Different Subsets Of CD4 Research Articles

Immunology of amyotrophic lateral sclerosis - role of the innate and adaptive immunity.

This review aims to summarize the latest evidence about the role of innate and adaptive immunity in Amyotrophic Lateral Sclerosis (ALS). ALS is a devastating neurodegenerative disease affecting upper and lower motor neurons, which involves essential cells of the immune system that play a basic role in innate or adaptive immunity, that can be neurotoxic or neuroprotective for neurons. However, distinguishing between the sole neurotoxic or neuroprotective function of certain cells such as astrocytes can be challenging due to intricate nature of these cells, the complexity of the microenvironment and the contextual factors. In this review, in regard to innate immunity we focus on the involvement of monocytes/macrophages, microglia, the complement, NK cells, neutrophils, mast cells, and astrocytes, while regarding adaptive immunity, in addition to humoral immunity the most important features and roles of T and B cells are highlighted, specifically different subsets of CD4+ as well as CD8+ T cells. The role of autoantibodies and cytokines is also discussed in distinct sections of this review.

Murine cytotoxic CD4+ T cells in the tumor microenvironment are at a hyper-maturation stage of Th1 CD4+ T cells sustained by IL-12.

The roles of tumor-infiltrating CD4+Foxp3- T cells are not well characterized due to their plasticity of differentiation, and varying levels of activation or exhaustion. To further clarify this issue, we used a model featuring subcutaneous murine colon cancer and analyzed the dynamic changes of phenotype and function of the tumor-associated CD4+ T-cell response. We found that, even at a late stage of tumor growth, the tumor-infiltrating CD4+Foxp3- T cells still expressed effector molecules, inflammatory cytokines and molecules that are expressed at reduced levels in exhausted cells. We used microarrays to examine the gene-expression profiles of different subsets of CD4+ T cells and revealed that the tumor-infiltrating CD4+Foxp3- T cells expressed not only type 1 helper (Th1) cytokines, but also cytolytic granules such as those encoded by Gzmb and Prf1. In contrast to CD4+ regulatory T cells, these cells exclusively co-expressed natural killer receptor markers and cytolytic molecules as shown by flow-cytometry studies. We used an ex vivo killing assay and proved that they could directly suppress CT26 tumor cells through granzyme B and perforin. Finally, we used pathway analysis and ex vivo stimulation to confirm that the CD4+Foxp3- T cells expressed higher levels of IL12rb1 genes and were activated by the IL-12/IL-27 pathway. In conclusion, this work finds that, in late-stage tumors, the tumor-infiltrating lymphocyte population of CD4+ cells harbored a sustained, hyper-maturated Th1 status with cytotoxic function supported by IL-12.

CD4+ T cell activation and inflammation in NASH-related fibrosis.

Liver fibrosis is a common pathological feature of end stage liver failure, a severe life-threatening disease worldwide. Nonalcoholic fatty liver disease (NAFLD), especially its more severe form with steatohepatitis (NASH), results from obesity, type 2 diabetes and metabolic syndrome and becomes a leading cause of liver fibrosis. Genetic factor, lipid overload/toxicity, oxidative stress and inflammation have all been implicated in the development and progression of NASH. Both innate immune response and adaptive immunity contribute to NASH-associated inflammation. Innate immunity may cause inflammation and subsequently fibrosis via danger-associated molecular patterns. Increasing evidence indicates that T cell-mediated adaptive immunity also provokes inflammation and fibrosis in NASH via cytotoxicity, cytokines and other proinflammatory and profibrotic mediators. Recently, the single-cell transcriptome profiling has revealed that the populations of CD4+ T cells, CD8+ T cells, γδ T cells, and TEMs are expanded in the liver with NASH. The activation of T cells requires antigen presentation from professional antigen-presenting cells (APCs), including macrophages, dendritic cells, and B-cells. However, since hepatocytes express MHCII molecules and costimulators, they may also act as an atypical APC to promote T cell activation. Additionally, the phenotypic switch of hepatocytes to proinflammatory cells in NASH contributes to the development of inflammation. In this review, we focus on T cells and in particular CD4+ T cells and discuss the role of different subsets of CD4+ T cells including Th1, Th2, Th17, Th22, and Treg in NASH-related liver inflammation and fibrosis.

