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IntroductionTumor-associated macrophages (TAMs) represent an important cell population within the tumor microenvironment, but little is known about the phenotype and function of these cells. The present study aims to characterize macrophages in high-grade serous ovarian cancer (HGSOC).MethodsPhenotype and expression of co-regulatory markers were assessed on TAMs derived from malignant ascites (MA) or peripheral blood (PB) by multiparametric flow cytometry. Samples were obtained from HGSOC patients (n=29) and healthy donors (HDs, n=16). Additional expression analysis was performed by RNAseq (n=192). Correlation with clinically relevant parameters was conducted and validated by a second patient cohort (n=517). Finally, the role of TIGIT in repolarization and phagocytosis was investigated in vitro.ResultsExpression of the M2-associated receptors CD163, CD204, and CD206, as well as of the co-regulatory receptors TIGIT, CD226, TIM-3, and LAG-3 was significantly more frequent on macrophages in HGSOC than in HDs. CD39 and CD73 were broadly expressed on (mainly M2) macrophages, but without a clear clustering in HGSOC. CD163 mRNA levels were higher in TAMs from patients with residual tumor mass after surgery and associated with a shorter overall survival. In addition, TIGIT expression was associated with a higher tumor grading, indicating a prognostic relevance of M2 infiltration in HGSOC. TIGIT blockade significantly reduced the frequency of M2 macrophages. Moreover, combined blockade of TIGIT and CD47 significantly increased phagocytosis of ovarian cancer cells by TAMs in comparison to a single blockade of CD47.ConclusionCombined blockade of TIGIT and CD47 represents a promising approach to enhance anti-CD47-facilitated phagocytosis.

Infections with hepatotropic viruses are associated with various immune phenomena. Hepatitis D virus (HDV) causes the most severe form of viral hepatitis. However, few recent data are available on non-disease-specific and non-organ-specific antibody (NOSA) titers and immunoglobulin G (IgG) levels in chronic hepatitis D (CHD) patients. Here, we examined the NOSA titers and IgG levels of 40 patients with CHD and different disease courses and compared them to 70 patients with chronic hepatitis B (CHB) infection. 43% of CHD patients had previously undergone treatment with pegylated interferon-α (IFN-α). The antibody display of 46 untreated patients diagnosed with autoimmune hepatitis (AIH) was used as a reference. The frequency of elevated NOSA titers (CHD 69% vs. CHB 43%, p

IntroductionThe nonstructural protein 12 (NSP12) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has a high sequence identity with common cold coronaviruses (CCC).MethodsHere, we comprehensively assessed the breadth and specificity of the NSP12-specific T-cell response after in vitro T-cell expansion with 185 overlapping 15-mer peptides covering the entire SARS-CoV-2 NSP12 at single-peptide resolution in a cohort of 27 coronavirus disease 2019 (COVID-19) patients. Samples of nine uninfected seronegative individuals, as well as five pre-pandemic controls, were also examined to assess potential cross-reactivity with CCCs.ResultsSurprisingly, there was a comparable breadth of individual NSP12 peptide-specific CD4+ T-cell responses between COVID-19 patients (mean: 12.82 responses; range: 0–25) and seronegative controls including pre-pandemic samples (mean: 12.71 responses; range: 0–21). However, the NSP12-specific T-cell responses detected in acute COVID-19 patients were on average of a higher magnitude. The most frequently detected CD4+ T-cell peptide specificities in COVID-19 patients were aa236–250 (37%) and aa246–260 (44%), whereas the peptide specificities aa686–700 (50%) and aa741–755 (36%), were the most frequently detected in seronegative controls. In CCC-specific peptide-expanded T-cell cultures of seronegative individuals, the corresponding SARS-CoV-2 NSP12 peptide specificities also elicited responses in vitro. However, the NSP12 peptide-specific CD4+ T-cell response repertoire only partially overlapped in patients analyzed longitudinally before and after a SARS-CoV-2 infection.DiscussionThe results of the current study indicate the presence of pre-primed, cross-reactive CCC-specific T-cell responses targeting conserved regions of SARS-CoV-2, but they also underline the complexity of the analysis and the limited understanding of the role of the SARS-CoV-2 specific T-cell response and cross-reactivity with the CCCs.

