Newborn and child-like molecular signatures in older adults stem from TCR shifts across human lifespan.

CD8+ T cells provide robust antiviral immunity, but how epitope-specific T cells evolve across the human lifespan is unclear. Here we defined CD8+ T cell immunity directed at the prominent influenza epitope HLA-A*02:01-M158-66 (A2/M158) across four age groups at phenotypic, transcriptomic, clonal and functional levels. We identify a linear differentiation trajectory from newborns to children then adults, followed by divergence and a clonal reset in older adults. Gene profiles in older adults closely resemble those of newborns and children, despite being clonally distinct. Only child-derived and adult-derived A2/M158+CD8+ T cells had the potential to differentiate into highly cytotoxic epitope-specific CD8+ T cells, which was linked to highly functional public T cell receptor (TCR)αß signatures. Suboptimal TCRαß signatures in older adults led to less proliferation, polyfunctionality, avidity and recognition of peptide mutants, although displayed no signs of exhaustion. These data suggest that priming T cells at different stages of life might greatly affect CD8+ T cell responses toward viral infections.

Targeting BMI-1 in B cells restores effective humoral immune responses and controls chronic viral infection.

Ineffective antibody-mediated responses are a key characteristic of chronic viral infection. However, our understanding of the intrinsic mechanisms that drive this dysregulation are unclear. Here, we identify that targeting the epigenetic modifier BMI-1 in mice improves humoral responses to chronic lymphocytic choriomeningitis virus. BMI-1 was upregulated by germinal center B cells in chronic viral infection, correlating with changes to the accessible chromatin landscape, compared to acute infection. B cell-intrinsic deletion of Bmi1 accelerated viral clearance, reduced splenomegaly and restored splenic architecture. Deletion of Bmi1 restored c-Myc expression in B cells, concomitant with improved quality of antibody and coupled with reduced antibody-secreting cell numbers. Specifically, BMI-1-deficiency induced antibody with increased neutralizing capacity and enhanced antibody-dependent effector function. Using a small molecule inhibitor to murine BMI-1, we could deplete antibody-secreting cells and prohibit detrimental immune complex formation in vivo. This study defines BMI-1 as a crucial immune modifier that controls antibody-mediated responses in chronic infection.

Thanks for the memories: Low-avidity T cells shine against escape variants.

T cell responses against foreign antigens are clonally diverse, but the significance of this diversity is unclear. In this issue of Immunity, Straub et al.1 show that recruitment of low-avidity T cells during primary infection can provide protection against subsequent encounter with escape variants.

The paradox of aging: Aging-related shifts in T cell function and metabolism.

T cell survival, differentiation after stimulation, and function are intrinsically linked to distinct cellular metabolic states. The ability of T cells to readily transition between metabolic states enables flexibility to meet the changing energy demands defined by distinct effector states or T cell lineages. Immune aging is characterized, in part, by the loss of naïve T cells, accumulation of senescent T cells, severe dysfunction in memory phenotype T cells in particular, and elevated levels of inflammatory cytokines, or 'inflammaging'. Here, we review our current understanding of the phenotypic and functional changes that occur with aging in T cells, and how they relate to metabolic changes in the steady state and after T cell activation. We discuss the apparent contradictions in the aging T cell phenotype - where enhanced differentiation states and metabolic profiles in the steady state can correspond to a diminished capacity to adapt metabolically and functionally after T cell activation. Finally, we discuss key recent studies that indicate the enormous potential for aged T cell metabolism to induce systemic inflammaging and organism-wide multimorbidity, resulting in premature death.

Reversed T Cell Receptor Docking on a Major Histocompatibility Class I Complex Limits Involvement in the Immune Response.

