T Follicular Helper Cell-Germinal Center B Cell Interaction Strength Regulates Entry into Plasma Cell or Recycling Germinal Center Cell Fate.

Higher- or lower-affinity germinal center (GC) B cells are directed either to plasma cell or GC recycling, respectively; however, how commitment to the plasma cell fate takes place is unclear. We found that a population of light zone (LZ) GC cells, Bcl6loCD69hi expressing a transcription factor IRF4 and higher-affinity B cell receptors (BCRs) or Bcl6hiCD69hi with lower-affinity BCRs, favored the plasma cell or recycling GC cell fate, respectively. Mechanistically, CD40 acted as a dose-dependent regulator for Bcl6loCD69hi cell formation. Furthermore, we found that expression of intercellular adhesion molecule 1 (ICAM-1) and signaling lymphocytic activation molecule (SLAM) in Bcl6loCD69hi cells was higher than in Bcl6hiCD69hi cells, thereby affording more stable T follicular helper (Tfh)-GC B cell contacts. These data support a model whereby commitment to the plasma cell begins in the GC and suggest that stability of Tfh-GC B cell contacts is key for plasma cell-prone GC cell formation.

Somatic mutations in tumor-infiltrating lymphocytes impact on antitumor immunity.

Immune checkpoint inhibitors (ICIs) exert clinical efficacy against various types of cancers by reinvigorating exhausted CD8+ T cells that can expand and directly attack cancer cells (cancer-specific T cells) among tumor-infiltrating lymphocytes (TILs). Although some reports have identified somatic mutations in TILs, their effect on antitumor immunity remains unclear. In this study, we successfully established 18 cancer-specific T cell clones, which have an exhaustion phenotype, from the TILs of four patients with melanoma. We conducted whole-genome sequencing for these T cell clones and identified various somatic mutations in them with high clonality. Among the somatic mutations, an SH2D2A loss-of-function frameshift mutation and TNFAIP3 deletion could activate T cell effector functions in vitro. Furthermore, we generated CD8+ T cell-specific Tnfaip3 knockout mice and showed that Tnfaip3 function loss in CD8+ T cell increased antitumor immunity, leading to remarkable response to PD-1 blockade in vivo. In addition, we analyzed bulk CD3+ T cells from TILs in additional 12 patients and identified an SH2D2A mutation in one patient through amplicon sequencing. These findings suggest that somatic mutations in TILs can affect antitumor immunity and suggest unique biomarkers and therapeutic targets.

HLA Class I Analysis Provides Insight Into the Genetic and Epigenetic Background of Immune Evasion in Colorectal Cancer With High Microsatellite Instability.

BACKGROUND & AIMS: A detailed understanding of antitumor immunity is essential for optimal cancer immune therapy. Although defective mutations in the B2M and HLA-ABC genes, which encode molecules essential for antigen presentation, have been reported in several studies, the effects of these defects on tumor immunity have not been quantitatively evaluated. METHODS: Mutations in HLA-ABC genes were analyzed in 114 microsatellite instability-high colorectal cancers using a long-read sequencer. The data were further analyzed in combination with whole-exome sequencing, transcriptome sequencing, DNA methylation array, and immunohistochemistry data. RESULTS: We detected 101 truncating mutations in 57 tumors (50%) and loss of 61 alleles in 21 tumors (18%). Based on the integrated analysis that enabled the immunologic subclassification of microsatellite instability-high colorectal cancers, we identified a subtype of tumors in which lymphocyte infiltration was reduced, partly due to reduced expression of HLA-ABC genes in the absence of apparent genetic alterations. Survival time of patients with such tumors was shorter than in patients with other tumor types. Paradoxically, tumor mutation burden was highest in the subtype, suggesting that the immunogenic effect of accumulating mutations was counterbalanced by mutations that weakened immunoreactivity. Various genetic and epigenetic alterations, including frameshift mutations in RFX5 and promoter methylation of PSMB8 and HLA-A, converged on reduced expression of HLA-ABC genes. CONCLUSIONS: Our detailed immunogenomic analysis provides information that will facilitate the improvement and development of cancer immunotherapy.

