TCR Avidity Research Articles

Monoclonal Invariant NKT (iNKT) Cell Mice Reveal a Role for Both Tissue of Origin and the TCR in Development of iNKT Functional Subsets.

Invariant NKT (iNKT) cell functional subsets are defined by key transcription factors and output of cytokines, such as IL-4, IFN-γ, IL-17, and IL-10. To examine how TCR specificity determines iNKT function, we used somatic cell nuclear transfer to generate three lines of mice cloned from iNKT nuclei. Each line uses the invariant Vα14Jα18 TCRα paired with unique Vβ7 or Vβ8.2 subunits. We examined tissue homing, expression of PLZF, T-bet, and RORγt, and cytokine profiles and found that, although monoclonal iNKT cells differentiated into all functional subsets, the NKT17 lineage was reduced or expanded depending on the TCR expressed. We examined iNKT thymic development in limited-dilution bone marrow chimeras and show that higher TCR avidity correlates with higher PLZF and reduced T-bet expression. iNKT functional subsets showed distinct tissue distribution patterns. Although each individual monoclonal TCR showed an inherent subset distribution preference that was evident across all tissues examined, the iNKT cytokine profile differed more by tissue of origin than by TCR specificity.

Prime-Boost Immunization Eliminates Metastatic Colorectal Cancer by Producing High-Avidity Effector CD8+ T Cells.

Heterologous prime-boost immunization with plasmid DNA and viral vector vaccines is an emerging approach to elicit CD8+ T cell-mediated immunity targeting pathogens and tumor Ags that is superior to either monotherapy. Yet, the mechanisms underlying the synergy of prime-boost strategies remain incompletely defined. In this study, we examine a DNA and adenovirus (Ad5) combination regimen targeting guanylyl cyclase C (GUCY2C), a receptor expressed by intestinal mucosa and universally expressed by metastatic colorectal cancer. DNA immunization efficacy was optimized by i.m. delivery via electroporation, yet it remained modest compared with Ad5. Sequential immunization with DNA and Ad5 produced superior antitumor efficacy associated with increased TCR avidity, whereas targeted disruption of TCR avidity enhancement eliminated GUCY2C-specific antitumor efficacy, without affecting responding T cell number or cytokine profile. Indeed, functional TCR avidity of responding GUCY2C-specific CD8+ T cells induced by various prime or prime-boost regimens correlated with antitumor efficacy, whereas T cell number and cytokine profile were not. Importantly, although sequential immunization with DNA and Ad5 maximized antitumor efficacy through TCR avidity enhancement, it produced no autoimmunity, reflecting sequestration of GUCY2C to intestinal apical membranes and segregation of mucosal and systemic immunity. Together, TCR avidity enhancement may be leveraged by prime-boost immunization to improve GUCY2C-targeted colorectal cancer immunotherapeutic efficacy and patient outcomes without concomitant autoimmune toxicity.

Dissection of CD8+ T cell immunodominance hierarchy in human influenza virus infection

Abstract Based on magnitude, virus-specific CD8+ cytotoxic T lymphocyte (CTL) responses can be grouped as immunodominant and subdominant. Such immunodominance hierarchies are governed by multiple factors, including naïve T cell precursor frequency, proliferative capacity and clonotypic diversity of Ag-specific CTLs. However, how these factors determine together inter/intra-individual immunodominance remains elusive. In this study, we dissected CTL immunodominance hierarchy for universally conserved influenza (IAV)-specific epitopes. Our data from 59 donors covering the HLAI types presenting universally conserved IAV-specific peptides, HLA-A* 0201, A*0301, B*2705, B*5701, B*1801 and B*0801, revealed that CTLs directed against HLA-A* 0201:M158 were immunodominant, except in A*0201+B*2705+ heterozygous individuals, where the HLA-B* 2705-NP383 recalls higher magnitude CTL responses. Subsequently, we further dissected the basis of B*2705 immundomination over A*0201 by analyses of T cell Receptor (TCRαβ) repertoire, TCR avidity and kinetics of killing and proliferation. Our data show that following in vitro peptide stimulation, B*2705:NP383 CTLs proliferate rapidly acquiring higher levels of polyfunctionality than A*02:M158 specific CTLs. Furthermore, ex vivo single-cell paired CDR3αβ analysis revealed that public A*0201:M158 TCRs characterized by Vβ19/Vα27 profiles in A*0201 individuals are significantly reduced/absent in A*0201+B*2705+ individuals. Clonal analysis of M158 dominant public TCRs suggested B*2705-mediated removal of self-reactive TCRs and is under investigation. Our study provides insights into the importance of human IAV-specific immunodominance at quantitative and qualitative levels.

