Limited maintenance of vaccine-induced simian immunodeficiency virus-specific CD8 T-cell receptor clonotypes after virus challenge

T-cell receptors (TCRs) govern the specificity, efficacy, and cross-reactivity of CD8 T cells. Here, we studied CD8 T-cell clonotypes from Mane-A*10(+) pigtail macaques responding to the simian immunodeficiency virus (SIV) Gag KP9 epitope in a setting of vaccination and subsequent viral challenge. We observed a diverse TCR repertoire after DNA, recombinant poxvirus, and live attenuated virus vaccination, with none of 59 vaccine-induced KP9-specific TCRs being identical between macaques. The KP9-specific TCR repertoires remained diverse after SIV or simian-human immunodeficiency virus challenge but, remarkably, exhibited substantially different clonotypic compositions compared to the corresponding populations prechallenge. Within serial samples from individual pigtail macaques, only a small subset (33.9%) of TCRs induced by vaccination were maintained or expanded after challenge. Most (66.1%) of the TCRs induced by vaccination were not detectable after challenge. Our results suggest that some CD8 T cells induced by vaccination are more efficient than others at responding to a viral challenge. These findings have implications for future AIDS virus vaccine studies, which should consider the "fitness" of vaccine-induced T cells in order to generate robust responses in the face of virus exposure.     

Different development of myelin basic protein agonist- and antagonist-specific human TCR transgenic T cells in the thymus and periphery

Myelin basic protein (MBP)-specific T cells are thought to play a role in the development of multiple sclerosis. MBP residues 111-129 compose an immunodominant epitope cluster restricted by HLA-DRB1*0401. The sequence of residues 111-129 of MBP (MBP(111-129)) differs in humans (MBP122:Arg) and mice (MBP122:Lys) at aa 122. We previously found that approximately 50% of human MBP(111-129) (MBP122:Arg)-specific T cell clones, including MS2-3C8 can proliferate in response to mouse MBP(111-129) (MBP122:Lys). However, the other half of T cell clones, including HD4-1C2, cannot proliferate in response to MBP(111-129) (MBP122:Lys). We found that MBP(111-129) (MBP122:Lys) is an antagonist for HD4-1C2 TCR, therefore, MS2-3C8 and HD4-1C2 TCRs are agonist- and antagonist-specific TCRs in mice, respectively. Therefore, we examined the development of HD4-1C2 TCR and MS2-3C8 TCR transgenic (Tg) T cells in the thymus and periphery. We found that dual TCR expression exclusively facilitates the development of MBP(111-129) TCR Tg T cells in the periphery of HD4-1C2 TCR/HLA-DRB1*0401 Tg mice although it is not required for their development in the thymus. We also found that MS2-3C8 TCR Tg CD8(+) T cells develop along with MS2-3C8 TCR Tg CD4(+) T cells, and that dual TCR expression was crucial for the development of MS2-3C8 TCR Tg CD4(+) and CD8(+) T cells in the thymus and periphery, respectively. These results suggest that thymic and peripheral development of MBP-specific T cells are different; however, dual TCR expression can facilitate their development.     

Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor

HIV's considerable capacity to vary its HLA-I-restricted peptide antigens allows it to escape from host cytotoxic T lymphocytes (CTLs). Nevertheless, therapeutics able to target HLA-I-associated antigens, with specificity for the spectrum of preferred CTL escape mutants, could prove effective. Here we use phage display to isolate and enhance a T-cell antigen receptor (TCR) originating from a CTL line derived from an infected person and specific for the immunodominant HLA-A(*)02-restricted, HIVgag-specific peptide SLYNTVATL (SL9). High-affinity (K(D) < 400 pM) TCRs were produced that bound with a half-life in excess of 2.5 h, retained specificity, targeted HIV-infected cells and recognized all common escape variants of this epitope. CD8 T cells transduced with this supraphysiologic TCR produced a greater range of soluble factors and more interleukin-2 than those transduced with natural SL9-specific TCR, and they effectively controlled wild-type and mutant strains of HIV at effector-to-target ratios that could be achieved by T-cell therapy.     

