Discovery of quinazoline HPK1 inhibitors with high cellular potency.

Hematopoietic progenitor kinase 1 (HPK1) is regarded as a highly validated target in pre-clinical immune oncology. HPK1 has been described as regulating multiple critical signaling pathway in both adaptive and innate cells. In support of this role, HPK1 KO T cells show enhanced sensitivity to TCR activation and HPK1 KO mice display enhanced anti-tumor activity. Taken together, inhibition of HPK1 has the potential to induce enhanced anti-tumor immune response. Herein, we described the discovery of highly potent HPK1 inhibitors starting form a weak HTS hit. Using a structure-based drug design, HPK1 inhibitors exhibiting excellent cellular single-digit nanomolar potency in both proximal (pSLP76) and distal (IL-2) biomarkers along with sustained elevation of IL-2 cytokine secretion were discovered.

Immune Checkpoint Blockade Restores HIV-Specific CD4 T Cell Help for NK Cells.

Immune exhaustion is an important feature of chronic infections, such as HIV, and a barrier to effective immunity against cancer. This dysfunction is in part controlled by inhibitory immune checkpoints. Blockade of the PD-1 or IL-10 pathways can reinvigorate HIV-specific CD4 T cell function in vitro, as measured by cytokine secretion and proliferative responses upon Ag stimulation. However, whether this restoration of HIV-specific CD4 T cells can improve help to other cell subsets impaired in HIV infection remains to be determined. In this study, we examine a cohort of chronically infected subjects prior to initiation of antiretroviral therapy (ART) and individuals with suppressed viral load on ART. We show that IFN-γ induction in NK cells upon PBMC stimulation by HIV Ag varies inversely with viremia and depends on HIV-specific CD4 T cell help. We demonstrate in both untreated and ART-suppressed individuals that dual PD-1 and IL-10 blockade enhances cytokine secretion of NK cells via restored HIV-specific CD4 T cell function, that soluble factors contribute to these immunotherapeutic effects, and that they depend on IL-2 and IL-12 signaling. Importantly, we show that inhibition of the PD-1 and IL-10 pathways also increases NK degranulation and killing of target cells. This study demonstrates a previously underappreciated relationship between CD4 T cell impairment and NK cell exhaustion in HIV infection, provides a proof of principle that reversal of adaptive immunity exhaustion can improve the innate immune response, and suggests that immune checkpoint modulation that improves CD4/NK cell cooperation can be used as adjuvant therapy in HIV infection.

Multiparametric characterization of rare HIV-infected cells using an RNA-flow FISH technique.

Efforts to cure HIV are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in individuals receiving antiretroviral therapy (ART). We describe a protocol for flow cytometric identification of viral reservoirs, based on concurrent detection of cellular HIV Gagpol mRNA by in situ RNA hybridization combined with antibody staining for the HIV Gag protein. By simultaneously detecting both HIV RNA and protein, the CD4 T cells harboring translation-competent virus can be identified. The HIVRNA/Gag method is 1,000-fold more sensitive than Gag protein staining alone, with a detection limit of 0.5-1 Gagpol mRNA+/Gag protein+ cells per million CD4 T cells. Uniquely, the HIVRNA/Gag assay also allows parallel phenotyping of viral reservoirs, including reactivated latent reservoirs in clinical samples. The assay takes 2 d, and requires antibody labeling for surface and intracellular markers, followed by mRNA labeling and multiple signal amplification steps.

Circulating CXCR5+CXCR3+PD-1lo Tfh-like cells in HIV-1 controllers with neutralizing antibody breadth.