Background and aimsThere is growing interest in T cell-based immune therapies for a functional cure of chronic HBV infection including check-point inhibition, T cell-targeted vaccines or TCR-grafted effector cells. All these approaches depend on recognition of HLA class I-presented viral peptides. The HBV core region 18-27 is an immunodominant target of CD8+ T cells and represents the prime target for T cell-based therapies. Here, a high-resolution analysis of the core18-27 specific CD8+ T cell and the selected escape pathways was performed.MethodsHLA class I typing and viral sequence analyses were performed for 464 patients with chronic HBV infection. HBV-specific CD8+ T-cell responses against the prototype and epitope variants were characterized by flow cytometry.ResultsConsistent with promiscuous presentation of the core18-27 epitope, antigen-specific T cells were detected in patients carrying HLA-A*02:01, HLA-B*35:01, HLA-B*35:03 or HLA-B*51:01. Sequence analysis confirmed reproducible selection pressure on the core18-27 epitope in the context of these alleles. Interestingly, the selected immune escape pathways depend on the presenting HLA-class I-molecule. Although cross-reactive T cells were observed, some epitope variants achieved functional escape by impaired TCR-interaction or disturbed antigen processing. Of note, selection of epitope variants was exclusively observed in HBeAg negative HBV infection and here, detection of variants associated with significantly greater magnitude of the CD8 T cell response compared to absence of variants.ConclusionThe core18-27 epitope is highly variable and under heavy selection pressure in the context of different HLA class I-molecules. Some epitope variants showed evidence for impaired antigen processing and reduced presentation. Viruses carrying such escape substitutions will be less susceptible to CD8+ T cell responses and should be considered for T cell-based therapy strategies.

IntroductionLittle is known about risk factors for both Long COVID and somatic symptoms that develop in individuals without a history of COVID-19 in response to the pandemic. There is reason to assume an interplay between pathophysiological mechanisms and psychosocial factors in the etiology of symptom persistence.ObjectiveTherefore, this study investigates specific risk factors for somatic symptom deterioration in a cohort of German adults with and without prior SARS-CoV-2 infection.MethodsGerman healthcare professionals underwent SARS-CoV-2 IgG antibody testing and completed self-rating questionnaires at baseline and 21 months later between April 2020 and February 2022. Differences in variables between the time points were analyzed and a regression analysis was performed to predict somatic symptom deterioration at follow-up.ResultsSeven hundred fifty-one adults completed both assessments. Until follow-up, n = 58 had contracted SARS-CoV-2 confirmed by serology. Between baseline and follow-up, signs of mental and physical strain increased significantly in the sample. Symptom expectations associated with COVID-19 and a self-reported history of COVID-19, but not serologically confirmed SARS-CoV-2 infection, significantly predicted somatic symptom deterioration at follow-up. A further predictor was baseline psychological symptom burden.ConclusionsThis study supports a disease-overarching biopsychosocial model for the development of burdensome somatic symptoms during the COVID-19 pandemic and supports research findings that symptom burden may be more related to the psychosocial effects of the pandemic than to infection itself. Future studies on Long COVID should include SARS-CoV-2 negative control groups and consider symptom burden prior to infection in order to avoid an overestimation of prevalence rates.

SARS-CoV-2 is still a major burden for global health despite effective vaccines. With the reduction of social distancing measures, infection rates are increasing in children, while data on the pediatric immune response to SARS-CoV-2 infection is still lacking. Although the typical disease course in children has been mild, emerging variants may present new challenges in this age group. Peripheral blood mononuclear cells (PBMC) from 51 convalescent children, 24 seronegative siblings from early 2020, and 51 unexposed controls were stimulated with SARS-CoV-2-derived peptide MegaPools from the ancestral and beta variants. Flow cytometric determination of activation-induced markers and secreted cytokines were used to quantify the CD4+ T cell response. The average time after infection was over 80 days. CD4+ T cell responses were detected in 61% of convalescent children and were markedly reduced in preschool children. Cross-reactive T cells for the SARS-CoV-2 beta variant were identified in 45% of cases after infection with an ancestral SARS-CoV-2 variant. The CD4+ T cell response was accompanied most predominantly by IFN-γ and Granzyme B secretion. An antiviral CD4+ T cell response was present in children after ancestral SARS-CoV-2 infection, which was reduced in the youngest age group. We detected significant cross-reactivity of CD4+ T cell responses to the more recently evolved immune-escaping beta variant. Our findings have epidemiologic relevance for children regarding novel viral variants of concern and vaccination efforts.

