Role Of CD4 Research Articles

Abstract 568: Activation of the RXR nuclear receptor regulates CD4 T cell activity in a murine model of HER2+ breast cancer

Abstract The role of CD4 T cells for anti-tumor responses is less recognized than CD8 cells. Evidence suggests that CD4 T cells can act directly as antitumor cells or direct the maturation and activation of dendritic cells (DCs). The retinoid X receptor (RXR) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors. RXR and its heterodimers have profound effects on the function of myeloid cells, but effects on T cells are less known. Treatment with the RXR agonist MSU42011 (100 mg/Kg diet) reduced tumor burden in a HER2+ model of breast cancer (p=0.0068) and in a KRAS-driven model of lung cancer (p=0.0015). However, when human lung cancer cells were implanted into immunodeficient mice, MSU42011 failed to reduce tumor burden, suggesting that the anti-tumor properties of MSU42011 are dependent on a functional immune system. After 10 days of treatment, the percentage of CD4, CD62L+ T cells in HER2+ mammary tumors treated with MSU42011 was significantly higher compared to the control group (2.41 vs 3.78, p=0.05), as analysed by flow cytometry. Additionally, tumor antigen presenting cells (CD11c+, CD11b+, IA-IE+) and DCs (CD11c+, IA-IE+) had increased expression of PDL1 in the MSU42011 group when compared with controls (MFI=4016 vs 5225, p=0.069; 5484 vs 7140, p=0.045, respectively). RT-PCR analysis of whole tumors showed a significant increase in mRNA expression of interferon (IFN; p=0.0026) and IL12a (p=0.0011) in treated mice. Similar increases in IFN and IL12a were found in the murine model of lung cancer. Next, T cells were depleted to determine if required for the anti-tumor effects of MSU42011. CD8 cells depletion did not reverse the anti-tumor efficacy of MSU42011. CD4 depletion in mice treated with MSU42011 rescued tumor growth (p=0.016 MSU42011 vs MSU42011+anti-CD4), as tumors of mice treated with MSU42011 and anti-CD4 antibodies for 10 days grew at the same rate as tumors treated with control plus or minus anti-CD4 antibodies. Tumors treated with MSU42011 and anti-CD4 antibodies also showed reduced levels of IL12a compared with MSU42011 alone (2.28 vs 0.87, p>0.05), as determined by RT-PCR.To dissect the effects of RXR activation on CD4 and DC cells, primary DCs, alone or co-cultured with T cells and conditioned media from cancer cells, were treated with MSU42011 (100 nM). This treatment increased PDL1 and IL12 mRNA expression in DCs. T cells, co-cultured with DC and conditioned media and treated with MSU42011, expressed higher IL2 mRNA levels. MSU42011 also increased expression of IFN in CD3 T cells. In conclusion, MSU42011 increased recruitment and activation of CD4 T cells in vitro and in HER2+ mammary tumors. These data, in combination with our previous work showing that RXR agonists reduce expression of tumor-promoting FOXP3 T cells in vitro and in vivo, confirm a strong correlation between RXR pharmacologic activation and CD4 T cell activation within the tumor microenvironment. Citation Format: Ana Sofia Leal, Karen T. Liby. Activation of the RXR nuclear receptor regulates CD4 T cell activity in a murine model of HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 568.

CD8αα expression on NK cells is associated with different K562 and MOLT4 killing capabilities of PBMC and different involvement CD8pos and CD8neg subsets in anti-viral response

