Tissue-resident immunity mediates host defense against pathogens and enables rapid adaptive memory responses. However, the study of tissue-resident immunity is hindered by a singular lack of experimental systems allowing pathogenic epithelial infection amidst the full spectrum of endogenous immune subsets. Particularly in lung, differing notions of transient versus sustained residency of tissue-resident memory T cells (TRM) have questioned the extent to which recall immunity to respiratory pathogens occurs locally or in concert with secondary lymphoid organs. We thus generated long-term adult human distal lung organoids from intact tissue fragments in 3D air-liquid interface (ALI) culture that co-preserved epithelial and stromal architecture alongside endogenous lung-resident immune cells (T, B, NK, myeloid). The organoid T cells exhibited persistent cytokine-assisted maintenance, expressed residency and memory markers, and preserved T cell receptor (TCR) repertoires of cognate fresh tissue. SARS-CoV-2 vigorously infected the organoid lung epithelium, stimulated inflammatory cytokine production, and crucially, induced widespread SARS-CoV-2-specific, tissue-resident T cell responses. Our studies introduce a robust adult human lung organoid experimental system containing a physiologic air interface and diverse resident immune subsets, demonstrate the organ-autonomous sufficiency of lung pathogen memory T cell responses, distinct from secondary lymphoid tissue, and provide a platform to investigate tissue-resident immunity in health and disease.

Human vaccine responses vary widely, but the determinants remain incompletely defined. Here we analyzed 66 cytokines across four inactivated influenza vaccine (IIV) cohorts over five seasons (n = 581) and identified baseline serum interleukin (IL)-18 and interferon (IFN)-β as correlates of day 28 antibody responses. To test causality, we evaluated 19 cytokines in human tonsil and spleen organoids and found that type I IFNs, IL-21 and IL-12, but not IL-18 or IFNγ, enhanced antibody production. The addition of IFNβ to IIV recapitulated key features of the live-vaccine cytokine program. IL-12 and IL-21 defined a parallel pathway independent of type I IFNs, with IL-12 inducing IL-21 in humans, unlike in mice. Delivery of IL-21 or IFNβ via mRNA lipid nanoparticles in vivo promoted long-lived plasma cell formation. Together, these findings define parallel pathways that regulate vaccine immunity. Our approach unites high-throughput organoid testing and human cohort studies, establishing a human-centric platform to identify adjuvant candidates.

Abstract Introduction Mycobacterium tuberculosis (Mtb) remains a leading cause of death worldwide. IFNγ Release Assay (IGRA) is widely used to diagnose Mtb infection by measuring the IFNγ response to Mtb antigens. However, healthy IGRA- individuals with high Mtb exposure (“resisters”) have shown evidence of infection and make Mtb-specific responses that differ from IGRA+ individuals. Human CD4 T cells are critical in controlling Mtb. IFNγ-expressing Th1 cells are largely believed to be the major protective subset. Resisters’ negative response to IGRA suggests alternative protective mechanisms of CD4 T cells. This study aims to profile the antigen-specific CD4+ T cell responses in Mtb-exposed IGRA- individuals. Methods We performed TCR sequencing on Ugandan resisters and IGRA+ individuals. TCR repertoires were analyzed with GLIPH3 algorithm we recently developed to identify resister-specific TCRs. Mtb antigenic ligands were discovered by a new T cell epitope discovery platform. Antigen-specific CD4+ T cells were then isolated using peptide-MHC multimers covering the discovered antigenic peptide, and characterized by single-cell multi-omics and Flow Cytometry. Results We identified 24 TCR specificity groups uniquely enriched in resisters. Two ligand peptides were decoded from Mtb antigens Rv2140c and ESAT6. In a parallel South African cohort, in IGRA- individuals, we detected T cell responses to ESAT6, the antigen used in IGRA test, and a robust response to Rv2140c. Notably, Rv2140c-specific CD4 T cells were predominantly follicular helper cells (Tfh), which correlated with protection from Mtb in mice, whereas IGRA+ individuals showed primarily Th1 responses. Conclusion We identified a novel Mtb antigen associated with protection, highlighting its potential as vaccine candidate. ESAT6-specific responses in IGRA- individuals confirmed underlying Mtb infection, indicating the limitations of IGRA tests. The predominance of Tfh cells reveals new protective human T cell mechanism against Mtb beyond the classic Th1 response. Funding Source Bill & Melinda Gates Foundation Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Abstract Introduction The immunodominance hierarchy of influenza antigens remains a major obstacle in developing broadly protective vaccines. Current flu vaccines primarily elicit antibody responses against variable head regions of hemagglutinin (HA), leading to strain-specific immunity and limited cross-protection. Cyclic dinucleotides (CDNs), potent agonists of the stimulator of interferon genes (STING) pathway, have emerged as promising adjuvants that can enhance both humoral and cellular immunity. In this study, we sought to investigate the capacity of CDN nanoparticle formulations to overcome immunodominance in influenza vaccination by combining CDN nanoparticles with influenza antigens. Methods Female C57BL/6 mice were immunized subcutaneously twice, with a 4-week interval between doses. Each group received either inactivated influenza vaccine (IIV) or hemagglutinin (HA) subunit antigen formulated with or without CDN nanoparticles. Serum samples were collected at defined time points to quantify antigen-specific IgG responses by ELISA. At the study endpoint, draining lymph nodes (inguinal lymph nodes) were harvested to assess germinal center B cell responses by flow cytometry, while bone marrow samples were collected to evaluate the frequencies of long-lived plasma cells reactive to influenza antigens by ELISpot. Results CDN nanoparticles not only enhanced the GC formation in draining lymph nodes, resulting in increased frequencies of stem-specific B cells within the GC, but also increased the frequencies of long-lived plasma cells (LLPCs) in the bone marrow. Consequently, serum antibody responses were broader and more durable. Conclusion CDN nanoparticle adjuvants broadened antibody specificity, enhanced germinal center reactions, and promoted durable long-lived plasma cell formation. This strategy overcomes the immunodominance of conventional vaccines and represents a promising approach for developing next-generation influenza vaccines with broader and longer-lasting protection. Funding Source NIH Topic Categories Vaccines and Immunotherapy (VAC)

