In Vivo CD4+ T Cell Differentiation and Function: Revisiting the Th1/Th2 Paradigm.

The discovery of CD4+ T cell subset-defining master transcription factors and framing of the Th1/Th2 paradigm ignited the CD4+ T cell field. Advances in in vivo experimental systems, however, have revealed that more complex lineage-defining transcriptional networks direct CD4+ T cell differentiation in the lymphoid organs and tissues. This review focuses on the layers of fate decisions that inform CD4+ T cell differentiation in vivo. Cytokine production by antigen-presenting cells and other innate cells influences the CD4+ T cell effector program [e.g., T helper type 1 (Th1), Th2, Th17]. Signals downstream of the T cell receptor influence whether individual clones bearing hallmarks of this effector program become T follicular helper cells, supporting development of B cells expressing specific antibody isotypes, or T effector cells, which activate microbicidal innate cells in tissues. These bifurcated, parallel axes allow CD4+ T cells to augment their particular effector program and prevent disease.

Tissue-Resident T Cells and Other Resident Leukocytes.

Resident memory T (Trm) cells stably occupy tissues and cannot be sampled in superficial venous blood. Trm cells are heterogeneous but collectively constitute the most abundant memory T cell subset. Trm cells form an integral part of the immune sensing network, monitor for local perturbations in homeostasis throughout the body, participate in protection from infection and cancer, and likely promote autoimmunity, allergy, and inflammatory diseases and impede successful transplantation. Thus Trm cells are major candidates for therapeutic manipulation. Here we review CD8+ and CD4+ Trm ontogeny, maintenance, function, and distribution within lymphoid and nonlymphoid tissues and strategies for their study. We briefly discuss other resident leukocyte populations, including innate lymphoid cells, macrophages, natural killer and natural killer T cells, nonclassical T cells, and memory B cells. Lastly, we highlight major gaps in knowledge and propose ways in which a deeper understanding could result in new methods to prevent or treat diverse human diseases.

Gut Microbiota Regulation of T Cells During Inflammation and Autoimmunity.

The intestinal microbiota plays a crucial role in influencing the development of host immunity, and in turn the immune system also acts to regulate the microbiota through intestinal barrier maintenance and immune exclusion. Normally, these interactions are homeostatic, tightly controlled, and organized by both innate and adaptive immune responses. However, a combination of environmental exposures and genetic defects can result in a break in tolerance and intestinal homeostasis. The outcomes of these interactions at the mucosal interface have broad, systemic effects on host immunity and the development of chronic inflammatory or autoimmune disease. The underlying mechanisms and pathways the microbiota can utilize to regulate these diseases are just starting to emerge. Here, we discuss the recent evidence in this area describing the impact of microbiota-immune interactions during inflammation and autoimmunity, with a focus on barrier function and CD4+ T cell regulation.

SARS-CoV-2 vaccination induces immunological T cell memory able to cross-recognize variants from Alpha to Omicron.

We address whether T cell responses induced by different vaccine platforms (mRNA-1273, BNT162b2, Ad26.COV2.S, and NVX-CoV2373) cross-recognize early SARS-CoV-2 variants. T cell responses to early variants were preserved across vaccine platforms. By contrast, significant overall decreases were observed for memory B cells and neutralizing antibodies. In subjects ∼6 months post-vaccination, 90% (CD4+) and 87% (CD8+) of memory T cell responses were preserved against variants on average by AIM assay, and 84% (CD4+) and 85% (CD8+) preserved against Omicron. Omicron RBD memory B cell recognition was substantially reduced to 42% compared with other variants. T cell epitope repertoire analysis revealed a median of 11 and 10 spike epitopes recognized by CD4+ and CD8+ T cells, with average preservation > 80% for Omicron. Functional preservation of the majority of T cell responses may play an important role as a second-level defense against diverse variants.

A local regulatory T cell feedback circuit maintains immune homeostasis by pruning self-activated T cells.

A fraction of mature T cells can be activated by peripheral self-antigens, potentially eliciting host autoimmunity. We investigated homeostatic control of self-activated T cells within unperturbed tissue environments by combining high-resolution multiplexed and volumetric imaging with computational modeling. In lymph nodes, self-activated T cells produced interleukin (IL)-2, which enhanced local regulatory T cell (Treg) proliferation and inhibitory functionality. The resulting micro-domains reciprocally constrained inputs required for damaging effector responses, including CD28 co-stimulation and IL-2 signaling, constituting a negative feedback circuit. Due to these local constraints, self-activated T cells underwent transient clonal expansion, followed by rapid death ("pruning"). Computational simulations and experimental manipulations revealed the feedback machinery's quantitative limits: modest reductions in Treg micro-domain density or functionality produced non-linear breakdowns in control, enabling self-activated T cells to subvert pruning. This fine-tuned, paracrine feedback process not only enforces immune homeostasis but also establishes a sharp boundary between autoimmune and host-protective T cell responses.

