CD8+ T Cell Priming in Established Chronic Viral Infection Preferentially Directs Differentiation of Memory-like Cells for Sustained Immunity.
ABSTRACT: CD8+ T cell exhaustion impedes control of chronic viral infection; yet how new T cell responses are mounted during chronic infection is unclear. Unlike T cells primed at the onset of infection that rapidly differentiate into effectors and exhaust, we demonstrate that virus-specific CD8+ T cells primed after establishment of chronic LCMV infection preferentially generate memory-like transcription factor TCF1+ cells that were transcriptionally and proteomically distinct, less exhausted, and more responsive to immunotherapy. Mechanistically, adaptations of antigen-presenting cells and diminished T cell signaling intensity promoted differentiation of the memory-like subset at the expense of rapid effector cell differentiation, which was now highly dependent on IL-21-mediated CD4+ T cell help for its functional generation. Chronic viral infection similarly redirected de novo differentiation of tumor-specific CD8+ T cells, ultimately preventing cancer control. Thus, targeting these T cell stimulatory pathways could enable strategies to control chronic infection, tumors, and enhance immunotherapeutic efficacy.
Project description:Chronic hepatitis C virus (HCV) infection causes generalized CD8<sup>+</sup> T cell impairment, not limited to HCV-specific CD8<sup>+</sup> T-cells. Liver-infiltrating monocyte-derived macrophages (MDMs) contribute to the local micro-environment and can interact with and influence cells routinely trafficking through the liver, including CD8<sup>+</sup> T-cells. MDMs can be polarized into M1 (classically activated) and M2a, M2b, and M2c (alternatively activated) phenotypes that perform pro- and anti-inflammatory functions, respectively. The impact of chronic HCV infection on MDM subset functions is not known. Our results show that M1 cells generated from chronic HCV patients acquire M2 characteristics, such as increased CD86 expression and IL-10 secretion, compared to uninfected controls. In contrast, M2 subsets from HCV-infected individuals acquired M1-like features by secreting more IL-12 and IFN-γ. The severity of liver disease was also associated with altered macrophage subset differentiation. In co-cultures with autologous CD8<sup>+</sup> T-cells from controls, M1 macrophages alone significantly increased CD8<sup>+</sup> T cell IFN-γ expression in a cytokine-independent and cell-contact-dependent manner. However, M1 macrophages from HCV-infected individuals significantly decreased IFN-γ expression in CD8<sup>+</sup> T-cells. Therefore, altered M1 macrophage differentiation in chronic HCV infection may contribute to observed CD8<sup>+</sup> T-cell dysfunction. Understanding the immunological perturbations in chronic HCV infection will lead to the identification of therapeutic targets to restore immune function in HCV<sup>+</sup> individuals, and aid in the mitigation of associated negative clinical outcomes.
Project description:During chronic infection and cancer, a self-renewing CD8<sup>+</sup> T cell subset maintains long-term immunity and is critical to the effectiveness of immunotherapy. These stem-like CD8<sup>+</sup> T cells diverge from other CD8<sup>+</sup> subsets early after chronic viral infection. However, pathways guarding stem-like CD8<sup>+</sup> T cells against terminal exhaustion remain unclear. Here, we show that the gene encoding transcriptional repressor BACH2 is transcriptionally and epigenetically active in stem-like CD8<sup>+</sup> T cells but not terminally exhausted cells early after infection. BACH2 overexpression enforced stem-like cell fate, whereas BACH2 deficiency impaired stem-like CD8<sup>+</sup> T cell differentiation. Single-cell transcriptomic and epigenomic approaches revealed that BACH2 established the transcriptional and epigenetic programs of stem-like CD8<sup>+</sup> T cells. In addition, BACH2 suppressed the molecular program driving terminal exhaustion through transcriptional repression and epigenetic silencing. Thus, our study reveals a new pathway that enforces commitment to stem-like CD8<sup>+</sup> lineage and prevents an alternative terminally exhausted cell fate.