Nerve fiber analysis of the lingual branch of the glossopharyngeal nerve in human adult subjects.

PURPOSE: Fibers of the glossopharyngeal part of the superior constrictor muscle are connected with fibers of the transverse lingual muscle, forming a ring of muscle at the base of the tongue. This group of muscles constrict the midpharyngeal cavity during retrusive movement of the tongue. The purpose of this study is to identify the contribution of the lingual branch of the glossopharyngeal nerve to the neuro-motor control of three muscles: the glossopharyngeal part of the superior pharyngeal constrictor muscle, the palatopharyngeal and the palatoglossus muscles. METHODS: Six en bloc samples (9 sides), including the tissue from the skull base to the hyoid bone were obtained from adult human cadavers. Nerve fiber of the lingual branch of the glossopharyngeal nerve (main root of the glossopharyngeal nerve) was examined by the use of a binocular stereomicroscope. RESULTS: We observed that, after branching to the stylopharyngeal muscle, the lingual branch of the glossopharyngeal nerve branched to the glossopharyngeal part of the superior pharyngeal constrictor muscle, the palatopharyngeal and the palatoglossus muscles before inserting into the space between the muscle layers of the superior and middle pharyngeal constrictors. CONCLUSION: These neuromuscular arrangements may suggest the presence of specialized constrictive movements of the midpharygeal cavity at the level of the base of the tongue with the retrusive movement of the tongue. The simultaneous contraction of the palatopharyngeal and palatoglossus muscles on the pharyngeal stage of deglutition may aid in the passage of bolus from the oral cavity to the midpharyngeal cavity by increasing pharyngeal pressure.

Characterization of SPP inhibitors suppressing propagation of HCV and protozoa.

Signal peptide peptidase (SPP) is an intramembrane aspartic protease involved in the maturation of the core protein of hepatitis C virus (HCV). The processing of HCV core protein by SPP has been reported to be critical for the propagation and pathogenesis of HCV. Here we examined the inhibitory activity of inhibitors for γ-secretase, another intramembrane cleaving protease, against SPP, and our findings revealed that the dibenzoazepine-type structure in the γ-secretase inhibitors is critical for the inhibition of SPP. The spatial distribution showed that the γ-secretase inhibitor compound YO-01027 with the dibenzoazepine structure exhibits potent inhibiting activity against SPP in vitro and in vivo through the interaction of Val223 in SPP. Treatment with this SPP inhibitor suppressed the maturation of core proteins of all HCV genotypes without the emergence of drug-resistant viruses, in contrast to the treatment with direct-acting antivirals. YO-01027 also efficiently inhibited the propagation of protozoa such as Plasmodium falciparum and Toxoplasma gondii These data suggest that SPP is an ideal target for the development of therapeutics not only against chronic hepatitis C but also against protozoiasis.

Allergic conversion of protective mucosal immunity against nasal bacteria in patients with chronic rhinosinusitis with nasal polyposis.

BACKGROUND: Chronic rhinosinusitis with nasal polyposis (CRSwNP) is characterized by eosinophilic inflammation and polyposis at the nose and paranasal sinus and a high concentration of IgE in nasal polyps (NPs). The causative antigen and pathogenesis of CRSwNP remain unknown. OBJECTIVE: We aimed to identify reactive allergens of IgE antibodies produced locally in NPs of patients with CRSwNP. We also attempted to unravel the differentiation pathway of IgE-producing B cells in NPs. METHODS: IgE reactivity of patients with CRSwNP was investigated by characterizing single cell-derived mAbs. T-cell response against identified allergens was investigated in vitro. NP-infiltrating lymphocytes were characterized by using flow cytometry. Immunoglobulins expressed in NPs were analyzed by using high-throughput DNA sequencing for immunoglobulin. RESULTS: About 20% of isolated IgE antibodies derived from NP-residing plasmablasts specifically recognized surface determinants of nasal bacteria, such as Staphylococcus aureus, Streptococcus pyogenes, and Haemophilus influenzae. A TH2 response against S pyogenes was observed in patients with CRSwNP. Flow cytometric analysis revealed sizable germinal center B-like cell and plasmablast subsets expressing IgE on the cell surface in NPs. High-throughput DNA sequencing immunoglobulin analysis highlighted the clonal connectivity of IgE with IgG and IgA1. The Iε-Cα1 circle transcript was detected in NPs. CONCLUSIONS: In patients with CRSwNP, nasal bacteria-reactive B cells differentiate into IgE-producing B cells through IgG/IgA1-IgE class switching, suggesting that allergic conversion of the mucosal response against nasal bacteria underlies disease pathogenesis.

