HLA-II immunopeptidome profiling and deep learning reveal features of antigenicity to inform antigen discovery.

CD4+ T cell responses are exquisitely antigen specific and directed toward peptide epitopes displayed by human leukocyte antigen class II (HLA-II) on antigen-presenting cells. Underrepresentation of diverse alleles in ligand databases and an incomplete understanding of factors affecting antigen presentation in vivo have limited progress in defining principles of peptide immunogenicity. Here, we employed monoallelic immunopeptidomics to identify 358,024 HLA-II binders, with a particular focus on HLA-DQ and HLA-DP. We uncovered peptide-binding patterns across a spectrum of binding affinities and enrichment of structural antigen features. These aspects underpinned the development of context-aware predictor of T cell antigens (CAPTAn), a deep learning model that predicts peptide antigens based on their affinity to HLA-II and full sequence of their source proteins. CAPTAn was instrumental in discovering prevalent T cell epitopes from bacteria in the human microbiome and a pan-variant epitope from SARS-CoV-2. Together CAPTAn and associated datasets present a resource for antigen discovery and the unraveling genetic associations of HLA alleles with immunopathologies.

Immune response induced by novel coronavirus infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has been prominent around the world since it was first discovered, affecting more than 100 million people. Although the symptoms of most infected patients are not serious, there is still a considerable proportion of patients who need hospitalization and even develop fatal symptoms such as cytokine storms, acute respiratory distress syndrome and so on. Cytokine storm is usually described as a collection of clinical manifestations caused by overactivation of the immune system, which plays an important role in tissue injury and multiorgan failure. The immune system of healthy individuals is composed of two interrelated parts, the innate immune system and the adaptive immune system. Innate immunity is the body's first line of defense against viruses; it can quickly perceive viruses through pattern recognition receptors and activate related inflammatory pathways to clear pathogens. The adaptive immune system is activated by specific antigens and is mainly composed of CD4+ T cells, CD8+ T cells and B cells, which play different roles in viral infection. Here, we discuss the immune response after SARS-CoV-2 infection. In-depth study of the recognition of and response of innate immunity and adaptive immunity to SARS-CoV-2 will help to prevent the development of critical cases and aid the exploration of more targeted treatments.

Impact of novel agent therapies on immune cell subsets and infectious complications in patients with relapsed/refractory multiple myeloma.

Introduction: Infections are a leading cause of morbidity and mortality in patients with multiple myeloma (MM). Methods: To examine the effects of modern second-generation novel agent therapy on immune cell subsets, in particular CD4+-T-cells, and infectious complications in patients with relapsed/refractory MM (RRMM), we conducted a prospective cohort study in 112 RRMM patients. Results: Substantially decreased CD4+-T-cells <200/µl before initiation of relapse therapy were detected in 27.7% of patients and were associated with a higher number of previous lines of therapy. Relapse therapy with carfilzomib or pomalidomide showed a significant further decrease of CD4+-T-cells. All novel agents led to a significant decrease of B-cell counts. Overall, infections were frequent with 21.3% of patients requiring antibacterial therapy within the first 3 months of relapse therapy, 5.6% requiring hospitalization. However, in the setting of standard antimicrobial prophylaxis in RRMM patients with very low CD4+-T-cells, no significant association of CD4+T-cell count and an increased risk of infection could be detected. Discussion: Our findings imply that reduced CD4+-T-cell numbers and infections are common in patients with RRMM. We also demonstrate an association with the number of previous therapies and certain substances suggesting an increased need for personalized prophylaxis strategies for opportunistic infections in this patient cohort.

Evidence for broad cross-reactivity of the SARS-CoV-2 NSP12-directed CD4+ T-cell response with pre-primed responses directed against common cold coronaviruses.

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 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. 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 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. 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.

Dynamics of CD4+ T-Cells and Neutralizing Antibody Responses to Three Consecutive Doses of Inactivated COVID-19 Vaccines in PLWH.