TCR recognition is a mediator in the control of HIV infection by CD8 T cells

Abstract CD8 T cell responses to HIV-specific MHC class I epitopes are critical for the control of HIV infection. The precise mechanisms that govern the control of HIV by CD8 T cells are largely unknown. Certain protective HLA alleles, such as, B*5701, are associated with long-term control of HIV-1. However, other mechanisms by CD8 T cells might be contributing to the control of HIV, because non-controllers with these protective alleles have been reported. Therefore, to determine the molecular mechanisms that underlie HIV control by CD8 T cells we assembled a cohort of HIV positive individuals, which consisted of controllers (viral load less than 2000 copies per mL), ART-naive non-controllers, and non-controllers on ART-therapy. Using PBMCs from our cohort we first stained the cells with a panel of thirteen T cell markers and then performed polychromatic single cell index sorting of CD3+CD8+ MHC class I tetramer-binding cells. Our tetramers consisted of HIV epitopes bound to HLA-B*5701, A*0201, B*0702 or B*0801. We then determined the TCR diversity for each tetramer-binding population by single cell TCR sequencing. We also reconstructed HIV-specific TCRs and expressed them in a reporter T cell line to measure TCR avidity to the tetramer. We found that HIV controllers have: (1) a diverse TCR repertoire for the KF11 epitope presented by B*5701, in contrast, to non-B*5701 alleles presenting HIV epitopes that are heavily skewed; (2) high avidity HIV-specific TCRs for HIV epitopes presented by B*5701; and (3) TCR-expanded clones with a novel CD27dimCD45ROneg T cell phenotype. Importantly, lack of any three of these parameters resulted in the inability to control HIV, indicating that no single parameter is sufficient for natural control.

Alterations in the thymic selection threshold skew the self-reactivity of the T cell receptor repertoire in neonates

Abstract Neonatal and adult T cells differ in their effector functions. Although it is known that cell-intrinsic differences in mature T cells contribute to this phenomenon, the factors involved remain unclear. Given emerging evidence that the binding strength of the TCR for self-peptide presented by MHC (self-pMHC) impacts T cell function, we sought to determine if altered thymic selection thresholds influence the self-reactivity of the TCR repertoire during ontogeny. We found that conventional and regulatory T cell subsets in the thymus of neonates and young mice expressed higher levels of cell-surface CD5, a surrogate marker for TCR avidity for self-pMHC, as compared to their adult counterparts and that this difference in self-reactivity was independent of their distinct TCR repertoire. The increased binding strength of the TCR repertoire for self-pMHC in neonates was not solely due to reported defects in clonal deletion. Rather, our data suggest that thymic selection is altered in young mice such that thymocytes bearing TCRs with low affinity for self-peptide are not efficiently selected into the neonatal repertoire and stronger TCR signals accompany both positive and agonist selection. Importantly, the differences in CD5 levels reflect physiologically relevant differences based on the preferential expansion of T cells from young mice to fill a lymphopenic environment. Therefore, differences in the thymic selection threshold in young versus adult mice skews the TCR repertoire, and the relatively higher self-reactivity of the T cell pool may contribute to the distinct immune responses observed in neonates.