HIV-1-specific broadly neutralizing antibodies (bnAbs) typically develop in individuals with continuous high-level viral replication and increased immune activation, conditions that cannot be reproduced during prophylactic immunization. Understanding mechanisms supporting bnAb development in the absence of high-level viremia may be important for designing bnAb-inducing immunogens. Here, we show that the breadth of neutralizing antibody responses in HIV-1 controllers was associated with a relative enrichment of circulating CXCR5+CXCR3+PD-1lo CD4+ T cells. These CXCR3+PD-1lo Tfh-like cells were preferentially induced in vitro by functionally superior dendritic cells from controller neutralizers, and able to secrete IL-21 and support B cells. In addition, these CXCR3+PD-1lo Tfh-like cells contained higher proportions of stem cell-like memory T cells, and upon antigenic stimulation differentiated into PD-1hi Tfh-like cells in a Notch-dependent manner. Together, these data suggest that CXCR5+CXCR3+PD-1lo cells represent a dendritic cell-primed precursor cell population for PD-1hi Tfh-like cells that may contribute to the generation of bnAbs in the absence of high-level viremia.

HIV Controllers Exhibit Enhanced Frequencies of Major Histocompatibility Complex Class II Tetramer+ Gag-Specific CD4+ T Cells in Chronic Clade C HIV-1 Infection.

Immune control of viral infections is heavily dependent on helper CD4+ T cell function. However, the understanding of the contribution of HIV-specific CD4+ T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibility complex (MHC) class II tetramers have emerged as a powerful tool for interrogating antigen-specific CD4+ T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4+ T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to the viral load of HIV-specific CD4+ T cell responses in a cohort of untreated HIV clade C-infected persons. We observed significantly higher frequencies of MHC class II tetramer-positive CD4+ T cells in HIV controllers than progressors (P = 0.0001), and these expanded Gag-specific CD4+ T cells in HIV controllers showed higher levels of expression of the cytolytic proteins granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control (r = -0.5, P = 0.02). These data identify an association between HIV-specific CD4+ T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of the use of class II tetramers in evaluating HIV-specific CD4+ T cell responses in natural infections.IMPORTANCE Increasing evidence suggests that virus-specific CD4+ T cells contribute to the immune-mediated control of clade B HIV-1 infection, yet there remains a relative paucity of data regarding the role of HIV-specific CD4+ T cells in shaping adaptive immune responses in individuals infected with clade C, which is responsible for the majority of HIV infections worldwide. Understanding the contribution of HIV-specific CD4+ T cell responses in clade C infection is particularly important for developing vaccines that would be efficacious in sub-Saharan Africa, where clade C infection is dominant. Here, we employed MHC class II tetramers designed to immunodominant Gag epitopes and used them to characterize CD4+ T cell responses in HIV-1 clade C infection. Our results demonstrate an association between the frequency of HIV-specific CD4+ T cell responses targeting an immunodominant DRB1*11-Gag41 complex and HIV control, highlighting the important contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infections.

Increased frequencies of CD8+CD57+ T cells are associated with antibody neutralization breadth against HIV in viraemic controllers.

INTRODUCTION: An effective prophylactic vaccine against HIV will need to elicit antibody responses capable of recognizing and neutralizing rapidly evolving antigenic regions. The immunologic milieu associated with development of neutralizing antibody breadth remains to be fully defined. In this study, we sought to identify immunological signatures associated with neutralization breadth in HIV controllers. We applied an immune monitoring approach to analyze markers of T cell and myeloid cell activation by flow cytometry, comparing broad neutralizers with low- and non-neutralizers using multivariate and univariate analyses. METHODS: Antibody neutralization breadth was determined, and cryopreserved peripheral blood mononuclear cells were stained for T cell and myeloid cell activation markers. Subjects were grouped according to neutralization breadth, and T cell and myeloid cell activation was analyzed by partial least squares discriminant analysis to determine immune signatures associated with high neutralization breadth. RESULTS: We show that neutralization breadth in HIV viraemic controllers (VC) was strongly associated with increased frequencies of CD8+CD57+ T cells and that this association was independent of viral load, CD4 count and time since HIV diagnosis. CONCLUSIONS: Our data show elevated frequencies of CD8+CD57+ T cells in VC who develop neutralization breadth against HIV. This immune signature could serve as a potential biomarker of neutralization breadth and should be further investigated in other HIV-positive cohorts and in HIV vaccine trials.

Single-Cell Characterization of Viral Translation-Competent Reservoirs in HIV-Infected Individuals.

