People living with HIV (PLWH) could be at risk of blunted immune responses to COVID-19 vaccination. We investigated factors associated with neutralizing antibody (NAb) responses against SARS-CoV-2 and variants of concern (VOCs), following two-dose and third booster monovalent COVID-19 mRNA vaccination in Japanese PLWH. NAb titers were assessed in polyclonal IgG fractions by lentiviral-based pseudovirus assays. Overall, NAb titers against Wuhan, following two-dose vaccination, were assessed in 82 PLWH on treatment, whereby 17/82 (20.73%) were classified as low-NAb participants. Within the low-NAb participants, the third booster vaccination enhanced NAb titers against Wuhan and VOCs, albeit to a significantly lower magnitude than the rest. In the multivariate analysis, NAb titers against Wuhan after two-dose vaccination correlated with age and days since vaccination, but not with CD4+ count, CD4+/CD8+ ratio, and plasma high-sensitivity C-Reactive protein (hsCRP). Interestingly, an extended analysis within age subgroups revealed NAb titers to correlate positively with the CD4+ count and negatively with plasma hsCRP in younger, but not older, participants. In conclusion, a third booster vaccination substantially enhances NAb titers, but the benefit may be suboptimal in subpopulations of PLWH exhibiting low titers at baseline. Considering clinical and immune parameters could provide a nuanced understanding of factors associated with vaccine responses in PLWH.

mRNA vaccines against the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) elicit strong T cell responses. However, a clonal-resolution analysis of T cell responses to mRNA vaccination has not been performed. Here, we temporally track the CD8+ T cell repertoire in individuals who received three shots of the BNT162b2 mRNA vaccine through longitudinal T cell receptor sequencing with peptide-human leukocyte antigen (HLA) tetramer analysis. We demonstrate a shift in T cell responses between the clonotypes with different kinetics: from early responders that expand rapidly after the first shot to main responders that greatly expand after the second shot. Although the main responders re-expand after the third shot, their clonal diversity is skewed, and newly elicited third responders partially replace them. Furthermore, this shift in clonal dominance occurs not only between, but also within, clonotypes specific for spike epitopes. Our study will be a valuable resource for understanding vaccine-induced T cell responses in general.

Determinants of HIV-1 latency establishment are yet to be elucidated. HIV reservoir comprises a rare fraction of infected cells that can survive host and virus-mediated killing. In vitro reporter models so far offered a feasible means to inspect this population, but with limited capabilities to dissect provirus silencing dynamics. Here, we describe a new HIV reporter model, HIV-Timer of cell kinetics and activity (HIV-Tocky) with dual fluorescence spontaneous shifting to reveal provirus silencing and reactivation dynamics. This unique feature allows, for the first time, identifying two latent populations: a directly latent, and a recently silenced subset, with the latter having integration features suggestive of stable latency. Our proposed model can help address the heterogeneous nature of HIV reservoirs and offers new possibilities for evaluating eradication strategies.

The gut microbiota has emerged as a key regulator of immune checkpoint inhibitor (ICI) efficacy. Therapeutic approaches aimed at manipulating the microbiota through targeted reconstitution to enhance cancer treatment outcomes have garnered considerable attention. A single live microbial biotherapeutic bacterium, Clostridium butyricum MIYAIRI 588 strain (CBM588), has been shown to enhance the effects of ICI monotherapy in patients with advanced lung cancer. However, whether CBM588 affects the outcomes of chemoimmunotherapy combinations in lung cancer remains unknown. We hypothesized that CBM588 augments the effect of chemoimmunotherapy combinations and restores diminished effectiveness in patients with non-small cell lung cancer (NSCLC) receiving dysbiosis-inducing drugs. To validate this hypothesis, we retrospectively analyzed 106 patients with stage IV or recurrent metastatic NSCLC consecutively treated with chemoimmunotherapy combinations. A survival analysis was performed employing univariate and multivariate Cox proportional hazard models with inverse probability of treatment weighting (IPTW) using propensity scores. Forty-five percent of patients received Clostridium butyricum therapy. CBM588 significantly extended overall survival in patients with NSCLC receiving chemoimmunotherapy. The favorable impact of CBM588 on the efficacy of chemoimmunotherapy combinations varied based on tumor-programmed cell death ligand 1 (PD-L1) expression. The survival benefit of CBM588 in the PD-L1 <1% cohort was higher than that in the PD-L1 1-49% and PD-L1 ≥ 50% cohorts. Furthermore, CBM588 was associated with improved overall survival in patients receiving proton pump inhibitors and/or antibiotics. CBM588-induced manipulation of the commensal microbiota holds the potential to enhance the efficacy of chemoimmunotherapy combinations, warranting further exploration of the synergy between CBM588 and immunotherapy.

