Detection of IFNγ-Secreting CD4+ and CD8+ Memory T Cells in COVID-19 Convalescents after Stimulation of Peripheral Blood Mononuclear Cells with Live SARS-CoV-2

Background: New coronavirus SARS-CoV-2, a causative agent of the COVID-19 pandemic, has been circulating among humans since November 2019. Multiple studies have assessed the qualitative and quantitative characteristics of virus-specific immunity in COVID-19 convalescents, however, some aspects of the development of memory T-cell responses after natural SARS-CoV-2 infection remain uncovered. Methods: In most of published studies T-cell immunity to the new coronavirus is assessed using peptides corresponding to SARS-CoV-1 or SARS-CoV-2 T-cell epitopes, or with peptide pools covering various parts of the viral proteins. Here, we determined the level of CD4⁺ and CD8⁺ memory T-cell responses in COVID-19 convalescents by stimulating PBMCs collected 1 to 6 months after recovery with sucrose gradient-purified live SARS-CoV-2. IFNγ production by the central and effector memory helper and cytotoxic T cells was assessed by intracellular cytokine staining assay and flow cytometry. Results: Stimulation of PBMCs with live SARS-CoV-2 revealed IFNγ-producing T-helper effector memory cells with CD4⁺CD45RA⁻CCR7⁻ phenotype, which persisted in circulation for up to 6 month after COVID-19. In contrast, SARS-CoV-2-specific IFNγ-secreting cytotoxic effector memory T cells were found at significant levels only shortly after the disease, but rapidly decreased over time. Conclusion: The stimulation of immune cells with live SARS-CoV-2 revealed a rapid decline in the pool of effector memory CD8⁺, but not CD4⁺, T cells after recovery from COVID-19. These data provide additional information on the development and persistence of cellular immune responses after natural infection, and can inform further development of T cell-based SARS-CoV-2 vaccines.     

Fine analysis of spontaneous MAGE-C1/CT7–specific immunity in melanoma patients

Cancer/testis (CT) antigens represent prime candidates for immunotherapy in cancer patients, because their expression is restricted to cancer cells and germ cells of the testis. MAGE-C1/CT7 is a CT antigen that is highly expressed in several types of cancers. Spontaneous occurrence of CT7-specific antibodies was previously detected by SEREX screen in a melanoma patient. However, naturally occurring CT7-specific T-cell responses have thus far not been detected. Peripheral blood mononuclear cells (PBMCs) from 26 metastatic melanoma patients expressing CT7 in their tumor lesions (CT7⁺) were analyzed for CT7-specific T-cell responses using overlapping peptides. CT7-specific CD4⁺ T-cell responses were detected in three patients (11.5%). These CT7-specific CD4⁺ T-cell responses were detectable in melanoma patients’ PBMCs exclusively from preexisting CD45RA⁻ memory CD4⁺ T-cell pool. Additional CT7-specific memory CD4⁺ T-cell responses were detected in CT7⁺ melanoma patients after depletion of CD4⁺CD25high Treg cells showing that Treg cells impact on CT7-specific CD4⁺ T cells in melanoma patients. CT7-specific CD4⁺ T-cell clones were generated and used to define minimal epitopes, restriction elements, and confirm the recognition of naturally processed antigen. Surprisingly, these clones were able to secrete perforin and exert cytotoxicity. This study shows that CT7 can induce specific cellular immunity in melanoma patients. Based on these findings, CT7 will be further explored as a potential vaccine for melanoma immunotherapy.     

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 (cT_FH) 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.     