The anti-viral T cell response is drawn from the naive T cell repertoire. During influenza infection, the CD8+ T cell response to an H-2Db-restricted nucleoprotein epitope (NP366) is characterized by preferential expansion of T cells bearing TRBV13+ T cell receptors (TCRs) and avoidance of TRBV17+ T cells, despite the latter dominating the naive precursor repertoire. We found two TRBV17+ TCRs that bound H-2Db-NP366 with a 180° reversed polarity compared to the canonical TCR-pMHC-I docking. The TRBV17 ß-chain dominated the interaction and, whereas the complementarity determining region-3 (CDR3) loops exclusively mediated contacts with the MHC-I, peptide specificity was attributable to germline-encoded recognition. Nevertheless, the TRBV17+ TCR exhibited moderate affinity toward H-2Db-NP366 and was capable of signal transduction. Thus, the naive CD8+ T cell pool can comprise TCRs adopting reversed pMHC-I docking modes that limit their involvement in the immune response.

Helminth Infection-Induced Increase in Virtual Memory CD8 T Cells Is Transient, Driven by IL-15, and Absent in Aged Mice.

CD8 virtual memory T (TVM) cells are Ag-naive CD8 T cells that have undergone partial differentiation in response to common γ-chain cytokines, particularly IL-15 and IL-4. TVM cells from young individuals are highly proliferative in response to TCR and cytokine stimulation but, with age, they lose TCR-mediated proliferative capacity and exhibit hallmarks of senescence. Helminth infection can drive an increase in TVM cells, which is associated with improved pathogen clearance during subsequent infectious challenge in young mice. Given the cytokine-dependent profile of TVM cells and their age-associated dysfunction, we traced proliferative and functional changes in TVM cells, compared with true naive CD8 T cells, after helminth infection of young and aged C57BL/6 mice. We show that IL-15 is essential for the helminth-induced increase in TVM cells, which is driven only by proliferation of existing TVM cells, with negligible contribution from true naive cell differentiation. Additionally, TVM cells showed the greatest proliferation in response to helminth infection and IL-15 compared with other CD8 T cells. Furthermore, TVM cells from aged mice did not undergo expansion after helminth infection due to both TVM cell-intrinsic and -extrinsic changes associated with aging.

Quantifiable predictive features define epitope-specific T cell receptor repertoires.

T cells are defined by a heterodimeric surface receptor, the T cell receptor (TCR), that mediates recognition of pathogen-associated epitopes through interactions with peptide and major histocompatibility complexes (pMHCs). TCRs are generated by genomic rearrangement of the germline TCR locus, a process termed V(D)J recombination, that has the potential to generate marked diversity of TCRs (estimated to range from 1015 (ref. 1) to as high as 1061 (ref. 2) possible receptors). Despite this potential diversity, TCRs from T cells that recognize the same pMHC epitope often share conserved sequence features, suggesting that it may be possible to predictively model epitope specificity. Here we report the in-depth characterization of ten epitope-specific TCR repertoires of CD8+ T cells from mice and humans, representing over 4,600 in-frame single-cell-derived TCRαß sequence pairs from 110 subjects. We developed analytical tools to characterize these epitope-specific repertoires: a distance measure on the space of TCRs that permits clustering and visualization, a robust repertoire diversity metric that accommodates the low number of paired public receptors observed when compared to single-chain analyses, and a distance-based classifier that can assign previously unobserved TCRs to characterized repertoires with robust sensitivity and specificity. Our analyses demonstrate that each epitope-specific repertoire contains a clustered group of receptors that share core sequence similarities, together with a dispersed set of diverse 'outlier' sequences. By identifying shared motifs in core sequences, we were able to highlight key conserved residues driving essential elements of TCR recognition. These analyses provide insights into the generalizable, underlying features of epitope-specific repertoires and adaptive immune recognition.

Dominant protection from HLA-linked autoimmunity by antigen-specific regulatory T cells.

