PD-1, PD-L1 and PD-L2 Gene Expression on T-Cells and Natural Killer Cells Declines in Conjunction with a Reduction in PD-1 Protein during the Intensive Phase of Tuberculosis Treatment

Background

The PD-1 axis is a cell intrinsic immunoregulatory pathway that mediates T cell exhaustion in chronic infection particularly in some viral infections. We hypothesized that PD-1, PD-L1 and PD-L2 would be highly expressed in untreated tuberculosis patients compared to controls due to their chronic infection and would decrease with successful TB treatment.

Materials and Methods

Untreated tuberculosis patients (n = 26) were recruited at diagnosis and followed up during treatment. Household contacts (n = 24) were recruited to establish baseline differences. Blood gene expression ex vivo was investigated using qRT-PCR. Flow cytometry was performed to establish protein expression patterns.

Results

PD-L1 gene expression was found to be elevated in active TB disease; however, this was not observed for PD-1 or PD-L2. The intensive phase of TB treatment was associated with a significant decline in PD-1, PD-L1 and PD-L2 gene expression. PD-1 protein expression on the surface of NK cells, CD8⁺ and CD4⁺ T cells was similar in patients with active TB disease compared to controls but declined with successful TB treatment, with the greatest decline occurring on the NK cells followed by CD8⁺ T cells and then CD4⁺ T cells. Granzyme B/PD-1 co-expression declined with successful intensive phase treatment.

Conclusion

Modulation of PD-1/PD-L1 pathway through TB treatment indicates changes in the peripheral T cell response caused by live Mycobacterium tuberculosis (Mtb) followed by the response to dead bacilli, antigen-release and immuno-pathology resolution. The PD-1 axis could be a host drug target for immunomodulatory treatments in the future.     

Complementary Role of HCV and HIV in T-Cell Activation and Exhaustion in HIV/HCV Coinfection

Objectives

To investigate whether T-cell activation and exhaustion is linked to HCV- and HIV disease parameters in HIV/HCV infected individuals, we studied T-cell characteristics in HIV/HCV coinfected patients and controls.

Methods

14 HIV/HCV coinfected, 19 HCV monoinfected, 10 HIV monoinfected patients and 15 healthy controls were included in this cross-sectional study. Differences in expression of activation and exhaustion markers (HLA-DR, CD38, PD-1, Tim-3 and Fas) and phenotypic markers on CD4⁺ and CD8⁺ T-cells were analysed by flow cytometry and were related to HCV disease parameters (HCV-viremia, ALT and liver fibrosis).

Results

Frequencies of activated CD4⁺ and CD8⁺ T-cells were higher in HIV/HCV-coinfected compared to healthy controls and HCV or HIV mono-infected individuals. Coinfected patients also showed high expression of the exhaustion marker PD-1 and death receptor Fas. In contrast, the exhaustion marker Tim-3 was only elevated in HIV-monoinfected patients. T-cell activation and exhaustion were correlated with HCV-RNA, suggesting that viral antigen influences T-cell activation and exhaustion. Interestingly, increased percentages of effector CD8⁺ T-cells were found in patients with severe (F3–F4) liver fibrosis compared to those with no to minimal fibrosis (F0–F2).

Conclusions

HIV/HCV coinfected patients display a high level of T-cell activation and exhaustion in the peripheral blood. Our data suggest that T-cell activation and exhaustion are influenced by the level of HCV viremia. Furthermore, high percentages of cytotoxic/effector CD8⁺ T-cells are associated with liver fibrosis in both HCV monoinfected and HIV/HCV coinfected patients.     

Combination CTLA-4 blockade and 4-1BB activation enhances tumor rejection by increasing T-cell infiltration, proliferation, and cytokine production

Background

The co-inhibitory receptor Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) attenuates immune responses and prevent autoimmunity, however, tumors exploit this pathway to evade the host T-cell response. The T-cell co-stimulatory receptor 4-1BB is transiently upregulated on T-cells following activation and increases their proliferation and inflammatory cytokine production when engaged. Antibodies which block CTLA-4 or which activate 4-1BB can promote the rejection of some murine tumors, but fail to cure poorly immunogenic tumors like B16 melanoma as single agents.

