The two enantiomers of 2-hydroxyglutarate differentially regulate cytotoxic T cell function.

2-Hydroxyglutarate (2HG) is a byproduct of the tricarboxylic acid (TCA) cycle and is readily detected in the tissues of healthy individuals. 2HG is found in two enantiomeric forms: S-2HG and R-2HG. Here, we investigate the differential roles of these two enantiomers in cluster of differentiation (CD)8+ T cell biology, where we find they have highly divergent effects on proliferation, differentiation, and T cell function. We show here an analysis of structural determinants that likely underlie these differential effects on specific α-ketoglutarate (αKG)-dependent enzymes. Treatment of CD8+ T cells with exogenous S-2HG, but not R-2HG, increased CD8+ T cell fitness in vivo and enhanced anti-tumor activity. These data show that S-2HG and R-2HG should be considered as two distinct and important actors in the regulation of T cell function.

Need to know which MHCs protect against COVID? There's an App for that!

In this article, we discuss the recent observation by Augusto et al. made using a novel mobile phone application-based COVID-19 Citizen Science Study that an HLA genetic variant, HLA-B*15:01, is associated with asymptomatic SARS-CoV-2 infection. To explain this association, Augusto et al. describe a cross-reactive memory CD8+ T-cell response in HLA-B*15:01+ SARS-CoV-2 unexposed individuals that retains high avidity for two structurally conserved epitopes found in SARS-CoV-2 and seasonal coronavirus strains. These observations provide an insight into potential molecular determinants that facilitate rapid, early clearance of virus.

Asymmetric cell division safeguards memory CD8 T cell development.

The strength of T cell receptor (TCR) stimulation and asymmetric distribution of fate determinants are both implied to affect T cell differentiation. Here, we uncover asymmetric cell division (ACD) as a safeguard mechanism for memory CD8 T cell generation specifically upon strong TCR stimulation. Using live imaging approaches, we find that strong TCR stimulation induces elevated ACD rates, and subsequent single-cell-derived colonies comprise both effector and memory precursor cells. The abundance of memory precursor cells emerging from a single activated T cell positively correlates with first mitosis ACD. Accordingly, preventing ACD by inhibition of protein kinase Cζ (PKCζ) during the first mitosis upon strong TCR stimulation markedly curtails the formation of memory precursor cells. Conversely, no effect of ACD on fate commitment is observed upon weak TCR stimulation. Our data provide relevant mechanistic insights into the role of ACD for CD8 T cell fate regulation upon different activation conditions.

T-cell heterogeneity, progenitor-progeny relationships, and function during latent and chronic viral infections.

Upon resolution of an acute viral infection, during latent-reactivating infection and during chronic active infections virus-specific T-cells differentiate into distinct subsets that differ in phenotype, longevity, transcriptional, metabolic, and epigenetic profiles, and effector functions. With recent advances in single-cell profiling, this substantial heterogeneity has become apparent and new subsets of virus-specific T cells, either of stable or transitory nature, are being identified. A unifying principle of T cells emerging in these different conditions is their precursor-progeny relationship. For acute and resolved viral infections, this relationship becomes apparent during re-challenge, whereas a constant differentiation of progenitor T cells into more differentiated cells occurs during latent-reactivating and active chronic viral infections. In this review, we summarize and discuss current knowledge about T-cell heterogeneity and progenitor-progeny relationships in the setting of persistent viral infections.

STING controls T cell memory fitness during infection through T cell-intrinsic and IDO-dependent mechanisms.