Susceptibility and protection against human autoimmune diseases, including type I diabetes, multiple sclerosis, and Goodpasture disease, is associated with particular human leukocyte antigen (HLA) alleles. However, the mechanisms underpinning such HLA-mediated effects on self-tolerance remain unclear. Here we investigate the molecular mechanism of Goodpasture disease, an HLA-linked autoimmune renal disorder characterized by an immunodominant CD4+ T-cell self-epitope derived from the α3 chain of type IV collagen (α3135-145). While HLA-DR15 confers a markedly increased disease risk, the protective HLA-DR1 allele is dominantly protective in trans with HLA-DR15 (ref. 2). We show that autoreactive α3135-145-specific T cells expand in patients with Goodpasture disease and, in α3135-145-immunized HLA-DR15 transgenic mice, α3135-145-specific T cells infiltrate the kidney and mice develop Goodpasture disease. HLA-DR15 and HLA-DR1 exhibit distinct peptide repertoires and binding preferences and present the α3135-145 epitope in different binding registers. HLA-DR15-α3135-145 tetramer+ T cells in HLA-DR15 transgenic mice exhibit a conventional T-cell phenotype (Tconv) that secretes pro-inflammatory cytokines. In contrast, HLA-DR1-α3135-145 tetramer+ T cells in HLA-DR1 and HLA-DR15/DR1 transgenic mice are predominantly CD4+Foxp3+ regulatory T cells (Treg cells) expressing tolerogenic cytokines. HLA-DR1-induced Treg cells confer resistance to disease in HLA-DR15/DR1 transgenic mice. HLA-DR15+ and HLA-DR1+ healthy human donors display altered α3135-145-specific T-cell antigen receptor usage, HLA-DR15-α3135-145 tetramer+ Foxp3- Tconv and HLA-DR1-α3135-145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes. Moreover, patients with Goodpasture disease display a clonally expanded α3135-145-specific CD4+ T-cell repertoire. Accordingly, we provide a mechanistic basis for the dominantly protective effect of HLA in autoimmune disease, whereby HLA polymorphism shapes the relative abundance of self-epitope specific Treg cells that leads to protection or causation of autoimmunity.

The Impact of MHC Class I Dose on Development and Maintenance of the Polyclonal Naive CD8+ T Cell Repertoire.

Naive CD8+ T cell survival in the periphery is critically dependent on tonic TCR signaling through peptide + MHC class I (MHCI) recognition; however, little is known about how natural variation in MHCI levels impacts the naive CD8+ T cell repertoire. Using mice that are hemizygous or homozygous for a single MHCI allele, we showed that despite a reduction in peripheral CD8+ T cell numbers of ∼50% in MHCI hemizygous mice, MHCI levels had no notable impact on the rate of thymic generation or emigration of CD8 single-positive T cells. Moreover, the peripheral T cell repertoire in hemizygous mice showed selective retention of T cell clonotypes with a greater competitive advantage as evidenced by increased expression of CD5 and IL-7Rα. The qualitative superiority of CD8+ T cells retained in hemizygous mice was also seen during influenza A virus infection, in which epitope-specific CD8+ T cells from hemizygous mice had a higher avidity for pMHCI and increased cytokine polyfunctionality, despite a reduced response magnitude. Collectively, this study suggests that natural variation in MHCI expression levels has a notable and biologically relevant impact on the maintenance, but not generation, of the naive CD8+ T cell repertoire.

Overlapping Peptides Elicit Distinct CD8+ T Cell Responses following Influenza A Virus Infection.

The presentation of pathogen-derived peptides on MHC class I molecules is essential for the initiation of adaptive CD8+ T cell immunity, which in turn is critical for effective control of many significant human infections. The identification of immunogenic pathogen-derived epitopes and a detailed understanding of how they are recognized by TCRs is essential for the design of effective T cell-based vaccines. In this study, we have characterized the T cell recognition and immune responses in mice to two naturally presented influenza A virus-derived peptides previously identified from virally infected cells via mass spectrometry. These neuraminidase-derived peptides, NA181-190 (SGPDNGAVAV) and NA181-191 (SGPDNGAVAVL), are completely overlapping with the exception of a 1 aa extension at the C terminus of the longer peptide. This minor peptidic difference results in the induction of two completely independent and non-cross-reactive T cell populations that show distinct functional characteristics after influenza A virus infection of B6 mice. We show that the unique TCR reactivity to the overlapping peptides is present in the naive repertoire prior to immune expansion in B6 mice. Moreover, we provide a structural explanation underlying the distinct CD8+ T cell reactivities, which reinforces the concept that peptide length is a key determinant of Ag specificity in CD8+ T cell responses.