Thymocyte selection-associated high mobility group box (TOX) has been described to be a key regulator in the formation of CD8+ T cell exhaustion. Hepatitis C virus (HCV) infection with different lengths of antigen exposure in acute, chronic, and after resolution of HCV infection is the ideal immunological model to study the expression of TOX in HCV-specific CD8+ T cells with different exposure to antigen. HCV-specific CD8+ T cells from 35 HLA-A*01:01, HLA-A*02:01, and HLA-A*24:02 positive patients were analyzed with a 16-color FACS-panel evaluating the surface expression of lineage markers (CD3, CD8), ectoenzymes (CD39, CD73), markers of differentiation (CD45RO, CCR7, CD127), and markers of exhaustion and activation (TIGIT, PD-1, KLRG1, CD226) and transcription factors (TOX, Eomesodermin, T-bet). Here, we defined on-target T cells as T cells against epitopes without escape mutations and off-target T cells as those against a “historical” antigen mutated in the autologous sequence. TOX+HCV-specific CD8+ T cells from patients with chronic HCV and on-target T cells displayed co-expression of Eomesodermin and were associated with the formation of terminally exhausted CD127-PD1hi, CD39hi, CD73low CD8+ T cells. In contrast, TOX+HCV-specific CD8+ T cells in patients with off-target T cells represented a progenitor memory Tex phenotype characterized by CD127hi expression and a CD39low and CD73hi phenotype. TOX+HCV-specified CD8+ T cells in patients with a sustained virologic response were characterized by a memory phenotype (CD127+, CD73hi) and co-expression of immune checkpoints and Eomesodermin, indicating a key structure in priming of HCV-specific CD8+ T cells in the chronic stage, which persisted as a residual after therapy. Overall, the occurrence of TOX+HCV-specific CD8+ T cells was revealed at each disease stage, which impacted the development of progenitor Tex, intermediate Tex, and terminally exhausted T cell through an individual molecular footprint. In sum, TOX is induced early during acute infection but is modulated by changes in viral sequence and antigen recognition. In the case of antigen persistence, the interaction with Eomesodermin leads to the formation of terminally exhausted virus-specific CD8+ T cells, and there was a direct correlation of the co-expression of TOX and Eomes and terminally exhausted phenotype of virus-specific CD8+ T cells.

Backgroundγδ T cells are unconventional T cells that have been demonstrated to be crucial for the pathogenesis and potentially for the cure of HIV-1 infection. The ectonucleotidase CD39 is part of the purinergic pathway that regulates immune responses by degradation of pro-inflammatory ATP in concert with CD73. Few studies on the expression of the ectoenzymes CD73 and CD39 on human γδ T cells in HIV have been performed to date.MethodsPBMC of n=86 HIV-1-infected patients were compared to PBMC of n=26 healthy individuals using 16-color flow cytometry determining the surface expression of CD39 and CD73 on Vδ1 and Vδ2 T cells in association with differentiation (CD45RA, CD28, CD27), activation and exhaustion (TIGIT, PD-1, CD38, and HLA-DR), and assessing the intracellular production of pro- and anti-inflammatory cytokines (IL-2, TGF-ß, TNF-α, Granzyme B, IL-10, IFN-γ) after in vitro stimulation with PMA/ionomycin.ResultsCD39 and CD73 expression on γδ T cells were inversed in HIV infection which correlated with HIV disease progression and immune activation. CD39, but not CD73 expression on γδ T cells of ART-treated patients returned to levels comparable with those of healthy individuals. Only a small subset (