Virus-specific and bystander CD8 T cells recruited during virus-induced encephalomyelitis

Neurotropic coronavirus-induced encephalitis was used to evaluate recruitment, functional activation, and retention of peripheral bystander memory CD8+ T cells. Mice were first infected with recombinant vaccinia virus expressing a non-cross-reactive human immunodeficiency virus (HIV) epitope, designated p18. Following establishment of an endogenous p18-specific memory CD8+ T-cell population, mice were challenged with coronavirus to directly compare recruitment, longevity, and activation characteristics of both primary coronavirus-specific and bystander memory populations trafficking into the central nervous system (CNS). HIV-specific memory CD8+ T cells were recruited early into the CNS as components of the innate immune response, preceding CD8+ T cells specific for the dominant coronavirus epitope, designated pN. Although pN-specific T-cell numbers gradually exceeded bystander p18-specific CD8+ T-cell numbers, both populations peaked concurrently within the CNS. Nevertheless, coronavirus-specific CD8+ T cells were preferentially retained. By contrast, bystander CD8+ T-cell numbers declined to background numbers following control of CNS virus replication. Furthermore, in contrast to highly activated pN-specific CD8+ T cells, bystander p18-specific CD8+ T cells recruited to the site of inflammation maintained a nonactivated memory phenotype and did not express ex vivo cytolytic activity. Therefore, analysis of host CD8+ T-cell responses to unrelated infections demonstrates that bystander memory CD8+ T cells can comprise a significant proportion of CNS inflammatory cells during virus-induced encephalitis. However, transient CNS retention and the absence of activation suggest that memory bystander CD8+ T cells may not overtly contribute to pathology in the absence of antigen recognition.     

Mutually exclusive T-cell receptor induction and differential susceptibility to human immunodeficiency virus type 1 mutational escape associated with a two-amino-acid difference between HLA class I subtypes

The relative contributions of HLA alleles and T-cell receptors (TCRs) to the prevention of mutational viral escape are unclear. Here, we examined human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) T-cell responses restricted by two closely related HLA class I alleles, B*5701 and B*5703, that differ by two amino acids but are both associated with a dominant response to the same HIV-1 Gag epitope KF11 (KAFSPEVIPMF). When this epitope is presented by HLA-B*5701, it induces a TCR repertoire that is highly conserved among individuals, cross-recognizes viral epitope variants, and is rarely associated with mutational escape. In contrast, KF11 presented by HLA-B*5703 induces an entirely different, more heterogeneous TCR beta-chain repertoire that fails to recognize specific KF11 escape variants which frequently arise in clade C-infected HLA-B*5703(+) individuals. These data show the influence of HLA allele subtypes on TCR selection and indicate that extensive TCR diversity is not a prerequisite to prevention of allowable viral mutations.     

Oligomannose-coated liposomes efficiently induce human T-cell leukemia virus-1-specific cytotoxic T lymphocytes without adjuvant

Human T-cell leukemia virus-1 (HTLV-1) causes adult T-cell leukemia/lymphoma, which is an aggressive peripheral T-cell neoplasm. Insufficient T-cell response to HTLV-1 is a potential risk factor in adult T-cell leukemia/lymphoma. Efficient induction of antigen-specific cytotoxic T lymphocytes is important for immunological suppression of virus-infected cell proliferation and oncogenesis, but efficient induction of antigen-specific cytotoxic T lymphocytes has evaded strategies utilizing poorly immunogenic free synthetic peptides. Here, we examined the efficient induction of an HTLV-1-specific CD8+ T-cell response by oligomannose-coated liposomes (OMLs) encapsulating the human leukocyte antigen (HLA)-A*0201-restricted HTLV-1 Tax-epitope (OML/Tax). Immunization of HLA-A*0201 transgenic mice with OML/Tax induced an HTLV-1-specific gamma-interferon reaction, whereas immunization with epitope peptide alone induced no reaction. Upon exposure of dendritic cells to OML/Tax, the levels of CD86, major histocompatibility complex class I, HLA-A02 and major histocompatibility complex class II expression were increased. In addition, our results showed that HTLV-1-specific CD8+ T cells can be efficiently induced by OML/Tax from HTLV-1 carriers compared with epitope peptide alone, and these HTLV-1-specific CD8+ T cells were able to lyse cells presenting the peptide. These results suggest that OML/Tax is capable of inducing antigen-specific cellular immune responses without adjuvants and may be useful as an effective vaccine carrier for prophylaxis in tumors and infectious diseases by substituting the epitope peptide.     