Background: Comprehensive characterization of safety and immune responses to vaccines is crucial for the prevention and treatment of COVID-19 among people living with HIV (PLWH). This study aimed to investigate the dynamic changes in SARS-CoV-2-specific CD4+ T-cell subsets and neutralizing antibody after three consecutive doses of inactivated COVID-19 vaccines (BBIBP-CorV) among PLWH. Methods: The blood samples were collected from 165 PLWH, including 66 PLWH in the 3-month interval between the second and third dose (cohort 1) and 99 PLWH in the 5-month interval (cohort 2). Blood collection for immunogenicity analysis was performed at 1-month post-2nd vaccination, pre-3rd vaccination, and within 2-month post-3rd vaccination. Wilcoxon matched-pairs signed-rank test was applied to compare the SARS-CoV-2-specific CD4+ T cell subsets and neutralizing antibody level at different time points. The relationship among CD4+ T-cells, Tregs subpopulations and SARS-CoV-2-specific neutralizing antibody level were evaluated with Spearman non-parametric correlation test. Results: No serious adverse reactions were found among PLWH. After two-dose or three-dose inactivated COVID-19 vaccination, the absolute counts of CD4+ T-cells and Tregs subpopulations (CD4+CD25HighCD127Low Tregs, CD45RA+ rTregs and CD45RO+ eTregs) increased in two cohorts. Satisfactory SARS-CoV-2-specific neutralizing antibody responses to the third-dose vaccination were found in two cohorts, including significantly enhanced neutralizing antibody level and high neutralizing antibody seroconversion rate. In addition, SARS-CoV-2-specific neutralizing antibody level were positively associated with the absolute counts of CD4+ T-cells and Tregs subpopulations as well as the frequency of CD45RO+ eTregs in PLWH after three doses of vaccinations. Conclusion: The three doses of inactivated COVID-19 vaccination were both safe and effective to increase SARS-CoV-2-specific CD4+ T-cells and neutralizing antibody in two PLWH cohorts with different inoculation intervals.

Adaptive immune system in severe COVID-19 patients in the first week of illness: A pilot study.

Introduction: The presentation of the course of COVID-19-related T-cell responses in the first week of the disease may be a more specific period for adaptive immune response assessment. This study aimed to clarify the relationship between changes in peripheral blood lymphocyte counts and death in patients with COVID-19 pneumonia. Methods: Thirty-three patients (14 females and 19 males) admitted for severe and desaturated COVID-19 pneumonia confirmed by polymerase chain reaction were included. Lymphocyte subsets and CD4+/CD8+ and CD16+/CD56+ rates were measured using flow cytometry from peripheral blood at admission and on the day of death or hospital discharge. Results: Twenty-eight patients survived and five died. On the day of admission, the CD4+ cell count was significantly higher and the saturation of O2 was significantly lower in the deceased patients compared to the survivors (P < 0.05). The CD16+/CD56+ rate was significantly lower on the day of death in the deceased patients than in discharge day for the survivors (P = 0.013). Conclusion: CD4+ lymphocyte percentages and O2 saturation in samples taken on the day of admission to the hospital and CD16+/CD56+ ratios taken at the time of discharge from the hospital were found to be associated with the mortality in patients with severe COVID-19.

Robust SARS-CoV-2 T cell responses with common TCRαß motifs toward COVID-19 vaccines in patients with hematological malignancy impacting B cells.

Immunocompromised hematology patients are vulnerable to severe COVID-19 and respond poorly to vaccination. Relative deficits in immunity are, however, unclear, especially after 3 vaccine doses. We evaluated immune responses in hematology patients across three COVID-19 vaccination doses. Seropositivity was low after a first dose of BNT162b2 and ChAdOx1 (∼26%), increased to 59%-75% after a second dose, and increased to 85% after a third dose. While prototypical antibody-secreting cells (ASCs) and T follicular helper (Tfh) cell responses were elicited in healthy participants, hematology patients showed prolonged ASCs and skewed Tfh2/17 responses. Importantly, vaccine-induced expansions of spike-specific and peptide-HLA tetramer-specific CD4+/CD8+ T cells, together with their T cell receptor (TCR) repertoires, were robust in hematology patients, irrespective of B cell numbers, and comparable to healthy participants. Vaccinated patients with breakthrough infections developed higher antibody responses, while T cell responses were comparable to healthy groups. COVID-19 vaccination induces robust T cell immunity in hematology patients of varying diseases and treatments irrespective of B cell numbers and antibody response.

Dynamic changes in radiological parameters, immune cells, selected miRNAs, and cytokine levels in peripheral blood of patients with severe COVID­19.