Methodology/Principal Findings

We find that combining αCTLA-4 and α4-1BB antibodies in the context of a Flt3-ligand, but not a GM-CSF, based B16 melanoma vaccine promoted synergistic levels of tumor rejection. 4-1BB activation elicited strong infiltration of CD8+ T-cells into the tumor and drove the proliferation of these cells, while CTLA-4 blockade did the same for CD4+ effector T-cells. Anti-4-1BB also depressed regulatory T-cell infiltration of tumors. 4-1BB activation strongly stimulated inflammatory cytokine production in the vaccine and tumor draining lymph nodes and in the tumor itself. The addition of CTLA-4 blockade further increased IFN-γ production from CD4+ effector T-cells in the vaccine draining node and the tumor. Anti 4-1BB treatment, with or without CTLA-4 blockade, induced approximately 75% of CD8+ and 45% of CD4+ effector T-cells in the tumor to express the killer cell lectin-like receptor G1 (KLRG1). Tumors treated with combination antibody therapy showed 1.7-fold greater infiltration by these KLRG1+CD4+ effector T-cells than did those treated with α4-1BB alone.

Conclusions/Significance

This study shows that combining T-cell co-inhibitory blockade with αCTLA-4 and active co-stimulation with α4-1BB promotes rejection of B16 melanoma in the context of a suitable vaccine. In addition, we identify KLRG1 as a useful marker for monitoring the anti-tumor immune response elicited by this therapy. These findings should aid in the design of future trials for the immunotherapy of melanoma.     

The effect of Curcumin on multi-level immune checkpoint blockade and T cell dysfunction in head and neck cancer

BackgroundDespite recent advances in understanding the complex immunologic dysfunction in the tumor microenvironment (TME), fewer than 20% of patients with head and neck squamous cell carcinoma (HNSCC) respond to immune checkpoint blockade (ICB). Thus, it is important to understand how inhibitory IC receptors maintain the suppressed dysfunctional TME, and to develop more effective combination immunotherapy. This study evaluated the immune-modulating effects of Curcumin, which has well-established anti-cancer and chemopreventive properties, and its long-term safety as a phytochemical drug.MethodsWe carried out the western blot and small interfering RNA (siRNA) transfection assay to evaluate the effects of Curcumin on IC ligands and IC ligands function in HNSCC. Through T-cell cytotoxicity assay and measurements of cytokine secretion, we assessed the effects of combination of Curcumin with programmed death-ligand 1 (PD-L1) Ab on cancer cell killing. Flow cytometry were used to analyze the effects of Curcumin on the expression of programmed cell death protein 1 (PD-1) and T-cell immunoglobulin and mucin-domain3 (TIM-3) on CD4, CD8 and Treg. Immunofluorescence, immunohistochemistry and western blot were used to detecte the cytokine (IFN-γ, Granzyme B), IC receptors (PD-1 and TIM-3) and its ligands (PD-L1, PD-L2, Galectin-9) in xenograft mouse model and 4-nitroquinoline-1-oxide (4-NQO) oral cancer model.ResultsWe found that Curcumin decreased the expression of IC ligands such as PD-L1, PD-L2, and Galectin-9 in HNSCC, leading to regulation of epithelial-to-mesenchymal transition-associated tumor invasion. Curcumin also effectively restored the ability of CD8⁺ cytotoxic T cells to lyse cancer cells. To evaluate the effect of Curcumin on the TME further, the 4-NQO oral cancer model was used. Curcumin increased T-cell proliferation, tumor-infiltrating lymphocytes (TILs), and effector cytokines, and decreased the expression of PD-1, TIM-3, suppressive IC receptors and their ligands (PD-L1, PD-L2, and Galectin-9) in the TME, implying reinvigoration of the exhausted CD8⁺ T cells. In addition, Curcumin inhibited expression of CD4⁺CD25⁺FoxP3⁺ Treg cells as well as PD-1 and TIM-3.ConclusionsThese results show that Curcumin reinvigorates defective T cells via multiple (PD-1 and TIM-3) and multi-level (IC receptors and its ligands) IC axis suppression, thus providing a rationale to combine Curcumin with conventional targeted therapy or ICB as a multi-faceted approach for treating patients with HNSCC.     

Synovial T cell hyporesponsiveness to myeloid dendritic cells is reversed by preventing PD-1/PD-L1 interactions

Introduction

The aim of this study was to investigate PD-1/PD-L1 involvement in the hyporesponsiveness of rheumatoid arthritis (RA) synovial fluid (SF) CD4 T cells upon stimulation by thymic stromal lymphopoietin (TSLP)–primed CD1c myeloid dendritic cells (mDCs).

