Human CD4 T-cell Research Articles

Overcoming Resistance of Human Non-Hodgkin's Lymphoma to CD19-CAR CTL Therapy by Celecoxib and Histone Deacetylase Inhibitors.

Patients with B-cell non-Hodgkin’s lymphoma (B-NHL) who fail to respond to first-line treatment regimens or develop resistance, exhibit poor prognosis. This signifies the need to develop alternative treatment strategies. CD19-chimeric antigen receptor (CAR) T cell-redirected immunotherapy is an attractive and novel option, which has shown encouraging outcomes in phase I clinical trials of relapsed/refractory NHL. However, the underlying mechanisms of, and approaches to overcome, acquired anti-CD19CAR CD8+ T cells (CTL)-resistance in NHL remain elusive. CD19CAR transduced primary human CTLs kill CD19+ human NHLs in a CD19- and caspase-dependent manner, mainly via the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) apoptotic pathway. To understand the dynamics of the development of resistance, we analyzed several anti-CD19CAR CTL-resistant NHL sublines (R-NHL) derived by serial exposure of sensitive parental lines to excessive numbers of anti-CD19CAR CTLs followed by a limiting dilution analysis. The R-NHLs retained surface CD19 expression and were efficiently recognized by CD19CAR CTLs. However, R-NHLs developed cross-resistance to CD19CAR transduced human primary CTLs and the Jurkat human T cell line, activated Jurkat, and lymphokine activated killer (LAK) cells, suggesting the acquisition of resistance is independent of CD19-loss and might be due to aberrant apoptotic machinery. We hypothesize that the R-NHL refractoriness to CD19CAR CTL killing could be partially rescued by small molecule sensitizers with apoptotic-gene regulatory effects. Chromatin modifiers and Celecoxib partially reversed the resistance of R-NHL cells to the cytotoxic effects of anti-CD19CAR CTLs and rhTRAIL. These in vitro results, though they require further examination, may provide a rational biological basis for combination treatment in the management of CD19CAR CTL-based therapy of NHL.

Human CD4 T-Cells With a Naive Phenotype Produce Multiple Cytokines During Mycobacterium Tuberculosis Infection and Correlate With Active Disease.

T-cell-mediated immune responses play a fundamental role in controlling Mycobacterium tuberculosis (M. tuberculosis) infection, and traditionally, this response is thought to be mediated by Th1-type CD4+ T-cells secreting IFN-γ. While studying the function and specificity of M. tuberculosis-reactive CD4+ T-cells in more detail at the single cell level; however, we found a human CD4+ T-cell population with a naive phenotype that interestingly was capable of producing multiple cytokines (TCNP cells). CD4+ TCNP cells phenotyped as CD95lo CD28int CD49dhi CXCR3hi and showed a broad distribution of T cell receptor Vβ segments. They rapidly secreted multiple cytokines in response to different M. tuberculosis antigens, their frequency was increased during active disease, but was comparable to latent tuberculosis infection in treated TB patients. These results identify a novel human CD4+ T-cell subset involved in the human immune response to mycobacteria, which is present in active TB patients’ blood. These results significantly expand our understanding of the immune response in infectious diseases.

Deregulated Mucosal Immune Surveillance through Gut-Associated Regulatory T Cells and PD-1+ T Cells in Human Colorectal Cancer.