Cross-reactive CD8+ T-cell immunity between the pandemic H1N1-2009 and H1N1-1918 influenza A viruses

Preexisting T-cell immunity directed at conserved viral regions promotes enhanced recovery from influenza virus infections, with there being some evidence of cross-protection directed at variable peptides. Strikingly, many of the immunogenic peptides derived from the current pandemic A(H1N1)-2009 influenza virus are representative of the catastrophic 1918 “Spanish flu” rather than more recent “seasonal” strains. We present immunological and structural analyses of cross-reactive CD8⁺ T-cell–mediated immunity directed at a variable (although highly cross-reactive) immunodominant NP_418–426 peptide that binds to a large B7 family (HLA-B*3501/03/0702) found throughout human populations. Memory CD8⁺ T-cell specificity was probed for 12 different NP₄₁₈ mutants that emerged over the 9 decades between the 1918 and 2009 pandemics. Although there is evidence of substantial cross-reactivity among seasonal NP₄₁₈ mutants, current memory T-cell profiles show no preexisting immunity to the 2009-NP₄₁₈ variant or the 1918-NP₄₁₈ variant. Natural infection with the A(H1N1)-2009 virus, however, elicits CD8⁺ T cells specific for the 2009-NP₄₁₈ and 1918-NP₄₁₈ epitopes. This analysis points to the potential importance of cross-reactive T-cell populations that cover the possible spectrum of T-cell variants and suggests that the identification of key residues/motifs that elicit cross-reactive T-cell sets could facilitate the evolution of immunization protocols that provide a measure of protection against unpredicted pandemic influenza viruses. Thus, it is worth exploring the potential of vaccines that incorporate peptide variants with a proven potential for broader immunogenicity, especially to those that are not recognized by the current memory T-cell pool generated by exposure to influenza variants that cause successive seasonal epidemics.     

T Cell Response following Anti-COVID-19 BNT162b2 Vaccination Is Maintained against the SARS-CoV-2 Omicron B.1.1.529 Variant of Concern

The progression of the COVID-19 pandemic has led to the emergence of variants of concern (VOC), which may compromise the efficacy of the currently administered vaccines. Antigenic drift can potentially bring about reduced protective T cell immunity and, consequently, more severe disease manifestations. To assess this possibility, the T cell responses to the wild-type Wuhan-1 SARS-CoV-2 ancestral spike protein and the Omicron B.1.1.529 spike protein were compared. Accordingly, peripheral blood mononuclear cells (PBMC) were collected from eight healthy volunteers 4–5 months following a third vaccination with BNT162b2, and stimulated with overlapping peptide libraries representing the spike of either the ancestral or the Omicron SARS-CoV-2 virus variants. Quantification of the specific T cells was carried out by a fluorescent ELISPOT assay, monitoring cells secreting interferon-gamma (IFNg), interleukin-10 (IL-10) and interleukin-4 (IL-4). For all the examined individuals, comparable levels of reactivity to both forms of spike protein were determined. In addition, a dominant Th1 response was observed, manifested mainly by IFNg-secreting cells and only limited numbers of IL-10- and IL-4-secreting cells. The data demonstrate stable T cell activity in response to the emerging Omicron variant in the tested individuals; therefore, the protective immunity to the variant following BNT162b2 vaccination is not significantly affected.     

Strong SARS-CoV-2 N-Specific CD8+ T Immunity Induced by Engineered Extracellular Vesicles Associates with Protection from Lethal Infection in Mice

SARS-CoV-2-specific CD8⁺ T cell immunity is expected to counteract viral variants in both efficient and durable ways. We recently described a way to induce a potent SARS-CoV-2 CD8⁺ T immune response through the generation of engineered extracellular vesicles (EVs) emerging from muscle cells. This method relies on intramuscular injection of DNA vectors expressing different SARS-CoV-2 antigens fused at their N-terminus with the Nef^mut protein, i.e., a very efficient EV-anchoring protein. However, quality, tissue distribution, and efficacy of these SARS-CoV-2-specific CD8⁺ T cells remained uninvestigated. To fill the gaps, antigen-specific CD8⁺ T lymphocytes induced by the immunization through the Nef^mut-based method were characterized in terms of their polyfunctionality and localization at lung airways, i.e., the primary targets of SARS-CoV-2 infection. We found that injection of vectors expressing Nef^mut/S1 and Nef^mut/N generated polyfunctional CD8⁺ T lymphocytes in both spleens and bronchoalveolar lavage fluids (BALFs). When immunized mice were infected with 4.4 lethal doses of 50% of SARS-CoV-2, all S1-immunized mice succumbed, whereas those developing the highest percentages of N-specific CD8⁺ T lymphocytes resisted the lethal challenge. We also provide evidence that the N-specific immunization coupled with the development of antigen-specific CD8⁺ T-resident memory cells in lungs, supporting the idea that the Nef^mut-based immunization can confer a long-lasting, lung-specific immune memory. In view of the limitations of current anti-SARS-CoV-2 vaccines in terms of antibody waning and efficiency against variants, our CD8⁺ T cell-based platform could be considered for a new combination prophylactic strategy.     