High avidity TCR-peptide interactions between CD4 lymphocytes and lymph node dendritic cells promote normal lymphocyte arrest, activation, proliferation, and differentiation independently of dendritic cell ICAM-1 and 2

Abstract The two main LFA-1 ligands ICAM-1 and ICAM-2 are constitutively co-expressed on antigen presenting cells (APCs) and on the lymph node stroma. We find abrogated antigen specific CD4 T cell priming in ICAM-1 and ICAM-2 deficient spleens. Nevertheless, the in vivo contribution of these ligands to lymphocyte activation proliferation and differentiation in lymph nodes has been difficult to dissect in total knockout mice due to their multiple roles in lymphocyte entry and priming in these organs. We therefore constructed lethally irradiated chimeric mice reconstituted with ICAM-1 and 2 double knock out (DKO) APCs in which lymphocyte entry into peripheral lymph nodes was reconstituted. Strikingly, these mice elicited normal antigen specific OT-II T cell activation, proliferation and differentiation into Th1 and Tfh effectors. OT-II T cells could also normally arrest on DCs deficient in ICAM-1 and 2. A compensatory role of VCAM-1 in ICAM deficient DCs was ruled out. Nevertheless, polyclonal T cell proliferation in response to endogenous self-antigens required the presence of ICAM-1 and ICAM-2 on endogenous lymph node DCs. Our results are the first indication that high avidity TCR peptide-MHC-II recognition can bypass integrin-mediated lymphocyte adhesions to DCs under specific settings.

Discrete TCR Binding Kinetics Control Invariant NKT Cell Selection and Central Priming.

Invariant NKT (iNKT) cells develop and differentiate in the thymus, segregating into iNKT1/2/17 subsets akin to Th1/2/17 classical CD4+ T cells; however, iNKT TCRs recognize Ags in a fundamentally different way. How the biophysical parameters of iNKT TCRs influence signal strength in vivo and how such signals affect the development and differentiation of these cells are unknown. In this study, we manipulated TCRs in vivo to generate clonotypic iNKT cells using TCR retrogenic chimeras. We report that the biophysical properties of CD1d-lipid-TCR interactions differentially impacted the development and effector differentiation of iNKT cells. Whereas selection efficiency strongly correlated with TCR avidity, TCR signaling, cell-cell conjugate formation, and iNKT effector differentiation correlated with the half-life of CD1d-lipid-TCR interactions. TCR binding properties, however, did not modulate Ag-induced iNKT cytokine production. Our work establishes that discrete TCR interaction kinetics influence iNKT cell development and central priming.

CD4 T cell epitope specificity determines follicular versus non-follicular helper differentiation in the polyclonal response to influenza infection or vaccination.

Follicular helper T cells (Tfh) are essential for B cell production of high-affinity, class-switched antibodies. Much interest in Tfh development focuses on the priming environment of CD4 T cells. Here we explored the role that peptide specificity plays in the partitioning of the polyclonal CD4 T cell repertoire between Tfh and NonTfh lineages during the response to influenza. Surprisingly, we found that CD4 T cells specific for different epitopes exhibited distinct tendencies to segregate into Tfh or NonTfh. To alter the microenvironment and abundance, viral antigens were introduced as purified recombinant proteins in adjuvant as native proteins. Also, the most prototypical epitopes were expressed in a completely foreign protein. In many cases, the epitope-specific response patterns of Tfh vs. NonTfh persisted. The functional TcR avidity of only a subset of epitope-specific cells correlated with the tendency to drive a Tfh response. Thus, we conclude that in a polyclonal CD4 T cell repertoire, features of TcR-peptide:MHC class II complex have a strong deterministic influence on the ability of CD4 T cells to become a Tfh or a NonTfh. Our data is most consistent with at least 2 checkpoints of Tfh selection that include both TcR affinity and B cell presentation.