Disturbed balance between immune surveillance and tolerance may lead to poor clinical outcomes in some malignancies. In paired analyses of adenocarcinoma and normal mucosa from 142 patients, we found a significant increase of the CD4/CD8 ratio and accumulation of regulatory T cells (Tregs) within the adenocarcinoma. The increased frequency of Tregs correlated with the local infiltration and extension of the tumor. There was concurrent maturation arrest, upregulation of programmed death-1 expression, and functional impairment in CD8+ T cells (CTLs) isolated from the adenocarcinoma. Adenocarcinoma-associated Tregs directly inhibit the function of normal human CTLs in vitro. With histopathological analysis, Foxp3+ Tregs were preferentially located in stroma. Concurrent transcriptome analysis of epithelial cells, stromal cells, and T cell subsets obtained from carcinomatous and normal intestinal samples from patients revealed a distinct gene expression signature in colorectal adenocarcinoma-associated Tregs, with overexpression of CCR1, CCR8, and TNFRSF9, whereas their ligands CCL4 and TNFSF9 were found upregulated in cancerous epithelium. Overexpression of WNT2 and CADM1, associated with carcinogenesis and metastasis, in cancer-associated stromal cells suggests that both cancer cells and stromal cells play important roles in the development and progression of colorectal cancer through the formation of a tumor microenvironment. The identification of CTL anergy by Tregs and the unique gene expression signature of human Tregs and stromal cells in colorectal cancer patients may facilitate the development of new therapeutics against malignancies.

292 - Regulation of Foxp3 expression in TGF-β induced regulatory T cells derived from human umbilical cord blood vs adult blood CD4 T cells

Natural T regulatory cells (nTreg) have shown the potential to safely and effectively induce immune tolerance in several in vivo applications. However, the limited proliferative capacity of nTreg remains a challenge to broader clinical applications. Inducible Treg (iTreg) can restore immune tolerance in settings where nTregs are decreased or defective. However, the instability of expression of the transcription factor Fork head box P3 (Foxp3) poses a significant barrier to successful immune therapy. Further understanding of Foxp3 binding partners and their function is emerging. The transcription factor BACH2 has been shown to repress effector programs to stabilize Treg-mediated immune homeostasis in murine KO studies. However, the potential of BACH2 to stabilize Foxp3 expression in human CD4 T-cells has not been explored. In the current studies, we compared the influence of BACH2 on Foxp3 expression in UCB vs. AB naive CD4 T-cells. UCB naïve CD4 T-cells highly express BACH2, 21-fold higher compared to AB naive CD4 T-cells, at the protein and mRNA levels (p < 0.01). Expression of Foxp3 is 2.5-fold higher in iTreg derived from UCB vs. AB naïve CD4 T-cells (p < 0.001). Also, the absolute number of Foxp3+ iTreg generated from UCB vs. AB naïve CD4 T-cells is 4-fold higher. Of particular interest, the suppressive function of UCB Foxp3+ iTreg is more potent in mixed lymphocyte cultures compared to AB Foxp3+ iTreg (p < 0.001). UCB iTreg also exhibit higher surface CD25 and CTLA-4 expression as measured by flow cytometry (p < 0.001). Transient BACH2 shRNA knockdown in UCB CD4 T-cells results in ~ 42% loss of Foxp3 expression (Empty shRNA: 78.8% vs. Bach2 shRNA: 46.2%, p < 0.001) and reduced expression of molecules down-stream of Foxp3, including CTLA-4, as measured by qPCR (p < 0.01) . Chromatin immunoprecipitation (ChIP) analysis revealed that BACH2 binds to the Foxp3 promoter in UCB iTreg but not AB iTreg (p < 0.01). Furthermore, Foxp3 luciferase reporter assays demonstrated that BACH2 is transcriptionally active at the Foxp3 promoter. Overall, our results suggest that BACH2 is an important transcription factor partner that stabilizes Foxp3 expression in UCB naïve CD4 T-cells.

An in vitro system of autologous lymphocytes culture that allows the study of homeostatic proliferation mechanisms in human naive CD4 T-cells

The size of peripheral T-cell pool is kept constant throughout life. However, a decline in lymphocyte numbers is a feature of several human disorders, in which fast and slow homeostatic proliferation play a crucial role. Several in vitro and in vivo models have been developed to study such processes. Nevertheless, self- and commensal- antigens, well-known triggers of homeostatic proliferation, have not been examined in these models. We have designed an in vitro culture of human T-cells exposed to rIL7 and autologous antigen-presenting cells (aAPC) that allows the simultaneous characterization of the different types of homeostatic proliferation. Using our model, we first confirmed that both rIL7 and aAPC are survival signals ultimately leading to homeostatic proliferation. In addition, we explored the modulation of different anti-apoptotic, proliferative, activation and homing markers during fast and slow homeostatic proliferation. Finally, different subsets of Treg were generated during homeostatic proliferation in our model. In summary, our in vitro system is able to simultaneously reproduce both types of homeostatic proliferation of human naive CD4 T-cells, and allows the characterization of these processes. Our in vitro system is a useful tool to explore specific features of human homeostatic proliferation in different human lymphopenia-related disorders and could be used as a cell therapy approach.

