Frequency of IRF5+ dendritic cells is associated with the TLR7-induced inflammatory cytokine response in SARS-CoV-2 infection.

The SARS-CoV-2 infection leads to enhanced inflammation driven by innate immune responses. Upon TLR7 stimulation, dendritic cells (DC) mediate the production of inflammatory cytokines, and in particular of type I interferons (IFN). Especially in DCs, IRF5 is a key transcription factor that regulates pathogen-induced immune responses via activation of the MyD88-dependent TLR signaling pathway. In the current study, the frequencies of IRF5+ DCs and the association with innate cytokine responses in SARS-CoV-2 infected individuals with different disease courses were investigated. In addition to a decreased number of mDC and pDC subsets, we could show reduced relative IRF5+ frequencies in mDCs of SARS-CoV-2 infected individuals compared with healthy donors. Functionally, mDCs of COVID-19 patients produced lower levels of IL-6 in response to in vitro TLR7 stimulation. IRF5+ mDCs more frequently produced IL-6 and TNF-α compared to their IRF5- counterparts upon TLR7 ligation. The correlation of IRF5+ mDCs with the frequencies of IL-6 and TNF-α producing mDCs were indicators for a role of IRF5 in the regulation of cytokine responses in mDCs. In conclusion, our data provide further insights into the underlying mechanisms of TLR7-dependent immune dysfunction and identify IRF5 as a potential immunomodulatory target in SARS-CoV-2 infection.

High-resolution analysis of individual spike peptide-specific CD4+ T-cell responses in vaccine recipients and COVID-19 patients.

Objectives: Potential differences in the breadth, distribution and magnitude of CD4+ T-cell responses directed against the SARS-CoV-2 spike glycoprotein between vaccinees, COVID-19 patients and subjects who experienced both ways of immunisation have not been comprehensively compared on a peptide level. Methods: Following virus-specific in vitro cultivation, we determined the T-cell responses directed against 253 individual overlapping 15-mer peptides covering the entire SARS-CoV-2 spike glycoprotein using IFN-γ ELISpot and intracellular cytokine staining. In vitro HLA binding was determined for selected peptides. Results: We mapped 955 single peptide-specific CD4+ T-cell responses in a cohort of COVID-19 patients (n = 8), uninfected vaccinees (n = 16) and individuals who experienced both infection and vaccination (n = 11). Patients and vaccinees (two-time and three-time vaccinees alike) had a comparable number of CD4+ T-cell responses (median 26 vs. 29, P = 0.7289). Most of these specificities were conserved in B.1.1.529 and the BA.4 and BA.5 sublineages. The highest magnitude of these in vitro IFN-γ CD4+ T-cell responses was observed in COVID-19 patients (median 0.35%), and three-time vaccinees showed a higher magnitude than two-time vaccinees (median 0.091% vs. 0.175%, P < 0.0001). Twelve peptide specificities were each detected in at least 40% of subjects. In vitro HLA binding showed promiscuous presentation by DRB1 molecules for several peptides. Conclusion: Both SARS-CoV-2 infection and vaccination prime broadly directed T-cell responses directed against the SARS-CoV-2 spike glycoprotein. This comprehensive high-resolution analysis of spike peptide specificities will be a useful resource for further investigation of spike-specific T-cell responses.

High and Sustained Ex Vivo Frequency but Altered Phenotype of SARS-CoV-2-Specific CD4+ T-Cells in an Anti-CD20-Treated Patient with Prolonged COVID-19.

Here, we longitudinally assessed the ex vivo frequency and phenotype of SARS-CoV-2 membrane protein (aa145-164) epitope-specific CD4+ T-cells of an anti-CD20-treated patient with prolonged viral positivity in direct comparison to an immunocompetent patient through an MHC class II DRB1*11:01 Tetramer analysis. We detected a high and stable SARS-CoV-2 membrane-specific CD4+ T-cell response in both patients, with higher frequencies of virus-specific CD4+ T-cells in the B-cell-depleted patient. However, we found an altered virus-specific CD4+ T-cell memory phenotype in the B-cell-depleted patient that was skewed towards late differentiated memory T-cells, as well as reduced frequencies of SARS-CoV-2-specific CD4+ T-cells with CD45RA- CXCR5+ PD-1+ circulating T follicular helper cell (cTFH) phenotype. Furthermore, we observed a delayed contraction of CD127- virus-specific effector cells. The expression of the co-inhibitory receptors TIGIT and LAG-3 fluctuated on the virus-specific CD4+ T-cells of the patient, but were associated with the inflammation markers IL-6 and CRP. Our findings indicate that, despite B-cell depletion and a lack of B-cell-T-cell interaction, a robust virus-specific CD4+ T-cell response can be primed that helps to control the viral replication, but which is not sufficient to fully abrogate the infection.

Analysis of Co-inhibitory Receptor Expression in COVID-19 Infection Compared to Acute Plasmodium falciparum Malaria: LAG-3 and TIM-3 Correlate With T Cell Activation and Course of Disease.

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.