Stimulator of interferon genes (STING) signaling has been extensively studied in inflammatory diseases and cancer, while its role in T cell responses to infection is unclear. Using Listeria monocytogenes strains engineered to induce different levels of c-di-AMP, we found that high STING signals impaired T cell memory upon infection via increased Bim levels and apoptosis. Unexpectedly, reduction of TCR signal strength or T cell-STING expression decreased Bim expression, T cell apoptosis, and recovered T cell memory. We found that TCR signal intensity coupled STING signal strength to the unfolded protein response (UPR) and T cell survival. Under strong STING signaling, Indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibition also reduced apoptosis and led to a recovery of T cell memory in STING sufficient CD8 T cells. Thus, STING signaling regulates CD8 T cell memory fitness through both cell-intrinsic and extrinsic mechanisms. These studies provide insight into how IDO and STING therapies could improve long-term T cell protective immunity.

Responsiveness to interleukin-15 therapy is shared between tissue-resident and circulating memory CD8+ T cell subsets.

Interleukin-15 (IL-15) is often considered a central regulator of memory CD8+ T cells, based primarily on studies of recirculating subsets. However, recent work identified IL-15-independent CD8+ T cell memory populations, including tissue-resident memory CD8+ T cells (TRM) in some nonlymphoid tissues (NLTs). Whether this reflects the existence of IL-15-insensitive memory CD8+ T cells is unclear. We report that IL-15 complexes (IL-15c) stimulate rapid proliferation and expansion of both tissue-resident and circulating memory CD8+ T cell subsets across lymphoid and nonlymphoid tissues with varying magnitude by tissue and memory subset, in some sites correlating with differing levels of the IL-2Rß. This was conserved for memory CD8+ T cells recognizing distinct antigens and elicited by different pathogens. Following IL-15c-induced expansion, divided cells contracted to baseline numbers and only slowly returned to basal proliferation, suggesting a mechanism to transiently amplify memory populations. Through parabiosis, we showed that IL-15c drive local proliferation of TRM, with a degree of recruitment of circulating cells to some NLTs. Hence, irrespective of homeostatic IL-15 dependence, IL-15 sensitivity is a defining feature of memory CD8+ T cell populations, with therapeutic potential for expansion of TRM and other memory subsets in an antigen-agnostic and temporally controlled fashion.

Expression of NAC1 Restrains the Memory Formation of CD8+ T Cells during Viral Infection.

Nucleus accumbens-associated protein 1 (NAC1) is a transcription co-factor that has been shown to possess multiple roles in stem cell and cancer biology. However, little is known about its roles in regulation of the immune system. In the current study, we observed that expression of NAC1 impacted the survival of CD8+ T cells in vitro. NAC1-/- CD8+ T cells displayed lower metabolism, including reduced glycolysis and oxidative phosphorylation. In vivo, compared with wild-type (WT) mice, NAC1-/- mice produced a lower response to vaccinia virus (VACV) infection, and viral antigen (Ag)-specific CD8+ T cells decreased more slowly. Additionally, we observed that the NAC1-/- mice demonstrated a stronger memory formation of viral Ag-specific CD8+ T cells post-viral infection. Mechanically, we identified that compared with WT CD8+ T cells, the Interferon Regulatory Factor 4 (IRF4), a key transcription factor in T cell development, was highly expressed in NAC1-/- CD8+ T cells, insinuating that IRF4 could be a critical regulatory target of NAC1 in the memory formation of CD8+ T cells. Our results indicate that NAC1 restrains the memory formation of CD8+ T cells by modulating IRF4, and targeting NAC1 may be exploited as a new approach to boosting CD8+ T cell memory.

PI3Kδ coordinates transcriptional, chromatin, and metabolic changes to promote effector CD8+ T cells at the expense of central memory.

