Role Of CD4 Research Articles

High circulating SDF-1and MCP-1 levels and genetic variations in CXCL12, CCL2 and CCR5: Prognostic signature of immune recovery status in treated HIV-positive patients.

BackgroundThe underlying mechanisms of incomplete immune reconstitution in treated HIV-positive patients are very complex and may be multifactorial, but perturbation of chemokine secretion could play a key role in CD4+T-cell turnover.MethodsWe evaluated the circulating baseline and 48-week follow-up concentrations of SDF-1/CXCL12, fractalkine/CX3CL1, MCP-1/CCL2, MIP-α/CCL3, MIP-β/CCL4 and RANTES/CCL5, and we estimated their association with CXCL12, CX3CR1, CCR2, CCL5 and CCR5 single nucleotide polymorphisms (SNPs) to investigate multiple chemokine-chemokine receptor signatures associated with immune dysregulation preceding poor immune recovery.FindingsThe circulating concentrations and gene expression patterns of SDF-1/CXCL12 (CXCL12 rs1801157) and MCP-1/CCL2 (CCR2 rs1799864_814) were associated with immune recovery status. CCR2 rs1799864_814 and CCR5 rs333_814 (Δ32) determine the baseline plasma RANTES and MIP-α concentrations, respectively, in participants with poor immune response.InterpretationSDF-1/CXCL12 and MCP-1/CCL2 could be considered prognostic markers of immune failure despite suppressive antiretroviral therapy. The strong linkage disequilibrium (LD) between CCR2 rs1799864_814 and CCR5 rs1800024 indicated that the alleles of each gene are inherited together more often than would be expected by chance.FundingThis work was supported by Fondo de Investigacion Sanitaria and SPANISH AIDS Research Network (ISCIII-FEDER); AGAUR and Gilead Fellowship. FV and YMP are supported by grants from the Programa de Intensificación (ISCIII) and Servicio Andaluz de Salud, respectively. JVG,EY and LR are supported by the 10.13039/501100004587Instituto de Salud Carlos III (ISCIII). AR is supported by Departament de Salut, Generalitat de Catalunya and by the Instituto de Salud Carlos III (ISCIII).

Single-Cell Analysis of the Classical Hodgkin Lymphoma Immune Environment Reveals a Clonally-Expanded CD8+ T Cell Population with a Cytotoxic Phenotype

Introduction: Classical Hodgkin lymphoma (cHL) is characterized by a robust and complex immune cell infiltrate and the rare presence of malignant Hodgkin-Reed-Sternberg (HRS) cells. At the genetic level, HRS cells recurrently acquire alterations that lead to defective antigen presentation (β2 microglobulin mutations) and mediate T cell dysfunction (PD-L1 copy gains/amplifications) in order to subvert host immune surveillance. The clinical relevance of PD-L1 protein over-expression in cHL is clear, as response rates to PD-1 blockade therapy are extremely high among patients with relapsed/refractory (r/r) disease. Despite its remarkable efficacy, the cells that mediate response to anti-PD-1 therapy in cHL remain undefined. Recent analyses have highlighted a possible role for CD4+ T cells in mediating the clinical activity of anti-PD-1 therapy in cHL. CD4+ T cells significantly outnumber CD8+ T cells in cHL lesions and are more frequently juxtaposed to HRS cells in situ. Furthermore, HLA class II expression on HRS cells predicted higher complete response rates to PD-1 blockade therapy in r/r cHL patients. However, a candidate T cell population capable of specific reactivity to antigens expressed by HRS cells has yet to be identified. This information is critical as such T cells might be functionally reinvigorated to mediate HRS cell elimination following PD-1 blockade therapy. In order to address this key knowledge gap, we analyzed data at single cell (sc) resolution using paired RNA and T cell receptor (TCR) sequencing in 9 diagnostic cHL and 5 reactive lymph node (RLN) specimens. Methods: Sequencing was performed using the 10x Genomics Chromium Single Cell 5' Gene Expression and V(D)J workflows. B-cell depletion of each sample was achieved using CD19 microbeads and negative selection to enrich T cell populations. Reads were analyzed and aligned with CellRanger (v3.1.0) and Seurat (v3.2.0) was used to conduct clustering by a shared nearest neighbor (SNN) graph on scRNA data. TCR sequencing data was integrated using scRepertoire (v1.0.0). Results: A detailed map of the immune cell states in cHL was created using scRNA-seq (10X) data on 79,085 cells from 9 cHL (52,602 cells) and 5 RLN samples (26,484 cells) expressing a total of 21,421 genes (mean 5649 cells/sample; mean 2849 mRNA reads/cell). Dimensionality reduction and unsupervised graph-based clustering revealed 21 distinct cell type and activation state clusters, including T cells, NK cells, macrophages, and dendritic cells (Fig 1A-B). A cluster identifying HRS cells was not observed, consistent with a recently published report. Ten T cell clusters were identified (47,573 cells), including naive- and memory-like T cells, effector/cytotoxic CD8+ T cells, regulatory T cells, and T follicular helper cells. Unexpectedly, a putative exhausted T cell cluster was not clearly observed. The relative contributions of cHL and RLNs cases to these clusters are shown in Fig 1C. Paired TCR sequencing was available for 23,943 cells. Overall TCR diversity was lower among cHL samples compared to RLN specimens (Fig 1D). In cHL samples, modest clonal expansion within regulatory T cell and memory CD4+ T cell clusters was observed, but the most striking clonal expansion occurred among cells assigned to effector/cytotoxic CD8+ T cell clusters - a finding not observed in most RLN specimens (Fig 1E). Clonally-expanded effector/cytotoxic CD8+ T cells displayed high expression of granzymes (GZMA, GZMH, GZMK), cytokines (TNF, IFNG) and chemokines (CCL4/CCL5), and modest expression of exhaustion markers (PDCD1, ENTPD1, HAVCR2, ITGAE), contrasting with data from single-cell analyses of solid tumors. Clonal expansion of effector/cytotoxic CD8+ T cells was particularly robust in EBV-positive cHLs, likely due to recognition of viral-derived epitopes displayed on HRS cells (Fig 1F). Phenotypic and functional validation of key immune cell clusters in cHL specimens using spectral cytometry is underway and will be reported at the meeting. Conclusions: For the first time, our data have unveiled the nature of the T cell repertoire in cHL at single cell resolution. Our results reveal a recurrent pattern of clonal expansion within effector CD8+ cells, which may be the HRS antigen-specific T cells that mediate response to PD-1 blockade. This hypothesis requires confirmation through similar analyses of pre- and on-treatment biopsies of cHL patients receiving anti-PD-1 therapy. Disclosures Godfrey: Gilead: Research Funding; Merck: Research Funding; Verastem: Research Funding. Venkataraman:EUSA Pharma: Speakers Bureau. Smith:Janssen: Consultancy; BMS: Consultancy; TG Therapeutics: Consultancy, Research Funding; Genentech/Roche: Consultancy, Other: Support of parent study and funding of editorial support, Research Funding; Karyopharm: Consultancy, Research Funding; FortySeven: Research Funding; Pharmacyclics: Research Funding; Acerta: Research Funding; Celgene: Consultancy, Research Funding. Kline:Kite/Gilead: Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Increase in inflammatory T cell subsets in atrial fibrillation: the missing link underlying inflammation in AF