Enhanced SARS-CoV-2-Specific CD4+ T Cell Activation and Multifunctionality in Late Convalescent COVID-19 Individuals

Background: Examination of CD4⁺ T cell responses during the natural course of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection offers useful information for the improvement of vaccination strategies against this virus and the protective effect of these T cells. Methods: We characterized the SARS-CoV-2-specific CD4⁺ T cell activation marker, multifunctional cytokine and cytotoxic marker expression in recovered coronavirus disease 2019 (COVID-19) individuals. Results: CD4⁺ T-cell responses in late convalescent (>6 months of diagnosis) individuals are characterized by elevated frequencies of activated as well as mono, dual- and multi-functional Th1 and Th17 CD4⁺ T cells in comparison to early convalescent (<1 month of diagnosis) individuals following stimulation with SARS-CoV-2-specific antigens. Similarly, the frequencies of cytotoxic marker expressing CD4⁺ T cells were also enhanced in late convalescent compared to early convalescent individuals. Conclusion: Our findings from a low-to middle-income country suggest protective adaptive immune responses following natural infection of SARS-CoV-2 are elevated even at six months following initial symptoms, indicating the CD4⁺ T cell mediated immune protection lasts for six months or more in natural infection.     

β-Catenin in dendritic cells exerts opposite functions in cross-priming and maintenance of CD8+ T cells through regulation of IL-10

