How Drivers of Seasonality in Respiratory Infections May Impact Vaccine Strategy: A Case Study in How Coronavirus Disease 2019 (COVID-19) May Help Us Solve One of Influenza's Biggest Challenges.

Vaccines against seasonal infections like influenza offer a recurring testbed, encompassing challenges in design, implementation, and uptake to combat a both familiar and ever-shifting threat. One of the pervading mysteries of influenza epidemiology is what causes the distinctive seasonal outbreak pattern. Proposed theories each suggest different paths forward in being able to tailor precision vaccines and/or deploy them most effectively. One of the greatest challenges in contrasting and supporting these theories is, of course, that there is no means by which to actually test them. In this communication we revisit theories and explore how the ongoing coronavirus disease 2019 (COVID-19) pandemic might provide a unique opportunity to better understand the global circulation of respiratory infections. We discuss how vaccine strategies may be targeted and improved by both isolating drivers and understanding the immunological consequences of seasonality, and how these insights about influenza vaccines may generalize to vaccines for other seasonal respiratory infections.

A requisite role for induced regulatory T cells in tolerance based on expanding antigen receptor diversity.

Although both natural and induced regulatory T (nTreg and iTreg) cells can enforce tolerance, the mechanisms underlying their synergistic actions have not been established. We examined the functions of nTreg and iTreg cells by adoptive transfer immunotherapy of newborn Foxp3-deficient mice. As monotherapy, only nTreg cells prevented disease lethality, but did not suppress chronic inflammation and autoimmunity. Provision of Foxp3-sufficient conventional T cells with nTreg cells reconstituted the iTreg pool and established tolerance. In turn, acute depletion of iTreg cells in rescued mice resulted in weight loss and inflammation. Whereas the transcriptional signatures of nTreg and in vivo-derived iTreg cells were closely matched, there was minimal overlap in their T cell receptor (TCR) repertoires. Thus, iTreg cells are an essential nonredundant regulatory subset that supplements nTreg cells, in part by expanding TCR diversity within regulatory responses.

Incorporating calendar effects to predict influenza seasonality in Milwaukee, Wisconsin.

Social outings can trigger influenza transmission, especially in children and elderly. In contrast, school closures are associated with reduced influenza incidence in school-aged children. While influenza surveillance modelling studies typically account for holidays and mass gatherings, age-specific effects of school breaks, sporting events and commonly celebrated observances are not fully explored. We examined the impact of school holidays, social events and religious observances for six age groups (all ages, ⩽4, 5-24, 25-44, 45-64, ⩾65 years) on four influenza outcomes (tests, positives, influenza A and influenza B) as reported by the City of Milwaukee Health Department Laboratory, Milwaukee, Wisconsin from 2004 to 2009. We characterised holiday effects by analysing average weekly counts in negative binomial regression models controlling for weather and seasonal incidence fluctuations. We estimated age-specific annual peak timing and compared influenza outcomes before, during and after school breaks. During the 118 university holiday weeks, average weekly tests were lower than in 140 school term weeks (5.93 vs. 11.99 cases/week, P < 0.005). The dampening of tests during Winter Break was evident in all ages and in those 5-24 years (RR = 0.31; 95% CI 0.22-0.41 vs. RR = 0.14; 95% CI 0.09-0.22, respectively). A significant increase in tests was observed during Spring Break in 45-64 years old adults (RR = 2.12; 95% CI 1.14-3.96). Milwaukee Public Schools holiday breaks showed similar amplification and dampening effects. Overall, calendar effects depend on the proximity and alignment of an individual holiday to age-specific and influenza outcome-specific peak timing. Better quantification of individual holiday effects, tailored to specific age groups, should improve influenza prevention measures.

Structural and Mechanistic Implications of Rearrangement Frequencies within Human TCRBV Genes.