Abstract Background The precise role of inflammation in the development and perpetuation of atrial fibrillation (AF) is yet to be fully uncovered. T lymphocytes have pivotal roles in orchestrating inflammation. Specialised subsets of lymphocytes either promote or prevent inflammation. We are investigating a unique subset of lymphocytes, the CD4+CD28null T cells that expand in patients with chronic inflammation. These cells secrete high levels of pro-inflammatory cytokines and have cytolytic function. CD4+CD28null T cells are normally maintained under control by regulatory T cells (Treg), a specialised subset of T lymphocytes with suppressive function that maintain immune homeostasis and prevent pathogenic immune responses. The role of CD4+CD28null and Treg cells has not been investigated in AF. Purpose We hypothesised that in AF the balance between pro-inflammatory and regulatory T lymphocytes is skewed in favour of inflammatory T cells, which may sustain inflammation in AF. Methods Circulating CD4+CD28null T lymphocytes and Tregs were quantified by flow cytometry in paroxysmal and persistent AF patients and healthy controls (n=30). Inflammatory cytokines were quantified in serum and the function of T lymphocyte subsets was investigated using ex vivo functional assays. Results CD4+CD28null T lymphocytes were significantly increased in the circulation of AF patients compared to controls. Of note, a higher proportion of patients with persistent AF showed an increase in inflammatory CD4+CD28null T lymphocytes compared to patients with paroxysmal AF. A marked reduction in Treg cells was present in AF patients compared to controls. Functional assays showed that IL-7 and IL-15 cytokines were responsible for CD4+CD28null T lymphocyte expansion in AF patients. Conclusions We show that patients with AF have marked changes in T lymphocytes subsets: pro-inflammatory CD4+CD28null T cells increase significantly, whilst anti-inflammatory Tregs are significantly reduced. We show for the first time that the cytokines IL-7 and IL-15 are the main drivers of CD4+CD28null T cell expansion in AF patients. These novel findings may reveal novel therapeutic strategies (e.g. cytokine blockade) to re-establish the balance between pro- and anti-inflammatory mechanisms at work in AF to improve patient outcomes. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation

Role of CD4- and CD8-Positive T Cells in Breast Cancer Progression and Outcome: A Pilot Study of 47 Cases in Central India Region

