Improvement of immune dysregulation in individuals with long COVID at 24-months following SARS-CoV-2 infection.

This study investigates the humoral and cellular immune responses and health-related quality of life measures in individuals with mild to moderate long COVID (LC) compared to age and gender matched recovered COVID-19 controls (MC) over 24 months. LC participants show elevated nucleocapsid IgG levels at 3 months, and higher neutralizing capacity up to 8 months post-infection. Increased spike-specific and nucleocapsid-specific CD4+ T cells, PD-1, and TIM-3 expression on CD4+ and CD8+ T cells were observed at 3 and 8 months, but these differences do not persist at 24 months. Some LC participants had detectable IFN-γ and IFN-ß, that was attributed to reinfection and antigen re-exposure. Single-cell RNA sequencing at the 24 month timepoint shows similar immune cell proportions and reconstitution of naïve T and B cell subsets in LC and MC. No significant differences in exhaustion scores or antigen-specific T cell clones are observed. These findings suggest resolution of immune activation in LC and return to comparable immune responses between LC and MC over time. Improvement in self-reported health-related quality of life at 24 months was also evident in the majority of LC (62%). PTX3, CRP levels and platelet count are associated with improvements in health-related quality of life.

Deficiency of factor-inhibiting HIF creates a tumor-promoting immune microenvironment.

Hypoxia signaling influences tumor development through both cell-intrinsic and -extrinsic pathways. Inhibiting hypoxia-inducible factor (HIF) function has recently been approved as a cancer treatment strategy. Hence, it is important to understand how regulators of HIF may affect tumor growth under physiological conditions. Here we report that in aging mice factor-inhibiting HIF (FIH), one of the most studied negative regulators of HIF, is a haploinsufficient suppressor of spontaneous B cell lymphomas, particular pulmonary B cell lymphomas. FIH deficiency alters immune composition in aged mice and creates a tumor-supportive immune environment demonstrated in syngeneic mouse tumor models. Mechanistically, FIH-defective myeloid cells acquire tumor-supportive properties in response to signals secreted by cancer cells or produced in the tumor microenvironment with enhanced arginase expression and cytokine-directed migration. Together, these data demonstrate that under physiological conditions, FIH plays a key role in maintaining immune homeostasis and can suppress tumorigenesis through a cell-extrinsic pathway.

The kynurenine pathway relates to post-acute COVID-19 objective cognitive impairment and PASC.

OBJECTIVE: To determine the prevalence and natural history of post-acute COVID-19 objective cognitive impairment and function, and their relationship to demographic, clinical factors, post-acute sequelae of COVID-19 (PASC), and biomarkers. METHODS: A total of 128 post-acute COVID-19 patients (age = 46 ± 15; 42% women, acute disease severity: not hospitalized: 38.6% mild: 0-1 symptoms, 52% 2+ symptoms; 9.4% hospitalized) completed standard cognition, olfaction, and mental health examinations 2-, 4-, and 12-month post diagnosis. Over the same time frame, WHO-defined PASC was determined. Blood cytokines, peripheral neurobiomarkers, and kynurenine pathway (KP) metabolites were measured. Objective cognitive function was demographically/practice corrected, and impairment prevalence was determined using the evidence-based Global Deficit Score method to detect at least mild cognitive impairment (GDS > 0.5). Linear mixed effect regression models with time effect (month post diagnosis) evaluated the relationships to cognition. RESULTS: Across the 12-month study period, mild to moderate cognitive impairment ranged from 16% to 26%, and 46.5% were impaired at least once. Impairment associated with poorer work capacity (p < 0.05), and 2-month objectively tested anosmia (p < 0.05). PASC with (p = 0.01) and without disability (p < 0.03) associated with acute COVID-19 severity. KP measures showed prolonged activation (2 to 8 months) (p < 0.0001) linked to IFN-beta in those with PASC. Of the blood analytes, only the KP metabolites (elevated quinolinic acid, 3-hydroxyanthranilic acid, kynurenine, the kynurenine/tryptophan ratio) associated (p < 0.001) with poorer cognitive performance and greater likelihood of impairment. PASC, independent of disability associated with abnormal kynurenine/tryptophan (p < 0.03). INTERPRETATION: The kynurenine pathway relates to post-acute COVID-19 objective cognitive impairment and PASC, thereby enabling biomarker and therapeutic possibilities.