T Cell Recognition of Disulfide Modified Antigens

Abstract Disulfide formation is one of the most common post-translational modifications and plays important in protein structures and functions. We discovered that the disulfide bridge was the epitope of a highly diabetogenic CD4 T cells. This is a shift from the paradigm that T cells commonly recognize the linear peptide antigens and showed a novel to create neo-epitopes. We identified in vivo that the different subsets of CD4 T cells could be stimulated by both reduced and oxidized form of the same antigens, IAPP. This suggested an imbalance in redox homeostasis may be important for T cell mediated pathogenesis. A disulfide bridge specific monoclonal antibody was created and showed extraordinary ability in preventing type 1 diabetes. The disulfide bridge specific T cells were able to be generalized another protein, CGRP. The lysosomal thiol reductase knock-out experiments showed that the reductase may be important for disulfide modified antigen generation during antigen processing. Elevating GSH levels through glutamate cysteine ligase pathway inhibited CD4 T cell response in a dose dependent manner. Although NOX attributed to the importance of hydrogen peroxide production in pancreatic beta cells, a Nox inhibitor effectively attenuated the BDC5.2.9 T cell activation. Our results shed light on how thiol-disulfide exchange reaction impacts the T cell responses. Supported by NIH R56 AI153488 NIH R21 AI149655

Bystander CD4+ T cells infiltrate human tumors and are phenotypically distinct

ABSTRACT Tumor-specific T cells likely underpin effective immune checkpoint-blockade therapies. Yet, most studies focus on Treg cells and CD8+ tumor-infiltrating lymphocytes (TILs). Here, we study CD4+ TILs in human lung and colorectal cancers and observe that non-Treg CD4+ TILs average more than 70% of total CD4+ TILs in both cancer types. Leveraging high dimensional analyses including mass cytometry, we reveal that CD4+ TILs are phenotypically heterogeneous, within each tumor and across patients. Consistently, we find different subsets of CD4+ TILs showing characteristics of effectors, tissue resident memory (Trm) or exhausted cells (expressing PD-1, CTLA-4 and CD39). In both cancer types, the frequencies of CD39– non-Treg CD4+ TILs strongly correlate with frequencies of CD39– CD8+ TILs, which we and others have previously shown to be enriched for cells specific for cancer-unrelated antigens (bystanders). Ex-vivo, we demonstrate that CD39– CD4+ TILs can be specific for cancer-unrelated antigens, such as HCMV epitopes. Overall, our findings highlight that CD4+ TILs can also recognize cancer-unrelated antigens and suggest measuring CD39 expression as a straightforward way to quantify or isolate bystander CD4+ T cells.

Programmed Cell Death Ligand (PD-L)-1 Contributes to the Regulation of CD4+ T Effector and Regulatory T Cells in Cutaneous Leishmaniasis.

Cutaneous Leishmaniasis (CL) affects up to one million people every year and treatments are costly and toxic. The regulation of the host immune response is complex and the knowledge of how CD4+ T cells are activated and maintained during Leishmania infection is still limited. Current therapies aim to target programmed cell death (PD)-1 and programmed cell death ligand (PD-L)-1 in order to boost T cell activity. However, the role of the PD-1/PD-L1 axis during Leishmania infection is still unclear. In this study, we found that patients with active and post-treatment CL displayed different subsets of CD4+PD-1+ T cells. Accordingly, L. major-infected mice upregulated PD-1 on activated CD4+ T effector cells and PD-L1 on resident macrophages and infiltrating monocytes at the site of infection. L. major-infected Pdl1−/− mice expressed lower levels of MHCII and higher levels of CD206 on macrophages and monocytes and, more importantly, the lack of PD-L1 contributed to a reduced frequency of CD4+Ly6Chi T effector cells and an increase of CD4+Foxp3+ regulatory T cells at the site of infection and in draining lymph nodes. Additionally, the lack of PD-L1 was associated with lower production of IL-27 by infiltrating monocytes and lower levels of the Th1 cytokines IFN-γ and TNF-α produced by CD4+ T effector cells. Pdl1−/− mice initially exhibited larger lesions despite having a similar parasite load. Our results describe for the first time how the interruption of the PD-1/PD-L1 axis influences the immune response against CL and suggests that this axis regulates the balance between CD4+Ly6Chi T effector cells and CD4+Foxp3+ regulatory T cells.

