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Background: Primary cutaneous acral CD8+ T-cell lymphoproliferative disorder (TLPD) is a rare and indolent lymphoma entity. Although TLPD was first identified many years ago, the molecular pathogenesis is still not fully understood., Objectives: In order to better understand the molecular pathogenesis of cutaneous acral CD8+ TLPD and to identify further discriminatory markers to differentiate this lymphoma subtype from other CD8+ cutaneous lymphomas, we analysed five cases of cutaneous acral CD8+ TLPD for putative molecular alterations., Methods: Somatic alterations were assessed using whole-exome and targeted sequencing of paraffin-embedded tissue. Results were evaluated using immunohistochemical staining of respective relevant proteins. CD8+ cutaneous T-cell lymphomas (n = 12) served as control for KIR3DL1 staining., Results: Copy number variation analysis revealed a homozygous deletion of the KIR3DL1 gene in two of the analysed cases. This resulted in loss of KIR3DL1 protein expression, which was observed in all cases of cutaneous acral CD8+ TLPD. In contrast, KIR3DL1 expression was more variable in other CD8+ cutaneous T-cell lymphomas with 50% of analysed cases (n = 12) found to be positive. In addition, one further case of acral CD8+ TLPD harboured a loss-of-function mutation in the PIK3R1 gene, presumably activating the phosphoinositide 3-kinase-AKT pathway., Conclusions: Alterations of the KIR3DL1 gene may be of pathogenetic relevance for acral CD8+ TLPD. Loss of KIR3DL1 protein expression may support the diagnosis of this indolent lymphoma entity; however, this is not a subtype-specific discriminative feature., Competing Interests: Conflicts of interest The authors declare they have no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Association of Dermatologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Inhibitors of epidermal growth factor receptor (EGFRi) or mitogen-activated kinase (MEKi) induce a folliculitis in 75-90% of patients, the pathobiology of which remains insufficiently understood., Objectives: To characterize changes in the skin immune status and global transcriptional profile of patients treated with EGFRi; to investigate whether EGFRi affects the hair follicle's (HF) immune privilege (IP); and to identify early proinflammatory signals induced by EGFRi/MEKi in human scalp HFs ex vivo., Methods: Scalp biopsies were taken from patients exhibiting folliculitis treated long term with EGFRi ('chronic EGFRi' group, n = 9) vs. healthy scalp skin (n = 9) and patients prior to commencing EGFRi treatment and after 2 weeks of EGFRi therapy ('acute EGFRi' group, n = 5). Healthy organ-cultured scalp HFs were exposed to an EGFRi (erlotinib, n = 5) or a MEKi (cobimetinib, n = 5). Samples were assessed by quantitative immunohistomorphometry, RNA sequencing (RNAseq) and in situ hybridization., Results: The 'chronic EGFRi' group showed CD8+ T-cell infiltration of the bulge alongside a partial collapse of the HF's IP, evidenced by upregulated major histocompatibility complex (MHC) class I, β2-microglobulin (B2 M) and MHC class II, and decreased transforming growth factor-β1 protein expression. Healthy HFs treated with EGFRi/MEKi ex vivo also showed partial HF IP collapse and increased transcription of human leucocyte antigen (HLA)-A, HLA-DR and B2 M transcripts. RNAseq analysis showed increased transcription of chemokines (CXCL1, CXCL13, CCL18, CCL3, CCL7) and interleukin (IL)-26 in biopsies from the 'chronic EGFRi' cohort, as well as increased IL-33 and decreased IL-37 expression in HF biopsies from the 'acute EGFRi' group and in organ-cultured HFs., Conclusions: The data show that EGFRi/MEKi compromise the physiological IP of human scalp HFs and suggest that future clinical management of EGFRi/MEKi-induced folliculitis requires HF IP protection and inhibition of IL-33., Competing Interests: Conflicts of interest D.R. is part funded by Roche/Genentech. R.P. is president of a contract research organization that also engages in analyses of drug skin toxicity (www.monasteriumlab.com), and chief executive officer of a company that develops new actives for skin and hair diseases (www.cutaneon.com), but declares no relevant conflicts of interest related to this study., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Association of Dermatologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Objectives: To analyse the immune cell and B-cell receptor (BCR) profiles of patients with SLE, with or without EBV infection, and identify the differences between them., Methods: We included two patients with SLE and positive detection of EBV infections (SLE-EBV+), four with SLE with negative detection of EBV infections (SLE-EBV-) and two healthy controls. Single-cell RNA sequencing was used to investigate the heterogeneity of cell populations by combining the transcriptomic profiles and BCR repertoires., Results: A total of 83 478 cells were obtained and divided into 31 subtypes. The proportion of CD8+ proliferation T cells was higher in the SLE-EBV+ group than in the SLE-EBV- group. The IFN-α/β pathways were upregulated in most T cells, monocytes and B cells in the SLE-EBV+ group, compared with the SLE-EBV- group. Moreover, T-cell trajectory indicated CD4+ Tregs may play crucial roles in SLE combined with EBV infection. In the BCR heavy chain, the IGHV3 and IGHV4 gene families were frequently present in all groups. Additionally, IgM was the largest component of five Ig isotypes, but its proportion was significantly decreased in the SLE-EBV+ group., Conclusion: This study provides a comprehensive characterization of the immune cell profiles and BCR repertoires of patients with SLE, both with and without concurrent EBV infections, contributing to a better understanding of the mechanism underlying the immune response to EBV infection in patients with SLE., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Enhancing patient response to immune checkpoint inhibitors (ICIs) is crucial in cancer immunotherapy. We aim to create a data-driven mathematical model of the tumor immune microenvironment (TIME) and utilize deep reinforcement learning (DRL) to optimize patient-specific ICI therapy combined with chemotherapy (ICC). Using patients' genomic and transcriptomic data, we develop an ordinary differential equations (ODEs)-based TIME dynamic evolutionary model to characterize interactions among chemotherapy, ICIs, immune cells, and tumor cells. A DRL agent is trained to determine the personalized optimal ICC therapy. Numerical experiments with real-world data demonstrate that the proposed TIME model can predict ICI therapy response. The DRL-derived personalized ICC therapy outperforms predefined fixed schedules. For tumors with extremely low CD8 + T cell infiltration ('extremely cold tumors'), the DRL agent recommends high-dosage chemotherapy alone. For tumors with higher CD8 + T cell infiltration ('cold' and 'hot tumors'), an appropriate chemotherapy dosage induces CD8 + T cell proliferation, enhancing ICI therapy outcomes. Specifically, for 'hot tumors', chemotherapy and ICI are administered simultaneously, while for 'cold tumors', a mid-dosage of chemotherapy makes the TIME 'hotter' before ICI administration. However, in several 'cold tumors' with rapid resistant tumor cell growth, ICC eventually fails. This study highlights the potential of utilizing real-world clinical data and DRL algorithm to develop personalized optimal ICC by understanding the complex biological dynamics of a patient's TIME. Our ODE-based TIME dynamic evolutionary model offers a theoretical framework for determining the best use of ICI, and the proposed DRL agent may guide personalized ICC schedules., (© The Author(s) 2024. Published by Oxford University Press.)