Project description:Differentiation and fate of virus-specific CD8<sup>+</sup> T cells after cessation of chronic antigen stimulation is unclear. Here we show that a TCF1<sup>+</sup>CD127<sup>+</sup>PD1<sup>+</sup> hepatitis C virus (HCV)-specific CD8<sup>+</sup> T-cell subset exists in chronically infected patients with phenotypic features of T-cell exhaustion and memory, both before and after treatment with direct acting antiviral (DAA) agents. This subset is maintained during, and for a long duration after, HCV elimination. After antigen re-challenge the less differentiated TCF1<sup>+</sup>CD127<sup>+</sup>PD1<sup>+</sup> population expands, which is accompanied by emergence of terminally exhausted TCF1-CD127-PD1<sup>hi</sup> HCV-specific CD8<sup>+</sup> T cells. These results suggest the TCF1<sup>+</sup>CD127<sup>+</sup>PD1<sup>+</sup> HCV-specific CD8<sup>+</sup> T-cell subset has memory-like characteristics, including antigen-independent survival and recall proliferation. We thus provide evidence for the establishment of memory-like virus-specific CD8<sup>+</sup> T cells in a clinically relevant setting of chronic viral infection and we uncover their fate after cessation of chronic antigen stimulation, implicating a potential strategy for antiviral immunotherapy.
Project description:Adoptive T cell therapy (ACT) requires lymphodepletion preconditioning to eliminate immune-suppressive elements and enable efficient engraftment of adoptively transferred tumor-reactive T cells. As anti-CD4 monoclonal antibody depletes CD4<sup>+</sup> immune-suppressive cells, the combination of anti-CD4 treatment and ACT has synergistic potential in cancer therapy. Here, we demonstrate a post-ACT conditioning regimen that involves transient anti-CD4 treatment (CD4<sup>post</sup>). Using murine melanoma, the combined effect of cyclophosphamide preconditioning (CTX<sup>pre</sup>), CD4<sup>post</sup>, and ex vivo primed tumor-reactive CD8<sup>+</sup> T-cell infusion is presented. CTX<sup>pre</sup>/CD4<sup>post</sup> increases tumor suppression and host survival by accelerating the proliferation and differentiation of ex vivo primed CD8<sup>+</sup> T cells and endogenous CD8<sup>+</sup> T cells. Endogenous CD8<sup>+</sup> T cells enhance effector profile and tumor-reactivity, indicating skewing of the TCR repertoire. Notably, enrichment of polyfunctional IL-18Rα<sup>hi</sup> CD8<sup>+</sup> T cell subset is the key event in CTX<sup>pre</sup>/CD4<sup>post</sup>-induced tumor suppression. Mechanistically, the anti-tumor effect of IL-18Rα<sup>hi</sup> subset is mediated by IL-18 signaling and TCR-MHC I interaction. This study highlights the clinical relevance of CD4<sup>post</sup> in ACT and provides insights regarding the immunological nature of anti-CD4 treatment, which enhances anti-tumor response of CD8<sup>+</sup> T cells.
Project description:Crosstalk between T and B cells is crucial for generating high-affinity, class-switched antibody responses. The roles of CD4<sup>+</sup> T cells in this process have been well-characterised. In contrast, regulation of antibody responses by CD8<sup>+</sup> T cells is significantly less defined. CD8<sup>+</sup> T cells are principally recognised for eliciting cytotoxic responses in peripheral tissues and forming protective memory. However, recent findings have identified a novel population of effector CD8<sup>+</sup> T cells that co-opt a differentiation program characteristic of CD4<sup>+</sup> T follicular helper (Tfh) cells, upregulate the chemokine receptor CXCR5 and localise to B cell follicles. While it has been shown that CXCR5<sup>+</sup>CD8<sup>+</sup> T cells mediate the removal of viral reservoirs in the context of follicular-trophic viral infections and maintain the response to chronic insults by virtue of progenitor/stem-like properties, it is not known if CXCR5<sup>+</sup>CD8<sup>+</sup> T cells arise during acute peripheral challenges in the absence of follicular infection and whether they influence B cell responses <i>in vivo</i> in these settings. Using the ovalbumin-specific T cell receptor transgenic (OT-I) system in an adoptive transfer-immunisation/infection model, this study demonstrates that CXCR5<sup>+</sup>CD8<sup>+</sup> T cells arise in response to protein immunisation and peripheral viral infection, displaying a follicular-homing phenotype, expression of cell surface molecules associated with Tfh cells and limited cytotoxic potential. Furthermore, studies assessing the B cell response in the presence of OT-I or <i>Cxcr5<sup>-/-</sup></i> OT-I cells revealed that CXCR5<sup>+</sup>CD8<sup>+</sup> T cells shape the antibody response to protein immunisation and peripheral viral infection, promoting class switching to IgG2c in responding B cells. Overall, the results highlight a novel contribution of CD8<sup>+</sup> T cells to antibody responses, expanding the functionality of the adaptive immune system.