The T cell repertoire is a function of thymic V(D)J rearrangement and of peripheral selection. The mature repertoire embodies TCR sequences that are important for survival and can identify important structural aspects of the TCR. Analysis of the circulating TCRBV19 CD8 T cell repertoire showed that a majority of NDN-encoded CDR3 amino acid motifs start at CDR3 position four, well within the V region. Rearrangement at this position indicates that the DNA hairpin loop is not opened at the position adjacent to the recombination signal sequence, but rather is trimmed back three or more bases. In this article, we show that the rearrangement frequency distribution within the V region reveals selection on CDR3 position four. The selection is already established in single-positive CD8 thymocytes. Crystal structures reveal a possible basis for this selection due to the location of this residue in a bend that positions the remaining portion of CDR3 to interact with the peptide and MHC. Examination of other TCRBV families also shows selection for rearrangement within the V region of a number of genes and for CD8 and CD4 cells. The exact profile of rearrangement within the V region appears to be V gene specific. The frequent observation of side chains associated with turn motifs at CDR3 positions three and four fits with the structural need for a bend. The data are discussed in terms of the generation of a structural turn motif, the rearrangement mechanism, and selection of the repertoire on the peptide and MHC.

Emergence of T cells that recognize nonpolymorphic antigens during graft-versus- host disease.

Chronic GVHD is a major cause of morbidity and mortality in allogeneic stem cell transplantation recipients and typically develops from antecedent acute GVHD. In contrast to acute GVHD, chronic GVHD has much broader tissue involvement and clinical manifestations that bear striking similarity to what is observed in autoimmune diseases. How autoimmunity arises out of alloimmunity has been a longstanding unresolved issue. To address this question, in the present study, we performed a comprehensive analysis of the clonotypic T-cell response using complementary murine models that simulate what occurs during the transition from acute to chronic GVHD. These studies revealed repertoire skewing and the presence of high-frequency clonotypes that had undergone significant in vivo expansion, indicating that GVHD-associated autoimmunity was characterized by antigen-driven expansion of a limited number of T-cell clones. Furthermore, we observed that T cells with identical TCRß CDR3 nucleotide sequences were capable of recognizing donor and host antigens, providing evidence that the loss of self-tolerance during acute GVHD leads to the emergence of self-reactive donor T cells that are capable of recognizing nonpolymorphic tissue or commensally derived antigens. These data provide a mechanistic framework for how autoimmunity develops within the context of preexisting GVHD and provide additional insight into the pathophysiology of chronic GVHD.

The TCR repertoires of regulatory and conventional T cells specific for the same foreign antigen are distinct.

The relationship between the TCR repertoires of natural regulatory T cells (nTregs) and conventional CD4(+) T cells (Tconv) capable of responding to the same antigenic epitope is unknown. In this study, we used TCRß-chain transgenic mice to generate polyclonal nTreg and Tconv populations specific for a foreign Ag. CD4(+) T cells from immunized 3.L2ß(+/-) TCRα(+/-) Foxp3(EGFP) mice were restimulated in culture to yield nTregs (EGFP(+)) and Tconv (EGFP(-)) defined by their antigenic reactivity. Relative to Tconv, nTreg expansion was delayed, although a higher proportion of viable nTregs had divided after 72 h. Spectratype analysis revealed that both the nTreg and Tconv responses were different and characterized by skewed distributions of CDR3 lengths. CDR3 sequences from nTregs displayed a divergent pattern of Jα usage, minimal CDR3 overlap (3.4%), and less diversity than did CDR3 sequences derived from Tconv. These data indicate that foreign Ag-specific nTregs and Tconv are clonally distinct and that foreign Ag-specific nTreg populations are constrained by a limited TCR repertoire.

Patients with juvenile idiopathic arthritis have decreased clonal diversity in the CD8+ T cell repertoire response to influenza vaccination.

