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Additional file 2: Figure S1. Gating strategy for flow cytometry analysis, related to Figs. 1���4. A Gating strategy for phenotypic identification of lymphocytic and monocytic cell populations and subsets by flow cytometry in peripheral blood. B Gating strategy for identification of functional lymphocytic cell populations by flow cytometry in peripheral blood. Figure S2. Lymphocytic and monocytic cell frequencies after BNT162b2 mRNA vaccine administration, related to Fig. 1. A Frequency of lymphocytic (T and B cells and their subsets) and monocytic cell subsets at Days 0, 10, 21 and 35 in the healthy individuals and immunocompromised patients receiving two doses of the BNT162b2 mRNA vaccine according to label. Healthy individuals (Day 0 n = 37, Day 10 n = 38, Day 21 n = 36, Day 35 n = 37), PLWH (Day 0 n = 48, Day 10 n = 46, Day 21 n = 44, Day 35 n = 44), PID (Day 0 n = 12, Day 10 n = 16, Day 21 n = 14, Day 35 n = 12), SOT (Day 0 n = 34, Day 10 n = 33, Day 21 n = 33, Day 35 n = 30). Kruskal���Wallis test followed by a Dunn���s multiple comparisons test. No statistical significance difference was observed within each respective group (i.e., when comparing days 0, 10, 21 and 35).

The Karolinska KI/K COVID-19 Immune Atlas project was conceptualized in March 2020 as a part of the academic research response to the developing SARS-CoV-2 pandemic. The aim was to rapidly provide a curated dataset covering the acute immune response towards SARS-CoV-2 infection in humans, as it occurred during the first wave. The Immune Atlas was built as an open resource for broad research and educational purposes. It contains a presentation of the response evoked by different immune and inflammatory cells in defined naïve patient-groups as they presented with moderate and severe COVID-19 disease. The present Resource Article describes how the Karolinska KI/K COVID-19 Immune Atlas allows scientists, students, and other interested parties to freely explore the nature of the immune response towards human SARS-CoV-2 infection in an online setting.

Additional file 1: Table S1. List of reagents used in the present study. Table S2. Antibodies used for Panel 1. Table S3. Antibodies used for Panel 2.

Additional file 1: Figure S1. MAIT cell associations with adaptive immune responses against the BNT162b2 mRNA vaccination in patients with primary and acquired immunodeficiency. Figure S2. MAIT cell phenotypic characteristics unchanged in patients with primary and acquired and immunodeficiency vaccinated with the BNT162b2 mRNA vaccine. Figure S3. No significant changes or associations between the overall non-MAIT T cell compartment and adaptive immune responses to the BNT162b2 mRNA vaccine. Figure S4. Preserved MAIT cell response to IL-12 and IL-18 co-stimulation throughout BNT162b2 mRNA vaccination. Table S1. Flow cytometry antibodies and reagents used in the phenotype panel. Table S2. Flow cytometry antibodies and reagent used in the functional panel. Table S3. Key resource table.

Significance Respiratory diseases are leading causes of death worldwide. However, the local immune cell composition in the human lung and individual outliers within the population still remain largely undescribed. We here identify adaptive-like NK cell expansions with tissue-resident traits in lung and blood in approximately 20% of individuals. This particular NK cell subset, which differed from adaptive-like CD16+ blood NK cells, was hyperresponsive to target cell stimulation. Individuals with such in vivo-primed, expanded NK cells will likely experience a different course of acute lung disease such as viral infections. Furthermore, we believe that target cell-hyperresponsive tissue-resident NK cells represent a future tool in the treatment of lung cancer., Human adaptive-like “memory” CD56dimCD16+ natural killer (NK) cells in peripheral blood from cytomegalovirus-seropositive individuals have been extensively investigated in recent years and are currently explored as a treatment strategy for hematological cancers. However, treatment of solid tumors remains limited due to insufficient NK cell tumor infiltration, and it is unknown whether large expansions of adaptive-like NK cells that are equipped for tissue residency and tumor homing exist in peripheral tissues. Here, we show that human lung and blood contains adaptive-like CD56brightCD16− NK cells with hallmarks of tissue residency, including expression of CD49a. Expansions of adaptive-like lung tissue-resident NK (trNK) cells were found to be present independently of adaptive-like CD56dimCD16+ NK cells and to be hyperresponsive toward target cells. Together, our data demonstrate that phenotypically, functionally, and developmentally distinct subsets of adaptive-like NK cells exist in human lung and blood. Given their tissue-related character and hyperresponsiveness, human lung adaptive-like trNK cells might represent a suitable alternative for therapies targeting solid tumors.