Methods

Expression of PD-1 on naïve (Tn), central memory (Tcm) and effector memory (Tem) CD4 T cell subsets was assessed by flow cytometry. PD-L1 expression and its regulation upon TSLP stimulation of mDCs from peripheral blood (PB) and SF of RA patients were investigated by quantitative RT-PCR and flow cytometry. The involvement of PD-1/PD-L1 interactions in SF T cell hyporesponsiveness upon (TSLP-primed) mDC activation was determined by cell culture in the presence of PD-1 blocking antibodies, with or without interleukin 7 (IL-7) as a recognized suppressor of PD-1 expression.

Results

PD-1 expression was increased on CD4 T cells derived from SF compared with PB of RA patients. TSLP increased PD-L1 mRNA expression in both PB and SF mDCs. PD-L1 protein expression was increased on SF mDCs compared with PB mDCs and was associated with T cell hyporesponsiveness. Blockade of PD-1, as well as IL-7 stimulation, during cocultures of memory T cells and (TSLP-primed) mDCs from RA patients significantly recovered T cell proliferation.

Conclusion

SF T cell hyporesponsiveness upon (TSLP-primed) mDC stimulation in RA joints is partially dependent on PD-1/PD-L1 interactions, as PD-1 and PD-L1 are both highly expressed on SF T cells and mDCs, respectively, and inhibiting PD-1 availability restores T cell proliferation. The potential of IL-7 to robustly reverse this hyporesponsiveness suggests that such proinflammatory cytokines in RA joints strongly contribute to memory T cell activation.     

Altered expression of T cell Immunoglobulin-Mucin (TIM) molecules in bronchoalveolar lavage CD4+ T cells in sarcoidosis

Background

Activated T helper (Th)-1 pulmonary CD4⁺ cells and their mediators are essential for the inflammation and granulomatous process in sarcoidosis. Recently, T-cell immunoglobulin and mucin domain (TIM) molecules were suggested to be important regulators of immune function. In this study, we wanted to investigate whether TIM molecules could play a role in sarcoidosis.

Methods

We used real-time polymerase chain reaction to investigate the differential gene expression of TIM-1 and TIM-3 as well as a few Th1 and Th2 cytokines (IL-2, IFN-γ, IL-4, IL-5 and IL-13) in CD4⁺ T cells isolated from bronchoalveolar lavage fluid (BALF) of patients (n = 28) and healthy controls (n = 8). Using flow cytometry, we were also able to analyse TIM-3 protein expression in 10 patients and 6 healthy controls.

Results

A decreased TIM-3 mRNA (p < 0.05) and protein (p < 0.05) expression was observed in patients, and the level of TIM-3 mRNA correlated negatively with the CD4/CD8 T cell ratio in BALF cells of patients. Compared to a distinct subgroup of patients i.e. those with Löfgren''s syndrome, BALF CD4⁺ T cells from non- Löfgren''s patients expressed decreased mRNA levels of TIM-1 (p < 0.05). mRNA expression of IL-2 was increased in patients (p < 0.01) and non-Löfgren''s patients expressed significantly higher levels of IFN-γ mRNA (p < 0.05) versus patients with Löfgren''s syndrome.

Conclusion

These findings are the first data on the expression of TIM-1 and TIM-3 molecules in sarcoidosis. The reduced TIM-3 expression in the lungs of patients may result in a defective T cell ability to control the Th1 immune response and could thus contribute to the pathogenesis of sarcoidosis. The down-regulated TIM-1 expression in non-Löfgren''spatients is in agreement with an exaggerated Th1 response in these patients.     

Regulatory Phenotype,PD-1 and TLR3 Expression in T Cells and Monocytes from HCV Patients Undergoing Antiviral Therapy: A Randomized Clinical Trial

Background & Aims

The cellular immunity has a profound impact on the status of hepatitis C virus (HCV) infection. However, the response of cellular immunity on the virological response in patients with antiviral treatment remains largely unclear. We aimed to clarify the response of peripheral T cells and monocytes in chronic hepatitis C patients with antiviral treatment.

Methods

Patients with chronic hepatitis C were treated either with interferon alpha-2b plus ribavirin (n = 37) or with pegylated interferon alpha-2a plus ribavirin (n = 33) for up to 24 weeks. Frequencies of peripheral regulatory T-cells (Tregs), programmed death-1 (PD-1) expressing CD4⁺ T-cells or CD8⁺ T-cells and toll-like receptor (TLR) 3 expressing CD14⁺ monocytes were evaluated by flow cytometry in patients at baseline, 12 and 24 weeks following treatment and in 20 healthy controls.