Recurrent exposures to a pathogenic antigen remodel the CD8+ T cell compartment and generate a functional memory repertoire that is polyclonal and complex. At the clonotype level, the response to the conserved influenza antigen, M158-66 has been well characterized in healthy individuals, but not in patients receiving immunosuppressive therapy or with aberrant immunity, such as those with juvenile idiopathic arthritis (JIA). Here we show that patients with JIA have a reduced number of M158-66 specific RS/RA clonotypes, indicating decreased clonal richness and, as a result, have lower repertoire diversity. By using a rank-frequency approach to analyze the distribution of the repertoire, we found several characteristics of the JIA T cell repertoire to be akin to repertoires seen in healthy adults, including an amplified RS/RA-specific antigen response, representing greater clonal unevenness. Unlike mature repertoires, however, there is more fluctuation in clonotype distribution, less clonotype stability, and more variable IFNy response of the M158-66 specific RS/RA clonotypes in JIA. This indicates that functional clonal expansion is altered in patients with JIA on immunosuppressive therapies. We propose that the response to the influenza M158-66 epitope described here is a general phenomenon for JIA patients receiving immunosuppressive therapy, and that the changes in clonal richness and unevenness indicate a retarded and uneven generation of a mature immune response.

Corrigendum: Patients with juvenile idiopathic arthritis have decreased clonal diversity in the CD8+ T cell repertoire response to influenza vaccination.

[This corrects the article DOI: 10.3389/fimmu.2024.1306490.].

Cross-reactive responses to modified M158-66 peptides by CD8⁺ T cells that use noncanonical BV genes can describe unknown repertoires.

Memory T-cell repertoires are populated by clonotypes selected by an individual's history of antigen exposures. Our previous analysis of middle-age CD8(+) T-cell memory repertoires to the influenza-derived epitope M1(58-66) , described a network of highly cross-reactive BV19 clonotypes responding to M1(58-66) and at least one peptide with a conservative amino acid substitution at either of two TCR contact positions. Here, we report that some substitutions abrogate BV19 responses and favor responses with different BV. Cross-reactive T cells using seven other BV families responded to 12 of 13 peptides tested. BV12 clonotypes define the most extensive cross-reactive network that encompasses seven peptides. We generated 3D networks based on the peptides recognized and BV family used and observed a cluster of five peptides that includes M1(58-66) and another cluster of five peptides that does not include M1(58-66) . The first cluster represents peptides structurally similar to M1(58-66) , and the second represents peptides with more considerable changes in epitope recognition surface. We hypothesize that the second cluster represents the cross-reactive network around another unknown epitope or epitopes. This data supports a model of stable CD8(+) T-cell memory networks that include a substantial contribution from cross-reactive T cells.

Cross-reactivity of T cells and its role in the immune system.

T-cell receptors recognize peptides presented by the major histocompatibility complex (MHC) on the surface of antigen-presenting cells (APC). The ability of the T-cell receptor (TCR) to recognize more than one peptide-MHC structure defines cross-reactivity. Cross-reactivity is a documented phenomenon of the immune system whose importance is still under investigation. There are a number of rational arguments for cross-reactivity. These include the discrepancy between the theoretical high number of pathogen-derived peptides and the lower diversity of the T-cell repertoire, the need for recognition of escape variants, and the intrinsic low affinity of this receptor-ligand pair. However, quantifying the phenomenon has been difficult, and its immunological importance remains unknown. In this review, we examined the cases for and against an important role for cross reactivity. We argue that it may be an essential feature of the immune system from the point of view of biological robustness.

The polyclonal CD8 T cell response to influenza M158-66 generates a fully connected network of cross-reactive clonotypes to structurally related peptides: a paradigm for memory repertoire coverage of novel epitopes or escape mutants.

Cross-reactivity of T cells is defined as recognition of two or more peptide-MHC complexes by the same T cell. Although examples of cross-reactivity have been reported, a detailed examination of cross-reactivity has not been performed. In this study, we took advantage of the high degree of polyclonality in the BV19 T cell repertoire responding to influenza M1(58-66) in HLA-A2 individuals to obtain a measure of simple cross-reactivity. We used substitutions that incrementally change the structure of the M1(58-66) peptide to measure how the HLA-A2-restricted response adapts to these changes. In three HLA-A2 adult subjects, we identified the BV19 clonotypes in the recall response to the influenza epitope M1(58-66) and 12 M1 peptides substituted at TCR contact position 63 or 65. The fraction of cross-reactive clonotypes in the M1(58-66) repertoire varied from 45-58% in the three donors. The extent of cross-reactivity, which is the additional number of peptides recognized by a single clonotype, is as high as six. We summarized the data using graph theory, with the cross-reactive clonotypes connecting the different HLA-A2 peptides recognized. The cross-reactive clonotypes form a well-connected network that could provide protection from virus-escape variants. We predict that any new pathogen with an epitope whose shape corresponds to that of the peptides that we studied would find a pre-existing repertoire ready to respond to it. We propose that in adult memory repertoires, previously encountered epitopes may have generated similar cross-reactive repertoires.