Abstract 4554: Functionally distinct T cell subsets contribute to ImmTAC-mediated anti-tumor response

Abstract Introduction: Bispecific T cell engagers hold promise for effective cancer treatment. The anti-tumor activity of these therapeutics molecules predominantly relies on their capacity to induce direct killing of tumor cells by engaging the cytotoxic machinery of polyclonal CD8+ T cells. However, the impact of additional functionally different T cell populations that can be engaged alongside CD8+ T cells remains unclear. ImmTAC molecules are a novel class of bispecific fusion proteins that use an affinity-enhanced monoclonal TCR to target tumor antigen-derived peptides presented by HLA, and an anti-CD3 monomeric antibody to engage T cells. Tebentafusp, our lead clinical candidate, targets a gp100-derived peptide and is under clinical investigation in metastatic melanoma. Preliminary data revealed T cell activation, trafficking and infiltration into the tumor bed in patients responding to treatment. Importantly not only CD8+ T cells, but also different subsets of CD4+ T cells appeared to be activated, mobilized and redirected to the tumor site following tebentafusp treatment, suggesting a potential contribution of these T cell subsets to the anti-tumor response. The aim of this study was to investigate in vitro the functional capacity of different T cell populations, besides CD8+ T cells, following ImmTAC-mediated redirection toward cancer cells. Methods: Th1, Th2, Th17 CD4+ T cells, γδ T cells and Mucosal-Associated Invariant T cells (MAIT) were isolated from healthy volunteers and their functional response following exposure to ImmTAC in the presence of target cells was analysed. Results: In addition to CD8+ T cells, other T cell populations, were activated in vitro by ImmTAC and elicited a range of effector functions that may differentially contribute to anti-tumor responses. For example, we found that γδ T cells were able to mount rapid and potent cytokine and cytotoxic responses against cancer cells upon redirection with ImmTAC. The mechanisms that underpin our in vitro observations and the potential correlations in patients treated with tebentafusp are under investigation. Conclusions: Multiple T cells populations display functionally distinct responses following ImmTAC-mediated redirection. These results will add valuable insight to our understanding of bispecific T cell engager mechanism of action. In addition, they will help guide rationale for improving the design of these therapeutic molecules and select appropriate combination strategies. Citation Format: Adel Benlahrech, David K. Cole, Christopher J. Holland, Rupert Kenefeck, Rahul C. Khanolkar, Cheryl McAlpine, Sion Lewis, Angela Valerio-Fernandes, Mariantonella Vardeu, David Berman, Marco Lepore. Functionally distinct T cell subsets contribute to ImmTAC-mediated anti-tumor response [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 4554.

Frequencies of Regulatory Subsets of CD4+ TH Cells in Peripheral Blood in Mycobacterium Tuberculosis-Infected Individuals and Healthy Contacts in a High-Burden Setting from Assam, Northeast India

AbstractBackground:Mycobacterium tuberculosis (Mtb) adapts many strategies to persist and replicate inside human tissue. One such strategy is the manipulation of CD4+ TH cells for subset interconversion to regulatory subsets. The aim of the present study is to get an insight of dynamic changes of CD4+ TH cells to regulatory subsets, CD4+ CD25+ forkhead box P3 (Foxp3)+ T-cells and CD4+ CD25+ Foxp3+ programmed death molecule-1 (Foxp3+) T-cells, in peripheral blood in Mtb-infected individuals and healthy contacts in a high-burden setting from Assam, Northeast India. Materials and Methods: A case–control study was conducted in newly diagnosed active pulmonary tuberculosis (APTBs) patients and 2 sets of controls: (i) individuals infected with latent tuberculosis infection (LTBI) and (ii) healthy close tuberculosis healthy contacts (HCs). The frequencies of different subsets of CD4+ cells with regulatory markers were measured in peripheral blood in 3 groups of study participants. Results and Observations: Frequencies of CD4+ CD25+ Foxp3+ T-cells (1.84 ± 1.40 vs. 4.32 ± 1.82 vs. 11.30 ± 3.66), CD4+ CD25+ Foxp3+ PD1+ T-cells (0.37 ± 1.28 vs. 2.99 ± 3.69 vs. 14.54 ± 5.10) and ligand (PD-L1)-positive CD4+ TH cells (0.80 ± 0.45 vs. 2.28 ± 0.95 vs. 7.13 ± 2.02) were significantly increased from HCs to LTBIs to APTB patients, respectively (P < 0.0001). No significant changes in frequencies of total CD4+ cells were observed between APTBs (29.51 ± 11.93), LTBIs (29.23 ± 8.16) and HCs (28.16 ± 9.73) whereas the mean ratios of CD4+ to CD4+ CD25+ FoxP3+ were significantly decreased from 34.34 ± 47.56 in HCs to 7.96 ± 5.8 in LTBIs to 3.12 ± 2.58 in APTBs (P < 0.0001). Significant decrease in mean ratios of CD4+ CD25+ FoxP3+ to CD4+ CD25+ FoxP3+ PD1+ were also observed from 4.97 ± 1.09 in HCs to 1.44 ± 0.49 in LTBIs to 0.78 ± 0.72 in APTBs. Conclusion: CD4+ TH cells change dynamically to regulatory subsets depending on the status of infection and a shift of response towards excessive regulatory T-cells, and PD-1/PD-L1 production may help in the development of active infection in latently infected individuals. These immunological parameters may be used, as potential biomarkers to see the changing dynamics of Mtb infection.

