Altered Basal Lipid Metabolism Underlies the Functional Impairment of Naive CD8+ T Cells in Elderly Humans.

Aging is associated with functional deficits in the naive T cell compartment, which compromise the generation of de novo immune responses against previously unencountered Ags. The mechanisms that underlie this phenomenon have nonetheless remained unclear. We found that naive CD8+ T cells in elderly humans were prone to apoptosis and proliferated suboptimally in response to stimulation via the TCR. These abnormalities were associated with dysregulated lipid metabolism under homeostatic conditions and enhanced levels of basal activation. Importantly, reversal of the bioenergetic anomalies with lipid-altering drugs, such as rosiglitazone, almost completely restored the Ag responsiveness of naive CD8+ T cells. Interventions that favor lipid catabolism may therefore find utility as adjunctive therapies in the elderly to promote vaccine-induced immunity against targetable cancers and emerging pathogens, such as seasonal influenza viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Impact of IgA isoforms on their ability to activate dendritic cells and to prime T cells.

Human IgA could be from different isotypes (IgA1/IgA2) and/or isoforms (monomeric, dimeric, or secretory). Monomeric IgA mainly IgA1 are considered as an anti-inflammatory isotype whereas dimeric/secretory IgA have clearly dual pro- and anti-inflammatory effects. Here, we show that IgA isotypes and isoforms display different binding abilities to FcαRI, Dectin-1, DC-SIGN, and CD71 on monocyte-derived dendritic cells (moDC). We describe that IgA regulate the expression of their own receptors and trigger modulation of moDC maturation. We also demonstrate that dimeric IgA2 and IgA1 induce different inflammatory responses leading to cytotoxic CD8+ T cells activation. moDC stimulation by dimeric IgA2 was followed by a strong pro-inflammatory effect. Our study highlights differences regarding IgA isotypes and isoforms in the context of DC conditioning. Further investigations are needed on the activation of adaptive immunity by IgA in the context of microbiota/IgA complexes during antibody-mediated immune selection.

New Insights into Lymphocyte Differentiation and Aging from Telomere Length and Telomerase Activity Measurements.

αß CD8+, γδ, and NK lymphocytes are fundamental effector cells against viruses and tumors. These cells can be divided into multiple subsets according to their phenotype. Based on progressive telomere attrition from naive to late effector memory cells, human CD8+ T cell subsets have been positioned along a pathway of differentiation, which is also considered as a process of lymphocyte aging or senescence. A similar categorization has not been clearly established for γδ and NK cell populations. Moreover, the distinction between the aging of these populations due to cellular differentiation or due to the chronological age of the donor has not been formally considered. In this study, we performed systematic measurements of telomere length and telomerase activity in human αß CD8+, γδ, and NK lymphocytes based on subset division and across age to address these points and better understand the dichotomy between differentiation and temporal aging. This approach enables us to position phenotypically distinct γδ or NK subsets along a putative pathway of differentiation, such as for CD8+ T cells. Moreover, our data show that both cellular differentiation and donor aging have profound but independent effects on telomere length and telomerase activity of lymphocyte subpopulations, implying distinct mechanisms and consequences on the immune system.

Cutting Edge: A Dual TLR2 and TLR7 Ligand Induces Highly Potent Humoral and Cell-Mediated Immune Responses.

TLR agonists are currently being developed and tested as adjuvants in various formulations to optimize the immunogenicity and efficacy of vaccines. The aim of this study was to evaluate the immunostimulatory properties of a novel compound incorporating covalently linked moieties designed to stimulate both TLR2 and TLR7. This dual TLR2/TLR7 agonist induced the maturation of dendritic cells and primed substantial populations of cytolytic and highly polyfunctional effector CD8+ T cells in vitro, and safely potentiated the immunogenic properties of a nanoparticulate Ag in vivo, eliciting humoral responses with a balanced TH1/TH2 profile in mice. Collectively, these data reveal the potential utility of chimeric adjuvants with synergistic activities mediated via TLRs.