Introduction. The CD8αα are present in a subset of T cells and NK cells, but its function is mostly unknown, as well as the role of CD8+ and CD8- NK cell subsets in physiological and pathological environments. Methods. We investigated 130 healthy individuals’ blood samples for the NK cell cytotoxicity against K562 and MOLT4 cell lines. We also analyzed patients after SarsCov2 infection and compared to healthy control. The NK cell phenotype and cytotoxicity were studied by the FACScan flow cytometer using BD monoclonal antibodies. Results. We confirmed that MOLT4 is significantly more resistant to the NK cell cytotoxicity compared to the “classical” K562. CD8+ NK cells are more effective at K562 killing compared to CD8- subsets. The correlation of lymphocyte levels with the specific K562 lysis was weaker for CD8- NK cell subsets (r = 0.37) than CD8+ NK cells (r = 0.45) or whole NK cells population (r = 0.46). However, we found that CD8+ NK cells mostly did not participate in the MOLT-4 killing. CD8- NK cells frequency correlates with MOLT4 lysis more significantly (r = 0.49) than CD8+ NK cells lymphocytes levels (r = 0.27) or whole NK cells population (r = 0.44). Also, we showed that HLA-DR and CD158a positive NK cell levels did not correlate with the MOLT4 and K562 killing, while HLA-DR and CD158a negative subsets levels did with the same significance as the whole NK cells population. Decreased of NK lymphocytes after SarsCov2 infection results to decrease NK population owing to CD8+NK decreased but not CD8neg. Conclusion. NK cell numbers determine NK cell cytotoxicity indirectly through the surface phenotype. CD8 expression on the NK cells is associated with the effective cytotoxicity against K562 but at the same time obstructs a response to MOLT4. CD8αα on NK cells might participate in HLA recognition or enhance response to HLA class-I negative target cells.

CD4 T Cell-Dependent and -Independent Roles for IFN-γ in Blood-Stage Malaria.

Production of IFN-γ by CD4 T cells is widely theorized to control Plasmodium parasite burden during blood-stage malaria. Surprisingly, the specific and crucial mechanisms through which this highly pleiotropic cytokine acts to confer protection against malarial disease remain largely untested invivo. Here we used a CD4 T cell-restricted Cre-Lox IFN-γ excision mouse model to test whether and how CD4 T cell-derived IFN-γ controls blood-stage malaria. Although complete absence of IFN-γ compromised control of the acute and the chronic, recrudescent blood-stage infections with P. c. chabaudi, we identified a specific, albeit modest, role for CD4 T cell-derived IFN-γ in limiting parasite burden only during the chronic stages of P. c. chabaudi malaria. CD4 T cell IFN-γ promoted IgG Ab class switching to the IgG2c isotype during P. c. chabaudi malaria in C57BL/6 mice. Unexpectedly, our data do not support gross defects in phagocytic activity in IFN-γ-deficient hosts infected with blood-stage malaria. Together, our data confirm CD4 T cell-dependent roles for IFN-γ but suggest CD4 T cell-independent roles for IFN-γ in immune responses to blood-stage malaria.

TCF-1 Is Required for CD4 T Cell Persistence Functions during AlloImmunity

The transcription factor T cell factor-1 (TCF-1) is encoded by Tcf7 and plays a significant role in regulating immune responses to cancer and pathogens. TCF-1 plays a central role in CD4 T cell development; however, the biological function of TCF-1 on mature peripheral CD4 T cell-mediated alloimmunity is currently unknown. This report reveals that TCF-1 is critical for mature CD4 T cell stemness and their persistence functions. Our data show that mature CD4 T cells from TCF-1 cKO mice did not cause graft versus host disease (GvHD) during allogeneic CD4 T cell transplantation, and donor CD4 T cells did not cause GvHD damage to target organs. For the first time, we showed that TCF-1 regulates CD4 T cell stemness by regulating CD28 expression, which is required for CD4 stemness. Our data showed that TCF-1 regulates CD4 effector and central memory formation. For the first time, we provide evidence that TCF-1 differentially regulates key chemokine and cytokine receptors critical for CD4 T cell migration and inflammation during alloimmunity. Our transcriptomic data uncovered that TCF-1 regulates critical pathways during normal state and alloimmunity. Knowledge acquired from these discoveries will enable us to develop a target-specific approach for treating CD4 T cell-mediated diseases.

Investigation of the role of TmMYB16/123 and their targets (TmMTP1/11) in the tolerance of Taxus media to cadmium.