HIV cure efforts are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in patients receiving antiretroviral therapy. We combine fluorescent in situ RNA hybridization with detection of HIV protein and flow cytometry, enabling detection of 0.5-1 gag-pol mRNA(+)/Gag protein(+)-infected cells per million. In the peripheral blood of untreated persons, active HIV replication correlated with viremia and occurred in CD4 T cells expressing T follicular helper cell markers and inhibitory co-receptors. In virally suppressed subjects, the approach identified latently infected cells capable of producing HIV mRNA and protein after stimulation with PMA/ionomycin and latency-reversing agents (LRAs). While ingenol-induced reactivation mirrored the effector and central/transitional memory CD4 T cell contribution to the pool of integrated HIV DNA, bryostatin-induced reactivation occurred predominantly in cells expressing effector memory markers. This indicates that CD4 T cell differentiation status differentially affects LRA effectiveness.

Elite controller CD8+ T cells exhibit comparable viral inhibition capacity, but better sustained effector properties compared to chronic progressors.

Mechanisms modulating HIV-specific CD8+ T cell-mediated viral inhibition are not well defined. To delineate features of effective control, we compared the ability of CD8+ T cells from HIV ECs and CPs to inhibit HIV ex vivo. ECs showed superior inhibition compared to HAART-treated or untreated CPs in a typical VIA in which CD8+ T cells are rested 3 d before use (P = 0.025). In contrast, comparable antiviral activity was observed in freshly thawed cells. Rested CD8+ T cells underwent apoptosis with preferential loss of HIV-specific cells. EC CD8+ T cells showed greater capacity to sustain polyfunctionality ex vivo compared with those of CPs, and incubation of CD8+ T cells with IL-15 augmented inhibition. These results indicate that superior ex vivo inhibition of viral replication by CD8+ T cells from ECs is associated with enhanced retention of functional qualities and that in vitro antiviral function is enhanced by IL-15.

Immunization of HIV-1-Infected Persons With Autologous Dendritic Cells Transfected With mRNA Encoding HIV-1 Gag and Nef: Results of a Randomized, Placebo-Controlled Clinical Trial.

BACKGROUND: HIV-1 eradication may require reactivation of latent virus along with stimulation of HIV-1-specific immune responses to clear infected cells. Immunization with autologous dendritic cells (DCs) transfected with viral mRNA is a promising strategy for eliciting HIV-1-specific immune responses. We performed a randomized controlled clinical trial to evaluate the immunogenicity of this approach in HIV-1-infected persons on antiretroviral therapy. METHODS: Fifteen participants were randomized 2:1 to receive intradermal immunization with HIV-1 Gag- and Nef-transfected DCs (vaccine) or mock-transfected DCs (placebo) at weeks 0, 2, 6, and 10. All participants also received DCs pulsed with keyhole limpet hemocyanin (KLH) to assess whether responses to a neo-antigen could be induced. RESULTS: After immunization, there were no differences in interferon-gamma enzyme-linked immunospot responses to HIV-1 Gag or Nef in the vaccine or placebo group. CD4 proliferative responses to KLH increased 2.4-fold (P = 0.026) and CD8 proliferative responses to KLH increased 2.5-fold (P = 0.053) after vaccination. There were increases in CD4 proliferative responses to HIV-1 Gag (2.5-fold vs. baseline, 3.4-fold vs. placebo, P = 0.054) and HIV-1 Nef (2.3-fold vs. baseline, 6.3-fold vs. placebo, P = 0.009) among vaccine recipients, but these responses were short-lived. CONCLUSION: Immunization with DCs transfected with mRNA encoding HIV-1 Gag and Nef did not induce significant interferon-gamma enzyme-linked immunospot responses. There were increases in proliferative responses to HIV-1 antigens and to a neo-antigen, KLH, but the effects were transient. Dendritic cell vaccination should be optimized to elicit stronger and long-lasting immune responses for this strategy to be effective as an HIV-1 therapeutic vaccine.

HIV-1 Antibody Neutralization Breadth Is Associated with Enhanced HIV-Specific CD4+ T Cell Responses.