Neutralizing potency of humoral immune responses induced by prior infection or vaccination is vital for protecting of individuals and population against severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). However, the emergence of viral variants that can evade neutralization by vaccine- or infection-induced immunity is a significant public health threat and requires continuous monitoring. Here, we have developed a novel scalable chemiluminescence-based assay for assessing SARS-CoV-2-induced cytopathic effect to quantify the neutralizing activity of antisera. The assay leverages the correlation between host cell viability and ATP levels in culture to measure the cytopathic effect on target cells induced by clinically isolated, replication-competent, authentic SARS-CoV-2. With this assay, we demonstrate that the recently arisen Omicron subvariants BQ.1.1 and XBB.1 display a significant decrease in sensitivity to neutralization by antibodies elicited from breakthrough infections with Omicron BA.5 and from receipt of three doses of mRNA vaccines. Thus, this scalable neutralizing assay provides a useful platform to assess the potency of acquired humoral immunity against newly emerging SARS-CoV-2 variants. IMPORTANCE The ongoing global pandemic of SARS-CoV-2 has emphasized the importance of neutralizing immunity in protecting individuals and populations against severe respiratory illness. In light of the emergence of viral variants with the potential to evade immunity, continuous monitoring is imperative. A virus plaque reduction neutralization test (PRNT) is a "gold standard" assay for analyzing neutralizing activity for authentic viruses that form plaques, like influenza virus, dengue virus, and SARS-CoV-2. However, this method is labor intensive and is not efficient for performing large-scale neutralization assays on patient specimens. The assay system established in this study allows for the detection of a patient's neutralizing activity by simply adding an ATP detection reagent, providing a simple evaluation system for neutralizing activity of antisera as an alternative to the plaque reduction method. Our extended analysis of the Omicron subvariants highlights their increasing capability to evade neutralization by both vaccine- and infection-induced humoral immunity.

MHC class I-related protein 1 (MR1) is a metabolite-presenting molecule that restricts MR1-reactive T cells including mucosal-associated invariant T (MAIT) cells. In contrast to MAIT cells, the function of other MR1-restricted T cell subsets is largely unknown. Here, we report that mice in which a T cell-specific transcription factor, B-cell lymphoma/leukemia 11B (Bcl11b), was ablated in immature thymocytes (Bcl11b∆iThy mice) develop chronic inflammation. Bcl11b∆iThy mice lack conventional T cells and MAIT cells, whereas CD4+IL-18R+ αβ T cells expressing skewed Traj33 (Jα33)+ T cell receptors (TCR) accumulate in the periphery, which are necessary and sufficient for the pathogenesis. The disorders observed in Bcl11b∆iThy mice are ameliorated by MR1-deficiency, transfer of conventional T cells, or germ-free conditions. We further show the crystal structure of the TCR expressed by Traj33+ T cells expanded in Bcl11b∆iThy mice. Overall, we establish that MR1-reactive T cells have pathogenic potential.