Preservation of Lymphopoietic Potential and Virus Suppressive Capacity by CD8+ T Cells in HIV-2-Infected Controllers.

Compared with HIV-1, HIV-2 infection is characterized by a larger proportion of slow or nonprogressors. A better understanding of HIV-2 pathogenesis should open new therapeutic avenues to establish control of HIV-1 replication in infected patients. In this study, we studied the production of CD8(+) T cells and their capacity for viral control in HIV-2 controllers from the French ANRS CO5 HIV-2 cohort. HIV-2 controllers display a robust capacity to support long-term renewal of the CD8(+) T cell compartment by preserving immune resources, including hematopoietic progenitors and thymic activity, which could contribute to the long-term maintenance of the CD8(+) T cell response and the avoidance of premature immune aging. Our data support the presence of HIV-2 Gag-specific CD8(+) T cells that display an early memory differentiation phenotype and robust effector potential in HIV-2 controllers. Accordingly, to our knowledge, we show for the first time that HIV-2 controllers possess CD8(+) T cells that show an unusually strong capacity to suppress HIV-2 infection in autologous CD4(+) T cells ex vivo, an ability that likely depends on the preservation of host immune resources. This effective and durable antiviral response probably participates in a virtuous circle, during which controlled viral replication permits the preservation of potent immune functions, thus preventing HIV-2 disease progression.

CD8 + T-cell priming is quantitatively but not qualitatively impaired in people with HIV-1 on antiretroviral therapy.

BACKGROUND: The induction of de novo CD8 + T-cell responses is essential for protective antiviral immunity, but this process is often impaired in people with HIV-1 (PWH). We investigated the extent to which the immune competence of naive CD8 + T cells, a key determinant of priming efficacy, could be preserved or restored in PWH via long-term antiretroviral therapy (ART). METHODS: We used flow cytometry, molecular analyses of gene transcription and telomere length, and a fully validated priming assay to characterize naive CD8 + T cells ex vivo and evaluate the induction of antigen-specific effector/memory CD8 + T cells in vitro , comparing age-matched healthy uninfected donors (HUDs), PWH on ART, and natural HIV-1 controllers (HICs). RESULTS: We found that naive CD8 + T cells were numerically reduced and exhibited a trend toward shorter telomere lengths in PWH on ART compared with HUDs and HICs. These features associated with impaired priming efficacy. However, we also found that naive CD8 + T cells were fully equipped proliferatively and transcriptionally in PWH on ART, enabling the generation of antigen-specific effector/memory CD8 + T cells with functional and phenotypic attributes comparable to those primed from HUDs. CONCLUSION: Our data suggest that naive CD8 + T cells in PWH on ART are intrinsically capable of generating functionally and phenotypically intact effector/memory CD8 + T cells in response to antigen, despite evidence of senescence and an overall numerical reduction that compromises priming efficacy relative to HUDs and HICs.

Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover.

The key attributes of CD8+ T cell protective immunity in human immunodeficiency virus (HIV) infection remain unclear. We report that CD8+ T cell responses specific for Gag and, in particular, the immunodominant p24 epitope KK10 correlate with control of HIV-1 replication in human histocompatibility leukocyte antigen (HLA)-B27 patients. To understand further the nature of CD8+ T cell-mediated antiviral efficacy, we performed a comprehensive study of CD8+ T cells specific for the HLA-B27-restricted epitope KK10 in chronic HIV-1 infection based on the use of multiparametric flow cytometry together with molecular clonotypic analysis and viral sequencing. We show that B27-KK10-specific CD8+ T cells are characterized by polyfunctional capabilities, increased clonal turnover, and superior functional avidity. Such attributes are interlinked and constitute the basis for effective control of HIV-1 replication. These data on the features of effective CD8+ T cells in HIV infection may aid in the development of successful T cell vaccines.

Exhausted cytotoxic control of Epstein-Barr virus in human lupus.