Patients with treated indolent lymphomas immunized with BNT162b2 have reduced anti-spike neutralizing IgG to SARS-CoV-2 variants, but preserved antigen-specific T cell responses.

Patients with indolent lymphoma undertaking recurrent or continuous B cell suppression are at risk of severe COVID-19. Patients and healthy controls (HC; N = 13) received two doses of BNT162b2: follicular lymphoma (FL; N = 35) who were treatment naïve (TN; N = 11) or received immunochemotherapy (ICT; N = 23) and Waldenström's macroglobulinemia (WM; N = 37) including TN (N = 9), ICT (N = 14), or treated with Bruton's tyrosine kinase inhibitors (BTKi; N = 12). Anti-spike immunoglobulin G (IgG) was determined by a high-sensitivity flow-cytometric assay, in addition to live-virus neutralization. Antigen-specific T cells were identified by coexpression of CD69/CD137 and CD25/CD134 on T cells. A subgroup (N = 29) were assessed for third mRNA vaccine response, including omicron neutralization. One month after second BNT162b2, median anti-spike IgG mean fluorescence intensity (MFI) in FL ICT patients (9977) was 25-fold lower than TN (245 898) and HC (228 255, p = .0002 for both). Anti-spike IgG correlated with lymphocyte count (r = .63; p = .002), and time from treatment (r = .56; p = .007), on univariate analysis, but only with lymphocyte count on multivariate analysis (p = .03). In the WM cohort, median anti-spike IgG MFI in BTKi patients (39 039) was reduced compared to TN (220 645, p = .0008) and HC (p < .0001). Anti-spike IgG correlated with neutralization of the delta variant (r = .62, p < .0001). Median neutralization titer for WM BTKi (0) was lower than HC (40, p < .0001) for early-clade and delta. All cohorts had functional T cell responses. Median anti-spike IgG decreased 4-fold from second to third dose (p = .004). Only 5 of 29 poor initial responders assessed after third vaccination demonstrated seroconversion and improvement in neutralization activity, including to the omicron variant.

Tracking the clonal dynamics of SARS-CoV-2-specific T cells in children and adults with mild/asymptomatic COVID-19.

Children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop less severe coronavirus disease 2019 (COVID-19) than adults. The mechanisms for the age-specific differences and the implications for infection-induced immunity are beginning to be uncovered. We show by longitudinal multimodal analysis that SARS-CoV-2 leaves a small footprint in the circulating T cell compartment in children with mild/asymptomatic COVID-19 compared to adult household contacts with the same disease severity who had more evidence of systemic T cell interferon activation, cytotoxicity and exhaustion. Children harbored diverse polyclonal SARS-CoV-2-specific naïve T cells whereas adults harbored clonally expanded SARS-CoV-2-specific memory T cells. A novel population of naïve interferon-activated T cells is expanded in acute COVID-19 and is recruited into the memory compartment during convalescence in adults but not children. This was associated with the development of robust CD4+ memory T cell responses in adults but not children. These data suggest that rapid clearance of SARS-CoV-2 in children may compromise their cellular immunity and ability to resist reinfection.

High titre neutralizing antibodies in response to SARS-CoV-2 infection require RBD-specific CD4 T cells that include proliferative memory cells.

Background: Long-term immunity to SARS-CoV-2 infection, including neutralizing antibodies and T cell-mediated immunity, is required in a very large majority of the population in order to reduce ongoing disease burden. Methods: We have investigated the association between memory CD4 and CD8 T cells and levels of neutralizing antibodies in convalescent COVID-19 subjects. Findings: Higher titres of convalescent neutralizing antibodies were associated with significantly higher levels of RBD-specific CD4 T cells, including specific memory cells that proliferated vigorously in vitro. Conversely, up to half of convalescent individuals had low neutralizing antibody titres together with a lack of receptor binding domain (RBD)-specific memory CD4 T cells. These low antibody subjects had other, non-RBD, spike-specific CD4 T cells, but with more of an inhibitory Foxp3+ and CTLA-4+ cell phenotype, in contrast to the effector T-bet+, cytotoxic granzymes+ and perforin+ cells seen in RBD-specific memory CD4 T cells from high antibody subjects. Single cell transcriptomics of antigen-specific CD4+ T cells from high antibody subjects similarly revealed heterogenous RBD-specific CD4+ T cells that comprised central memory, transitional memory and Tregs, as well as cytotoxic clusters containing diverse TCR repertoires, in individuals with high antibody levels. However, vaccination of low antibody convalescent individuals led to a slight but significant improvement in RBD-specific memory CD4 T cells and increased neutralizing antibody titres. Interpretation: Our results suggest that targeting CD4 T cell epitopes proximal to and within the RBD-region should be prioritized in booster vaccines.