CD4+ T help promotes influenza virus-specific CD8+ T cell memory by limiting metabolic dysfunction.

There is continued interest in developing novel vaccine strategies that induce establish optimal CD8+ cytotoxic T lymphocyte (CTL) memory for pathogens like the influenza A viruses (IAVs), where the recall of IAV-specific T cell immunity is able to protect against serologically distinct IAV infection. While it is well established that CD4+ T cell help is required for optimal CTL responses and the establishment of memory, when and how CD4+ T cell help contributes to determining the ideal memory phenotype remains unclear. We assessed the quality of IAV-specific CD8+ T cell memory established in the presence or absence of a concurrent CD4+ T cell response. We demonstrate that CD4+ T cell help appears to be required at the initial priming phase of infection for the maintenance of IAV-specific CTL memory, with "unhelped" memory CTL exhibiting intrinsic dysfunction. High-throughput RNA-sequencing established that distinct transcriptional signatures characterize the helped vs. unhelped IAV-specific memory CTL phenotype, with the unhelped set showing a more "exhausted T cell" transcriptional profile. Moreover, we identify that unhelped memory CTLs exhibit defects in a variety of energetic pathways, leading to diminished spare respiratory capacity and diminished capacity to engage glycolysis upon reactivation. Hence, CD4+ T help at the time of initial priming promotes molecular pathways that limit exhaustion by channeling metabolic processes essential for the rapid recall of memory CD8+ T cells.

Most viral peptides displayed by class I MHC on infected cells are immunogenic.

CD8+ T cells are essential effectors in antiviral immunity, recognizing short virus-derived peptides presented by MHC class I (pMHCI) on the surface of infected cells. However, the fraction of viral pMHCI on infected cells that are immunogenic has not been shown for any virus. To approach this fundamental question, we used peptide sequencing by high-resolution mass spectrometry to identify more than 170 vaccinia virus pMHCI presented on infected mouse cells. Next, we screened each peptide for immunogenicity in multiple virus-infected mice, revealing a wide range of immunogenicities. A surprisingly high fraction (>80%) of pMHCI were immunogenic in at least one infected mouse, and nearly 40% were immunogenic across more than half of the mice screened. The high number of peptides found to be immunogenic and the distribution of responses across mice give us insight into the specificity of antiviral CD8+ T cell responses.

Characterisation of clinical and immune reactivity to barley and rye ingestion in children with coeliac disease.

OBJECTIVE: Barley and rye are major components of the Western diet, and historic feeding studies indicate that they cause clinical effects in patients with coeliac disease (CD). This toxicity has been attributed to sequence homology with immunogenic wheat sequences, but in adults with CD, these cereals stimulate unique T cells, indicating a critical contribution to gluten immunity independent of wheat. Clinical and immune feeding studies with these grains in children with CD are sparse. We undertook a barley and rye feeding study to characterise the clinical and T-cell responses in children with CD. DESIGN: 42 children with human leucocyte antigen (HLA)-DQ2.5+ (aged 3-17 years) consumed barley or rye for 3 days. Blood-derived gluten-specific T cells were tested for reactivity against a panel of barley (hordein) and rye (secalin) peptides. Hordein and secalin-specific T-cell clones were generated and tested for grain cross-reactivity. T-cell receptor sequencing was performed on sorted single cells. T-cell responses were compared with those observed in adults with CD. RESULTS: 90% of the children experienced adverse symptoms, mostly GI, and 61% had detectable gluten-specific T-cell responses targeting peptides homologous to those immunogenic in adults. Deamidation was important for peptide reactivity. Homozygosity for HLA-DQ2.5 predicted a stronger T-cell response. Gluten-specific T cells showed striking similarities in their cross-reactivity between children and adults. CONCLUSIONS: Barley and rye induce a consistent range of clinical and T-cell responses in children with CD. The findings highlight the importance of a series of dominant hordein and secalin peptides pathogenic in children with CD, some independent of wheat, which closely correspond to those seen in adults.