Selective T cell expansion during aging of CD8 memory repertoires to influenza revealed by modeling.

The aging of T cell memory is often considered in terms of senescence, a process viewed as decay and loss of memory T cells. How senescence would affect memory is a function of the initial structure of the memory repertoire and whether the clonotypes that make up the repertoire decay at random. We examine this issue using the T cell memory generated to the conserved influenza A epitope M1(58-66), which induces a strong, focused, but polyclonal CD8 T cell response in HLA-A2 individuals. We analyzed the CD8 T cell memory repertoires in eight healthy middle-aged and eight healthy older blood donors representing an average age difference of ∼ 27 y. Although the repertoires show broadly similar clonotype distributions, the number of observable clonotypes decreases significantly. This decrease disproportionally affects low-frequency clonotypes. Rank frequency analysis shows the same two-component clonotype distribution described earlier for these repertoires. The first component includes lower frequency clonotypes for which distribution can be described by a power law. The slope of this first component is significantly steeper in the older cohort. Generating a representative repertoire for each healthy cohort allowed agent-based modeling of the aging process. Interestingly, simple senescence of middle-aged repertoires is insufficient to describe the older clonotype distribution. Rather, a selective clonotype expansion must be included to achieve the best fit. We propose that responses to periodic virus exposure may drive such expansion, ensuring that the remaining clonotypes are optimized for continued protection.

Naive T cell repertoire skewing in HLA-A2 individuals by a specialized rearrangement mechanism results in public memory clonotypes.

How the naive T cell repertoire arises and forms the memory repertoire is still poorly understood. This relationship was analyzed by taking advantage of the focused TCR usage in HLA-A2-restricted CD8 memory T cell responses to influenza M1(58-66). We analyzed rearranged BV19 genes from CD8 single-positive thymocytes, a surrogate for the naive repertoire, from 10 HLA-A2 individuals. CDR3 amino acid sequences associated with response to influenza were observed at higher frequencies than expected by chance, an indicator of preselection. We propose that a rearrangement mechanism involving long P-nucleotide addition from the J2.7 region explains part of this increase. Special rearrangement mechanisms can result in identical T cells in different individuals, referred to as public responses. Indeed, the rearrangements utilizing long P nucleotide additions were commonly observed in the response to the M1(58-66) epitope in 30 HLA-A2 middle-aged adults. Thus, in addition to negative and positive selection, special rearrangement mechanisms may influence the composition of the naive repertoire, resulting in more robust responses to a pathogen in some individuals.

Cutting edge: HLA-DM-mediated peptide exchange functions normally on MHC class II-peptide complexes that have been weakened by elimination of a conserved hydrogen bond.

The mechanism by which HLA-DM (DM) promotes exchange of peptides bound to HLA-DR (DR) is still unclear. We have shown that peptide interaction with DR1 can be considered a folding process as evidenced by cooperativity. However, in DM-mediated ligand exchange, prebound peptide release is noncooperative, which could be a function of the breaking of a critical interaction. The hydrogen bond (H-bond) between beta-chain His(81) and the peptide backbone at the -1 position is a candidate for such a target. In this study, we analyze the exchange of peptides bound to a DR1 mutant in which formation of this H-bond is impaired. We observe that DM still functions normally. However, as expected of a cooperative model, this H-bond contributes to the overall energetics of the complex and its disruption impacts the ability of the exchange ligand to fold with the binding groove into a stable complex.

Peptide-MHC class II complex stability governs CD4 T cell clonal selection.

