Effector memory-type regulatory T cells display phenotypic and functional instability.

Regulatory T cells (Treg cells) hold promise for sustainable therapy of immune disorders. Recent advancements in chimeric antigen receptor development and genome editing aim to enhance the specificity and function of Treg cells. However, impurities and functional instability pose challenges for the development of safe gene-edited Treg cell products. Here, we examined different Treg cell subsets regarding their fate, epigenomic stability, transcriptomes, T cell receptor repertoires, and function ex vivo and after manufacturing. Each Treg cell subset displayed distinct features, including lineage stability, epigenomics, surface markers, T cell receptor diversity, and transcriptomics. Earlier-differentiated memory Treg cell populations, including a hitherto unidentified naïve-like memory Treg cell subset, outperformed late-differentiated effector memory-like Treg cells in regulatory function, proliferative capacity, and epigenomic stability. High yields of stable, functional Treg cell products could be achieved by depleting the small effector memory-like Treg cell subset before manufacturing. Considering Treg cell subset composition appears critical to maintain lineage stability in the final cell product.

Long-term cellular immunity of vaccines for Zaire Ebola Virus Diseases.

Recent Ebola outbreaks underscore the importance of continuous prevention and disease control efforts. Authorized vaccines include Merck's Ervebo (rVSV-ZEBOV) and Johnson & Johnson's two-dose combination (Ad26.ZEBOV/MVA-BN-Filo). Here, in a five-year follow-up of the PREVAC randomized trial (NCT02876328), we report the results of the immunology ancillary study of the trial. The primary endpoint is to evaluate long-term memory T-cell responses induced by three vaccine regimens: Ad26-MVA, rVSV, and rVSV-booster. Polyfunctional EBOV-specific CD4+ T-cell responses increase after Ad26 priming and are further boosted by MVA, whereas minimal responses are observed in the rVSV groups, declining after one year. In-vitro expansion for eight days show sustained EBOV-specific T-cell responses for up to 60 months post-prime vaccination with both Ad26-MVA and rVSV, with no decline. Cytokine production analysis identify shared biomarkers between the Ad26-MVA and rVSV groups. In secondary endpoint, we observed an elevation of pro-inflammatory cytokines at Day 7 in the rVSV group. Finally, we establish a correlation between EBOV-specific T-cell responses and anti-EBOV IgG responses. Our findings can guide booster vaccination recommendations and help identify populations likely to benefit from revaccination.

Tissue-resident memory T cells in epicardial adipose tissue comprise transcriptionally distinct subsets that are modulated in atrial fibrillation.

Atrial fibrillation (AF) is the most common sustained arrhythmia and carries an increased risk of stroke and heart failure. Here we investigated how the immune infiltrate of human epicardial adipose tissue (EAT), which directly overlies the myocardium, contributes to AF. Flow cytometry analysis revealed an enrichment of tissue-resident memory T (TRM) cells in patients with AF. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell T cell receptor (TCR) sequencing identified two transcriptionally distinct CD8+ TRM cells that are modulated in AF. Spatial transcriptomic analysis of EAT and atrial tissue identified the border region between the tissues to be a region of intense inflammatory and fibrotic activity, and the addition of TRM populations to atrial cardiomyocytes demonstrated their ability to differentially alter calcium flux as well as activate inflammatory and apoptotic signaling pathways. This study identified EAT as a reservoir of TRM cells that can directly modulate vulnerability to cardiac arrhythmia.

mRNA-1273 vaccination induces polyfunctional memory CD4 and CD8 T cell responses in patients with solid cancers undergoing immunotherapy or/and chemotherapy.