Abstract 3235: Activation-inducible TNFR family receptor (AITR) signaling mediates the polarization of CD4+ T cells into Th1, Th2, and Th17, converts Treg to Teff and eradicates solid tumors

Abstract AITR (activation-inducible TNFR family receptor, human GITR), an inducible costimulatory receptor of human T cells, is expressed constitutively on Treg and inducible on Teff upon antigen engagement. AITR co-stimulates T cell activation and recruits certain members of TRAF family upon interaction with its ligand. We examined whether the stimulation of distinct regions of extracellular domain of AITR can generate signals that promote different subsets of CD4+ T cells. We found that crosslinking AITR by agonistic anti-AITR Abs (A27, A41, and A35), that recognize three distinct regions of the extracellular domain, polarize CD4+ T cells into Th1, Th2 and Th17, respectively. The subset polarization evoked by these antibodies was the result of recruitment of specific TRAFs, phosphorylation of distinct STATs and expression of Th subset fate-determining master regulatory genes. Stimulation with A27 recruited TRAF-1 and -2, activated STAT-1, -4 and JNK1/2, and induced transcription factor T-bet. On the other hand, activation with A35 recruited TRAF-6, activated STAT-3 and p38 MAPK, and induced RORγt. In contrast, A41 recruited TRAF-3 and -5, activated STAT-5, -6 and ERK1/2, and produced GATA-3. Remarkably, A27 induced the conversion of CD25+Foxp3+ Treg cells to IFN-γ-producing Th1 cells with a concomitant suppression of Foxp3 and TGF-β expression both in healthy individuals as well as cancer patients. A35 also, but not A41, suppressed the expression of Foxp3 and induced IL-17 in Treg cells. A27 alone, but not others produced a strong anti-tumor effect in a human tumor xenograft model of humanized mice in an IFN-γ- and Treg conversion-dependent mechanism. Taken together, the data that AITR regulates Th subset polarization and eradicates solid tumors can be a target for broad-spectrum immune check point therapeutics against cancers and autoimmune diseases. Citation Format: Byoung S. Kwon, Seung J. Lee, Young H. Kim, Joong W. Lee, Sun H. Hwang, Dass S. Vinay. Activation-inducible TNFR family receptor (AITR) signaling mediates the polarization of CD4+ T cells into Th1, Th2, and Th17, converts Treg to Teff and eradicates solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3235.

Evidence for Innate and Adaptive Immune Responses in a Cohort of Intractable Pediatric Epilepsy Surgery Patients.

Brain-infiltrating lymphocytes (BILs) were isolated from resected brain tissue from 10 pediatric epilepsy patients who had undergone surgery for Hemimegalencephaly (HME) (n = 1), Tuberous sclerosis complex (TSC) (n = 2), Focal cortical dysplasia (FCD) (n = 4), and Rasmussen encephalitis (RE) (n = 3). Peripheral blood mononuclear cells (PBMCs) were also isolated from blood collected at the time of the surgery. Cells were immunostained with a panel of 20 antibody markers, and analyzed by mass cytometry. To identify and quantify the immune cell types in the samples, an unbiased clustering method was applied to the entire data set. More than 85 percent of the CD45+ cells isolated from resected RE brain tissue comprised T cells; by contrast NK cells and myeloid cells constituted 80–95 percent of the CD45+ cells isolated from the TSC and the FCD brain specimens. Three populations of myeloid cells made up >50 percent of all of the myeloid cells in all of the samples of which a population of HLA-DR+ CD11b+ CD4− cells comprised the vast majority of myeloid cells in the BIL fractions from the FCD and TSC cases. CD45RA+ HLA-DR− CD11b+ CD16+ NK cells constituted the major population of NK cells in the blood from all of the cases. This subset also comprised the majority of NK cells in BILs from the resected RE and HME brain tissue, whereas NK cells defined as CD45RA− HLA-DR+ CD11b− CD16− cells comprised 86–96 percent of the NK cells isolated from the FCD and TSC brain tissue. Thirteen different subsets of CD4 and CD8 αβ T cells and γδ T cells accounted for over 80% of the CD3+ T cells in all of the BIL and PBMC samples. At least 90 percent of the T cells in the RE BILs, 80 percent of the T cells in the HME BILs and 40–66 percent in the TSC and FCD BILs comprised activated antigen-experienced (CD45RO+ HLA-DR+ CD69+) T cells. We conclude that even in cases where there is no evidence for an infection or an immune disorder, activated peripheral immune cells may be present in epileptogenic areas of the brain, possibly in response to seizure-driven brain inflammation.