The toxicity and stress caused by heavy metal contamination has become an important constraint to the growth and flourishing of trees. In particular, species belonging to the genus Taxus, which are the only natural source for the anti-tumor medicine paclitaxel, are known to be highly sensitive to environmental changes. To investigate the response of Taxus spp. to heavy metal stress, we analyzed the transcriptomic profiles of Taxus media trees exposed to cadmium (Cd2+). In total, six putative genes from the metal tolerance protein (MTP) family were identified in T. media, including two Cd2+ stress inducible TMP genes (TmMTP1, TmMTP11 and Taxus media). Secondary structure analyses predicted that TmMTP1 and TmMTP11, which are members of the Zn-CDF and Mn-CDF subfamily proteins, respectively, contained six and four classic transmembrane domains, respectively. The introduction of TmMTP1/11 into the ∆ycf1 yeast cadmium-sensitive mutant strain showed that TmMTP1/11 might regulate the accumulation of Cd2+ to yeast cells. To screen the upstream regulators, partial promoter sequences of the TmMTP1/11 genes were isolated using the chromosome walking method. Several myeloblastosis (MYB) recognition elements were identified in the promoters of these genes. Furthermore, two Cd2+-induced R2R3-MYB TFs, TmMYB16 and TmMYB123, were identified. Both in vitro and in vivo assays confirmed that TmMTB16/123 play a role in Cd2+ tolerance by activating and repressing the expression of TmMTP1/11 genes. The present study elucidated new regulatory mechanisms underlying the response to Cd stress and can contribute to the breeding of Taxus species with high environmental adaptability.

The role of CD8+ T-cell clones in immune thrombocytopenia

AbstractImmune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multidimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterized patients with ITP and compared them with age-matched controls using immunophenotyping, next-generation sequencing of T-cell receptor (TCR) genes, single-cell RNA sequencing, and functional T-cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L−) expressing intracellular interferon gamma, tumor necrosis factor α, and granzyme B, defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the TCR showed expanded T-cell clones in patients with ITP. T-cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon gamma, and trigger platelet activation and apoptosis via the TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP.

Multicytokine-producing CD4+ T cells characterize the livers of patients with NASH.

A role of CD4+ T cells during the progression from nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) has been suggested, but which polarization state of these cells characterizes this progression and the development of fibrosis remain unclear. In addition, a gut-liver axis has been suggested to play a role in NASH, but the role of CD4+ T cells in this axis has just begun to be investigated. Combining single-cell RNA sequencing and multiple-parameter flow cytometry, we provide the first cell atlas to our knowledge focused on liver-infiltrating CD4+ T cells in patients with NAFLD and NASH, showing that NASH is characterized by a population of multicytokine-producing CD4+ T cells. Among these cells, only those with a Th17 polarization state were enriched in patients with advanced fibrosis. In parallel, we observed that Bacteroides appeared to be enriched in the intestine of NASH patients and to correlate with the frequency of multicytokine-producing CD4+ T cells. In short, we deliver a CD4+ T cell atlas of NAFLD and NASH, providing the rationale to target CD4+ T cells with a Th17 polarization state to block fibrosis development. Finally, our data offer an early indication to test whether multicytokine-producing CD4+ T cells are part of the gut-liver axis characterizing NASH.

Structural Analyses of a Dominant Cryptosporidium parvum Epitope Presented by H-2Kb Offer New Options To Combat Cryptosporidiosis.