Introduction: Research has confirmed the safety and comparable seroconversion rates following SARS-CoV-2 vaccination in patients with solid cancers. However, the impact of cancer treatment on vaccine-induced T cell responses remains poorly understood. Methods: In this study, we expand on previous findings within the VOICE trial by evaluating the functional and phenotypic composition of mRNA-1273-induced T cell responses in patients with solid tumors undergoing immunotherapy, chemotherapy, or both, compared to individuals without cancer. We conducted an ELISpot analysis on 386 participants to assess spike-specific T cell responses 28 days after full vaccination. Further in-depth characterization of using flow cytometry was performed on a subset of 63 participants to analyze the functional phenotype and differentiation state of spike-specific T cell responses. Results: ELISpot analysis showed robust induction of spike-specific T cell responses across all treatment groups, with response rates ranging from 75% to 80%. Flow cytometry analysis revealed a distinctive cytokine production pattern across cohorts, with CD4 T cells producing IFNγ, TNF, and IL-2, and CD8 T cells producing IFNγ, TNF, and CCL4. Variations were observed in the proportion of monofunctional CD4 T cells producing TNF, particularly higher in individuals without cancer and patients treated with chemotherapy alone, while those treated with immunotherapy or chemoimmunotherapy predominantly produced IFNγ. Despite these differences, polyfunctional spike-specific memory CD4 and CD8 T cell responses were comparable across cohorts. Notably, immunotherapy-treated patients exhibited an expansion of spike-specific CD4 T cells with a terminally differentiated effector memory phenotype. Discussion: These findings demonstrate that systemic treatment in patients with solid tumors does not compromise the quality of polyfunctional mRNA-1273-induced T cell responses. This underscores the importance of COVID-19 vaccination in patients with solid cancers undergoing systemic treatment.

Single cell RNA-sequencing delineates CD8+ tissue resident memory T cells maintaining rejection in liver transplantation.

Rationale: Understanding the immune mechanisms associated with liver transplantation (LT), particularly the involvement of tissue-resident memory T cells (TRMs), represents a significant challenge. Methods: This study employs a multi-omics approach to analyse liver transplant samples from both human (n = 17) and mouse (n = 16), utilizing single-cell RNA sequencing, bulk RNA sequencing, and immunological techniques. Results: Our findings reveal a comprehensive T cell-centric landscape in LT across human and mouse species, involving 235,116 cells. Notably, we found a substantial increase in CD8+ TRMs within rejected grafts compared to stable ones. The elevated presence of CD8+ TRMs is characterised by a distinct expression profile, featuring upregulation of tissue-residency markers (CD69, CXCR6, CD49A and CD103+/-,), immune checkpoints (PD1, CTLA4, and TIGIT), cytotoxic markers (GZMB and IFNG) and proliferative markers (PCNA and TOP2A) during rejection. Furthermore, there is a high expression of transcription factors such as EOMES and RUNX3. Functional assays and analyses of cellular communication underscore the active role of CD8+ TRMs in interacting with other tissue-resident cells, particularly Kupffer cells, especially during rejection episodes. Conclusions: These insights into the distinctive activation and interaction patterns of CD8+ TRMs suggest their potential utility as biomarkers for graft rejection, paving the way for novel therapeutic strategies aimed at enhancing graft tolerance and improving overall transplant outcomes.

Preschool-age children maintain a distinct memory CD4+ T cell and memory B cell response after SARS-CoV-2 infection.

The development of the human immune system lasts for several years after birth. The impact of this maturation phase on the quality of adaptive immunity and the acquisition of immunological memory after infection at a young age remains incompletely defined. Here, using an antigen-reactive T cell (ARTE) assay and multidimensional flow cytometry, we profiled circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-reactive CD3+CD4+CD154+ T cells in children and adults before infection, during infection, and 11 months after infection, stratifying children into separate age groups and adults according to disease severity. During SARS-CoV-2 infection, children younger than 5 years old displayed a lower antiviral CD4+ T cell response, whereas children older than 5 years and adults with mild disease had, quantitatively and phenotypically, comparable virus-reactive CD4+ T cell responses. Adults with severe disease mounted a response characterized by higher frequencies of virus-reactive proinflammatory and cytotoxic T cells. After SARS-CoV-2 infection, preschool-age children not only maintained neutralizing SARS-CoV-2-reactive antibodies postinfection comparable to adults but also had phenotypically distinct memory T cells displaying high inflammatory features and properties associated with migration toward inflamed sites. Moreover, preschool-age children had markedly fewer circulating virus-reactive memory B cells compared with the other cohorts. Collectively, our results reveal unique facets of antiviral immunity in humans at a young age and indicate that the maturation of adaptive responses against SARS-CoV-2 toward an adult-like profile occurs in a progressive manner.

Generation of antigen-specific memory CD4 T cells by heterologous immunization enhances the magnitude of the germinal center response upon influenza infection.