Structural Basis for the Presentation of Tumor-Associated MHC Class II-Restricted Phosphopeptides to CD4 T Cells

Dysregulated protein phosphorylation is a hallmark of malignant transformation. Transformation can generate major histocompatibility complex (MHC)-bound phosphopeptides that are differentially displayed on tumor cells for specific recognition by T cells. To understand how phosphorylation alters the antigenic identity of self-peptides and how MHC class II molecules present phosphopeptides for CD4⁺ T-cell recognition, we determined the crystal structure of a phosphopeptide derived from melanoma antigen recognized by T cells-1 (pMART-1), selectively expressed by human melanomas, in complex with HLA-DR1. The structure revealed that the phosphate moiety attached to the serine residue at position P5 of pMART-1 is available for direct interactions with T-cell receptor (TCR) and that the peptide N-terminus adopts an unusual conformation orienting it toward TCR. This structure, combined with measurements of peptide affinity for HLA-DR1 and of peptide-MHC recognition by pMART-1-specific T cells, suggests that TCR recognition is focused on the N-terminal portion of pMART-1. This recognition mode appears to be distinct from that of foreign antigen complexes but is remarkably reminiscent of the way autoreactive TCRs engage self- or altered self-peptides, consistent with the tolerogenic nature of tumor-host immune interactions.     

Effect of MHC class I diversification on influenza epitope-specific CD8+ T cell precursor frequency and subsequent effector function

Earlier studies of influenza-specific CD8(+) T cell immunodominance hierarchies indicated that expression of the H2K(k) MHC class I allele greatly diminishes responses to the H2D(b)-restriced D(b)PA(224) epitope (acid polymerase, residues 224-233 complexed with H2D(b)). The results suggested that the presence of H2K(k) during thymic differentiation led to the deletion of a prominent Vβ7(+) subset of D(b)PA(224)-specific TCRs. The more recent definition of D(b)PA(224)-specific TCR CDR3β repertoires in H2(b) mice provides a new baseline for looking again at this possible H2K(k) effect on D(b)PA(224)-specific TCR selection. We found that immune responses to several H2D(b)- and H2K(b)-restricted influenza epitopes were indeed diminished in H2(bxk) F(1) versus homozygous mice. In the case of D(b)PA(224), lower numbers of naive precursors were part of the explanation, though a similar decrease in those specific for the D(b)NP(366) epitope did not affect response magnitude. Changes in precursor frequency were not associated with any major loss of TCR diversity and could not fully account for the diminished D(b)PA(224)-specific response. Further functional and phenotypic characterization of influenza-specific CD8(+) T cells suggested that the expansion and differentiation of the D(b)PA(224)-specific set is impaired in the H2(bxk) F(1) environment. Thus, the D(b)PA(224) response in H2(bxk) F(1) mice is modulated by factors that affect the generation of naive epitope-specific precursors and the expansion and differentiation of these T cells during infection, rather than clonal deletion of a prominent Vβ7(+) subset. Such findings illustrate the difficulties of predicting and defining the effects of MHC class I diversification on epitope-specific responses.     

Detection of virus-specific T cells and CD8+ T-cell epitopes by acquisition of peptide-HLA-GFP complexes: analysis of T-cell phenotype and function in chronic viral infections

Antigen-specific CD8+ T cells acquire peptide-major histocompatibility complex (MHC) clusters through T-cell receptor (TCR)-mediated endocytosis after specific antigen stimulation. We generated an antigen-presenting cell (APC) expressing human leukocyte antigen (HLA)-A*201 coupled to the enhanced green fluorescent protein (GFP), which delivered GFP to an antigen-specific T cell when pulsed with antigenic peptide. We quantitatively identified human T-cell lymphotropic virus type I (HTLV-I) Tax(11-19) peptide-specific T-cell populations in peripheral blood mononuclear cells (PBMCs) from patients with HTLV-I-associated neurologic disease and defined a new CD8+ T-cell epitope in the HTLV-I envelope region. Acquisition of peptide-HLA-GFP complexes by antigen-specific T cells could distinguish, with respect to phenotype and perforin production, T cells from the chronic viral infections cytomegalovirus and HTLV-I. This approach will be a powerful tool in understanding the role of antigen-specific T-cell responses in health and disease.     