Background: Within a year of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, vaccines inducing a robust humoral and cellular immune response were implemented worldwide. However, emergence of novel variants and waning vaccine-induced immunity led to implementation of additional vaccine boosters., Methods: This prospective study evaluated the temporal profile of cellular and serological responses in a cohort of 639 SARS-CoV-2-vaccinated participants, of whom a large proportion experienced a SARS-CoV-2 infection. All participants were infection naïve at the time of their first vaccine dose. Proportions of SARS-CoV-2 spike-specific T cells were determined after each vaccine dose using the activation-induced marker assay, while levels of circulating SARS-CoV-2 antibodies were determined by the Meso Scale serology assay., Results: We found a significant increase in SARS-CoV-2 spike-specific CD4+ and CD8+ T-cell responses following the third dose of a SARS-CoV-2 messenger RNA vaccine as well as enhanced CD8+ T-cell responses after the fourth dose. Furthermore, increased age was associated with a poorer response. Finally, we observed that SARS-CoV-2 infection boosts both the cellular and humoral immune response, relative to vaccine-induced immunity alone., Conclusions: Our findings highlight the boosting effect on T-cell immunity of repeated vaccine administration. The combination of multiple vaccine doses and SARS-CoV-2 infections maintains population T-cell immunity, although with reduced levels in the elderly., Competing Interests: Potential conflicts of interest . T. B. reports the following grants from the past 36 months: unrestricted grant to his institution from Novo Nordisk Foundation, Simonsen Foundation, Lundbeck Foundation, Kai Foundation, Erik and Susanna Olesen’s Charitable Fund; unrestricted grant to his institution, principal investigator [PI]/clinical trial, advisory board from Pfizer, MSD, GSK, and Gilead Sciences; and grant for PI/clinical trial from Boehringer Ingelheim, Roche, Novartis, Kancera AB, Janssen, and AstraZeneca. T. B. is board member in Pentabase; serves on advisory boards for Janssen and AstraZeneca; and reports receiving payment or honoraria for lectures at GSK, Pfizer, Gilead Sciences, Boehringer Ingelheim, AbbVie, and AstraZeneca. L. W. M. reports receiving travel support from AbbVie in September 2023. N. B. S. reports receiving funding from Danish Ministry of Health for the manuscript and consulting fees on study regarding respiratory syncytial virus from Pfizer and serving as PI in clinical studies sponsored by Pfizer, Bavarian Nordic, Moderna, and AstraZeneca. O. S. S. reports receiving funding from Danish Ministry of Health for the manuscript and consulting fees from UNION therapeutics and participation on a data and safety monitoring board or advisory board with Immunocore, ViiV Healthcare, and Gilead. M.T. reports funding from Danish Ministry of Health for the manuscript. All other authors report no conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: COVID-19 remains a global public health challenge due to new immune-evasive SARS-CoV-2 variants and heterogeneous immunity., Methods: In this cross-sectional study, we evaluated the adaptive immune responses in US active duty personnel who completed a COVID-19 primary vaccine series and had heterogenous SARS-CoV-2 vaccination and infection histories to 3 previously dominant variants (ancestral, Delta, BA.5) and 3 circulating variants (XBB.1.5, EG.5, and BA.2.86) in late 2023. Analyses were based on the most recent exposure in terms of timing (within or beyond 12 months) and type (vaccine or infection)., Results: Significant reduction was observed in binding antibodies, neutralization antibodies, memory B cells, and CD8+ T cells against circulating variants when compared with previous variants. The reduction in antibody response was more pronounced in those whose most recent exposure was >12 months from enrollment. In contrast, the CD4+ T-cell response was largely consistent across all tested variants. The type of most recent exposure was not a significant factor in determining the magnitude of current immune responses., Conclusions: Administration of the XBB.1.5-based booster is likely to enhance cross-reactive humoral responses against SARS-CoV-2 circulating lineages. Ongoing surveillance of immune responses to emerging variants is needed for informing vaccine composition and timing., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2024.)

Background: Coinfection of human immunodeficiency virus type 1 (HIV-1) is the most significant risk factor for tuberculosis (TB). The immune responses of the lung are essential to restrict the growth of Mycobacterium tuberculosis and avoid the emergence of the disease. Nevertheless, there is still limited knowledge about the local immune response in people with HIV-1-TB coinfection., Methods: We employed single-cell RNA sequencing (scRNA-seq) on bronchoalveolar lavage fluid from 9 individuals with HIV-1-TB coinfection and 10 with pulmonary TB., Results: A total of 19 058 cells were grouped into 4 major cell types: myeloid cells, T/natural killer (NK) cells, B cells, and epithelial cells. The myeloid cells and T/NK cells were further divided into 10 and 11 subsets, respectively. The proportions of dendritic cell subsets, CD4+ T cells, and NK cells were lower in the HIV-1-TB coinfection group compared to the TB group, while the frequency of CD8+ T cells was higher. Additionally, we identified numerous differentially expressed genes between the CD4+ and CD8+ T-cell subsets between the 2 groups., Conclusions: HIV-1 infection not only affects the abundance of immune cells in the lungs but also alters their functions in patients with pulmonary TB., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The expanding number of rare immunodeficiency syndromes offers an opportunity to understand key genes that support immune defense against infectious diseases. However, analysis of these in patients is complicated by their treatments and comorbid infections, requiring the use of mouse models for detailed investigations. We developed a mouse model of DOCK2 immunodeficiency and herein demonstrate that these mice have delayed clearance of herpes simplex virus type 1 (HSV-1) infections. We also uncovered a critical, cell-intrinsic role of DOCK2 in the priming of antiviral CD8+ T cells and in particular their initial expansion, despite apparently normal early activation of these cells. When this defect was overcome by priming in vitro, DOCK2-deficient CD8+ T cells were surprisingly protective against HSV-1 disease, albeit not as effectively as wild-type cells. These results shed light on a cellular deficiency that is likely to impact antiviral immunity in DOCK2-deficient patients., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Objective: Obesity and type 2 diabetes (DM) are risk factors for severe coronavirus disease 2019 (COVID-19) outcomes, which disproportionately affect South Asian populations. This study aims to investigate the humoral and cellular immune responses to SARS-CoV-2 in adult COVID-19 survivors with overweight/obesity (Ov/Ob, BMI ≥ 23 kg/m2) and DM in Bangladesh., Methods: In this cross-sectional study, SARS-CoV-2-specific antibody and T-cell responses were investigated in 63 healthy and 75 PCR-confirmed COVID-19 recovered individuals in Bangladesh, during the pre-vaccination first wave of the COVID-19 pandemic in 2020., Results: In COVID-19 survivors, SARS-CoV-2 infection induced robust antibody and T-cell responses, which correlated with disease severity. After adjusting for age, sex, DM status, disease severity, and time since onset of symptoms, Ov/Ob was associated with decreased neutralizing antibody titers, and increased SARS-CoV-2 spike-specific IFN-γ response along with increased proliferation and IL-2 production by CD8 + T cells. In contrast, DM was not associated with SARS-CoV-2-specific antibody and T-cell responses after adjustment for obesity and other confounders., Conclusion: Ov/Ob is associated with lower neutralizing antibody levels and higher T-cell responses to SARS-CoV-2 post-COVID-19 recovery, while antibody or T-cell responses remain unaltered in DM., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology.)