In a span of less than 3 years since the declaration of the coronavirus pandemic, numerous SARS-CoV-2 variants of concern have emerged all around the globe, fueling a surge in the number of cases and deaths that caused severe strain on the health care system. A major concern is whether viral evolution eventually promotes greater fitness advantages, transmissibility, and immune escape.

Although the Omicron variant of the SARS-CoV-2 virus shows resistance to neutralizing antibody, it retains susceptibility to the cellular immune response. Here we characterize vaccine-induced T cells specific for various SARS-CoV-2 variants and identified HLA-A*24:02-restricted CD8+ T cells that strongly suppress Omicron BA.1 replication in vitro. Mutagenesis analyses revealed that a G446S mutation, located just outside the N-terminus of the cognate epitope, augmented TCR recognition of this variant. In contrast, no enhanced suppression of replication is observed against cells infected with the prototype, Omicron BA.2, and Delta variants that express G446. The enhancing effect of the G446S mutation is lost when target cells are treated with inhibitors of tripeptidyl peptidase II, a protein that mediates antigen processing. These ex vivo analysis and in vitro results demonstrate that the G446S mutation in the Omicron BA.1 variant affects antigen processing/presentation and potentiates antiviral activity by vaccine-induced T cells, leading to enhanced T cell recognition towards emerging variants.

Many SARS-CoV-2 variants with naturally acquired mutations have emerged. These mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has been investigated, sensitivity to human leukocyte antigen (HLA)-restricted cellular immunity remains largely unexplored. Here, we demonstrate that two recently emerging mutations in the receptor-binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429 and B.1.617) and Y453F (in B.1.1.298), confer escape from HLA-A24-restricted cellular immunity. These mutations reinforce affinity toward the host entry receptor ACE2. Notably, the L452R mutation increases spike stability, viral infectivity, viral fusogenicity, and thereby promotes viral replication. These data suggest that HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes and that a further threat of the SARS-CoV-2 pandemic is escape from cellular immunity.

Administration of convalescent plasma or neutralizing monoclonal antibodies (mAbs) is a potent therapeutic option for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, SARS-CoV-2 variants with mutations in the spike protein have emerged in many countries. To evaluate the efficacy of neutralizing antibodies induced in convalescent patients against emerging variants, we isolate anti-spike mAbs from two convalescent COVID-19 patients infected with prototypic SARS-CoV-2 by single-cell sorting of immunoglobulin-G-positive (IgG(+)) memory B cells. Anti-spike antibody induction is robust in these patients, and five mAbs have potent neutralizing activities. The efficacy of most neutralizing mAbs and convalescent plasma samples is maintained against B.1.1.7 and mink cluster 5 variants but is significantly decreased against variants B.1.351 from South Africa and P.1 from Brazil. However, mAbs with a high affinity for the receptor-binding domain remain effective against these neutralization-resistant variants. Rapid spread of these variants significantly impacts antibody-based therapies and vaccine strategies against SARS-CoV-2.

Many studies have investigated how trehalose glycolipid structures can be modified to improve their Macrophage inducible C-type lectin (Mincle)-mediated adjuvanticity. However, in all instances, the ester-linkage of α,ά-trehalose to the lipid of choice remained. We investigated how changing this ester-linkage to an amide influences Mincle signalling and agonist activity and demonstrated that Mincle tolerates this functional group change. In in vivo vaccination studies in murine and ovine model systems, using OVA or Mannheimia haemolytica and Mycoplasma ovipneumoniae as vaccine antigens, respectively, it was demonstrated that a representative trehalose diamide glycolipid was able to enhance antibody-specific immune responses. Notably, IgG titres against M. ovipneumoniae were significantly greater when using trehalose dibehenamide (A-TDB) compared to trehalose dibehenate (TDB). This is particularly important as infection with M. ovipneumoniae predisposes sheep to pneumonia.

