Differentiation marker-negative CD4+ T cells persist after yellow fever virus vaccination and contribute to durable memory.

Factors that contribute to durable immunological memory remain incompletely understood. In our longitudinal analyses of CD4+ T cell responses to the yellow fever virus (YFV) vaccine by peptide-MHC tetramers, we unexpectedly found naïve phenotype virus-specific CD4+ T cells that persisted months to years after immunization. These Marker negative T cells (TMN) lacked CD95, CXCR3, CD11a, and CD49d surface protein expression, distinguishing them from previously discovered stem-cell memory T cells. Functionally, they resembled genuine naïve T cells upon in vitro stimulation. Single-cell TCR sequencing detected expanded clonotypes within the TMN subset and identified a shared repertoire with memory and effector T cells. T cells expressing TMN-associated TCRs were rare before vaccination, suggesting their expansion following vaccination. Longitudinal tracking of YFV-specific responses over the subsequent years revealed superior stability of the TMN subset and their association with the longevity of the overall population. The identification of these long-lived, antigen-experienced T cells may inform the design of durable T cell-based vaccines and engineered T cell therapies.

Pre-existing T Cell Memory to Novel Pathogens.

Immunological experiences lead to the development of specific T and B cell memory, which readies the host for a later pathogen rechallenge. Currently, immunological memory is best understood as a linear process whereby memory responses are generated by and directed against the same pathogen. However, numerous studies have identified memory cells that target pathogens in unexposed individuals. How "pre-existing memory" forms and impacts the outcome of infection remains unclear. In this review, we discuss differences in the composition of baseline T cell repertoire in mice and humans, factors that influence pre-existing immune states, and recent literature on their functional significance. We summarize current knowledge on the roles of pre-existing T cells in homeostasis and perturbation and their impacts on health and disease.

SARS-CoV-2-specific T cells in unexposed adults display broad trafficking potential and cross-react with commensal antigens.

The baseline composition of T cells directly affects later response to pathogens, but the complexity of precursor states remains poorly defined. Here, we examined the baseline state of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells in unexposed individuals. SARS-CoV-2-specific CD4+ T cells were identified in prepandemic blood samples by major histocompatibility complex (MHC) class II tetramer staining and enrichment. Our data revealed a substantial number of SARS-CoV-2-specific T cells that expressed memory phenotype markers. Integrated phenotypic analyses demonstrated diverse preexisting memory states that included cells with distinct polarization features and trafficking potential to barrier tissues. T cell clones generated from tetramer-labeled cells cross-reacted with antigens from commensal bacteria in the skin and gastrointestinal tract. Direct ex vivo tetramer staining for one spike-specific population showed a similar level of cross-reactivity to sequences from endemic coronavirus and commensal bacteria. These data highlight the complexity of precursor T cell repertoire and implicate noninfectious exposures to common microbes as a key factor that shapes human preexisting immunity to SARS-CoV-2.

SARS-CoV-2-specific T cells in unexposed adults display broad trafficking potential and cross-react with commensal antigens.

The baseline composition of T cells directly impacts later response to a pathogen, but the complexity of precursor states remains poorly defined. Here we examined the baseline state of SARS-CoV-2 specific T cells in unexposed individuals. SARS-CoV-2 specific CD4 + T cells were identified in pre-pandemic blood samples by class II peptide-MHC tetramer staining and enrichment. Our data revealed a substantial number of SARS-CoV-2 specific T cells that expressed memory phenotype markers, including memory cells with gut homing receptors. T cell clones generated from tetramer-labeled cells cross-reacted with bacterial peptides and responded to stool lysates in a MHC-dependent manner. Integrated phenotypic analyses revealed additional precursor diversity that included T cells with distinct polarized states and trafficking potential to other barrier tissues. Our findings illustrate a complex pre-existing memory pool poised for immunologic challenges and implicate non-infectious stimuli from commensal colonization as a factor that shapes pre-existing immunity. ONE SENTENCE SUMMARY: Pre-existing immunity to SARS-CoV-2 contains a complex pool of precursor lymphocytes that include differentiated cells with broad tissue tropism and the potential to cross-react with commensal antigens.

Vaccination reshapes the virus-specific T cell repertoire in unexposed adults.

We examined how baseline CD4+ T cell repertoire and precursor states impact responses to pathogen infection in humans using primary immunization with yellow fever virus (YFV) vaccine. YFV-specific T cells in unexposed individuals were identified by peptide-MHC tetramer staining and tracked pre- and post-vaccination by tetramers and TCR sequencing. A substantial number of YFV-reactive T cells expressed memory phenotype markers and contained expanded clones in the absence of exposure to YFV. After vaccination, pre-existing YFV-specific T cell populations with low clonal diversity underwent limited expansion, but rare populations with a reservoir of unexpanded TCRs generated robust responses. These altered dynamics reorganized the immunodominance hierarchy and resulted in an overall increase in higher avidity T cells. Thus, instead of further increasing the representation of dominant clones, YFV vaccination recruits rare and more responsive T cells. Our findings illustrate the impact of vaccines in prioritizing T cell responses and reveal repertoire reorganization as a key component of effective vaccination.