Immunoglobulin A nephropathy is a complex autoimmune disease with various underlying causes and significant clinical heterogeneity. There are large individual differences in its development, and the etiology and pathogenesis are still poorly understood. While it is known that immunobiological factors play a significant role in the pathophysiology of immunoglobulin A nephropathy, the specific nature of these factors has yet to be fully elucidated. Numerous investigations have verified that CD4+ and CD8+ T lymphocytes are involved in the immunopathogenesis of immunoglobulin A nephropathy. Furthermore, certain data also point to γδT cells' involvement in the pathophysiology of immunoglobulin A nephropathy. By thoroughly examining the mechanisms of action of these T cells in the context of immunoglobulin A nephropathy, this review sheds light on the immunopathogenesis of the disease and its associated factors. The review is intended to provide reference value for the future research in this field and promising treatment clues for clinical patients., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Although tumor cell-derived microparticles (MPs) vaccines have reportedly induced antitumor immune reactions for various cancers, the mechanism by which MPs derived from Hepa1-6 cells are taken up by dendritic cells (DCs) and provide the MPs antigens message to CD8+ T cells to exert their anti-hepatocellular carcinoma (HCC) effects remain unclear. Furthermore, the role of MPs in combination with the small-molecule drug MSI-1436, an inhibitor of protein tyrosine phosphatase 1B (PTP1B), in HCC has not yet been reported. In this study, protein mass spectrometry combined with cytology revealed that MPs are mainly taken up by DCs via the clathrin-mediated endocytosis and phagocytosis pathway and localized mainly in lysosomes. High concentration of tumor necrosis factor-α and interferon-γ was detected in CD8+ T cells stimulated with MPs-loaded DCs. Moreover, MPs combined with MSI-1436 further suppressed the proliferation of HCC cells in C57BL/6 tumor-bearing mice, which was closely correlated with CD4+/CD8+ T cells counts in peripheral blood, spleen, and the tumor microenvironment. Mechanistically, the combination of MPs and MSI-1436 exerts a more powerful anti-HCC effect, which may be related to the further inhibition of the expression of PTP1B. Overall, MPs combined with MSI-1436 exerted stronger antitumor effects than MPs or MSI-1436 alone. Therefore, the combination of MPs and MSI-1436 may be a promising means of treating HCC., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Licensed vaccines against the Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging pathogen of concern, are lacking. The modified vaccinia virus Ankara vector-based vaccine MVA-MERS-S, expressing the MERS-CoV-spike glycoprotein (MERS-S), is one of 3 candidate vaccines in clinical development and elicits robust humoral and cellular immunity. Here, we identified for the first time a MERS-S-specific CD8+ T-cell epitope in an HLA-A*03:01/HLA-B*35:01-positive vaccinee using a screening assay, intracellular cytokine staining, and in silico epitope prediction. As evidence from MERS-CoV infection suggests a protective role of long-lasting CD8+ T-cell responses, the identification of epitopes will facilitate longitudinal analyses of vaccine-induced T-cell immunity., Competing Interests: Potential conflicts of interest. T. W. received consulting fees and honoraria from Gilead Sciences and Merck Sharp Dome. All other authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Psoriasis is a T cell-mediated chronic inflammatory skin condition characterized by the interaction of T cells with various cell types, forming an inflammatory microenvironment that sustains psoriatic inflammation. Homeostasis of these tissue-resident T cells is supported by fibroblasts, the primary structural cells in the dermis. In psoriasis, there is increased expression of matrix metalloproteinase 2 (MMP2), mediating structural alterations in skin tissues and modulating inflammation. Additionally, the CD100-plexin-B2 (PLXNB2) axis is known to enhance psoriasis inflammation via keratinocytes, and CD103 levels are associated with the severity of psoriasis upon relapse., Objectives: To elucidate the role of fibroblasts and the MMP2-CD100 axis in modulating psoriasis inflammation., Methods: CD100 expression and function in psoriasis were assessed using immunofluorescence, enzyme-linked immunosorbent assay, single-cell transcriptome sequencing, cellular interaction analyses and quantitative reverse transcriptase polymerase chain reaction. CD8+ T cells from people with psoriasis were isolated using magnetic beads, to investigate the regulatory effect of MMP2 on CD100 expression on their membranes. Single-cell transcriptome sequencing, spatial transcriptome sequencing, mimetic timing analysis, immunofluorescence and flow cytometry were used to determine the origin of MMP2 and its impact on CD103+ CD8+ T cells. The hypotheses were further validated in vivo using MMP2 and CD100 inhibitors., Results: Soluble CD100 (sCD100) was significantly upregulated in both psoriatic lesions and peripheral blood, amplifying psoriasis inflammation by promoting the production of inflammatory cytokines by keratinocytes, fibroblasts and endothelial cells via the sCD100-PLXNB2 axis. Fibroblasts that highly expressed MMP2 (MMP2hi) exacerbated psoriasis symptoms by facilitating CD100 shedding from CD8+ T-cell membranes. Additionally, it was shown that fibroblasts enhance the upregulation of the CD8+ T-cell residency factor CD103 in co-cultures with CD8+ T cells. Inhibitors targeting MMP2 and CD100 were effective in reducing inflammation in an imiquimod-induced psoriasis model., Conclusions: Our findings underscore the pivotal role of MMP2hi fibroblasts in the amplification and recurrence of inflammatory responses in psoriasis. These fibroblasts augment psoriasis inflammation through the CD100-PLXNB2 axis by facilitating CD100 shedding on CD8+ T-cell membranes and by upregulating CD103, thereby enhancing CD8+ T-cell residency., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Association of Dermatologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: We aimed to analyze the clinical characteristics of peripheral Epstein-Barr virus (EBV)-infected lymphocyte subtypes in children with chronic active EBV infection (CAEBV)., Methods: The levels of peripheral EBV infection of CD4+ T cells, CD8+ T cells, and CD56+ natural killer (NK) cells were determined by flow cytometry and quantitative polymerase chain reaction (qPCR) in patients with CAEBV from July 2017 to July 2022., Results: In total, 112 children with CAEBV were evaluated. Of these, CD4+ type, CD8+ type, and CD56+ type were defined in 44, 21, and 47 patients, respectively. Patients with CD8+ T-cell type had a significantly higher frequency of rash, while hepatomegaly was more common in patients with CD4+ T-cell type. Generally, patients with CD8+ T-cell type had the lowest overall survival rate (P = .017). Patients treated with chemotherapy and hematopoietic stem cell transplantation (HSCT) had a better prognosis (P = .001). In multivariate analysis, rash, hemophagocytic lymphohistiocytosis, CD8+ T-cell type, and no decrease of plasma EBV-DNA after treatment were independent indicators of poor prognosis (P = .002, .024, .022, and .012, respectively)., Conclusions: In children with CAEBV, rash was more frequent in patients with CD8+ T-cell type, whereas patients with CD4+ T-cell type were more likely to develop hepatomegaly. Patients with CD8+ T-cell type had a poor prognosis despite receiving chemotherapy or further HSCT., Competing Interests: Potential conflicts of interest . All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes a variety of clinical manifestations, many of which originate from altered immune responses, either locally or systemically. Immune cell cross-talk occurs mainly in lymphoid organs. However, systemic cell interaction specific to coronavirus disease 2019 has not been well characterized. Here, by employing single-cell RNA sequencing and imaging flow cytometry analysis, we unraveled, in peripheral blood, a heterogeneous group of cell complexes formed by the adherence of CD14+ monocytes to different cytotoxic lymphocytes, including SARS-CoV-2-specific CD8+ T cells, γδ T cells, and natural killer T cells. These lymphocytes attached to CD14+ monocytes that showed enhanced inflammasome activation and pyroptosis-induced cell death in progression stage; in contrast, in the convalescent phase, CD14+ monocytes with elevated antigen presentation potential were targeted by cytotoxic lymphocytes, thereby restricting the excessive immune activation. Collectively, our study reports previously unrecognized cell-cell interplay in the SARS-CoV-2-specific immune response, providing new insight into the intricacy of dynamic immune cell interaction representing antiviral defense., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Systematic investigation of tumor-infiltrating immune (TII) cells is important to the development of immunotherapies, and the clinical response prediction in cancers. There exists complex transcriptional regulation within TII cells, and different immune cell types display specific regulation patterns. To dissect transcriptional regulation in TII cells, we first integrated the gene expression profiles from single-cell datasets, and proposed a computational pipeline to identify TII cell type-specific transcription factor (TF) mediated activity immune modules (TF-AIMs). Our analysis revealed key TFs, such as BACH2 and NFKB1 play important roles in B and NK cells, respectively. We also found some of these TF-AIMs may contribute to tumor pathogenesis. Based on TII cell type-specific TF-AIMs, we identified eight CD8+ T cell subtypes. In particular, we found the PD1 + CD8+ T cell subset and its specific TF-AIMs associated with immunotherapy response. Furthermore, the TII cell type-specific TF-AIMs displayed the potential to be used as predictive markers for immunotherapy response of cancer patients. At the pan-cancer level, we also identified and characterized six molecular subtypes across 9680 samples based on the activation status of TII cell type-specific TF-AIMs. Finally, we constructed a user-friendly web interface CellTF-AIMs (http://bio-bigdata.hrbmu.edu.cn/CellTF-AIMs/) for exploring transcriptional regulatory pattern in various TII cell types. Our study provides valuable implications and a rich resource for understanding the mechanisms involved in cancer microenvironment and immunotherapy., (© The Author(s) 2024. Published by Oxford University Press.)