Modified Vaccinia Virus Ankara Can Induce Optimal CD8+ T Cell Responses to Directly Primed Antigens Depending on Vaccine Design.

A variety of strains of vaccinia virus (VACV) have been used as recombinant vaccine vectors with the aim of inducing robust CD8+ T cell immunity. While much of the pioneering work was done with virulent strains, such as Western Reserve (WR), attenuated strains such as modified vaccinia virus Ankara (MVA) are more realistic vectors for clinical use. To unify this literature, side-by-side comparisons of virus strains are required. Here, we compare the form of antigen that supports optimal CD8+ T cell responses for VACV strains WR and MVA using equivalent constructs. We found that for multiple antigens, minimal antigenic constructs (epitope minigenes) that prime CD8+ T cells via the direct presentation pathway elicited optimal responses from both vectors, which was surprising because this finding contradicts the prevailing view in the literature for MVA. We then went on to explore the discrepancy between current and published data for MVA, finding evidence that the expression locus and in some cases the presence of the viral thymidine kinase may influence the ability of this strain to prime optimal responses from antigens that require direct presentation. This extends our knowledge of the design parameters for VACV vectored vaccines, especially those based on MVA.IMPORTANCE Recombinant vaccines based on vaccinia virus and particularly attenuated strains such as MVA are in human clinical trials, but due to the complexity of these large vectors much remains to be understood about the design parameters that alter their immunogenicity. Previous work had found that MVA vectors should be designed to express stable protein in order to induce robust immunity by CD8+ (cytotoxic) T cells. Here, we found that the primacy of stable antigen is not generalizable to all designs of MVA and may depend where a foreign antigen is inserted into the MVA genome. This unexpected finding suggests that there is an interaction between genome location and the best form of antigen for optimal T cell priming in MVA and thus possibly other vaccine vectors. It also highlights that our understanding of antigen presentation by even the best studied of vaccine vectors remains incomplete.

Hiding in Plain Sight: Virtually Unrecognizable Memory Phenotype CD8+ T cells.

Virtual memory T (TVM) cells are a recently described population of conventional CD8+ T cells that, in spite of their antigen inexperience, express markers of T cell activation. TVM cells exhibit rapid responsiveness to both antigen-specific and innate stimuli in youth but acquire intrinsic antigen-specific response defects in the elderly. In this article, we review how the identification of TVM cells necessitates a re-evaluation of accepted paradigms for conventional memory T (TMEM) cells, the potential for heterogeneity within the TVM population, and the defining characteristics of TVM cells. Further, we highlight recent literature documenting the development of TVM cells as a distinct CD8+ T cell lineage as well their biological significance in the context of disease.

Heightened self-reactivity associated with selective survival, but not expansion, of naïve virus-specific CD8+ T cells in aged mice.

In advanced age, decreased CD8(+) cytotoxic T-lymphocyte (CTL) responses to novel pathogens and cancer is paralleled by a decline in the number and function of naïve CTL precursors (CTLp). Although the age-related fall in CD8(+) T-cell numbers is well established, neither the underlying mechanisms nor the extent of variation for different epitope specificities have been defined. Furthermore, naïve CD8(+) T cells expressing high levels of CD44 accumulate with age, but it is unknown whether this accumulation reflects their preferential survival or an age-dependent driver of CD8(+) T-cell proliferation. Here, we track the number and phenotype of four influenza A virus (IAV)-specific CTLp populations in naïve C57BL/6 (B6) mice during aging, and compare T-cell receptor (TCR) clonal diversity for the CD44hi and CD44lo subsets of one such population. We show differential onset of decline for several IAV-specific CD8(+) T-cell populations with advanced age that parallel age-associated changes in the B6 immunodominance hierarchy, suggestive of distinct impacts of aging on different epitope-specific populations. Despite finding no evidence of clonal expansions in an aged, epitope-specific TCR repertoire, nonrandom alterations in TCR usage were observed, along with elevated CD5 and CD8 coreceptor expression. Collectively, these data demonstrate that naïve CD8(+) T cells expressing markers of heightened self-recognition are selectively retained, but not clonally expanded, during aging.