Cryptosporidium parvum has gained much attention as a major cause of diarrhea in the world, particularly in those with compromised immune systems. The data currently available on how the immune system recognizes C. parvum are growing rapidly, but we lack data on the interactions among host major histocompatibility complex (MHC) diversity and parasitic T-cell epitopes. To identify antigenic epitopes in a murine model, we performed systematic profiling of H-2Kb-restricted peptides by screening the dominant Cryptosporidium antigens. The results revealed that the glycoprotein-derived epitope Gp40/15-SVF9 induced an immunodominant response in C. parvum-recovered C57BL/6 mice, and injection of the cytotoxic-T-lymphocyte (CTL) peptide with the adjuvant activated peptide-specific CD8+ T cells. Notably, the SVF9 epitope was highly conserved across Cryptosporidium hominis, C. parvum, and many other Cryptosporidium species. SVF9 also formed stable peptide-MHC class I (MHC I) complexes with HLA-A*0201, suggesting cross-reactivity between H-2Kb and human MHC I specificities. Crystal structure analyses revealed that the interactions of peptide-MHC surface residues of H-2Kb and HLA-A*0201 are highly conserved. The hydrogen bonds of H-2Kb-SVF9 are similar to those of a dominant epitope presented by HLA-A*0201, which can be recognized by a public human T-cell receptor (TCR). Notably, we found double conformations in position 4 (P4), 5 (P5) of the SVF9 peptide, which showed high flexibility, and multiple peptide conformations generated more molecular surfaces that can potentially be recognized by TCRs. Our findings demonstrate that an immunodominant C. parvum epitope and its homologs from different Cryptosporidium species and subtypes can benefit vaccine development to combat cryptosporidiosis. IMPORTANCE Adaptive immune responses and T lymphocytes have been implicated as important mechanisms of parasite-induced protection. However, the role of CD8+ T lymphocytes in the resolution of C. parvum infection is largely unresolved. Our results revealed that the glycoprotein-derived epitope Gp40/15-SVF9 induced an immunodominant CD8+ T-cell response in C57BL/6 mice. Crystal structure analyses revealed that the interactions of the H-2Kb-SVF9 peptide are similar to those of a dominant epitope presented by HLA-A*0201, which can be recognized by human TCRs. In addition, we found double conformations of the SVF9 peptide, which showed high flexibility and multiple peptide conformations that can potentially be recognized by TCRs.

The Role of CD4 T Cell Help in CD8 T Cell Differentiation and Function During Chronic Infection and Cancer.

CD4 and CD8 T cells are key players in the immune response against both pathogenic infections and cancer. CD4 T cells provide help to CD8 T cells via multiple mechanisms, including licensing dendritic cells (DCs), co-stimulation, and cytokine production. During acute infection and vaccination, CD4 T cell help is important for the development of CD8 T cell memory. However, during chronic viral infection and cancer, CD4 helper T cells are critical for the sustained effector CD8 T cell response, through a variety of mechanisms. In this review, we focus on T cell responses in conditions of chronic Ag stimulation, such as chronic viral infection and cancer. In particular, we address the significant role of CD4 T cell help in promoting effector CD8 T cell responses, emerging techniques that can be utilized to further our understanding of how these interactions may take place in the context of tertiary lymphoid structures, and how this key information can be harnessed for therapeutic utility against cancer.

The ligation of CD4 molecules, expressed on monocytes by an anti-CD4 monoclonal antibody, inhibits T cell activation and monocyte mobility.

CD4, a leukocyte surface glycoprotein, is mainly expressed on CD4+ T cells, but is also expressed on monocytes. The difference in the expression level and structure of CD4 on T cells and monocytes predicts the different functions of this molecule in both cell types. Although the function of CD4 on T cells is well characterized, little is known about that expressed on primary monocytes. In this study, we investigated the immunoregulation function of CD4 on peripheral blood monocytes. Methods: CD4 molecule on monocyte was ligated by anti-CD4 monoclonal antibody (mAb), MT4/3. The effect of mAb MT4/3 on T cell proliferation, cytokine production, the expression of monocyte costimulatory molecules, monocyte migration, and macrophage differentiation were investigated. Moreover, the molecular weight of CD4 on peripheral blood monocyte was carried out by Western immunoblotting. We demonstrated that mAb MT4/3 inhibited anti-CD3 induced T cell proliferation, cytokine production, and the expression of monocyte costimulatory molecules. The ligation of only CD4 on monocytes was sufficient to inhibit T cell activation. Moreover, mAb MT4/3 could inhibit monocyte migration in a transwell migration assay, but not affect the differentiation of monocytes to macrophages. Using purified primary monocytes, the molecular weight of CD4 expressed on monocytes was identified as 55 kDa. The CD4 molecule expressed on monocytes might play an important role in the regulation of immune responses in both innate and adaptive immunity. Understanding the novel role of CD4 on monocytes in immunoregulation is valuable in the development of new therapeutic approaches.