CD8 T cells are crucial adaptive immune cells with cytotoxicity to fight against pathogens or abnormal self-cells via major histocompatibility complex class I-dependent priming pathways. The composition of the memory CD8 T-cell pool is influenced by various factors. Physiological aging, chronic viral infection, and autoimmune diseases promote the accumulation of CD8 T cells with highly differentiated memory phenotypes. Accumulating studies have shown that some of these memory CD8 T cells also exhibit innate-like cytotoxicity and upregulate the expression of receptors associated with natural killer (NK) cells. Further analysis shows that these NK-like CD8 T cells have transcriptional profiles of both NK and CD8 T cells, suggesting the transformation of CD8 T cells into NK cells. However, the specific induction mechanism underlying NK-like transformation and the implications of this process for CD8 T cells are still unclear. This review aimed to deduce the possible differentiation model of NK-like CD8 T cells, summarize the functions of major NK-cell receptors expressed on these cells, and provide a new perspective for exploring the role of these CD8 T cells in health and disease., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: CD8+ tumor-infiltrating lymphocyte (TIL) predicts response to anti-PD-(L)1 therapy. However, there remains no standardized method to assess CD8+ TIL in melanoma, and developing a specific, cost-effective, reproducible, and clinically actionable biomarker to anti-PD-(L)1 remains elusive. We report on the development of automatic CD8+ TIL density quantification via whole slide image (WSI) analysis in advanced melanoma patients treated with front-line anti-PD-1 blockade, and correlation immunotherapy response., Methods: Seventy-eight patients treated with PD-1 inhibitors in the front-line setting between January 2015 and May 2023 at the University of Pittsburgh Cancer Institute were included. CD8+ TIL density was quantified using an image analysis algorithm on digitized WSI. Targeted next-generation sequencing (NGS) was performed to determine tumor mutation burden (TMB) in a subset of 62 patients. ROC curves were used to determine biomarker cutoffs and response to therapy. Correlation between CD8+ TIL density and TMB cutoffs and response to therapy was studied., Results: Higher CD8+ TIL density was significantly associated with improved response to front-line anti-PD-1 across all time points measured. CD8+ TIL density ≥222.9 cells/mm2 reliably segregated responders and non-responders to front-line anti-PD-1 therapy regardless of when response was measured. In a multivariate analysis, patients with CD8+ TIL density exceeding cutoff had significantly improved PFS with a trend toward improved OS. Similarly, increasing TMB was associated with improved response to anti-PD-1, and a cutoff of 14.70 Mut/Mb was associated with improved odds of response. The correlation between TMB and CD8+ TIL density was low, suggesting that each represented independent predictive biomarkers of response., Conclusions: An automatic digital analysis algorithm provides a standardized method to quantify CD8+ TIL density, which predicts response to front-line anti-PD-1 therapy. CD8+ TIL density and TMB are independent predictors of response to anti-PD-1 blockade., (© The Author(s) 2024. Published by Oxford University Press.)

Platelets engage in HIV-1 infection by interacting with immune cells, which has been realized broadly. However, the potential interaction between platelets and CD8+ T cells remains unidentified. Here, treatment-naive individuals with HIV-1, complete immunological responders to antiretroviral therapy, and healthy controls were enrolled. First, we found that treatment-naive individuals with HIV-1 had low platelet numbers and high CD8+ T-cell counts when compared with complete immunological responders to antiretroviral therapy and healthy controls, leading to a low platelet/CD8+ T-cell ratio in peripheral blood, which could effectively differentiate the status of HIV-1 infection. Moreover, cytokines that may have been derived from platelets were higher in the plasma of people with HIV-1 despite viral suppression. Furthermore, we demonstrated that platelet-CD8+ T-cell aggregates were elevated in treatment-naive individuals with HIV-1, which positively correlated with HIV-1 viral load but negatively correlated with CD4+ T-cell count and CD4/CD8 ratio. Finally, we revealed that platelet-CD8+ T-cell aggregates correlate with enhanced activation/exhaustion and pyroptosis/apoptosis compared with free CD8+ T cells. Moreover, platelet-induced caspase 1 activation of CD8+ T cells correlated with IL-1β and IL-18 plasma levels. In brief, we reveal the importance of platelets in HIV-1 infection, which might secrete more cytokines and mediate CD8+ T-cell phenotypic characteristics by forming platelet-CD8+ T-cell aggregates, which are related to poor prognosis., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The immune response to SARS-CoV-2 has been extensively studied following the pandemic outbreak in 2020; however, the presence of specific T cells against SARS-CoV-2 before vaccination has not been evaluated in Mexico. In this study, we estimated the frequency of T CD4+ and T CD8+ cells that exhibit a specific response to S (spike) and N (nucleocapsid) proteins in a Mexican population. We collected 78 peripheral blood samples from unvaccinated subjects, and the presence of antibodies against spike (RBD) and N protein was determined. Peripheral blood mononuclear cells were isolated and stimulated with a pool of S or N protein peptides (Wuhan-Hu-1 strain). IL-1β, IL-4, IL-6, IL-10, IL-2, IL-8, TNF-α, IFN-γ, and GM-CSF levels were quantified in the supernatant of the activated cells, and the cells were stained to assess the activation and memory phenotypes. Differential activation frequency dependent on serological status was observed in CD4+ cells but not in CD8+ cells. The predominantly activated population was the central memory T CD4+ cells. Only 10% of the population exhibited the same phenotype with respect to the response to nucleocapsid peptides. The cytokine profile differed between the S and N responses. S peptides induced a more proinflammatory response compared with the N peptides. In conclusion, in a Mexican cohort before vaccination, there was a significant response to the S and N SARS-CoV-2 proteins resulting from previous infections with seasonal coronaviruses or previous undetected exposure to SARS-CoV-2., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Donor-reactive memory cells represent a barrier to long-term kidney graft survival. A better understanding of regulatory mechanisms that counterbalance alloreactive memory responses may help to identify patients with operational tolerance. This prospective study investigated the equilibrium between memory T-cell subsets and regulatory T or B cells (Tregs, Bregs) in peripheral blood of kidney transplant recipients with operational tolerance (N = 8), chronic rejection (N = 8), and different immunosuppressive treatment regimens (N = 81). Patients on hemodialysis and healthy individuals served as controls (N = 50). In addition, the expression of Treg- and Breg-associated molecule genes was analyzed. Patients with chronic rejection showed a disrupted memory T-cell composition with a significantly higher frequency of circulating CD8+ terminally differentiated effector memory (TEMRA) T cells than patients with operational tolerance, patients on hemodialysis, or healthy controls (P < 0.001). Low frequency of CD8+ TEMRA and high frequency of Tregs and transitional Bregs were found in operationally tolerant patients. Consequently, operationally tolerant patients showed, as compared to all other transplant recipients with different immunosuppressive regiments, the lowest ratios between CD8+ TEMRA T cells and Tregs or Bregs (for both P < 0.001). Moreover, a specific peripheral blood transcription pattern was found in operationally tolerant patients with an increased expression of Breg- and Treg-associated genes CD22 and FoxP3 and a decreased FcγRIIA/FcγRIIB transcript ratio (for all P < 0.001). In conclusion, monitoring the balance between circulating CD8+ TEMRA T cells and regulatory cell subsets and their transcripts may help to distinguish transplant recipients with operational tolerance from recipients at risk of graft loss., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Developing new adjuvants that can effectively induce humoral and cellular immune responses while broadening the immune response is of great value. In this study, we aimed to develop single-stranded RNA adjuvants expressing (1) granulocyte monocyte colony-stimulating factor or (2) interleukin 18 based on the encephalomyocarditis virus internal ribosome entry site; we also tested their efficacy in combination with ovalbumin or inactivated influenza vaccines. Notably, cytokine-expressing RNA adjuvants increased the expression of antigen-presenting cell activation markers in mice. Specifically, when combined with ovalbumin, RNA adjuvants expressing granulocyte monocyte colony-stimulating factor increased CD4+ T-cell responses, while those expressing interleukin 18 increased CD8+ T-cell responses. Cytokine-expressing RNA adjuvants further increased the frequency of polyclonal T cells with the influenza vaccine and reduced the clinical illness scores and weight loss of mice after viral challenge. Collectively, our results suggest that cytokine-expressing RNA adjuvants can be applied to protein-based or inactivated vaccines to increase their efficacy., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Primary Sjogren's syndrome (pSS) is a complex chronic autoimmune disease in which local tissue damage in exocrine glands is combined with broader systemic involvement across the body in tissues including the skin. These combined manifestations negatively impact patient health and quality of life. While studies have previously reported differences in immune cell composition in the peripheral blood of pSS patients relative to healthy control subjects, a detailed immune cell landscape of the damaged exocrine glands of these patients remains lacking. Through single-cell transcriptomics and repertoire sequencing of immune cells in paired peripheral blood samples and salivary gland biopsies, we present here a preliminary picture of adaptive immune response in pSS. We characterize a number of points of divergence between circulating and glandular immune responses that have been hitherto underappreciated, and identify a novel population of CD8+ CD9+ cells with tissue-residential properties that are highly enriched in the salivary glands of pSS patients. Through comparative analyses with other sequencing data, we also observe a potential connection between these cells and the tissue-resident memory cells found in cutaneous vasculitis lesions. Together, these results indicate a potential role for CD8+ CD9+ cells in mediating glandular and systemic effects associated with pSS and other autoimmune disorders., Competing Interests: Conflict of interest statement. The authors have no conflicts of interest to disclose., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Inborn errors of immunity are a group of rare genetically determined diseases that impair immune system development or function. Many of these diseases include immune dysregulation, autoimmunity, or autoinflammation as prominent clinical features. In some children diagnosed with very early onset inflammatory bowel disease (VEOIBD), monogenic inborn errors of immune dysregulation underlie disease. We report a case of VEOIBD caused by a novel homozygous loss of function mutation in IL10RB. We use cytometry by time-of-flight with a broad panel of antibodies to interrogate the immunophenotype of this patient and detect reduced frequencies of CD4 and CD8 T cells with additional defects in some populations of T helper cells, innate-like T cells, and memory B cells. Finally, we identify the patient's mutation as a founder allele in an isolated indigenous population and estimate the age of this variant by studying the shared ancestral haplotype., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We have demonstrated previously that TNF-α-producing CD8+ T cells mediate chlamydial pathogenesis, likely in an antigen (Ag)-specific fashion. Here we hypothesize that inhibition of Ag-specific CD8+ T cell response after immunization and/or challenge would correlate with protection against oviduct pathology induced by a protective vaccine regimen. Intranasal (i.n.) live chlamydial elementary body (EB), intramuscular (i.m.) live EB, or i.n. irrelevant antigen, bovine serum albumin (BSA), immunized animals induced near-total protection, 50% protection, or no protection, respectively against oviduct pathology following i.vag. C. muridarum challenge. In these models, we evaluated Ag-specific CD8+ T cell cytokine response at various time-periods after immunization or challenge. The results show protective efficacy of vaccine regimens correlated with reduction of Ag-specific CD8+ T cell TNF-α responses following i.vag. chlamydial challenge, not after immunization. Depletion of CD4+ T cells abrogated, whereas adoptive transfer of Ag-specific CD4+ T cells induced the significant reduction of Ag-specific CD8+ T cell TNF-α response after chlamydial challenge. In conclusion, protective anti-chlamydial vaccine regimens induce Ag-specific CD4+ T cell response that mediate early inhibition of pathogenic CD8+ T cell response following challenge and may serve as a predictive biomarker of protection against Chlamydia -induced chronic pathologies., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Simultaneous targeting multiple genes is a big advantage of CRISPR (clustered regularly interspaced short palindromic repeats) genome editing but challenging to achieve in CRISPR screening. The crosstalk among genes or gene products is a common and fundamental mechanism to ensure cellular stability and functional diversity. However, the screening approach to map high-order gene combinations to the interesting phenotype is still lacking. Here, we developed a universal in-library ligation strategy and applied it to generate multiplexed CRISPR library, which could perturb four pre-designed targets in a cell. We conducted in vivo CRISPR screening for potential guide RNA (gRNA) combinations inducing anti-tumor immune responses. Simultaneously disturbing a combination of three checkpoints in CD8+ T cells was demonstrated to be more effective than disturbing Pdcd1 only for T cell activation in the tumor environment. This study developed a novel in-library ligation strategy to facilitate the multiplexed CRISPR screening, which could extend our ability to explore the combinatorial outcomes from coordinated gene behaviors., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The activation of dendritic cells (DC) during respiratory viral infections is central to directing the immune response and the pathologic outcome. In these studies, the effect of RSV infection on development of ER stress responses and the impact on innate immunity was examined. The upregulation of ER stress was closely associated with the PERK pathway through the upregulation of CHOP in RSV infected DC. The inhibition of PERK corresponded with decreased EIF2a phosphorylation but had no significant effect on Nrf2 in DC, two primary pathways regulated by PERK. Subsequent studies identified that by blocking PERK activity in infected DC an altered ER stress response and innate cytokine profile was observed with the upregulation of IFNβ and IL-12, coincident to the down regulation of IL-1β. When mitochondria respiration was assessed in PERK deficient DC there were increased dysfunctional mitochondria after RSV infection that resulted in reduced oxygen consumption rates (OCR) and ATP production indicating altered cellular metabolism. Use of a CD11c targeted genetic deleted murine model, RSV infection was characterized by reduced inflammation and diminished mucus staining as well as reduced mucus-associated gene gob5 expression. The assessment of the cytokine responses showed decreased IL-13 and IL-17 along with diminished IL-1β in the lungs of PERK deficient infected mice. When PERK-deficient animals were assessed in parallel for lung leukocyte numbers, animals displayed significantly reduced myeloid and activated CD4 and CD8 T cell numbers. Thus, the PERK activation pathway may provide a rational target for altering the severe outcome of an RSV infection through modifying immune responses., (©2021 Society for Leukocyte Biology.)

