PD-1highCXCR5-CD4+ peripheral helper T cells promote CXCR3+ plasmablasts in human acute viral infection.

T cell-B cell interaction is the key immune response to protect the host from severe viral infection. However, how T cells support B cells to exert protective humoral immunity in humans is not well understood. Here, we use COVID-19 as a model of acute viral infections and analyze CD4+ T cell subsets associated with plasmablast expansion and clinical outcome. Peripheral helper T cells (Tph cells; denoted as PD-1highCXCR5-CD4+ T cells) are significantly increased, as are plasmablasts. Tph cells exhibit "B cell help" signatures and induce plasmablast differentiation in vitro. Interestingly, expanded plasmablasts show increased CXCR3 expression, which is positively correlated with higher frequency of activated Tph cells and better clinical outcome. Mechanistically, Tph cells help B cell differentiation and produce more interferon γ (IFNγ), which induces CXCR3 expression on plasmablasts. These results elucidate a role for Tph cells in regulating protective B cell response during acute viral infection.

A sex-biased imbalance between Tfr, Tph, and atypical B cells determines antibody responses in COVID-19 patients.

Sex-biased humoral immune responses to COVID-19 patients have been observed, but the cellular basis for this is not understood. Using single-cell proteomics by mass cytometry, we find disrupted regulation of humoral immunity in COVID-19 patients, with a sex-biased loss of circulating follicular regulatory T cells (cTfr) at a significantly greater rate in male patients. In addition, a male sex-associated cellular network of T-peripheral helper, plasma blasts, proliferating and extrafollicular/atypical CD11c+ memory B cells was strongly positively correlated with neutralizing antibody concentrations and negatively correlated with cTfr frequency. These results suggest that sex-specific differences to the balance of cTfr and a network of extrafollicular antibody production-associated cell types may be a key factor in the altered humoral immune responses between male and female COVID-19 patients.

The HDAC inhibitor zabadinostat is a systemic regulator of adaptive immunity.

Protein acetylation plays a key role in regulating cellular processes and is subject to aberrant control in diverse pathologies. Although histone deacetylase (HDAC) inhibitors are approved drugs for certain cancers, it is not known whether they can be deployed in other therapeutic contexts. We have explored the clinical HDAC inhibitor, zabadinostat/CXD101, and found that it is a stand-alone regulator of the adaptive immune response. Zabadinostat treatment increased expression of MHC class I and II genes in a variety of cells, including dendritic cells (DCs) and healthy tissue. Remarkably, zabadinostat enhanced the activity of DCs, and CD4 and CD8 T lymphocytes. Using an antigenic peptide presented to the immune system by MHC class I, zabadinostat caused an increase in antigen-specific CD8 T lymphocytes. Further, mice immunised with covid19 spike protein and treated with zabadinostat exhibit enhanced covid19 neutralising antibodies and an increased level of T lymphocytes. The enhanced humoral response reflected increased activity of T follicular helper (Tfh) cells and germinal centre (GC) B cells. Our results argue strongly that zabadinostat has potential to augment diverse therapeutic agents that act through the immune system.

The Differentiation and Maintenance of SARS-CoV-2-Specific Follicular Helper T Cells.

Upon acute viral infection, virus-specific CD4+ T cells differentiate into either TH1 cells or follicular helper T (TFH) cells. The molecular pathways governing such bimodal cell fate commitment remain elusive. Additionally, effector virus-specific TFH cells further differentiate into corresponding memory population, which confer long-term protection against re-infection of same viruses by providing immediate help to virus-specific memory B cells. Currently, the molecular mechanisms underlying the long-term maintenance of memory TFH cells are largely unknown. In this review, we discuss current understanding of early differentiation of virus-specific effector TFH cells and long-term maintenance of virus-specific memory TFH cells in mouse models of viral infection and patients of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Transient and durable T cell reactivity after COVID-19.