Current influenza vaccine strategies have yet to overcome significant obstacles, including rapid antigenic drift of seasonal influenza viruses, in generating efficacious long-term humoral immunity. Due to the necessity of germinal center formation in generating long-lived high affinity antibodies, the germinal center has increasingly become a target for the development of novel or improvement of less-efficacious vaccines. However, there remains a major gap in current influenza research to effectively target T follicular helper cells during vaccination to alter the germinal center reaction. In this study, we used a heterologous infection or immunization priming strategy to seed an antigen-specific memory CD4+ T cell pool prior to influenza infection in mice to evaluate the effect of recalled memory T follicular helper cells in increased help to influenza-specific primary B cells and enhanced generation of neutralizing antibodies. We found that heterologous priming with intranasal infection with acute lymphocytic choriomeningitis virus (LCMV) or intramuscular immunization with adjuvanted recombinant LCMV glycoprotein induced increased antigen-specific effector CD4+ T and B cellular responses following infection with a recombinant influenza strain that expresses LCMV glycoprotein. Heterologously primed mice had increased expansion of secondary Th1 and Tfh cell subsets, including increased CD4+ TRM cells in the lung. However, the early enhancement of the germinal center cellular response following influenza infection did not impact influenza-specific antibody generation or B cell repertoires compared to primary influenza infection. Overall, our study suggests that while heterologous infection or immunization priming of CD4+ T cells is able to enhance the early germinal center reaction, further studies to understand how to target the germinal center and CD4+ T cells specifically to increase long-lived antiviral humoral immunity are needed.

Excess generation and activation of naturally arising memory-phenotype CD4+ T lymphocytes are inhibited by regulatory T cells in steady state.

Conventional CD4+ T lymphocytes consist of naïve, foreign antigen-specific memory, and self-antigen-driven memory-phenotype (MP) cell compartments at homeostasis. We recently showed that MP cells tonically proliferate in response to self-antigens and differentiate into the T-bet+ subset in steady state. How excess proliferation and differentiation of MP cells are inhibited remains unclear. Given immunosuppressive function of regulatory T cells (Tregs), it is possible that they are also involved in inhibition of spontaneous MP cell activation. Here we show using Foxp3-diphtheria toxin receptor-transgenic mice that both MP and naïve CD4+ T cells spontaneously proliferate and differentiate into Th1 cells upon acute Treg depletion. At an early time point post Treg depletion, MP as compared to naïve CD4+ T cells are preferentially activated while at a later stage, the response is dominated by activated cells originated from the naïve pool. Moreover, we argue that MP cell proliferation is driven by TCR and CD28 signaling whereas Th1 differentiation mediated by IL-2. Furthermore, our data indicate that such activation of MP and naïve CD4+ T lymphocytes contribute to development of multi-organ inflammation at early and later time points, respectively, after Treg ablation. Together our findings reveal that Tregs tonically inhibit early, spontaneous proliferation and Th1 differentiation of MP CD4+ T lymphocytes as well as late activation of naïve cells, thereby contributing to maintenance of T cell homeostasis.

Contribution of memory T cells to the generation of long-lasting immunity against COVID-19.

Humans are exposed every day to innumerable external stimuli, both environmental and microbial. Immunological memory recalls each specific stimulus and mounts a secondary response that is faster and of a larger magnitude than the primary response; this process constitutes the basis for vaccine development. The COVID-19 pandemic offers a unique opportunity to study the development of immune memory against an emergent microorganism. Memory T cells have an important role in the resolution of COVID-19, and they are key pillars of immunological memory. In this review, we summarize the main findings regarding anti-SARS-CoV-2 memory T cells after infection, after vaccination, and after the combination of these two events ("hybrid immunity"), and analyze how these cells can contribute to long-term protection against the infection with SARS-CoV-2 variants.

Los humanos se exponen cada día a innumerables estímulos externos, tanto ambientales como microbianos. La memoria inmunológica registra de manera específica un estímulo y articula una respuesta secundaria más rápida y de mayor magnitud que la respuesta primaria; este proceso constituye la base del desarrollo de vacunas. La pandemia de COVID-19 ofreció la oportunidad de estudiar el desarrollo de la memoria inmunológica contra un microorganismo emergente. Las células T de memoria tienen un papel importante en la resolución de COVID-19 y son pilares importantes de la memoria inmunológica. En esta revisión se resumen los principales hallazgos de la respuesta de las células T de memoria contra la infección por SARS-CoV-2, a la vacunación o a la combinación de ambos procesos ("inmunidad híbrida"), y se discute cómo estas células pueden contribuir a la protección a largo plazo contra distintas variantes del virus.