Background: Evaluating age as a risk factor for susceptibility to infectious diseases, particularly coronavirus disease 2019 (COVID-19), is critical. Cytomegalovirus (CMV) serologic prevalence increases with age and associates with inflammatory-mediated diseases in the elderly. However, little is known regarding the subclinical impact of CMV and risk it poses to healthy older adults. Prior to the COVID-19 pandemic we conducted a study to determine the association of CMV to biologic age and immune dysregulation., Methods: Community-dwelling, healthy adults older than 60 years were evaluated using DNA methylation assays to define epigenetic age (EpiAge) and T-cell immunophenotyping to assess immune dysregulation., Results: All subjects were healthy and asymptomatic. Those CMV seropositive had more lymphocytes, CD8 T cells, CD28- T cells, decreased CD4:CD8 cell ratios, and had higher average EpiAge (65.34 years) than those CMV seronegative (59.53 years). Decreased percent CD4 (P = .003) and numbers of CD4 T cells (P = .0199) correlated with increased EpiAge., Conclusions: Our novel findings distinguish altered immunity in the elderly based on CMV status. Chronic CMV infection in healthy, older adults is associated with indicators of immune dysregulation, both of which correlate to differences in EpiAge., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The immune system plays a crucial role in the response against severe acute respiratory syndrome coronavirus 2 with significant differences among patients. The study investigated the relationships between lymphocyte subsets, cytokines, and disease outcomes in patients with coronavirus disease 2019 (COVID-19). The measurements of peripheral blood lymphocytes subsets and cytokine levels were performed by flow cytometry for 57 COVID-19 patients. Patients were categorized into two groups according to the severity of the disease (nonsevere vs. severe). Total lymphocytes, T cells, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells were decreased in COVID-19 patients and statistical differences were found among different severity of illness and survival states (P ˂ 0.01). The levels of IL-6 and IL-10 were significantly higher in severe and death groups and negatively correlated with lymphocyte subsets counts. The percentages of Th17 in the peripheral blood of patients were higher than those of healthy controls whereas the percentages of Th2 were lower. For the severe cases, the area under receiver operating characteristic (ROC) curve of IL-6 was the largest among all the immune parameters (0.964; 95% confidence interval: 0.927-1.000, P < 0.0001). In addition, the preoperative IL-6 concentration of 77.38 pg/ml was the optimal cutoff value (sensitivity: 84.6%, specificity: 100%). Using multivariate logistic regression analysis and ROC curves, IL-6 > 106.44 pg/ml and CD8+ T cell counts <150 cells/μl were found to be associated with mortality. Measuring the immune parameters and defining a risk threshold can segregate patients who develop a severe disease from those with a mild pathology. The identification of these parameters may help clinicians to predict the outcome of the patients with high risk of unfavorable progress of the disease., (©2021 Society for Leukocyte Biology.)

Obesity is an independent risk factor for morbidity and mortality in response to influenza infection. However, the underlying mechanisms by which obesity impairs immunity are unclear. Herein, we investigated the effects of diet-induced obesity on pulmonary CD8 + T cell metabolism, cytokine production, and transcriptome as a potential mechanism of impairment during influenza virus infection in mice. Male C57BL/6J lean and obese mice were infected with sub-lethal mouse-adapted A/PR/8/34 influenza virus, generating a pulmonary anti-viral and inflammatory response. Extracellular metabolic flux analyses revealed pulmonary CD8 + T cells from obese mice, compared with lean controls, had suppressed oxidative and glycolytic metabolism at day 10 post-infection. Flow cytometry showed the impairment in pulmonary CD8 + T cell metabolism with obesity was independent of changes in glucose or fatty acid uptake, but concomitant with decreased CD8 + GrB + IFNγ + populations. Notably, the percent of pulmonary effector CD8 + GrB + IFNγ + T cells at day 10 post-infection correlated positively with total CD8 + basal extracellular acidification rate and basal oxygen consumption rate. Finally, next-generation RNA sequencing revealed complex and unique transcriptional regulation of sorted effector pulmonary CD8 + CD44 + T cells from obese mice compared to lean mice following influenza infection. Collectively, the data suggest diet-induced obesity increases influenza virus pathogenesis, in part, through CD8 + T cell-mediated metabolic reprogramming and impaired effector CD8 + T cell function., (©2021 Society for Leukocyte Biology.)