Background: γδ T cells represent a unique T cell subpopulation due to their ability to recognize cancer cells in a T cell receptor- (TCR) dependent manner, but also in a non-major histocompatibility complex- (MHC) restricted way via natural killer receptors (NKRs). Endowed with these features, they represent attractive effectors for immuno-therapeutic strategies with a better safety profile and a more favorable anti-tumor efficacy in comparison to conventional αβ T cells. Also, remarkable progress has been achieved re-activating exhausted T lymphocytes with inhibitors of co-regulatory receptors e.g., programmed cell death protein 1 (PD-1), T cell immunoreceptor with Ig and ITIM domains (TIGIT) and of the adenosine pathway (CD39, CD73). Regarding γδ T cells, little evidence is available. This study aimed to immunophenotypically characterize γδ T cells from patients with diagnosed acute myeloid leukemia (AML) in comparison to patients with multiple myeloma (MM) and healthy donors (HD).Methods: The frequency, differentiation, activation, and exhaustion status of bone marrow- (BM) derived γδ T cells from patients with AML (n = 10) and MM (n = 11) were assessed in comparison to corresponding CD4+ and CD8+ T cells and peripheral blood- (PB) derived γδ T cells from HDs (n = 16) using multiparameter flow cytometry.Results: BM-infiltrating Vδ1 T cells showed an increased terminally differentiated cell population (TEMRAs) in AML and MM in comparison to HDs with an aberrant subpopulation of CD27−CD45RA++ cells. TIGIT, PD-1, TIM-3, and CD39 were more frequently expressed by γδ T cells in comparison to the corresponding CD4+ T cell population, with expression levels that were similar to that on CD8+ effector cells in both hematologic malignancies. In comparison to Vδ2 T cells, the increased frequency of PD-1+-, TIGIT+-, TIM-3+, and CD39+ cells was specifically observed on Vδ1 T cells and related to the TEMRA Vδ1 population with a significant co-expression of PD-1 and TIM-3 together with TIGIT.Conclusion: Our results revealed that BM-resident γδ T cells in AML and MM express TIGIT, PD-1, TIM-3 and CD39. As effector population for autologous and allogeneic strategies, inhibition of co-inhibitory receptors on especially Vδ1 γδ T cells may lead to re-invigoration that could further increase their cytotoxic potential.

Background: Progressive multifocal leukoencephalopathy (PML) caused by JCV is a rare but frequently fatal disease of the central nervous system, usually affecting immunocompromised individuals. Our study aims to expand the data on patient characteristics, diagnosis, clinical course, possible PML-directed treatment, and outcome of patients with PML at a German tertiary-care hospital.Methods:In this single-center observational cohort study, 37 consecutive patients with a confirmed diagnosis of PML seen at the University Medical Center Hamburg-Eppendorf from 2013 until 2019 were retrospectively analyzed by chart review with a special focus on demographics, risk factors, and clinical aspects as well as PML-directed treatment and survival.Results:We identified 37 patients with definite, probable, and possible PML diagnosis. 36 patients (97%) had underlying immunosuppressive disorders such as HIV/AIDS (n = 17; 46%), previous treatment with monoclonal antibodies (n = 6; 16%), hematological or oncological malignancies (n = 6; 16%), sarcoidosis (n = 5; 14%), solid organ transplantation (n = 1; 3%), and diagnosis of mixed connective tissue disease (n = 1; 3%). In only one patient no evident immunocompromised condition was detected (n = 1; 3%). Treatment attempts to improve the outcome of PML were reported in 13 patients (n = 13; 35%). Twenty seven percent of patients were lost to follow-up (n = 10). Twenty four-month survival rate after diagnosis of PML was 56% (n = 15).Conclusion: This interdisciplinary retrospective study describes epidemiology, risk factors, clinical course, and treatment trials in patients with PML at a German tertiary-care hospital. Acquired immunosuppression due to HIV-1 constituted the leading cause of PML in this monocenter cohort.

The ectoenzymes CD39 and CD73 play a major role in controlling tissue inflammation by regulating the balance between adenosine triphosphate (ATP) and adenosine. Still, little is known about the role of these two enzymes and ATP and its metabolites in the pathophysiology of inflammatory bowel disease (IBD). We isolated mononuclear cells from peripheral blood and lamina propria of the large intestine of patients diagnosed with IBD and of healthy volunteers. We then comprehensively analyzed the CD39 and CD73 expression patterns together with markers of activation (HLA-DR, CD38), differentiation (CCR7, CD45RA) and tissue-residency (CD69, CD103, CD49a) on CD4+, CD8+, γδ+ T cells and mucosa-associated invariant T cells using flow cytometry. CD39 expression levels of γδ+ and CD8+ T cells in lamina propria lymphocytes (LPL) were much higher compared to peripheral blood mononuclear cells. Moreover, the frequency of CD39+ CD4+ and CD8+, but not γδ+ LPL positively correlated with T-cell activation. The frequency of CD39+ cells among tissue-resident memory LPL (Trm) was higher compared to non-Trm for all subsets, confirming that CD39 is a marker for the tissue-resident memory phenotype. γδ+ Trm also showed a distinct cytokine profile upon stimulation – the frequency of IFN-γ+ and IL-17A+ cells was significantly lower in γδ+ Trm compared to non-Trm. Interestingly, we observed a decreased frequency of CD39+ γδ+ T cells in IBD patients compared to healthy controls (p = 0.0049). Prospective studies need to elucidate the exact role of this novel CD39+ γδ+ T-cell population with tissue-resident memory phenotype and its possible contribution to the pathogenesis of IBD and other inflammatory disorders.