The profile and prognostic significance of bone marrow T-cell differentiation subsets in adult AML at diagnosis.

Background: T lymphocytes in tumor microenvironment play a pivotal role in the anti-tumor immunity, and the memory of T cells contributes to the long-term protection against tumor antigens. Compared to solid tumors, studies focusing on the T-cell differentiation in the acute myeloid leukemia (AML) bone marrow (BM) microenvironment remain limited. Patients and methods: Fresh BM specimens collected from 103 adult AML patients at diagnosis and 12 healthy donors (HDs) were tested T-cell differentiation subsets by multi-parameter flow cytometry. Results: CD4 and CD8 T-cell compartments had different constituted profiles of T-cell differentiated subsets, which was similar between AML patients and HDs. Compared to HDs, AML patients as a whole had a significantly higher proportion of CD8 effector T cells (Teff, P = 0.048). Moreover, the T-cell compartment of AML patients with no DNMT3A mutations skewed toward terminal differentiation at the expense of memory T cells (CD4 Teff: P = 0.034; CD8 Teff: P = 0.030; CD8 memory T: P = 0.017), whereas those with mutated DNMT3A had a decrease in CD8 naïve T (Tn) and CD4 effector memory T cells (Tem) as well as an increase in CD4 central memory T cells (Tcm) (P = 0.037, 0.053 and 0.053). Adverse ELN genetic risk correlated with a lower proportion of CD8 Tn. In addition, the low proportions of CD4 Tem and CD8 Tn independently predicted poorer relapse-free survival (RFS, HR [95%CI]: 5.7 (1.4-22.2), P = 0.017 and 4.8 [1.3-17.4], P = 0.013) and event-free survival (EFS, HR [95% CI]: 3.3 (1.1-9.5), P = 0.029; 4.0 (1.4-11.5), P = 0.010), respectively. Conclusions: AML patients had abnormal profiles of BM T-cell differentiation subsets at diagnosis, which was related to DNMT3A mutations. The low proportions of CD4 Tem and CD8 Tn predicted poor outcomes.

Resident memory T cells and cancer.

Tissue-resident memory T (TRM) cells positively correlate with cancer survival, but the anti-tumor mechanisms underlying this relationship are not understood. This review reconciles these observations, summarizing concepts of T cell immunosurveillance, fundamental TRM cell biology, and clinical observations on the role of TRM cells in cancer and immunotherapy outcomes. We also discuss emerging strategies that utilize TRM-phenotype cells for patient diagnostics, staging, and therapy. Current challenges are highlighted, including a lack of standardized T cell nomenclature and our limited understanding of relationships between T cell markers and underlying tumor biology. Existing findings are integrated into a summary of the field while emphasizing opportunities for future research.

Crystalline silica-induced recruitment and immuno-imbalance of CD4+ tissue resident memory T cells promote silicosis progression.

Occupational crystalline silica (CS) particle exposure leads to silicosis. The burden of CS-associated disease remains high, and treatment options are limited due to vague mechanisms. Here we show that pulmonary CD4+ tissue-resident memory T cells (TRM) accumulate in response to CS particles, mediating the pathogenesis of silicosis. The TRM cells are derived from peripheral lymphocyte recruitment and in situ expansion. Specifically, CD69+CD103+ TRM-Tregs depend more on circulating T cell replenishment. CD69 and CD103 can divide the TRM cells into functionally distinct subsets, mirroring the immuno-balance within CD4+ TRM cells. However, targeting CD103+ TRM-Tregs do not mitigate disease phenotype since the TRM subsets exert immunosuppressive but not pro-fibrotic roles. After identifying pathogenic CD69+CD103- subsets, we highlight IL-7 for their maintenance and function, that present a promising avenue for mitigating silicosis. Together, our findings highlight the distinct role of CD4+ TRM cells in mediating CS-induced fibrosis and provide potential therapeutic strategies.