The outbreak of coronavirus disease 2019 (COVID-19) has now become a pandemic, and the etiologic agent is the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). T cell mediated immune responses play an important role in virus controlling; however, the understanding of the viral protein immunogenicity and the mechanisms of the induced responses are still limited. So, identification of specific epitopes and exploring their immunogenic properties would provide valuable information. In our study, we utilized the Immune Epitope Database and Analysis Resource and NetMHCpan to predict HLA-A2 restricted CD8 + T cell epitopes in structural proteins of SARS-CoV-2, and screened out 23 potential epitopes. Among them, 18 peptides showed strong or moderate binding with HLA-A2 with a T2A2 cell binding model. Next, the mixed peptides induced the increased expression of CD69 and highly expressed levels of IFN-γ and granzyme B in CD8 + T cells, indicating effective activation of specific CD8 + T cells. In addition, the peptide-activated CD8 + T cells showed significantly increased killing to the target cells. Furthermore, tetramer staining revealed that the activated CD8 + T cells mainly recognized seven epitopes. All together, we identified specific CD8 + T cell epitopes in SARS-CoV-2 structural proteins, which could induce the production of specific immune competent CD8 + T cells. Our work contributes to the understanding of specific immune responses and vaccine development for SARS-CoV-2., (© 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals LLC on behalf of Society for Leukocyte Biology.)

Hi-C is a genome-wide assay based on Chromosome Conformation Capture and high-throughput sequencing to decipher 3D chromatin organization in the nucleus. However, computational methods to detect functional interactions utilizing Hi-C data face challenges including the correction for various sources of biases and the identification of functional interactions with low counts of interacting fragments. We present Chrom-Lasso, a lasso linear regression model that removes complex biases assumption-free and identifies functional interacting loci with increased power by combining information of local reads distribution surrounding the area of interest. We showed that interacting regions identified by Chrom-Lasso are more enriched for 5C validated interactions and functional GWAS hits than that of GOTHiC and Fit-Hi-C. To further demonstrate the ability of Chrom-Lasso to detect interactions of functional importance, we performed time-series Hi-C and RNA-seq during T cell activation and exhaustion. We showed that the dynamic changes in gene expression and chromatin interactions identified by Chrom-Lasso were largely concordant with each other. Finally, we experimentally confirmed Chrom-Lasso's finding that Erbb3 was co-regulated with distinct neighboring genes at different states during T cell activation. Our results highlight Chrom-Lasso's utility in detecting weak functional interaction between cis-regulatory elements, such as promoters and enhancers., (© The Author(s) 2021. Published by Oxford University Press.)

Background: We previously reported T-cell senescence to be similar in people with human immunodeficiency virus (PWH) with suppressed viremia (predominantly men who have sex with men [MSM]) and human immunodeficiency virus (HIV)-negative otherwise comparable controls but greater than in healthy blood donors. This led us to compare CD4+ and CD8+ T-cell counts and CD4+/CD8+ ratios between HIV-negative MSM and men who only have sex with women (MSW) and relate observed differences in behavioral factors and infectious exposures, including cytomegalovirus (CMV) infection., Methods: In 368 HIV-negative MSM and 72 HIV-negative MSW, T lymphocyte phenotyping was performed 3 times biennially. Baseline CMV serology and sexually transmitted infection (STI) incidence and/or STI seroprevalence, sexual, and substance-use behavior data were collected during study visits., Results: Men who have sex with men, compared with MSW, had higher CD8+ counts (551 vs 437 cells/mm3, P < .001), similar CD4+ counts (864 vs 880 cells/mm3, P = .5), and lower CD4+/CD8+ ratios (1.84 vs 2.47, P < .001). Differences were most pronounced for MSM with >10 recent sex partners and partly explained by higher CMV seroprevalence in MSM., Conclusions: These findings suggest that factors other than HIV may, in both PWH and certain HIV-negative MSM, contribute to a low CD4+/CD8+ ratio. Whether this, like in PWH, contributes to comorbidity risk in HIV-negative MSM requires further study., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Coronavirus disease 2019 (COVID-19) continues to be a major public health challenge globally. The identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived T-cell epitopes is of critical importance for peptide vaccines or diagnostic tools of COVID-19., Methods: In this study, several SARS-CoV-2-derived human leukocyte antigen (HLA)-I binding peptides were predicted by NetMHCpan-4.1 and selected by Popcover to achieve pancoverage of the Chinese population. The top 5 ranked peptides derived from each protein of SARS-CoV-2 were then evaluated using peripheral blood mononuclear cells from unexposed individuals (negative for SARS-CoV-2 immunoglobulin G)., Results: Seven epitopes derived from 4 SARS-CoV-2 proteins were identified. It is interesting to note that most (5 of 7) of the SARS-CoV-2-derived peptides with predicted affinities for HLA-I molecules were identified as HLA-II-restricted epitopes and induced CD4+ T cell-dependent responses. These results complete missing pieces of pre-existing SARS-CoV-2-specific T cells and suggest that pre-existing T cells targeting all SARS-CoV-2-encoded proteins can be discovered in unexposed populations., Conclusions: In summary, in the current study, we present an alternative and effective strategy for the identification of T-cell epitopes of SARS-CoV-2 in healthy subjects, which may indicate an important role in the development of peptide vaccines for COVID-19., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Since December 2019, Coronavirus disease-19 (COVID-19) has spread rapidly throughout the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as cancer patients who recently underwent a haematopoietic stem cell transplantation. Immunological protection against COVID-19 is mediated by both short-lived neutralizing antibodies and long-lasting virus-reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Here, we identify T cells from COVID-19 patients with a potentially protective response to two major antigens of the SARS-CoV-2 virus, Spike and Nucleocapsid protein. By generating clones of highly virus-reactive CD4 + T cells, we were able to confirm a set of nine immunodominant epitopes and characterize T cell responses against these. Accordingly, the sensitivity of T cell clones for their specific epitope, as well as the extent and focus of their cytokine response was examined. Moreover, using an advanced T cell receptor (TCR) sequencing approach, we determined the paired TCR-αβ sequences of clones of interest. While these data on a limited population require further expansion for universal application, the results presented here form a crucial first step towards TCR-transgenic CD4 + T cell therapy of COVID-19., (© 2021 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

IFNγ released from CD8 + T cells or natural killer cells plays a crucial role in antitumor host immunity. Several studies have found that IFNγ is involved in regulating tumor cell proliferation and apoptosis. However, few studies have examined its role in cell ferroptosis. Here, we found that IFNγ treatment enhanced glutathione depletion, promoted cell cycle arrested in G0/G1 phase, increased lipid peroxidation, and sensitized cells to ferroptosis activators. Additionally, IFNγ down-regulated the mRNA and protein levels of SLC3A2 and SLC7A11, two subunits of the glutamate-cystine antiporter system xc - via activating the JAK/STAT pathway in hepatocellular carcinoma (HCC) cell lines. Furthermore, IFNγ increased reactive oxygen species levels and decreased mitochondiral membrane potential in Bel7402 and HepG2 cells. These changes were accompanied by decreased system xc - activity. Cancer cells exposed to TGFβ1 for 48 h showed sensitization to IFNγ + erastin-induced ferroptosis, with decreased system xc - expression. In conclusion, IFNγ repressed system xc - activation via activating JAK/STAT signaling. Additionally, enhanced lipid peroxidation was associated with altered mitochondrial function in HCC cells. Our findings identified a role for IFNγ in sensitizing HCC cells to ferroptosis, which provided new insights for applying IFNγ as a cancer treatment., (©2021 Society for Leukocyte Biology.)