Immune response plays an important role in cancer progression. In humans, the study of the inflammatory infiltrate in malignancies, mainly T lymphocytes, has been a subject of great interest. The study was conducted to correlate CD4- and CD8-positive lymphocyte expression with other prognostic factors in invasive breast cancer, such as clinical stage, histological grade, tumour size and clinical course. It was a retrospective cross-sectional study. In this study, 47 cases of breast carcinoma which were diagnosed at least 12 months back from the initiating point of study were included. The immunohistochemical expression of CD4 and CD8 in lymphocytes infiltrating the intratumoural, adjacent or distant stroma sites was studied and correlated with other prognostic factors. Less occurrence of adverse outcome was found with the presence of CD8+ T cells anywhere in the vicinity of tumour (only 27%, 7 out of 26 cases) as compared to the group in which both CD4+ and CD8+ T cells were absent where adverse outcome occurred in 78% (7 of 9 cases, p = 0.007). High histological grade was found to be associated with negative CD8+ T cells staining in 21 out of 30 cases with grade III tumours at distant stroma site (p = 0.026). Presence of CD8 cells at intratumoural site had reduced disease-free survival (DFS) (8 months) as compared to their absence at the same site (DFS = 31 months, log rank p = 0.001). CD4 and CD8 staining did not correlate with clinical staging (p > 0.05). Infiltration around the primary tumour by CD8+ T cells in carcinoma breast is a sign of good prognosis, while their absence indicates more adverse outcomes. On the other hand, intratumoural infiltration is associated with rapid advancement of disease. CD4+ cells did not correlate well with prognosis.

Apoptosis in idiopathic inflammatory myopathies with partial invasion; a role for CD8+ cytotoxic T cells?

Polymyositis and inclusion body myositis are idiopathic inflammatory myopathies, with a pathology characterized by partial invasion of non-necrotic muscle fibres by CD8+ cytotoxic T-cells, leading to fibre degeneration. Although the main effector pathway of CD8+ T-cells is to induce apoptosis of target cells, it has remained unclear if apoptosis occurs in these diseases, and if so, if it is mediated by CD8+ T-cells. In consecutive biopsy sections from 10 patients with partial invasion, muscle fibres and inflammatory cells were assessed by immunohistochemistry and apoptotic nuclei by the TUNEL assay. Analysis of muscle fibre morphology, staining pattern and quantification were performed on digital images, and they were compared with biopsies from 10 dermatomyositis patients and 10 controls without muscle disease. Apoptotic myonuclei were found in muscle with partial invasion, but not in the invaded fibres. Fibres with TUNEL positive nuclei were surrounded by CD8+ T-cells, granzyme B+ cells and macrophages, but lacked FAS receptor expression. In contrast, apoptotic myonuclei were rare in dermatomyositis and absent in controls. The findings confirm that apoptosis occurs in idiopathic inflammatory myopathies and support that it is mediated by CD8+ cytotoxic T- cells, acting in parallel to the process of partial invasion.

Two identified subsets of CD8 T cells in obstructed kidneys play different roles in inflammation and fibrosis.

Inflammation plays a crucial role in initiating renal fibrosis after injury. The infiltration of inflammatory cells, such as CD4+ T cells and macrophages, contributes to renal fibrosis following ureteric obstruction. However, the function of CD8+ T cells in obstructed kidneys remains unclear. Although CD8+ T cell depletion intensifies renal fibrosis by decreasing IFN-γ and increasing IL-4 in the kidneys, the change and role of CD8 T cell populations following environmental changes during renal fibrosis are largely unknown. Here, we identified two CD8 T cell subsets in mouse obstructed kidneys with unilateral ureteric obstruction and revealed their different functions in building an inflammatory or profibrotic environment. Following renal fibrosis, the phenotypes of infiltrated CD8 T cells were mainly Tc1 (CD44+CD25−CD62L−) at the early inflammation stage and then changed to Tc2 (CD44+CD25highCD62Llow). Tc1 and Tc2 secreted IFN-γ, contributing to the decrease in the Th2-induced over-polarization of M2 macrophages and fibrosis. Moreover, Tc2 secreted pro- and anti-inflammation factors and decreased the inflammatory responses of other cells to control inflammation and fibrosis. This work and our previous study showed that CD8 T cells could balance out inflammation by controlling its level in renal fibrosis.

CD4 + T-Cell Endogenous Cystathionine γ Lyase–Hydrogen Sulfide Attenuates Hypertension by Sulfhydrating Liver Kinase B1 to Promote T Regulatory Cell Differentiation and Proliferation

