The CD4 Versus CD8 T Cell Fate Decision: A Multiomics-Informed Perspective.

The choice of developing thymocytes to become CD8+ cytotoxic or CD4+ helper T cells has been intensely studied, but many of the underlying mechanisms remain to be elucidated. Recent multiomics approaches have provided much higher resolution analysis of gene expression in developing thymocytes than was previously achievable, thereby offering a fresh perspective on this question. Focusing on our recent studies using CITE-seq (cellular indexing of transcriptomes and epitopes) analyses of mouse thymocytes, we present a detailed timeline of RNA and protein expression changes during CD8 versus CD4 T cell differentiation. We also revisit our current understanding of the links between T cell receptor signaling and expression of the lineage-defining transcription factors ThPOK and RUNX3. Finally, we propose a sequential selection model to explain the tight linkage between MHC-I versus MHC-II recognition and T cell lineage choice. This model incorporates key aspects of previously proposed kinetic signaling, instructive, and stochastic/selection models. Expected final online publication date for the Annual Review of Immunology, Volume 42 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Commensal bacteria maintain a Qa-1b-restricted unconventional CD8+ T population in gut epithelium.

Intestinal intraepithelial lymphocytes (IELs) are characterized by an unusual phenotype and developmental pathway, yet their specific ligands and functions remain largely unknown. Here by analysis of QFL T cells, a population of CD8+ T cells critical for monitoring the MHC I antigen processing pathway, we established that unconventional Qa-1b-restricted CD8+ T cells are abundant in intestinal epithelium. We found that QFL T cells showed a Qa-1b-dependent unconventional phenotype in the spleen and small intestine of naïve wild-type mice. The splenic QFL T cells showed innate-like functionality exemplified by rapid response to cytokines or antigens, while the gut population was refractory to stimuli. Microbiota was required for the maintenance, but not the initial gut homing of QFL T cells. Moreover, monocolonization with Pediococcus pentosaceus, which expresses a peptide that cross-activated QFL T cells, was sufficient to maintain QFL T cells in the intestine. Thus, microbiota is critical for shaping the Qa-1b-restricted IEL landscape.

The promiscuous development of an unconventional Qa1b-restricted T cell population.

MHC-E restricted CD8 T cells show promise in vaccine settings, but their development and specificity remain poorly understood. Here we focus on a CD8 T cell population reactive to a self-peptide (FL9) bound to mouse MHC-E (Qa-1b) that is presented in response to loss of the MHC I processing enzyme ERAAP, termed QFL T cells. We find that mature QFL thymocytes are predominantly CD8αß+CD4-, show signs of agonist selection, and give rise to both CD8αα and CD8αß intraepithelial lymphocytes (IEL), as well as memory phenotype CD8αß T cells. QFL T cells require the MHC I subunit ß-2 microglobulin (ß2m), but do not require Qa1b or classical MHC I for positive selection. However, QFL thymocytes do require Qa1b for agonist selection and full functionality. Our data highlight the relaxed requirements for positive selection of an MHC-E restricted T cell population and suggest a CD8αß+CD4- pathway for development of CD8αα IELs.

Single-cell multiomic analysis of thymocyte development reveals drivers of CD4+ T cell and CD8+ T cell lineage commitment.

The development of CD4+ T cells and CD8+ T cells in the thymus is critical to adaptive immunity and is widely studied as a model of lineage commitment. Recognition of self-peptide major histocompatibility complex (MHC) class I or II by the T cell antigen receptor (TCR) determines the CD8+ or CD4+ T cell lineage choice, respectively, but how distinct TCR signals drive transcriptional programs of lineage commitment remains largely unknown. Here we applied CITE-seq to measure RNA and surface proteins in thymocytes from wild-type and T cell lineage-restricted mice to generate a comprehensive timeline of cell states for each T cell lineage. These analyses identified a sequential process whereby all thymocytes initiate CD4+ T cell lineage differentiation during a first wave of TCR signaling, followed by a second TCR signaling wave that coincides with CD8+ T cell lineage specification. CITE-seq and pharmaceutical inhibition experiments implicated a TCR-calcineurin-NFAT-GATA3 axis in driving the CD4+ T cell fate. Our data provide a resource for understanding cell fate decisions and implicate a sequential selection process in guiding lineage choice.

Commensal Bacteria Maintain a Qa-1 b -restricted Unconventional CD8 + T Population in Gut Epithelium.