The present study aimed to investigate the dynamic changes in peripheral blood leucocyte subpopulations, cytokine and miRNA levels, and changes in computed tomography (CT) scores in patients with severe coronavirus disease 2019 (COVID-19) (n=14) and age-matched non-COVID-19 volunteers (n=17), which were included as a reference control group. All data were collected on the day of patient admission (day 0) and on the 7th, 14th and 28th days of follow-up while CT of the lungs was performed on weeks 2, 8, 24 and 48. On day 0, lymphopenia and leucopenia were detected in most patients with COVID-19, as well as an increase in the percentage of banded neutrophils, B cells, and CD4+ Treg cells, and a decrease in the content of PD-1low T cells, classical, plasmacytoid, and regulatory dendritic cells. On day 7, the percentage of T and natural killer cells decreased with a concurrent increase in B cells, but returned to the initial level after treatment discharge. The content of different T and dendritic cell subsets among CD45+ cells increased during two weeks and remained elevated, suggesting the activation of an adaptive immune response. The increase of PD-1-positive subpopulations of T and non-T cells and regulatory CD4 T cells in patients with COVID-19 during the observation period suggests the development of an inflammation control mechanism. The levels of interferon γ-induced protein 10 (IP-10), tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 decreased on day 7, but increased again on days 14 and 28. C-reactive protein and granulocyte colony-stimulating factor (G-CSF) levels decreased gradually throughout the observation period. The relative expression levels of microRNA (miR)-21-5p, miR-221-3p, miR-27a-3p, miR-146a-5p, miR-133a-3p, and miR-126-3p were significantly higher at the beginning of hospitalization compared to non-COVID-19 volunteers. The plasma levels of all miRs, except for miR-126-3p, normalized within one week of treatment. At week 48, CT scores were most prominently correlated with the content of lymphocytes, senescent memory T cells, CD127+ T cells and CD57+ T cells, and increased concentrations of G-CSF, IP-10, and macrophage inflammatory protein-1α.

T HELPER CELL SUBSETS AND RELATED TARGET CELLS IN ACUTE COVID-19.

Current review presents a brief overview of the immune system dysregulation during acute COVID-19 and illustrates the main alterations in peripheral blood CD4+ T-cell (Th) subsets as well as related target cells. Effects of dendritic cell dysfunction induced by SARS-CoV-2 exhibited decreased expression of cell-surface HLA-DR, CCR7 as well as co-stimulatory molecules CD80 and CD86, suggesting reduced antigen presentation, migratory and activation capacities of peripheral blood dendritic cells. SARS-CoV-2-specific Th cells could be detected as early as days 2-4 post-symptom onset, whereas the prolonged lack of SARS-CoV-2-specific Th cells was associated with severe and/or poor COVID-19 outcome. Firstly, in acute COVID-19 the frequency of Th1 cell was comparable with control levels, but several studies have reported about upregulated inhibitory immune checkpoint receptors and exhaustion-associated molecules (TIM3, PD-1, BTLA, TIGIT etc.) on circulating CD8+ T-cells and NK-cells, whereas the macrophage count was increased in bronchoalveolar lavage (BAL) samples. Next, type 2 immune responses are mediated mainly by Th2 cells, and several studies have revealed a skewing towards dominance of Th2 cell subset in peripheral blood samples from patients with acute COVID-19. Furthermore, the decrease of circulating main Th2 target cells - basophiles and eosinophils - were associated with severe COVID-19, whereas the lung tissue was enriched with mast cells and relevant mediators released during degranulation. Moreover, the frequency of peripheral blood Th17 cells was closely linked to COVID-19 severity, so that low level of Th17 cells was observed in patients with severe COVID-19, but in BAL the relative number of Th17 cells as well as the concentrations of relevant effector cytokines were dramatically increased. It was shown that severe COVID-19 patients vs. healthy control had higher relative numbers of neutrophils if compared, and the majority of patients with COVID-19 had increased frequency and absolute number of immature neutrophils with altered ROS production. Finally, the frequency of Tfh cells was decreased during acute COVID-19 infection. Elevated count of activated Tfh were found as well as the alterations in Tfh cell subsets characterized by decreased "regulatory" Tfh1 cell and increased "pro-inflammatory" Tfh2 as well as Tfh17 cell subsets were revealed. Descriptions of peripheral blood B cells during an acute SARS-CoV-2 infection werev reported as relative B cell lymphopenia with decreased frequency of "naive" and memory B cell subsets, as well as increased level of CD27hiCD38hiCD24- plasma cell precursors and atypical CD21low B cells. Thus, the emerging evidence suggests that functional alterations occur in all Th cell subsets being linked with loss-of-functions of main Th cell subsets target cells. Furthermore, recovered individuals could suffer from long-term immune dysregulation and other persistent symptoms lasting for many months even after SARS-CoV-2 elimination, a condition referred to as post-acute COVID-19 syndrome.Copyright © 2022 Saint Petersburg Pasteur Institute. All rights reserved.