The role of Th9 and other IL-9 producing cells in allergic asthma

The incidence of allergic asthma has been increasing year by year. It is estimated that there are more than 300 million illnesses. It is associated with increased IgE, mast cell activation, airway hyperreactivity, excessive mucus production, and airway remodeling. The traditional view is that allergic asthma is associated with allergic reactions involving T helper type 2 (Th2) cells. Recent studies have found that different subsets of CD4+ T cells (Th17, Th9) as well as innately immunized cells such as mast cells and innate lymphocyte type 2 (ILC2s) are able to produce cytokine IL-9, leading to asthma. Th9 cells develop from nave T cells to IL-9 producing cells in the presence of IL-4 and TGF-β. IRF4, GATA3, a basic leucine zipper transcription factor, several transcription factors downstream of BATF and IL-4 such as signal transduction and STAT6-activated NFAT are activated. In addition, the transcription factor PU.1 downstream of TGF-β signaling appears to be also critical for the development of Th9 cells. IL-9 is a pleiotropic cytokine that affects the function of different target cells such as T cells, B cells, mast cells and airway epithelial cells by activating STAT1, STAT3 and STAT5. Due to its versatility, IL-9 has been shown to be involved in several diseases such as cancer, autoimmunity and other pathogenmediated immunomodulatory diseases. This article reviews the role of Th9 and IL-9producing cells in allergic asthma.

Regulation of lymphocyte trafficking in central nervous system autoimmunity.

CD4+ T helper (Th) cells play a central role in orchestrating protective immunity but also in autoimmunity. Multiple Sclerosis (MS) is a human autoimmune disease of the central nervous system (CNS) characterized by the infiltration of inflammatory lymphocytes and myeloid cells into the brain and spinal cord, leading to demyelination, axonal damage, and progressive loss of motor functions. The release of T cells in the circulation and their migration in the central nervous system are key and tightly regulated processes which have been targeted to decrease CD4+ T cell presence in the CNS and limit disease progression. Here, we review two of these pathways and discuss how their blockade modulate different subsets of CD4+ T cells.

Circulating Th1, Th2, Th9, Th17, Th22, and Treg Levels in Aortic Dissection Patients.

Background Previous studies demonstrated that the subsets of CD4+ T helper (Th) cells are closely related to vascular diseases, including atherosclerosis and hypertension. This study is aimed at investigating the circulating Th1, Th2, Th9, Th17, Th22, and Treg levels in aortic dissection (AD) patients. Methods Blood samples from AD (n = 56) and non-AD (NAD, n = 24) patients were collected, and the circulating levels of Th1, Th2, Th9, Th17, Th22, and Treg cells and their transcription factors and functional cytokines were measured by flow cytometric analysis, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assays, respectively. In addition, the human aortic vascular smooth muscle cells (HASMCs) were treated with saline, angiotensin II (Ang II), or plasma from AD patients. Results Compared with the levels in the NAD group, the Th1, Th9, Th17, Th22, and their transcription factor levels were increased and the Th2, Treg, and their transcription factor levels exhibited a decreasing trend in AD patients. In addition, higher IFN-γ, IL-9, IL-17, and IL-22 levels and lower IL-4 and IL-35 levels were observed in AD patients. Simple linear regression analysis and binary logistic regression analysis suggested that Th1/IFN-γ, IL-9, Th17/IL-17, and Th22/IL-22 positively regulated the occurrence of AD, while Th2/IL-4 and Treg/IL-35 negatively regulated the occurrence of AD. Plasma from AD patients further increased Bax mRNA levels but decreased Bcl2 and α-SMA mRNA levels in Ang II-treated HASMCs. Conclusions Changes in Th1, Th2, Th9, Th17, Th22, and Treg activity are associated with the onset of AD. Different subsets of CD4+ T cells play different roles in the presence of AD.