Immunological dysfunction persists for 8 months following initial mild-to-moderate SARS-CoV-2 infection.

A proportion of patients surviving acute coronavirus disease 2019 (COVID-19) infection develop post-acute COVID syndrome (long COVID (LC)) lasting longer than 12 weeks. Here, we studied individuals with LC compared to age- and gender-matched recovered individuals without LC, unexposed donors and individuals infected with other coronaviruses. Patients with LC had highly activated innate immune cells, lacked naive T and B cells and showed elevated expression of type I IFN (IFN-ß) and type III IFN (IFN-λ1) that remained persistently high at 8 months after infection. Using a log-linear classification model, we defined an optimal set of analytes that had the strongest association with LC among the 28 analytes measured. Combinations of the inflammatory mediators IFN-ß, PTX3, IFN-γ, IFN-λ2/3 and IL-6 associated with LC with 78.5-81.6% accuracy. This work defines immunological parameters associated with LC and suggests future opportunities for prevention and treatment.

Maintenance of broad neutralizing antibodies and memory B cells 1 year post-infection is predicted by SARS-CoV-2-specific CD4+ T cell responses.

Understanding the long-term maintenance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity is critical for predicting protection against reinfection. In an age- and gender-matched cohort of 24 participants, the association of disease severity and early immune responses on the maintenance of humoral immunity 12 months post-infection is examined. All severely affected participants maintain a stable subset of SARS-CoV-2 receptor-binding domain (RBD)-specific memory B cells (MBCs) and good neutralizing antibody breadth against the majority of the variants of concern, including the Delta variant. Modeling these immune responses against vaccine efficacy data indicate a 45%-76% protection against symptomatic infection (variant dependent). Overall, these findings indicate durable humoral responses in most participants after infection, reasonable protection against reinfection, and implicate baseline antigen-specific CD4+ T cell responses as a predictor of maintenance of antibody neutralization breadth and RBD-specific MBC levels at 12 months post-infection.

Human MAIT cells respond to and suppress HIV-1.

Human MAIT cells sit at the interface between innate and adaptive immunity, are polyfunctional and are capable of killing pathogen infected cells via recognition of the Class IB molecule MR1. MAIT cells have recently been shown to possess an antiviral protective role in vivo and we therefore sought to explore this in relation to HIV-1 infection. There was marked activation of MAIT cells in vivo in HIV-1-infected individuals, which decreased following ART. Stimulation of THP1 monocytes with R5 tropic HIVBAL potently activated MAIT cells in vitro. This activation was dependent on IL-12 and IL-18 but was independent of the TCR. Upon activation, MAIT cells were able to upregulate granzyme B, IFNγ and HIV-1 restriction factors CCL3, 4, and 5. Restriction factors produced by MAIT cells inhibited HIV-1 infection of primary PBMCs and immortalized target cells in vitro. These data reveal MAIT cells to be an additional T cell population responding to HIV-1, with a potentially important role in controlling viral replication at mucosal sites.

Characterizing Hepatitis C Virus-Specific CD4+ T Cells Following Viral-Vectored Vaccination, Directly Acting Antivirals, and Spontaneous Viral Cure.