Neoantigen-driven B cell and CD4 T follicular helper cell collaboration promotes anti-tumor CD8 T cell responses.

CD4 T follicular helper (TFH) cells support B cells, which are critical for germinal center (GC) formation, but the importance of TFH-B cell interactions in cancer is unclear. We found enrichment of TFH cell transcriptional signature correlates with GC B cell signature and with prolonged survival in individuals with lung adenocarcinoma (LUAD). We further developed a murine LUAD model in which tumor cells express B cell- and T cell-recognized neoantigens. Interactions between tumor-specific TFH and GC B cells, as well as interleukin (IL)-21 primarily produced by TFH cells, are necessary for tumor control and effector CD8 T cell function. Development of TFH cells requires B cells and B cell-recognized neoantigens. Thus, tumor neoantigens can regulate the fate of tumor-specific CD4 T cells by facilitating their interactions with tumor-specific B cells, which in turn promote anti-tumor immunity by enhancing CD8 T cell effector functions.

Humans with inherited T cell CD28 deficiency are susceptible to skin papillomaviruses but are otherwise healthy.

We study a patient with the human papilloma virus (HPV)-2-driven "tree-man" phenotype and two relatives with unusually severe HPV4-driven warts. The giant horns form an HPV-2-driven multifocal benign epithelial tumor overexpressing viral oncogenes in the epidermis basal layer. The patients are unexpectedly homozygous for a private CD28 variant. They have no detectable CD28 on their T cells, with the exception of a small contingent of revertant memory CD4+ T cells. T cell development is barely affected, and T cells respond to CD3 and CD2, but not CD28, costimulation. Although the patients do not display HPV-2- and HPV-4-reactive CD4+ T cells in vitro, they make antibodies specific for both viruses in vivo. CD28-deficient mice are susceptible to cutaneous infections with the mouse papillomavirus MmuPV1. The control of HPV-2 and HPV-4 in keratinocytes is dependent on the T cell CD28 co-activation pathway. Surprisingly, human CD28-dependent T cell responses are largely redundant for protective immunity.

Bifidobacteria-mediated immune system imprinting early in life.

Immune-microbe interactions early in life influence the risk of allergies, asthma, and other inflammatory diseases. Breastfeeding guides healthier immune-microbe relationships by providing nutrients to specialized microbes that in turn benefit the host's immune system. Such bacteria have co-evolved with humans but are now increasingly rare in modern societies. Here we show that a lack of bifidobacteria, and in particular depletion of genes required for human milk oligosaccharide (HMO) utilization from the metagenome, is associated with systemic inflammation and immune dysregulation early in life. In breastfed infants given Bifidobacterium infantis EVC001, which expresses all HMO-utilization genes, intestinal T helper 2 (Th2) and Th17 cytokines were silenced and interferon ß (IFNß) was induced. Fecal water from EVC001-supplemented infants contains abundant indolelactate and B. infantis-derived indole-3-lactic acid (ILA) upregulated immunoregulatory galectin-1 in Th2 and Th17 cells during polarization, providing a functional link between beneficial microbes and immunoregulation during the first months of life.

Translocated microbiome composition determines immunological outcome in treated HIV infection.

The impact of the microbiome on HIV disease is widely acknowledged although the mechanisms downstream of fluctuations in microbial composition remain speculative. We detected rapid, dynamic changes in translocated microbial constituents during two years after cART initiation. An unbiased systems biology approach revealed two distinct pathways driven by changes in the abundance ratio of Serratia to other bacterial genera. Increased CD4 T cell numbers over the first year were associated with high Serratia abundance, pro-inflammatory innate cytokines, and metabolites that drive Th17 gene expression signatures and restoration of mucosal integrity. Subsequently, decreased Serratia abundance and downregulation of innate cytokines allowed re-establishment of systemic T cell homeostasis promoting restoration of Th1 and Th2 gene expression signatures. Analyses of three other geographically distinct cohorts of treated HIV infection established a more generalized principle that changes in diversity and composition of translocated microbial species influence systemic inflammation and consequently CD4 T cell recovery.

The endogenous repertoire harbors self-reactive CD4+ T cell clones that adopt a follicular helper T cell-like phenotype at steady state.