A decline in specific antibody responses is a hallmark of human aging, yet the differential contributions of B and T lymphocytes remain unclear. CXCL13 is a chemokine that shapes germinal center (GC) organization, but the regulation of human-specific CXCL13+ T follicular helper (Tfh) cells during aging is not known. Using human tonsil organoids, single-cell RNA sequencing, and CRISPR perturbations, we mapped age-associated changes in Tfh cells, the cell type that provides help to B cells in GCs. Tonsil organoids from older donors generate weaker influenza-specific antibody responses, which we trace to Tfh cell defects rather than B cells. Single-cell profiling revealed a selective loss of mature CXCL13+ GC-Tfh cells accompanied by accumulation of precursor states. Trajectory analysis shows that aging arrests Tfh maturation at the early activated precursor transition, and CRISPR perturbations identify BACH2 and SOX4 as regulators of differentiation reduced with age. These findings reveal a human-specific mechanism of immune aging with implications for strategies to restore humoral immunity.

Despite advances in vaccine and antiviral drug development, the prevention of respiratory viral infection and transmission remains a substantial challenge worldwide. One obvious limitation of these approaches is that they do not provide robust protection at the initial site of infection, which is the respiratory mucosa. Currently, strategies to enhance mucosal immunity against respiratory pathogens remain lacking. Here we engineered mucus-tethering bispecific nanobodies designed to provide the simultaneous neutralization of viruses by binding to their surface proteins and the entrapment of viruses within the mucus by securing them to mucin. Compared with conventional non-mucus-tethering nanobodies, these mucus-tethering bispecific nanobodies demonstrated increased retention in the respiratory tract, provided enhanced protection against influenza viral infection in mice and reduced SARS-CoV-2 transmission in hamsters. Together, our findings represent a promising strategy for enhancing mucosal defences against respiratory viruses by blocking viral entry and limiting onward transmission. Engineered mucus-tethering bispecific nanobodies neutralize and entrap viruses to enhance mucosal immunity, preventing influenza infection and limiting SARS-CoV-2 transmission.