Clinical significance of PD-L1 levels in plasma-derived exosomes in head and neck cancer.

117 Background: Despite the broad spectrum of available therapies, HNSCC patients have poor outcome due to locoregional recurrence. Mechanisms responsible for immune suppression include activities of checkpoint receptors/ligands. HNSCCs with high expression levels of PD-L1 have especially poor outcome. Thus, PD-1 and PD-L1 inhibitors show potential as anticancer strategies. However, many patients with PD-L1+ HNSCC don’t benefit from checkpoint inhibitor therapy, suggesting that PDL-1 expression on tumors is not the only factor predictive of response. Tumor derived exosomes carry numerous immunosuppressive molecules and deliver them to recipient immune cells. We have demonstrated that elevated levels of circulating immunosuppressive exosomes play a key role in immune suppression and disease progression. Here, we show that surface PD-L1 on exosomes is responsible for these effects. Methods: Exosomes were isolated from plasma of 40 HNSCC patients by mini size exclusion chromatography, captured on beads using anti-CD63 Abs, stained for PD-1 and PD-L1 and analyzed by flow cytometry. Percentages of PD-L1+ and PD-1+ exosome/bead complexes were correlated with clinicopathological data. PD-L1high or PD-L1low exosomes were incubated with activated human CD8 T-cells ± PD-1 inhibitor and CD69 expression levels on T-cells were measured. Patients’ plasma was tested for soluble PD-L1 by ELISA. Results: The PD-L1 surface expression on exosomes correlated with patient’s disease activity, UICC stage and lymph node status with p values ranging from 0.0008-0.013. In contrast, plasma PD-L1 levels didn't correlate with any parameters. Exosome PD-1 levels were not informative. T-cell activation was inhibited (p &lt; 0.03) by co-incubation with PD-L1high but not PD-L1low exosomes. This inhibition was reversed by adding a PD-1 inhibitor to T-cells prior to their incubation with exosomes. Conclusions: We show that PD-L1 levels on exosomes, but not plasma levels, correlated with clinicopathological data in HNSCC patients. Blocking of PD-L1+ exosomes signaling to PD-1+ T-cells with anti-PD-1 Ab attenuated immune suppression. Altogether, PD-L1+ exosomes serve as useful metrics of disease and immune activity in HNSCC patients.

A novel Epstein\u2013Barr virus-latent membrane protein-1-specific T-cell receptor for TCR gene therapy

Background:Adoptive transfer of genetically engineered T-cells to express antigen-specific T-cell receptor (TCR) is a feasible and effective therapeutic approach for numerous types of cancers, including Epstein–Barr virus (EBV)-associated malignancies. Here, we describe a TCR gene transfer regimen to rapidly and reliably generate T-cells specific to EBV-encoded latent membrane protein-1 (LMP1), which is a potential target for T-cell-based immunotherapy.Methods:A novel TCR specific to LMP1 (LMP1-TCR) was isolated from HLA-A*0201 transgenic mice that were immunised with the minimal epitope LMP1166 (TLLVDLLWL), and LMP1-TCR-transduced peripheral blood lymphocytes were evaluated for functional specificities.Results:Both human CD8 and CD4 T-cells expressing the LMP1-TCR provoked high levels of cytokine secretion and cytolytic activity towards peptide-pulsed and LMP1-expressing tumour cells. Notably, recognition of these T-cells to peptide-pulsed cells was maintained at low concentration of peptide, implying that the LMP1-TCR has high avidity. Infusion of these engineered T-cells revealed remarkable therapeutic effects and inhibition of tumour growth in a preclinical xenogeneic model. We observed explosive ex vivo proliferation of functional TCR-transduced T-cells with artificial antigen-presenting cells that express co-stimulatory molecules CD80 and 4-1BBL.Conclusions:These data suggest that the novel TCR-targeting LMP1 might allow the potential design of T-cell-based immunotherapeutic strategies against EBV-positive malignancies.