Ageing dramatically affects number and function of both innate and adaptive arms of immune system, particularly T cell subsets, contributing to reduced vaccination efficacy, decreased resistance to infections and increased prevalence of cancer in older people. In the present paper, we analysed the age-related changes in the absolute number of lymphocytes in 214 Sicilian subjects, and in the percentages of T and natural killer (NK) cells in a subcohort of donors. We compared these results with the immunophenotype of the oldest living Italian supercentenarian (aged 111 years). The results were also sorted by gender. The correlation between number/percentage of cells and age in all individuals. and separately in males and females, was examined using a simple linear regression analysis. We did not record the increase in the rate of inversion of the CD4/CD8 ratio, frequently reported as being associated with ageing in literature. Our observation was the direct consequence of a flat average trend of CD4 + and CD8 + T cell percentages in ageing donors, even when gender differences were included. Our results also suggest that CD4 + and CD8 + subsets are not affected equally by age comparing females with males, and we speculated that gender may affect the response to cytomegalovirus (CMV) infection. The supercentenarian showed a unique immunophenotypic signature regarding the relative percentages of her T cell subsets, with CD4 + and CD8 + T cell percentages and CD4 + naive T cell values in line with those recorded for the octogenarian subjects. This suggests that the supercentenarian has a naive 'younger' T cell profile comparable to that of a >80-year-old female., (© 2021 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

Although millions of patients have clinically recovered from COVID-19, little is known about the immune status of lymphocytes in these individuals. In this study, the peripheral blood mononuclear cells of a clinically recovered (CR) cohort were comparatively analyzed with those of an age- and sex-matched healthy donor cohort. We found that CD8+ T cells in the CR cohort had higher numbers of effector T cells and effector memory T cells but lower Tc1 (IFN-γ+), Tc2 (IL-4+), and Tc17 (IL-17A+) cell frequencies. The CD4+ T cells of the CR cohort were decreased in frequency, especially the central memory T cell subset. Moreover, CD4+ T cells in the CR cohort showed lower programmed cell death protein 1 (PD-1) expression and had lower frequencies of Th1 (IFN-γ+), Th2 (IL-4+), Th17 (IL-17A+), and circulating follicular helper T (CXCR5+PD-1+) cells. Accordingly, the proportion of isotype-switched memory B cells (IgM-CD20hi) among B cells in the CR cohort showed a significantly lower proportion, although the level of the activation marker CD71 was elevated. For CD3-HLA-DR- lymphocytes in the CR cohort, in addition to lower levels of IFN-γ, granzyme B and T-bet, the correlation between T-bet and IFN-γ was not observed. Additionally, by taking into account the number of days after discharge, all the phenotypes associated with reduced function did not show a tendency toward recovery within 4‒11 weeks. The remarkable phenotypic alterations in lymphocytes in the CR cohort suggest that  severe acute respiratory syndrome coronavirus 2 infection profoundly affects lymphocytes and potentially results in dysfunction even after clinical recovery., (© The Author(s) (2021). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

Herein we measured CD4+ T-cell responses against common cold coronaviruses (CCC) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in high-risk health care workers (HCW) and community controls. We observed higher levels of CCC-reactive T cells in SARS-CoV-2-seronegative HCW compared to community donors, consistent with potential higher occupational exposure of HCW to CCC. We further show that SARS-CoV-2 T-cell reactivity of seronegative HCW was higher than community controls and correlation between CCC and SARS-CoV-2 responses is consistent with cross-reactivity and not associated with recent in vivo activation. Surprisingly, CCC T-cell reactivity was decreased in SARS-CoV-2-infected HCW, suggesting that exposure to SARS-CoV-2 might interfere with CCC responses, either directly or indirectly. This result was unexpected, but consistently detected in independent cohorts derived from Miami and San Diego. CD4+ T-cell responses against common cold coronaviruses (CCC) are elevated in SARS-CoV-2 seronegative high-risk health care workers (HCW) compared to COVID-19 convalescent HCW, suggesting that exposure to SARS-CoV-2 might interfere with CCC responses and/or cross-reactivity associated with a protective effect., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Arthritis is a common clinical feature of systemic lupus erythematosus (SLE) and is usually non-erosive, as opposed to rheumatoid arthritis (RA). While RA synovial pathology has been extensively studied, little is known about the pathophysiology of lupus arthritis. Here, we aimed to explore the cytokine and cellular compartments in synovial fluids of SLE patients with arthritic manifestations. Acellular synovial fluid and paired serum samples from SLE patients (n = 17) were analyzed with cytokine bead array for T helper-associated cytokines. From two SLE patients, synovial fluid mononuclear cells (SFMC) could also be captured and were analyzed by multiparameter flow cytometry to dissect T cell, B cell, monocyte and dendritic cell phenotypes. SLE-derived SFMC were further stimulated in vitro to measure their capacity for producing interferon (IFN)-γ and interleukin (IL)-17A. All patients fulfilled the ACR 1982 classification criteria for SLE. Clinical records were reviewed to exclude the presence of co-morbidities such as osteoarthritis or overlap with RA. IL-17A and IL-6 levels were high in SLE synovial fluid. A clear subset of the synovial CD4 + T cells expressed CCR6 + , a marker associated with T helper type 17 (Th17) cells. IL-17A-production was validated among CD4 + CCR6 + T cells following in-vitro stimulation. Furthermore, a strong IFN-γ production was observed in both CD4 + and CD8 + cells. Our study shows high IL-17A and IL-6 levels in synovial fluids of patients with lupus arthritis. The Th17 pathway has been implicated in several aspects of SLE disease pathogenesis and our data also point to Th17 involvement for lupus arthritis., (© 2021 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

High expression of the inhibitory receptor programmed cell death ligand 1 (PD-L1) on tumor cells and tumor stromal cells have been found to play a key role in tumor immune evasion in several human malignancies. However, the expression of PD-L1 on bone marrow mesenchymal stem cells (BMSCs) and whether the programmed cell death 1 (PD-1)/PD-L1 signal pathway is involved in the BMSCs versus T cell immune response in multiple myeloma (MM) remains poorly defined. In this study, we explored the expression of PD-L1 on BMSCs from newly diagnosed MM (NDMM) patients and the role of PD-1/PD-L1 pathway in BMSC-mediated regulation of CD8 + T cells. The data showed that the expression of PD-L1 on BMSCs in NDMM patients was significantly increased compared to that in normal controls (NC) (18·81 ± 1·61 versus 2·78± 0·70%; P < 0·001). Furthermore, the PD-1 expression on CD8 + T cells with NDMM patients was significantly higher than that in normal controls (43·22 ± 2·98 versus 20·71 ± 1·08%; P < 0·001). However, there was no significant difference in PD-1 expression of CD4 + T cells and natural killer (NK) cells between the NDMM and NC groups. Additionally, the co-culture assays revealed that BMSCs significantly suppressed CD8 + T cell function. However, the PD-L1 inhibitor effectively reversed BMSC-mediated suppression in CD8 + T cells. We also found that the combination of PD-L1 inhibitor and pomalidomide can further enhance the killing effect of CD8 + T cells on MM cells. In summary, our findings demonstrated that BMSCs in patients with MM may induce apoptosis of CD8 + T cells through the PD-1/PD-L1 axis and inhibit the release of perforin and granzyme B from CD8 + T cells to promote the immune escape of MM., (© 2021 British Society for Immunology.)

IL-26 is a newly discovered IL-10 cytokine family member mainly secreted by Th17 cells. However, the relationship between IL-26 and lung cancer remains unclear. The present study reported that IL-26 is involved in the production and promotion of malignant pleural effusion (MPE) for the first time. The concentrations of IL-26 and several Th17-related cytokines in MPE and peripheral blood (PB) from MPE patients were measured. IL-26, IL-10, and IL-6 were elevated in MPE compared to PB. The cell resource of IL-26 was primary Th17 cells measured by flow cytometry, whereas Tc17 cells and macrophages could also contribute to higher concentration of IL-26 in MPE. Abundant IL-6 and IL-23 in MPE could promote the frequency of IL-26 expressed by CD4 + T cells through phosphorylating STAT3 signaling pathway and promoting the expression of a specific Th17 lineage marker RORγt subsequently. IL-26 could selectively increase Th22 proportion through up-regulating the percentage of Ki-67 expressed by CD4 + T cells and the expression of IL-22 secreted by memory CD4 + T cells. In addition, IL-26 could decrease secretion of granzyme B. The tumor-killing activity of CD8 + T cells were inhibited as well when cocultured with malignant cells. Furthermore, the accumulation of IL-26 protein in MPE predicted poor patient survival. In summary, our results indicated that IL-26 was involved in the pathogenesis of MPE by exerting its impacts on both CD4 + T cells and CD8 + T cells., (©2021 Society for Leukocyte Biology.)