Effect of Macrophages and Latent Reservoirs on the Dynamics of HTLV-I and HIV-1 Coinfection

Human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type I (HTLV-I) are two retroviruses that have a similar fashion of transmission via sharp objects contaminated by viruses, transplant surgery, transfusion, and sexual relations. Simultaneous infections with HTLV-I and HIV-1 usually occur in areas where both viruses have become endemic. CD4+T cells are the main targets of HTLV-I, while HIV-1 can infect CD4+T cells and macrophages. It is the aim of this study to develop a model of HTLV-I and HIV-1 coinfection that describes the interactions of nine compartments: susceptible cells of both CD4+T cells and macrophages, HIV-1-infected cells that are latent/active in both CD4+T cells and macrophages, HTLV-I-infected CD4+T cells that are latent/active, and free HIV-1 particles. The well-posedness, existence of equilibria, and global stability analysis of our model are investigated. The Lyapunov function and LaSalle's invariance principle were used to study the global asymptotic stability of all equilibria. The theoretically predicted outcomes were verified by utilizing numerical simulations. The effect of including the macrophages and latent reservoirs in the HTLV-I and HIV-1 coinfection model is discussed. We show that the presence of macrophages makes a coinfection model more realistic when the case of the coexistence of HIV-1 and HTLV-I is established. Moreover, we have shown that neglecting the latent reservoirs in HTLV-I and HIV-1 coinfection modeling will lead to the design of an overflow of anti-HIV-1 drugs.

T-cell activation-induced marker assays in health and disease.

Activation-induced marker (AIM) assays have proven to be an accessible and rapid means of antigen-specific T-cell detection. The method typically involves short-term incubation of whole blood or peripheral blood mononuclear cells with antigens of interest, where autologous antigen-presenting cells process and present peptides in complex with major histocompatibility complex (MHC) molecules. Recognition of peptide-MHC complexes by T-cell receptors then induces upregulation of activation markers on the T cells that can be detected by flow cytometry. In this review, we highlight the most widely used activation markers for assays in the literature while identifying nuances and potential downfalls associated with the technique. We provide a summary of how AIM assays have been used in both discovery science and clinical studies, including studies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity. This review primarily focuses on AIM assays using human blood or peripheral blood mononuclear cell samples, with some considerations noted for tissue-derived T cells and nonhuman samples. AIM assays are a powerful tool that enables detailed analysis of antigen-specific T-cell frequency, phenotype and function without needing to know the precise antigenic peptides and their MHC restriction elements, enabling a wider analysis of immunity generated following infection and/or vaccination.

T follicular helper cell responses to SARS-CoV-2 vaccination among healthy and immunocompromised adults.

The worldwide rollout of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccinations in the last 2 years has produced a multitude of studies investigating T-cell responses in the peripheral blood and a limited number in secondary lymphoid tissues. As a key component to an effective immune response, vaccine-specific T follicular helper (Tfh) cells are localized in the draining lymph node (LN) and assist in the selection of highly specific B-cell clones for the production of neutralizing antibodies. While these cells have been noted in the blood as circulating Tfh (cTfh) cells, they are not often taken into consideration when examining effective CD4+ T-cell responses, particularly in immunocompromised groups. Furthermore, site-specific analyses in locations such as the LN have recently become an attractive area of investigation. This is mainly a result of improved sampling methods via ultrasound-guided fine-needle biopsy (FNB)/fine-needle aspiration (FNA), which are less invasive than LN excision and able to be performed longitudinally. While these studies have been undertaken in healthy individuals, data from immunocompromised groups are lacking. This review will focus on both Tfh and cTfh responses after SARS-CoV-2 vaccination in healthy and immunocompromised individuals. This area of investigation could identify key characteristics of a successful LN response required for the prevention of infection and viral clearance. This furthermore may highlight responses that could be fine-tuned to improve vaccine efficacy within immunocompromised groups that are at a risk of more severe disease.