Coronavirus disease 2019 (COVID-19) which is caused by the novel SARS-CoV-2 virus is a severe flu-like illness which is associated with hyperinflammation and immune dysfunction. The virus induces a strong T and B cell response but little is known about the immune pathology of this viral infection. Acute Plasmodium falciparum malaria also causes acute clinical illness and is characterized by hyperinflammation due to the strong production of pro-inflammatory cytokines and a massive activation of T cells. In malaria, T cells express a variety of co-inhibitory receptors which might be a consequence of their activation but also might limit their overwhelming function. Thus, T cells are implicated in protection as well as in pathology. The outcome of malaria is thought to be a consequence of the balance between co-activation and co-inhibition of T cells. Following the hypothesis that T cells in COVID-19 might have a similar, dual function, we comprehensively characterized the differentiation (CCR7, CD45RO) and activation status (HLA-DR, CD38, CD69, CD226), the co-expression of co-inhibitory molecules (PD1, TIM-3, LAG-3, BTLA, TIGIT), as well as the expression pattern of the transcription factors T-bet and eomes of CD8+ and CD4+ T cells of PBMC of n = 20 SARS-CoV-2 patients compared to n = 10 P. falciparum infected patients and n = 13 healthy controls. Overall, acute COVID-19 and malaria infection resulted in a comparably elevated activation and altered differentiation status of the CD8+ and CD4+ T cell populations. T effector cells of COVID-19 and malaria patients showed higher frequencies of the inhibitory receptors T-cell immunoglobulin mucin-3 (TIM-3) and Lymphocyte-activation gene-3 (LAG-3) which was linked to increased activation levels and an upregulation of the transcription factors T-bet and eomes. COVID-19 patients with a more severe disease course showed higher levels of LAG-3 and TIM-3 than patients with a mild disease course. During recovery, a rapid normalization of these inhibitory receptors could be observed. In summary, comparing the expression of different co-inhibitory molecules in CD8+ and CD4+ T cells in COVID-19 vs. malaria, there is a transient increase of the expression of certain inhibitory receptors like LAG-3 and TIM-3 in COVID-19 in the overall context of acute immune activation.

Background: T cells are thought to play a major role in conferring immunity against malaria. This study aimed to comprehensively define the breadth and specificity of the Plasmodium falciparum (P. falciparum)-specific CD4+ T cell response directed against the exported protein 1 (EXP1) in a cohort of patients diagnosed with acute malaria.Methods: Peripheral blood mononuclear cells of 44 patients acutely infected with P. falciparum, and of one patient infected with P. vivax, were stimulated and cultured in vitro with an overlapping set of 31 P. falciparum-specific 13-17-mer peptides covering the entire EXP1 sequence. EXP1-specific T cell responses were analyzed by ELISPOT and intracellular cytokine staining for interferon-γ production after re-stimulation with individual peptides. For further characterization of the epitopes, in silico and in vitro human leukocyte antigen (HLA) binding studies and fine mapping assays were performed.Results: We detected one or more EXP1-specific CD4+ T cell responses (mean: 1.09, range 0–5) in 47% (21/45) of our patients. Responses were directed against 15 of the 31 EXP1 peptides. Peptides EXP1-P13 (aa60-74) and P15 (aa70-85) were detected by 18% (n = 8) and 27% (n = 12) of the 45 patients screened. The optimal length, as well as the corresponding most likely HLA-restriction, of each of these two peptides was assessed. Interestingly, we also identified one CD4+ T cell response against peptide EXP1-P15 in a patient who was infected with P. vivax but not falciparum.Conclusions: This first detailed characterization of novel EXP1-specific T cell epitopes provides important information for future analysis with major histocompatibility complex-multimer technology as well as for immunomonitoring and vaccine design.