Patients with activated phosphatidylinositol 3-kinase delta (PI3Kδ) syndrome (APDS) present with sinopulmonary infections, lymphadenopathy, and cytomegalvirus (CMV) and/or Epstein-Barr virus (EBV) viremia, yet why patients fail to clear certain chronic viral infections remains incompletely understood. Using patient samples and a mouse model (Pik3cdE1020K/+ mice), we demonstrate that, upon activation, Pik3cdE1020K/+ CD8+ T cells exhibit exaggerated features of effector populations both in vitro and after viral infection that are associated with increased Fas-mediated apoptosis due to sustained FoxO1 phosphorylation and Fasl derepression, enhanced mTORC1 and c-Myc signatures, metabolic perturbations, and an altered chromatin landscape. Conversely, Pik3cdE1020K/+ CD8+ cells fail to sustain expression of proteins critical for central memory, including TCF1. Strikingly, activated Pik3cdE1020K/+ CD8+ cells exhibit altered transcriptional and epigenetic circuits characterized by pronounced interleukin-2 (IL-2)/STAT5 signatures and heightened IL-2 responses that prevent differentiation to memory-like cells in IL-15. Our data position PI3Kδ as integrating multiple signaling nodes that promote CD8+ T cell effector differentiation, providing insight into phenotypes of patients with APDS.

Runx proteins and transcriptional mechanisms that govern memory CD8 T cell development.

Adaptive immunity to intracellular pathogens and tumors is mediated by antigen-experienced CD8 T cells. Individual naive CD8 T cells have the potential to differentiate into a diverse array of antigen-experienced subsets that exhibit distinct effector functions, life spans, anatomic positioning, and potential for regenerating an entirely new immune response during iterative pathogenic exposures. The developmental process by which activated naive cells undergo diversification involves regulation of chromatin structure and transcription but is not entirely understood. This review examines how alterations in chromatin structure, transcription factor binding, extracellular signals, and single-cell gene expression explain the differential development of distinct effector (TEFF ) and memory (TMEM ) CD8 T cell subsets. Special emphasis is placed on how Runx proteins function with additional transcription factors to pioneer changes in chromatin accessibility and drive transcriptional programs that establish the core attributes of cytotoxic T lymphocytes, subdivide circulating and non-circulating TMEM cell subsets, and govern terminal differentiation. The discussion integrates the roles of specific cytokine signals, transcriptional circuits and how regulation of individual nucleosomes and RNA polymerase II activity can contribute to the process of differentiation. A model that integrates many of these features is discussed to conceptualize how activated CD8 T cells arrive at their fates.

Sustained IL-2R signaling of limited duration by high-dose mIL-2/mCD25 fusion protein amplifies tumor-reactive CD8+ T cells to enhance antitumor immunity.

High-dose IL-2 induces cancer regression but its therapeutic use is limited due to high toxicities resulting from its broad cell targeting. In one strategy to overcome this limitation, IL-2 has been modified to selectively target the intermediate affinity IL-2R that broadly activates memory-phenotypic CD8+ T and NK cells, while minimizing Treg-associated tolerance. In this study, we modeled an alternative strategy to amplify tumor antigen-specific TCR transgenic CD8+ T cells through limited application of a long-acting IL-2 fusion protein, mIL-2/mCD25, which selectively targets the high-affinity IL-2R. Here, mice were vaccinated with a tumor antigen and high-dose mIL-2/mCD25 was applied to coincide with the induction of the high affinity IL-2R on tumor-specific T cells. A single high dose of mIL-2/mCD25, but not an equivalent amount of IL-2, amplified the frequency and function of tumor-reactive CD8+ T effector (Teff) and memory cells. These mIL-2/mCD25-dependent effects relied on distinctive requirements for TLR signals during priming of CD8+ tumor-specific T cells. The mIL-2/mCD25-amplified tumor-reactive effector and memory T cells supported long-lasting antitumor responses to B16-F10 melanoma. This regimen only transiently increased Tregs, yielding a favorable Teff-Treg ratio within the tumor microenvironment. Notably, mIL-2/mCD25 did not increase non-tumor-specific Teff or NK cells within tumors, further substantiating the specificity of mIL-2/mCD25 for tumor antigen-activated T cells. Thus, the selectivity and persistence of mIL-2/mCD25 in conjunction with a tumor vaccine supports antitumor immunity through a mechanism that is distinct from recombinant IL-2 or IL-2-based biologics that target the intermediate affinity IL-2R.