BACKGROUND AND AIMS: Induction of functional helper CD4+ T cells is the hallmark of a protective immune response against hepatitis C virus (HCV), associated with spontaneous viral clearance. Heterologous prime/boost viral vectored vaccination has demonstrated induction of broad and polyfunctional HCV-specific CD8+ T cells in healthy volunteers; however, much less is known about CD4+ T-cell subsets following vaccination. APPROACH AND RESULTS: We analyzed HCV-specific CD4+ T-cell populations using major histocompatibility complex class II tetramers in volunteers undergoing HCV vaccination with recombinant HCV adenoviral/modified vaccinia Ankara viral vectors. Peptide-specific T-cell responses were tracked over time, and functional (proliferation and cytokine secretion) and phenotypic (cell surface and intranuclear) markers were assessed using flow cytometry. These were compared to CD4+ responses in 10 human leukocyte antigen-matched persons with HCV spontaneous resolution and 21 chronically infected patients treated with directly acting antiviral (DAA) therapy. Vaccination induced tetramer-positive CD4+ T cells that were highest 1-4 weeks after boosting (mean, 0.06%). Similar frequencies were obtained for those tracked following spontaneous resolution of disease (mean, 0.04%). In addition, the cell-surface phenotype (CD28, CD127) memory subset markers and intranuclear transcription factors, as well as functional capacity of peptide-specific CD4+ T-cell responses characterized after vaccination, are comparable to those following spontaneous viral resolution. In contrast, helper responses in chronic infection were infrequently detected and poorly functional and did not consistently recover following HCV cure. CONCLUSIONS: Helper CD4+ T-cell phenotype and function following HCV viral vectored vaccination resembles "protective memory" that is observed following spontaneous clearance of HCV. DAA cure does not promote resurrection of exhausted CD4+ T-cell memory in chronic infection.

Maintenance of Functional CD57+ Cytolytic CD4+ T Cells in HIV+ Elite Controllers.

Cytolytic CD4+ T cells play a prominent role in chronic viral infection. CD4+ CTLs clones specific for HIV-1 Nef and Gag are capable of killing HIV-1 infected CD4+ T cells and macrophages. Additionally, HIV-specific cytolytic CD4+ T cell responses in acute HIV infection are predictive of disease progression. CD57 expression on CD4s identifies cytolytic cells. These cells were dramatically increased in chronic HIV infection. CD57 expression correlated with cytolytic granules, granzyme B and perforin expression. They express lower CCR5 compared to CD57- cells, have less HIV total DNA, and were a minor component of the HIV reservoir. A small percentage of CD57+ CD4+ CTLs from EC were HIV-specific, could upregulate IFNγ with Gag peptide stimulation, express cytolytic granule markers and maintain TbethighEomes+ transcription factor phenotype. This was not observed in viraemic controllers. The maintenance of HIV-specific CD4 cytolytic function in Elite controllers together with CD8 CTLs may be important for the control of HIV viraemia and of potential relevance to cure strategies.

Single-cell transcriptome analysis of CD8 + T-cell memory inflation.

Background: Persistent viruses such as murine cytomegalovirus (MCMV) and adenovirus-based vaccines induce strong, sustained CD8 + T-cell responses, described as memory "inflation". These retain functionality, home to peripheral organs and are associated with a distinct transcriptional program. Methods: To further define the nature of the transcriptional mechanisms underpinning memory inflation at different sites we used single-cell RNA sequencing of tetramer-sorted cells from MCMV-infected mice, analyzing transcriptional networks in virus-specific populations in the spleen and gut intra-epithelial lymphocytes (IEL). Results: We provide a transcriptional map of T-cell memory and define a module of gene expression, which distinguishes memory inflation in spleen from resident memory T-cells (T RM) in the gut. Conclusions: These data indicate that CD8 + T-cell memory in the gut epithelium induced by persistent viruses and vaccines has a distinct quality from both conventional memory and "inflationary" memory which may be relevant to protection against mucosal infections.

Perturbation of mucosal-associated invariant T cells and iNKT cells in HIV infection.

PURPOSE OF REVIEW: To analyze the possible role that the 'unconventional' T-cell populations mucosal-associated invariant T cell (MAIT) and iNKT cells play during HIV infection and following antiretroviral therapy (ART) treatment. RECENT FINDINGS: A substantial body of evidence now demonstrates that both MAIT and iNKT cells are depleted in blood during HIV infection. The depletion and dysfunction of MAIT and iNKT cells are only partially restored by suppressive ART, potentially contributing to HIV-related comorbidities. SUMMARY: The deficiency and dysfunction of MAIT and iNKT T-cell subsets likely impact on immunity to important coinfections including Mycobacterium tuberculosis. This underscores the importance of research on restoring these unconventional T cells during HIV infection. Future studies in this field should address the challenge of studying tissue-resident cells, particularly in the gut, and better defining the determinants of MAIT/iNKT cell dysfunction. Such studies could have a significant impact on improving the immune function of HIV-infected individuals.

CD32-Expressing CD4 T Cells Are Phenotypically Diverse and Can Contain Proviral HIV DNA.