This study analyzed whole blood samples (n = 56) retrieved from 30 patients at 1 to 21 (median 9) mo after verified COVID-19 to determine the polarity and duration of antigen-specific T cell reactivity against severe acute respiratory syndrome coronavirus 2-derived antigens. Multimeric peptides spanning the entire nucleocapsid protein triggered strikingly synchronous formation of interleukin (IL)-4, IL-12, IL-13, and IL-17 ex vivo until ∼70 d after confirmed infection, whereafter this reactivity was no longer inducible. In contrast, levels of nucleocapsid-induced IL-2 and interferon-γ remained stable and highly correlated at 3 to 21 mo after infection. Similar cytokine dynamics were observed in unvaccinated, convalescent patients using whole-blood samples stimulated with peptides spanning the N-terminal portion of the spike 1 protein. These results unravel two phases of T cell reactivity following natural COVID-19: an early, synchronous response indicating transient presence of multipolar, antigen-specific T helper (TH) cells followed by an equally synchronous and durable TH1-like reactivity reflecting long-lasting T cell memory.

B cells promote CD8 T cell primary and memory responses to subunit vaccines.

The relationship between B cells and CD4 T cells has been carefully studied, revealing a collaborative effort in which B cells promote the activation, differentiation, and expansion of CD4 T cells while the so-called "helper" cells provide signals to B cells, influencing their class switching and fate. Interactions between B cells and CD8 T cells are not as well studied, although CD8 T cells exhibit an accelerated contraction after certain infections in B-cell-deficient mice. Here, we find that B cells significantly enhance primary CD8 T cell responses after vaccination. Moreover, memory CD8 numbers and function are impaired in B-cell-deficient animals, leading to increased susceptibility to bacterial challenge. We also show that interleukin-27 production by B cells contributes to their impact on primary, but not memory, CD8 responses. Better understanding of the interactions between CD8 T cells and B cells may aid in the design of more effective future vaccine strategies.

T helper profile in pregnant women recovered from COVID-19.

T helper (Th) cell subsets play distinct and important roles during pregnancy. This work was focused on investigating the Th and cytokine profile in pregnant women recovered from COVID-19. To this aim, the frequency of Th1, Th2, Th17 subsets and the level of associated cytokines were analysed in pregnant women recovered from COVID-19 and in matched non-pregnant women. Principal component analysis highlighted a significant impact of pregnancy on Th profile with an increase of ex-Th17 subset and a parallel decrease of Th1 population. These modulations may participate in both preserving the pregnancy and reducing the risk of severe infection.

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.

Emerging Paradigms in Type 2 Immunity.

A principal purpose of type 2 immunity was thought to be defense against large parasites, but it also functions in the restoration of homeostasis, such as toxin clearance following snake bites. In other cases, like allergy, the type 2 T helper (Th2) cytokines and cells present in the environment are detrimental and cause diseases. In recent years, the recognition of cell heterogeneity within Th2-associated cell populations has revealed specific functions of cells with a particular phenotype or gene signature. In addition, here we discuss the recent data regarding heterogeneity of type 2 immunity-related cells, as well as their newly identified role in a variety of processes ranging from involvement in respiratory viral infections [especially in the context of the recent COVID-19 (coronavirus disease 2019) pandemic] to control of cancer development or of metabolic homeostasis.

Establishment and recall of SARS-CoV-2 spike epitope-specific CD4+ T cell memory.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination elicit CD4+ T cell responses to the spike protein, including circulating follicular helper T (cTFH) cells that correlate with neutralizing antibodies. Using a novel HLA-DRB1*15:01/S751 tetramer to track spike-specific CD4+ T cells, we show that primary infection or vaccination induces robust S751-specific CXCR5- and cTFH cell memory responses. Secondary exposure induced recall of CD4+ T cells with a transitory CXCR3+ phenotype, and drove expansion of cTFH cells transiently expressing ICOS, CD38 and PD-1. In both contexts, cells exhibited a restricted T cell antigen receptor repertoire, including a highly public clonotype and considerable clonotypic overlap between CXCR5- and cTFH populations. Following a third vaccine dose, the rapid re-expansion of spike-specific CD4+ T cells contrasted with the comparatively delayed increase in antibody titers. Overall, we demonstrate that stable pools of cTFH and memory CD4+ T cells established by infection and/or vaccination are efficiently recalled upon antigen reexposure and may contribute to long-term protection against SARS-CoV-2.