Systemic Lupus Erythematosus (SLE) pathology has long been associated with an increased Epstein-Barr Virus (EBV) seropositivity, viremia and cross-reactive serum antibodies specific for both virus and self. It has therefore been postulated that EBV triggers SLE immunopathology, although the mechanism remains elusive. Here, we investigate whether frequent peaks of EBV viral load in SLE patients are a consequence of dysfunctional anti-EBV CD8+ T cell responses. Both inactive and active SLE patients (n = 76 and 42, respectively), have significantly elevated EBV viral loads (P = 0.003 and 0.002, respectively) compared to age- and sex-matched healthy controls (n = 29). Interestingly, less EBV-specific CD8+ T cells are able to secrete multiple cytokines (IFN-γ, TNF-α, IL-2 and MIP-1ß) in inactive and active SLE patients compared to controls (P = 0.0003 and 0.0084, respectively). Moreover, EBV-specific CD8+ T cells are also less cytotoxic in SLE patients than in controls (CD107a expression: P = 0.0009, Granzyme B release: P = 0.0001). Importantly, cytomegalovirus (CMV)-specific responses were not found significantly altered in SLE patients. Furthermore, we demonstrate that EBV-specific CD8+ T cell impairment is a consequence of their Programmed Death 1 (PD-1) receptor up-regulation, as blocking this pathway reverses the dysfunctional phenotype. Finally, prospective monitoring of lupus patients revealed that disease flares precede EBV reactivation. In conclusion, EBV-specific CD8+ T cell responses in SLE patients are functionally impaired, but EBV reactivation appears to be an aggravating consequence rather than a cause of SLE immunopathology. We therefore propose that autoimmune B cell activation during flares drives frequent EBV reactivation, which contributes in a vicious circle to the perpetuation of immune activation in SLE patients.

Primary role of type I interferons for the induction of functionally optimal antigen-specific CD8+ T cells in HIV infection.

BACKGROUND: CD8+ T cells equipped with a full arsenal of antiviral effector functions are critical for effective immune control of HIV-1. It has nonetheless remained unclear how best to elicit such potent cellular immune responses in the context of immunotherapy or vaccination. HIV-2 has been associated with milder disease manifestations and more commonly elicits functionally replete virus-specific CD8+ T cell responses compared with HIV-1. We aimed to learn from this immunological dichotomy and to develop informed strategies that could enhance the induction of robust CD8+ T cell responses against HIV-1. METHODS: We developed an unbiased in vitro system to compare the de novo induction of antigen-specific CD8+ T cell responses after exposure to HIV-1 or HIV-2. The functional properties of primed CD8+ T cells were assessed using flow cytometry and molecular analyses of gene transcription. FINDINGS: HIV-2 primed functionally optimal antigen-specific CD8+ T cells with enhanced survival properties more effectively than HIV-1. This superior induction process was dependent on type I interferons (IFNs) and could be mimicked via the adjuvant delivery of cyclic GMP-AMP (cGAMP), a known agonist of the stimulator of interferon genes (STING). CD8+ T cells elicited in the presence of cGAMP were polyfunctional and highly sensitive to antigen stimulation, even after priming from people living with HIV-1. INTERPRETATION: HIV-2 primes CD8+ T cells with potent antiviral functionality by activating the cyclic GMP-AMP synthase (cGAS)/STING pathway, which results in the production of type I IFNs. This process may be amenable to therapeutic development via the use of cGAMP or other STING agonists to bolster CD8+ T cell-mediated immunity against HIV-1. FUNDING: This work was funded by INSERM, the Institut Curie, and the University of Bordeaux (Senior IdEx Chair) and by grants from Sidaction (17-1-AAE-11097, 17-1-FJC-11199, VIH2016126002, 20-2-AEQ-12822-2, and 22-2-AEQ-13411), the Agence Nationale de la Recherche sur le SIDA (ECTZ36691, ECTZ25472, ECTZ71745, and ECTZ118797), and the Fondation pour la Recherche Médicale (EQ U202103012774). D.A.P. was supported by a Wellcome Trust Senior Investigator Award (100326/Z/12/Z).

