Experimental validation of immunogenic SARS-CoV-2 T cell epitopes identified by artificial intelligence

During the COVID-19 pandemic we utilized an AI-driven T cell epitope prediction tool, the NEC Immune Profiler (NIP) to scrutinize and predict regions of T cell immunogenicity (hotspots) from the entire SARS-CoV-2 viral proteome. These immunogenic regions offer potential for the development of universally protective T cell vaccine candidates. Here, we validated and characterized T cell responses to a set of minimal epitopes from these AI-identified universal hotspots. Utilizing a flow cytometry-based T cell activation-induced marker (AIM) assay, we identified 59 validated screening hits, of which 56% (33 peptides) have not been previously reported. Notably, we found that most of these novel epitopes were derived from the non-spike regions of SARS-CoV-2 (Orf1ab, Orf3a, and E). In addition, ex vivo stimulation with NIP-predicted peptides from the spike protein elicited CD8+ T cell response in PBMC isolated from most vaccinated donors. Our data confirm the predictive accuracy of AI platforms modelling bona fide immunogenicity and provide a novel framework for the evaluation of vaccine-induced T cell responses

People who use drugs show no increase in pre-existing T-cell cross-reactivity toward SARS-CoV-2 but develop a normal polyfunctional T-cell response after standard mRNA vaccination

People who use drugs (PWUD) are at a high risk of contracting and developing severe coronavirus disease 2019 (COVID-19) and other infectious diseases due to their lifestyle, comorbidities, and the detrimental effects of opioids on cellular immunity. However, there is limited research on vaccine responses in PWUD, particularly regarding the role that T cells play in the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we show that before vaccination, PWUD did not exhibit an increased frequency of preexisting cross-reactive T cells to SARS-CoV-2 and that, despite the inhibitory effects that opioids have on T-cell immunity, standard vaccination can elicit robust polyfunctional CD4+ and CD8+ T-cell responses that were similar to those found in controls. Our findings indicate that vaccination stimulates an effective immune response in PWUD and highlight targeted vaccination as an essential public health instrument for the control of COVID-19 and other infectious diseases in this group of high-risk patients

Immune complexes, innate immunity, and NETosis in ChAdOx1 vaccine-induced thrombocytopenia

Aims - We recently reported five cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) 7–10 days after receiving the first dose of the ChAdOx1 nCoV-19 adenoviral vector vaccine against corona virus disease 2019 (COVID-19). We aimed to investigate the pathogenic immunological responses operating in these patients. Methods and results - We assessed circulating inflammatory markers by immune assays and immune cell phenotyping by flow cytometry analyses and performed immunoprecipitation with anti-platelet factor (PF)4 antibody in plasma samples followed by mass spectrometry from all five patients. A thrombus was retrieved from the sinus sagittal superior of one patient and analysed by immunohistochemistry and flow cytometry. Precipitated immune complexes revealed multiple innate immune pathway triggers for platelet and leucocyte activation. Plasma contained increased levels of innate immune response cytokines and markers of systemic inflammation, extensive degranulation of neutrophils, and tissue and endothelial damage. Blood analyses showed activation of neutrophils and increased levels of circulating H3Cit, dsDNA, and myeloperoxidase–DNA complex. The thrombus had extensive infiltration of neutrophils, formation of neutrophil extracellular traps (NETs), and IgG deposits. Conclusions - The results show that anti-PF4/polyanion IgG-mediated thrombus formation in VITT patients is accompanied by a massive innate immune activation and particularly the fulminant activation of neutrophils including NETosis. These results provide novel data on the immune response in this rare adenoviral vector-induced VITT

The Colocalization Potential of HIV-Specific CD8+ and CD4+ T-Cells is Mediated by Integrin β7 but Not CCR6 and Regulated by Retinoic Acid