Higher levels of serum interleukin-35 are associated with the severity of pulmonary fibrosis and Th2 responses in patients with systemic sclerosis.

The aim of the study is to investigate the levels of serum interleukin-35 (IL-35) before and after treatment and its relationship with clinical parameters as well as the frequency of different subsets of CD4+ T cells in patients with systemic sclerosis (SSc) to explore the role of IL-35 in the pathogenesis of SSc. The levels of serum IL-35, interferon-γ (IFN-γ), IL-4, IL-17A, and IL-10 in 49 patients with SSc and 50 age- and gender-matched healthy subjects were measured by enzyme-linked immunosorbent assay (ELISA). The percentages of peripheral blood Th1, Th2, Th17 cells and Tregs in 49 SSc patients and 20 healthy controls were characterized by flow cytometry. The relationship between the levels of serum IL-35 and the percentages of different subsets of CD4+ T cells, disease duration, the values of forced vital capacity (FVC), modified Rodnan skin scores (MRSS) or high-resolution computed tomography (HRCT) scores was analyzed in patients with SSc. The levels of serum IL-35 in SSc patients were significantly higher than that of healthy controls (P < 0.001), but significantly reduced after treatment for 3months (P = 0.001). The levels of serum IL-35 were positively correlated with the HRCT scores in SSc patients (P = 0.014) and with disease duration in patients with diffuse cutaneous SSc (P = 0.03), but negatively correlated with the FVC values in SSc patients (P = 0.034). In comparison with that in the healthy controls, significantly decreased percentages of Th1, but increased Th2 and Th17 cells were detected in patients, leading to reduced ratios of Th1/Th2 and increased ratios of Th17/Tregs (P < 0.001). The levels of serum IL-35 were positively correlated with IL-4 (P = 0.036) and tended to be positively associated with the frequency of Tregs in SSc patients (P = 0.054). Higher levels of serum IL-35 may be associated with the development of SSc and severity of pulmonary fibrosis in SSc patients.

A Novel Single-Cell FISH-Flow Assay Identifies Effector Memory CD4+ T cells as a Major Niche for HIV-1 Transcription in HIV-Infected Patients.

ABSTRACTCells that actively transcribe HIV-1 have been defined as the “active viral reservoir” in HIV-infected individuals. However, important technical limitations have precluded the characterization of this specific viral reservoir during both treated and untreated HIV-1 infections. Here, we used a novel single-cell RNA fluorescence in situ hybridization-flow cytometry (FISH-flow) assay that requires only 15 million unfractionated peripheral blood mononuclear cells (PBMCs) to characterize the specific cell subpopulations that transcribe HIV RNA in different subsets of CD4+ T cells. In samples from treated and untreated HIV-infected patients, effector memory CD4+ T cells were the main cell population supporting HIV RNA transcription. The number of cells expressing HIV correlated with the plasma viral load, intracellular HIV RNA, and proviral DNA quantified by conventional methods and inversely correlated with the CD4+ T cell count and the CD4/CD8 ratio. We also found that after ex vivo infection of unstimulated PBMCs, HIV-infected T cells upregulated the expression of CD32. In addition, this new methodology detected increased numbers of primary cells expressing viral transcripts and proteins after ex vivo viral reactivation with latency reversal agents. This RNA FISH-flow technique allows the identification of the specific cell subpopulations that support viral transcription in HIV-1-infected individuals and has the potential to provide important information on the mechanisms of viral pathogenesis, HIV persistence, and viral reactivation.

Abstract 4633: Crosstalk between MDSCs and Th17

Abstract Expansion of myeloid-derived suppressor cells (MDSCs) within the tumor is a known barrier to successful cancer immunotherapy. MDSCs are heterogeneous population of immature myeloid cells with potent immunosuppressive activity. Emerging evidences in the recent years define the role of immunosuppressive activity of MDSCs on different subsets of CD4+ T cells, however the exact effect of MDSCs on inflammatory Th17 cells during tumor progression is not clear. Here we demonstrate the immunosuppressive effect of MDSCs on Th17 cells. We further show that the interaction between MDSCs and Th17 is not unilateral and reveal that Th17 cells can promote the immunosuppressive effect of MDSCs. MDSCs, either isolated from tumor bearing mice or generated in vitro from bone marrow (BM-derived MDSCs), can both suppress IL-17A expression. Our data also demonstrate that IFNg, produced by Th17 cells is essential for the immunosuppressive activity of BM-derived MDSCs on IL-17A expression and further that the IFNgR1-driven signaling is important for Nos2-mediated immunosuppressive effect of BM-derived MDSCs on IL-17A expression. Understanding the molecular mechanism of the interplay between MDSCs and Th17 cells may help to develop more specific targeted therapy for cancer immunotherapy and improve therapeutic efficacy of the existing therapies against cancer. Note: This abstract was not presented at the meeting. Citation Format: Brijendra Singh. Crosstalk between MDSCs and Th17 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4633. doi:10.1158/1538-7445.AM2017-4633

LPS-treated bone marrow-derived dendritic cells induce immune tolerance through modulating differentiation of CD4+ regulatory T cell subpopulations mediated by 3G11 and CD127.