UNLABELLED: Antigen-specific CD4(+) T helper cell responses have long been recognized to be a critical component of effective vaccine immunity. CD4(+) T cells are necessary to generate and maintain humoral immune responses by providing help to antigen-specific B cells for the production of antibodies. In HIV infection, CD4(+) T cells are thought to be necessary for the induction of Env-specific broadly neutralizing antibodies. However, few studies have investigated the role of HIV-specific CD4(+) T cells in association with HIV neutralizing antibody activity in vaccination or natural infection settings. Here, we conducted a comprehensive analysis of HIV-specific CD4(+) T cell responses in a cohort of 34 untreated HIV-infected controllers matched for viral load, with and without neutralizing antibody breadth to a panel of viral strains. Our results show that the breadth and magnitude of Gag-specific CD4(+) T cell responses were significantly higher in individuals with neutralizing antibodies than in those without neutralizing antibodies. The breadth of Gag-specific CD4(+) T cell responses was positively correlated with the breadth of neutralizing antibody activity. Furthermore, the breadth and magnitude of gp41-specific, but not gp120-specific, CD4(+) T cell responses were significantly elevated in individuals with neutralizing antibodies. Together, these data suggest that robust Gag-specific CD4(+) T cells and, to a lesser extent, gp41-specific CD4(+) T cells may provide important intermolecular help to Env-specific B cells that promote the generation or maintenance of Env-specific neutralizing antibodies. IMPORTANCE: One of the earliest discoveries related to CD4(+) T cell function was their provision of help to B cells in the development of antibody responses. Yet little is known about the role of CD4(+) T helper responses in the setting of HIV infection, and no studies to date have evaluated the impact of HIV-specific CD4(+) T cells on the generation of antibodies that can neutralize multiple different strains of HIV. Here, we addressed this question by analyzing HIV-specific CD4(+) T cell responses in untreated HIV-infected persons with and without neutralizing antibodies. Our results indicate that HIV-infected persons with neutralizing antibodies have significantly more robust CD4(+) T cell responses targeting Gag and gp41 proteins than individuals who lack neutralizing antibodies. These associations suggest that Gag- and gp41-specific CD4(+) T cell responses may provide robust help to B cells for the generation or maintenance of neutralizing antibodies in natural HIV-infection.

Sex Differences in Plasmacytoid Dendritic Cell Levels of IRF5 Drive Higher IFN-α Production in Women.

Increased IFN-α production contributes to the pathogenesis of infectious and autoimmune diseases. Plasmacytoid dendritic cells (pDCs) from females produce more IFN-α upon TLR7 stimulation than pDCs from males, yet the mechanisms underlying this difference remain unclear. In this article, we show that basal levels of IFN regulatory factor (IRF) 5 in pDCs were significantly higher in females compared with males and positively correlated with the percentage of IFN-α-secreting pDCs. Delivery of recombinant IRF5 protein into human primary pDCs increased TLR7-mediated IFN-α secretion. In mice, genetic ablation of the estrogen receptor 1 (Esr1) gene in the hematopoietic compartment or DC lineage reduced Irf5 mRNA expression in pDCs and IFN-α production. IRF5 mRNA levels furthermore correlated with ESR1 mRNA levels in human pDCs, consistent with IRF5 regulation at the transcriptional level by ESR1. Taken together, these data demonstrate a critical mechanism by which sex differences in basal pDC IRF5 expression lead to higher IFN-α production upon TLR7 stimulation in females and provide novel targets for the modulation of immune responses and inflammation.

Co-infection with Mycobacterium tuberculosis impairs HIV-Specific CD8+ and CD4+ T cell functionality.