Background: Cytomegalovirus (CMV) can cause congenital infection and is the leading cause of nongenetic newborn disabilities. V160, a conditionally replication-defective virus, is an investigational vaccine under evaluation for prevention of congenital CMV. The vaccine was well tolerated and induced both humoral and cellular immunity in CMV-seronegative trial participants. T-cell-mediated immunity is important for immune control of CMV. Here we describe efforts to understand the quality attributes of the T-cell responses induced by vaccination., Methods: Using multicolor flow cytometry, we analyzed vaccine-induced T cells for memory phenotype, antigen specificity, cytokine profiles, and cytolytic potential. Moreover, antigen-specific T cells were sorted from 4 participants, and next-generation sequencing was used to trace clonal lineage development during the course of vaccination using T-cell receptor β-chain sequences as identifiers., Results: The results demonstrated that vaccination elicited polyfunctional CD4 and CD8 T cells to 2 dominant antigens, pp65 and IE1, with a predominantly effector phenotype. Analysis of T-cell receptor repertoires showed polyclonal expansion of pp65- and IE1-specific T cells after vaccination., Conclusion: V160 induced a genetically diverse and polyfunctional T-cell response and the data support further clinical development of V160 for prevention of CMV infection and congenital transmission., Clinical Trials Registration: NCT01986010., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The mechanisms that drive systemic lupus erythematosus (SLE) patients to achieve remission are unknown; one possible explanation might be T cell exhaustion. The aim of the present study was to measure CD4 + and CD8 + T cell exhaustion in SLE patients in prolonged remission (PR-SLE) and compared them with patients with active SLE (Act-SLE) and healthy subjects. We included 15 PR-SLE patients, 15 Act-SLE and 29 healthy subjects. T cell exhaustion was determined by flow cytometry according to the expression of programmed cell death 1 (PD)-1, T cell immunoglobulin and mucin 3 (Tim-3), natural killer cell receptor (2B4), eomesodermin (EOMES) and T-box transcription factor TBX21 (T-bet) in CD4 + and CD8 + T cells. Dimensionality reduction using the T-distributed stochastic neighbor-embedding algorithm and clustering analysis was used for the identification of relevant populations. Percentages of CD3 + , CD4 + and CD8 + T cells were similar among groups. We identified five subpopulations of CD8 + and seven of CD4 + cells. The CD4 + T-bet + CD45RO + cells identified in the unsupervised analysis were significantly increased in PR-SLE versus Act-SLE [median = 0·20, interquartile range (IQR) = 1·74-30·50 versus 1·68, IQR = 0·4-2·83; P < 0·01]. CD4 + EOMES + cells were also increased in PR-SLE versus Act-SLE (5·24, IQR = 3·38-14·70 versus 1·39, IQR = 0·48-2·87; P < 0·001). CD8 + EOMES + cells were increased in PR-SLE versus Act-SLE (37·6, IQR = 24·9-53·2 versus 8·13, IQR = 2·33-20·5; P < 0·001). Exhausted and activated T cells presented an increased frequency of PD-1, CD57 and EOMES in SLE patients versus healthy subjects. Some subpopulations of T cells expressing markers associated with exhaustion are increased in patients in remission, supporting T cell exhaustion as a tolerance mechanism in SLE. Exhaustion of specific populations of T cells might represent a potential therapeutic tool that will contribute to the goal of achieving sustained remission in these patients., (© 2021 British Society for Immunology.)

Across the world, many commercial poultry flocks and captive birds are threatened by infection with Aspergillus fumigatus. Susceptibility to aspergillosis varies among birds; among galliform birds specifically, morbidity and mortality rates seem to be greater in turkeys than in chickens. Little is known regarding the features of avian immune responses after inhalation of Aspergillus conidia, and to date, scarce information on inflammatory responses during aspergillosis exists. Thus, in the present study, we aimed to improve our understanding of the interactions between A. fumigatus and economically relevant galliform birds in terms of local innate immune responses. Intra-tracheal aerosolization of A. fumigatus conidia in turkey and chicken poults led to more severe clinical signs and lung lesions in turkeys, but leukocyte recovery from lung lavages was higher in chickens at 1dpi only. Interestingly, only chicken CD8+ T lymphocyte proportions increased after infection. Furthermore, the lungs of infected chickens showed an early upregulation of pro-inflammatory cytokines, including IL-1β, IFN-γ and IL-6, whereas in turkeys, most of these cytokines showed a downregulation or a delayed upregulation. These results confirmed the importance of an early pro-inflammatory response to ensure the development of an appropriate anti-fungal immunity to avoid Aspergillus dissemination in the respiratory tract. In conclusion, we show for the first time that differences in local innate immune responses between chickens and turkeys during aspergillosis may determine the outcome of the disease., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

When Thetis dipped her son Achilles into the River Styx to make him immortal, she held him by the heel, which was not submerged, and thus created a weak spot that proved deadly for Achilles. Millennia later, Achilles heel is part of today's lexicon meaning an area of weakness or a vulnerable spot that causes failure. Also implied is that an Achilles heel is often missed, forgotten or under-appreciated until it is under attack, and then failure is fatal. Paris killed Achilles with an arrow 'guided by the Gods'. Understanding the pathogenesis of type 1 diabetes (T1D) in order to direct therapy for prevention and treatment is a major goal of research into T1D. At the International Congress of the Immunology of Diabetes Society, 2018, five leading experts were asked to present the case for a particular cell/element that could represent 'the Achilles heel of T1D'. These included neutrophils, B cells, CD8 + T cells, regulatory CD4 + T cells, and enteroviruses, all of which have been proposed to play an important role in the pathogenesis of type 1 diabetes. Did a single entity emerge as 'the' Achilles heel of T1D? The arguments are summarized here, to make this case., (© 2021 British Society for Immunology.)

Background: BK polyomavirus (BKPyV) remains a significant cause of premature kidney transplant failure. In the absence of effective antivirals, current treatments rely on reducing immunosuppression to regain immune control over BKPyV replication. Increasing BKPyV-specific CD8 T cells correlate with clearance of BKPyV DNAemia in kidney transplant patients. We characterized a novel approach for expanding BKPyV-specific CD8 T cells in vitro using 27mer-long synthetic BKPyV peptides, different types of antigen-presenting cells, and CD4 T cells., Methods: Langerhans cells and immature or mature monocyte-derived dendritic cells (Mo-DCs) were generated from peripheral blood mononuclear cells of healthy blood donors, pulsed with synthetic peptide pools consisting of 36 overlapping 27mers (27mP) or 180 15mers (15mP). BKPyV-specific CD8 T-cell responses were assessed by cytokine release assays using 15mP or immunodominant 9mers., Results: BKPyV-specific CD8 T cells expanded using 27mP and required mature Mo-DCs (P = .0312) and CD4 T cells (P = .0156) for highest responses. The resulting BKPyV-specific CD8 T cells proliferated, secreted multiple cytokines including interferon γ and tumor necrosis factor α, and were functional (CD107a+/PD1-) and cytotoxic., Conclusions: Synthetic 27mP permit expanding BKPyV-specific CD8 T-cell responses when pulsing mature Mo-DCs in presence of CD4 T cells, suggesting novel and safe approaches to vaccination and adoptive T-cell therapies for patients before and after kidney transplantation., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Metabolic regulation has been proven to play a critical role in T cell antitumor immunity. However, cholesterol metabolism as a key component of this regulation remains largely unexplored. Herein, we found that the low-density lipoprotein receptor (LDLR), which has been previously identified as a transporter for cholesterol, plays a pivotal role in regulating CD8 + T cell antitumor activity. Besides the involvement of cholesterol uptake which is mediated by LDLR in T cell priming and clonal expansion, we also found a non-canonical function of LDLR in CD8 + T cells: LDLR interacts with the T-cell receptor (TCR) complex and regulates TCR recycling and signaling, thus facilitating the effector function of cytotoxic T-lymphocytes (CTLs). Furthermore, we found that the tumor microenvironment (TME) downregulates CD8 + T cell LDLR level and TCR signaling via tumor cell-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) which binds to LDLR and prevents the recycling of LDLR and TCR to the plasma membrane thus inhibits the effector function of CTLs. Moreover, genetic deletion or pharmacological inhibition of PCSK9 in tumor cells can enhance the antitumor activity of CD8 + T cells by alleviating the suppressive effect on CD8 + T cells and consequently inhibit tumor progression. While previously established as a hypercholesterolemia target, this study highlights PCSK9/LDLR as a potential target for cancer immunotherapy as well.