Results

Frequencies of Tregs, PD-1 and TLR3 expressing cells were higher in patients than those in control subjects (P<0.05). Patients with complete early virological response (cEVR) showed lower Tregs, PD-1 expressing CD4⁺ or CD8⁺ T-cells than those without cEVR at 12 weeks (P<0.05). Patients with low TLR3 expressing CD14⁺ monocytes at baseline had a high rate of cEVR (P<0.05).

Conclusions

Low peripheral TLR3 expressing CD14⁺ monocytes at baseline could serve as a predictor for cEVR of antiviral therapy in chronic HCV-infected patients. The cEVR rates were significantly increased in the patients with reduced circulating Tregs, PD-1 expressing CD4⁺ or CD8⁺ T-cells.

Trial Registration

Chinese Clinical Trial Registry ChiCTR10001090.     

Dendritic Cells in Chronic Mycobacterial Granulomas Restrict Local Anti-Bacterial T Cell Response in a Murine Model

Background

Mycobacterium-induced granulomas are the interface between bacteria and host immune response. During acute infection dendritic cells (DCs) are critical for mycobacterial dissemination and activation of protective T cells. However, their role during chronic infection in the granuloma is poorly understood.

Methodology/Principal Findings

We report that an inflammatory subset of murine DCs are present in granulomas induced by Mycobacteria bovis strain Bacillus Calmette-guerin (BCG), and both their location in granulomas and costimulatory molecule expression changes throughout infection. By flow cytometric analysis, we found that CD11c⁺ cells in chronic granulomas had lower expression of MHCII and co-stimulatory molecules CD40, CD80 and CD86, and higher expression of inhibitory molecules PD-L1 and PD-L2 compared to CD11c⁺ cells from acute granulomas. As a consequence of their phenotype, CD11c⁺ cells from chronic lesions were unable to support the reactivation of newly-recruited, antigen 85B-specific CD4⁺IFNγ⁺ T cells or induce an IFNγ response from naïve T cells in vivo and ex vivo. The mechanism of this inhibition involves the PD-1:PD-L signaling pathway, as ex vivo blockade of PD-L1 and PD-L2 restored the ability of isolated CD11c⁺ cells from chronic lesions to stimulate a protective IFNγ T cell response.

Conclusions/Significance

Our data suggest that DCs in chronic lesions may facilitate latent infection by down-regulating protective T cell responses, ultimately acting as a shield that promotes mycobacterium survival. This DC shield may explain why mycobacteria are adapted for long-term survival in granulomatous lesions.     

High-Throughput 13-Parameter Immunophenotyping Identifies Shifts in the Circulating T-Cell Compartment Following Reperfusion in Patients with Acute Myocardial Infarction

Rationale

With the advent of primary PCI (PPCI), reperfusion is achieved in almost all patients presenting with acute myocardial infarction. However, despite multiple trials, reperfusion injury has not been successfully dealt with so far. In mouse models, CD4⁺ T lymphocytes (T cells) have been shown to be crucial instigators of reperfusion injury.

Objective

Our goal was to investigate the role of CD4⁺ T cells during myocardial reperfusion following PPCI by developing a protocol for high-throughput multiplexed flow cytometric analysis and multivariate flow clustering.

Methods and Results

13-parameter immunophenotyping and hierarchical cluster analysis (HCA) identified a unique CD4⁺CD57⁺ T-cell population in PPCI patients that reflected acute proliferation in the CD4⁺ T-cell compartment. CD4⁺CCR7⁺ T cells were specifically depleted from peripheral blood during the first 30 min of myocardial reperfusion after PPCI, suggesting a potential role for the chemokine receptor CCR7 in T-cell redistribution to either peripheral tissues or migration to the infarcted heart during ischemia/reperfusion following PPCI.

Conclusions

High-throughput polychromatic flow cytometry and HCA are capable of objective, time and cost efficient assessment of the individual T-cell immune profile in different stages of coronary heart disease and have broad applications in clinical trials.     

T cells from Programmed Death-1 deficient mice respond poorly to Mycobacterium tuberculosis infection

Background

Programmed Death-1 (PD-1; CD279) receptor molecule is widely believed to be a negative regulator predominantly expressed by exhausted/activated mouse T cells. Upon interaction with its ligands, PD-L1 and PD-L2, PD-1 inhibits activation of T cells and cytokine production, which has been documented in various viral and fungal infections as well as in vitro studies. Therefore, inhibition of T cell responses by PD-1 resulted in disease resistance in a variety of mouse infection models studied heretofore.