Project description:<h4>Background</h4>HIV-1-specific CD8<sup>+</sup> T cells are required for immune suppression of HIV-1 replication and elimination of the associated viral reservoirs. However, effective induction of functional HIV-1-specific CD8<sup>+</sup> T cells from naïve cells remains problematic in the setting of human vaccine trials. In this study, we investigated priming of functional HIV-1-specific CD8<sup>+</sup> T cells from naïve cells.<h4>Methods</h4>HIV-1-specific CD8<sup>+</sup> T cells were primed from naïve T cells of HIV-1-seronegative individuals using TLR4 ligand LPS or STING ligand 3'3'-cGAMP in vitro. We established HIV-1-specific CD8<sup>+</sup> T cell lines from primed T cells and then investigated functional properties of these cells.<h4>Findings</h4>HIV-1-specific CD8<sup>+</sup> T cells primed with LPS failed to suppress HIV-1. In contrast, 3'3'-cGAMP effectively primed HIV-1-specific CD8<sup>+</sup> T cells with strong ability to suppress HIV-1. 3'3'-cGAMP-primed T cells had higher expression levels of perforin and granzyme B than LPS-primed ones. The expression levels of granzyme B and perforin and viral suppression ability of 3'3'-cGAMP-primed T cells were positively correlated with the production level of type I IFN from PBMCs stimulated with 3'3'-cGAMP.<h4>Interpretation</h4>The present study demonstrates the potential of 3'3'-cGAMP to induce HIV-1-specific CD8<sup>+</sup> T cells with strong effector function from naïve cells via a strong type I IFN production and suggests that this STING ligand may be useful for AIDS vaccine and cure treatment.
Project description:Acquired T cell dysfunction is a hallmark of chronic lymphocytic leukemia (CLL), and is linked to an increased risk of infections, but also reduced immune surveillance and disappointing responses to autologous T cell-based immunotherapy. The mechanisms of T cell dysfunction in CLL are not well understood. Studying immunity against chronic viruses allows for detailed analysis of the effect of CLL on T cells chronically exposed to a specific antigen. Cytomegalovirus (CMV) reactivations are rare in CLL, which corroborates with preserved CMV-specific T cell function. Epstein-Barr virus (EBV) is another herpesvirus that results in chronic infection, but unlike CMV, is characterized by subclinical reactivations in CLL patients. Since both herpesviruses induce strong CD8<sup>+</sup> T cell responses, but have different clinical outcomes, studying these specific T cells may shed light on the mechanisms of CLL-induced T cell dysfunction. We first analyzed the phenotype of EBV-specific CD8<sup>+</sup> T cells in CLL and healthy controls, and found that in CLL EBV-specific CD8<sup>+</sup> T cells are in an advanced differentiation state with higher expression of inhibitory receptors. Secondly, CLL-derived EBV-specific CD8<sup>+</sup> T cells show reduced cytotoxic potential, in contrast to CMV-specific T cells. Finally, we performed transcriptome analysis to visualize differential modulation by CLL of these T cell subsets. While T cell activation and differentiation genes are unaffected, in EBV-specific T cells expression of genes involved in synapse formation and T cell exhaustion is altered. Our findings on the heterogeneity of antigen specific T cell function in CLL aids in understanding immune-dysregulation in this disease.