Selective synthesis of zeolites A and X from two industrial wastes: Crushed stone powder and aluminum ash.

Crushed stone powder and aluminum ash are industrial wastes, and effective utilization of these wastes has been highly expected. Since the main components of the two wastes are Si, Al and O, those wastes can be used as starting materials for synthesis of zeolites of which some types have been commercialized as catalysts and ion-exchangers. In this study, zeolites A and X well-known as practical materials were successfully synthesized with high purity using the two industrial wastes by a mild process based on two hydrothermal treatments with intermediate acid treatment. In the first hydrothermal treatment at 150 °C, quartz in the crushed stone powder was dissolved and acid-soluble hydroxysodalite (Na8(AlSiO4)6(H2O)2(OH)2) with Si/Al = 1 and sodium aluminosilicate (Na6(AlSiO4)6) were formed. Those compounds were dissolved with HCl aq. solution. The zeolites were successfully synthesized from the second hydrothermal treatment of the yellow dried filtrates at 80 °C in NaOH aq. solution. In the process proposed, removal of Ca from the crushed stone powder was effective to formation of zeolites A and/or X. Selective synthesis of zeolites A and X was achieved by controlling the acid treatment conditions. Furthermore, the effect of the drying condition of the filtrate obtained after the acid treatment was also investigated on the differences in the product phase.

RabGDIα is a negative regulator of interferon-γ-inducible GTPase-dependent cell-autonomous immunity to Toxoplasma gondii.

IFN-γ orchestrates cell-autonomous host defense against various intracellular vacuolar pathogens. IFN-γ-inducible GTPases, such as p47 immunity-related GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), are recruited to pathogen-containing vacuoles, which is important for disruption of the vacuoles, culminating in the cell-autonomous clearance. Although the positive regulation for the proper recruitment of IRGs and GBPs to the vacuoles has been elucidated, the suppressive mechanism is unclear. Here, we show that Rab GDP dissociation inhibitor α (RabGDIα), originally identified as a Rab small GTPase inhibitor, is a negative regulator of IFN-γ-inducible GTPases in cell-autonomous immunity to the intracellular pathogen Toxoplasma gondii. Overexpression of RabGDIα, but not of RabGDIß, impaired IFN-γ-dependent reduction of T. gondii numbers. Conversely, RabGDIα deletion in macrophages and fibroblasts enhanced the IFN-γ-induced clearance of T. gondii. Furthermore, upon a high dose of infection by T. gondii, RabGDIα-deficient mice exhibited a decreased parasite burden in the brain and increased resistance in the chronic phase than did control mice. Among members of IRGs and GBPs important for the parasite clearance, Irga6 and Gbp2 alone were more frequently recruited to T. gondii-forming parasitophorous vacuoles in RabGDIα-deficient cells. Notably, Gbp2 positively controlled Irga6 recruitment that was inhibited by direct and specific interactions of RabGDIα with Gbp2 through the lipid-binding pocket. Taken together, our results suggest that RabGDIα inhibits host defense against T. gondii by negatively regulating the Gbp2-Irga6 axis of IFN-γ-dependent cell-autonomous immunity.

Hepatocyte Factor JMJD5 Regulates Hepatitis B Virus Replication through Interaction with HBx.