Role of Regulatory T Cell and Effector T Cell Exhaustion in Liver-Mediated Transgene Tolerance in Muscle.

The pro-tolerogenic environment of the liver makes this tissue an ideal target for gene replacement strategies. In other peripheral tissues such as the skeletal muscle, anti-transgene immune response can result in partial or complete clearance of the transduced fibers. Here, we characterized liver-induced transgene tolerance after simultaneous transduction of liver and muscle. A clinically relevant transgene, α-sarcoglycan, mutated in limb-girdle muscular dystrophy type 2D, was fused with the SIINFEKL epitope (hSGCA-SIIN) and expressed with adeno-associated virus vectors (AAV-hSGCA-SIIN). Intramuscular delivery of AAV-hSGCA-SIIN resulted in a strong inflammatory response, which could be prevented and reversed by concomitant liver expression of the same antigen. Regulatory T cells and upregulation of checkpoint inhibitor receptors were required to establish and maintain liver-mediated peripheral tolerance. This study identifies the fundamental role of the synergy between Tregs and upregulation of checkpoint inhibitor receptors in the liver-mediated control of anti-transgene immunity triggered by muscle-directed gene transfer.

Dual muscle-liver transduction imposes immune tolerance for muscle transgene engraftment despite preexisting immunity.

Immune responses to therapeutic transgenes are a potential hurdle to treat monogenic muscle disorders. These responses result from the neutralizing activity of transgene-specific B cells and cytotoxic T cells recruited upon gene transfer. We explored here how dual muscle-liver expression of a foreign transgene allows muscle transgene engraftment after adenoassociated viral vector delivery. We found in particular that induction of transgene-specific tolerance is imposed by concurrent muscle and liver targeting, resulting in the absence of CD8+ T cell responses to the transgene. This tolerance can be temporally decoupled, because transgene engraftment can be achieved in muscle weeks after liver transduction. Importantly, transgene-specific CD8+ T cell tolerance can be established despite preexisting immunity to the transgene. Whenever preexisting, transgene-specific CD4+ and CD8+ memory T cell responses are present, dual muscle-liver transduction turns polyclonal, transgene-specific CD8+ T cells into typically exhausted T cells with high programmed cell death 1 (PD-1) expression and lack of IFN-γ production. Our results demonstrate that successful transduction of muscle tissue can be achieved through liver-mediated control of humoral and cytotoxic T cell responses, even in the presence of preexisting immunity to the muscle-associated transgene.

Cross-Presentation of Skin-Targeted Recombinant Adeno-associated Virus 2/1 Transgene Induces Potent Resident Memory CD8+ T Cell Responses.

A key aspect to consider for vaccinal protection is the induction of a local line of defense consisting of nonrecirculating tissue-resident memory T cells (TRM), in parallel to the generation of systemic memory CD8+ T cell responses. The potential to induce TRM has now been demonstrated for a number of pathogens and viral vectors. This potential, however, has never been tested for recombinant adeno-associated virus (rAAV) vectors, which are weakly inflammatory and poor transducer of dendritic cells. Using a model rAAV2/1-based vaccine, we determined that a single intradermal immunization with rAAV2/1 vectors in mice induces fully functional TRM at the local site of immunization. The optimal differentiation of rAAV-induced transgene-specific skin TRM was dependent on local transgene expression and additional CD4+ T cell help. Transgene expression in dendritic cells, however, appeared to be dispensable for the priming of transgene-specific skin TRM, suggesting that this process solely depends on the cross-presentation of transgene products. Overall, this study provides needed information to properly assess rAAV vectors as T cell-inducing vaccine carriers.IMPORTANCE rAAVs display numerous characteristics that could make them extremely attractive as vaccine carriers, including an excellent safety profile in humans and great flexibility regarding serotypes and choice of target tissue. Studies addressing the ability of rAAV to induce protective T cell responses, however, are scarce. Notably, the potential to induce a tissue-resident memory T cell response has never been described for rAAV vectors, strongly limiting further interest for their use as vaccine carriers. Using a model rAAV2/1 vaccine delivered to the skin, our study demonstrated that rAAV vectors can induce bona fide skin resident TRM and provides additional clues regarding the cellular mechanisms underlying this process. These results will help widen the field of rAAV applications.