Antigen sensitivity is a major determinant of CD8+ T-cell polyfunctionality and HIV-suppressive activity.

CD8(+) T cells are major players in the immune response against HIV. However, recent failures in the development of T cell-based vaccines against HIV-1 have emphasized the need to reassess our basic knowledge of T cell-mediated efficacy. CD8(+) T cells from HIV-1-infected patients with slow disease progression exhibit potent polyfunctionality and HIV-suppressive activity, yet the factors that unify these properties are incompletely understood. We performed a detailed study of the interplay between T-cell functional attributes using a bank of HIV-specific CD8(+) T-cell clones isolated in vitro; this approach enabled us to overcome inherent difficulties related to the in vivo heterogeneity of T-cell populations and address the underlying determinants that synthesize the qualities required for antiviral efficacy. Conclusions were supported by ex vivo analysis of HIV-specific CD8(+) T cells from infected donors. We report that attributes of CD8(+) T-cell efficacy against HIV are linked at the level of antigen sensitivity. Highly sensitive CD8(+) T cells display polyfunctional profiles and potent HIV-suppressive activity. These data provide new insights into the mechanisms underlying CD8(+) T-cell efficacy against HIV, and indicate that vaccine strategies should focus on the induction of HIV-specific T cells with high levels of antigen sensitivity to elicit potent antiviral efficacy.

Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections.

The viruses HIV-1, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and hepatitis C virus (HCV) are characterized by the establishment of lifelong infection in the human host, where their replication is thought to be tightly controlled by virus-specific CD8+ T cells. Here we present detailed studies of the differentiation phenotype of these cells, which can be separated into three distinct subsets based on expression of the costimulatory receptors CD28 and CD27. Whereas CD8+ T cells specific for HIV, EBV and HCV exhibit similar characteristics during primary infection, there are significant enrichments at different stages of cellular differentiation in the chronic phase of persistent infection according to the viral specificity, which suggests that distinct memory T-cell populations are established in different virus infections. These findings challenge the current definitions of memory and effector subsets in humans, and suggest that ascribing effector and memory functions to subsets with different differentiation phenotypes is no longer appropriate.

HIV-specific cytotoxic T cells from long-term survivors select a unique T cell receptor.

HIV-specific cytotoxic T lymphocytes (CTL) are important in controlling HIV replication, but the magnitude of the CTL response does not predict clinical outcome. In four donors with delayed disease progression we identified Vbeta13.2 T cell receptors (TCRs) with very similar and unusually long beta-chain complementarity determining region 3 (CDR3) regions in CTL specific for the immunodominant human histocompatibility leukocyte antigens (HLA)-B8-restricted human immunodeficiency virus-1 (HIV-1) nef epitope, FLKEKGGL (FL8). CTL expressing Vbeta13.2 TCRs tolerate naturally arising viral variants in the FL8 epitope that escape recognition by other CTL. In addition, they expand efficiently in vitro and are resistant to apoptosis, in contrast to FL8-specific CTL using other TCRs. Selection of Vbeta13.2 TCRs by some patients early in the FL8-specific CTL response may be linked with better clinical outcome.

Upregulation of interleukin 7 receptor alpha and programmed death 1 marks an epitope-specific CD8+ T-cell response that disappears following primary Epstein-Barr virus infection.