CD4+ T-cells from gut-associated lymphoid tissues (GALT) are major targets for HIV-1 infection. Recruitment of excess effector CD8+ T-cells in the proximity of target cells is critical for the control of viral replication. Here, we investigated the colocalization potential of HIV-specific CD8+ and CD4+ T-cells into the GALT and explored the role of retinoic acid (RA) in regulating this process in a cohort of HIV-infected subjects with slow disease progression. The expression of the gut-homing molecules integrin β7, CCR6, and CXCR3 was identified as a “signature” for HIV-specific but not CMV-specific CD4+ T-cells thus providing a new explanation for their enhanced permissiveness to infection in vivo. HIV-specific CD8+ T-cells also expressed high levels of integrin β7 and CXCR3; however CCR6 was detected at superior levels on HIV-specific CD4+ versus CD8+ T-cells. All trans RA (ATRA) upregulated the expression of integrin β7 but not CCR6 on HIV-specific T-cells. Together, these results suggest that HIV-specific CD8+ T-cells may colocalize in excess with CD4+ T-cells into the GALT via integrin β7 and CXCR3, but not via CCR6. Considering our previous findings that CCR6+CD4+ T-cells are major cellular targets for HIV-DNA integration in vivo, a limited ability of CD8+ T-cells to migrate in the vicinity of CCR6+CD4+ T-cells may facilitate HIV replication and dissemination at mucosal sites

Facteurs de croissance et progéniteurs hématopoïetiques autologues (de nouveaux outils pour la restauration de la tolérance au soi dans le diabète auto-immun)

La rupture de la tolérance au soi s'accompagne d'un déséquilibre fonctionnel entre cellules T régulatrices et pathogènes. Les stratégies thérapeutiques visent soit à limiter la progression des cellules T effectrices soit à expandre les Treg. Nous avons fait l'hypothèse que la stimulation de l'hematopoïèse favoriserait l'émergence de Treg. Un traitement par le G-CSF prévient en effet la survenue du diabète de type 1 chez la souris NOD par l'émergence de cellules dendritiques tolérogènes semi-matures qui recrutent des Treg. En outre, associé au Flt-3ligand, le G-CSF engendre le développement de progéniteurs hématopoïetiques tolérogènes dont l'expression de Jagged-2 favorise l'expansion directe des Treg en augmentant leur expression membranaire du récepteur Notch3. L'émergence de cellules T régulatrices par la greffe de cellules souches hématopoïétiques mobilisées autologue pourrait jouer un rôle crucial dans les rémissions observées dans les maladies auto-immunes.Expansion of regulatory T cells in vivo is a promising strategy to cure auto-immune diseases. Here we demonstrate that stimulation of hematopoiesis either by the growth factor G-CSF or by transplantation of mobilised hematopoietic progenitors (HPC), but not of medullary hematopoietic stem cells, increases CD4+CD25+Foxp3+ regulatory T cell (Treg) numbers in peripancreatic lymph nodes and provides protection against type 1 diabetes in the NOD mouse. Protection is based on the following mechanisms : G-CSF promotes the differentiation of semi-mature plasmacytoid dendritic cells which in turn recruit Treg whereas HPC directly expand Treg. The HPC-Treg interaction requires cell contact and activates the Notch signalling pathway via Jagged2, selectively expressed on HPC, which increases Notch3 expression on Treg. This selective expression of jagged2 on mobilised progenitors needs to be confirmed in human and could represent a novel way to distinguish immunogenic from tolerogenic progenitors.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Notch activation on effector T cells increases their sensitivity to Treg cell-mediated suppression through upregulation of TGF-βRII expression.

International audienceNotch proteins play an important role in embryonic development and cell-fate decisions. Notch influences also the activation and differentiation of peripheral T cells. Here, we investigated whether Notch signaling modulates the response of effector T cells to regulatory T (Treg) cells. Pre-exposure of CD4(+) CD25(-) effector T cells to the Notch ligands Delta-4 and Jagged-1, but not Delta-1, increases significantly effector T-cell sensitivity to Treg cell-mediated suppression through upregulation of TGF-βRII expression and increased levels of the phosphorylated form of the Smad 3 protein. This effect is relieved by anti-TGF-β Abs. We demonstrate that HES (hairy and enhancer of split), the main transcription factor downstream of Notch, induces strong transactivation of TGF-ßRII by binding the TGF-βRII promoter through its DNA-binding domain. Thus, the crosstalk between Notch and the TGF-β pathway leads to potentiation of the suppressive effect of Treg cells

CMV induces expansion of highly polyfunctional CD4+ T cell subset coexpressing CD57 and CD154.