Recent studies have demonstrated that β-catenin in DCs serves as a key mediator in promoting both CD4⁺ and CD8⁺ T-cell tolerance, although how β-catenin exerts its functions remains incompletely understood. Here we report that activation of β-catenin in DCs inhibits cross-priming of CD8⁺ T cells by up-regulating mTOR-dependent IL-10, suggesting blocking β-catenin/mTOR/IL-10 signaling as a viable approach to augment CD8⁺ T-cell immunity. However, vaccination of DC–β-catenin^−/− (CD11c-specific deletion of β-catenin) mice surprisingly failed to protect them against tumor challenge. Further studies revealed that DC–β-catenin^−/− mice were deficient in generating CD8⁺ T-cell immunity despite normal clonal expansion, likely due to impaired IL-10 production by β-catenin^−/− DCs. Deletion of β-catenin in DCs or blocking IL-10 after clonal expansion similarly led to reduced CD8⁺ T cells, suggesting that β-catenin in DCs plays a positive role in CD8⁺ T-cell maintenance postclonal expansion through IL-10. Thus, our study has not only identified mTOR/IL-10 as a previously unidentified mechanism for β-catenin–dependent inhibition of cross-priming, but also uncovered an unexpected positive role that β-catenin plays in maintenance of CD8⁺ T cells. Despite β-catenin’s opposite functions in regulating CD8⁺ T-cell responses, selectively blocking β-catenin with a pharmacological inhibitor during priming phase augmented DC vaccine-induced CD8⁺ T-cell immunity and improved antitumor efficacy, suggesting manipulating β-catenin signaling as a feasible therapeutic strategy to improve DC vaccine efficacy.As the initiators of antigen-specific immune responses, dendritic cells (DCs) play a central role in regulating both T-cell immunity and tolerance (1). β-Catenin, a major component in Wnt signaling pathway, has emerged as a key factor in DC differentiation and function (2). Previous studies have shown that β-catenin regulates DC-mediated CD4⁺ T-cell responses and promotes CD4⁺ T-cell tolerance in murine models of autoimmune diseases (3, 4). Consistently, activation of β-catenin in DCs has recently been shown to suppress CD8⁺ T-cell immunity in a DC-targeted vaccine model (5), suggesting that β-catenin in DCs might similarly serve as a tolerizing signal that shifts the balance between CD8⁺ T-cell immunity and tolerance. Although the underlying mechanisms of how β-catenin mediates CD8⁺ T-cell tolerance remain largely unclear, we have shown that activation of β-catenin in DCs genetically or induced by tumors suppresses CD8⁺ T-cell immunity by inhibiting cross-priming (5). Exploiting their ability to potentiate host effector and memory CD8⁺ T-cell responses, DC vaccines have emerged as a leading strategy for cancer immunotherapy (6). However, one major obstacle for their success is host DC-mediated immunosuppression (7–9). Given that cross-priming plays a major role in generating antitumor CD8⁺ T-cell immunity (7, 10), activation of β-catenin in DCs might be a key mechanism for tumors to achieve immunosuppression. Thus, manipulating β-catenin function in cross-priming might be a viable approach to overcome DC-mediated immunosuppression and improve DC vaccine efficacy. However, The underlying mechanisms of how β-catenin in DCs achieves immunosuppression, in particular how β-catenin negatively regulates cross-priming to suppress CD8⁺ T-cell immunity, remain poorly understood.Although the mechanisms for DC-mediated priming of antitumor CD8⁺ T cells through cross-presentation remain incompletely understood, DC subsets, DC maturation status and cytokines have been shown to possibly affect their capacity in cross-priming (7, 10, 11). Although the role of cytokines in cross-priming has not been directly tested, cytokines as “signal 3” have been shown in principal to play a critical role in priming and effector differentiation of antitumor CD8⁺ T cells (12). β-Catenin in DCs has been shown to play a critical role in regulating cytokine induction (3, 4), thus suggesting that β-catenin might regulate DC cytokine production to achieve its effects on cross-priming.In this report we have identified mTOR/IL-10 signaling as a mechanism for β-catenin–dependent inhibition of cross-priming. Activation of β-catenin in DCs inhibited cross-priming of CD8⁺ T cells by up-regulating mTOR-dependent IL-10, and blocking mTOR or IL-10 led to restored cross-priming by β-catenin^active DCs. Surprisingly, mice with DC-specific deletion of β-catenin (DC–β-catenin^−/− mice) exhibited reduced antitumor immunity upon vaccination, despite the fact that deletion of β-catenin in DCs abrogated tumor-induced inhibition of cross-priming. Further studies showed that DC–β-catenin^−/− mice were deficient in generating CD8⁺ T-cell immunity despite normal clonal expansion, and β-catenin in DCs was required to maintain primed CD8⁺ T cells postclonal expansion. Thus, β-catenin in DCs exerts negative and positive functions in cross-priming and maintenance of CD8⁺ T cells, respectively. Importantly, we have demonstrated blocking β-catenin selectively at priming phase as a feasible strategy to improve DC vaccine efficacy.     

Steep Decline in Binding Capability of SARS-CoV-2 Omicron Variant (B.1.1.529) RBD to the Antibodies in Early COVID-19 Convalescent Sera and Inactivated Vaccine Sera

A new SARS-CoV-2 variant B.1.1.529 was named by the WHO as Omicron and classified as a Variant of Concern (VOC) on 26 November 2021. Because this variant has more than 50 mutations, including 30 mutations on the spike, it has generated a lot of concerns on the potential impacts of the VOC on COVID-19. Here through ELISA assays using the recombinant RBD proteins with sequences the same to that of SARS-CoV-2 WIV04 (lineage B.1), the Delta variant and the Omicron variant as the coating antigens, the binding capabilities between the RBDs and the antibodies in COVID-19 convalescent sera and vaccine sera after two doses of the inactivated vaccine produced by Sinopharm WIBP are compared with each other. The results showed that the Omicron variant may evade antibodies induced by the ancestral strain and by the inactivated vaccine, with significant reduction in the binding capability of its RBD much greater than that of the Delta variant.     