The T cell repertoire of healthy mice and humans harbors self-reactive CD4+ conventional T (Tconv) cells capable of inducing autoimmunity. Using T cell receptor profiling paired with in vivo clonal analysis of T cell differentiation, we identified Tconv cell clones that are recurrently enriched in non-lymphoid organs following ablation of Foxp3+ regulatory T (Treg) cells. A subset of these clones was highly proliferative in the lymphoid organs at steady state and exhibited overt reactivity to self-ligands displayed by dendritic cells, yet were not purged by clonal deletion. These clones spontaneously adopted numerous hallmarks of follicular helper T (TFH) cells, including expression of Bcl6 and PD-1, exhibited an elevated propensity to localize within B cell follicles at steady state, and produced interferon-γ in non-lymphoid organs following sustained Treg cell depletion. Our work identifies a naturally occurring population of self-reactive TFH-like cells and delineates a previously unappreciated fate for self-specific Tconv cells.

Aging-associated HELIOS deficiency in naive CD4+ T cells alters chromatin remodeling and promotes effector cell responses.

Immune aging combines cellular defects in adaptive immunity with the activation of pathways causing a low-inflammatory state. Here we examined the influence of age on the kinetic changes in the epigenomic and transcriptional landscape induced by T cell receptor (TCR) stimulation in naive CD4+ T cells. Despite attenuated TCR signaling in older adults, TCR activation accelerated remodeling of the epigenome and induced transcription factor networks favoring effector cell differentiation. We identified increased phosphorylation of STAT5, at least in part due to aberrant IL-2 receptor and lower HELIOS expression, as upstream regulators. Human HELIOS-deficient, naive CD4+ T cells, when transferred into human-synovium-mouse chimeras, infiltrated tissues more efficiently. Inhibition of IL-2 or STAT5 activity in T cell responses of older adults restored the epigenetic response pattern to the one seen in young adults. In summary, reduced HELIOS expression in non-regulatory naive CD4+ T cells in older adults directs T cell fate decisions toward inflammatory effector cells that infiltrate tissue.

T cell-mediated host immune defenses in the lung.

Evidence has increasingly shown that the lungs are a major site of immune regulation. A robust and highly regulated immune response in the lung protects the host from pathogen infection, whereas an inefficient or deleterious response can lead to various pulmonary diseases. Many cell types, such as epithelial cells, dendritic cells, macrophages, neutrophils, eosinophils, and B and T lymphocytes, contribute to lung immunity. This review focuses on the recent advances in understanding how T lymphocytes mediate pulmonary host defenses against bacterial, viral, and fungal pathogens.

The biology of recent thymic emigrants.

The generation of the TCRαß lineage of T cells occurs in the thymus through a series of orchestrated developmental events that result in a carefully selected population of CD4 or CD8 lineage-committed TCR(+) thymocytes capable of recognizing foreign antigen in the context of self MHC. T cells first exit the thymus in a phenotypically and functionally immature state and require an approximately 3-week period of post-thymic maturation before transitioning into the mature T cell compartment. A greater understanding of recent thymic emigrant biology has come with the development of methods to exclusively identify and isolate this population for further characterization. I now review current knowledge about the phenotype and function of this key but understudied population of peripheral T cells.

Coordinated Cellular Neighborhoods Orchestrate Antitumoral Immunity at the Colorectal Cancer Invasive Front.

Antitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We re-engineered co-detection by indexing (CODEX) for paraffin-embedded tissue microarrays, enabling simultaneous profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers. We identified nine conserved, distinct cellular neighborhoods (CNs)-a collection of components characteristic of the CRC iTME. Enrichment of PD-1+CD4+ T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferior outcomes. This study provides a framework for interrogating how complex biological processes, such as antitumoral immunity, occur through concerted actions of cells and spatial domains.

HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis.

The HLA-DR15 haplotype is the strongest genetic risk factor for multiple sclerosis (MS), but our understanding of how it contributes to MS is limited. Because autoreactive CD4+ T cells and B cells as antigen-presenting cells are involved in MS pathogenesis, we characterized the immunopeptidomes of the two HLA-DR15 allomorphs DR2a and DR2b of human primary B cells and monocytes, thymus, and MS brain tissue. Self-peptides from HLA-DR molecules, particularly from DR2a and DR2b themselves, are abundant on B cells and thymic antigen-presenting cells. Furthermore, we identified autoreactive CD4+ T cell clones that can cross-react with HLA-DR-derived self-peptides (HLA-DR-SPs), peptides from MS-associated foreign agents (Epstein-Barr virus and Akkermansia muciniphila), and autoantigens presented by DR2a and DR2b. Thus, both HLA-DR15 allomorphs jointly shape an autoreactive T cell repertoire by serving as antigen-presenting structures and epitope sources and by presenting the same foreign peptides and autoantigens to autoreactive CD4+ T cells in MS.

Quick COVID-19 Healers Sustain Anti-SARS-CoV-2 Antibody Production.