Hydrogen sulfide (H2S) has antihypertension and anti-inflammatory effects, and its endogenous-generation key enzyme cystathionine γ lyase (CSE) is expressed in CD4+ T cells. However, the role of CD4+ T-cell endogenous CSE/H2S in the development of hypertension is unclear. Peripheral blood lymphocytes were isolated from hypertensive patients or spontaneously hypertensive rats, then H2S production and expression of its generation enzymes, cystathionine β synthase and CSE, were measured to determine the major H2S generation system changes in hypertension. Mice with CSE-specific knockout in T cells (conditional knockout, by CD4cre mice hybridization) and CD4 null mice were generated for investigating the pathophysiological relevance of the CSE/H2S system. In lymphocytes, H2S from CSE, but not cystathionine β synthase, responded to blood pressure changes, supported by lymphocyte CSE protein changes and a negative correlation between H2S production with systolic blood pressure and diastolic blood pressure, but positive correlation with the serum level of interleukin 10 (an anti-inflammatory cytokine). Deletion of CSE in T cells elevated BP (5-8 mm Hg) under the physiological condition and exacerbated angiotensin II-induced hypertension. In keeping with hypertension, mesenteric artery dilation impaired association with arterial inflammation, an effect attributed to reduced immunoinhibitory T regulatory cell (Treg) numbers in the blood and kidney, thus causing excess CD4+ and CD8+ T cell infiltration in perivascular adipose tissues and kidney. CSE knockout CD4+ T cell transfer into CD4 null mice, also showed the similar phenotypes' confirming the role of endogenous CSE/H2S action. Adoptive transfer of Tregs (to conditional knockout mice) reversed hypertension, vascular relaxation impairment, and immunocyte infiltration, which confirmed that conditional knockout-induced hypertension was attributable, in part, to the reduced Treg numbers. Mechanistically, endogenous CSE/H2S promoted Treg differentiation and proliferation by activating AMP-activated protein kinase. In part, it depended on activation of its upstream kinase, liver kinase B1, by sulfhydration to facilitate its substrate binding and phosphorylation. The constitutive sulfhydration of liver kinase B1 by CSE-derived H2S activates its target kinase, AMP-activated protein kinase, and promotes Treg differentiation and proliferation, which attenuates the vascular and renal immune-inflammation, thereby preventing hypertension.

The Role of CD3, CD20, CD30, and Ki67 Immunohistochemistries in Establishing Mediastinal Gray Zone Lymphoma Diagnosis in 24-years-old Female Patient

Title: The Role of CD3, CD20, CD30, and Ki67 Immunohistochemistries in Establishing  Mediastinal Gray Zone Lymphoma Diagnosis in 24-years-old Female PatientAuthor: Frenky Hardiyanto, MD. Pulmonology and Respiratory Medicine Department, Brawijaya University, Malang, Indonesia. Supervisor: Suryanti Dwi Pratiwi, MD, Pulmonologist Consultant, Artono I, MD, Cardiovascular and Thorax Surgeon, Dini Rachma Erawati, MD, Radiologist Consultant, Diah Prabawati R, MD, Patology Anatomy Specialist, Herwindo P, MD, Internal Medicine Specialist.Introduction:. A woman with complaints of shortness of breath, cough, fever, night sweating, decrease of body weight and swelling in face, neck and right arm. Chest x-rays and ct thorax revealed a mass in mediastinum.Case Description: Patient is a 24 years old female with complaints of shortness of breath, cough, fever, night sweating, decrease of body weight and swelling in face, neck and right arm. Chest x rays and CT thorax revealed a mass in mediastinum. Open biopsy on right neck mass and transthoracic FNAB resulting of chronic inflammation, so the patient was diagnosed with glandular tuberculosis and Mediastinal Tumor. Patient was getting worse after one month consuming OAT. Patient was consulted to Cardiovascular and thorax Surgery Department. Patient was performed thoracotomy and debulking. Anatomical pathology was examined from debulking mass, the result of immunohistochemistry was found nodular mediastinal gray zone lymphoma.Discussion: this case it is very difficult to enforce the diagnosis. Patients have done neck FNAB, transthoracic FNAB, open biopsy right neck mass and FOB have not been able to establish diagnosis. Until finally done thoracotomy and debulking in this patient, from immunohistochemistry results obtained nodular sclerosis Hodgkin lymphoma. Collaboration from pulmonologist, radiologist, cardiovascular and thorax surgeon, and patology anatomy specialist is needed to confirm the diagnosis in these patients.Keywords: mediastinal tumor, mediastinal gray zone lymphoma

Effector CD8 T Cell-Dependent Zika Virus Control in the CNS: A Matter of Time and Numbers.

Zika virus (ZIKV), a mosquito-borne flavivirus, came into the spotlight in 2016 when it was found to be associated with an increased rate of microcephalic newborns in Brazil. The virus has further been recognized to cause neurologic complications in children and adults in the form of myelitis, encephalitis, acute disseminated encephalomyelitis (ADEM) and Guillain Barre Syndrome in a fraction of infected individuals. With the ultimate goal of identifying correlates of protection to guide the design of an effective vaccine, the study of the immune response to ZIKV infection has become the focus of research worldwide. Both innate and adaptive immune responses seem to be essential for controlling the infection. Induction of sufficient levels of neutralizing antibodies has been strongly correlated with protection against reinfection in various models, while the role of CD8 T cells as antiviral effectors in the CNS has been controversial. In an attempt to improve our understanding regarding the role of ZIKV-induced CD8 T cells in protective immunity inside the CNS, we have expanded on previous studies in intracranially infected mice. In a recent study, we have demonstrated that, peripheral ZIKV infection in adult C57BL/6 mice induces a robust CD8 T cell response that peaks within a week. In the present study, we used B cell deficient as well as wild-type mice to show that there is a race between CXCR3-dependent recruitment of the effector CD8 T cells and local ZIKV replication, and that CD8 T cells are capable of local viral control if they arrive in the brain early after viral invasion, in appropriate numbers and differentiation state. Our data highlight the benefits of considering this subset when designing vaccines against Zika virus.