Study Objectives:Onsets of Narcolepsy type-1 (NT1) increased following A/H1N1 vaccination with Pandemrix® in Europe and with A/H1N1pdm2009 infections in China and other countries. To test if other strains could trigger narcolepsy, we measured strain-specific antibodies in patients with recent onset NT1 compared to controls. Methods:Antibodies against hemagglutinin (HA) and neuraminidase (NA) were tested in 62 patients with very recent onset (onset and blood collection following a single flu season, mean ± SEM: 0.44 ± 0.06 years since onset) and 100 controls matched by age, sex, season and year of collection (2000-2025). Results were next extended to 181 recent onset patients (mean± SEM: 1.00 ± 0.05 years) versus 260 controls, matched by sex, season and year, but having a slightly higher mean age. HA inhibition (HAI) and NA inhibition (NAI) assays were conducted using flu strains known to circulate during the corresponding flu seasons. HAI results are shown as % positive (titers ≥ 40) and NAI results as geometric mean titers. Odds ratio (OR) and β coefficient were used to compare antibody titers in NT1 versus controls. The contribution of each assay to prediction was finally quantified in the larger sample set using Shapley decomposition. Results:NT1 patients had increased anti-HA and anti-NA antibodies against A/H1N1pdm2009 (anti-HA OR= 3.86, anti-NA β= 0.35) and B/Victoria (anti-HA OR=1.90, anti-NA β=0.22), but not A/H1N1pre2009, A/H3N2, or B/Yamagata, independent of HLA-DQB1*06:02 status, age, sex, and flu season. Correlations between anti-HA and anti-NA antibodies titers were weak to moderate but significant (r 2 =-0.10 to 0.34). Multivariable model outperformed age-only baseline (McFadden R 2 = 0.19 vs. 0.03; AUC = 0.79 vs. 0.64; likelihood-ratio test χ 2 = 51, p<10 -9 ), with anti-HA against A/H1N1pdm2009 (β = 0.78, p < 10 -6 ) and anti-NA against B/Victoria (β = 0.69, p < 10 -5 ) emerging as the strongest independent predictors. Conclusions:A/H1N1pdm2009 and B/Victoria, but not other strains can trigger the autoimmune process leading to orexin cell loss in narcolepsy.

Over 65 million individuals worldwide are estimated to have Long COVID (LC), a complex multisystemic condition marked by fatigue, post-exertional malaise, and other symptoms resembling myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). With no clinically approved treatments or reliable diagnostic markers, there is an urgent need to define the molecular underpinnings of these conditions. By studying bioenergetic characteristics of peripheral blood lymphocytes in 25 healthy controls, 27 ME/CFS, and 20 LC donors, we find both ME/CFS and LC donors exhibit signs of elevated oxidative stress, especially in the memory subset. Using a combination of flow cytometry, RNA-seq, mass spectrometry, and systems chemistry analysis, we observed aberrations in reactive oxygen species (ROS) clearance pathways including elevated glutathione levels, decreases in mitochondrial superoxide dismutase protein levels, and glutathione peroxidase 4-mediated lipid oxidative damage. Strikingly, these redox pathways changes show sex-specific trends. While ME/CFS females exhibit higher total ROS and mitochondrial calcium levels, males have normal ROS levels, with pronounced mitochondrial lipid oxidative damage. In females, these higher ROS levels correlate with T cell hyperproliferation, consistent with the known role of elevated ROS in initiating proliferation. This hyperproliferation can be attenuated by metformin, suggesting this Food and Drug Administration (FDA)-approved drug as a possible treatment, as also suggested by a recent clinical study of LC patients. Moreover, these results suggest a shared mechanistic basis for the systemic phenotypes of ME/CFS and LC, which can be detected by quantitative blood cell measurements, and that effective, patient-tailored drugs might be discovered using standard lymphocyte stimulation assays.

Studies show that human vaccine responses widely vary. Here, we analyzed data measuring 66 cytokines from 4 different inactivated influenza vaccine (IIV) cohorts over 5 seasons (N=581) and identified a significant correlation between baseline/day 0 serum IL-18 and IFN-β concentrations and the vaccine-specific antibody response on day 28, suggesting these factors may have an adjuvant-like effect. To investigate this further, we tested the impact of 19 cytokines on the development of anti-influenza antibody response when administered together with the IIV vaccine in human tonsil and spleen organoids. We found that Type I IFNs (IFN-β and others), IL-21, IL-12, IL-10, but not IL-18 or IFN-γ, enhanced the antibody response. The live attenuated influenza vaccine (LAIV) induced a stronger antibody response than the inactivated one in organoids. Adding a single cytokine, IFN-β, to IIV stimulation recapitulated most of the live vaccine-specific cytokine activation program. It increased the antibody response of the inactivated vaccine to that of the LAIV. Two other antibody-boosting cytokines, IL-12 and IL-21, were induced by LAIV but not by Type I IFNs, indicating different cytokines can affect different pathways leading to a robust antibody response. We then generated cytokine mRNA lipid nanoparticles (LNPs) to test the effect of cytokines on IIV response in a mouse model of immunization. We found that IL-21-LNPs augmented the quantity and breadth of the antibody responses, while IFN-β LNPs enhanced durability. These findings identified parallel cytokine pathways regulating human vaccine responses and provide a rationale for using cytokines as adjuvants to mimic the effectiveness of live-attenuated vaccines without the risk of viral replication. ### Competing Interest Statement The authors have declared no competing interest.