Intestinal intraepithelial lymphocytes (IELs) are characterized by an unusual phenotype and developmental pathway, yet their specific ligands and functions remain largely unknown. Here by analysis of QFL T cells, a population of CD8 + T cells critical for monitoring the MHC I antigen processing pathway, we established that unconventional Qa-1 b -restricted CD8 + T cells are abundant in intestinal epithelium. We found that QFL T cells showed a Qa-1 b -dependent unconventional phenotype in the spleen and small intestine of naïve wild-type mice. The splenic QFL T cells showed innate-like functionality exemplified by rapid response to cytokines or antigen, while the gut population was refractory to stimuli. Microbiota was required for the maintenance, but not the initial gut homing of QFL T cells. Moreover, monocolonization with Pediococcus pentosaceus, which expresses a peptide that cross-activated QFL T cells, was sufficient to maintain QFL T cells in the intestine. Thus, microbiota is critical for shaping the Qa-1 b -restricted IEL landscape.

Murine cytomegalovirus downregulates ERAAP and induces an unconventional T cell response to self.

The endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) plays a crucial role in shaping the peptide-major histocompatibility complex (MHC) class I repertoire and maintaining immune surveillance. While murine cytomegalovirus (MCMV) has multiple strategies for manipulating the antigen processing pathway to evade immune responses, the host has also developed ways to counter viral immune evasion. In this study, we find that MCMV modulates ERAAP and induces an interferon γ (IFN-γ)-producing CD8+ T cell effector response that targets uninfected ERAAP-deficient cells. We observe that ERAAP downregulation during infection leads to the presentation of the self-peptide FL9 on non-classical Qa-1b, thereby eliciting Qa-1b-restricted QFL T cells to proliferate in the liver and spleen of infected mice. QFL T cells upregulate effector markers upon MCMV infection and are sufficient to reduce viral load after transfer to immunodeficient mice. Our study highlights the consequences of ERAAP dysfunction during viral infection and provides potential targets for anti-viral therapies.

Studying T Cell Development in Neonatal and Adult Thymic Slices.

T cell development occurs in the thymus and is coordinated temporally and spatially within the highly complex thymic microenvironment. Therefore, T cell selection and maturation events cannot be fully recapitulated using traditional two-dimensional tissue culture in vitro. The thymic slice system provides a highly versatile system for studying T cell development ex vivo while preserving three-dimensional thymic architecture. Using the thymic slice system, T cell selection and maturation events can be visualized by live imaging and quantified by flow cytometry. Here we describe the process for generating slices from neonatal and adult mice.

Thymic macrophages consist of two populations with distinct localization and origin.

Tissue-resident macrophages are essential to protect from pathogen invasion and maintain organ homeostasis. The ability of thymic macrophages to engulf apoptotic thymocytes is well appreciated, but little is known about their ontogeny, maintenance, and diversity. Here, we characterized the surface phenotype and transcriptional profile of these cells and defined their expression signature. Thymic macrophages were most closely related to spleen red pulp macrophages and Kupffer cells and shared the expression of the transcription factor (TF) SpiC with these cells. Single-cell RNA sequencing (scRNA-Seq) showed that the macrophages in the adult thymus are composed of two populations distinguished by the expression of Timd4 and Cx3cr1. Remarkably, Timd4+ cells were located in the cortex, while Cx3cr1+ macrophages were restricted to the medulla and the cortico-medullary junction. Using shield chimeras, transplantation of embryonic thymuses, and genetic fate mapping, we found that the two populations have distinct origins. Timd4+ thymic macrophages are of embryonic origin, while Cx3cr1+ macrophages are derived from adult hematopoietic stem cells. Aging has a profound effect on the macrophages in the thymus. Timd4+ cells underwent gradual attrition, while Cx3cr1+ cells slowly accumulated with age and, in older mice, were the dominant macrophage population in the thymus. Altogether, our work defines the phenotype, origin, and diversity of thymic macrophages.

Peptide Centric Vß Specific Germline Contacts Shape a Specialist T Cell Response.

Certain CD8 T cell responses are particularly effective at controlling infection, as exemplified by elite control of HIV in individuals harboring HLA-B57. To understand the structural features that contribute to CD8 T cell elite control, we focused on a strongly protective CD8 T cell response directed against a parasite-derived peptide (HF10) presented by an atypical MHC-I molecule, H-2Ld. This response exhibits a focused TCR repertoire dominated by Vß2, and a representative TCR (TG6) in complex with Ld-HF10 reveals an unusual structure in which both MHC and TCR contribute extensively to peptide specificity, along with a parallel footprint of TCR on its pMHC ligand. The parallel footprint is a common feature of Vß2-containing TCRs and correlates with an unusual Vα-Vß interface, CDR loop conformations, and Vß2-specific germline contacts with peptides. Vß2 and Ld may represent "specialist" components for antigen recognition that allows for particularly strong and focused T cell responses.

CXCR3 regulates stem and proliferative CD8+ T cells during chronic infection by promoting interactions with DCs in splenic bridging channels.