Cross-talk between iNKT cells and CD8 T cells in the spleen requires the IL-4/CCL17 axis for the generation of short-lived effector cells.

Mounting an effective immune response relies critically on the coordinated interactions between adaptive and innate compartments. How and where immune cells from these different compartments interact is still poorly understood. Here, we demonstrate that the cross-talk between invariant natural killer T cells (iNKT) and CD8+ T cells in the spleen, essential for initiating productive immune responses, is biphasic and occurs at 2 distinct sites. Codelivery of antigen and adjuvant to antigen-presenting cells results in: 1) initial short-lived interactions (0 to 6 h), between CD8+ T cells, dendritic cells (DCs), and iNKT cells recruited outside the white pulp; 2) followed by long-lasting contacts (12 to 24 h) between iNKT cells, DCs, and CD8+ T cells occurring in a 3-way interaction profile within the white pulp. Both CXCR3 and CCR4 are essential to orchestrate this highly dynamic process and play nonredundant in T cell memory generation. While CXCR3 promotes memory T cells, CCR4 supports short-lived effector cell generation. We believe our work provides insights into the initiation of T cell responses in the spleen and their consequences for T cell differentiation.

Keratinocyte-Mediated Activation of the Cytokine TGF-ß Maintains Skin Recirculating Memory CD8+ T Cells.

Regulated activation of the cytokine TGF-ß by integrins αvß6 and αvß8 expressed on keratinocytes is required for residence of epidermal-resident memory T cells, but whether skin-derived signals also affect recirculating memory cells in the skin remains unclear. Here, we show that after resolution of skin vaccinia virus (VV) infection, antigen-specific circulating memory CD8+ T cells migrated into skin. In mice lacking αvß6 and αvß8 integrins (Itgb6-/-Itgb8fl/fl-K14-cre), the absence of epidermal-activated TGF-ß resulted in a gradual loss of E- or P-selectin-binding central and peripheral memory populations, which were rescued when skin entry was inhibited. Skin recirculating memory cells were required for optimal host defense against skin VV infection. These data demonstrate that skin migration can persist after resolution of local skin infection and that the cytokine environment within this nonlymphoid tissue shapes the differentiation state and persistence of the central and peripheral memory-T-cell pool.

Biological sex affects vaccine efficacy and protection against influenza in mice.

Biological sex affects adaptive immune responses, which could impact influenza infection and vaccine efficacy. Infection of mice with 2009 H1N1 induced antibody responses, CD4+ T cell and CD8+ T cell memory responses that were greater in females than males; both sexes, however, were equally protected against secondary challenge with an H1N1 drift variant virus. To test whether greater antibody in females is sufficient for protection against influenza, males and females were immunized with an inactivated H1N1 vaccine that induced predominantly antibody-mediated immunity. Following vaccination, females had greater antibody responses and protection against challenge with an H1N1 drift variant virus than males. Antibody derived from vaccinated females was better at protecting both naïve males and females than antibody from males, and this protection was associated with increased antibody specificity and avidity to the H1N1 virus. The expression of Tlr7 was greater in B cells from vaccinated females than males and was associated with reduced DNA methylation in the Tlr7 promoter region, higher neutralizing antibody, class switch recombination, and antibody avidity in females. Deletion of Tlr7 reduced sex differences in vaccine-induced antibody responses and protection following challenge and had a greater impact on responses in females than males. Taken together, these data illustrate that greater TLR7 activation and antibody production in females improves the efficacy of vaccination against influenza.

Potential killers exposed: tracking endogenous influenza-specific CD8+ T cells.