Efforts to both characterize and eradicate the HIV reservoir have been limited by the rarity of latently infected cells and the absence of a specific denoting biomarker. CD32a (FcγRIIa) has been proposed to be a marker for an enriched CD4 T cell HIV reservoir, but this finding remains controversial. Here, we explore the expression of CD32 on CD3+CD4+ cells in participants from two primary HIV infection studies and identify at least three distinct phenotypes (CD32low, CD32+CD14+, and CD32high). Of note, CD4 negative enrichment kits remove the majority of CD4+CD32+ T cells, potentially skewing subsequent analyses if used. CD32high CD4 T cells had higher levels of HLA-DR and HIV co-receptor expression than other subsets, compatible with their being more susceptible to infection. Surprisingly, they also expressed high levels of CD20, TCRαß, IgD, and IgM (but not IgG), markers for both T cells and naïve B cells. Compared with other populations, CD32low cells had a more differentiated memory phenotype and high levels of immune checkpoint receptors, programmed death receptor-1 (PD-1), Tim-3, and TIGIT. Within all three CD3+CD4+CD32+ phenotypes, cells could be identified in infected participants, which contained HIV DNA. CD32 expression on CD4 T cells did not correlate with HIV DNA or cell-associated HIV RNA (both surrogate measures of overall reservoir size) or predict time to rebound viremia following treatment interruption, suggesting that it is not a dominant biomarker for HIV persistence. Our data suggest that while CD32+ T cells can be infected with HIV, CD32 is not a specific marker of the reservoir although it might identify a population of HIV enriched cells in certain situations.

Human MAIT cells show metabolic quiescence with rapid glucose-dependent upregulation of granzyme B upon stimulation.

Mucosal-associated invariant T (MAIT) cells are a well-characterized innate-like T cell population abundant in the human liver, peripheral tissues and blood. MAIT cells serve in the first line of defense against infections, through engagement of their T cell receptor, which recognizes microbial metabolites presented on MR1, and through cytokine-mediated triggering. Typically, they show a quiescent memory phenotype but can undergo rapid upregulation of effector functions including cytolysis upon stimulation. T cells profoundly change their cellular metabolism during their maturation and activation. We sought to determine how MAIT cell metabolism may facilitate both the long-term memory phase in tissue and the transition to rapid effector function. Here, we show, by flow cytometric metabolism assays and extracellular flux analysis that, despite an effector-memory profile, human MAIT cells are metabolically quiescent in a resting state comparable to naïve and central memory T cells. Upon stimulation, they rapidly increase uptake of glucose and show a concomitant upregulation of the effector molecules notably granzyme B, which is impaired by inhibition of glycolysis with 2-deoxyglucose. These findings suggest that MAIT cells share some metabolic characteristics of both resting and effector T cell subsets, with a rapid transition upon triggering. Metabolic programming of this cell type may be of interest in understanding and modulating their function in infectious diseases and cancer.

HIV-1 and SIV Predominantly Use CCR5 Expressed on a Precursor Population to Establish Infection in T Follicular Helper Cells.

BACKGROUND: T follicular helper (Tfh) cells are increasingly recognized as a major reservoir of HIV infection that will likely need to be addressed in approaches to curing HIV. However, Tfh express minimal CCR5, the major coreceptor for HIV-1, and the mechanism by which they are infected is unclear. We have previously shown that macaque Tfh lack CCR5, but are infected in vivo with CCR5-using SIV at levels comparable to other memory CD4+ T cells. Similarly, human splenic Tfh cells are highly infected with HIV-1 DNA. Therefore, we set out to examine the mechanism of infection of Tfh cells. METHODOLOGY: Tfh and other CD4+ T cell subsets from macaque lymph nodes and spleens, splenic Tfh from HIV+ subjects, and tonsillar Tfh from HIV-uninfected subjects were isolated by cell sorting prior to cell surface and molecular characterization. HIV proviral gp120 sequences were submitted to genotypic and phenotypic tropism assays. Entry of CCR5- and CXCR4-using viruses into Tfh from uninfected tonsillar tissue was measured using a fusion assay. RESULTS: Phylogenetic analysis, genotypic, and phenotypic analysis showed that splenic Tfh cells from chronic HIV+ subjects were predominantly infected with CCR5-using viruses. In macaques, purified CCR5+PD-1intermediate(int)+ memory CD4+ T cells were shown to include pre-Tfh cells capable of differentiating in vitro to Tfh by upregulation of PD-1 and Bcl6, confirmed by qRT-PCR and single-cell multiplex PCR. Infected PD-1int cells survive, carry SIV provirus, and differentiate into PD-1hi Tfh after T cell receptor stimulation, suggesting a pathway for SIV infection of Tfh. In addition, a small subset of macaque and human PD-1hi Tfh can express low levels of CCR5, which makes them susceptible to infection. Fusion assays demonstrated CCR5-using HIV-1 entry into CCR5+ Tfh and pre-Tfh cells from human tonsils. CONCLUSION: The major route of infection of Tfh in macaques and humans appears to be via a CCR5-expressing pre-Tfh population. As the generation of Tfh are important for establishing effective immune responses during primary infections, Tfh are likely to be an early target of HIV-1 following transmission, creating an important component of the reservoir that has the potential to expand over time.