Relationship between CD8-dependent antigen recognition,T cell functional avidity,and tumor cell recognition

Effective immunotherapy using T cell receptor (TCR) gene-modified T cells requires an understanding of the relationship between TCR affinity and functional avidity of T cells. In this study, we evaluate the relative affinity of two TCRs isolated from HLA-A2-restricted, gp100-reactive T cell clones with extremely high functional avidity. Furthermore, one of these T cell clones, was CD4⁻CD8⁻ indicating that antigen recognition by this clone was CD8 independent. However, when these TCRs were expressed in CD8⁻ Jurkat cells, the resulting Jurkat cells recognized gp100:209–217 peptide loaded T2 cells and had high functional avidity, but could not recognize HLA-A2⁺ melanoma cells expressing gp100. Tumor cell recognition by Jurkat cells expressing these TCRs could not be induced by exogenously loading the tumor cells with the native gp100:209–217 peptide. These results indicate that functional avidity of a T cell does not necessarily correlate with TCR affinity and CD8-independent antigen recognition by a T cell does not always mean its TCR will transfer CD8-independence to other effector cells. The implications of these findings are that T cells can modulate their functional avidity independent of the affinity of their TCRs. Companion Paper: “Characterization of MHC class-I restricted TCRαβ⁺ CD4⁻ CD8⁻ double negative T cells recognizing the gp100 antigen from a melanoma patient after gp100 vaccination” by Simon Voelkl, Tamson V. Moore, Michael Rehli, Michael I. Nishimura, Andreas Mackensen, and Karin Fischer. doi:.     

Multimolecular associations of the T-cell antigen receptor

T cells are activated when the T-cell receptor for antigen (TCR) interacts with an antigenic peptide bound to a major histocompatibility complex (MHC) molecule on the surface of another cell. It is often assumed that T-cell activation is induced by the crosslinking of TCRs. In this article, Albertus Beyers, Louise Spruyt and Alan Williams argue that this mechanism is not generally applicable. They hypothesize that the key event in T-cell activation is the formation of multimolecular complexes consisting of the TCR and several other polypeptides, including CD4 or CD8, CD2, CD5 and the associated tyrosine kinases p59(fyn) and p56(lck).     

Structural basis of biased T cell receptor recognition of an immunodominant HLA-A2 epitope of the SARS-CoV-2 spike protein

CD8⁺ T cells play an important role in vaccination and immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although numerous SARS-CoV-2 CD8⁺ T cell epitopes have been identified, the molecular basis underpinning T cell receptor (TCR) recognition of SARS-CoV-2-specific T cells remains unknown. The T cell response directed toward SARS-CoV-2 spike protein–derived S^269–277 peptide presented by the human leukocyte antigen (HLA)-A∗02:01 allomorph (hereafter the HLA-A2^S269–277 epitope) is, to date, the most immunodominant SARS-CoV-2 epitope found in individuals bearing this allele. As HLA-A2^S269–277-specific CD8⁺ T cells utilize biased TRAV12 gene usage within the TCR α-chain, we sought to understand the molecular basis underpinning this TRAV12 dominance. We expressed four TRAV12⁺ TCRs which bound the HLA-A2^S269–277 complex with low micromolar affinity and determined the crystal structure of the HLA-A2^S269–277 binary complex, and subsequently a ternary structure of the TRAV12⁺ TCR complexed to HLA-A2^S269–277. We found that the TCR made extensive contacts along the entire length of the S^269–277 peptide, suggesting that the TRAV12⁺ TCRs would be sensitive to sequence variation within this epitope. To examine this, we investigated cross-reactivity toward analogous peptides from existing SARS-CoV-2 variants and closely related coronaviruses. We show via surface plasmon resonance and tetramer studies that the TRAV12⁺ T cell repertoire cross-reacts poorly with these analogous epitopes. Overall, we defined the structural basis underpinning biased TCR recognition of CD8⁺ T cells directed at an immunodominant epitope and provide a framework for understanding TCR cross-reactivity toward viral variants within the S^269–277 peptide.     