Control of malaria is an important global health issue and there is still an urgent need for the development of an effective prophylactic vaccine. Multiple studies have provided strong evidence that Plasmodium falciparum-specific MHC class I-restricted CD8+ T cells are important for sterile protection against Plasmodium falciparum infection. Here, we present an interactive epitope map of all P. falciparum-specific CD8+ T cell epitopes published to date, based on a comprehensive data base (IEDB), and literature search. The majority of the described P. falciparum-specific CD8+ T cells were directed against the antigens CSP, TRAP, AMA1, and LSA1. Notably, most of the epitopes were discovered in vaccine trials conducted with malaria-naïve volunteers. Only few immunological studies of P. falciparum-specific CD8+ T cell epitopes detected in patients suffering from acute malaria or in people living in malaria endemic areas have been published. Further detailed immunological mappings of P. falciparum-specific epitopes of a broader range of P. falciparum proteins in different settings and with different disease status are needed to gain a more comprehensive understanding of the role of CD8+ T cell responses for protection, and to better guide vaccine design and to study their efficacy.

CD8+ T cells are key players during infection with the malaria parasite Plasmodium berghei ANKA (PbA). While they cannot provide protection against blood-stage parasites, they can cause immunopathology, thus leading to the severe manifestation of cerebral malaria. Hence, the tight control of CD8+ T cell function is key in order to prevent fatal outcomes. One major mechanism to control CD8+ T cell activation, proliferation and effector function is the integration of co-inhibitory and co-stimulatory signals. In this study, we show that one such pathway, the HVEM-CD160 axis, significantly impacts CD8+ T cell regulation and thereby the incidence of cerebral malaria. Here, we show that the co-stimulatory molecule HVEM is indeed required to maintain CD8+ T effector populations during infection. Additionally, by generating a CD160−/− mouse line, we observe that the HVEM ligand CD160 counterbalances stimulatory signals in highly activated and cytotoxic CD8+ T effector cells, thereby restricting immunopathology. Importantly, CD160 is also induced on cytotoxic CD8+ T cells during acute Plasmodium falciparum malaria in humans. In conclusion, CD160 is specifically expressed on highly activated CD8+ T effector cells that are harmful during the blood-stage of malaria.

Inhibitory KIRs play a central role in regulating NK cell activity. KIR2DL2/3 bind to HLA-C molecules, but the modulation of these interactions by viral infections and presentation of viral epitopes is not well-understood. We investigated whether the frequencies of KIR2DL2/3+ NK cells recognizing HLA-C*03:04/viral peptide complexes were impacted by YFV vaccination or HIV-1 and HCV infection. Ex vivo HLA class I tetramer staining of primary human NK cells derived from YFV-vaccinated individuals, or HIV-1- or HCV-infected individuals revealed that the YFV/HLA-C*03:04-NS2A4−13-tetramer bound to a larger proportion of KIR2DL2/3+ NK cells compared to HIV-1/HLA-C*03:04-Gag296−304- or HCV/HLA-C*03:04-Core136−144-tetramers. The YFV/HLA-C*03:04-NS2A4−13-tetramer also exhibited a stronger avidity to KIR2DL2/3 compared to the other tested tetramers. The proportional frequencies of KIR2DL2/3+ NK cells binding to the three tested HLA-C*03:04 tetramers were identical between YFV-vaccinated individuals or HIV-1- or HCV-infected individuals, and remained stable following YFV vaccination. These data demonstrate consistent hierarchies in the frequency of primary KIR2DL2/3+ NK cells binding HLA-C*03:04/peptide complexes that were determined by the HLA-C-presented peptide and not modulated by the underlying viral infection or vaccination.

Introduction: Tumor-associated macrophages (TAMs) represent an important cell population within the tumor microenvironment, but little is known about the phenotype and function of these cells. The present study aims to characterize macrophages in high-grade serous ovarian cancer (HGSOC). Methods: Phenotype and expression of co-regulatory markers were assessed on TAMs derived from malignant ascites (MA) or peripheral blood (PB) by multiparametric flow cytometry. Samples were obtained from HGSOC patients (n=29) and healthy donors (HDs, n=16). Additional expression analysis was performed by RNAseq (n=192). Correlation with clinically relevant parameters was conducted and validated by a second patient cohort (n=517). Finally, the role of TIGIT in repolarization and phagocytosis was investigated in vitro. Results: Expression of the M2-associated receptors CD163, CD204, and CD206, as well as of the co-regulatory receptors TIGIT, CD226, TIM-3, and LAG-3 was significantly more frequent on macrophages in HGSOC than in HDs. CD39 and CD73 were broadly expressed on (mainly M2) macrophages, but without a clear clustering in HGSOC. CD163 mRNA levels were higher in TAMs from patients with residual tumor mass after surgery and associated with a shorter overall survival. In addition, TIGIT expression was associated with a higher tumor grading, indicating a prognostic relevance of M2 infiltration in HGSOC. TIGIT blockade significantly reduced the frequency of M2 macrophages. Moreover, combined blockade of TIGIT and CD47 significantly increased phagocytosis of ovarian cancer cells by TAMs in comparison to a single blockade of CD47. Conclusion: Combined blockade of TIGIT and CD47 represents a promising approach to enhance anti-CD47-facilitated phagocytosis. [ABSTRACT FROM AUTHOR]