CD8 Treg Cells Inhibit B-Cell Proliferation and Immunoglobulin Production

Aim: The role of CD4+ Treg in immune responses has been well established. More recently, a role of CD8+ T regulatory cells (CD8 Treg) in the regulation of immune responses in health and autoimmune diseases has been investigated. Furthermore, different investigators have used different markers to define CD8 Treg. Finally, regulatory effects of CD8 Treg have been studied against T-cell responses; however, their role in regulating B-cell proliferation and immunoglobulin production has not been evaluated. Therefore, in this study we examined the effect of two types of CD8 Treg on B-cell proliferation and immunoglobulin production. Methods: Purified CD8+ T cells were activated with anti-CD3/CD28 for 48 h and then sorted into two different types of CD8 Treg as defined by two different sets of markers, CD8+CD183+CD197+CD45RA− and CD8+CD183+CD25^highCD278+. Purified B cells were cocultured with sorted CD8 Treg at 1:1, 1:1/2, and 1:1/4 ratios and activated with anti-CD40 and CpG. B-cell proliferation was assessed by the CFSE dye dilution assay and immunoglobulin production by the ELISA assay. Results: Our data show CD183+CD197+CD45RA−CD8 Treg significantly inhibited B-cell proliferation and inhibited IgM and IgG production but not IgA production at 1:1 ratio only. However, CD183+CD25^highCD278+CD8 Treg inhibited significantly B-cell proliferation at 1:1 and 1:1/2 ratios and IgM, IgG, and IgA production at all ratios. Conclusion: CD8 Treg regulate B-cell responses, and CD183+CD25^highCD278+CD8 Treg are more powerful regulators of B-cell proliferation and immunoglobulin production than CD183+CD197+CD45RA−CD8 Treg and, therefore, may be used as preferred markers for CD8 Treg.

Abstract 927: Deep proteomic profiling of CD4+ and CD8+ T cell-depleted tumor models in PD-1 treatment

Abstract Background Immunotherapies targeting the PD-1/PD-L1 axis have been shown to be effective in only around 20% of cancer patients and the mechanism of action (MOA) underlying the differences between responders and non-responders remains poorly understood. It is therefore critical to understand the roles of different lineages of immune cells play mediating PD-1 response, while gaining an overall understanding of the interplay between tumor microenvironment (TME) and immune cell populations. To address this, tumor-bearing syngeneic mice were treated with an anti-PD-1 antibody in combination with targeted CD4+ or CD8+ T cell depletion. Tumor tissues were subsequently investigated with an unbiased proteomics workflow based on data-independent acquisition (DIA) mass spectrometry to provide insights into TME in different treatment contexts. Methods T cell subpopulations were depleted in two subcutaneous murine syngeneic models (MC38, and Hepa 1-6) followed by anti-PD-1 treatment (10mg/kg). The depletion of CD4+ and CD8+ T cells were accomplished with anti-CD4 (GK1.5) and anti-CD8 (2.43). The effectiveness of each depletion was assessed by tumor growth, followed by flow cytometry analysis of tumor infiltrating lymphocytes. FFPE tumor tissue samples, taken at the end of study (17-20 days), were digested using standard procedures and analyzed using 4h gradients on a C18 column coupled to a Thermo Scientific Q Exactive HF-X mass spectrometer. Data were extracted using Spectronaut™ (Biognosys) with a sample specific library. Results Consistent with expectation, depletion of CD8+ T cells significantly attenuated the antitumor effects of anti-PD-1 treatment in both tumor models, confirming the crucial roles of CD8+ T cells in tumor cell killing and inhibition of growth. However, depletion of CD4+ T cells showed opposing effects in the two models tested with a significant enhancement of anti-PD-1 efficacy in MC38 tumors and a reduction of anti-PD-1 efficacy in Hepa1-6 tumors. These effects were further studied by a deep proteome analysis (>9'000 proteins quantified. Statistical analysis showed large differences on proteome level between the two models (>3,000 proteins differentially expressed). We observed a strong upregulation of IFNγ inducible proteins upon anti-PD-1 therapy in the MC38 model, which is boosted upon CD4+ depletion, while many IFNγ pathway related markers were downregulated in the combo group of anti-PD-1 and anti-CD4 in Hepa 1-6. The data of the proteome analysis will be correlated to additional data from RNAseq analysis. Conclusions This study shows an unequivocal role of CD8+ T cells in anti-PD-1 induced tumor growth inhibition whereas the role of CD4+ T cells depends on the specific tumor type and TME. In our study we demonstrate how a deep proteome analysis improves the understanding of the interdependency of immune cells and the TME. Citation Format: Jan Muntel, Ying Jin, Haochen Yu, Nicholas Dupuis, Yongli Shan, Annie X. An, Wubin Qian, Davy Ouyang, Kristina Beeler, Roland Bruderer. Deep proteomic profiling of CD4+ and CD8+ T cell-depleted tumor models in PD-1 treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 927.