Molecular analysis of TCR and peptide/MHC interaction using P18-I10-derived peptides with a single D-amino acid substitution

For the structural analysis of T-cell receptor (TCR) and peptide/MHC interaction, a series of peptides with a single amino acid substitution by a corresponding D-amino acid, having the same weight, size, and charge, within P18-I10 (aa318-327: RGPGRAFVTI), an immunodominant epitope of HIV-1 IIIB envelope glycoprotein, restricted by the H-2Dd class I MHC molecule, has been synthesized. Using those peptides, we have observed that the replacement at positions 324F, 325V, 326T, and 327I with each corresponding D-amino acid induced marked reduction of the potency to sensitize targets for P18-I10-specific murine CD8+ cytotoxic T lymphocytes (CTLs), LINE-IIIB, recognition. To analyze further the role of amino acid at position 325, the most critical site for determining epitope specificity, we have developed a CTL line [LINE-IIIB(325D)] and its offspring clones specific for the epitope I-10(325v) having a D-valine (v) at position 325. Taking advantage of two distinct sets of CD8+ CTLs restricted by the same Dd, three-dimensional structural analysis on TCR and peptide/MHC complexes by molecular modeling was performed, which indicates that the critical amino acids within the TCRs for interacting with 325V or 325v appear to belong to the complementarity-determining region 1 but not to the complementarity-determining region 3 of Vbeta chain.     

The molecular basis for public T-cell responses?

Public T-cell responses, in which T cells bearing identical T-cell receptors (TCRs) are observed to dominate the response to the same antigenic epitope in multiple individuals, have long been a focus of immune T-cell repertoire studies. However, the mechanism that enables the survival of a specific TCR from the diverse repertoire produced in the thymus through to its involvement in a public immune response remains unclear. In this Opinion article, we propose that the frequency of production of T cells bearing different TCRs during recombination has an important role in the sharing of TCRs in an immune response, with variable levels of 'convergent recombination' driving production frequencies.     

Impaired specific CD8 T cell response with aging is not due to decreased expression of CD90 on TCR transgenic T cells

Background

CD90 (Thy-1) is a small glycoprotein that is particularly abundant on the surface of mouse thymocytes and peripheral T cells, and is often used as a marker in adoptive transfer experiments to distinguish donor and recipient T cells with different CD90 subtypes. We have performed adoptive transfer experiments with T cell receptor transgenic (TCR Tg) mice to study the impaired CD8 T cell response with aging.

Findings

After stimulation with a CD8 T cell epitope, HA_518-524, the response of TCR Tg CD8 T cells from aged mice was decreased compared to the response of TCR Tg T cells from young mice. CD90 expression was also substantially decreased on the TCR Tg CD8 T cells of aged mice. However, the responses of CD90^hi and CD90^low CD8 T cells of the aged mice were similar in both early activation and proliferation, demonstrating that the impaired Tg T cell response with aging is not associated with the altered CD90 expression on CD8 T cells.

Conclusions

The impaired Tg CD8 T cell response in aged mice is not due to age-associated changes in CD90 expression on Tg CD8 T cells.