Methodology/Principal Findings

Here, we report that PD-1 deficient (PD-1^−/−) mice infected with Mycobacterium tuberculosis (M. tb) H37Rv by the aerosol route have increased susceptibility as compared with their wild type littermates. Surprisingly, M. tb antigen-specific T cell proliferation was dramatically reduced in PD-1 deficient animals compared with wild-type littermates, and this was due to increased numbers of regulatory T cells (Tregs) and recruitment of mesenchymal stem cells. Furthermore, PD-1^−/− mice exhibited decreases in the autophagy-induced LC3-B marker protein in macrophages.

Conclusions/Significance

Our findings suggest that PD-1 does not play an inhibitory role during M. tb infection and instead promotes mycobacterial clearance in mice.     

Decreased Frequencies of Circulating CD4+ T Follicular Helper Cells Associated with Diminished Plasma IL-21 in Active Pulmonary Tuberculosis

Background

Circulating T follicular helper (Tfh) cells represent a distinct subset of CD4⁺ T cells and are important in immunity to infections. Although they have been shown to play a role in experimental models of tuberculosis infection, their role in human tuberculosis remains unexplored.

Aims/Methodology

To determine the distribution of circulating Tfh cells in human TB, we measured the frequencies of Tfh cells ex vivo and following TB - antigen or polyclonal stimulation in pulmonary TB (PTB; n = 30) and latent TB (LTB; n = 20) individuals, using the markers CXCR5, PD-1 and ICOS.

Results

We found that both ex vivo and TB - antigen induced frequencies of Tfh cell subsets was significantly lower in PTB compared to LTB individuals. Similarly, antigen induced frequencies of Tfh cells expressing IL-21 was also significantly lower in PTB individuals and this was reflected in diminished circulating levels of IL-21 and IFNγ. This was not accompanied by diminished frequencies of activated or memory B cell subsets. Finally, the diminution in frequency of Tfh cells in PTB individuals was dependent on IL-10, CTLA-4 and PD-L1 in vitro.

Conclusions

Thus, PTB is characterized by adiminution in the frequency of Tfh cell subsets.     

Focal Radiation Therapy Combined with 4-1BB Activation and CTLA-4 Blockade Yields Long-Term Survival and a Protective Antigen-Specific Memory Response in a Murine Glioma Model

Background

Glioblastoma (GBM) is the most common malignant brain tumor in adults and is associated with a poor prognosis. Cytotoxic T lymphocyte antigen -4 (CTLA-4) blocking antibodies have demonstrated an ability to generate robust antitumor immune responses against a variety of solid tumors. 4-1BB (CD137) is expressed by activated T lymphocytes and served as a co-stimulatory signal, which promotes cytotoxic function. Here, we evaluate a combination immunotherapy regimen involving 4-1BB activation, CTLA-4 blockade, and focal radiation therapy in an immune-competent intracranial GBM model.

Methods

GL261-luciferace cells were stereotactically implanted in the striatum of C57BL/6 mice. Mice were treated with a triple therapy regimen consisted of 4-1BB agonist antibodies, CTLA-4 blocking antibodies, and focal radiation therapy using a small animal radiation research platform and mice were followed for survival. Numbers of brain-infiltrating lymphocytes were analyzed by FACS analysis. CD4 or CD8 depleting antibodies were administered to determine the relative contribution of T helper and cytotoxic T cells in this regimen. To evaluate the ability of this immunotherapy to generate an antigen-specific memory response, long-term survivors were re-challenged with GL261 glioma en B16 melanoma flank tumors.

Results

Mice treated with triple therapy had increased survival compared to mice treated with focal radiation therapy and immunotherapy with 4-1BB activation and CTLA-4 blockade. Animals treated with triple therapy exhibited at least 50% long-term tumor free survival. Treatment with triple therapy resulted in a higher density of CD4⁺ and CD8⁺ tumor infiltrating lymphocytes. Mechanistically, depletion of CD4⁺ T cells abrogated the antitumor efficacy of triple therapy, while depletion of CD8⁺ T cells had no effect on the treatment response.

Conclusion

Combination therapy with 4-1BB activation and CTLA-4 blockade in the setting of focal radiation therapy improves survival in an orthotopic mouse model of glioma by a CD4⁺ T cell dependent mechanism and generates antigen-specific memory.     