Current influenza A virus (IAV) vaccines stimulate antibody responses that are directed against variable regions of the virus, and are therefore ineffective against divergent strains. As CD8+ T cells target the highly conserved, internal IAV proteins, they have the potential to increase heterosubtypic immunity. Early T-cell priming events influence lasting memory, which is required for long-term protection. However, the early responding, IAV-specific cells are difficult to monitor because of their low frequencies. Here, we tracked the dissemination of endogenous IAV-specific CD8+ T cells during the initial phases of the immune response following IAV infection. We exposed a significant population of recently activated, CD25+ CD43+ IAV-specific T cells that were not detected by tetramer staining. By tracking this population, we found that initial T-cell priming occurred in the mediastinal lymph nodes, which gave rise to the most expansive IAV-specific CD8+ T-cell population. Subsequently, IAV-specific CD8+ T cells dispersed to the bronchoalveolar lavage and blood, followed by spleen and liver, and finally to the lung. These data provide important insight into the priming and tissue dispersion of an endogenous CD8+ T-cell response. Importantly, the CD25+ CD43+ phenotype identifies an inclusive population of early responding CD8+ T cells, which may provide insight into TCR repertoire selection and expansion. A better understanding of this response is critical for designing improved vaccines that target CD8+ T cells.

The Fate Choice Between Effector and Memory T Cell Lineages: Asymmetry, Signal Integration, and Feedback to Create Bistability.

CD8+ T cells clear primary infections with intracellular pathogens and provide long-term immunity against reinfection. Two different types of CD8+ T cells are responsible for these functions: short-lived effector T cells and memory T cells. The cellular relationship between these two types of CD8+ T cells has been subject to much investigation. Both cell types can derive from a single naïve CD8+ T cell precursor. Their generation requires a fate choice early during a T cell response. As a result, two populations of T cells emerge. One of these consists of terminally differentiated short-lived effector T cells. The other contains cells able to develop into long-lived memory T cells. A foundation for development of these two populations may be laid during the first division of an activated naïve T cell precursor, as a consequence of asymmetric segregation of fate-determining factors into the daughter cells. Nonetheless, the binary choice between the two lineages is strongly influenced by signals, which ensure that the differentiation process is matched with the needs posed by the infection. Here, we will discuss the genetic and metabolic programs governing differentiation of these two lineages as well as the processes leading to their induction and consolidation to create bistability. These processes involve extensive lateral inhibition between the programs as well as positive feedback between the genetic programs and the signaling pathways responsible for their induction. These features will be highlighted by discussing the role of the Notch signaling pathway in guiding the decision between the two lineages.

In Vitro-Generated Tc17 Cells Present a Memory Phenotype and Serve As a Reservoir of Tc1 Cells In Vivo.

Memory CD8+ T cells are ideal candidates for cancer immunotherapy because they can mediate long-term protection against tumors. However, the therapeutic potential of different in vitro-generated CD8+ T cell effector subsets to persist and become memory cells has not been fully characterized. Type 1 CD8+ T (Tc1) cells produce interferon-γ and are endowed with high cytotoxic capacity, whereas IL-17-producing CD8+ T (Tc17) cells are less cytotoxic but display enhanced self-renewal capacity. We sought to evaluate the functional properties of in vitro-generated Tc17 cells and elucidate their potential to become long lasting memory cells. Our results show that in vitro-generated Tc17 cells display a greater in vivo persistence and expansion in response to secondary antigen stimulation compared to Tc1 cells. When transferred into recipient mice, Tc17 cells persist in secondary lymphoid organs, present a recirculation behavior consistent with central memory T cells, and can shift to a Tc1 phenotype. Accordingly, Tc17 cells are endowed with a higher mitochondrial spare respiratory capacity than Tc1 cells and express higher levels of memory-related molecules than Tc1 cells. Together, these results demonstrate that in vitro-generated Tc17 cells acquire a central memory program and provide a lasting reservoir of Tc1 cells in vivo, thus supporting the use of Tc17 lymphocytes in the design of novel and more effective therapies.

Regulation of Effector and Memory CD8 T Cell Differentiation by IL-2-A Balancing Act.