Is a Human CD8 T-Cell Vaccine Possible, and if So, What Would It Take? CD8 T-Cell-Mediated Protective Immunity and Vaccination against Enteric Bacteria.

Although induction of CD8+ responses is widely accepted as critical in clearing viral infections and necessary for effective vaccines against viruses, much less is known regarding the role of these cells in bacterial and other infections, particularly those that enter the host via the gastrointestinal tract. In this commentary, I discuss the likelihood that CD8+ responses are also important in protection from intestinal Gram-negative bacteria, as well as the many factors that should be taken into consideration during the development of vaccines, based on eliciting long-term protection predominantly mediated by CD8+ responses against these organisms.

Is a Human CD8 T-Cell Vaccine Possible, and if So, What Would It Take? Could a CD8+ T-Cell Vaccine Prevent Persistent HIV Infection?

Vaccines that stimulate CD8+ T cells could clear early virus infection or control ongoing infection and prevent disease. This could be valuable to combat human immunodeficiency virus type 1 (HIV-1) where it has not yet been possible to generate broadly reacting neutralizing antibodies with a vaccine. However, HIV-1 vaccines aimed at stimulating CD8+ T cells have had no success. In contrast, a cytomegalovirus vectored simian immunodeficiency virus (SIV) vaccine enabled clearance of early SIV infection. This may open the door to the design of an effective HIV vaccine.

Is a Human CD8 T-Cell Vaccine Possible, and if So, What Would It Take? CD8 T-Cell Vaccines: To B or Not to B?

Although CD8 T-cell vaccines do not have the record of success of humoral-mediated vaccines, they do not receive the same degree of effort. Many diseases, including malaria, tuberculosis, and acquired immune deficiency syndrome (AIDS) have not yielded to vaccines, and intrinsic barriers may impede approaches limited solely to generating antibodies. Moreover, population growth and modernization are driving an increased pace of new emerging global health threats (human immunodeficiency virus [HIV] is a recent example), which will create unpredictable challenges for vaccinologists. Vaccine-elicited CD8 T cells may contribute to protective modalities, although their development will require a more thorough understanding of CD8 T-cell biology, practices for manufacturing and delivering CD8 T-cell-eliciting vectors that have acceptable safety profiles, and, ultimately, the political will and faith of those that make vaccine research funding decisions.

Enhanced TH17 Responses in Patients with IL10 Receptor Deficiency and Infantile-onset IBD.

IL10 receptor (IL10R) deficiency causes severe infantile-onset inflammatory bowel disease. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell functions in mice. We have previously reported a key role of IL10 in the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4 T-cell function. Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4 T-cell subsets, naive T-cell proliferation, regulatory T cell (Treg)-mediated suppression, and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time polymerase chain reaction. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa. Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared with control subjects. In addition, in vitro Treg suppression of CD4 T-cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naive cells exhibited higher proliferative capacity, a strong TH17 signature, and an increase in polarization toward TH17 cells, compared with controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1β leads to enhanced production of IL17A. IL10R signaling regulates TH17 polarization and T-cell proliferation in humans but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation.

IL-7-Induced Proliferation of Human Naive CD4 T-Cells Relies on Continued Thymic Activity.