Involvement of Galectin-9/TIM-3 Pathway in the Systemic Inflammatory Response in Early-Onset Preeclampsia

Background

Preeclampsia is a common obstetrical disease affecting 3-5% of pregnancies and representing one of the leading causes of both maternal and fetal mortality. Maternal symptoms occur as an excessive systemic inflammatory reaction in response to the placental factors released by the oxidatively stressed and functional impaired placenta. The T-cell immunoglobulin domain and mucin domain (TIM) family is a relatively newly described group of molecules with a conserved structure and important immunological functions. Identification of Galectin-9 as a ligand for TIM-3 has established the Galectin-9/TIM-3 pathway as an important regulator of Th1 immunity and tolerance induction.

Methods

The aim of our study was to investigate the expression and function of Galectin-9 and TIM-3 molecules by peripheral blood mononuclear cells and the possible role of Galectin-9/TIM-3 pathway in the immunoregulation of healthy pregnancy and early-onset preeclampsia. We determined TIM-3 and Gal-9 expression and cytotoxicicty of peripheral lymphocytes of early-onset preeclamptic women and healthy pregnant woman using flow cytometry.

Results

Investigating peripheral lymphocytes of women with early-onset preeclampsia, our results showed a decreased TIM-3 expression by T cells, cytotoxic T cells, NK cells and CD56^dim NK cells compared to healthy pregnant women. Interestingly, we found a notably increased frequency of Galectin-9 positive cells in each investigated lymphocyte population in the case of early-onset preeclamptic patients. We further demonstrated increased cytotoxic activity by cytotoxic T and CD56^dim NK cells in women with early-onset preeclampsia. Our findings showed that the strongest cellular cytotoxic response of lymphocytes occurred in the TIM-3 positive subpopulations of different lymphocytes subsets in early-onset preeclampsia.

Conclusion

These data suggest that Gal-9/TIM-3 pathway could play an important role in the immune regulation during pregnancy and the altered Galectin-9 and TIM-3 expression could result an enhanced systemic inflammatory response including the activation of Th1 lymphocytes in preeclampsia.     

The CTLA-4 and PD-1/PD-L1 Inhibitory Pathways Independently Regulate Host Resistance to Plasmodium-induced Acute Immune Pathology

The balance between pro-inflammatory and regulatory immune responses in determining optimal T cell activation is vital for the successful resolution of microbial infections. This balance is maintained in part by the negative regulators of T cell activation, CTLA-4 and PD-1/PD-L, which dampen effector responses during chronic infections. However, their role in acute infections, such as malaria, remains less clear. In this study, we determined the contribution of CTLA-4 and PD-1/PD-L to the regulation of T cell responses during Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM) in susceptible (C57BL/6) and resistant (BALB/c) mice. We found that the expression of CTLA-4 and PD-1 on T cells correlates with the extent of pro-inflammatory responses induced during PbA infection, being higher in C57BL/6 than in BALB/c mice. Thus, ECM develops despite high levels of expression of these inhibitory receptors. However, antibody-mediated blockade of either the CTLA-4 or PD-1/PD-L1, but not the PD-1/PD-L2, pathways during PbA-infection in ECM-resistant BALB/c mice resulted in higher levels of T cell activation, enhanced IFN-γ production, increased intravascular arrest of both parasitised erythrocytes and CD8⁺ T cells to the brain, and augmented incidence of ECM. Thus, in ECM-resistant BALB/c mice, CTLA-4 and PD-1/PD-L1 represent essential, independent and non-redundant pathways for maintaining T cell homeostasis during a virulent malaria infection. Moreover, neutralisation of IFN-γ or depletion of CD8⁺ T cells during PbA infection was shown to reverse the pathologic effects of regulatory pathway blockade, highlighting that the aetiology of ECM in the BALB/c mice is similar to that in C57BL/6 mice. In summary, our results underscore the differential and complex regulation that governs immune responses to malaria parasites.     

Reconstitution of Intestinal CD4 and Th17 T Cells in Antiretroviral Therapy Suppressed HIV-Infected Subjects: Implication for Residual Immune Activation from the Results of a Clinical Trial

Introduction

During HIV infection the severe depletion of intestinal CD4⁺ T-cells is associated with microbial translocation, systemic immune activation, and disease progression. This study examined intestinal and peripheral CD4⁺ T-cell subsets reconstitution under combined antiretroviral therapy (cART), and systemic immune activation markers.