Project description:Vaccines against Zika virus (ZIKV) infection that target CD8<sup>+</sup> T cells are of considerable interest because Abs may enhance infection susceptibility. However, whether CD8<sup>+</sup> T cells are protective or promote susceptibility to clinical infection symptoms remains uncertain. To more precisely investigate ZIKV-specific CD8<sup>+</sup> T cells in isolation, we engineered a <i>Listeria monocytogenes</i>-based vector to express a single MHC class I-restricted immune dominant peptide, E294-302, from ZIKV envelope protein. We show accumulation of activated ZIKV-specific CD8<sup>+</sup> T cells primed by recombinant <i>L. monocytogenes</i> is associated with reductions in circulating virus levels after ZIKV challenge in type I IFN receptor-deficient mice and wildtype mice administered neutralizing Abs against type I IFN receptor. Interestingly, susceptibility to ZIKV clinical infection including weight loss and mortality each persists and is neither significantly improved nor worsened compared with isogenic <i>L. monocytogenes</i>-primed control mice. These data demonstrating persistent ZIKV clinical susceptibility despite reduced viral burden in mice with expanded virus-specific CD8<sup>+</sup> T cells highlights the need for targeting other adaptive immune components in developing vaccines against ZIKV infection.
Project description:Mutations in <i>SH2D1A</i> gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency disease defined by exquisite sensitivity to the B-lymphotropic Epstein-Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we investigate the hypothesis that SAP is critical for CD8<sup>+</sup> T cell immune surveillance of antigen (Ag)-expressing B cells or B lymphoma cells under conditions of defined T cell receptor (TCR) signaling. <i>Sh2d1a<sup>-</sup><sup>/</sup><sup>-</sup></i> CD8<sup>+</sup> T cells exhibited greatly diminished proliferation relative to wild type when Ag-presenting-B cells or -B lymphoma cells served as the primary Ag-presenting cell (APC). By contrast, <i>Sh2d1a<sup>-</sup><sup>/</sup><sup>-</sup></i> CD8<sup>+</sup> T cells responded equivalently to wild-type CD8<sup>+</sup> T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed Ag presentation. Through application of signaling lymphocyte activation molecule (SLAM) family receptor blocking antibodies or SLAM family receptor-deficient CD8<sup>+</sup> T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the Ag-driven CD8<sup>+</sup> T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naive CD8<sup>+</sup> T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas.
Project description:Some viruses have established an equilibrium with their host. African green monkeys (AGM) display persistent high viral replication in the blood and intestine during Simian immunodeficiency virus (SIV) infection but resolve systemic inflammation after acute infection and lack intestinal immune or tissue damage during chronic infection. We show that NKG2<sub>a/c</sub> <sup>+</sup>CD8<sup>+</sup> T cells increase in the blood and intestine of AGM in response to SIVagm infection in contrast to SIVmac infection in macaques, the latter modeling HIV infection. NKG2<sub>a/c</sub> <sup>+</sup>CD8<sup>+</sup> T cells were not expanded in lymph nodes, and CXCR5<sup>+</sup>NKG2<sub>a/c</sub> <sup>+</sup>CD8<sup>+</sup> T cell frequencies further decreased after SIV infection. Genome-wide transcriptome analysis of NKG2<sub>a/c</sub> <sup>+</sup>CD8<sup>+</sup> T cells from AGM revealed the expression of NK cell receptors, and of molecules with cytotoxic effector, gut homing, and immunoregulatory and gut barrier function, including CD73. NKG2<sub>a/c</sub> <sup>+</sup>CD8<sup>+</sup> T cells correlated negatively with IL-23 in the intestine during SIVmac infection. The data suggest a potential regulatory role of NKG2<sub>a/c</sub> <sup>+</sup>CD8<sup>+</sup> T cells in intestinal inflammation during SIV/HIV infections.