Production of effector CD8+ T cells during persistent infection requires a stable pool of stem-like cells that can give rise to effector cells via a proliferative intermediate population. In infection models marked by T cell exhaustion, this process can be transiently induced by checkpoint blockade but occurs spontaneously in mice chronically infected with the protozoan intracellular parasite Toxoplasma gondii. We observe distinct locations for parasite-specific T cell subsets, implying a link between differentiation and anatomical niches in the spleen. Loss of the chemokine receptor CXCR3 on T cells does not prevent white pulp-to-red pulp migration but reduces interactions with CXCR3 ligand-producing dendritic cells (DCs) and impairs memory-to-intermediate transition, leading to a buildup of memory T cells in the red pulp. Thus, CXCR3 increases T cell exposure to differentiation-inducing signals during red pulp migration, providing a dynamic mechanism for modulating effector differentiation in response to environmental signals.

T cell self-reactivity during thymic development dictates the timing of positive selection.

Functional tuning of T cells based on their degree of self-reactivity is established during positive selection in the thymus, although how positive selection differs for thymocytes with relatively low versus high self-reactivity is unclear. In addition, preselection thymocytes are highly sensitive to low-affinity ligands, but the mechanism underlying their enhanced T cell receptor (TCR) sensitivity is not fully understood. Here we show that murine thymocytes with low self-reactivity experience briefer TCR signals and complete positive selection more slowly than those with high self-reactivity. Additionally, we provide evidence that cells with low self-reactivity retain a preselection gene expression signature as they mature, including genes previously implicated in modulating TCR sensitivity and a novel group of ion channel genes. Our results imply that thymocytes with low self-reactivity downregulate TCR sensitivity more slowly during positive selection, and associate membrane ion channel expression with thymocyte self-reactivity and progress through positive selection.

Factors that influence the thymic selection of CD8αα intraepithelial lymphocytes.

Thymocytes bearing αß T cell receptors (TCRαß) with high affinity for self-peptide-MHC complexes undergo negative selection or are diverted to alternate T cell lineages, a process termed agonist selection. Among thymocytes bearing TCRs restricted to MHC class I, agonist selection can lead to the development of precursors that can home to the gut and give rise to CD8αα-expressing intraepithelial lymphocytes (CD8αα IELs). The factors that influence the choice between negative selection versus CD8αα IEL development remain largely unknown. Using a synchronized thymic tissue slice model that supports both negative selection and CD8αα IEL development, we show that the affinity threshold for CD8αα IEL development is higher than for negative selection. We also investigate the impact of peptide presenting cells and cytokines, and the migration patterns associated with these alternative cell fates. Our data highlight the roles of TCR affinity and the thymic microenvironments on T cell fate.

Robust intrathymic development of regulatory T cells in young NOD mice is rapidly restrained by recirculating cells.

Regulatory T lymphocytes (Treg) play a vital role in the protection of the organism against autoimmune pathology. It is therefore paradoxical that comparatively large numbers of Treg were found in the thymus of type I diabetes-prone NOD mice. The Treg population in the thymus is composed of newly developing cells and cells that had recirculated from the periphery back to the thymus. We here demonstrate that exceptionally large numbers of Treg develop in the thymus of young, but not adult, NOD mice. Once emigrated from the thymus, an unusually large proportion of these Treg is activated in the periphery, which causes a particularly abundant accumulation of recirculating Treg in the thymus. These cells then rapidly inhibit de novo development of Treg. The proportions of developing Treg thus reach levels similar to or lower than those found in most other, type 1 diabetes-resistant, inbred mouse strains. Thus, in adult NOD mice the particularly large Treg-niche is actually composed of mostly recirculating cells and only few newly developing Treg.

An Unusual MHC Molecule Generates Protective CD8+ T Cell Responses to Chronic Infection.

The CD8+ T cell response to the intracellular parasite Toxoplasma gondii varies dramatically between mouse strains, resulting in stark differences in control of the parasite. Protection in BALB/c mice can be attributed to an unusually strong and protective MHC-1 Ld-restricted CD8+ T cell response directed against a peptide derived from the parasite antigen GRA6. The MHC-1 Ld molecule has limited peptide binding compared to conventional MHC molecules such as Kb or Db, which correlates with polymorphisms associated with "elite control" of HIV in humans. To investigate the link between the unusual MHC-1 molecule Ld and the generation of "elite controller" CD8+ T cell responses, we compared the GRA6-Ld specific T cell response to the well-studied OVA-Kb specific response, and demonstrated that GRA6-Ld specific T cells are significantly more protective and resistant to exhaustion in chronic T. gondii infection. To further investigate the connection between limited peptide presentation and robust T cell responses, we used CRISPR/Cas9 to generate mice with a point mutation (W97R) in the peptide-binding groove of Ld that results in broader peptide binding. We investigated the effect of this Ld W97R mutation on another robust Ld-restricted response against the IE1 peptide during Murine Cytomegalovirus (MCMV) infection. This mutation leads to an increase in exhaustion markers in the IE1-Ld specific CD8+ T cell response. Our results indicate that limited peptide binding by MHC-1 Ld correlates with the development of robust and protective CD8+ T cell responses that may avoid exhaustion during chronic infection.