Antibodies are key immune effectors that confer protection against pathogenic threats. The nature and longevity of the antibody response to SARS-CoV-2 infection are not well defined. We charted longitudinal antibody responses to SARS-CoV-2 in 92 subjects after symptomatic COVID-19. Antibody responses to SARS-CoV-2 are unimodally distributed over a broad range, with symptom severity correlating directly with virus-specific antibody magnitude. Seventy-six subjects followed longitudinally to ∼100 days demonstrated marked heterogeneity in antibody duration dynamics. Virus-specific IgG decayed substantially in most individuals, whereas a distinct subset had stable or increasing antibody levels in the same time frame despite similar initial antibody magnitudes. These individuals with increasing responses recovered rapidly from symptomatic COVID-19 disease, harbored increased somatic mutations in virus-specific memory B cell antibody genes, and had persistent higher frequencies of previously activated CD4+ T cells. These findings illuminate an efficient immune phenotype that connects symptom clearance speed to differential antibody durability dynamics.

Intratumoral CD4+ T Cells Mediate Anti-tumor Cytotoxicity in Human Bladder Cancer.

Responses to anti-PD-1 immunotherapy occur but are infrequent in bladder cancer. The specific T cells that mediate tumor rejection are unknown. T cells from human bladder tumors and non-malignant tissue were assessed with single-cell RNA and paired T cell receptor (TCR) sequencing of 30,604 T cells from 7 patients. We find that the states and repertoires of CD8+ T cells are not distinct in tumors compared with non-malignant tissues. In contrast, single-cell analysis of CD4+ T cells demonstrates several tumor-specific states, including multiple distinct states of regulatory T cells. Surprisingly, we also find multiple cytotoxic CD4+ T cell states that are clonally expanded. These CD4+ T cells can kill autologous tumors in an MHC class II-dependent fashion and are suppressed by regulatory T cells. Further, a gene signature of cytotoxic CD4+ T cells in tumors predicts a clinical response in 244 metastatic bladder cancer patients treated with anti-PD-L1.

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals.

Understanding adaptive immunity to SARS-CoV-2 is important for vaccine development, interpreting coronavirus disease 2019 (COVID-19) pathogenesis, and calibration of pandemic control measures. Using HLA class I and II predicted peptide "megapools," circulating SARS-CoV-2-specific CD8+ and CD4+ T cells were identified in ∼70% and 100% of COVID-19 convalescent patients, respectively. CD4+ T cell responses to spike, the main target of most vaccine efforts, were robust and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. The M, spike, and N proteins each accounted for 11%-27% of the total CD4+ response, with additional responses commonly targeting nsp3, nsp4, ORF3a, and ORF8, among others. For CD8+ T cells, spike and M were recognized, with at least eight SARS-CoV-2 ORFs targeted. Importantly, we detected SARS-CoV-2-reactive CD4+ T cells in ∼40%-60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating "common cold" coronaviruses and SARS-CoV-2.

Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity.

Limited knowledge is available on the relationship between antigen-specific immune responses and COVID-19 disease severity. We completed a combined examination of all three branches of adaptive immunity at the level of SARS-CoV-2-specific CD4+ and CD8+ T cell and neutralizing antibody responses in acute and convalescent subjects. SARS-CoV-2-specific CD4+ and CD8+ T cells were each associated with milder disease. Coordinated SARS-CoV-2-specific adaptive immune responses were associated with milder disease, suggesting roles for both CD4+ and CD8+ T cells in protective immunity in COVID-19. Notably, coordination of SARS-CoV-2 antigen-specific responses was disrupted in individuals ≥ 65 years old. Scarcity of naive T cells was also associated with aging and poor disease outcomes. A parsimonious explanation is that coordinated CD4+ T cell, CD8+ T cell, and antibody responses are protective, but uncoordinated responses frequently fail to control disease, with a connection between aging and impaired adaptive immune responses to SARS-CoV-2.

Hierarchical regulation of the resting and activated T cell epigenome by major transcription factor families.

T cell activation, a key early event in the adaptive immune response, is subject to elaborate transcriptional control. In the present study, we examined how the activities of eight major transcription factor (TF) families are integrated to shape the epigenome of naive and activated CD4 and CD8 T cells. By leveraging extensive polymorphisms in evolutionarily divergent mice, we identified the 'heavy lifters' positively influencing chromatin accessibility. Members of Ets, Runx and TCF/Lef TF families occupied the vast majority of accessible chromatin regions, acting as 'housekeepers', 'universal amplifiers' and 'placeholders', respectively, at sites that maintained or gained accessibility upon T cell activation. In addition, a small subset of strongly induced immune response genes displayed a noncanonical TF recruitment pattern. Our study provides a key resource and foundation for the understanding of transcriptional and epigenetic regulation in T cells and offers a new perspective on the hierarchical interactions between critical TFs.