Interleukin-2 (IL-2) regulates key aspects of CD8 T cell biology-signaling through distinct pathways IL-2 triggers critical metabolic and transcriptional changes that lead to a spectrum of physiological outcomes such as cell survival, proliferation, and effector differentiation. In addition to driving effector differentiation, IL-2 signals are also critical for formation of long-lived CD8 T cell memory. This review discusses a model of rheostatic control of CD8 T cell effector and memory differentiation by IL-2, wherein the timing, duration, dose, and source of IL-2 signals are considered in fine-tuning the balance of key transcriptional regulators of cell fate.

CD103+ CD8 T Cells in the Toxoplasma-Infected Brain Exhibit a Tissue-Resident Memory Transcriptional Profile.

During chronic infection, memory T cells acquire a unique phenotype and become dependent on different survival signals than those needed for memory T cells generated during an acute infection. The distinction between the role of effector and memory T cells in an environment of persistent antigen remains unclear. Here, in the context of chronic Toxoplasma gondii infection, we demonstrate that a population of CD8 T cells exhibiting a tissue-resident memory (TRM) phenotype accumulates within the brain. We show that this population is distributed throughout the brain in both parenchymal and extraparenchymal spaces. Furthermore, this population is transcriptionally distinct and exhibits a transcriptional signature consistent with the TRM observed in acute viral infections. Finally, we establish that the CD103+ TRM population has an intrinsic capacity to produce both IFN-γ and TNF-α, cytokines critical for parasite control within the central nervous system (CNS). The contribution of this population to pro-inflammatory cytokine production suggests an important role for TRM in protective and ongoing immune responses in the infected CNS. Accession number: GSE95105.

NFκB-Pim-1-Eomesodermin axis is critical for maintaining CD8 T-cell memory quality.

T-cell memory is critical for long-term immunity. However, the factors involved in maintaining the persistence, function, and phenotype of the memory pool are undefined. Eomesodermin (Eomes) is required for the establishment of the memory pool. Here, we show that in T cells transitioning to memory, the expression of high levels of Eomes is not constitutive but rather requires a continuum of cell-intrinsic NFκB signaling. Failure to maintain NFκB signals after the peak of the response led to impaired Eomes expression and a defect in the maintenance of CD8 T-cell memory. Strikingly, we found that antigen receptor [T-cell receptor (TCR)] signaling regulates this process through expression of the NFκB-dependent kinase proviral integration site for Moloney murine leukemia virus-1 (PIM-1), which in turn regulates NFκB and Eomes. T cells defective in TCR-dependent NFκB signaling were impaired in late expression of Pim-1, Eomes, and CD8 memory. These defects were rescued when TCR-dependent NFκB signaling was restored. We also found that NFκB-Pim-1 signals were required at memory to maintain memory CD8 T-cell longevity, effector function, and Eomes expression. Hence, an NFκB-Pim-1-Eomes axis regulates Eomes levels to maintain memory fitness.

Interrupting CD28 costimulation before antigen rechallenge affects CD8(+) T-cell expansion and effector functions during secondary response in mice.

The role of CD28-mediated costimulation in secondary CD8(+) T-cell responses remains controversial. Here, we have used two tools - blocking mouse anti-mouse CD28-specific antibodies and inducible CD28-deleting mice - to obtain definitive answers in mice infected with ovalbumin-secreting Listeria monocytogenes. We report that both blockade and global deletion of CD28 reveal its requirement for full clonal expansion and effector functions such as degranulation and IFN-γ production during the secondary immune response. In contrast, cell-intrinsic deletion of CD28 in transferred TCR-transgenic CD8(+) T cells before primary infection leads to impaired clonal expansion but an increase in cells able to express effector functions in both primary and secondary responses. We suggest that the proliferation-impaired CD8(+) T cells respond to CD28-dependent help from their environment by enhanced functional differentiation. Finally, we report that cell-intrinsic deletion of CD28 after the peak of the primary response does not affect the establishment, maintenance, or recall of long-term memory. Thus, if given sufficient time, the progeny of primed CD8(+) T cells adapt to the absence of this costimulator.