Here we analyzed the relative contributions of CD4+ regulatory T cells expressing Forkhead box protein P3 (FOXP3) and CD8+ regulatory T cells expressing killer cell immunoglobulin-like receptors to the control of autoreactive T and B lymphocytes in human tonsil-derived immune organoids. FOXP3 and GZMB respectively encode proteins FOXP3 and granzyme B, which are critical to the suppressive functions of CD4+ and CD8+ regulatory T cells. Using CRISPR-Cas9 gene editing, we were able to achieve a reduction of ~90-95% in the expression of these genes. FOXP3 knockout in tonsil T cells led to production of antibodies against a variety of autoantigens and increased the affinity of influenza-specific antibodies. By contrast, GZMB knockout resulted in an increase in follicular helper T cells, consistent with the ablation of CD8+ regulatory T cells observed in mouse models, and a marked expansion of autoreactive CD8+ and CD4+ T cells. These findings highlight the distinct yet complementary roles of CD8+ and CD4+ regulatory T cells in regulating cellular and humoral responses to prevent autoimmunity.

It has recently emerged that a small subset of CD8+ T cells in humans or mice plays a key role in controlling autoimmunity. However, how these cells function compared to the much better-known CD4+ regulatory T cells is not well understood. Here, we analyzed the relative contribution of CD4 + (FOXP3+) and CD8 + (KIR+) regulatory T cells to the control of autoreactive T and B lymphocytes in human tonsil-derived immune organoids. FOXP3 and GZMB are essential genes in the suppressive functions of CD4+ and CD8+ regulatory T cells, respectively, and we were able to reduce the expression of these genes by ∼90-95% using CRISPR-Cas9 gene editing. Knocking out FOXP3 in tonsil T cells produced antibodies to a variety of autoantigens and also increased the affinity of influenza-specific antibodies. In contrast, a knocking out GZMB resulted in an increase in follicular helper T cells, consistent with the ablation of CD8+ regulatory T cells in mouse models, and a marked increase in autoreactive CD8+ and CD4+ T cells. Thus, CD8+ and CD4+ regulatory T cells have distinct and complementary roles in regulating cellular and humoral responses to prevent autoimmunity. Supported by the Howard Hughes Medical Institute, Open Philanthropy, The Bill and Melinda Gates Foundation, and NIAID (AI057229) Basic Autoimmunity (BA)

While it has been known for many years that children under five years old are much more vulnerable to most infectious diseases than older children or adults, we know very little about the specific immunological reasons. Thus, we leveraged our recently developed tonsil organoid model, a high-resolution in vitro system of human immunity to vaccination, to compare tonsils from children as young as 2 years old to those from young adults. After stimulation with the live attenuated influenza vaccine, toddlers exhibited lower levels of influenza-specific IgA and IgG antibodies, limited T-independent response, and fewer activated cytotoxic CD8+T cells, all critical components supporting influenza defense. Additionally, toddlers showed reduced levels of key cytokine signaling proteins, including FLT3L, IL2, IL17, TACI, which are important in antibody class switching. Conversely, toddlers produced more of the pro-inflammatory cytokines CCL2 and PAI1, both associated with more severe influenza infection. We observed fewer interactions between T and B cells and diminished TLR and T-bet signaling in toddlers than in adults. Further analysis identified distinct metabolic disadvantages in toddlers, particularly within germinal centers, observed in a time-dependent manner. Machine learning analyses of our multi-omic data highlighted dominant variables and key predictors that distinguish diverse immune responses among groups. Our study used systems approaches to underscore critical deficits in cellular compositions, cytokine profiles, intracellular signaling, cell-cell interactions, and metabolic programs in young children's immune systems under vaccine/viral stimulation, offering valuable guidance for future vaccine development and therapies.