Mucosal-associated invariant T (MAIT) cells are an abundant subset of innate-like T lymphocytes. MAIT cells are activated by microbial riboflavin-derived antigens, such as 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), when presented by the major histocompatibility complex (MHC) class I-related protein (MR1). We have synthesized all stereoisomers of 5-OP-RU to investigate the effects of its stereochemistry on the MR1-dependent MAIT cell activation and MR1 upregulation. The analysis of MAIT cell activation by these 5-OP-RU isomers revealed that the stereocenters at the 2’- and 3’-OH groups in the ribityl tail are crucial for the recognition of MAIT-TCR, whereas that of 4’-OH group does not significantly affect the regulation of MAIT cell activity. Furthermore, kinetic analysis of complex formation between the ligands and MR1 suggested that 5-OP-RU forms a covalent bond to MR1 in cells within 1 hour. These findings provide guidelines for designing ligands that regulate MAIT cell functions.

Mucosal-associated invariant T (MAIT) are a subset of innate-like T cells that are activated by uracil ligands presented by MR1. For the first time, we demonstrate that changes to the 6-aminoalkyl chain on uracil agonist 5-OP-RU can determine agonistic or antagonistic MAIT cell activity. Insomuch, a simplified agonist with a functional profile similar to 5-OP-RU, and a new structural class of antagonist that exhibits similar activity to known MAIT cell antagonist Ac-6-FP, were identified.

Populations of CD8+ lung-resident memory T (TRM) cells persist in the interstitium and epithelium (airways) following recovery from respiratory virus infections. While it is clear that CD8+ TRM cells in the airways are dynamically maintained via the continuous recruitment of new cells, there is a vigorous debate about whether tissue-circulating effector memory T (TEM) cells are the source of these newly recruited cells. Here we definitively demonstrate that CD8+ TRM cells in the lung airways are not derived from TEM cells in the circulation, but are seeded continuously by TRM cells from the lung interstitium. This process is driven by CXCR6 that is expressed uniquely on TRM cells but not TEM cells. We further demonstrate that the lung interstitium CD8+ TRM cell population is also maintained independently of TEM cells via a homeostatic proliferation mechanism. Taken together, these data show that lung memory CD8+ TRM cells in the lung interstitium and airways are compartmentally separated from TEM cells and clarify the mechanisms underlying their maintenance.

C-type lectin receptors (CLRs) comprise a large family of immunoreceptors that recognize polysaccharide ligands exposed on pathogen surfaces and are conserved among mammals. However, interspecies differences in their ligand spectrums are not fully understood. Dectin-1 is a well-characterized CLR that recognizes -glucan. We report here that seaweed-derived fucan activates cells expressing human Dectin-1 but not mouse Dectin-1. Low-valency -glucan components within fucan appeared to be responsible for this activation, as the ligand activity was eliminated by -glucanase treatment. The low-valency -glucan laminarin also acted as an agonist for human Dectin-1 but not for mouse Dectin-1, whereas the high-valency -glucan curdlan activated both human and mouse Dectin-1. Reciprocal mutagenesis analysis revealed that the ligand-binding domain of human Dectin-1 does not determine its unique sensitivity to low-valency -glucan. Rather, we found that its intracellular domain renders human Dectin-1 reactive to low-valency -glucan ligand. Substitution with two amino acids, Glu(2) and Pro(5), located in the human Dectin-1 intracellular domain was sufficient to confer sensitivity to low-valency -glucan in mouse Dectin-1. Conversely, the introduction of mouse-specific amino acids, Lys(2) and Ser(5), to human Dectin-1 reduced the reactivity to low-valency -glucan. Indeed, low-valency ligands induced a set of proinflammatory genes in human but not mouse dendritic cells. These results suggest that the intracellular domain, not ligand-binding domain, of Dectin-1 determines the species-specific ligand profile.