Comparative Analysis of NF-κB in the MyD88-Mediated Pathway After Implantation of Titanium Alloy and Stainless Steel and the Role of Regulatory T Cells

Development of immunologically smart implants, integrated to biological systems, is a key aim to minimize the inflammatory response of the host to biomaterial implants. The aim of this study is to investigate the influence of titanium alloy and stainless steel implants on immunological responses in rats by comparative analysis of nuclear factor kappa B (NF-κB) profiles in the activation of inflammatory signaling pathways and the role of CD4+CD25+Foxp3+. Both Ti alloy and stainless steel alloy group implantation affect Toll-like receptors-4 pathways and CD4+CD25+ regulatory T cells in different ways. Results show that NF-κB/p65 and NF-κB1/p50 possess potential as a therapeutic target in the prevention of adverse reactions to metal, especially for controlling inflammation after the implantation.

Regulatory T Cells Expressing Tumor Necrosis Factor Receptor Type 2 Play a Major Role in CD4+ T-Cell Impairment During Sepsis.

Sepsis causes inflammation-induced immunosuppression with lymphopenia and alterations of CD4+ T-cell functions that renders the host prone to secondary infections. Whether and how regulatory T cells (Treg) are involved in this postseptic immunosuppression is unknown. We observed in vivo that early activation of Treg during Staphylococcus aureus sepsis induces CD4+ T-cell impairment and increases susceptibility to secondary pneumonia. The tumor necrosis factor receptor 2 positive (TNFR2pos) Treg subset endorsed the majority of effector immunosuppressive functions, and TNRF2 was particularly associated with activation of genes involved in cell cycle and replication in Treg, probably explaining their maintenance. Blocking or deleting TNFR2 during sepsis decreased the susceptibility to secondary infection. In humans, our data paralleled those in mice; the expression of CTLA-4 was dramatically increased in TNFR2pos Treg after culture in vitro with S. aureus. Our findings describe in vivo mechanisms underlying sepsis-induced immunosuppression and identify TNFR2pos Treg as targets for therapeutic intervention.

Immunological role of CD4 and CD154 in patients infected with S.typhi in Al-Najaf Governorate, Iraq.

Immunological role of CD4 and CD154 in patients infected with S.typhi in Al-Najaf Governorate, Iraq.

THU0325 REDUCED OF TREG CELLS ASSOCIATED WITH THE DISEASE ACTIVITY OF ANCA-ASSOCIATED VASCULITIS

Background:Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune disease that can cause systemic organ damage, characterized with the presence of abnormal antibodies (ANCAs) in the circulation and the small- and medium-vessel vasculitis[1].However,the etiology of AAV remained unclear. Several observations have showed that the breakdown of immune tolerance caused by many complex interactions was involved in the pathogenesis of AAV[2].It has been confirmed that the disorder of the CD4+T cell,especially the imbalance of Th17 and Treg cells can destroy the immune tolerance and cause many autoimmune disease[3]. But the relationship between the Th17/Treg and AAV is unknown.Objectives:We investigated the absolute numbers of CD4+T subsets cells in peripheral blood of patients with AAV and healthy adults,and then compared them in different disease activity of AAV to explore the role of CD4+T subsets cells in the pathogenesis and development of AAV.Methods:49 patients with AAV,hospitalized at the Second Hospital of Shanxi Medical University from the May 2016 to the November 2019 were enrolled, and 31 age and gender-matched healthy adults were anticipated as controls.According to BVAS, the patients were divided into disease-activity group (BVAS≥15, n=27) and non-disease-activity group (BVAS<15, n=22). The absolute numbers of CD4+T subsets cells including Th17 and Tregs in peripheral blood of these individuals were detected by flow cytometry.We analyzed whether there was difference of CD4+T subsets between the patients and healthy controls,and between disease-activity group and non-disease-activity group.Results:There was significant decreased level of Treg cells in the patients with AAV compared with healthy controls,especially in the disease-activity group. The absolute numbers of Treg cells was decreased in the patients with AAV compared with healthy controls (P<0.001) leading to a higher Th17/Treg ratio in the patients (P<0.01).Similarly,the absolute number of Treg cells was decreased in the disease- activity group (P<0.01) compared with the non-disease-activity group, and the absolute number of Treg cells was significant negative correlation with the disease activity indexes such as BVAS (r=-0.342,P=0.016), erythrocyte sedimentation rate(ESR) (r=-0.315,P=0.027) and C-reactive protein(CRP) (r=-0.305,P=0.033). But there was no statistically significant in the absolute number of Th17 cells between the patients and healthy controls, and between disease-activity group and non-disease-activity group.Conclusion:The results we investigated here suggested that the decreased number of Treg cells failed to control autoimmune inflammatory response and maintain immune tolerance, and the disease activity of AAV was associated with the reduced number of Treg cells.Figure 1.(A-C) Characteristics of the absolute number of Th17 cells and Treg cells in peripheral blood of healthy controls (n=31) and the patients with AAV (n=49). There was significant decreased level of Treg cells in the patients with AAV compared with healthy controls leading to a higher Th17/Treg ratio in the patients with AAV. (D-F) The absolute number of Treg cells was decreased in the disease- activity group (n=27) compared with the non-disease-activity group (n=21). The absolute number of Th17 cells and Treg cells was detected by flow cytometry. Statistical analyses were performed by the Mann-Whitney U test. *p<0.05,**p<0.01, ***p<0.001.

