The TLR9 ligand CpG ODN 2006 is a poor adjuvant for the induction of de novo CD8+ T-cell responses in vitro.

Toll-like receptor 9 (TLR9) agonists have gained traction in recent years as potential adjuvants for the induction of adaptive immune responses. It has nonetheless remained unclear to what extent such ligands can facilitate the priming events that generate antigen-specific effector and/or memory CD8+ T-cell populations. We used an established in vitro model to prime naive precursors from human peripheral blood mononuclear cells in the presence of various adjuvants, including CpG ODN 2006, a synthetic oligonucleotide TLR9 ligand (TLR9L). Unexpectedly, we found that TLR9L induced a suboptimal inflammatory milieu and promoted the antigen-driven expansion and functional maturation of naive CD8+ T cells ineffectively compared with either ssRNA40 or 2'3'-cGAMP, which activate other pattern recognition receptors (PRRs). TLR9L also inhibited the priming efficacy of 2'3'-cGAMP. Collectively, these results suggest that TLR9L is unlikely to be a good candidate for the optimal induction of de novo CD8+ T-cell responses, in contrast to adjuvants that operate via discrete PRRs.

Priming of HIV-1-specific CD8+ T cells with strong functional properties from naïve T cells.

BACKGROUND: HIV-1-specific CD8+ 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+ 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+ T cells from naïve cells. METHODS: HIV-1-specific CD8+ 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+ T cell lines from primed T cells and then investigated functional properties of these cells. FINDINGS: HIV-1-specific CD8+ T cells primed with LPS failed to suppress HIV-1. In contrast, 3'3'-cGAMP effectively primed HIV-1-specific CD8+ 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. INTERPRETATION: The present study demonstrates the potential of 3'3'-cGAMP to induce HIV-1-specific CD8+ 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.

Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors.

The human leukocyte antigen-A2 (HLA-A2)-restricted zinc transporter 8186-194 (ZnT8186-194) and other islet epitopes elicit interferon-γ secretion by CD8+ T cells preferentially in type 1 diabetes (T1D) patients compared with controls. We show that clonal ZnT8186-194-reactive CD8+ T cells express private T cell receptors and display equivalent functional properties in T1D and healthy individuals. Ex vivo analyses further revealed that CD8+ T cells reactive to ZnT8186-194 and other islet epitopes circulate at similar frequencies and exhibit a predominantly naïve phenotype in age-matched T1D and healthy donors. Higher frequencies of ZnT8186-194-reactive CD8+ T cells with a more antigen-experienced phenotype were detected in children versus adults, irrespective of disease status. Moreover, some ZnT8186-194-reactive CD8+ T cell clonotypes were found to cross-recognize a Bacteroides stercoris mimotope. Whereas ZnT8 was poorly expressed in thymic medullary epithelial cells, variable thymic expression levels of islet antigens did not modulate the peripheral frequency of their cognate CD8+ T cells. In contrast, ZnT8186-194-reactive cells were enriched in the pancreata of T1D patients versus nondiabetic and type 2 diabetic individuals. Thus, islet-reactive CD8+ T cells circulate in most individuals but home to the pancreas preferentially in T1D patients. We conclude that the activation of this common islet-reactive T cell repertoire and progression to T1D likely require defective peripheral immunoregulation and/or a proinflammatory islet microenvironment.

Divergent roles for antigenic drive in the aetiology of primary versus dasatinib-associated CD8+ TCR-Vß+ expansions.

CD8+ T-cell expansions are the primary manifestation of T-cell large granular lymphocytic leukemia (T-LGLL), which is frequently accompanied by neutropenia and rheumatoid arthritis, and also occur as a secondary phenomenon in leukemia patients treated with dasatinib, notably in association with various drug-induced side-effects. However, the mechanisms that underlie the genesis and maintenance of expanded CD8+ T-cell receptor (TCR)-Vß+ populations in these patient groups have yet to be fully defined. In this study, we performed a comprehensive phenotypic and clonotypic assessment of expanded (TCR-Vß+) and residual (TCR-Vß-) CD8+ T-cell populations in T-LGLL and dasatinib-treated chronic myelogenous leukemia (CML) patients. The dominant CD8+ TCR-Vß+ expansions in T-LGLL patients were largely monoclonal and highly differentiated, whereas the dominant CD8+ TCR-Vß+ expansions in dasatinib-treated CML patients were oligoclonal or polyclonal, and displayed a broad range of memory phenotypes. These contrasting features suggest divergent roles for antigenic drive in the immunopathogenesis of primary versus dasatinib-associated CD8+ TCR-Vß+ expansions.

Fine-tuning of CD8(+) T-cell effector functions by targeting the 2B4-CD48 interaction.

Polyfunctionality and cytotoxic activity dictate CD8(+) T-cell efficacy in the eradication of infected and malignant cells. The induction of these effector functions depends on the specific interaction between the T-cell receptor (TCR) and its cognate peptide-MHC class I complex, in addition to signals provided by co-stimulatory or co-inhibitory receptors, which can further regulate these functions. Among these receptors, the role of 2B4 is contested, as it has been described as either co-stimulatory or co-inhibitory in modulating T-cell functions. We therefore combined functional, transcriptional and epigenetic approaches to further characterize the impact of disrupting the interaction of 2B4 with its ligand CD48, on the activity of human effector CD8(+) T-cell clones. In this setting, we show that the 2B4-CD48 axis is involved in the fine-tuning of CD8(+) T-cell effector function upon antigenic stimulation. Blocking this interaction resulted in reduced CD8(+) T-cell clone-mediated cytolytic activity, together with a subtle drop in the expression of genes involved in effector function regulation. Our results also imply a variable contribution of the 2B4-CD48 interaction to the modulation of CD8(+) T-cell functional properties, potentially linked to intrinsic levels of T-bet expression and TCR avidity. The present study thus provides further insights into the role of the 2B4-CD48 interaction in the fine regulation of CD8(+) T-cell effector function upon antigenic stimulation.