In immunocompetent individuals, the stability of the herpesvirus-host balance limits opportunities to study the disappearance of a virus-specific CD8(+) T-cell response. However, we noticed that in HLA-A 0201-positive infectious mononucleosis (IM) patients undergoing primary Epstein-Barr virus (EBV) infection, the initial CD8 response targets three EBV lytic antigen-derived epitopes, YVLDHLIVV (YVL), GLCTLVAML (GLC), and TLDYKPLSV (TLD), but only the YVL and GLC reactivities persist long-term; the TLD response disappears within 10 to 27 months. While present, TLD-specific cells remained largely indistinguishable from YVL and GLC reactivities in many phenotypic and functional respects but showed unique temporal changes in two markers of T-cell fate, interleukin 7 receptor alpha (IL-7Ralpha; CD127) and programmed death 1 (PD-1). Thus, following the antigen-driven downregulation of IL-7Ralpha seen on all populations in acute IM, in every case, the TLD-specific population recovered expression unusually quickly post-IM. As well, in four of six patients studied, TLD-specific cells showed very strong PD-1 upregulation in the last blood sample obtained before the cells' disappearance. Our data suggest that the disappearance of this individual epitope reactivity from an otherwise stable EBV-specific response (i) reflects a selective loss of cognate antigen restimulation (rather than of IL-7-dependent signals) and (ii) is immediately preceded, and perhaps mediated, by PD-1 upregulation to unprecedented levels.

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.

New chimeric TLR7/NOD2 agonist is a potent adjuvant to induce mucosal immune responses.

BACKGROUND: PRR (Pattern Recognition Receptor) agonists have been widely tested as potent vaccine adjuvants. TLR7 (Toll-Like Receptor 7) and NOD2 (nucleotide-binding oligomerization domain 2) are key innate receptors widely expressed at mucosal levels. METHODS: Here, we evaluated the immunostimulatory properties of a novel hybrid chemical compound designed to stimulate both TLR7 and NOD2 receptors. FINDING: The combined TLR7/NOD2 agonist showed increase efficacy than TLR7L or NOD2L agonists alone or combined in different in vitro models. Dual TLR7/NOD2 agonist efficiently stimulates TLR7 and NOD2, and promotes the maturation and reprogramming of human dendritic cells, as well as the secretion of pro-inflammatory or adaptive cytokines. This molecule also strongly induces autophagy in human cells which is a major intracellular degradation system that delivers cytoplasmic constituents to lysosomes in both MHC class I and II-restricted antigen presentation. In vivo, TLR7/NOD2L agonist is a potent adjuvant after intranasal administration with NP-p24 HIV vaccine, inducing high-quality humoral and adaptive responses both in systemic and mucosal compartments. Use of TLR7/NOD2L adjuvant improves very significantly the protection of mice against an intranasal challenge with a vaccinia virus expressing the p24. INTERPRETATION: Dual TLR7/NOD2L agonist is a very potent and versatile vaccine adjuvant and promote very efficiently both systemic and mucosal immunity. FUNDING: This work was supported by Sidaction.

The STING ligand cGAMP potentiates the efficacy of vaccine-induced CD8+ T cells.

Pathogen recognition receptor (PRR) agonists are currently being developed and tested as adjuvants in various formulations to optimize the immunogenicity and efficacy of vaccines. Using an original in vitro approach to prime naive precursors from unfractionated human peripheral blood mononuclear cells, we assessed the influence of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a ligand for the stimulator of interferon genes (STING), on the induction of antigen-specific CD8+ T cells. We found that 2'3'-cGAMP and 3'3'-cGAMP were especially potent adjuvants in this system, driving the expansion and maturation of functionally replete antigen-specific CD8+ T cells via the induction of type I IFNs. The biological relevance of these findings was confirmed in vivo using two mouse models, in which 2'3'-cGAMP-adjuvanted vaccination elicited protective antitumor or antiviral CD8+ T cell responses. These results identify particular isoforms of cGAMP as effective adjuvants that may find utility in the development of novel immunotherapies and vaccines.

Elderly human hematopoietic progenitor cells express cellular senescence markers and are more susceptible to pyroptosis.