A role for phagocytosis in inducing cell death during thymocyte negative selection.

Autoreactive thymocytes are eliminated during negative selection in the thymus, a process important for establishing self-tolerance. Thymic phagocytes serve to remove dead thymocytes, but whether they play additional roles during negative selection remains unclear. Here, using a murine thymic slice model in which thymocytes undergo negative selection in situ, we demonstrate that phagocytosis promotes negative selection, and provide evidence for the escape of autoreactive CD8 T cells to the periphery when phagocytosis in the thymus is impaired. We also show that negative selection is more efficient when the phagocyte also presents the negative selecting peptide. Our findings support a model for negative selection in which the death process initiated following strong TCR signaling is facilitated by phagocytosis. Thus, the phagocytic capability of cells that present self-peptides is a key determinant of thymocyte fate.

CD8+ T Cell Responses to Toxoplasma gondii: Lessons from a Successful Parasite.

Toxoplasma gondii infection in mice provides an excellent model for the study of CD8+ T cell responses. Natural and engineered T. gondii antigens have led the way to understanding the factors regulating antigen presentation from vacuolar pathogens. T. gondii infection of resistant and sensitive mouse strains provides unique models to study both effective CD8+ T cell function and protection in a well-controlled infection attributed to a novel T cell population, and T cell exhaustion in a progressing chronic infection. Additionally, the long-term persistence of the parasite in the brain provides a unique model of neurotropic infection used to study CD8+ T cell entry, retention, and function in the brain. Here we discuss recent advances in each of these areas.

Robust Control of a Brain-Persisting Parasite through MHC I Presentation by Infected Neurons.

Control of CNS pathogens by CD8 T cells is key to avoid fatal neuroinflammation. Yet, the modalities of MHC I presentation in the brain are poorly understood. Here, we analyze the antigen presentation mechanisms underlying CD8 T cell-mediated control of the Toxoplasma gondii parasite in the CNS. We show that MHC I presentation of an efficiently processed model antigen (GRA6-OVA), even when not expressed in the bradyzoite stage, reduces cyst burden and dampens encephalitis in C57BL/6 mice. Antigen presentation assays with infected primary neurons reveal a correlation between lower MHC I presentation of tachyzoite antigens by neurons and poor parasite control in vivo. Using conditional MHC I-deficient mice, we find that neuronal MHC I presentation is required for robust restriction of T. gondii in the CNS during chronic phase, showing the importance of MHC I presentation by CNS neurons in the control of a prevalent brain pathogen.

Central CD4+ T cell tolerance: deletion versus regulatory T cell differentiation.

The diversion of MHC class II-restricted thymocytes into the regulatory T (Treg) cell lineage is driven by intrathymic encounter of agonist self-antigens in a similar manner to the clonal deletion of thymocytes. Somewhat paradoxically, it thus seems that the expression of an autoreactive T cell receptor is a shared characteristic of T cells that are subject to clonal deletion and T cells that are diverted into the Treg cell lineage. Here, we discuss how thymocyte-intrinsic and thymocyte-extrinsic determinants may specify the choice between these two fundamentally different T cell fates.

Continuous Effector CD8(+) T Cell Production in a Controlled Persistent Infection Is Sustained by a Proliferative Intermediate Population.

Highly functional CD8(+) effector T (Teff) cells can persist in large numbers during controlled persistent infections, as exemplified by rare HIV-infected individuals who control the virus. Here we examined the cellular mechanisms that maintain ongoing T effector responses using a mouse model for persistent Toxoplasma gondii infection. In mice expressing the protective MHC-I molecule, H-2L(d), a dominant T effector response against a single parasite antigen was maintained without a contraction phase, correlating with ongoing presentation of the dominant antigen. Large numbers of short-lived Teff cells were continuously produced via a proliferative, antigen-dependent intermediate (Tint) population with a memory-effector hybrid phenotype. During an acute, resolved infection, decreasing antigen load correlated with a sharp drop in the Tint cell population and subsequent loss of the ongoing effector response. Vaccination approaches aimed at the development of Tint populations might prove effective against pathogens that lead to chronic infection.