Introduction: The nonstructural protein 12 (NSP12) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has a high sequence identity with common cold coronaviruses (CCC). Methods: Here, we comprehensively assessed the breadth and specificity of the NSP12-specific T-cell response after in vitro T-cell expansion with 185 overlapping 15-mer peptides covering the entire SARS-CoV-2 NSP12 at singlepeptide resolution in a cohort of 27 coronavirus disease 2019 (COVID-19) patients. Samples of nine uninfected seronegative individuals, as well as five pre-pandemic controls, were also examined to assess potential cross-reactivity with CCCs. Results: Surprisingly, there was a comparable breadth of individual NSP12 peptide-specific CD4+ T-cell responses between COVID-19 patients (mean: 12.82 responses; range: 0-25) and seronegative controls including prepandemic samples (mean: 12.71 responses; range: 0-21). However, the NSP12-specific T-cell responses detected in acute COVID-19 patients were on average of a higher magnitude. The most frequently detected CD4+ T-cell peptide specificities in COVID-19 patients were aa236-250 (37%) and aa246-260 (44%), whereas the peptide specificities aa686-700 (50%) and aa741-755 (36%), were the most frequently detected in seronegative controls. In CCCspecific peptide-expanded T-cell cultures of seronegative individuals, the corresponding SARS-CoV-2 NSP12 peptide specificities also elicited responses in vitro. However, the NSP12 peptide-specific CD4+ T-cell response repertoire only partially overlapped in patients analyzed longitudinally before and after a SARS-CoV-2 infection. Discussion: The results of the current study indicate the presence of pre-primed, cross-reactive CCC-specific T-cell responses targeting conserved regions of SARS-CoV-2, but they also underline the complexity of the analysis and the limited understanding of the role of the SARS-CoV-2 specific T-cell response and cross-reactivity with the CCCs. [ABSTRACT FROM AUTHOR]

Background and aims: There is growing interest in T cell-based immune therapies for a functional cure of chronic HBV infection including check-point inhibition, T cell-targeted vaccines or TCR-grafted effector cells. All these approaches depend on recognition of HLA class I-presented viral peptides. The HBV core region 18-27 is an immunodominant target of CD8+ T cells and represents the prime target for T cell-based therapies. Here, a high-resolution analysis of the core18-27 specific CD8+ T cell and the selected escape pathways was performed. Methods: HLA class I typing and viral sequence analyses were performed for 464 patients with chronic HBV infection. HBV-specific CD8+ T-cell responses against the prototype and epitope variants were characterized by flow cytometry. Results: Consistent with promiscuous presentation of the core18-27 epitope, antigen-specific T cells were detected in patients carrying HLA-A*02:01, HLA-B*35:01, HLA-B*35:03 or HLA-B*51:01. Sequence analysis confirmed reproducible selection pressure on the core18-27 epitope in the context of these alleles. Interestingly, the selected immune escape pathways depend on the presenting HLA-class I-molecule. Although cross-reactive T cells were observed, some epitope variants achieved functional escape by impaired TCR-interaction or disturbed antigen processing. Of note, selection of epitope variants was exclusively observed in HBeAg negative HBV infection and here, detection of variants associated with significantly greater magnitude of the CD8 T cell response compared to absence of variants. Conclusion: The core18-27 epitope is highly variable and under heavy selection pressure in the context of different HLA class I-molecules. Some epitope variants showed evidence for impaired antigen processing and reduced presentation. Viruses carrying such escape substitutions will be less susceptible to CD8+ T cell responses and should be considered for T cell-based therapy strategies. [ABSTRACT FROM AUTHOR]