Human T-Cell Leukemia Virus Type 1 Envelope Protein: Post-Entry Roles in Viral Pathogenesis

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL), an aggressive and fatal CD4⁺ T-cell malignancy, and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic neurological disease. Disease progression in infected individuals is the result of HTLV-1-driven clonal expansion of CD4⁺ T-cells and is generally associated with the activities of the viral oncoproteins Tax and Hbz. A closely related virus, HTLV-2, exhibits similar genomic features and the capacity to transform T-cells, but is non-pathogenic. In vitro, HTLV-1 primarily immortalizes or transforms CD4⁺ T-cells, while HTLV-2 displays a transformation tropism for CD8⁺ T-cells. This distinct tropism is recapitulated in infected people. Through comparative studies, the genetic determinant for this divergent tropism of HTLV-1/2 has been mapped to the viral envelope (Env). In this review, we explore the emerging roles for Env beyond initial viral entry and examine current perspectives on its contributions to HTLV-1-mediated disease development.     

SARS-CoV-2 T Cell Responses Elicited by COVID-19 Vaccines or Infection Are Expected to Remain Robust against Omicron

Omicron, the most recent SARS-CoV-2 variant of concern (VOC), harbours multiple mutations in the spike protein that were not observed in previous VOCs. Initial studies suggest Omicron to substantially reduce the neutralizing capability of antibodies induced from vaccines and previous infection. However, its effect on T cell responses remains to be determined. Here, we assess the effect of Omicron mutations on known T cell epitopes and report data suggesting T cell responses to remain broadly robust against this new variant.     

Omicron mutations enhance infectivity and reduce antibody neutralization of SARS-CoV-2 virus-like particles

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant contains extensive sequence changes relative to the earlier-arising B.1, B.1.1, and Delta SARS-CoV-2 variants that have unknown effects on viral infectivity and response to existing vaccines. Using SARS-CoV-2 virus-like particles (VLPs), we examined mutations in all four structural proteins and found that Omicron and Delta showed 4.6-fold higher luciferase delivery overall relative to the ancestral B.1 lineage, a property conferred mostly by enhancements in the S and N proteins, while mutations in M and E were mostly detrimental to assembly. Thirty-eight antisera samples from individuals vaccinated with Pfizer/BioNTech, Moderna, or Johnson & Johnson vaccines and convalescent sera from unvaccinated COVID-19 survivors had 15-fold lower efficacy to prevent cell transduction by VLPs containing the Omicron mutations relative to the ancestral B.1 spike protein. A third dose of Pfizer vaccine elicited substantially higher neutralization titers against Omicron, resulting in detectable neutralizing antibodies in eight out of eight subjects compared to one out of eight preboosting. Furthermore, the monoclonal antibody therapeutics casirivimab and imdevimab had robust neutralization activity against B.1 and Delta VLPs but no detectable neutralization of Omicron VLPs, while newly authorized bebtelovimab maintained robust neutralization across variants. Our results suggest that Omicron has similar assembly efficiency and cell entry compared to Delta and that its rapid spread is due mostly to reduced neutralization in sera from previously vaccinated subjects. In addition, most currently available monoclonal antibodies will not be useful in treating Omicron-infected patients with the exception of bebtelovimab.

Understanding the molecular determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral fitness is central to effective vaccine and therapeutic development. The emergence of viral variants including Delta and Omicron underscores the need to assess both infectivity and antibody neutralization, but biosafety level 3 handling requirements slow the pace of research on intact SARS-CoV-2. Although vesicular stomatitis virus and lentivirus pseudotyped with the SARS-CoV-2 spike (S) protein enable evaluation of S-mediated cell binding and entry via the ACE2 and TMPRSS2 receptors, they cannot determine effects of mutations outside the S gene (1, 2). To address these challenges, we developed SARS-CoV-2 virus-like particles (SC2-VLPs) comprising the S, N, M, and E structural proteins and a packaging signal-containing messenger RNA (mRNA) that together form RNA-loaded capsids capable of spike-dependent cell transduction (3). This system faithfully reflects the impact of mutations in structural proteins that are observed in infections with virus isolates, enabling rapid testing of SARS-CoV-2 structural gene variants for their impact on both infection efficiency and antibody or antiserum neutralization.     