Increased Pro Th1 And Th17 Transcriptional Activity In Patients With Severe COVID-19.

Background: COVID-19 is known to disrupt immune response and induce hyperinflammation that could potentially induce fatal outcome of the disease. Until now, it is known that interplay among cytokines is rather important for clinical presentation and outcome of COVID-19. The aim of this study was to determine transcriptional activity and functional phenotype of T cells and the relationship between pro- and anti-inflammatory cytokines and clinical parameters of COVID-19 severity. Methods: All recruited patients met criteria for COVID-19 are were divided in four groups according to disease severity. Serum levels of IL-12, IFN-γ, IL-17 and IL-23 were measured, and flow cytometry analysis of T cells from peripheral blood was performed. Results: Significant elevation of IL-12, IFN-γ, IL-17 and IL-23 in stage IV of the disease has been revealed. Further, strong intercorrelation between IL-12, IFN-γ, IL-17 and IL-23 was also found in stage IV of the disease, marking augmented Th1 and Th17 response. Analyses of T cells subsets indicate a noticeable phenotype change. CD4+, but not CD8+ T cells expressed increased transcriptional activity through increased expression of Tbet and RORγT, accompanied with increased percentage of IFN-γ and IL-17 producing T cells. Conclusion: Our results pose a novel hypothesis of the underlying mechanism behind deteriorating immune response in severe cases of COVID-19.

Emerging Effects of IL-33 on COVID-19.

Since the start of COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more than 6 million people have lost their lives worldwide directly or indirectly. Despite intensified efforts to clarify the immunopathology of COVID-19, the key factors and processes that trigger an inflammatory storm and lead to severe clinical outcomes in patients remain unclear. As an inflammatory storm factor, IL-33 is an alarmin cytokine, which plays an important role in cell damage or infection. Recent studies have shown that serum IL-33 is upregulated in COVID-19 patients and is strongly associated with poor outcomes. Increased IL-33 levels in severe infections may result from an inflammatory storm caused by strong interactions between activated immune cells. However, the effects of IL-33 in COVID-19 and the underlying mechanisms remain to be fully elucidated. In this review, we systematically discuss the biological properties of IL-33 under pathophysiological conditions and its regulation of immune cells, including neutrophils, innate lymphocytes (ILCs), dendritic cells, macrophages, CD4+ T cells, Th17/Treg cells, and CD8+ T cells, in COVID-19 phagocytosis. The aim of this review is to explore the potential value of the IL-33/immune cell pathway as a new target for early diagnosis, monitoring of severe cases, and clinical treatment of COVID-19.

Is the Sars-CoV-2 virus a possible trigger agent for the development of achalasia?

BACKGROUND: Achalasia is an autoimmune disease whose probable causal agent is a neurotropic virus that chronically infects the myenteric plexus of the esophagus and induces the disease in a genetically susceptible host. The association between achalasia and coronaviruses has not been reported. AIMS: To evaluate the presence of the SARS-CoV-2 virus, the ACE2 expression, the tissue architecture, and immune response in the lower esophageal sphincter muscle (LESm) of achalasia patients who posteriorly had SARS-CoV-2 (achalasia-COVID-19) infection before laparoscopic Heller myotomy (LHM) and compare the findings with type II achalasia patients and transplant donors (controls) without COVID-19. METHODS: The LESm of 7 achalasia-COVID-19 patients (diagnosed by PCR), ten achalasia patients, and ten controls without COVID-19 were included. The presence of the virus was evaluated by in situ PCR and immunohistochemistry. ACE2 receptor expression and effector CD4 T cell and regulatory subsets were determined by immunohistochemistry. KEY RESULTS: Coronavirus was detected in 6/7 patients-COVID-19. The SARS-CoV-2 was undetectable in the LESm of the achalasia patients and controls. ACE2 receptor was expressed in all the patients and controls. One patient developed achalasia type II post-COVID-19. The percentage of Th22/Th17/Th1/pDCreg was higher in achalasia and achalasia-COVID-19 pre-HLM vs. controls. The Th2/Treg/Breg cell percentages were higher only in achalasia vs. controls. CONCLUSION & INFERENCES: SARS-CoV2 and its receptor expression in the LESm of achalasia patients who posteriorly had COVID-19 but not in the controls suggests that it could affect the myenteric plexus. Unlike achalasia, patients-COVID-19 have an imbalance between effector CD4 T cells and the regulatory mechanisms.