Naive CD4 T-cell maintenance is critical for immune competence. We investigated here the fine-tuning of homeostatic mechanisms of the naive compartment to counteract the loss of de novo CD4 T-cell generation. Adults thymectomized in early childhood during corrective cardiac surgery were grouped based on presence or absence of thymopoiesis and compared with age-matched controls. We found that the preservation of the CD31− subset was independent of the thymus and that its size is tightly controlled by peripheral mechanisms, including prolonged cell survival as attested by Bcl-2 levels. Conversely, a significant contraction of the CD31+ naive subset was observed in the absence of thymic activity. This was associated with impaired responses of purified naive CD4 T-cells to IL-7, namely, in vitro proliferation and upregulation of CD31 expression, which likely potentiated the decline in recent thymic emigrants. Additionally, we found no apparent constraint in the differentiation of naive cells into the memory compartment in individuals completely lacking thymic activity despite upregulation of DUSP6, a phosphatase associated with increased TCR threshold. Of note, thymectomized individuals featuring some degree of thymopoiesis were able to preserve the size and diversity of the naive CD4 compartment, further arguing against complete thymectomy in infancy. Overall, our data suggest that robust peripheral mechanisms ensure the homeostasis of CD31− naive CD4 pool and point to the requirement of continuous thymic activity to the maintenance of IL-7-driven homeostatic proliferation of CD31+ naive CD4 T-cells, which is essential to secure T-cell diversity throughout life.

Comprehensive Analysis of Activation and Proliferation Kinetics and Effector Functions of Human Lymphocyte Subsets in Xenogeneic GvHD Model

To better understand the biology of graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation (HSCT) and develop novel treatment strategies, accurate and clinically relevant experimental animal models are indispensable. The majority of our understanding of this potentially lethal complication is based on mouse models of bone marrow transplantation in major histocompatibility complex antigens (MHC) and/or minor histocompatibility antigen mismatched settings. These mouse models of GvHD provide us with an immeasurable wealth of information; however, many findings obtained from mouse models are not necessarily correlated with clinical GVHD in humans. In addition, these mouse models are not suitable for predicting the effectiveness of recent human-specific therapies such as monoclonal anti-human antibodies and adaptive cell immunotherapies. To overcome these limitations, xenogeneic GvHD models using severe immunodeficient mice are currently being used in worldwide and allow us to investigate in vivo human immune reactions.The major aim of our study is to clarify the precise mechanism of immune response in vivo and to determine the principle components responsible for xenogeneic GvHD. We first observed that immunodeficient NOG mice receiving either no or sublethal irradiation consistently showed gradual body weight loss and eventual severe GvHD following injection of human unmanipulated peripheral blood mononuclear cells (PBMCs). Histopathology performed at the late phase of GvHD showed extensive infiltration of human CD45+ mononuclear cells, most of which were T-cells, and tissue destruction in lungs, bone marrow, liver, and spleen, with a smaller number detected in gut and skin unlike in human GvHD. We identified the infiltration of human T-cells exclusively in lungs and spleen before the onset of GvHD. Flow cytometric analysis showed a marked reduce of naïve (CD45RA+CCR7+) and central memory (CD45RA-CCR7+) T-cells and an increase of effector memory (CD45RA-CCR7-) T-cells within CD4+ subset in lungs, suggesting that xenogeneic response by human cells in lungs occur preceding systemic inflammation. Of note, the distribution of human cells was similar between intraperitoneal and intravenous injection. These results indicated that the acute lung injury is not associated with cell trapping within mouse pulmonary microvasculature by intravenous delivery.We next subdivided human lymphocyte subsets by magnetic cell sorting before adoptive transfer to characterize human cells responsive for xenogeneic GvHD development. CD4+ T-cells mediated more rapid and severe GvHD than did an equal number of CD8+ T-cells, whereas neither natural killer cells nor γδT cells caused any symptoms of GvHD. All antigen presenting cells (APCs)-depleted PBMCs mediated GvHD as well, suggesting that human T-cells are activated independently of their own APCs in this model.CFSE cell division assay showed more rapid proliferation of CD4+ rather than CD8+ T-cells. It is a noteworthy that the proliferation of and the expression of early activation marker CD69 on CD8+ T-cells was accelerated in the presence of CD4+ T-cells, indicating that CD4+ T-cells are required for the rapid and more efficacious response of CD8+ T-cells. We also found a strong increase of human IL-2, IFN-γ, and TNF-α in serum of mice injected with whole T-cells. In contrast, we observed very low levels of these inflammatory cytokines in mice injected with isolated CD8+ T-cells alone.Taken together, these results suggest that the early activation of human naïve CD4 T-cells by recognition of foreign antigen on mouse APCs following cytokine production play a critical role in triggering systemic inflammation in xenogeneic GvHD model. This in vivo response was confirmed in vitro by demonstrating that human CD4 T-cells rapidly proliferated and expressed CD69 with significantly increase of IL-2 and IFN-γ in response to mouse dendritic cells. These findings are helpful for adequate evaluation of immune response and creation of optimal experimental conditions in xenogeneic GvHD model. DisclosuresKanda:Otsuka Pharmaceutical: Honoraria, Research Funding.