In-Silico Analysis of Monoclonal Antibodies against SARS-CoV-2 Omicron

Omicron was designated by the WHO as a VOC on 26 November 2021, only 4 days after its sequence was first submitted. However, the impact of Omicron on current antibodies and vaccines remains unknown and evaluations are still a few weeks away. We analysed the mutations in the Omicron variant against epitopes. In our database, 132 epitopes of the 120 antibodies are classified into five groups, namely NTD, RBD-1, RBD-2, RBD-3, and RBD-4. The Omicron mutations impact all epitopes in NTD, RBD-1, RBD-2, and RBD-3, with no antibody epitopes spared by these mutations. Only four out of 120 antibodies may confer full resistance to mutations in the Omicron spike, since all antibodies in these three groups contain one or more epitopes that are affected by these mutations. Of all antibodies under EUA, the neutralisation potential of Etesevimab, Bamlanivimab, Casirivimab, Imdevima, Cilgavimab, Tixagevimab, Sotrovimab, and Regdanvimab might be dampened to varying degrees. Our analysis suggests the impact of Omicron on current therapeutic antibodies by the Omicron spike mutations may also apply to current COVID-19 vaccines.     

Reproducible selection of high avidity CD8+ T-cell clones following secondary acute virus infection

The recall of memory CD8⁺ cytotoxic T lymphocytes (CTLs), elicited by prior virus infection or vaccination, is critical for immune protection. The extent to which this arises as a consequence of stochastic clonal expansion vs. active selection of particular clones remains unclear. Using a parallel adoptive transfer protocol in combination with single cell analysis to define the complementarity determining region (CDR) 3α and CDR3β regions of individual T-cell receptor (TCR) heterodimers, we characterized the antigen-driven recall of the same memory CTL population in three individual recipients. This high-resolution analysis showed reproducible enrichment (or diminution) of particular TCR clonotypes across all challenged animals. These changes in clonal composition were TCRα− and β chain–dependent and were directly related to the avidity of the TCR for the virus-derived peptide (p) + major histocompatibility complex class I molecule. Despite this shift in clonotype representation indicative of differential selection, there was no evidence of overall repertoire narrowing, suggesting a strategy to optimize CTL responses while safeguarding TCR diversity.Virus-specific CD8⁺ cytotoxic T lymphocytes (CTLs) are key for effective pathogen clearance. To exert their antiviral effects, naïve CD8⁺ CTL precursors (CTLps) must first be activated through the specific recognition of virus-derived peptides (p) in the context of major histocompatibility complex class I molecules (MHCI) expressed on the surface of dendritic cells. Ligation of these pMHCI epitopes is mediated via specific T-cell receptor (TCR) αβ heterodimers, leading to the recruitment, proliferation, and activation of antigen-specific CTLs. Subsequent to virus clearance, CTL populations contract to form a stable pool of resting memory cells, typically at around 5–10% of their acute phase numbers (1, 2). On secondary virus encounter, this memory pool of CD8⁺ T cells is able to expand rapidly, providing potent immune protection (2).An understanding of CD8⁺ T-cell recruitment/expansion into the recall response has significant implications for effective vaccine strategies. If recruitment and expansion occur stochastically, a memory population in which highly effective clones predominate is desirable. Alternatively, if selection is deterministic, the basic requirement would be the presence of high-quality clones in the memory population for selective expansion by antigen-driven mechanisms. Moreover, different mechanisms of memory recruitment have different long-term implications for the clonal diversity of epitope-specific populations, especially with repeated virus exposure. Repeated selection of particular clones inherent in deterministic clonal selection has the potential consequence of narrowing the CTL repertoire, whereas stochastic recruitment/expansion seems more likely to maintain CTL diversity, shown to be beneficial for virus control (3–7). Despite the implications, there remains conjecture about how epitope-specific CD8⁺ T cells are recruited from the memory pool. Studies have indicated a focusing of the recall response relative to the memory pool, arising from only a subpopulation of cells being recruited or expanded (8), whereas others have indicated that primary and secondary responses are either highly similar or randomly different, prompting the conclusion that selection from the memory pool likely occurs as a result of stochastic T-cell selection (9–13).Using secondary virus challenge of influenza A virus–primed B6 mice as a model of acute localized infection, we investigated the recruitment/expansion of memory CD8⁺ T-cell clones specific for the immunodominant D^bNP₃₆₆ epitope. We used a parallel adoptive transfer method, in which a population of memory CD8⁺ T cells from one individual is rechallenged with virus in three independent recipients, to definitively ascertain whether changes in clonal prevalence between memory and recall populations occur in a stochastic or deterministic fashion. Using high-resolution single cell TCRβ or αβ analysis, our data show evidence of active selection for particular clones following secondary infection, without a reduction in overall TCR diversity. Further, the selection for particular T-cell clones appears to be based on the avidity of the TCR–pMHCI interaction. Thus, it seems that the recall response may be optimized without diminishing the breadth of TCR use that may be critical for effective virus control.     