Methods

This longitudinal single-arm pilot study evaluates CD4⁺ T cells, including Th1 and Th17, in gut and blood and soluble markers for inflammation in HIV-infected individuals before (M0) and after eight (M8) months of cART. From January 2010 to December 2011, 10 HIV-1 naïve patients were screened and 9 enrolled. Blood and gut CD4⁺ T-cells subsets and cellular immune activation were determined by flow-cytometry and plasma soluble CD14 by ELISA. CD4⁺ Th17 cells were detected in gut biopsies by immunohistochemistry. Microbial translocation was measured by limulus-amebocyte-lysate assay to detect bacterial lipopolysaccharide (LPS) and PCR Real Time to detect plasma bacterial 16S rDNA.

Results

Eight months of cART increased intestinal CD4⁺ and Th17 cells and reduced levels of T-cell activation and proliferation. The magnitude of intestinal CD4⁺ T-cell reconstitution correlated with the reduction of plasma LPS. Importantly, the magnitude of Th17 cells reconstitution correlated directly with blood CD4⁺ T-cell recovery.

Conclusion

Short-term antiretroviral therapy resulted in a significant increase in the levels of total and Th17 CD4⁺ T-cells in the gut mucosa and in decline of T-cell activation. The observation that pre-treatment levels of CD4⁺ and of CD8⁺ T-cell activation are predictors of the magnitude of Th17 cell reconstitution following cART provides further rationale for an early initiation of cART in HIV-infected individuals.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02097381","term_id":"NCT02097381"}}NCT02097381     

TIM-3 expression characterizes regulatory T cells in tumor tissues and is associated with lung cancer progression

Background

T cell immunoglobulin-3 (TIM-3) has been established as a negative regulatory molecule and plays a critical role in immune tolerance. TIM-3 is upregulated in exhausted CD8⁺ T cells in both chronic infection and tumor. However, the nature of TIM-3⁺CD4⁺ T cells in the tumor microenvironment is unclear. This study is to characterize TIM-3 expressing lymphocytes within human lung cancer tissues and establish clinical significance of TIM-3 expression in lung cancer progression.

Methodology

A total of 51 human lung cancer tissue specimens were obtained from pathologically confirmed and newly diagnosed non-small cell lung cancer (NSCLC) patients. Leukocytes from tumor tissues, distal normal lung tissues, and peripheral blood mononuclear cells (PBMC) were analyzed for TIM-3 surface expression by flow cytometry. TIM-3 expression on tumor-infiltrating lymphocytes (TILs) was correlated with clinicopathological parameters.

Conclusions

TIM-3 is highly upregulated on both CD4⁺ and CD8⁺ TILs from human lung cancer tissues but negligibly expressed on T cells from patients'' peripheral blood. Frequencies of IFN-γ⁺ cells were reduced in TIM-3⁺CD8⁺ TILs compared to TIM-3⁻CD8⁺ TILs. However, the level of TIM-3 expression on CD8⁺ TILs failed to associate with any clinical pathological parameter. Interestingly, we found that approximately 70% of TIM-3⁺CD4⁺ TILs expressed FOXP3 and about 60% of FOXP3⁺ TILs were TIM-3⁺. Importantly, TIM-3 expression on CD4⁺ T cells correlated with poor clinicopathological parameters of NSCLC such as nodal metastasis and advanced cancer stages. Our study reveals a new role of TIM-3 as an important immune regulator in the tumor microenvironment via its predominant expression in regulatory T cells.     

Differential response of BDCA-1+ and BDCA-3+ myeloid dendritic cells to respiratory syncytial virus infection

Background

Respiratory syncytial virus (RSV) is the leading cause of respiratory infections in children, elderly, and immunocompromised individuals. Severe infection is associated with short- and long-term morbidity including pneumonia, recurrent wheezing, and abnormal pulmonary function, and several lines of evidence indicate that impaired adaptive immune responses during infection are critical in the pathophysiology of RSV-mediated disease. Myeloid Dendritic cells (mDCs) play a pivotal role in shaping antiviral immune responses in the respiratory tract; however, few studies have examined the interactions between RSV and individual mDC subsets. In this study, we examined the effect of RSV on the functional response of primary mDC subsets (BDCA-1⁺ and BDCA-3⁺) isolated from peripheral blood.