The ability of antigen-specific T cells to simultaneously produce multiple cytokines is thought to correlate with the functional capacity and efficacy of T cells. These 'polyfunctional' T cells have been associated with control of HIV. We aimed to assess the impact of co-infection with Mycobacterium tuberculosis (MTB) on HIV-specific CD8+ and CD4+ T cell function. We assessed T cell functionality in 34 South African adults by investigating the IFN-y, IL-2, TNF-α, IL-21 and IL-17 cytokine secretion capacity, using polychromatic flow cytometry, following HIV Gag-specific stimulation of peripheral blood mononuclear cells. We show that MTB is associated with lower HIV-specific T cell function in co-infected as compared to HIV mono-infected individuals. This decline in function was greatest in co-infection with active Tuberculosis (TB) compared to co-infection with latent MTB (LTBI), suggesting that mycobacterial load may contribute to this loss of function. The described impact of MTB on HIV-specific T cell function may be a mechanism for increased HIV disease progression in co-infected subjects as functionally impaired T cells may be less able to control HIV.

High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry.

Fluorescent in situ hybridization (FISH) is a method that uses fluorescent probes to detect specific nucleic acid sequences at the single-cell level. Here we describe optimized protocols that exploit a highly sensitive FISH method based on branched DNA technology to detect mRNA and miRNA in human leukocytes. This technique can be multiplexed and combined with fluorescent antibody protein staining to address a variety of questions in heterogeneous cell populations. We demonstrate antigen-specific upregulation of IFNγ and IL-2 mRNAs in HIV- and CMV-specific T cells. We show simultaneous detection of cytokine mRNA and corresponding protein in single cells. We apply this method to detect mRNAs for which flow antibodies against the corresponding proteins are poor or are not available. We use this technique to show modulation of a microRNA critical for T-cell function, miR-155. We adapt this assay for simultaneous detection of mRNA and proteins by ImageStream technology.

Tuberculosis distorts the inhibitory impact of interleukin-10 in HIV infection.

OBJECTIVES: This study aimed to assess how Mycobacterium tuberculosis (MTB) coinfection alters the impact of interleukin-10 in chronic HIV infection. DESIGN: We assessed plasma cytokine levels (interleukin-10, interferon-γ, tumor necrosis factor-α, interleukin-2, interleukin-6 and interleukin-13) in 82 individuals presenting with HIV monoinfection, HIV-LTBI (latent MTB infection) coinfection or HIV-TB (active tuberculosis) coinfection. We also assessed the influence of MTB on the functional impact of interleukin-10 receptor alpha (interleukin-10Rα) blockade on HIV and MTB-specific CD4(+) T cells. METHODS: Plasma cytokine levels were measured by high sensitivity Luminex. We used an ex-vivo interleukin-10Rα blockade assay to assess if functional enhancement of HIV and MTB-specific CD4(+) T cells was possible following a 48-h stimulation with HIV gag or pooled ESAT-6 (6 kDa early secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein) peptides. Cell supernatant was collected 48 h after stimulation and the cytokine profile was measured by Luminex. RESULTS: Plasma interleukin-10 levels were elevated in HIV-TB as compared with HIV monoinfection (P < 0.05) and HIV-LTBI (P < 0.05). Plasma interleukin-10 levels correlated to HIV viral load in HIV monoinfection (P = 0.016) and HIV-LTBI (P = 0.042), but not HIV-TB. Ex-vivo blockade of interleukin-10Rα significantly enhanced MTB and HIV-specific CD4(+) T-cell function in HIV-LTBI individuals but not in HIV-TB individuals. CONCLUSION: Tuberculosis disrupts the correlation between interleukin-10 and markers of HIV disease progression. In addition, HIV-TB is associated with a more inflammatory cytokine milieu compared with HIV monoinfection. Interestingly, interleukin-10Rα blockade can enhance both HIV and MTB-specific T-cell function in HIV-LTBI, but not in HIV-TB coinfection.

Differential impact of PD-1 and/or interleukin-10 blockade on HIV-1-specific CD4 T cell and antigen-presenting cell functions.