UNLABELLED: Hepatitis B virus (HBV) is a causative agent for chronic liver diseases such as hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBx protein encoded by the HBV genome plays crucial roles not only in pathogenesis but also in replication of HBV. Although HBx has been shown to bind to a number of host proteins, the molecular mechanisms by which HBx regulates HBV replication are largely unknown. In this study, we identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner of HBx interacting in the cytoplasm. DNA microarray analysis revealed that JMJD5-knockout (JMJD5KO) Huh7 cells exhibited a significant reduction in the expression of transcriptional factors involved in hepatocyte differentiation, such as HNF4A, CEBPA, and FOXA3. We found that hydroxylase activity of JMJD5 participates in the regulation of these transcriptional factors. Moreover, JMJD5KO Huh7 cells exhibited a severe reduction in HBV replication, and complementation of HBx expression failed to rescue replication of a mutant HBV deficient in HBx, suggesting that JMJD5 participates in HBV replication through an interaction with HBx. We also found that replacing Gly(135) with Glu in JMJD5 abrogates binding with HBx and replication of HBV. Moreover, the hydroxylase activity of JMJD5 was crucial for HBV replication. Collectively, these results suggest that direct interaction of JMJD5 with HBx facilitates HBV replication through the hydroxylase activity of JMJD5. IMPORTANCE: HBx protein encoded by hepatitis B virus (HBV) plays important roles in pathogenesis and replication of HBV. We identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner to HBx. JMJD5 was shown to regulate several transcriptional factors to maintain hepatocyte function. Although HBx had been shown to support HBV replication, deficiency of JMJD5 abolished contribution of HBx in HBV replication, suggesting that HBx-mediated HBV replication is largely dependent on JMJD5. We showed that hydroxylase activity of JMJD5 in the C terminus region is crucial for expression of HNF4A and replication of HBV. Furthermore, a mutant JMJD5 with Gly(135) replaced by Glu failed to interact with HBx and to rescue the replication of HBV in JMJD5-knockout cells. Taken together, our data suggest that interaction of JMJD5 with HBx facilitates HBV replication through the hydroxylase activity of JMJD5.

Complete genome sequences of two highly divergent Japanese isolates of Plantago asiatica mosaic virus.

Plantago asiatica mosaic virus (PlAMV) is a member of the genus Potexvirus and has an exceptionally wide host range. It causes severe damage to lilies. Here we report on the complete nucleotide sequences of two new Japanese PlAMV isolates, one from the eudicot weed Viola grypoceras (PlAMV-Vi), and the other from the eudicot shrub Nandina domestica Thunb. (PlAMV-NJ). Their genomes contain five open reading frames (ORFs), which is characteristic of potexviruses. Surprisingly, the isolates showed only 76.0-78.0 % sequence identity with each other and with other PlAMV isolates, including isolates from Japanese lily and American nandina. Amino acid alignments of the replicase coding region encoded by ORF1 showed that the regions between the methyltransferase and helicase domains were less conserved than other regions, with several insertions and/or deletions. Phylogenetic analyses of the full-length nucleotide sequences revealed a moderate correlation between phylogenetic clustering and the original host plants of the PlAMV isolates. This study revealed the presence of two highly divergent PlAMV isolates in Japan.

Quantifying sequence and structural features of protein-RNA interactions.

Increasing awareness of the importance of protein-RNA interactions has motivated many approaches to predict residue-level RNA binding sites in proteins based on sequence or structural characteristics. Sequence-based predictors are usually high in sensitivity but low in specificity; conversely structure-based predictors tend to have high specificity, but lower sensitivity. Here we quantified the contribution of both sequence- and structure-based features as indicators of RNA-binding propensity using a machine-learning approach. In order to capture structural information for proteins without a known structure, we used homology modeling to extract the relevant structural features. Several novel and modified features enhanced the accuracy of residue-level RNA-binding propensity beyond what has been reported previously, including by meta-prediction servers. These features include: hidden Markov model-based evolutionary conservation, surface deformations based on the Laplacian norm formalism, and relative solvent accessibility partitioned into backbone and side chain contributions. We constructed a web server called aaRNA that implements the proposed method and demonstrate its use in identifying putative RNA binding sites.