The maintenance of effective immunity over time is dependent on the capacity of hematopoietic stem cells (HSCs) to sustain the pool of immunocompetent mature cells. Decline of immune competence with old age may stem from HSC defects, including reduced self-renewal potential and impaired lymphopoiesis, as suggested in murine models. To obtain further insights into aging-related alteration of hematopoiesis, we performed a comprehensive study of blood hematopoietic progenitor cells (HPCs) from older humans. In the elderly, HPCs present active oxidative phosphorylation and are pressed to enter cell cycling. However, p53-p21 and p15 cell senescence pathways, associated with telomerase activity deficiency, strong telomere attrition, and oxidative stress, are engaged, thus limiting cell cycling. Moreover, survival of old HPCs is impacted by pyroptosis, an inflammatory form of programmed cell death. Lastly, telomerase activity deficiency and telomere length attrition of old HPCs may be passed on to progeny cells such as naive T lymphocytes, further highlighting the poor hematopoietic potential of the elderly. This pre-senescent profile is characteristic of the multiple intrinsic and extrinsic factors affecting HPCs in elderly individuals and represents a major obstacle in terms of immune reconstitution and efficacy with advanced age.

Naïve CD8+ T-Cells Engage a Versatile Metabolic Program Upon Activation in Humans and Differ Energetically From Memory CD8+ T-Cells.

Background: Characterization of the intracellular biochemical processes that regulate the generation and maintenance of effector and memory CD8+ T-cells from naïve precursors is essential for our understanding of adaptive immune responses and the development of immunotherapies. However, the metabolic determinants of antigen-driven activation and differentiation remain poorly defined, especially in humans. Methods: We used a variety of different approaches, including gene expression profiling and measurements of nutrient flux, to characterize the basal and activation-induced energetic requirements of naïve and phenotypically-defined subsets of human memory CD8+ T-cells. Findings: Profound metabolic differences were apparent as a function of differentiation status, both at rest and in response to stimulation via the T cell receptor (TCR). Of particular note, resting naïve CD8+ T cells were largely quiescent, but rapidly upregulated diverse energetic pathways after ligation of surface-expressed TCRs. Moreover, autophagy and the mechanistic target of rapamycin (mTOR)-dependent glycolytic pathway were identified as critical mediators of antigen-driven priming in the naïve CD8+ T cell pool, the efficiency of which was dampened by the presence of neutral lipids and fatty acids. Interpretation: These observations provide a metabolic roadmap of the CD8+ T-cell compartment in humans and reveal potentially selective targets for novel immunotherapies.

Cell permeabilization for the assessment of T lymphocyte polyfunctional capacity.

The rapid progress in flow cytometry means that an increasing number of parameters can be looked at simultaneously, which is highly relevant for the assessment of lymphocyte characteristics, in particular their multifunctional profile. However, procedures using less complex technology need now optimization in order to take into account the new reagents and applications related to polychromatic flow cytometry. Through optimization of an immunomonitoring protocol to assess the functional profile of antigen specific T-cells using 9-10 colour flow cytometry, we tested the efficacy of three distinct standardized permeabilization buffers for the staining of relevant intracellular molecules. We show significant discrepancies in staining sensitivity for cytokine and cytotoxic factor expression from one permeabilization kit to another, which can lead to different data and interpretation. It is important to be aware of this potential bias and to design specific application/experimental procedures in order to obtain optimal results.

Accelerated immune senescence and HIV-1 infection.

A recent consensus has emerged regarding the association between chronic immune activation and poor outcome in HIV-1 infection. However, its basis remains unclear. Accumulating evidence suggests that the cells of the immune system may have a limited replicative lifespan in vivo. In this context, persistent activation during chronic HIV infection may lead to an exhaustion of immune resources. This may occur at two levels: Clonal and Global. Some HIV-1-specific CD8+ T-cells start expressing the senescence marker CD57 soon after primary infection. Persistently activated HIV-1-specific T-cell clones may eventually reach stages of replicative senescence and disappear, resulting in the specific loss of CD8+ T-cell populations important to control viral replication. In addition, HIV-1 infected individuals are characterized by the accumulation of highly differentiated CD8+ and CD4+ T-cells overtime. Together with the decline of T-cell renewal capacities, this may reflect a general ageing of the lymphocyte population. Similar observations have been done in HIV non-infected elderly individuals, which suggests that premature immunosenescence occurs in HIV-1 infection, as a result of persistent immune activation.