Introduction: Little is known about risk factors for both Long COVID and somatic symptoms that develop in individuals without a history of COVID-19 in response to the pandemic. There is reason to assume an interplay between pathophysiological mechanisms and psychosocial factors in the etiology of symptom persistence. Objective: Therefore, this study investigates specific risk factors for somatic symptom deterioration in a cohort of German adults with and without prior SARS-CoV-2 infection. Methods: German healthcare professionals underwent SARS-CoV-2 IgG antibody testing and completed self-rating questionnaires at baseline and 21 months later between April 2020 and February 2022. Dierences in variables between the time points were analyzed and a regression analysis was performed to predict somatic symptom deterioration at follow-up. Results: Seven hundred fifty-one adults completed both assessments. Until follow-up, n = 58 had contracted SARS-CoV-2 confirmed by serology. Between baseline and follow-up, signs of mental and physical strain increased significantly in the sample. Symptom expectations associated with COVID19 and a self-reported history of COVID-19, but not serologically confirmed SARS-CoV-2 infection, significantly predicted somatic symptom deterioration at follow-up. A further predictor was baseline psychological symptom burden. Conclusions: This study supports a disease-overarching biopsychosocial model for the development of burdensome somatic symptoms during the COVID-19 pandemic and supports research findings that symptom burden may be more related to the psychosocial eects of the pandemic than to infection itself. Future studies on Long COVID should include SARS-CoV-2 negative control groups and consider symptom burden prior to infection in order to avoid an overestimation of prevalence rate. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 is still a major burden for global health despite effective vaccines. With the reduction of social distancing measures, infection rates are increasing in children, while data on the pediatric immune response to SARS-CoV-2 infection is still lacking. Although the typical disease course in children has been mild, emerging variants may present new challenges in this age group. Peripheral blood mononuclear cells (PBMC) from 51 convalescent children, 24 seronegative siblings from early 2020, and 51 unexposed controls were stimulated with SARS-CoV-2-derived peptide MegaPools from the ancestral and beta variants. Flow cytometric determination of activation-induced markers and secreted cytokines were used to quantify the CD4+ T cell response. The average time after infection was over 80 days. CD4+ T cell responses were detected in 61% of convalescent children and were markedly reduced in preschool children. Cross- reactive T cells for the SARS-CoV-2 beta variant were identified in 45% of cases after infection with an ancestral SARS-CoV-2 variant. The CD4+ T cell response was accompanied most predominantly by IFN-g and Granzyme B secretion. An antiviral CD4+ T cell response was present in children after ancestral SARS-CoV-2 infection, which was reduced in the youngest age group. We detected significant cross-reactivity of CD4+ T cell responses to the more recently evolved immune-escaping beta variant. Our findings have epidemiologic relevance for children regarding novel viral variants of concern and vaccination efforts. [ABSTRACT FROM AUTHOR]

Inhibitory KIRs play a central role in regulating NK cell activity. KIR2DL2/3 bind to HLA-C molecules, but the modulation of these interactions by viral infections and presentation of viral epitopes is not well-understood. We investigated whether the frequencies of KIR2DL2/3+ NK cells recognizing HLA-C∗03:04/viral peptide complexes were impacted by YFV vaccination or HIV-1 and HCV infection. Ex vivo HLA class I tetramer staining of primary human NK cells derived from YFV-vaccinated individuals, or HIV-1- or HCV-infected individuals revealed that the YFV/HLA-C∗03:04-NS2A4−13-tetramer bound to a larger proportion of KIR2DL2/3+ NK cells compared to HIV-1/HLA-C∗03:04-Gag296−304- or HCV/HLA-C∗03:04-Core136−144-tetramers. The YFV/HLA-C∗03:04-NS2A4−13-tetramer also exhibited a stronger avidity to KIR2DL2/3 compared to the other tested tetramers. The proportional frequencies of KIR2DL2/3+ NK cells binding to the three tested HLA-C∗03:04 tetramers were identical between YFV-vaccinated individuals or HIV-1- or HCV-infected individuals, and remained stable following YFV vaccination. These data demonstrate consistent hierarchies in the frequency of primary KIR2DL2/3+ NK cells binding HLA-C∗03:04/peptide complexes that were determined by the HLA-C-presented peptide and not modulated by the underlying viral infection or vaccination. [ABSTRACT FROM AUTHOR]