Autoreactive and immunoregulatory T-cell subsets in insulindependent diabetes mellitus

Aims/hypothesis. Type I (insulin-dependent) diabetes mellitus is a T-cell mediated autoimmune disease. Several subsets of T-cells, in particular CD4⁺ and in vivo activate CD45RA⁺RO⁺ T-cells, have been shown to be increased at disease onset. The functional implications of these relative increases in CD4 T-cells were investigated. Methods. Subsets of T-cells were sorted on the basis of their activation status (CD45RA⁺ naïve cells, CD45RA⁺RO⁺ recently activated cells and CD45RO⁺ memory cells) and stimulated with autoantigens or recall antigen in vitro. Results. Proliferative responses to tetanus toxoid were primarily or exclusively observed in resting memory T-cells (CD45RO⁺). Autoimmune T-cell responses were, however, primarily measured in activated T-cells (CD45RA⁺RO⁺) in newly diagnosed Type I diabetic patients, whereas those with longer disease duration reacted to autoantigens with memory T-cells (CD45RO⁺) (p < 0.004). Interestingly, in non-diabetic control subjects not responding to autoantigens in the regular assay, considerable autoreactive T-cell responses were detectable after sorting in the CD45RO⁺ or CD45RA⁺RO⁺ lymphocyte subsets. Remixing these subsets showed that these autoimmune responses in activated cells could be down-modulated by CD45RA⁺ lymphocytes, whereas resting memory cells appeared unaffected by the suppressive CD45RA subset. Conclusion/interpretation. These results show that autoimmune T-cell responses can be linked to particular subsets which differ depending on clinical status. Furthermore, the CD45RA T-cell subset harbours lymphocytes potentially capable of suppressing autoimmune T-cell responses. The changes in responsiveness to exogenous insulin may help to unravel the mechanism by which isohormonal therapy could prevent the onset of Type I diabetes. [Diabetologia (1999) 42: 443–449]     

Immune Escape Associated with RBD Omicron Mutations and SARS-CoV-2 Evolution Dynamics

The evolution and the emergence of new mutations of viruses affect their transmissibility and/or pathogenicity features, depending on different evolutionary scenarios of virus adaptation to the host. A typical trade-off scenario of SARS-CoV-2 evolution has been proposed, which leads to the appearance of an Omicron strain with lowered lethality, yet enhanced transmissibility. This direction of evolution might be partly explained by virus adaptation to therapeutic agents and enhanced escape from vaccine-induced and natural immunity formed by other SARS-CoV-2 strains. Omicron’s high mutation rate in the Spike protein, as well as its previously described high genome mutation rate (Kandeel et al., 2021), revealed a gap between it and other SARS-CoV-2 strains, indicating the absence of a transitional evolutionary form to the Omicron strain. Therefore, Omicron has emerged as a new serotype divergent from the evolutionary lineage of other SARS-CoV-2 strains. Omicron is a rapidly evolving variant of high concern, whose new subvariants continue to manifest. Its further understanding and the further monitoring of key mutations that provide virus immune escape and/or high affinity towards the receptor could be useful for vaccine and therapeutic development in order to control the evolutionary direction of the COVID-19 pandemic.     