UNLABELLED: Antigen persistence in chronic infections and cancer upregulates inhibitory networks, such as the PD-1 and interleukin-10 (IL-10) pathways, that impair immunity and lead to disease progression. These pathways are attractive targets for immunotherapy, as demonstrated by recent clinical trials of PD-1/PD-L1 blockade in cancer patients. However, in HIV-1 infection not all subjects respond to inhibition of either pathway and the mechanistic interactions between these two networks remain to be better defined. Here we demonstrate that in vitro blockade of PD-L1 and/or IL-10Rα results in markedly different profiles of HIV-1-specific CD4 T cell restoration. Whereas PD-L1 blockade leads to balanced increase in gamma interferon (IFN-γ), IL-2, and IL-13 secretion, IL-10Rα blockade preferentially restores IFN-γ production. In viremic subjects, combined PD-L1/IL-10Rα blockade results in a striking 10-fold increase in IFN-γ secretion by HIV-1-specific CD4 T cells that is not observed in subjects with spontaneous (elite controllers) or therapy-induced control of viral replication. In contrast to the dramatic increase in IFN-γ production, concurrent blockade has a marginal additive effect on IL-2 production, IL-13 secretion, and HIV-1-specific CD4 T cell proliferation. IFN-γ produced by Thelper cells upregulates PD-L1, HLA I/II, and IL-12 expression by monocytes. The effect of combined blockade on IFN-γ was dependent on reciprocal reinforcement through IL-12. These studies provide crucial information on the different immunoregulatory qualities of PD-1 and IL-10 in progressive disease and link exhausted virus-specific CD4 T cells and monocytes in the regulation of IFN-γ and IL-12 secretion. IMPORTANCE: Infection with HIV results in most people in uncontrolled viral replication and progressive weakening of the body defenses. In the absence of antiviral therapy, this process results in clinical disease, or AIDS. An important reason why HIV continues to multiply is that a population of white blood cells called CD4 T cells that targets the virus fails to work properly. At least part of this impairment is under the control of inhibitory mechanisms that can be blocked to improve the function of these CD4 T cells. In this report, we show that blocking one or two of the molecules involved, called PD-1 and IL-10, has different effects on the individual functions of these cells and that one is strongly improved. We investigate how these effects are caused by interactions between CD4 T cells and antigen-presenting cells. These observations can have implications for new therapeutic approaches in HIV infection.

In vitro assay to evaluate the impact of immunoregulatory pathways on HIV-specific CD4 T cell effector function.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects. Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10Rα and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 °C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

Simultaneous TCR and CD244 signals induce dynamic downmodulation of CD244 on human antiviral T cells.

Various cosignaling molecules on T cells can contribute to activation, inhibition, or exhaustion, depending on context. The surface receptor signaling lymphocytic activation molecule (SLAM) family receptor CD244 (2B4/SLAMf4) has been shown to be capable of either inhibitory or enhancing effects upon engagement of its ligand CD48 (SLAMf2). We examined phenotypes of CD8 T cells from HIV(+) and HIV(neg) human donors, specific for HIV and/or respiratory syncytial virus. Cultured and ex vivo CD8 T cells expressed PD-1, CD244, and TIM-3. We found that ex vivo CD8 T cells downregulated CD244 in response to superantigen. Furthermore, cognate peptide induced rapid downregulation of both CD244 and TIM-3, but not PD-1, on CD8 T cell clones. CD244 downmodulation required simultaneous signaling via both TCR and CD244 itself. Using a pH-sensitive fluorophore conjugated to avidin-Ab tetramers, we found that CD244 crosslinking in the presence of TCR signaling resulted in rapid transport of CD244 to an acidic intracellular compartment. Downregulation was not induced by PMA-ionomycin, or prevented by PI3K inhibition, implicating a TCR-proximal signaling mechanism. CD244 internalization occurred within hours of TCR stimulation and required less peptide than was required to induce IFN-γ production. The degree of CD244 internalization varied among cultured CD8 T cell lines of different specificities, and correlated with the enhancement of IFN-γ production in response to CD48 blockade in HIV(+), but not HIV(neg), subjects. Our results indicate that rapid CD244 internalization is induced by a two-signal mechanism and plays a role in modulation of antiviral CD8 T cell responses by CD48-CD244 signaling.

Cutting edge: Prolonged exposure to HIV reinforces a poised epigenetic program for PD-1 expression in virus-specific CD8 T cells.