CD4+ T-Cells cause cognitive dysfunction in preeclampsia or COVID-19 infection in a pregnant rat model

Preeclampsia (PE), new onset hypertension (HTN) during pregnancy, is associated with increased cerebral blood flow (CBF), impaired cognitive function, and memory loss. We have shown adoptive transfer of placental CD4+ T cells from PE women into athymic nude pregnant rats causes HTN and increased circulating factors associated with PE. COVID-19 (CV) during pregnancy is associated with increased diagnosis of PE. However, the role of CD4+ T cells stimulated in response to CV in contributing to the PE phenotype in patients with a Hx of CV during pregnancy is unknown.

De Novo transcriptome combined with physiological analyses revealed key genes for cadmium accumulation in Zhe-Maidong (Ophiopogon japonicus).

Cadmium (Cd) is a toxic heavy metal that severely threatens safe food production. Zhe-Maidong, a well-known Chinese traditional herbal medicine, is susceptible to Cd stress. However, the characteristics of Cd transformation and migration, as well as the regulatory system for genes conferring Cd accumulation of Zhe-Maidong, remains an essential issue to be addressed. Zhe-Maidong seedling growth in Cd-contaminated and uncontaminated soil was conducted for 90 days. The Cd concentration was determined by inductively coupled plasma-mass spectrometry, and the Cd2+ fluorescence probe detected Cd distributions. The root transcriptome of Zhe-Maidong was then evaluated using various Cd stress hydroponic treatments designated Cd-0, Cd-M, and Cd-H. The enrichment factor (EF) value in the root was four times that of the leaves, indicating that the root has a high ability to absorb and accumulate Cd. The Cd2+ were mainly distributed in the root hair and the epidermis in both roots and leaves, revealing that the epidermal cells of roots may collect Cd2+ and also have an outstanding role in Cd2+ uptake. A total of 50 DEGs involved in Cd translocation and accumulation were identified. Among these DEGs, ANN, ABCC2/4, HMA1- 5, and CCX gene expression were positively correlated with EF-root, EF-leaf, EF-total, Cd-leaf, Cd-root, and Cd-plant, indicating their role in Cd transport and accumulation under Cd-stress. These data could be helpful in uncovering the Cd accumulation characteristics in Zhe-Maidong, as well as provide a bioinformatic foundation for investigations on finding gene functions and the screening of candidate genes related to Cd accumulation.

The Role of CD4+ T Cells in the Immunotherapy of Brain Disease by Secreting Different Cytokines.

Upon different stimulation, naïve CD4+ T cells differentiate into various subsets of T helper (Th) cells, including Th1, Th2, Th17, and Tregs. They play both protective and pathogenic roles in the central nervous system (CNS) by secreting different cytokines. Failure of the homeostasis of the subgroups in the CNS can result in different brain diseases. Recently, immunotherapy has drawn more and more attention in the therapy of various brain diseases. Here, we describe the role of different CD4+ T cell subsets and their secreted cytokines in various brain diseases, as well as the ways in which by affecting CD4+ T cells in therapy of the CNS diseases. Understanding the role of CD4+ T cells and their secreted cytokines in the immunotherapy of brain disease will provide new targets and therapeutics for the treatment of brain disease. The role of CD4 + T cell subtypes in different diseases and their associated regulatory genes, proteins, and enzymes. CD4 + T cell subtypes play both protective (green) and pathogenic (red) roles in different brain diseases. The immune regulatory effects of CD4 + T cells and their subtypes are promoted or inhibited by different genes, proteins, and enzymes.

CD103 fate mapping reveals that intestinal CD103- tissue-resident memory T cells are the primary responders to secondary infection.