Tremendous progress has been made in understanding the role of T cell immunity in acute and convalescent COVID-19 infection. Here we shed light on the "known unknowns" of pre-existing and acquired T cell responses in relation to acute and convalescent SARS-CoV-2 infection.

Given the synergistic nature of TIGIT and PD-1, the coexpression of those inhibitory receptors should be considered when evaluating T-cell pathogenesis, developing immunomodulatory therapies or vaccines for HIV, and when using immunotherapy or vaccination for other causes in HIV-infected patients. HIV-mediated T-cell exhaustion influences the patient´s disease progression, immune system and subsequently non-AIDS complications, and efficacy of vaccinations against other pathogens. Consequently, the possibilities of interfering with exhaustion are numerous. Expanding the use of immunomodulatory therapies to include HIV treatment depends on information about possible targets and their role in the deterioration of the immune system. Furthermore, the rise of immunotherapies against cancer and elevated cancer incidence in HIV-infected patients together increase the need for detailed knowledge of T-cell exhaustion and possible interactions. A broader approach to counteract immune exhaustion to alleviate complications and improve efficacy of other vaccines also promises to increase patients’ health and quality of life., While the relationship of protective human leukocyte antigen (HLA) class I alleles and HIV progression is well defined, the interaction of HLA-mediated protection and CD8 T-cell exhaustion is less well characterized. To gain insight into the influence of HLA-B*57:01 on the deterioration of CD8 T-cell responses during HIV infection in the absence of antiretroviral treatment, we compared HLA-B*57:01-restricted HIV-specific CD8 T-cell responses to responses restricted by other HLA class I alleles longitudinally after control of peak viremia. Detailed characterization of polyfunctionality, differentiation phenotypes, transcription factor, and inhibitory receptor expression revealed progression of CD8 T-cell exhaustion over the course of the infection in both patient groups. However, early effects on the phenotype of the total CD8 T-cell population were apparent only in HLA-B*57-negative patients. The HLA-B*57:01-restricted, HIV epitope-specific CD8 T-cell responses showed beneficial functional patterns and significantly lower frequencies of inhibitory receptor expression, i.e., PD-1 and coexpression of PD-1 and TIGIT, within the first year of infection. Coexpression of PD-1 and TIGIT was correlated with clinical markers of disease progression and declining percentages of the T-bethi Eomesdim CD8 T-cell population. In accordance with clinical and immunological deterioration in the HLA-B*57:01 group, the difference in PD-1 and TIGIT receptor expression did not persist to later stages of the disease. IMPORTANCE Given the synergistic nature of TIGIT and PD-1, the coexpression of those inhibitory receptors should be considered when evaluating T-cell pathogenesis, developing immunomodulatory therapies or vaccines for HIV, and when using immunotherapy or vaccination for other causes in HIV-infected patients. HIV-mediated T-cell exhaustion influences the patient´s disease progression, immune system and subsequently non-AIDS complications, and efficacy of vaccinations against other pathogens. Consequently, the possibilities of interfering with exhaustion are numerous. Expanding the use of immunomodulatory therapies to include HIV treatment depends on information about possible targets and their role in the deterioration of the immune system. Furthermore, the rise of immunotherapies against cancer and elevated cancer incidence in HIV-infected patients together increase the need for detailed knowledge of T-cell exhaustion and possible interactions. A broader approach to counteract immune exhaustion to alleviate complications and improve efficacy of other vaccines also promises to increase patients’ health and quality of life.