Antidiabetogenic MHC class II promotes the differentiation of MHC-promiscuous autoreactive T cells into FOXP3+ regulatory T cells

Polymorphisms in MHC class II molecules, in particular around β-chain position-57 (β57), afford susceptibility/resistance to multiple autoimmune diseases, including type 1 diabetes, through obscure mechanisms. Here, we show that the antidiabetogenic MHC class II molecule I-A^b affords diabetes resistance by promoting the differentiation of MHC-promiscuous autoreactive CD4⁺ T cells into disease-suppressing natural regulatory T cells, in a β56–67-regulated manner. We compared the tolerogenic and antidiabetogenic properties of CD11c promoter-driven transgenes encoding I-A^b or a form of I-A^b carrying residues 56–67 of I-Aβ^g7 (I-A^b-g7) in wild-type nonobese diabetic (NOD) mice, as well as NOD mice coexpressing a diabetogenic and I-A^g7–restricted, but MHC-promiscuous T-cell receptor (4.1). Both I-A transgenes protected NOD and 4.1-NOD mice from diabetes. However, whereas I-A^b induced 4.1-CD4⁺ thymocyte deletion and 4.1-CD4⁺Foxp3⁺ regulatory T-cell development, I-A^b-g7 promoted 4.1-CD4⁺Foxp3⁺ Treg development without inducing clonal deletion. Furthermore, non–T-cell receptor transgenic NOD.CD11cP-I-A^b and NOD.CD11cP-IA^b-g7 mice both exported regulatory T cells with superior antidiabetogenic properties than wild-type NOD mice. We propose that I-A^b, and possibly other protective MHC class II molecules, afford disease resistance by engaging a naturally occurring constellation of MHC-promiscuous autoreactive T-cell clonotypes, promoting their deviation into autoregulatory T cells.     

Activated CD4+ and CD8+ T-cell subsets in Wegener's granulomatosis

Several lines of evidence argue in favour of an involvement of T cells in the pathogenesis of Wegener's granulomatosis (WG). These include the presence of highly specific IgG autoantibodies to proteinase 3, perivascular T-cell infiltrates and elevated amounts of soluble interleukin-2 (IL-2) receptors in patient's serum. In order to further address this question we evaluated by double immunoflourescence and flow cytometry the expression of several cell surface molecules associated with T-cell activation. As compared to healthy controls (n=15), the CD4⁺ subset was significantly diminished, while the percentage of CD8⁺ T cells was elevated in WG patients (n=24). Within the CD4⁺ T-cell subset we found a highly significant increase in activation/memory markers (CD25, CD29, HLA-DR). Within the CD8⁺ T-cell subset the expression of CD11b, CD29 and CD57 was significantly elevated, while the expression of VD28 was reduced. The use of 10 V-, 1 V-and 1 V-specific monoclonal reagents failed to reveal any significant bias in the peripheral T-cell receptor V-gene repertoire of WG patients. There was also no correlation between T-cell activation markers and laboratory parameters [C-reactive protein (CRP), ESR], disease duration or therapy. A significant correlation was found only for the degree of organ involvement and the increase in CD4⁺ T cells coexpressing HLA-DR, as well as the increase in CD57 expression on CD8⁺ T cells. In conclusion, both CD4⁺ and CD8⁺ T-cell subsets were activated in WG. Cytotoxic CD8⁺ CD57⁺ CD11b⁺ CD28^– T cells may directly contribute to damage of vascular endothelium.