CD8(+) tissue-resident memory T cells (T-RM cells) reside permanently in nonlymphoid tissues and provide a first line of protection against invading pathogens. However, the precise localization of CD8(+) T-RM cells in the lung, which physiologically consists of a markedly scant interstitium compared with other mucosa, remains unclear. In this study, we show that lung CD8(+) T-RM cells localize predominantly in specific niches created at the site of regeneration after tissue injury, whereas peripheral tissue-circulating CD8(+) effector memory T cells (T-EM cells) are widely but sparsely distributed in unaffected areas. Although CD69 inhibited sphingosine 1-phosphate receptor 1-mediated egress of CD8(+) T cells immediately after their recruitment into lung tissues, such inhibition was not required for the retention of cells in the T-RM niches. Furthermore, despite rigid segregation of T-EM cells from the T-RM niche, prime-pull strategy with cognate antigen enabled the conversion from T-EM cells to T-RM cells by creating de novo T-RM niches. Such damage site-specific localization of CD8(+) T-RM cells may be important for efficient protection against secondary infections by respiratory pathogens.

The non-obese diabetic mouse model of type 1 diabetes continues to be an important tool for delineating the role of T-cell mediated destruction of pancreatic beta-cells. However, little is known about the molecular mechanisms that enable this disease pathway. We show that insulin reactivity by a CD8(+) T-cell clone, known to induce type 1 diabetes, is characterized by weak T-cell antigen receptor binding to a relatively unstable peptide-MHC. The structure of the native 9- and 10-mer insulin epitopes demonstrated that peptide residues 7 and 8 form a prominent solvent-exposed bulge that could potentially be the main focus of T-cell receptor binding. The C terminus of the peptide governed peptide-MHC stability. Unexpectedly, we further demonstrate a novel mode of flexible peptide presentation in which the MHC peptide-binding groove is able to "open the back door" to accommodate extra C-terminal peptide residues.

Emerging data indicate that particular major histocompatibility complex (MHC)-bound antigenic peptides can be recognized by identical or near-identical T cell receptors (TCRs) in different individuals. To establish the functional relevance of this phenomenon, we artificially paired and chains from closely related TCRs specific for the human leucocyte antigen (HLA)-B*35:01-restricted HIV-1 negative regulatory factor (Nef)-derived epitope VY8 (VPLRPMTY, residues 74-81). Several hybrid TCRs generated in this manner failed to express at the cell surface, despite near homology with naturally isolated chain combinations. Moreover, a substantial proportion of those TCRs that did express lost specificity for the index VY8 peptide sequence. One such hybrid pair gained neo-variant specificity in the context of the VY8 backbone. Collectively, these data show that clonotypically similar TCRs can display profound differences in surface expression, antigen specificity and cross-reactivity with potential relevance for the control of mutable viruses.

Overlapping peptides of different lengths from a certain immunodominant region can be presented by the same HLA class I molecule and elicit different T cell responses. However, how peptide-length specificity of antigen-specific CD8(+)T lymphocytes influence cross-reactivity profiles of these cells remains elusive. This question is particularly important in the face of highly variable pathogens such as HIV-1. Here, we examined this problem by using HLA-B*35:01-restricted CD8(+) T lymphocytes specific for Nef epitopes, i.e., RY11 (RPQVPLRPMTY), VY8 (VPLRPMTY), and RM9 (RPQVPLRPM), in which VY8 and RM9 were contained entirely within RY11, in combination with a T cell receptor (TCR) reconstruction system as well as HLA-B35 tetramers and a set of a single-variant peptide library. The TCR reactivity toward the peptide-length variants was classified into three types: mutually exclusive specificity toward (1) RY11 or (2) VY8 and (3) cross-recognition toward RM9 and RY11. TCR cross-reactivity toward variant peptides was similar within the same peptide-length reactivity type but was markedly different between the types. Thus, TCRs showing similar peptide-length reactivity have shared peptide recognition footprints and thereby similar weakness to antigenic variations, providing us with further insight into the antiviral vaccine design. (C) 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