The functional properties of circulating CD8+ T cells have been associated with immune control of HIV. However, viral replication occurs predominantly in secondary lymphoid tissues, such as lymph nodes (LNs). We used an integrated single-cell approach to characterize effective HIV-specific CD8+ T cell responses in the LNs of elite controllers (ECs), defined as individuals who suppress viral replication in the absence of antiretroviral therapy (ART). Higher frequencies of total memory and follicle-homing HIV-specific CD8+ T cells were detected in the LNs of ECs compared with the LNs of chronic progressors (CPs) who were not receiving ART. Moreover, HIV-specific CD8+ T cells potently suppressed viral replication without demonstrable cytolytic activity in the LNs of ECs, which harbored substantially lower amounts of CD4+ T cell-associated HIV DNA and RNA compared with the LNs of CPs. Single-cell RNA sequencing analyses further revealed a distinct transcriptional signature among HIV-specific CD8+ T cells from the LNs of ECs, typified by the down-regulation of inhibitory receptors and cytolytic molecules and the up-regulation of multiple cytokines, predicted secreted factors, and components of the protein translation machinery. Collectively, these results provide a mechanistic framework to expedite the identification of novel antiviral factors, highlighting a potential role for the localized deployment of noncytolytic functions as a determinant of immune efficacy against HIV.

Current paradigms of CD8+ T cell-mediated protection in HIV infection center almost exclusively on studies of peripheral blood, which is thought to provide a window into immune activity at the predominant sites of viral replication in lymphoid tissues (LTs). Through extensive comparison of blood, thoracic duct lymph (TDL), and LTs in different species, we show that many LT memory CD8+ T cells bear phenotypic, transcriptional, and epigenetic signatures of resident memory T cells (TRMs). Unlike their circulating counterparts in blood or TDL, most of the total and follicular HIV-specific CD8+ T cells in LTs also resemble TRMs Moreover, high frequencies of HIV-specific CD8+ TRMs with skewed clonotypic profiles relative to matched blood samples are present in LTs of individuals who spontaneously control HIV replication in the absence of antiretroviral therapy (elite controllers). Single-cell RNA sequencing analysis confirmed that HIV-specific TRMs are enriched for effector-related immune genes and signatures compared with HIV-specific non-TRMs in elite controllers. Together, these data indicate that previous studies in blood have largely failed to capture the major component of HIV-specific CD8+ T cell responses resident within LTs.

HIV-specific CD8 T cells display an accumulation of inhibitory receptors that are associated with poor effector functions. Expression of the inhibitory receptor, T cell immunoglobulin and ITIM domain (TIGIT), its co-stimulatory receptor CD226 and their ligand poliovirus receptor (PVR) are altered during chronic viral infections and cancer. Here we show that the TIGIT/CD226/PVR axis is dysregulated during HIV infection and linked to poor polyfunctional activity, increased expression of inhibitory receptors and an altered effector transcriptional programming. We report that TIGIT expression is increased on CD8 T cells in untreated HIV infection. Additionally, a longitudinal increase in TIGIT expression was demonstrated despite early initiation of antiretroviral therapy during acute HIV infection. The HIV-specific TIGIT+ CD8 T cells co-expressed PD-1, CD160 and 2B4 and had an inverse expression profile of the T-box transcription factors T-bet and Eomes. The HIV-specific CD8 T cells were almost exclusively TIGIThiCD226neg/dim and the frequency of TIGIThi cells was inversely correlated with polyfunctionality. Furthermore, the TIGIT/CD226 ligand PVR was increased on CD4 T cells, especially T follicular helper (Tfh) cells in HIV-infected lymph nodes. These results demonstrate a preferential skewing of the TIGIT/CD226 axis towards the inhibitory receptor TIGIT on CD8 T cells during HIV-1 infection, which is linked to severe T cell dysfunction. The findings highlight the importance of the TIGIT/CD226/PVR axis as an immune checkpoint barrier that potentially could hinder future therapeutic “cure” strategies that require potent HIV-specific CD8 T cells and/or the clearance of HIV-1 infected Tfh cells.