Structures and interface mapping of the TIR domain-containing adaptor molecules involved in interferon signaling.

Homotypic and heterotypic interactions between Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors (TLRs) and downstream adaptors are essential to evoke innate immune responses. However, such oligomerization properties present intrinsic difficulties in structural studies of TIR domains. Here, using BB-loop mutations that disrupt homotypic interactions, we determined the structures of the monomeric TIR domain-containing adaptor molecule (TICAM)-1 and TICAM-2 TIR domains. Docking of the monomeric structures, together with yeast two hybrid-based mutagenesis assays, reveals that the homotypic interaction between TICAM-2 TIR is indispensable to present a scaffold for recruiting the monomeric moiety of the TICAM-1 TIR dimer. This result proposes a unique idea that oligomerization of upstream TIR domains is crucial for binding of downstream TIR domains. Furthermore, the bivalent nature of each TIR domain dimer can generate a large signaling complex under the activated TLRs, which would recruit downstream signaling molecules efficiently. This model is consistent with previous reports that BB-loop mutants completely abrogate downstream signaling.

Kotai Antibody Builder: automated high-resolution structural modeling of antibodies.

MOTIVATION: Kotai Antibody Builder is a Web service for tertiary structural modeling of antibody variable regions. It consists of three main steps: hybrid template selection by sequence alignment and canonical rules, 3D rendering of alignments and CDR-H3 loop modeling. For the last step, in addition to rule-based heuristics used to build the initial model, a refinement option is available that uses fragment assembly followed by knowledge-based scoring. Using targets from the Second Antibody Modeling Assessment, we demonstrate that Kotai Antibody Builder generates models with an overall accuracy equal to that of the best-performing semi-automated predictors using expert knowledge. AVAILABILITY AND IMPLEMENTATION: Kotai Antibody Builder is available at http://kotaiab.org CONTACT: standley@ifrec.osaka-u.ac.jp.

RNA-LIM: a novel procedure for analyzing protein/single-stranded RNA propensity data with concomitant estimation of interface structure.

RNA-LIM is a procedure that can analyze various pseudo-potentials describing the affinity between single-stranded RNA (ssRNA) ribonucleotides and surface amino acids to produce a coarse-grained estimate of the structure of the ssRNA at the protein interface. The search algorithm works by evolving an ssRNA chain, of known sequence, as a series of walks between fixed sites on a protein surface. Optimal routes are found by application of a set of minimal "limiting" restraints derived jointly from (i) selective sampling of the ribonucleotide amino acid affinity pseudo-potential data, (ii) limited surface path exploration by prior determination of surface arc lengths, and (iii) RNA structural specification obtained from a statistical potential gathered from a library of experimentally determined ssRNA structures. We describe the general approach using a NAST (Nucleic Acid Simulation Tool)-like approximation of the ssRNA chain and a generalized pseudo-potential reflecting the location of nucleic acid binding residues. Minimum and maximum performance indicators of the methodology are established using both synthetic data, for which the pseudo-potential defining nucleic acid binding affinity is systematically degraded, and a representative real case, where the RNA binding sites are predicted by the amplified antisense RNA (aaRNA) method. Some potential uses and extensions of the routine are discussed. RNA-LIM analysis programs along with detailed instructions for their use are available on request from the authors.

High-resolution modeling of antibody structures by a combination of bioinformatics, expert knowledge, and molecular simulations.

In the second antibody modeling assessment, we used a semiautomated template-based structure modeling approach for 11 blinded antibody variable region (Fv) targets. The structural modeling method involved several steps, including template selection for framework and canonical structures of complementary determining regions (CDRs), homology modeling, energy minimization, and expert inspection. The submitted models for Fv modeling in Stage 1 had the lowest average backbone root mean square deviation (RMSD) (1.06 Å). Comparison to crystal structures showed the most accurate Fv models were generated for 4 out of 11 targets. We found that the successful modeling in Stage 1 mainly was due to expert-guided template selection for CDRs, especially for CDR-H3, based on our previously proposed empirical method (H3-rules) and the use of position specific scoring matrix-based scoring. Loop refinement using fragment assembly and multicanonical molecular dynamics (McMD) was applied to CDR-H3 loop modeling in Stage 2. Fragment assembly and McMD produced putative structural ensembles with low free energy values that were scored based on the OSCAR all-atom force field and conformation density in principal component analysis space, respectively, as well as the degree of consensus between the two sampling methods. The quality of 8 out of 10 targets improved as compared with Stage 1. For 4 out of 10 Stage-2 targets, our method generated top-scoring models with RMSD values of less than 1 Å. In this article, we discuss the strengths and weaknesses of our approach as well as possible directions for improvement to generate better predictions in the future.