CD8(+) CTL responses directed toward the HLA-B*51:01-restricted HIV-RT128-135 epitope TAFTIPSI (TI8) are associated with long-term nonprogression to AIDS. Clonotypic analysis of responses to B51-TI8 revealed a public clonotype using TRAV17/TRBV7-3 TCR genes in six out of seven HLA-B*51:01(+) patients. Structural analysis of a TRAV17/TRBV7-3 TCR in complex with HLA-B51-TI8, to our knowledge the first human TCR complexed with an 8-mer peptide, explained this bias, as the unique combination of residues encoded by these genes was central to the interaction. The relatively featureless peptide-MHC (pMHC) was mainly recognized by the TCR CDR1 and CDR2 loops in an MHC-centric manner. A highly conserved residue Arg(97) in the CDR3 alpha loop played a major role in recognition of peptide and MHC to form a stabilizing ball-and-socket interaction with the MHC and peptide, contributing to the selection of the public TCR clonotype. Surface plasmon resonance equilibrium binding analysis showed the low affinity of this public TCR is in accordance with the only other 8-mer interaction studied to date (murine 2C TCR-H-2K(b)-dEV8). Like pMHC class II complexes, 8-mer peptides do not protrude out the MHC class I binding groove like those of longer peptides. The accumulated evidence suggests that weak affinity might be a common characteristic of TCR binding to featureless pMHC landscapes.

Antigen cross-reactivity is an inbuilt feature of the T cell compartment. However, little is known about the flexibility of T cell recognition in the context of genetically variable pathogens such as HIV-1. In this study, we used a combinatorial library containing 24 billion octamer peptides to characterize the cross-reactivity profiles of CD8(+) T cells specific for the immunodominant HIV-1 subtype B Nef epitope VY8 (VPLRPMTY) presented by HLA-B*35:01. In conjunction, we examined naturally occurring antigenic variations within the VY8 epitope. Sequence analysis of plasma viral RNA isolated from 336 HIV-1-infected individuals revealed variability at position (P) 3 and P8 of VY8; Phe at P8, but not Val at P3, was identified as an HLA-B*35:01-associated polymorphism. VY8-specific T cells generated from several different HIV-1-infected patients showed unique and clonotype-dependent cross-reactivity footprints. Nonetheless, all T cells recognized both the index Leuandmutant Val atP3 equally well. In contrast, competitive titration assays revealed that the Tyr to Phe substitution at P8 reduced T cell recognition by 50-130 fold despite intact peptide binding to HLA-B*35:01. These findings explain the preferential selection of Phe at the C-terminus of VY8 in HLA-B*35:01(+) individuals and demonstrate that HIV-1 can exploit the limitations of T cell recognition in vivo.

Untreated human immunodeficiency virus (HIV) infections usually lead to death from AIDS, although the rate of the disease progression varies widely among individuals. The cytotoxic T lymphocyte (CTL) response, which is restricted by highly polymorphic MHC class I alleles, plays a central role in controlling HIV replication. It is now recognized that the antiviral efficacy of CTLs at the single cell level is dependent on their antigen specificity and is important in determining the quality of host response to viruses so that the individual will remain asymptomatic. However, because of the extreme mutational plasticity of HIV, HIV-specific CTL responses are continuously and dynamically changing. In order to rationally design an effective vaccine, the questions as to what constitutes an effective antiviral CTL response and what characterizes a potent antigenic peptide to induce such responses are becoming highlighted as needing to be answered.