Methods

BDCA-1⁺ and BDCA-3⁺ mDCs were isolated from the peripheral blood of healthy adults using FACS sorting. Donor-matched BDCA-1⁺ and BDCA-3⁺ mDCs were infected with RSV at a multiplicity of infection (MOI) of 5 for 40 hours. After infection, cells were analyzed for the expression of costimulatory molecules (CD86, CD80, and PD-L1), cytokine production, and the ability to stimulate allogenic CD4⁺ T cell proliferation.

Results

Both BDCA-1⁺ and BDCA-3⁺ mDCs were susceptible to infection with RSV and demonstrated enhanced expression of CD86, and the inhibitory costimulatory molecules CD80 and PD-L1. Compared to BDCA-3⁺ mDCs, RSV-infected BDCA-1⁺ mDC produced a profile of cytokines and chemokines predominantly associated with pro-inflammatory responses (IL-1β, IL-6, IL-12, MIP-1α, and TNF-α), and both BDCA-1⁺ and BDCA-3⁺ mDCs were found to produce IL-10. Compared to uninfected mDCs, RSV-infected BDCA-1⁺ and BDCA-3⁺ mDCs demonstrated a reduced capacity to stimulate T cell proliferation.

Conclusions

RSV infection induces a distinct pattern of costimulatory molecule expression and cytokine production by BDCA-1⁺ and BDCA-3⁺ mDCs, and impairs their ability to stimulate T cell proliferation.The differential expression of CD86 and pro-inflammatory cytokines by highly purified mDC subsets in response to RSV provides further evidence that BDCA-1⁺ and BDCA-3⁺ mDCs have distinct roles in coordinating the host immune response during RSV infection. Findings of differential expression of PD-L1 and IL-10 by infected mDCs, suggests possible mechanisms by which RSV is able to impair adaptive immune responses.     

Programmed Death-1 expression on Epstein Barr virus specific CD8+ T cells varies by stage of infection, epitope specificity, and T-cell receptor usage

Background

Programmed Death-1 (PD-1) is an inhibitory member of the CD28 family of molecules expressed on CD8+ T cells in response to antigenic stimulation. To better understand the role of PD-1 in antiviral immunity we examined the expression of PD-1 on Epstein-Barr virus (EBV) epitope-specific CD8+ T cells during acute infectious mononucleosis (AIM) and convalescence.

Methodology/Principal Findings

Using flow cytometry, we observed higher frequencies of EBV-specific CD8+ T cells and higher intensity of PD-1 expression on EBV-specific CD8+ T cells during AIM than during convalescence. PD-1 expression during AIM directly correlated with viral load and with the subsequent degree of CD8+ T cell contraction in convalescence. Consistent differences in PD-1 expression were observed between CD8+ T cells with specificity for two different EBV lytic antigen epitopes. Similar differences were observed in the degree to which PD-1 was upregulated on these epitope-specific CD8+ T cells following peptide stimulation in vitro. EBV epitope-specific CD8+ T cell proliferative responses to peptide stimulation were diminished during AIM regardless of PD-1 expression and were unaffected by blocking PD-1 interactions with PD-L1. Significant variability in PD-1 expression was observed on EBV epitope-specific CD8+ T cell subsets defined by V-beta usage.

Conclusions/Significance

These observations suggest that PD-1 expression is not only dependent on the degree of antigen presentation, but also on undefined characteristics of the responding cell that segregate with epitope specificity and V-beta usage.     

Autologous HIV-1 Clade-B Nef Peptides Elicit Increased Frequency,Breadth and Function of CD8+ T-Cells Compared to Consensus Peptides

Objective

To determine the function and phenotype of CD8⁺ T-cells targeting consensus and autologous sequences of entire HIV-1 Nef protein.

Methods

Multiparameter flow cytometry-based analysis was used to evaluate the responses of two treatment naïve HIV-infected individuals, during primary and the chronic phases of infection.

Results

A greater breadth and magnitude of CD8 IFN-γ responses to autologous compared to clade-B consensus peptides was observed in both subjects. Cross recognition between autologous and consensus peptides decreased in both subjects during progression from primary to chronic infection. The frequencies of TEMRA and TEM CD8⁺ T-cells targeting autologous peptides were higher than those targeting consensus peptides and were more polyfunctional (IFN-γ⁺ Gr-B⁺ CD107a⁺).

Conclusions

Our data indicate superior sensitivity and specificity of autologous peptides. The functional and maturational aspects of “real” versus “cross-recognized” responses were also found to differ, highlighting the importance of a sequence-specific approach towards understanding HIV immune response.