High-affinity, neutralizing antibodies to SARS-CoV-2 can be made without T follicular helper cells.

T follicular helper (TFH) cells are the conventional drivers of protective, germinal center (GC)­based antiviral antibody responses. However, loss of TFH cells and GCs has been observed in patients with severe COVID-19. As T cell­B cell interactions and immunoglobulin class switching still occur in these patients, noncanonical pathways of antibody production may be operative during SARS-CoV-2 infection. We found that both TFH-dependent and -independent antibodies were induced against SARS-CoV-2 infection, SARS-CoV-2 vaccination, and influenza A virus infection. Although TFH-independent antibodies to SARS-CoV-2 had evidence of reduced somatic hypermutation, they were still high affinity, durable, and reactive against diverse spike-derived epitopes and were capable of neutralizing both homologous SARS-CoV-2 and the B.1.351 (beta) variant of concern. We found by epitope mapping and B cell receptor sequencing that TFH cells focused the B cell response, and therefore, in the absence of TFH cells, a more diverse clonal repertoire was maintained. These data support an alternative pathway for the induction of B cell responses during viral infection that enables effective, neutralizing antibody production to complement traditional GC-derived antibodies that might compensate for GCs damaged by viral inflammation.

Follicular T cells optimize the germinal center response to SARS-CoV-2 protein vaccination in mice.

Follicular helper T (Tfh) cells promote, whereas follicular regulatory T (Tfr) cells restrain, germinal center (GC) reactions. However, the precise roles of these cells in the complex GC reaction remain poorly understood. Here, we perturb Tfh or Tfr cells after SARS-CoV-2 spike protein vaccination in mice. We find that Tfh cells promote the frequency and somatic hypermutation (SHM) of Spike-specific GC B cells and regulate clonal diversity. Tfr cells similarly control SHM and clonal diversity in the GC but do so by limiting clonal competition. In addition, deletion of Tfh or Tfr cells during primary vaccination results in changes in SHM after vaccine boosting. Aged mice, which have altered Tfh and Tfr cells, have lower GC responses, presenting a bimodal distribution of SHM. Together, these data demonstrate that GC responses to SARS-CoV-2 spike protein vaccines require a fine balance of positive and negative follicular T cell help to optimize humoral immunity.

Multicolor Flow Cytometry and High-Dimensional Data Analysis to Probe Complex Questions in Vaccinology.

Vaccines induce a highly complex immune reaction in secondary lymphoid organs to generate immunological memory against an antigen or antigens of interest. Measurement of post immunization immune responses generated by specialized lymphocyte subsets requires time-dependent sampling, usually of the blood. Several T and B cell subsets are involved in the reaction, including CD4 and CD8 T cells, T follicular helper cells (Tfh), and germinal center B cells alongside their circulating (c) counterparts; cTfh and antibody secreting cells. Multicolor flow cytometry of peripheral blood mononuclear cells (PBMC) coupled with high-dimensional analysis offers an opportunity to study these cells in detail. Here we demonstrate a method by which such data can be generated and analysed using software that renders multidimensional data on a two dimensional map to identify rare vaccine-induced T and B cell subsets.

T-Helper Cell Subset Response Is a Determining Factor in COVID-19 Progression.

The immune response type organized against viral infection is determinant in the prognosis of some infections. This work has aimed to study Th polarization in acute COVID-19 and its possible association with the outcome through an observational prospective study. Fifty-eight COVID-19 patients were recruited in the Medicine Department of the hospital "12 de Octubre," 55 patients remaining after losses to follow-up. Four groups were established according to maximum degree of disease progression. T-helper cell percentages and phenotypes, analyzed by flow cytometer, and serum cytokines levels, analyzed by Luminex, were evaluated when the microbiological diagnosis (acute phase) of the disease was obtained. Our study found a significant reduction of %Th1 and %Th17 cells with higher activated %Th2 cells in the COVID-19 patients compared with reference population. A higher percent of senescent Th2 cells was found in the patients who died than in those who survived. Senescent Th2 cell percentage was an independent risk factor for death (OR: 13.88) accompanied by the numbers of total lymphocytes (OR: 0.15) with an AUC of 0.879. COVID-19 patients showed a profile of pro-inflammatory serum cytokines compared to controls, with higher levels of IL-2, IL-6, IL-15, and IP-10. IL-10 and IL-13 were also elevated in patients compared to controls. Patients who did not survive presented significantly higher levels of IL-15 than those who recovered. No significant differences were observed according to disease progression groups. The study has shown that increased levels of IL-15 and a high Th2 response are associated with a fatal outcome of the disease.