SARS-CoV-2 reinfection broadens the antibody responses and promotes the phenotypic differentiation of virus-specific memory T cells in adolescents.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants raises concerns regarding the effectiveness of immunity acquired from previous Omicron subvariants breakthrough infections (BTIs) or reinfections (RIs) against the current circulating Omicron subvariants. In this study, we prospectively investigate the dynamic changes of virus-specific antibody and T cell responses among 77 adolescents following Omicron BA.2.3 BTI with or without subsequent Omicron BA.5 RI. Notably, the neutralizing antibodies (NAbs) titers against various detected SARS-CoV-2 variants, especially the emerging Omicron CH.1.1, XBB.1.5, XBB.1.16, EG.5.1, and JN.1 subvariants, exhibited a significant decrease along the time. A lower level of IgG and NAbs titers post-BTI was found to be closely associated with subsequent RI. Elevated NAbs levels and shortened antigenic distances were observed following Omicron BA.5 RI. Robust T cell responses against both Omicron BA.2- and CH.1.1-spike peptides were observed at each point visited. The exposure to Omicron BA.5 promoted phenotypic differentiation of virus-specific memory T cells, even among the non-seroconversion adolescents. Therefore, updated vaccines are needed to provide effective protection against newly emerging SARS-CoV-2 variants among adolescents.

Frequent detection of IFN-gamma -producing memory effector and effector T cells in patients with progressive multifocal leukoencephalopathy.

Introduction: Progressive Multifocal Leukoencephalopathy (PML) is a rare and deadly demyelinating disease caused by JC virus (JCV) replication in the central nervous system. PML occurs exclusively in patients with severe underlying immune deficiencies, including AIDS and hematological malignancies. PML has also emerged as a significant threat to patients on potent new immunosuppressive biologics, including natalizumab in multiple sclerosis. Methods: Here, we developed an IFN-γ release assay (IGRA) that mainly detects JCV-specific effector memory T cells and effectors T cells in the blood. Results: This assay was frequently positive in patients with active PML (with a positive JCV PCR in CSF) of various underlying immunosuppression causes (84% sensitivity). Only 3% of healthy donors had a positive response (97% specificity). The frequency of positivity also increased in multiple sclerosis patients according to the time on natalizumab (up to 36% in patients treated for more than 48 months, who are considered at a higher risk of PML). Discussion: The results show this assay's frequent or increased positivity in patients with PML or an increased risk of PML, respectively. The assay may help to stratify the risk of PML.

Stem cell memory EBV-specific T cells control EBV tumor growth and persist in vivo.

Adoptive T cell therapy (ACT), the therapeutic transfer of defined T cell immunity to patients, offers great potential in the fight against different human diseases including difficult-to-treat viral infections, but persistence and longevity of the cells are areas of concern. Very-early-differentiated stem cell memory T cells (TSCMs) have superior self-renewal, engraftment, persistence, and anticancer efficacy, but their potential for antiviral ACT remains unknown. Here, we developed a clinically scalable protocol for expanding Epstein-Barr virus (EBV)-specific TSCM-enriched T cells with high proportions of CD4+ T cells and broad EBV antigen coverage. These cells showed tumor control in a xenograft model of EBV-induced lymphoma and were superior to previous ACT protocols in terms of tumor infiltration, in vivo proliferation, persistence, proportion of functional CD4+ T cells, and diversity of EBV antigen specificity. Thus, our protocol may pave the way for the next generation of potent unmodified antigen-specific cell therapies for EBV-associated diseases, including tumors, and other indications.

PPARß/δ-orchestrated metabolic reprogramming supports the formation and maintenance of memory CD8+ T cells.

The formation of memory T cells is a fundamental feature of adaptative immunity, allowing the establishment of long-term protection against pathogens. Although emerging evidence suggests that metabolic reprogramming is crucial for memory T cell differentiation and survival, the underlying mechanisms that drive metabolic rewiring in memory T cells remain unclear. Here, we found that up-regulation of the nuclear receptor peroxisome proliferator-activated receptor ß/δ (PPARß/δ) instructs the metabolic reprogramming that occurs during the establishment of central memory CD8+ T cells. PPARß/δ-regulated changes included suppression of aerobic glycolysis and enhancement of oxidative metabolism and fatty acid oxidation. Mechanistically, exposure to interleukin-15 and expression of T cell factor 1 facilitated activation of the PPARß/δ pathway, counteracting apoptosis induced by antigen clearance and metabolic stress. Together, our findings indicate that PPARß/δ is a master metabolic regulator orchestrating a metabolic switch that may be favorable for T cell longevity.

Alloreactive memory CD4 T cells promote transplant rejection by engaging DCs to induce innate inflammation and CD8 T cell priming.