The clonal composition of the T cell response can affect its ability to mediate infection control or to induce autoimmunity, but the mechanisms regulating the responding TCR repertoire remain poorly defined. In this study, we immunized mice with wild-type or mutated peptides displaying varying binding half-lives with MHC class II molecules to measure the impact of peptide-MHC class II stability on the clonal composition of the CD4 T cell response. We found that, although all peptides elicited similar T cell response size on immunization, the clonotypic diversity of the CD4 T cell response correlated directly with the half-life of the immunizing peptide. Peptides with short half-lives focused CD4 T cell response toward high-affinity clonotypes expressing restricted public TCR, whereas peptides with longer half-lives broadened CD4 T cell response by recruiting lower-affinity clonotypes expressing more diverse TCR. Peptides with longer half-lives did not cause the elimination of high-affinity clonotypes, and at a low dose, they also skewed CD4 T cell response toward higher-affinity clonotypes. Taken collectively, our results suggest the half-life of peptide-MHC class II complexes is the primary parameter that dictates the clonotypic diversity of the responding CD4 T cell compartment.

Role of cross-reactivity in cellular immune targeting of influenza A M158-66 variant peptide epitopes.

The immunologic significance of cross-reactivity of TCR recognition of peptide:MHC complexes is still poorly understood. We have described TCR cross-reactivity in a system involving polyclonal CD8 T cell recognition of the well characterized influenza viral M158-66 epitope. While M158-66 is generally conserved between influenza A isolates, error-prone transcription generates stable variant RNA during infection which could act as novel epitopes. If packaged and viable, variant genomic RNA generates an influenza quasispecies. The stable RNA variants would generate a new transmissible epitope that can select a specific repertoire, which itself should have cross-reactive properties. We tested two candidate peptides in which Thr65 is changed to Ala (A65) or Ser (S65) using recall responses to identify responding T cell clonotypes. Both peptides generated large polyclonal T cell repertoires of their own with repertoire characteristics and cross-reactivity patterns like that observed for the M158-66 repertoire. Both substitutions could be present in viral genomes or mRNA at sufficient frequency during an infection to drive immunity. Peptides from the resulting protein would be a target for CD8 cells irrespective of virus viability or transmissibility. These data support the hypothesis that cross-reactivity is important for immunity against RNA virus infections.

Autoreactive CD8 T cells in NOD mice exhibit phenotypic heterogeneity but restricted TCR gene usage.

Type 1 diabetes (T1D) is an autoimmune disorder defined by CD8 T cell-mediated destruction of pancreatic ß cells. We have previously shown that diabetogenic CD8 T cells in the islets of non-obese diabetic mice are phenotypically heterogeneous, but clonal heterogeneity remains relatively unexplored. Here, we use paired single-cell RNA and T-cell receptor sequencing (scRNA-seq and scTCR-seq) to characterize autoreactive CD8 T cells from the islets and spleens of non-obese diabetic mice. scTCR-seq demonstrates that CD8 T cells targeting the immunodominant ß-cell epitope IGRP206-214 exhibit restricted TCR gene usage. scRNA-seq identifies six clusters of autoreactive CD8 T cells in the islets and six in the spleen, including memory and exhausted cells. Clonal overlap between IGRP206-214-reactive CD8 T cells in the islets and spleen suggests these cells may circulate between the islets and periphery. Finally, we identify correlations between TCR genes and T-cell clonal expansion and effector fate. Collectively, our work demonstrates that IGRP206-214-specific CD8 T cells are phenotypically heterogeneous but clonally restricted, raising the possibility of selectively targeting these TCR structures for therapeutic benefit.

Single-cell immune profiling reveals a developmentally distinct CD4+ GM-CSF+ T-cell lineage that induces GI tract GVHD.