The antiviral activity of HIV-specific CTL is not equally potent but rather is dependent on their specificity. But what characteristic of targeted peptides influences CTL antiviral activity remains elusive. We addressed this issue based on HLA-B35-restrieted CTLs specific for two overlapping immunodominant Nef epitopes, VY8 (VPLRPMTY) and RY11 (RPQVPLRPMTY). VY8-specific CTLs were more potently cytotoxic toward HIV-infected primary CD4(+) cells than RY11-specific CTLs. Reconstruction of their TCR revealed no substantial difference in their functional avidity toward cognate Ags. Instead, the decay analysis of the peptide-MHC complex (pMHC) revealed that the VY8/HLA-B35 complex could maintain its capacity to sensitize T cells much longer than its RY11 counterpart. Corroboratively, the introduction of a mutation in the epitopes; that substantially delayed pMHC decay rendered Nef-expressing target cells more susceptible to CTL, killing. Moreover, by using differential scanning calorimetry and circular dichroism analyses, we found that the susceptible pMHC ligands for CTL, killing showed interdependent and cooperative, rather than separate or sequential, transitions within their heterotrimer components under the thermally induced unfolding process. Collectively, our results highlight the significant effects of intrinsic peptide factors that support cooperative thermodynamics within pMHC on the efficient CTL. killing of HIV-infected cells, thus providing us better insight into vaccine design. The Journal of Immunology, 2009, 182: 5528-5536.

HIV-1 Nef plays multiple roles in modulating immune responses, even though it is a dominant CTL target itself. How Nef accomplishes the balance between such conflicting selective pressures remains elusive. By genetic and functional studies, we found that Arg(75)Thr and Tyr(85)Phe mutations, located in a well-conserved proline-rich region in Nef, were differently associated with escape from CTL responses specific for two overlapping HLA-B35-restricted epitopes. CTLs specific for an epitope, that selected Tyr(85)Phe, were elicited earlier and had more potent functional avidities than did those that selected Arg(75)Thr. Although the double mutant could escape from both CTLs, the mutations are rarely observed in combination naturally. Introduction of both mutations reduced Nef's HLA class I down-regulation activity and increased the susceptibility of virus-infected cells to recognition by CTLs targeting other epitopes. Moreover, the mutant Nef was impaired in the association with activated cellular kinases and in the enhancement of viral replication. These results highlight CTL immunosurveillance as important modulators of Nef's biological activity in the infected host.

The mutational escape of HIV-1 from established CTL responses is becoming evident. However, it is not yet clear whether antigenic variations of HIV-1 may have an additional effect on the differential antiviral effectiveness of HIV-specific CTLs. Herein, we characterized HIV-specific CTL responses toward Pol, Env, and Nef optimal epitopes presented by HLA-B*35 during a chronic phase of HIV-1 infection. We found CTL escape variants within Pol and Nef epitopes that affected recognition by TCRs, although there was no mutation within the Env epitope. An analysis of peptide-HLA tetrameric complexes revealed that CD8 T cells exclusively specific for the Nef variant were generated following domination by the variant viruses. The variant-specific cells were capable of killing target cells and producing antiviral cytokines but showed impaired Ag-specific proliferation ex vivo, whereas wild-type specific cells had potent activities. Moreover, clonotypic CD8 T cells specific for the Pol variant showed diminished proliferation, whereas Env-specific ones had no functional heterogeneity. Taken together, our data indicate that antigenic variations that abolished TCR recognition not only resulted in escape from established CTL responses but also eventually generated another subset of variant-specific CTLs having decreased antiviral activity, causing an additional negative effect on antiviral immune responses during a chronic HIV infection.