Stem-like progenitor and terminally differentiated TFH-like CD4+ T cell exhaustion in the tumor microenvironment.

Immune checkpoint inhibitors exert clinical efficacy against various types of cancer through reinvigoration of exhausted CD8+ T cells that attack cancer cells directly in the tumor microenvironment (TME). Using single-cell sequencing and mouse models, we show that CXCL13, highly expressed in tumor-infiltrating exhausted CD8+ T cells, induces CD4+ follicular helper T (TFH) cell infiltration, contributing to anti-tumor immunity. Furthermore, a part of the TFH cells in the TME exhibits cytotoxicity and directly attacks major histocompatibility complex-II-expressing tumors. TFH-like cytotoxic CD4+ T cells have high LAG-3/BLIMP1 and low TCF1 expression without self-renewal ability, whereas non-cytotoxic TFH cells express low LAG-3/BLIMP1 and high TCF1 with self-renewal ability, closely resembling the relationship between terminally differentiated and stem-like progenitor exhaustion in CD8+ T cells, respectively. Our findings provide deep insights into TFH-like CD4+ T cell exhaustion with helper progenitor and cytotoxic differentiated functions, mediating anti-tumor immunity orchestrally with CD8+ T cells.

CD106 in Tumor-Specific Exhausted CD8+ T Cells Mediates Immunosuppression by Inhibiting TCR Signaling.

T-cell exhaustion is a major contributor to immunosuppression in the tumor microenvironment (TME). Blockade of key regulators of T-cell exhaustion, such as programmed death 1, can reinvigorate tumor-specific T cells and activate antitumor immunity in various types of cancer. In this study, we identified that CD106 was specifically expressed in exhausted CD8+ T cells in the TME using single-cell RNA sequencing. High CD106 expression in the TME in clinical samples corresponded to improved response to cancer immunotherapy. CD106 in tumor-specific T cells suppressed antitumor immunity both in vitro and in vivo, and loss of CD106 in CD8+ T cells suppressed tumor growth and improved response to programmed death 1 blockade. Mechanistically, CD106 inhibited T-cell receptor (TCR) signaling by interacting with the TCR/CD3 complex and reducing its surface expression. Together, these findings provide insights into the immunosuppressive role of CD106 expressed in tumor-specific exhausted CD8+ T cells, identifying it as a potential biomarker and therapeutic target for cancer immunotherapy. Significance: CD106 is specifically expressed in tumor-specific exhausted CD8+ T cells and inhibits the TCR signaling pathway by reducing surface expression of the TCR/CD3 complex to suppress antitumor immunity.

Sema6D forward signaling impairs T cell activation and proliferation in head and neck cancer.

Immune checkpoint inhibitors (ICIs) are indicated for a diverse range of cancer types, and characterizing the tumor immune microenvironment is critical for optimizing therapeutic strategies, including ICIs. T cell infiltration and activation status in the tumor microenvironment greatly affects the efficacy of ICIs. Here, we show that semaphorin 6D (Sema6D) forward signaling, which is reportedly involved in coordinating the orientation of cell development and migration as a guidance factor, impaired the infiltration and activation of tumor-specific CD8+ T cells in murine oral tumors. Sema6D expressed by nonhematopoietic cells was responsible for this phenotype. Plexin-A4, a receptor for Sema6D, inhibited T cell infiltration and partially suppressed CD8+ T cell activation and proliferation induced by Sema6D stimulation. Moreover, mouse oral tumors, which are resistant to PD-1-blocking treatment in wild-type mice, showed a response to the treatment in Sema6d-KO mice. Finally, analyses of public data sets of human head and neck squamous cell carcinoma, pan-cancer cohorts, and a retrospective cohort study showed that SEMA6D was mainly expressed by nonhematopoietic cells such as cancer cells, and SEMA6D expression was significantly negatively correlated with CD8A, PDCD1, IFNG, and GZMB expression. Thus, targeting Sema6D forward signaling is a promising option for increasing ICI efficacy.