Pretreatment of activated human CD8 T cells with IL-12 leads to enhanced TCR-induced signaling and cytokine production.

During the immune response to pathogens and autoantigens, CD8T cells are exposed to numerous inflammatory agents including the cytokine IL-12. Previous studies have focused on how IL-12 regulates T cell functions when present during or after the activation of the T cell receptor (TCR). However, recent studies suggest that prior exposure to IL-12 also alters the TCR responsiveness of murine T cells. Whether similar phenomena occur in human activated CD8T cells and the mechanisms mediating these effects remain unexplored. In this study, we observed that pretreatment of human activated CD8T cells with IL-12 results in increased cytokine mRNA and protein production following subsequent TCR challenge. The potentiation of TCR-mediated cytokine release was transient and required low doses of IL-12 for at least 24h. Mechanistically, prior exposure to IL-12 increased the TCR induced activation of select MAPKs and AKT without altering the activation of more proximal TCR signaling molecules, suggesting that the IL-12 mediated changes in TCR signaling are responsible for the increased production of cytokines. Our data suggest that prior treatment with IL-12 potentiates human CD8T cell responses at sites of infection and inflammation, expanding our understanding of the function of this clinically important cytokine.

Red blood cell alloimmunization: new findings at the bench and new recommendations for the bedside.

To summarize recent discoveries from clinical studies and animal models that contribute to understanding the alloimmune response to non-ABO blood group antigens. Several studies have confirmed high rates of alloimmunization among patients requiring chronic red blood cell (RBC) transfusion. Moreover, 'triggers' for alloantibody development in the transfusion setting have been identified, with a number of investigations linking recipient inflammation to a higher likelihood of alloimmunization. Additional associations between human leukocyte antigen expression and CD4 T-cell markers in 'responder' or 'nonresponder' humans have been revealed. Recent animal studies have described novel mechanistic properties by which the alloimmune response is governed, including the critical role played by dendritic cells in transfusion-associated alloimmunization. New light has also been shed on the properties of alloantibodies developed as a result of pregnancy, as well as mechanisms through which such alloimmunization may be prevented. Many of the clinical/biological factors that contribute to the RBC alloimmune response have been further elucidated. This knowledge will be applied to identify individuals most likely to mount an immune response to RBC antigens, such that appropriate resources and strategies for preventing alloimmunization (or mitigating its harmful effects) can be implemented.

Regulation of the IL-7 receptor alpha chain gene in primary human CD8 T-cells

Regulation of the IL-7 receptor alpha chain gene in primary human CD8 T-cells

Molecular Mechanisms Regulating T Helper 1 versus T Follicular Helper Cell Differentiation in Humans

IL-12 is important for the differentiation of T follicular helper (Tfh) cells, as well as Th1 cells in humans. Still, how IL-12 signals regulate Tfh versus Th1 cell differentiation remains poorly characterized. Here we aimed to determine the molecular mechanisms that regulate the differentiation and the function of IL-12-stimulated human naive CD4(+) Tcells. We found that T-bet promoted the expression of CXCR5 in human CD4(+) Tcells. We provide evidence that T-bet does not strongly inhibit the Tfh cell differentiation program per se but diminishes the functions to provide help to B cells. This study also suggests that IRF4 plays an important role in driving the early differentiation of IL-12-stimulated CD4(+) Tcells toward Tfh and away from Th1 by inhibiting the expression of Eomesodermin. Thus, the fate of IL-12-stimulated CD4(+) Tcells is determined through interplay of multiple transcription factors at early stages.