Alloreactive memory T cells have been implicated as central drivers of transplant rejection. Perplexingly, innate cytokines, such as IL-6, IL-1ß, and IL-12, are also associated with rejection of organ transplants. However, the pathways of innate immune activation in allogeneic transplantation are unclear. While the role of microbial and cell death products has been previously described, we identified alloreactive memory CD4 T cells as the primary triggers of innate inflammation. Memory CD4 T cells engaged MHC II-mismatched dendritic cells (DCs), leading to the production of innate inflammatory cytokines. This innate inflammation was independent of several pattern recognition receptors and was primarily driven by TNF superfamily ligands expressed by alloreactive memory CD4 T cells. Blocking of CD40L and TNFα resulted in dampened inflammation, and mice genetically deficient in these molecules exhibited prolonged survival of cardiac allografts. Furthermore, myeloid cell and CD8 T cell infiltration into cardiac transplants was compromised in both CD40L- and TNFα-deficient recipients. Strikingly, we found that priming of naive alloreactive CD8 T cells was dependent on licensing of DCs by memory CD4 T cells. This study unravels the key mechanisms by which alloreactive memory CD4 T cells contribute to destructive pathology and transplant rejection.

Single cell RNA-sequencing identifies the effect of Normothermic ex vivo liver perfusion on liver-resident T cells.

BACKGROUND: Normothermic ex vivo liver perfusion (NEVLP) is an exciting strategy to preserve livers prior to transplant, however, the effects of NEVLP on the phenotype of tissue-resident immune cells is largely unknown. The presence of tissue-resident memory T cells (TRM) in the liver may protect against acute rejection and decrease allograft dysfunction. Therefore, we investigated the effects of NEVLP on liver TRMs and assessed the ability of anti-inflammatory cytokines to reduce TRM activation during NEVLP. METHODS: Rat livers underwent NEVLP with or without the addition of IL-10 and TGF-ß. Naïve and cold storage livers served as controls. Following preservation, TRM T cell gene expression profiles were assessed through single cell RNA sequencing (scRNA-seq). Differential gene expression analysis was performed with Wilcoxon rank sum test to identify differentially expressed genes (DEGs) associated with a specific treatment group. Using the online Database for Annotation, Visualization and Integrated Discovery (DAVID), gene set enrichment was then conducted with Fisher's exact test on DEGs to highlight differentially regulated pathways and functional terms associated with treatment groups. RESULTS: Through scRNA-seq analysis, an atlas of liver-resident memory T cell subsets was created for all livers. TRM T cells could be identified in all livers, and through scRNA-seq, DEG was identified with Wilcoxon rank sum test at FDR < 0.05. Based on the gene set enrichment analysis of DEGs using Fisher's exact test, NEVLP is associated with downregulation of multiple gene enrichment pathways associated with surface proteins. Furthermore, NEVLP with anti-inflammatory cytokines was associated with down regulation of 52 genes in TRM T cells when compared to NEVLP alone (FDR <0.05), most of which are pro-inflammatory. CONCLUSION: This is the first study to create an atlas of liver TRM T cells in the rat liver undergoing NEVLP and demonstrate the effects of NEVLP on liver TRM T cells at the single cell gene expression level.

Heterogeneous IL-9 Production by Circulating Skin-Tropic and Extracutaneous Memory T Cells in Atopic Dermatitis Patients.

Interleukin (IL)-9 is present in atopic dermatitis (AD) lesions and is considered to be mainly produced by skin-homing T cells expressing the cutaneous lymphocyte-associated antigen (CLA). However, its induction by AD-associated triggers remains unexplored. Circulating skin-tropic CLA+ and extracutaneous/systemic CLA- memory T cells cocultured with autologous lesional epidermal cells from AD patients were activated with house dust mite (HDM) and staphylococcal enterotoxin B (SEB). Levels of AD-related mediators in response to both stimuli were measured in supernatants, and the cytokine response was associated with different clinical characteristics. Both HDM and SEB triggered heterogeneous IL-9 production by CLA+ and CLA- T cells in a clinically homogenous group of AD patients, which enabled patient stratification into IL-9 producers and non-producers, with the former group exhibiting heightened HDM-specific and total IgE levels. Upon allergen exposure, IL-9 production depended on the contribution of epidermal cells and class II-mediated presentation; it was the greatest cytokine produced and correlated with HDM-specific IgE levels, whereas SEB mildly induced its release. This study demonstrates that both skin-tropic and extracutaneous memory T cells produce IL-9 and suggests that the degree of allergen sensitization reflects the varied IL-9 responses in vitro, which may allow for patient stratification in a clinically homogenous population.