Tissue-resident memory T (TRM) cells remain poised in the tissue and mediate robust protection from secondary infection. TRM cells within the intestine and other tissues are heterogeneous in their phenotype and function; however, the contributions of these TRM subsets to secondary infection remain poorly defined. To address the plasticity of intestinal TRM subsets and their role in local and systemic immunity, we generated mice to fate map intestinal CD103+ TRM cells and track their location and function during secondary infection with Yersinia pseudotuberculosis. We found that CD103+ TRM cells remained lodged in the tissue and were poorly reactivated during secondary challenge. CD103- TRM cells were the primary responders to secondary infection and expanded within the tissue, with limited contribution from circulating memory T cells. The transcriptional profile of CD103- TRM cells demonstrated maintenance of a gene signature similar to circulating T cells along with increased cytokine production and migratory potential. CD103- TRM cells also expressed genes associated with T cell receptor (TCR) activation and displayed enhanced TCR-mediated reactivation both in vitro and in vivo compared with their CD103+ counterparts. These studies reveal the limited recall potential of CD103+ TRM subsets and the role of CD103- TRM cells as central memory-like T cells within peripheral tissues.

Role of CD8+ T cell exhaustion in the progression and prognosis of acute respiratory distress syndrome induced by sepsis: a prospective observational study

BackgroundCD8+ T cells are important for protective immunity against intracellular pathogens. Excessive amounts of antigen and/or inflammatory signals often lead to the gradual deterioration of CD8+ T cell function, a state called “exhaustion”. However, the association between CD8+ T cell exhaustion and acute respiratory distress syndrome (ARDS) has not been studied. This study was conducted to elucidate how CD8+ T cells and inhibitory receptors were related to the clinical prognosis of ARDS.MethodsA prospective observational study in an emergency department enrolled patients who were diagnosed with sepsis-associated ARDS according to the sepsis-3 criteria and Berlin definition. Peripheral blood samples were collected within 24 h post recruitment. CD8+ T cell count, proliferation ratio, cytokine secretion, and the expression of coinhibitory receptors were assayed.ResultsSixty-two patients with ARDS met the inclusion criteria. CD8+ T cell counts and proliferation rates were dramatically decreased in non-surviving ARDS patients. Increasing programmed cell death 1 (PD-1) expression on the CD8+ T cell surface was seen in patients with worse organ function, while an increasing level of T cell immunoglobulin mucin-3 (Tim-3) was associated with a longer duration of the shock. Kaplan–Meier analysis showed that low CD8+ T cell percentages and increased inhibitory molecule expression were significantly associated with a worse survival rate.ConclusionsCD8+ T cells and coinhibitory receptors are promising independent prognostic markers of sepsis-induced ARDS, and increased CD8+ T cell exhaustion is significantly correlated with poor prognosis.