During pregnancy, immune responses must balance protection from infections with tolerance of the semiallogeneic fetus. However, the mechanisms regulating maternal-fetal tolerance remain poorly understood. Recently, we identified CD8 + T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) as a regulatory subset important for suppressing self-reactivity in human autoimmune and infectious diseases. To better understand what other roles these cells might play, we asked whether they are active during pregnancy. We first observed an increased frequency of KIR + CD8 + T cells in the peripheral blood of pregnant people in the second trimester, especially in those carrying a male fetus. In vitro, KIR + CD8 + T cells inhibited the alloreactive responses of maternal T cells against irradiated cord blood cells and selectively suppressed CD8 + T cells targeting male-specific proteins in mothers with male pregnancies. Therefore, the higher induction of KIR + CD8 + T cells in mothers carrying a male fetus may help suppress additional allogeneic responses triggered by male-specific alloantigens. Longitudinal analysis showed that KIR + CD8 + T cells undergo expansion and differentiate into functional cytotoxic cells during pregnancy. Single-cell RNA sequencing of decidual CD8 + T cells from early pregnancy revealed elevated numbers and increased expression of activation markers in KIR + CD8 + T cells at the maternal-fetal interface. In addition, a higher frequency of KIR + CD8 + T cells was associated with spontaneous abortion and preeclampsia. Together, our findings suggest that KIR + CD8 + T cells may contribute to maternal tolerance by modulating fetal-specific alloreactive T cell responses. They may also be useful as candidate biomarkers or therapeutic targets for human pregnancy disorders.

More than 65 million individuals worldwide are estimated to have Long COVID (LC), a complex multisystemic condition, wherein patients of all ages report fatigue, post-exertional malaise, and other symptoms resembling myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS). With no current treatments or reliable diagnostic markers, there is an urgent need to define the molecular underpinnings of these conditions. By studying bioenergetic characteristics of peripheral blood lymphocytes in over 16 healthy controls, 15 ME/CFS, and 15 LC, we find both ME/CFS and LC donors exhibit signs of elevated oxidative stress, relative to healthy controls, especially in the memory subset. Using a combination of flow cytometry, bulk RNA-seq analysis, mass spectrometry, and systems chemistry analysis, we also observed aberrations in ROS clearance pathways including elevated glutathione levels, decreases in mitochondrial superoxide dismutase levels, and glutathione peroxidase 4 mediated lipid oxidative damage. Critically, these changes in redox pathways show striking sex-specific trends. While females diagnosed with ME/CFS exhibit higher total ROS and mitochondrial calcium levels, males with an ME/CFS diagnosis have normal ROS levels, but larger changes in lipid oxidative damage. Further analyses show that higher ROS levels correlates with hyperproliferation of T cells in females, consistent with the known role of elevated ROS levels in the initiation of proliferation. This hyperproliferation of T cells can be attenuated by metformin, suggesting this FDA-approved drug as a possible treatment, as also suggested by a recent clinical study of LC patients. Thus, we report that both ME/CFS and LC are mechanistically related and could be diagnosed with quantitative blood cell measurements. We also suggest that effective, patient tailored drugs might be discovered using standard lymphocyte stimulation assays.

γδ T cells are essential for immune defense and modulating physiological processes. While they have the potential to recognize large numbers of antigens through somatic gene rearrangement, the antigens which trigger most γδ T cell response remain unidentified, and the role of antigen recognition in γδ T cell function is contentious. Here, we show that some γδ T cell receptors (TCRs) exhibit polyspecificity, recognizing multiple ligands of diverse molecular nature. These ligands include haptens, metabolites, neurotransmitters, posttranslational modifications, as well as peptides and proteins of microbial and host origin. Polyspecific γδ T cells are enriched among activated cells in naive mice and the responding population in infection. They express diverse TCR sequences, have different functional potentials, and include the innate-like γδ T cells, such as the major IL-17 responders in various pathological/physiological conditions. We demonstrate that encountering their antigenic microbiome metabolite maintains their homeostasis and functional response, indicating that their ability to recognize multiple ligands is essential for their function. Human γδ T cells with similar polyspecificity also respond to various immune challenges. This study demonstrates that polyspecificity is a prevalent feature of γδ T cell antigen recognition, which enables rapid and robust T cell responses to a wide range of challenges, highlighting a unique function of γδ T cells.