Single-cell profiling of lineage determining transcription factors in antigen-specific CD4+ T cells reveals unexpected complexity in recall responses during immune reconstitution.

Recent studies of protein and gene expression at the single-cell level have revealed that the memory T-cell compartment is more heterogeneous than previously acknowledged. Identifying different T helper subsets involved in memory responses at the single-cell level is thus necessary to understand the level of heterogeneity within this population. Antigen-specific CD4+ T cells were measured using the CD25/OX40 assay together with a qualitative multiplex single-cell RT-PCR assay. Transcription profiles and subset proportions within the antigen-specific CD4+ T-cell population were dissected. Cytomegalovirus (CMV)-specific CD4+ T-cell responses skewed toward a Th1 response, whereas Tetanus toxoid responses skewed toward a Th2 type response. Fluctuations in CD4+ T-cell subsets were observed within the HIV-Gag-specific response during ongoing antiretroviral therapy. Strong effector responses (Th1) were observed in early treatment, however with ongoing therapy this effector response significantly decreased in combination with an increase in Tregs and circulating Tfh-like BCL-6+ memory cells. The apparent increase in Tcm in peripheral blood after a several weeks of antiretroviral therapy may be due to Tfh-like cell egress from germinal centers into the periphery.

The Role of CD4+ T Follicular Helper Cells in HIV Infection: From the Germinal Center to the Periphery.

T follicular helper cells (TFh) are key components of the adaptive immune system; they are primarily found in germinal centers (GCs) where their interaction with B cells supports humoral immune responses and efficient antibody production. They are defined by the expression of CXC receptor 5, program death-1, ICOS, and secretion of IL-21. Their differentiation is regulated by B-cell lymphoma 6. The relationship and function of circulating TFh to bona fide TFh resident in the GC is much debated. HIV infection impacts the TFh response with evidence of aberrant TFh function observed in acute and chronic infection. Effective TFh responses are associated with the development of broadly neutralizing antibody responses to HIV and may be important for viral control. In addition, TFh are preferentially infected and act as a key reservoir for latent HIV infection. This review explores recent developments in our understanding of TFh differentiation, regulation, function, and the relationship between cTFh and those in GCs, and the complex interaction between TFh and HIV infection.

Detecting Antigen-Specific T Cell Responses: From Bulk Populations to Single Cells.

A new generation of sensitive T cell-based assays facilitates the direct quantitation and characterization of antigen-specific T cell responses. Single-cell analyses have focused on measuring the quality and breadth of a response. Accumulating data from these studies demonstrate that there is considerable, previously-unrecognized, heterogeneity. Standard assays, such as the ICS, are often insufficient for characterization of rare subsets of cells. Enhanced flow cytometry with imaging capabilities enables the determination of cell morphology, as well as the spatial localization of the protein molecules within a single cell. Advances in both microfluidics and digital PCR have improved the efficiency of single-cell sorting and allowed multiplexed gene detection at the single-cell level. Delving further into the transcriptome of single-cells using RNA-seq is likely to reveal the fine-specificity of cellular events such as alternative splicing (i.e., splice variants) and allele-specific expression, and will also define the roles of new genes. Finally, detailed analysis of clonally related antigen-specific T cells using single-cell TCR RNA-seq will provide information on pathways of differentiation of memory T cells. With these state of the art technologies the transcriptomics and genomics of Ag-specific T cells can be more definitively elucidated.