CD25 As Feedback Regulator of Oncogenic TCR Signaling in T-Cell Lymphoma

Introduction: CD25, also known as interleukin-2 receptor α chain (IL-2Rα), forms a heterotrimer with IL-2Rβ and common γ chain, and mediates IL-2 signaling. IL-2 signaling is essential for T- and NK-cell survival and proliferation. Additionally, CD25 is required for development of thymic regulatory T cells (Treg) and their suppressive function. Interestingly, CD25 is upregulated in a variety of lymphoid and myeloid malignancies, which do not depend on IL-2 signaling, suggesting IL-2-independent roles of CD25. Here, we seek to investigate whether CD25 has previously unrecognized functions in T cells and T-cell lymphomas. Results: Our previous study in B cells has discovered the cytoplasmic tail of CD25 as an essential structural element for site-specific recruitment of inhibitory phosphatases. CD25-mediated shuttling of inhibitory phosphatases balanced strength of oncogenic B-cell receptor (BCR)-signaling and was essential for cell-survival in B-cell malignancies. Interestingly, we found that CD25 expressed on B cells was monomeric and showed dynamic recruitment to BCR or its oncogenic mimics within lipid rafts. Studying gene expression data across different hematopoietic lineages, we found CD25 was strongly induced in T cell receptor (TCR)-activated T cells, as well as in T-cell malignancies that harbor oncogenic TCR-mimics, such as PTCL. Our mechanistic studies discovered that the serine residue 268 (S268) in the cytoplasmic tail of CD25 was a principal substrate of protein kinase C (PKC), and that S268 phosphorylation by PKC was critical for cell surface expression of CD25. The proximity-based labeling assays (Bio-ID) revealed that CD25-S268 but not its phospho-dead mutant recruits PKC and its adapter RACK1, which scaffold the inhibitory phosphatases SHIP1 and SHP1 for site-specific activation in lipid rafts. CRISPR-mediated knock-in of CD25-S268A that abrogates the formation of inhibitory phosphatase complexes in primary human T cells enabled us to specifically interrogate the functional relevance of CD25-S268 in the regulation of TCR signaling. By using single stranded homology-directed repair templates (ssHDRT) that contain a Cas9 shuttling sequence, we were able to achieve 70% knock-in efficiency in primary human T cells (Figure 1). Signaling studies with these cells revealed that S268A mutation significantly augmented TCR signaling strength. Additionally, S268A mutation resulted in acute inactivation of inhibitory phosphatases including SHP1. Consistent with these results, CD25 shuttling to TCR complex was promptly induced upon TCR stimulation and was mitigated by S268 mutation. Furthermore, immunophenotyping showed that loss of S268 phosphorylation resulted in increased expression of T-cell activation markers such as CD44 and CD69. Based on the ex vivo results, we generated a transgenic mouse model with germline knock-in of CD25-S268A, which would allow us to study S268-specific roles in CD25-mediated feedback regulation of TCR and oncogenic signaling in vivo. Conclusions: The role of CD25 in mediating IL-2 signaling has been extensively studied. However, increasing evidence suggests that CD25 plays a more sophisticated role in TCR signaling and T-lymphomagenesis. Here we discovered a previously unrecognized function of CD25 as a feedback regulator of TCR signaling. TCR activation or oncogenic TCR-mimics induce CD25-mediated recruitment of inhibitory phosphatases in lipid rafts to mitigate TCR signaling or curb excessive oncogenic signaling. Figure 1. CRISPR-mediated knock-out of CD25 alone, or followed by knock-in of CD25-S268-GFP or CD25-A268-GFP, in primary human T cells. The proportion of CD25-expressing GFP+ cells are analyzed by flow cytometry on day 4 and day 30 post CRISPR. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Excessive IL-15 promotes cytotoxic CD4 + CD28− T cell-mediated renal injury in lupus nephritis

BackgroundPatients with systemic lupus erythematosus (SLE) are highly susceptible to infection and cardiovascular events, suggesting that chronic antigenic stimulation may accelerate premature aging in SLE patients. Premature aging in SLE is often accompanied with the expansion of cytotoxic CD4 + CD28−T cells. Damage caused by CD4 + CD28− T cells enhances the progressive aging of the tissue function and loss of organism’s fitness. The high serum level of IL-15 has been implicated in the pathogenesis of SLE, but its role in CD4 + CD28−T cell-mediated cytotoxicity in nephritic SLE remains unclear. The aim of this study was to investigate the effect of IL-15 on functional properties and associated renal damage of cytotoxic CD4 + CD28− T cell in lupus nephritis (LN).ResultsFlow cytometry showed that the number of circulating innate-like CD4 + CD28− T cells was increased in patients with nephritic SLE. Immunofluorescence showed CD4 + CD28− T cell infiltration in the kidney of LN patients, which was correlated with multiple clinicopathological features including estimated glomerular filtration rate (eGFR), proteinuria, the proportion of glomerulosclerosis and the degree of renal chronicity. In addition, a high level of IL-15 and IL15-expressing macrophage infiltration was detected in the periglomerular and intraglomerular tissues of LN patients, which enhanced the innate features, cytokine secretion and migratory capability of CD4 + CD28− T cells, and finally exerted direct TCR-independent cytotoxicity on glomerular endothelial cells in an IL-15-dependent manner in vitro.ConclusionOur study demonstrated that excessive IL-15 potentially promoted cytotoxic CD4 + CD28− T cell-mediated renal damage in LN. This finding may provide new insights into the potential association of premature aging and tissue damage in LN.