The seasonal influenza vaccine contains strains of viruses from distinct subtypes that are grown independently and then combined. However, most individuals exhibit a more robust response to one of these strains and thus are vulnerable to infection by others. By studying a monozygotic twin cohort, we found that although prior exposure is a factor, host genetics are a stronger driver of subtype bias to influenza viral strains. We found that covalent coupling of heterologous hemagglutinin (HA) from different viral strains could largely eliminate subtype bias in an animal model and in a human tonsil organoid system. We proposed that coupling of heterologous antigens improves antibody responses across influenza strains by broadening T cell help, and we found that using this approach substantially improved the antibody response to avian influenza HA.

Anti-IgLON5 disease is a rare and likely underdiagnosed subtype of autoimmune encephalitis. The disease displays a heterogeneous phenotype that includes sleep, movement and bulbar-associated dysfunction. The presence of IgLON5-antibodies in CSF/serum, together with a strong association with HLA-DRB1*10:01∼DQB1*05:01, supports an autoimmune basis. In this study, a multicentric human leukocyte antigen (HLA) study of 87 anti-IgLON5 patients revealed a stronger association with HLA-DQ than HLA-DR. Specifically, we identified a predisposing rank-wise association with HLA-DQA1*01:05∼DQB1*05:01, HLA-DQA1*01:01∼DQB1*05:01 and HLA-DQA1*01:04∼DQB1*05:03 in 85% of patients. HLA sequences and binding cores for these three DQ heterodimers were similar, unlike those of linked DRB1 alleles, supporting a causal link to HLA-DQ. This association was further reflected in an increasingly later age of onset across each genotype group, with a delay of up to 11 years, while HLA-DQ-dosage dependent effects were also suggested by reduced risk in the presence of non-predisposing DQ1 alleles. The functional relevance of the observed HLA-DQ molecules was studied with competition binding assays. These proof-of-concept experiments revealed preferential binding of IgLON5 in a post-translationally modified, but not native, state to all three risk-associated HLA-DQ receptors. Further, a deamidated peptide from the Ig2-domain of IgLON5 activated T cells in two patients, compared with one control carrying HLA-DQA1*01:05∼DQB1*05:01. Taken together, these data support a HLA-DQ-mediated T-cell response to IgLON5 as a potentially key step in the initiation of autoimmunity in this disease.

The ideal vaccine against viruses such as influenza and SARS-CoV-2 must provide a robust, durable and broad immune protection against multiple viral variants. However, antibody responses to current vaccines often lack robust cross-reactivity. Here we describe a polymeric Toll-like receptor 7 agonist nanoparticle (TLR7-NP) adjuvant, which enhances lymph node targeting, and leads to persistent activation of immune cells and broad immune responses. When mixed with alum-adsorbed antigens, this TLR7-NP adjuvant elicits cross-reactive antibodies for both dominant and subdominant epitopes and antigen-specific CD8 + T-cell responses in mice. This TLR7-NP-adjuvanted influenza subunit vaccine successfully protects mice against viral challenge of a different strain. This strategy also enhances the antibody response to a SARS-CoV-2 subunit vaccine against multiple viral variants that have emerged. Moreover, this TLR7-NP augments antigen-specific responses in human tonsil organoids. Overall, we describe a nanoparticle adjuvant to improve immune responses to viral antigens, with promising implications for developing broadly protective vaccines.

T cells are a critical component of the response to SARS-CoV-2, but their kinetics after infection and vaccination are insufficiently understood. Using "spheromer" peptide-MHC multimer reagents, we analyzed healthy subjects receiving two doses of the Pfizer/BioNTech BNT162b2 vaccine. Vaccination resulted in robust spike-specific T cell responses for the dominant CD4+ (HLA-DRB1∗15:01/S191) and CD8+ (HLA-A∗02/S691) T cell epitopes. Antigen-specific CD4+ and CD8+ T cell responses were asynchronous, with the peak CD4+ T cell responses occurring 1 week post the second vaccination (boost), whereas CD8+ T cells peaked 2 weeks later. These peripheral T cell responses were elevated compared with COVID-19 patients. We also found that previous SARS-CoV-2 infection resulted in decreased CD8+ T cell activation and expansion, suggesting that previous infection can influence the T cell response to vaccination.