Intravenous transfer of LPS-treated bone marrow-derived dendritic cells blocks development of autoimmunity induced by CD4+ T cells in vivo. However, cellular mechanisms of dendritic cell-mediated immune tolerance have not yet been fully elucidated. Here, we report that there are two new subpopulations of CD4+CD25+FoxP3+GITR+ regulatory T cells (CD127+3G11+ and CD127+3G11- cells). LPS-treated dendritic cells facilitate development of CD4+CD127+3G11- regulatory T cells but inhibit that of CD4+CD127+3G11+ regulatory T cells. LPS-induced tolerogenic dendritic cells may cause immune tolerance through modulating balance of different subsets of CD4+ regulatory T cells mediated by CD127 and 3G11. Our results imply a new potential cellular mechanism of dendritic cell-mediated immune tolerance.

In Virologically Suppressed HIV+ Patients, High CD4/CD8 Ratio and Length of Treatment Independently Correlate to the Intracellular Content of Proviral HIV-DNA in Different Types of CD4+ T Cells

Introduction:Potent antiretroviral drugs block the progression of HIV infection and inhibit virus integration into the host DNA. Response to therapy is typically monitored by counting CD4+ T cells and measuring plasma viral load, which becomes undetectable in most patients. However, at present HIV cannot be eradicated and persists in the host. Robust data are needed to understand the importance and clinical meaning of the residual activity of the virus, and it is crucial to investigate the role of intracellular HIV reservoirs in patients with undetectable viremia. Since HIV establishes latent infection at different degrees within central memory (CM), effector memory (EM) or naïve (TN) CD4+ T cells, measuring the content of HIV-DNA in different lymphocyte populations is a novel approach to follow the infection. Similarly, the residual capacity of the host to reconstitute the immune system can be accurately monitored by measuring: i) the amount of cells expressing signal-joint (sj) T cell receptor (TCR) rearrangement excision circles (sjTREC), that are a marker of homeostatic proliferation, and ii) telomere length, for evaluating cell senescence. Thus, using flow cytometry and cell sorting along with a molecular biology approach based on droplet digital PCR (ddPCR), we have quantified proviral HIV DNA, the amount of sjTREC+ cells and telomere length in different subsets of CD4+ T cells, such as TN, CM and EM.Methods:According to the Declaration of Helsinki, after informed consent and approval by the local Ethical Committee, we enrolled 32 HIV+ patients (mean age 49.0±7.2 years, 11 females) successfully treated for >2 years, with a CD4+ T cell count >500 cells/uL and plasma viremia undetectable from at least one year. After staining with fluorochrome-labeled mAbs, TN, CM and EM CD4+ T cells were sorted with a S3e sorter (Bio-Rad, CA, USA) equipped with a specifically designed biosafety containment hood (Biobubble, UK). Cell purity was always >95%. Proviral HIV-DNA and sjTREC were quantified in each lymphocyte subset with QX200 droplet digital PCR (Bio-Rad); telomere length, expressed as relative T/S (the ratio between telomere length and single gene copy, related to CD178, i.e., Fas Ligand) was quantified with CFX9600 Real time PCR (Bio-Rad). Viro-immunological parameters such as CD4+ and CD8+ T cell counts and percentages, and viral load were collected for each withdrawal together with the clinical characteristics of patients.Results:Considering all patients, HIV proviral DNA, measured as LTR copies/1,000 cells, was significantly lower in TN cells (mean±SEM: 0.77±0.23) compared to CM (2.42±0.38) or to EM (2.34±0.33), with p<0.0001 in both cases. Conversely, TN cells contained a higher number of sjTREC copies/1,000 cells (11.62±1.54) compared to CM (0.99±0.23) or to EM (1.26±0.44); p<0.0001 in both comparisons. No appreciable changes were observed in T/S ratio among CD4+ subsets. The stratification of patients revealed that HIV content was significantly higher in TN and CM T cells of patients with shorter time of treatment. Similarly, stratifying for CD4/CD8 ratio revealed that those with lower ratio had more intracellular HIV. A similar trend was observed also in EM cells, even if it was not statistically significant. The CD4+ T cell count pre-therapy or CD4+ T cell nadir did not influence the HIV reservoir.Conclusions: The importance of recovering a good CD4/CD8 ratio after successful antiretroviral therapy is further underlined by the fact that different amounts of virus were present and well measurable in different CD4+ T cell populations from patients with high or low ratio. The length of treatment was also significantly correlated to a better virological outcome. It is to note that in our patients homeostatic proliferation or cell senescence likely did not affect HIV reservoir. Linking highly purified cell sorting to a molecular biology approach based on ddPCR is crucial to understand what happens to the HIV reservoir when plasma viremia is suppressed. The measure of HIV DNA in selected lymphocyte populations not only will lead to a better comprehension of the features of HIV reservoir and its monitoring, but could also be useful for the development of eradication strategies. DisclosuresNo relevant conflicts of interest to declare.