Breadth and Durability of SARS-CoV-2-Specific T Cell Responses following Long-Term Recovery from COVID-19.

T cell immunity is crucial for long-term immunological memory, but the profile of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific memory T cells in individuals who recovered from COVID-19 (COVID-19-convalescent individuals) is not sufficiently assessed. In this study, the breadth and magnitude of SARS-CoV-2-specific T cell responses were determined in COVID-19-convalescent individuals in Japan. Memory T cells against SARS-CoV-2 were detected in all convalescent individuals, and those with more severe disease exhibited a broader T cell response relative to cases with mild symptoms. Comprehensive screening of T cell responses at the peptide level was conducted for spike (S) and nucleocapsid (N) proteins, and regions frequently targeted by T cells were identified. Multiple regions in S and N proteins were targeted by memory T cells, with median numbers of target regions of 13 and 4, respectively. A maximum of 47 regions were recognized by memory T cells for an individual. These data indicate that SARS-CoV-2-convalescent individuals maintain a substantial breadth of memory T cells for at least several months following infection. Broader SARS-CoV-2-specific CD4+ T cell responses, relative to CD8+ T cell responses, were observed for the S but not the N protein, suggesting that antigen presentation is different between viral proteins. The binding affinity of predicted CD8+ T cell epitopes to HLA class I molecules in these regions was preserved for the Delta variant and at 94 to 96% for SARS-CoV-2 Omicron subvariants, suggesting that the amino acid changes in these variants do not have a major impact on antigen presentation to SARS-CoV-2-specific CD8+ T cells. IMPORTANCE RNA viruses, including SARS-CoV-2, evade host immune responses through mutations. As broader T cell responses against multiple viral proteins could minimize the impact of each single amino acid mutation, the breadth of memory T cells would be one essential parameter for effective protection. In this study, breadth of memory T cells to S and N proteins was assessed in COVID-19-convalescent individuals. While broad T cell responses were induced against both proteins, the ratio of N to S proteins for breadth of T cell responses was significantly higher in milder cases. The breadth of CD4+ and CD8+ T cell responses was also significantly different between S and N proteins, suggesting different contributions of N and S protein-specific T cells for COVID-19 control. Most CD8+ T cell epitopes in the immunodominant regions maintained their HLA binding to SARS-CoV-2 Omicron subvariants. Our study provides insights into understanding the protective efficacy of SARS-CoV-2-specific memory T cells against reinfection.

Antibody feedback contributes to facilitating the development of Omicron-reactive memory B cells in SARS-CoV-2 mRNA vaccinees.

In contrast to a second dose of the SARS-CoV-2 mRNA vaccine, a third dose elicits potent neutralizing activity against the Omicron variant. To address the underlying mechanism for this differential antibody response, we examined spike receptor-binding domain (RBD)-specific memory B cells in vaccinated individuals. Frequency of Omicron-reactive memory B cells increased ∼9 mo after the second vaccine dose. These memory B cells show an altered distribution of epitopes from pre-second memory B cells, presumably due to an antibody feedback mechanism. This hypothesis was tested using mouse models, showing that an addition or a depletion of RBD-induced serum antibodies results in a concomitant increase or decrease, respectively, of Omicron-reactive germinal center (GC) and memory B cells. Our data suggest that pre-generated antibodies modulate the selection of GC and subsequent memory B cells after the second vaccine dose, accumulating more Omicron-reactive memory B cells over time, which contributes to the generation of Omicron-neutralizing antibodies elicited by the third vaccine dose.