TCR clonotypes: molecular determinants of T-cell efficacy against HIV.

Because of the enormous complexity and breadth of the overall HIV-specific CD8(+) T-cell response, invaluable information regarding important aspects of T-cell efficacy against HIV can be sourced from studies performed on individual clonotypes. Data gathered from ex vivo and in vitro analyses of T-cell responses and viral evolution bring us one step closer towards deciphering the correlates of protection against HIV. HIV-responsive CD8(+) T-cell populations are characterized by specific clonotypic immunodominance patterns and public TCRs. The TCR endows T-cells with two key features, important for the effective control of HIV: avidity and crossreactivity. While TCR avidity is a major determinant of CD8(+) T-cell functional efficacy against the virus, crossreactivity towards wildtype and mutant viral epitopes is crucial for adaptation to HIV evolution. The properties of CD4(+) T-cell responses in HIV controllers appear also to be shaped by high avidity public TCR clonotypes. The molecular nature of the TCR, together with the clonotypic composition of the HIV-specific T-cell response, emerge as major determinants of anti-viral efficacy.

Reduced naïve CD8(+) T-cell priming efficacy in elderly adults.

Aging is associated with impaired vaccine efficacy and increased susceptibility to infectious and malignant diseases. CD8(+) T-cells are key players in the immune response against pathogens and tumors. In aged mice, the dwindling naïve CD8(+) T-cell compartment is thought to compromise the induction of de novo immune responses, but no experimental evidence is yet available in humans. Here, we used an original in vitro assay based on an accelerated dendritic cell coculture system in unfractioned peripheral blood mononuclear cells to examine CD8(+) T-cell priming efficacy in human volunteers. Using this approach, we report that old individuals consistently mount quantitatively and qualitatively impaired de novo CD8(+) T-cell responses specific for a model antigen. Reduced CD8(+) T-cell priming capacity in vitro was further associated with poor primary immune responsiveness in vivo. This immune deficit likely arises as a consequence of intrinsic cellular defects and a reduction in the size of the naïve CD8(+) T-cell pool. Collectively, these findings provide new insights into the cellular immune insufficiencies that accompany human aging.

Priming of Qualitatively Superior Human Effector CD8+ T Cells Using TLR8 Ligand Combined with FLT3 Ligand.

The quality of Ag-specific CD8(+) T cell responses is central to immune efficacy in infectious and malignant settings. Inducing effector CD8(+) T cells with potent functional properties is therefore a priority in the field of immunotherapy. However, the optimal assessment of new treatment strategies in humans is limited by currently available testing platforms. In this study, we introduce an original model of in vitro CD8(+) T cell priming, based on an accelerated dendritic cell coculture system, which uses unfractionated human PBMCs as the starting material. This approach enables the rapid evaluation of adjuvant effects on the functional properties of human CD8(+) T cells primed from Ag-specific naive precursors. We demonstrate that a selective TLR8 agonist, in combination with FLT3L, primes high-quality CD8(+) T cell responses. TLR8L/FLT3L-primed CD8(+) T cells displayed enhanced cytotoxic activity, polyfunctionality, and Ag sensitivity. The acquisition of this superior functional profile was associated with increased T-bet expression induced via an IL-12-dependent mechanism. Collectively, these data validate an expedited route to vaccine delivery or optimal T cell expansion for adoptive cell transfer.

The link between CD8⁺ T-cell antigen-sensitivity and HIV-suppressive capacity depends on HLA restriction, target epitope and viral isolate.

BACKGROUND: Although it is established that CD8 T-cell immunity is critical for the control of HIV replication in vivo, the key factors that determine antiviral efficacy are yet to be fully elucidated. Antigen-sensitivity and T-cell receptor (TCR) avidity have been identified as potential determinants of CD8⁺ T-cell efficacy. However, there is no general consensus in this regard because the relationship between these parameters and the control of HIV infection has been established primarily in the context of immunodominant CD8⁺ T-cell responses against the Gag263₋272 KK10 epitope restricted by human leukocyte antigen (HLA)-B27. METHODS: To investigate the relationship between antigen-sensitivity, TCR avidity and HIV-suppressive capacity in vitro across epitope specificities and HLA class I restriction elements, we used a variety of techniques to study CD8⁺ T-cell clones specific for Nef73₋82 QK10 and Gag20₋29 RY10, both restricted by HLA-A3, alongside CD8⁺ T-cell clones specific for Gag263₋272 KK10. RESULTS: For each targeted epitope, the linked parameters of antigen-sensitivity and TCR avidity correlated directly with antiviral efficacy. However, marked differences in HIV-suppressive capacity were observed between epitope specificities, HLA class I restriction elements and viral isolates. CONCLUSIONS: Collectively, these data emphasize the central role of the TCR as a determinant of CD8⁺ T-cell efficacy and demonstrate that the complexities of antigen recognition across epitope and HLA class I boundaries can confound simple relationships between TCR engagement and HIV suppression.

T-cell receptor-optimized peptide skewing of the T-cell repertoire can enhance antigen targeting.

Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8(+) T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A(27-35) (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8(+) T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8(+) T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201(+) individuals that were capable of efficient HLA A*0201(+) melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.