Gastrointestinal (GI) tract involvement is a major determinant for subsequent morbidity and mortality arising during graft-versus-host disease (GVHD). CD4+ T cells that produce granulocyte-macrophage colony stimulating factor (GM-CSF) have emerged as central mediators of inflammation in this tissue site as GM-CSF serves as a critical cytokine link between the adaptive and innate arms of the immune system. However, cellular heterogeneity within the CD4+ GM-CSF+ T-cell population due to the concurrent production of other inflammatory cytokines has raised questions as to whether these cells have a common ontology or if a unique CD4+ GM-CSF+ subset exists that differs from other defined T helper subtypes. Using single-cell RNA sequencing analysis (scRNAseq), we identified two CD4+ GM-CSF+ T-cell populations that arose during GVHD and were distinguishable according to the presence or absence of interferon-γ (IFN-γ) coexpression. CD4+ GM-CSF+ IFN-γ- T cells, which emerged preferentially in the colon, had a distinct transcriptional profile, used unique gene regulatory networks, and possessed a nonoverlapping T-cell receptor repertoire compared with CD4+ GM-CSF+ IFN-γ+ T cells as well as all other transcriptionally defined CD4+ T-cell populations in the colon. Functionally, this CD4+ GM-CSF+ T-cell population contributed to pathologic damage in the GI tract that was critically dependent on signaling through the interleukin-17 (IL-7) receptor but was independent of type 1 interferon signaling. Thus, these studies help to unravel heterogeneity within CD4+ GM-CSF+ T cells that arise during GVHD and define a developmentally distinct colitogenic T helper subtype GM-CSF+ subset that mediates immunopathology.

Two compensatory pathways maintain long-term stability and diversity in CD8 T cell memory repertoires.

The time-dependent changes of human memory T cell repertoires are still poorly understood. We define a T cell memory repertoire as the pool of clonotypic lineages participating in a recall response to the influenza M1(58-66) epitope. In HLA-A2 individuals, this response predominantly uses BV19 chains with Arg-Ser (RS) in the CDR3 loop. We previously showed that the repertoire is polyclonal with a large fraction of clonotype that are only observed once. In this study, we perform longitudinal analyses of memory repertoires in three middle-aged individuals at times that spanned from 7 to 10 years. In these individuals, who are well into thymic involution, a substantial number of clonotypes were stable, e.g., detected at two times. The shape of the repertoire was stable over time as reflected by a number of repertoire characteristics, including singletons, i.e., the fraction of clonotypes observed only once, and repertoire diversity. However, the RS-clonotype subset showed a significant decline in the fraction of singletons and in clonotypic diversity. Thus, repertoire structure is maintained over time by a recruitment of non-RS-clonotypes and a shift of existing RS-clonotypes into higher frequencies. The recruitment of new clonotypes into the low-frequency component of the repertoire implies a role for these clonotypes.

The transcription factor RFX protects MHC class II genes against epigenetic silencing by DNA methylation.

Classical and nonclassical MHC class II (MHCII) genes are coregulated by the transcription factor RFX (regulatory factor X) and the transcriptional coactivator CIITA. RFX coordinates the assembly of a multiprotein "enhanceosome" complex on MHCII promoters. This enhanceosome serves as a docking site for the binding of CIITA. Whereas the role of the enhanceosome in recruiting CIITA is well established, little is known about its CIITA-independent functions. A novel role of the enhanceosome was revealed by the analysis of HLA-DOA expression in human MHCII-negative B cell lines lacking RFX or CIITA. HLA-DOA was found to be reactivated by complementation of CIITA-deficient but not RFX-deficient B cells. Silencing of HLA-DOA was associated with DNA methylation at its promoter, and was relieved by the demethylating agent 5-azacytidine. Surprisingly, DNA methylation was also established at the HLA-DRA and HLA-DQB loci in RFX-deficient cells. This was a direct consequence of the absence of RFX, as it could be reversed by restoring RFX function. DNA methylation at the HLA-DOA, HLA-DRA, and HLA-DQB promoters was observed in RFX-deficient B cells and fibroblasts, but not in CIITA-deficient B cells and fibroblasts, or in wild-type fibroblasts, which lack CIITA expression. These results indicate that RFX and/or enhanceosome assembly plays a key CIITA-independent role in protecting MHCII promoters against DNA methylation. This function is likely to be crucial for retaining MHCII genes in an open chromatin configuration permissive for activation in MHCII-negative cells, such as the precursors of APC and nonprofessional APC before induction with IFN-gamma.