     

Highly protective in vivo function of cytomegalovirus IE1 epitope-specific memory CD8 T cells purified by T-cell receptor-based cell sorting

Reconstitution of antiviral CD8 T cells is essential for controlling cytomegalovirus (CMV) infection after bone marrow transplantation. Accordingly, polyclonal CD8 T cells derived from BALB/c mice infected with murine CMV protect immunocompromised adoptive transfer recipients against CMV disease. The protective population comprises CD8 T cells with T-cell receptors (TCRs) specific for defined and for as-yet-unknown viral epitopes, as well as a majority of nonprotective cells with unrelated specificities. Defined epitopes include IE1/m123 and m164, which are immunodominant in terms of the magnitude of the CD8 T-cell response, and a panel of subordinate epitopes (m04, m18, M45, M83, and M84). While cytolytic T-lymphocyte lines (CTLLs) were shown to be protective regardless of the immunodominance of the respective epitope, the individual contributions of in vivo resident epitope-specific CD8 T cells to the antiviral control awaited investigation. The IE1 peptide 168-YPHFMPTNL-176 is generated from the immediate-early protein 1 (IE1) (pp89/76) of murine CMV and is presented by the major histocompatibility complex class I (MHC-I) molecule Ld. To quantitate its contribution to the protective potential of a CD8-T memory (CD8-TM) cell population, IE1-TCR+ and IE1-TCR- CD8-TM cells were purified by epitope-specific cell sorting with IE1 peptide-loaded MHC-immunoglobulin G1 dimers as ligands of cognate TCRs. Of relevance for clinical approaches to an adoptive cellular immunotherapy, sorted IE1 epitope-specific CD8-TM cells were found to be exceedingly protective upon adoptive transfer. Compared with CTLLs specific for the same epitope and of comparable avidity and TCR beta-chain variable region (Vbeta)-defined polyclonality, sorted CD8-TM cells proved to be superior by more than 2 orders of magnitude.     

Targeting hepatitis B virus-infected cells with a T-cell receptor-like antibody

Virus-specific CD8 T cells are activated when their T-cell receptors (TCRs) recognize the specific viral peptide/major histocompatibility complex (MHC) class I (pMHC) complexes present on the surface of infected cells. Antibodies able to recognize the specific pMHC can mimic TCR specificity and both represent a valuable biological tool to visualize pMHC complexes on infected cells and serve as a delivery system for highly targeted therapies. To evaluate these possibilities, we created a monoclonal antibody able to specifically recognize a hepatitis B virus (HBV) envelope epitope (Env at positions 183 to 91 [Env183-91]) presented by the HLA-A201 molecule, and we tested its ability to recognize HBV-infected hepatocytes and to deliver a cargo to a specific target. We demonstrate that this antibody detects and visualizes the processed product of HBV proteins produced in naturally HBV-infected cells, is not inhibited by soluble HBV proteins present in patient sera, and mediates the intracellular delivery of a fluorescent molecule to target cells. Additionally, compared to CD8 T cells specific for the same HBV epitope, the TCR-like antibody has both a superior sensitivity and a specificity focused on distinct amino acids within the epitope. These data demonstrate that a T-cell receptor-like antibody can be used to determine the quantitative relationship between HBV replication and specific antigen presentation to CD8 T cells and serves as a novel therapeutic delivery platform for personalized health care for HBV-infected patients.     

T-cell receptor tetramer binding or the lack there of does not necessitate antigen reactivity in T-cell receptor transduced T cells

Genetic transfer of T-cell receptor (TCR) chains provides a means of transferring tumor antigen specificity onto an alternate T-cell population. To determine which tumor reactive TCRs are best suitable for such adoptive transfer, careful evaluation of the resulting TCR modified populations need to be performed. We have previously cloned, and expressed TCRs from melanoma, EBV, HCV, and HPV reactive T-cell clones and found that several routine indicators of T-cell function do not always predict the relative strength of a TCR. Using a combination of tetramer binding assays and antigen recognition assays, we identified TCRs that fall into three classes. One class of TCR did not bind tetramers yet resulted in cells with high avidity for antigen. A second TCR class bound tetramer but did not secrete cytokines in response to antigen. Finally, the third class of TCRs bound tetramer and reacted to antigen as would be expected. We conclude that tetramer binding is not always a good indicator of the function of a cloned TCR or the avidity of a TCR gene modified T cell. Furthermore, our data indicate that the use of tetramer binding alone to identify antigen reactive TCRs may result in the exclusion of TCRs that may be highly reactive or cross reactive to the relevant tumor antigen.