Correlation of antigen-specific immune response with disease severity among COVID-19 patients in Bangladesh.

Coronavirus disease 2019 (COVID-19) is a protean disease causing different degrees of clinical severity including fatality. In addition to humoral immunity, antigen-specific T cells may play a critical role in defining the protective immune response against SARS-CoV-2, the virus that causes this disease. As a part of a longitudinal cohort study in Bangladesh to investigate B and T cell-specific immune responses, we sought to evaluate the activation-induced marker (AIM) and the status of different immune cell subsets during a COVID-19 infection. We analyzed a total of 115 participants, which included participants with asymptomatic, mild, moderate, and severe clinical symptoms. We observed decreased mucosal-associated invariant T (MAIT) cell frequency on the initial days of the COVID-19 infection in symptomatic patients compared to asymptomatic patients. However, natural killer (NK) cells were found to be elevated in symptomatic patients just after the onset of the disease compared to both asymptomatic patients and healthy individuals. Moreover, we found a significant increase of AIM+ (both OX40+CD137+ and OX40+CD40L+) CD4+ T cells in moderate and severe COVID-19 patients in response to SARS-CoV-2 peptides (especially spike peptides) compared to pre-pandemic controls who are unexposed to SARS-CoV-2. Notably, we did not observe any significant difference in the CD8+ AIMs (CD137+CD69+), which indicates the exhaustion of CD8+ T cells during a COVID-19 infection. These findings suggest that patients who recovered from moderate and severe COVID-19 were able to mount a strong CD4+ T-cell response against shared viral determinants that ultimately induced T cells to mount further immune responses to SARS-CoV-2.

Resolving SARS-CoV-2 CD4+ T cell specificity via reverse epitope discovery.

The current strategy to detect immunodominant T cell responses focuses on the antigen, employing large peptide pools to screen for functional cell activation. However, these approaches are labor and sample intensive and scale poorly with increasing size of the pathogen peptidome. T cell receptors (TCRs) recognizing the same epitope frequently have highly similar sequences, and thus, the presence of large sequence similarity clusters in the TCR repertoire likely identify the most public and immunodominant responses. Here, we perform a meta-analysis of large, publicly available single-cell and bulk TCR datasets from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals to identify public CD4+ responses. We report more than 1,200 αßTCRs forming six prominent similarity clusters and validate histocompatibility leukocyte antigen (HLA) restriction and epitope specificity predictions for five clusters using transgenic T cell lines. Collectively, these data provide information on immunodominant CD4+ T cell responses to SARS-CoV-2 and demonstrate the utility of the reverse epitope discovery approach.

Staining of activated ß2-integrins in combination with CD137 and CD154 for sensitive identification of functional antigen-specific CD4+ and CD8+ T cells.

Common flow cytometry-based methods used for functional assessment of antigen-specific T cells rely on de novo expression of intracellular cytokines or cell surface activation induced markers. They come with some limitations such as complex experimental setting, loss of cell viability and often high unspecific background which impairs assay sensitivity. We have previously shown that staining of activated ß2-integrins either with multimers of their ligand ICAM-1 or with a monoclonal antibody can serve as a functional marker detectable on T cells after minutes (CD8+) or few hours (CD4+) of activation. Here, we present a simple method for detection of activated ß2-integrins in combination with established cell surface activation induced markers. We observed that activated ß2-integrins were still detectable after 14 hours of stimulation, allowing their detection together with CD137 and CD154. Combinatorial gating of cells expressing activated ß2-integrins and CD137 or CD154 reduced background in unstimulated samples, increasing the signal-to-noise ratio and allowing improved assessment of low-frequency T cell responses. Extracellular staining of these markers highly correlated with production of intracellular cytokines IL-2, TNF or IFNγ in CD4+ and CD8+ T cells. As an exemplary application, SARS-CoV-2 spike-specific T cell responses were assessed in individuals after COVID-19 vaccination. This method should be useful for epitope discovery projects and for the simultaneous monitoring of low-frequency antigen-specific CD4+ and CD8+ T cell responses in various physiological situations.