T細胞抗原受容体 (TCR) を用いたがんのTCR-T細胞療法は、免疫チェックポイント阻害剤療法を補完する免疫学的がん治療法のひとつとして注目されている。課題として、治療に使われる細胞傷害性CD8陽性T細胞のTCRが主要組織適合抗原 (HLA) クラスI分子に拘束されており、HLAクラスI分子の多型性のために、1人の患者から取得した腫瘍特異的TCRを、HLAの異なる別の患者の治療に用いることができないことがある。我々はこれまでに乳癌患者の腫瘍浸潤CD8陽性T細胞のTCRを単一細胞レベルで解析し、その中から患者HLAクラスI分子に関係なく腫瘍細胞に反応するTCRを見出した。本研究の目的として、取得したHLAクラスI非拘束性腫瘍反応性TCRの抗原提示分子を明らかにし、抗原分子を同定することが掲げられている。令和3年度は抗原提示分子を明らかにするためにHLAクラスI分子、β2-ミクログロブリン分子、MR1分子をそれぞれノックアウトした乳がん細胞株を作製し、各TCRの反応性を解析した。その結果、取得した全てのHLAクラスI非拘束性TCRはβ2-ミクログロブリン及びMR1分子をノックアウトした乳がん細胞株に反応することができなかった。このことより、我々が取得したHLAクラスI非拘束性TCRはMR１分子に提示された抗原を認識することがわかった。MR1拘束性のT細胞としてMucosal associated invariant T (MAIT)細胞が知られており、そのTCRに結合する抗原/MR1分子複合体テトラマーが作製されている。我々はそのテトラマーが同定したTCRに結合するか調べたが、結合しなかった。以上の結果より、我々が同定したHLAクラスI非拘束性TCRはMR1分子に提示される抗原を認識するが、既知のMAIT細胞の抗原とは異なるものを認識することがわかった。

Although the activating signals are balanced by inhibitory receptor-mediated signals, which maintains immune homeostasis, ligand recognitions and functions via inhibitory innate-receptors bearing ITIMs (Immunoreceptor tyrosine-based inhibitory motifs) remains unclear. To identify their endogenous ligands, we employed nuclear factor of activated T-cells (NFAT)-GFP reporter cells expressing chimeric inhibitory CLRs fused to CD3ζ and analyzed the reporter activity in response to various components. We identified the structure of their lipid ligands from plasma membrane and Mycobacteria. Mycobacteria infection experiment revealed that T cell recall responses were substantially enhanced in mice lacking the inhibitory receptor. These data indicate that blockade of the inhibitory receptor interaction and signaling potentiate cellular immunity against mycobacteria.

Although the activating signals are balanced by inhibitory receptor-mediated signals, which maintains immune homeostasis, ligand recognitions and functions via inhibitory immune receptor remains unclear. In this study, we identified a novel ligand recognized by an inhibitory immune receptor and revealed the inhibitory function.

Populations of CD8 lung-resident memory T (TRM) cells persist in the interstitium and airways following recovery from respiratory virus infections. While it is clear that CD8 TRM cells in the airways are dynamically maintained via the continuous recruitment of new cells, there is a vigorous debate whether tissue-circulating effector memory T (TEM) cells are the source of these newly recruited cells. Here we definitively demonstrate that CD8 TRM in the lung airways are not derived from TEM in the circulation, but are seeded continuously by TRM from the lung interstitium. This process is driven by CXCR6 that is expressed on TRM, but not TEM. We further demonstrate that the lung interstitium CD8 TRM population is also maintained independently of TEM via a homeostatic proliferation mechanism. Taken together, these data show that lung memory CD8 TRM cells in the lung interstitium and airways are compartmentally separated from TEM and clarify the mechanisms underlying their maintenance.

TLR7 is expressed on several types of cell including dendritic cells and B cells. In this study, we analyzed the requirement of B-cell intrinsic TLR7 for the protection of retrovirus-induced leukemia. Mice lacking of B cell-intrinsic TLR7 produced virus-neutralizing antibodies as efficiently as the wild-type mice by priming of T helper cells, and rapid elimination of exogenous retroviruses were observed. Nevertheless,the B cell-intrinsic TLR7 lacking mice later died of leukemia. These results suggest that B cell-intrinsic TLR7 is essential to regulate the appearance of endogenous and/or recombinant retroviruses even when the virus-specific Th cells are efficiently activated.

Understanding functional characteristics of T-cell responses associated with effective elimination of pathogenic retroviruses or tumors is crucial for the development of vaccines. In this study, we demonstrated that the use of an appropriate epitope preferentially induces functional T cell responses in the early phase of acute retroviral infection that contribute to the effective antiviral protection, providing a better information for the development of vaccine.