Targeted deletion of Traf2 allows immunosuppression-free islet allograft survival in mice.

AIMS/HYPOTHESIS: Administration of anti-CD40 ligand (CD40L) antibodies has been reported to allow long-term islet allograft survival in non-human primates without the need for exogenous immunosuppression. However, the use of anti-CD40L antibodies was associated with thromboembolic complications. Targeting downstream intracellular components shared between CD40 and other TNF family co-stimulatory molecules could bypass these complications. TNF receptor associated factor 2 (TRAF2) integrates multiple TNF receptor family signalling pathways that are critical for T cell activation and may be a central node of alloimmune responses. METHODS: T cell-specific Traf2-deficient mice (Traf2TKO) were generated to define the role of TRAF2 in CD4+ T cell effector responses that mediate islet allograft rejection in vivo. In vitro allograft responses were tested using mixed lymphocyte reactions and analysis of IFN-γ and granzyme B effector molecule expression. T cell function was assessed using anti-CD3/CD28-mediated proliferation and T cell polarisation studies. RESULTS: Traf2TKO mice exhibited permanent survival of full MHC-mismatched pancreatic islet allografts without exogenous immunosuppression. Traf2TKO CD4+ T cells exhibited reduced proliferation, activation and acquisition of effector function following T cell receptor stimulation; however, both Traf2TKO CD4+ and CD8+ T cells exhibited impaired alloantigen-mediated proliferation and acquisition of effector function. In polarisation studies, Traf2TKO CD4+ T cells preferentially converted to a T helper (Th)2 phenotype, but exhibited impaired Th17 differentiation. Without TRAF2, thymocytes exhibited dysregulated TNF-mediated induction of c-Jun N-terminal kinase (JNK) and canonical NFκB pathways. Critically, targeting TRAF2 in T cells did not impair the acute phase of CD8-dependent viral immunity. These data highlight a specific requirement for a TRAF2-NFκB and TRAF2-JNK signalling cascade in T cell activation and effector function in rejecting islet allografts. CONCLUSION/INTERPRETATION: Targeting TRAF2 may be useful as a therapeutic approach for immunosuppression-free islet allograft survival that avoids the thromboembolic complications associated with the use of anti-CD40L antibodies.

T cell mediated immunity to influenza: mechanisms of viral control.

Infection with influenza A virus (IAV) is a major cause of worldwide morbidity and mortality. Recent findings indicate that T cell immunity is key to limiting severity of disease arising from IAV infection, particularly in instances where antibody immunity is ineffective. As such, there is a need to understand better the mechanisms that mediate effective IAV-specific cellular immunity, especially given that T cell immunity must form an integral part of any vaccine designed to elicit crossreactive immunity against existing and new strains of influenza virus. Here, we review the current understanding of cellular immunity to IAV, highlighting recent findings that demonstrate important roles for both CD4+ and CD8+ T cell immunity in protection from IAV-mediated disease.

Modelling cross-reactivity and memory in the cellular adaptive immune response to influenza infection in the host.

The cellular adaptive immune response plays a key role in resolving influenza infection. Experiments where individuals are successively infected with different strains within a short timeframe provide insight into the underlying viral dynamics and the role of a cross-reactive immune response in resolving an acute infection. We construct a mathematical model of within-host influenza viral dynamics including three possible factors which determine the strength of the cross-reactive cellular adaptive immune response: the initial naive T cell number, the avidity of the interaction between T cells and the epitopes presented by infected cells, and the epitope abundance per infected cell. Our model explains the experimentally observed shortening of a second infection when cross-reactivity is present, and shows that memory in the cellular adaptive immune response is necessary to protect against a second infection.