Novel regulation of ILC by memory CD4 T cells during protective responses against influenza A virus

Abstract The primary goal of vaccination is to protect individuals from the morbidity and mortality of pathogen exposure by generating immunological memory. Our current understanding is that memory CD4 T cells, commonly regarded as the regulators of memory cytotoxic CD8 T cells and/or antibody-producing B cells, provide protection by hastening pathogen clearance through ‘faster, bigger, and better’ immune responses. It is now clear, however, that many different subsets of CD4 T cells, many with specialized functions other than providing ‘help’, are generated during immune responses against pathogens. In studies designed to ascertain the full functional potential of memory CD4 T cells responding against influenza A virus (IAV), we found that virus induced TH1-like, as well as in vitro polarized TH1 or TH17 memory CD4 T cells, enhance early innate inflammatory responses that correlate with better and earlier control of IAV in infected lungs. In further studies, we found innate lymphoid cells (ILC), which are not normally associated with anti-viral responses but rather associated with healing and wound repair, are also mobilized by memory CD4 T cells during the early stage of the response against IAV. Unexpectedly, the protection afforded by memory CD4 T cells against lethal IAV challenge is significantly compromised when ILCs are depleted. These findings reveal a previously unappreciated beneficial role of memory CD4 T cells in regulating tissue homeostasis during recall responses against pathogens through the regulation of ILC subsets.

The Circulating Treg/Th17 Cell Ratio Is Correlated with Relapse and Treatment Response in Pulmonary Sarcoidosis Patients after Corticosteroid Withdrawal.

ObjectivesPulmonary sarcoidosis is an immune-mediated disease, and some patients can be effectively treated with corticosteroids. However, nearly half of all sarcoidosis patients relapse after corticosteroid withdrawal. Different subsets of CD4+ helper T cells participate in the immunopathogenesis of sarcoidosis. Thus, the aims of our study were to investigate whether the circulating subsets of CD4+ helper T cells were associated with sarcoidosis relapse and with its remission after retreatment. Additionally, we identified a useful biomarker for predicting the relapse and remission of sarcoidosis patients.MethodsForty-two patients were enrolled in the present study who had previously been diagnosed with pulmonary sarcoidosis and treated with corticosteroids. The patients were allocated into either a stable group if they exhibited sustained remission (n = 22) or a relapse group if they experienced clinical or radiological recurrence after treatment withdrawal (n = 20). Peripheral blood cells were collected from these patients and analyzed to determine the frequencies of subsets of circulating CD4+ helper T cells by flow cytometry. The patients in the relapse group were retreated with corticosteroids and immunosuppressive agents and were then reevaluated to determine the frequencies of dynamic subsets of circulating CD4+ helper T cells after remission.ResultsThe frequencies of circulating Tregs were significantly increased concomitant with a decrease in the circulating Th17 cell frequency in the relapsed patients compared with the stable patients. The Treg/Th17 ratio was negatively correlated with sarcoidosis activity and was sensitive to retreatment. In addition, the percentage of isolated CD45RO+Ki67+ Tregs was higher in the patients who were stable and in those who recovered after retreatment than in those who relapsed.ConclusionsAn imbalance between Tregs and Th17 cells is associated with pulmonary sarcoidosis relapse after corticosteroid withdrawal. The circulating Treg/Th17 ratio could serve as an alternative marker for monitoring pulmonary sarcoidosis relapse after the end of corticosteroid treatment and for rapidly predicting the response to retreatment.