A COVID-19 peptide vaccine for the induction of SARS-CoV-2 T cell immunity.

T cell immunity is central for the control of viral infections. CoVac-1 is a peptide-based vaccine candidate, composed of SARS-CoV-2 T cell epitopes derived from various viral proteins1,2, combined with the Toll-like receptor 1/2 agonist XS15 emulsified in Montanide ISA51 VG, aiming to induce profound SARS-CoV-2 T cell immunity to combat COVID-19. Here we conducted a phase I open-label trial, recruiting 36 participants aged 18-80 years, who received a single subcutaneous CoVac-1 vaccination. The primary end point was safety analysed until day 56. Immunogenicity in terms of CoVac-1-induced T cell response was analysed as the main secondary end point until day 28 and in the follow-up until month 3. No serious adverse events and no grade 4 adverse events were observed. Expected local granuloma formation was observed in all study participants, whereas systemic reactogenicity was absent or mild. SARS-CoV-2-specific T cell responses targeting multiple vaccine peptides were induced in all study participants, mediated by multifunctional T helper 1 CD4+ and CD8+ T cells. CoVac-1-induced IFNγ T cell responses persisted in the follow-up analyses and surpassed those detected after SARS-CoV-2 infection as well as after vaccination with approved vaccines. Furthermore, vaccine-induced T cell responses were unaffected by current SARS-CoV-2 variants of concern. Together, CoVac-1 showed a favourable safety profile and induced broad, potent and variant of concern-independent T cell responses, supporting the presently ongoing evaluation in a phase II trial for patients with B cell or antibody deficiency.

COVID-19 genetic risk variants are associated with expression of multiple genes in diverse immune cell types.

Common genetic polymorphisms associated with COVID-19 illness can be utilized for discovering molecular pathways and cell types driving disease pathogenesis. Given the importance of immune cells in the pathogenesis of COVID-19 illness, here we assessed the effects of COVID-19-risk variants on gene expression in a wide range of immune cell types. Transcriptome-wide association study and colocalization analysis revealed putative causal genes and the specific immune cell types where gene expression is most influenced by COVID-19-risk variants. Notable examples include OAS1 in non-classical monocytes, DTX1 in B cells, IL10RB in NK cells, CXCR6 in follicular helper T cells, CCR9 in regulatory T cells and ARL17A in TH2 cells. By analysis of transposase accessible chromatin and H3K27ac-based chromatin-interaction maps of immune cell types, we prioritized potentially functional COVID-19-risk variants. Our study highlights the potential of COVID-19 genetic risk variants to impact the function of diverse immune cell types and influence severe disease manifestations.

Sterilizing Immunity against COVID-19: Developing Helper T cells I and II activating vaccines is imperative.

Six months after the publication of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) sequence, a record number of vaccine candidates were listed, and quite a number of them have since been approved for emergency use against the novel coronavirus disease 2019 (COVID-19). This unprecedented pharmaceutical feat did not only show commitment, creativity and collaboration of the scientific community, but also provided a swift solution that prevented global healthcare system breakdown. Notwithstanding, the available data show that most of the approved COVID-19 vaccines protect only a proportion of recipients against severe disease but do not prevent clinical manifestation of COVID-19. There is therefore the need to probe further to establish whether these vaccines can induce sterilizing immunity, otherwise, COVID-19 vaccination would have to become a regular phenomenon. The emergence of SARS-CoV-2 variants could further affect the capability of the available COVID-19 vaccines to prevent infection and protect recipients from a severe form of the disease. These notwithstanding, data about which vaccine(s), if any, can confer sterilizing immunity are unavailable. Here, we discuss the immune responses to viral infection with emphasis on COVID-19, and the specific adaptive immune response to SARS-CoV-2 and how it can be harnessed to develop COVID-19 vaccines capable of conferring sterilizing immunity. We further propose factors that could be considered in the development of COVID-19 vaccines capable of stimulating sterilizing immunity. Also, an old, but effective vaccine development technology that can be applied in the development of COVID-19 vaccines with sterilizing immunity potential is reviewed.

Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.

CCR2 Regulates Vaccine-Induced Mucosal T-Cell Memory to Influenza A Virus.

Elicitation of lung tissue-resident memory CD8 T cells (TRMs) is a goal of T cell-based vaccines against respiratory viral pathogens, such as influenza A virus (IAV). C-C chemokine receptor type 2 (CCR2)-dependent monocyte trafficking plays an essential role in the establishment of CD8 TRMs in lungs of IAV-infected mice. Here, we used a combination adjuvant-based subunit vaccine strategy that evokes multifaceted (TC1/TC17/TH1/TH17) IAV nucleoprotein-specific lung TRMs to determine whether CCR2 and monocyte infiltration are essential for vaccine-induced TRM development and protective immunity to IAV in lungs. Following intranasal vaccination, neutrophils, monocytes, conventional dendritic cells (DCs), and monocyte-derived dendritic cells internalized and processed vaccine antigen in lungs. We found that basic leucine zipper ATF-like transcription factor 3 (BATF3)-dependent DCs were essential for eliciting T cell responses, but CCR2 deficiency enhanced the differentiation of CD127hi, KLRG-1lo, OX40+ve CD62L+ve, and mucosally imprinted CD69+ve CD103+ve effector and memory CD8 T cells in lungs and airways of vaccinated mice. Mechanistically, increased development of lung TRMs induced by CCR2 deficiency was linked to dampened expression of T-bet but not altered TCF-1 levels or T cell receptor signaling in CD8 T cells. T1/T17 functional programming, parenchymal localization of CD8/CD4 effector and memory T cells, recall T cell responses, and protective immunity to a lethal IAV infection were unaffected in CCR2-deficient mice. Taken together, we identified a negative regulatory role for CCR2 and monocyte trafficking in mucosal imprinting and differentiation of vaccine-induced TRMs. Mechanistic insights from this study may aid the development of T-cell-based vaccines against respiratory viral pathogens, including IAV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IMPORTANCE While antibody-based immunity to influenza A virus (IAV) is type and subtype specific, lung- and airway-resident memory T cells that recognize conserved epitopes in the internal viral proteins are known to provide heterosubtypic immunity. Hence, broadly protective IAV vaccines need to elicit robust T cell memory in the respiratory tract. We have developed a combination adjuvant-based IAV nucleoprotein vaccine that elicits strong CD4 and CD8 T cell memory in lungs and protects against H1N1 and H5N1 strains of IAV. In this study, we examined the mechanisms that control vaccine-induced protective memory T cells in the respiratory tract. We found that trafficking of monocytes into lungs might limit the development of antiviral lung-resident memory T cells following intranasal vaccination. These findings suggest that strategies that limit monocyte infiltration can potentiate vaccine-induced frontline T-cell immunity to respiratory viruses, such as IAV and SARS-CoV-2.

SARS-CoV-2 Spike Impairs DNA Damage Repair and Inhibits V(D)J Recombination In Vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the coronavirus disease 2019 (COVID-19) pandemic, severely affecting public health and the global economy. Adaptive immunity plays a crucial role in fighting against SARS-CoV-2 infection and directly influences the clinical outcomes of patients. Clinical studies have indicated that patients with severe COVID-19 exhibit delayed and weak adaptive immune responses; however, the mechanism by which SARS-CoV-2 impedes adaptive immunity remains unclear. Here, by using an in vitro cell line, we report that the SARS-CoV-2 spike protein significantly inhibits DNA damage repair, which is required for effective V(D)J recombination in adaptive immunity. Mechanistically, we found that the spike protein localizes in the nucleus and inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site. Our findings reveal a potential molecular mechanism by which the spike protein might impede adaptive immunity and underscore the potential side effects of full-length spike-based vaccines.