P0532CD8+CD103+ INDUCED REGULATORY T CELLS BUT NOT NATURE REGULATORY T CELLS AMELIORATE IR-INDUCED KIDNEY INJURY

Abstract Background and Aims Acute kidney injury (AKI) is one of the most common complications in clinical practice, but current approaches offer no cures for AKI. Inflammatory response is the key mechanism of ischemia reperfusion (IR)-induce AKI, known as hypoxia/reoxygenation injury. T lmphocytes are crucial mediators of IR-induced AKI. It is reported that regulatory T cell (Treg) has potential ability to ameliorate IR-induced AKI. Tregs consist of nature Treg (nTreg) and induced Treg (iTreg), which are well established to blunt immune responses. We confirmed that CD8+CD103+iTreg remain steadily and barely transfer into Th17, which produce proinflammatory response, under inflammatory response. It is studied that nTreg tends to transfer into Th17 under inflammatory condition, which clarifies the instability of nTreg. Thus, the instability of nTreg under inflammatory response hints us that nTreg are not suitable for recovery from IR-induced AKI. Accordingly, we studied the role of CD8+CD103+iTreg in repair after IR-induced kidney. Method In vitro study, we test the expression of CD103, Foxp3 and IL-17a in CD8+CD103+iTreg under 1% O2 concentration. In vivo study, SPF C57BL/6J male mice (8-10 weeks old, body weight 20-25g) were divided into four groups, sham-operated control group, AKI group, iTreg treatment group and nTreg treatment group, with 6 mice in each group. On the day of surgery, we anesthetize the mice (pen-tobarbital sodium, 50 mg/kg body weight). Mice in the AKI group were reperfused after 25 minutes of bilateral renal artery ischemia. We injected CD8+CD103+iTreg (2 × 106/mouse) or nTreg (2 × 106/mouse) intraperitoneally after 24 hours of modeling to the treatment group and euthanized the mice on the third day of anesthesia. Results We discovered that the expression of CD103 is stable in CD8+iTreg in vitro under 1% O2 concentration (Figure 1, 2). In addition, their inhibiting abilities of T cells proliferation in vitro remain steadily (Figure not shown). However, CD8+CD103+iTreg seldom transfer into Th17, but remain Foxp3 under hypoxic condition (Figure 2). Adoptively transferring CD8+CD103+iTregs, a new subpopulation of CD8+iTreg we have identified, to AKI mice, we found that these cells can ameliorate the development of AKI by mitigating the level of serum creatine, alleviating acute tubular necrosis (ATN) and decreasing the mortality of AKI. Conclusion Therefore, we confirmed that CD8+CD103+iTreg is stable under inflammatory(hypoxic) environment. Thus, CD8+CD103+iTreg targeting may be a novel therapeutic approach to enhance recovery from IR-induced AKI.

Influences of CD8+ Tregs on Peripheral Blood Mononuclear Cells from Allergic Rhinitis Patients.

CD8+ (or CD4+ ) CD25+ fork-head box transcription factor (Foxp3)+ regulatory T cells (CD8+ or CD4+ Tregs) all play a significant role in immune homeostasis and tolerance. However, the role of CD8+ Tregs in allergic rhinitis (AR) have not been clearly elucidated. The present study was aimed to assess the influence of CD8+ Tregs on peripheral blood mononuclear cells (PBMCs) from AR patients. Prospective cross-sectional study. Patients with AR were enrolled. PBMCs were obtained, and CD4+ and CD8+ Tregs were separated from PBMCs and cultured in vitro. We examined percentages of these Tregs in total CD4+ or CD8+ T cells, respectively. After that, we evaluated levels of interleukin (IL)-10 and transforming growth factor (TGF)-β in Tregs cultures. Finally, we administered CD4+ and CD8+ Tregs from AR patients into PBMCs cultures and examined contents of IL-4 and IL-5. The percentages of CD4+ or CD8+ Tregs in the total CD4+ or CD8+ T cells from PBMCs in AR patients were reduced compared to normal subjects. However, IL-10 and TGF-β and their mRNAs were increased in CD4+ and CD8+ Tregs cultures from AR patients, and there were no significant differences in their levels between these two Tregs cultures. IL-4 and IL-5 were increased in AR subjects' PBMCs compared to normal ones and decreased after the AR CD4+ or CD8+ Tregs administration. However, there were no statistical differences in IL-4 and IL-5 concentrations between these two Tregs treatments. The findings demonstrate that CD8+ Tregs may alleviate inflammatory responses in AR condition. 3 Laryngoscope, 131:E316-E323, 2021.

