cells in the lungs of ATO-treated Fra-2TG mice, was observed when compared to PBS group Fra-2Tg mice. RNA-seq analysis of ATO-treated mouse lungs revealed a downregulation of biological pathways associated with immune activity and inflammation, such as T-cell activation, regulation of leucocyte activation, leucocyte cell–cell adhesion, and regulation of lymphocyte activation.ConclusionsOur results suggest the clinical relevance of ATO treatment in SSc. Using the Fra2TG mouse model, we observed significant lung histological changes, a trend towards a decrease in various fibrotic makers, and a strong reduction in vascular remodeling. The mechanism of action of ATO appears to involve a marked counteraction of the immune activation characteristic of SSc, particularly T-cell involvement. These findings pave the way for further studies in SSc.

Sirtuin 1 overexpression contributes to the expansion of follicular helper T cells in systemic lupus erythematosus and may serve as an accessible therapeutic target.

SIRT1, an NAD+-dependent deacetylase, is upregulated in CD4+ T cells from SLE patients and MRL/lpr lupus-like mice. This study aimed to explore the role of SIRT1 in follicular helper T (Tfh) cell expansion and its potential value as a therapeutic target for SLE. Frequencies of CD4+CXCR5+PD-1+ Tfh cells in peripheral blood from SLE patients and their expression of SIRT1 and B cell lymphoma 6 (BCL-6) were determined with flow cytometry. Naïve CD4+ T cells were transfected with SIRT1-expressing lentivirus and small interfering RNA (siRNA) targeting SIRT1, respectively, and then cultured under Tfh-polarizing conditions to study the impact of SIRT1 on Tfh cell differentiation. This impact was also evaluated in both CD4+ T cells and naïve CD4+ T cells by treatment with SIRT1 inhibitors (EX527 and nicotinamide) in vitro. MRL/lpr mice and pristane-induced lupus mice were treated with continuous daily intake of nicotinamide, and their lupus phenotypes (including skin rash, arthritis, proteinuria and serum anti-dsDNA autoantibodies) were compared with those of controls. Expression of SIRT1 was elevated in Tfh cells from SLE patients and was positively correlated with Tfh cell frequencies. SIRT1 expression gradually increased during Tfh cell differentiation. Overexpression of SIRT1 by lentiviral vectors significantly promoted Tfh cell differentiation/proliferation. Reciprocally, suppressing expression of SIRT1 by siRNA and inhibiting SIRT1 activity by EX-527 or nicotinamide hindered Tfh cell expansion. Continuous daily intake of nicotinamide alleviated lupus-like phenotypes and decreased serum CXCL13 in the two mouse models. SIRT1 overexpression contributed to the expansion of Tfh cells in SLE and may serve as a potential target for treatment.

Mitochondrial ROS-dependent CD4+PD-1+T cells are pathological expansion in patients with primary immune thrombocytopenia.

Aberrant-activated T cells, especially CD4+T cells, play a crucial part in the pathogenetic progress of immune thrombocytopenia (ITP). PD-1-mediated signals play a negative part in the activation of CD4+T cells. However, knowledge is limited on the pathogenic characteristics and function of CD4+PD-1+T cells in ITP. The frequency and phenotype including cell activation, apoptosis, and cytokine production of CD4+PD-1+T cells were evaluated by flow cytometry. PD-1 Ligation Assay was performed to assess the function of PD-1 pathway in CD4+T cells. Mitochondrial reactive oxygen species (mtROS) were detected by MitoSOX Red probe. Compared with healthy controls (HC), the frequencies of CD4+PD-1+T cells were significantly increased in ITP patients. However, these cells are not exhausted despite PD-1 expression. Besides retaining cytokine-producing potential, these CD4+PD-1+T cells also had a possible B-cell helper function including expressing ICOS, CD84, and CD40L. Moreover, the CD4+PD-1+T cell subset contained higher levels of mitochondrial ROS than CD4+PD-1-T cell subset in patients with ITP. And mtROS inhibition could reduce the secretion of the inflammatory cytokines and regulate the function of CD4+PD-1+T cells. Upon in-vitro T cell receptor (TCR) stimulation of CD4+T cells in the presence of plate-bound PD-L1 fusion protein (PD-L1-Ig), CD4+T cells from ITP patients appeared resistant to such PD-1-mediated inhibition of interferon (IFN)-γ secretion. The CD4+PD-1+T cells were more abundant in patients with ITP. Additionally, this CD4+PD-1+T cell subset may be a potential etiology of ITP and a potential immune therapeutic target for ITP patients in the future.