Synovial Macrophages Expression of OX40L Is Required for Follicular Helper T Cells Differentiation in the Joint Microenvironment.

Signaling via the OX40/OX40L axis plays a key role in CD4+ T cell development, and OX40L expression is primarily restricted to antigen-presenting cells (APCs). This study was designed to assess the role of APC-mediated OX40L expression in the context of the development of rheumatoid arthritis (RA)-associated CD4+ T cell subsets. For these analyses, clinical samples were harvested from patients with osteoarthritis and RA, with additional analyses performed using OX40−/− mice and mice harboring monocyte/macrophage-specific deletions of OX40L. Together, these analyses revealed tissue-specific roles for OX40/OX40L signaling in RA. Specifically, higher levels of synovial macrophage OX40L expression were associated with the enhanced development of T follicular helper cells in the joint microenvironment, thereby contributing to the pathogenesis of RA. This Tfh differentiation was found to be OX40/OX40L-dependent in this synovial setting. Overall, these results indicate that the expression of OX40L by synovia macrophages is necessary to support Tfh differentiation in the joint tissues, thus offering new insight regarding the etiological basis for RA progression.

Research Highlights

Research Highlights

Patients With Severe Multiple Sclerosis Exhibit Functionally Altered CD8+ Regulatory T Cells.

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the CNS. Studies of immune dysfunction in MS have mostly focused on CD4+ Tregs, but the role of CD8+ Tregs remains largely unexplored. We previously evidenced the suppressive properties of rat and human CD8+CD45RClow/neg Tregs from healthy individuals, expressing Forkhead box P3 (FOXP3) and acting through interferon-gamma (IFN-γ), transforming growth factor beta (TGFβ), and interleukin-34 (IL-34). secretions to regulate immune responses and control diseases such as transplant rejection. To better understand CD8+CD45RClow/neg Tregs contribution to MS pathology, we further investigated their phenotype, function, and transcriptome in patients with MS. We enrolled adults with relapsing-remitting MS and age-matched and sex-matched healthy volunteers (HVs). CD8+ T cells were segregated based on low or lack of expression of CD45RC. First, the frequency in CSF and blood, phenotype, transcriptome, and function of CD8+CD45RClow and neg were investigated according to exacerbation status and secondarily, according to clinical severity based on the MS severity score (MSSS) in patients with nonexacerbating MS. We then induced active MOG35-55 EAE in C57Bl/6 mice and performed adoptive transfer of fresh and expanded CD8+CD45RCneg Tregs to assess their ability to mitigate neuroinflammation in vivo. Thirty-one untreated patients with relapsing-remitting MS were compared with 40 age-matched and sex-matched HVs. We demonstrated no difference of CSF CD8+CD45RClow and CD8+CD45RCneg proportions, but blood CD8+CD45RClow frequency was lower in patients with MS exacerbation when compared with that in HVs. CD8+CD45RCneg Tregs but not CD8+CD45RClow showed higher suppressive capacities in vitro in MS patients with exacerbation than in patients without acute inflammatory attack. In vitro functional assays showed a compromised suppression capacity of CD8+CD45RClow Tregs in patients with nonexacerbating severe MS, defined by the MSSS. We then characterized murine CD8+CD45RCneg Tregs and demonstrated the potential of CD45RCneg cells to migrate to the CNS and mitigate experimental autoimmune encephalomyelitis in vivo. Altogether, these results suggest a defect in the number and function of CD8+CD45RClow Tregs during MS relapse and an association of CD8+CD45RClow Tregs dysfunction with MS severity. Thus, CD8+CD45RClow/neg T cells might bring new insights into the pathophysiology and new therapeutic approaches of MS.