CD4+ T cells are essential for human CMV infection control. CMV-specific CD4+ T cells possess antiviral functions and participate in anti-CMV humoral/cellular responses. In the elderly, CMV infection impairs immunity to other viruses and has been traditionally associated with T cell senescence; however, recent results suggest that, in younger people, CMV confers immune protection against other pathogens (heterologous immunity). To shed light on this controversy, we analyzed latent CMV infection effects on the quality of young individuals' immune response, specifically, the presence of polyfunctional T cells through an extensive phenotypic and functional characterization of the CD4+ T cell subset. CD154 expression, degranulation (CD107a), and cytokine production (IFN-γ, TNF-α, and IL-2) as well as T cell phenotype markers (CD57, CD28, and CD27) were analyzed. We demonstrate that CD4+ T cells that coexpress CD57 and CD154, which are exclusively present in CMV-positive individuals, are the most polyfunctional CD4+ subset, whereas CD4+CD27+CD28- T cells associate with lower polyfunctionality. Conversely, the frequency of CD4+CD28+ T cells correlates with higher polyfunctionality of CD4+CD57- T cells from CMV-seronegative individuals and CD4+CD57+CD154+ T cells from CMV-seropositive individuals. Thus, polyfunctionality is a property of central memory CD4+ T cells in CMV-seronegative individuals, whereas after CMV infection, polyfunctional T cells become highly differentiated, which allows efficient eradication of infections. We extend previous observations of the impact of CMV on CD8+ T cell functionality to the CD4+ T cell compartment, revealing CD57 as a polyfunctionality marker of T cells which expands after CMV infection. CD57+ T cells have been associated with inflammatory conditions, but their potential role in the response against infectious disease and vaccination should now be investigated

CD127 Expression, Exhaustion Status and Antigen Specific Proliferation Predict Sustained Virologic Response to IFN in HCV/HIV Co-Infected Individuals

<div><p>Hepatitis C virus (HCV) infection is a major cause of morbidity and mortality in the HIV co-infected population. Interferon-alpha (IFN-α) remains a major component of anti-HCV therapy despite its deleterious effects on the immune system. Furthermore, IFN-α was recently shown to diminish the size of the latent HIV reservoir. The objectives of this study were to monitor the impact of IFN-α on T cell phenotype and proliferation of HIV and HCV-specific T cells during IFN therapy, and to identify immune markers that can predict the response to IFN in HICV/HIV co-infected patients. We performed longitudinal analyses of T cell numbers, phenotype and function in co-infected patients undergoing IFN-α therapy with different outcomes including IFN-α non-responders (NR) (n = 9) and patients who achieved sustained virologic response (SVR) (n = 19). We examined the expression of activation (CD38, HLA-DR), functional (CD127) and exhaustion markers (PD1, Tim-3, CD160 and CD244) on total CD4 and CD8 T cells before, during and after therapy. In addition, we examined the HIV- and HCV-specific proliferative responses against HIV-p24 and HCV-NS3 proteins. Frequencies of CD127⁺ CD4 T cells were higher in SVR than in NR patients at baseline. An increase in CD127 expression on CD8 T cells was observed after IFN-α therapy in all patients. In addition, CD8 T cells from NR patients expressed a higher exhaustion status at baseline. Finally, SVR patients exhibited higher proliferative response against both HIV and HCV antigens at baseline. Altogether, SVR correlated with higher expression of CD127, lower T cell exhaustion status and better HIV and HCV proliferative responses at baseline. Such factors might be used as non-invasive methods to predict the success of IFN–based therapies in co-infected individuals.</p></div

Non response to IFN-α therapy correlates with increased CD8 T cell exhaustion at baseline.

<p>Expression of the indicated activation/exhaustion molecules on CD8 T cells was monitored longitudinally in NR (n = 8) and SVR (n = 10) HCV/HIV co-infected patients before, during and after HCV therapy. (A) Representative flow cytometry plots for one patient from each group NR (top panel) and SVR (bottom panel) demonstrating expression of the different markers on total CD8 T cells (gated on viable CD8⁺CD3⁺ lymphocytes). (B-E) CD8 activation/exhaustion was measured by the absence of CD127 and the expression of the activation/exhaustion markers HLA-DR, PD-1, CD160, CD244 and Tim-3. P-values were calculated using a two-tailed Mann Whitney U test. Open symbols represent patients of group A and closed symbols represent patients of group B. (* p<0.05, ** p<0.01, *** p<0.001).</p