R-DOTAP Cationic Lipid Nanoparticles Outperform Squalene-Based Adjuvant Systems in Elicitation of CD4 T Cells after Recombinant Influenza Hemagglutinin Vaccination.

It is clear that new approaches are needed to promote broadly protective immunity to viral pathogens, particularly those that are prone to mutation and escape from antibody-mediated immunity. Prototypic pathogens of this type are influenza and SARS-CoV-2, where the receptor-binding protein exhibits extremely high variability in its receptor-binding regions. T cells, known to target many viral proteins, and within these, highly conserved peptide epitopes, can contribute greatly to protective immunity through multiple mechanisms but are often poorly recruited by current vaccine strategies. Here, we have studied a promising novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP), which was previously recognized for its ability to generate anti-tumor immunity through the induction of potent cytotoxic CD8 T cells. Using a preclinical mouse model, we have assessed an R-DOTAP nanoparticle adjuvant system for its ability to promote CD4 T cell responses to vaccination with recombinant influenza protein. Our studies revealed that R-DOTAP consistently outperformed a squalene-based adjuvant emulsion, even when it was introduced with a potent TLR agonist CpG, in the ability to elicit peptide epitope-specific CD4 T cells when quantified by IFN-γ and IL-2 ELISpot assays. Clinical testing of R-DOTAP containing vaccines in earlier work by others has demonstrated an acceptable safety profile. Hence, R-DOTAP can offer exciting opportunities as an immune stimulant for next-generation prophylactic recombinant protein-based vaccines.

T Helper Cell Subsets and Related Target Cells in Acute Covid-19

Current review presents a brief overview of the immune system dysregulation during acute COVID-19 and illustrates the main alterations in peripheral blood CD4(+) T-cell (Th) subsets as well as related target cells. Effects of dendritic cell dysfunction induced by SARS-CoV-2 exhibited decreased expression of cell-surface HLA-DR, CCR7 as well as co-stimulatory molecules CD80 and CD86, suggesting reduced antigen presentation, migratory and activation capacities of peripheral blood dendritic cells. SARS-CoV-2- specific Th cells could be detected as early as days 2-4 post-symptom onset, whereas the prolonged lack of SARS-CoV-2-specific Th cells was associated with severe and/or poor COVID-19 outcome. Firstly, in acute COVID-19 the frequency of Th1 cell was comparable with control levels, but several studies have reported about upregulated inhibitory immune checkpoint receptors and exhaustion-associated molecules (TIM3, PD-1, BTLA, TIGIT etc.) on circulating CD8(+) T-cells and NK-cells, whereas the macrophage count was increased in bronchoalveolar lavage (BAL) samples. Next, type 2 immune responses are mediated mainly by Th2 cells, and several studies have revealed a skewing towards dominance of Th2 cell subset in peripheral blood samples from patients with acute COVID-19. Furthermore, the decrease of circulating main Th2 target cells - basophiles and eosinophils - were associated with severe COVID-19, whereas the lung tissue was enriched with mast cells and relevant mediators released during degranulation. Moreover, the frequency of peripheral blood Th17 cells was closely linked to COVID-19 severity, so that low level of Th17 cells was observed in patients with severe COVID-19, but in BAL the relative number of Th17 cells as well as the concentrations of relevant effector cytokines were dramatically increased. It was shown that severe COVID-19 patients vs. healthy control had higher relative numbers of neutrophils if compared, and the majority of patients with COVID-19 had increased frequency and absolute number of immature neutrophils with altered ROS production. Finally, the frequency of Tfh cells was decreased during acute COVID-19 infection. Elevated count of activated Tfh were found as well as the alterations in Tfh cell subsets characterized by decreased "regulatory" Tfh1 cell and increased "pro-inflammatory" Tfh2 as well as Tfh17 cell subsets were revealed. Descriptions of peripheral blood B cells during an acute SARS- CoV-2 infection werev reported as relative B cell lymphopenia with decreased frequency of "naive" and memory B cell subsets, as well as increased level of CD27(hi)CD38(hi)CD24(-) plasma cell precursors and atypical CD21(low) B cells. Thus, the emerging evidence suggests that functional alterations occur in all Th cell subsets being linked with loss-of-functions of main Th cell subsets target cells. Furthermore, recovered individuals could suffer from long-term immune dysregulation and other persistent symptoms lasting for many months even after SARS-CoV-2 elimination, a condition referred to as post-acute COVID-19 syndrome.