Role of CD8+ T-cell infiltration in breast tumors of Black women compared to White women.

e13608 Background: Literature regarding racial differences in tumor immune responses in breast cancer remains sparse. To address this research gap, we assessed CD8+ T-cells in breast tumor samples from the Women’s Circle of Health Study (WCHS) population of predominantly Black breast cancer patients. We characterized and compared the density of CD8+ T cells, their prognostic value and their association with pharmacologic beta-blockers. Prior studies suggest that reducing adrenergic signaling through beta blockers can stimulate CD8+ T-cells. Methods: Tumor-infiltrating CD8+ T-cells were assessed by IHC staining of tissue microarray cores from 688 breast cancer patients, including 550 Blacks and 138 Whites. CD8+ T cells were scored with digital image analysis. Comparisons of demographic and clinical variables (including CD8+ T cell density) were made using the Mann-Whitney U or Kruskal-Wallis and Fisher’s exact tests. Associations with overall survival (OS) and disease-specific (DSS) survival were evaluated using the log-rank test of Cox regression. Analyses were performed in the overall sample and by disease sub-type. Results: Higher CD8+ T cell density was seen in Black women compared to White, with mean values of 756.2/mm2 and 292.4/mm2 respectively (p < 0.001). Within the overall population and in black women, CD8+ T cell density was significantly higher in younger patients, patients with high grade, and ER negative tumors. No significant associations were observed between CD8+ T cell density and OS or DSS. However, when stratified by subtype, Black patients with triple negative breast cancer and high CD8+ T cell density showed a trend towards improved OS in comparison to patients with low CD8+ T cell density (p = 0.065). 170 patients with information on beta blocker usage were analyzed. No significant associations were noted between CD8+ T cell density and beta blocker use. Conclusions: We observe a significantly higher CD8+ T cell density in Blacks compared to Whites, but this does not confer a survival advantage. Additionally, beta-blockers did not seem to enhance CD8+ T cell infiltration in tumors from Black patients. Our data raise the possibility that distinct mechanisms of immune cell action may occur in different racial groups. Future studies are needed to determine the functional properties of CD8+ T cells in Black women and to characterize additional immune cell subtypes that may also play a role.

Pivotal role of CD103 in the development of psoriasiform dermatitis

The integrin αE known as CD103 binds integrin β7 to form the complete heterodimeric integrin molecule αEβ7. CD103 is mainly expressed by lymphocytes within epithelial tissues of intestine, lung, and skin as well as subsets of mucosal and dermal conventional dendritic cells (cDCs). CD103 has been originally implicated in the attachment of lymphocytes to epithelium in the gut and skin through the interaction with E-cadherin expressed on intestinal epithelial cells, keratinocytes, and Langerhans cells (LCs). However, an impact of CD103 on the cutaneous immune responses and the development of inflammatory skin diseases remains elusive. Here, we report that CD103 regulates the development of psoriasiform dermatitis through the control of the function of cDCs. Deficiency in CD103 exacerbates psoriasiform dermatitis, accompanied by excessive epidermal hyperplasia and infiltration of inflammatory leukocytes. Furthermore, deficiency in CD103 not only accelerates the production of proinflammatory cytokines in psoriatic lesions but also promotes the generation of lymphocytes producing interleukin (IL)-17 in the skin-draining peripheral lymph nodes (PLNs). Under the deficiency in CD103, cDCs localized in PLNs enhance cytokine production following activation. Thus, our findings reveal a pivotal role for CD103 in the control of the function of cDCs to regulate cutaneous inflammation in psoriasiform dermatitis.

Teriflunomide inhibits activation-induced CD25 expression on T cells and may affect Foxp3-expressing regulatory T cells

Teriflunomide (TER) is an immunomodulatory agent. Although the first reports on the use of TER in dogs have already appeared, immune mechanisms underlying the immunomodulatory effect of TER do not seem to have been fully elucidated yet. There were two aspects of this study. First, further insight into the mode of action of TER was gained by investigating its effect on the expression of IL-2 receptor α-chain (CD25) and Forkhead box P3 (Foxp3) by CD4+ and CD8+ T cells and apoptosis of these cells. Second, in view in the earlier lack of data on the effect of TER on T cells in dogs, the results of this study filled in this gap. TER at a concentration which can be achieved in vivo prevented or reduced the activation-induced CD25 expression on CD4+ and CD8+ T cells, respectively. Taking into consideration the role of CD25 in T cell proliferation, this effect may constitute an additional mechanism responsible for the antiproliferative effect of the drug. Under stimulation conditions, TER induced Foxp3 expression in Foxp3-negative CD4+ and CD8+ T cells, while down-regulating it under unstimulated conditions. These results suggest that TER may generate iTreg cells, but this process requires cell activation. TER was not found to affect on the absolute count and apoptosis of CD4+ and CD8+ T cells. The results suggest that the impairment of CD25 expression during T cell activation and generation of iTreg cells may constitute additional mechanisms, besides the principal one, underlying the immunomodulatory effect of TER.