Can sugarcoated fingerprints be used to identify lurking viruses?

Comparative proteomics is increasingly used to detect biomarkers and therapeutic targets that differ between healthy and diseased populations; however, differences in posttranslational modifications have received less attention. In this issue, Yang etal. (Proteomics 2016, 16, 1872-1880) present data indicating that a glycoproteomics approach can detect N-glycosylated membrane protein differences between non-HIV-infected and latently infected human CD4(+) T-cell lines, identifying 172 proteins differentially expressed by these cells. Latently infected CD4(+) Tcells are thought to represent the major barrier to eventual HIV cure, but do not express detectable levels of viral protein and have not been shown to express biomarkers that can distinguish them from the vastly more abundant uninfected CD4(+) T-cell population. The findings of Yang etal. suggest that glycoproteomic analyses may have untapped potential to identify novel biomarkers and therapeutic targets in cell populations not readily distinguishable be standard proteomic analyses.

5-Hydroxymethylcytosine Remodeling Precedes Lineage Specification during Differentiation of Human CD4(+) T Cells.

5-methylcytosine (5mC) is converted to 5-hydroxymethylcytosine (5hmC) by the TET family of enzymes as part of a recently discovered active DNA de-methylation pathway. 5hmC plays important roles in regulation of gene expression and differentiation and has been implicated in Tcell malignancies and autoimmunity. Here, we report early and widespread 5mC/5hmC remodeling during human CD4(+) Tcell differentiation exvivo at genes and cell-specific enhancers with known Tcell function. We observe similar DNA de-methylation in CD4(+) memory Tcells invivo, indicating thatearly remodeling events persist long term in differentiated cells. Underscoring their important function, 5hmC loci were highly enriched for genetic variants associated with Tcell diseases andT-cell-specific chromosomal interactions. Extensive functional validation of 22 risk variants revealed potentially pathogenic mechanisms in diabetes and multiple sclerosis. Our results support 5hmC-mediated DNA de-methylation as a key component of CD4(+) Tcell biology in humans, with important implications for gene regulation and lineage commitment.

Sympathetic neural signaling via the β2-adrenergic receptor suppresses T-cell receptor-mediated human and mouse CD8(+) T-cell effector function.

Postganglionic sympathetic neurons innervate secondary lymphoid organs and secrete norepinephrine (NE) as the primary neurotransmitter. NE binds and signals through five distinct members of the adrenergic receptor family. In this study, we show elevated expression of the β2-adrenergic receptor (ADRB2) on primary human CD8(+) effector memory T cells. Treatment of both human and murine CD8(+) T cells with NE decreased IFN-γ and TNF-α secretion and suppressed their cytolytic capacity in response to T-cell receptor (TCR) activation. The effects of NE were specifically reversed by β2-specific antagonists. Adrb2(-/-) CD8(+) T cells were completely resistant to the effects of NE. Further, the ADRB2-specific pharmacological ligand, albuterol, significantly suppressed effector functions in both human and mouse CD8(+) T cells. While both TCR activation and stimulation with IL-12 + IL-18 were able to induce inflammatory cytokine secretion, NE failed to suppress IFN-γ secretion in response to IL-12 + IL18. Finally, the long-acting ADRB2-specific agonist, salmeterol, markedly reduced the cytokine secretion capacity of CD8(+) T cells in response to infection with vesicular stomatitis virus. This study reveals a novel intrinsic role for ADRB2 signaling in CD8(+) T-cell function and underscores the novel role this pathway plays in adaptive T-cell responses to infection.