Ag-specific CD8 T cells play a critical role in controlling HIV infection but eventually lose antiviral functions in part because of expression and signaling through the inhibitory programmed death-1 (PD-1) receptor. To better understand the impact of prolonged TCR ligation on regulation of PD-1 expression in HIV-specific CD8 T cells, we investigated the capacity of virus-specific CD8 T cells to modify the PD-1 epigenetic program after reduction in viral load. We observed that the transcriptional regulatory region was unmethylated in the PD-1(hi) HIV-specific CD8 T cells, whereas it remained methylated in donor-matched naive cells at acute and chronic stages of infection. Surprisingly, the PD-1 promoter remained unmethylated in HIV-specific CD8 T cells from subjects with a viral load controlled by antiviral therapy for >2 y or from elite controllers. Together, these data demonstrate that the epigenetic program at the PD-1 locus becomes fixed after prolonged exposure to HIV virus.

High-dimensional immunomonitoring models of HIV-1-specific CD8 T-cell responses accurately identify subjects achieving spontaneous viral control.

UNLABELLED: The development of immunomonitoring models to determine HIV-1 vaccine efficacy is a major challenge. Studies suggest that HIV-1­specific CD8 T cells play a critical role in subjects achieving spontaneous viral control (HIV-1 controllers) and that they will be important in immune interventions. However, no single CD8 T-cell function is uniquely associated with controller status and the heterogeneity of responses targeting different epitopes further complicates the discovery of determinants of protective immunity. In the present study, we describe immunomonitoring models integrating multiple functions of epitope-specific CD8 T cells that distinguish controllers from subjects with treated or untreated progressive infection. Models integrating higher numbers of variables and trained with the least absolute shrinkage and selection operator (LASSO) variant of logistic regression and 10-fold cross-validation produce "diagnostic tests" that display an excellent capacity to delineate subject categories. The test accuracy reaches 75% area under the receiving operating characteristic curve in cohorts matched for prevalence of protective alleles. Linear mixed-effects model analyses show that the proliferative capacity, cytokine production, and kinetics of cytokine secretion are associated with HIV-1 control. Although proliferative capacity is the strongest single discriminant, integrated modeling of different dimensions of data leverages individual associations. This strategy may have important applications in predictive model development and immune monitoring of HIV-1 vaccine trials. KEY POINTS: Immune monitoring models integrating multiple functions of HIV-1-specific CD8 T cells distinguish controllers from subjects with progressive HIV-1 infection. This strategy may have important applications in predictive model development and immune monitoring of HIV-1 vaccine trials.

Prognostic value of tgfb1 protein in endometrioid adenocarcinoma.

BACKGROUND: Angiogenesis is a prerequisite for tumour development, progression and metastasis; however, its underlying molecular mechanisms in endometrial carcinoma are poorly understood. DESIGN: In this study, the mRNA and protein expression profiles of two key regulators of angiogenesis, vascular endothelial growth factor (VEGF) and transforming growth factor beta-1 (TGFB1), were evaluated by real-time PCR and western blot analysis in 23 endometrial cancer tissue-paired specimens (malignant vs. adjacent normal tissues). We aimed to investigate whether VEGF and TGFB1 serve as markers of the malignant transformation of the endometrium and whether VEGF or TGFB1 expression can constitute a useful prognostic marker of survival in patients with endometrial carcinoma. RESULTS: Tissue-pair analysis revealed VEGF transcriptional up-regulation and TGFB1 mRNA down-regulation as the most frequent transcriptional features. VEGF and TGFB1 mRNA were positively correlated (P < 0·001). VEGF protein levels were higher in endometrioid-type tissue pairs (P = 0·047). TGFB1 protein and mRNA levels were negatively correlated (P = 0·042). TGFB1 protein expression was related to survival only in patients with endometrioid adenocarcinoma (P = 0·045). CONCLUSIONS: Tissue-pair mRNA and protein analysis reveals VEGF transcriptional up-regulation and TGFB1 down-regulation that are correlated with the malignant transformation of the endometrium, while post-transcriptional mechanisms control VEGF and TGFB1 protein. TGFB1 protein demonstrated a prognostic value only in endometrioid adenocarcinoma.