The fate of CD8+ T cells during infection is linked to their developmental origin

Abstract Key features of the CD8+ T cell response to infection are the robust clonal expansion and phenotypic diversification of naïve precursors into effector and memory cells. However, mechanisms that regulate the fate of naïve CD8+ T cells after microbial challenge remain a subject of controversy. Previous studies have suggested heterogeneity in the effector pool is driven by differences in TCR avidity, asymmetric cell division, and environmental cues. However, one variable that has not been closely examined is the developmental origin of naïve CD8+ T cells. Notably, the pool of naïve CD8+ T cells in the adult is comprised of clonotypes that were produced during various stages of life. To determine whether the developmental origin of naïve CD8+ T cells in adults plays a deterministic role in their fate after infection, we developed a system to ‘timestamp’ CD8+ T cells from various stages of development (1 day, 1 week, and 4 weeks) and examined their phenotype and behavior in 8 week-old adult mice. Interestingly, we found naïve CD8+ T cells made early in life predominantly display a CD44hi CD122hi memory phenotype whereas cells made later in life are CD44lo CD122lo. In vitro stimulation of stamped populations showed that fetal- and neonatal-derived CD8+ T cells are hypersensitive to IL12/IL18 and proliferate more rapidly than cells made in older animals. In response to infection in adulthood, both polyclonal and monoclonal CD8+ T cells produced in early life skew towards a terminally differentiated short-lived effector phenotype (KLRG1hiCD127lo), indicating this phenomenon is TCR independent. These data indicate that the spectrum of CD8+ T cell differentiation observed after infection is influenced by when the responding cells were initially made.

Human memory CD4+ T cell response to the major dog allergen Can f 5, prostatic kallikrein.

Human CD4+ T cell responses to important animal allergens are still insufficiently understood. To comprehensively characterize invitro and exvivo the peripheral blood memory CD4+ T cell responses of subjects with and without allergy to the major dog allergen Can f 5, the only known animal allergen in the kallikrein family of proteins. Can f 5-specific memory CD4+ T cell lines (TCLs) were established from the peripheral blood of 12 subjects with and 12 subjects without allergy to Can f 5 and characterized for their functional and phenotypic properties. The results were evaluated with those obtained exvivo with a novel CD154 enrichment method. The epitopes recognized by the Can f 5-specific TCLs were determined with 72 overlapping 16-mer peptides covering the sequence of the allergen. Can f 5-specific TCLs were obtained at about tenfold higher frequency from allergic than from non-allergic subjects. Functionally, the TCLs of allergic subjects displayed a Th2-biased cytokine phenotype and increased T cell receptor avidity, whereas the TCLs of non-allergic subjects displayed a Th1-/Th0-biased cytokine phenotype and lower TCR avidity. The higher frequency and the Th2 phenotype of Can f 5-specific memory CD4+ T cells in allergic subjects were confirmed by the CD154 enrichment method exvivo. Six distinct T cell epitope regions of Can f 5 were predominantly recognized by the TCLs from allergic subjects. Can f 5-specific memory CD4+ T cell responses differ considerably between subjects with and without allergy, as assessed by both invitro and exvivo approaches. Peptides containing the dominant T cell epitopes of Can f 5 can be employed for developing peptide-based immunotherapy for dog allergy.

Rapid Evolution of the CD8+ TCR Repertoire in Neonatal Mice.

Currently, there is little consensus regarding the most appropriate animal model to study acute infection and the virus-specific CD8(+) T cell (CTL) responses in neonates. TCRβ high-throughput sequencing in naive CTL of differently aged neonatal mice was performed, which demonstrated differential Vβ family gene usage. Using an acute influenza infection model, we examined the TCR repertoire of the CTL response in neonatal and adult mice infected with influenza type A virus. Three-day-old mice mounted a greatly reduced primary NP(366-374)-specific CTL response when compared with 7-d-old and adult mice, whereas secondary CTL responses were normal. Analysis of NP(366-374)-specific CTL TCR repertoire revealed different Vβ gene usage and greatly reduced public clonotypes in 3-d-old neonates. This could underlie the impaired CTL response in these neonates. To directly test this, we examined whether controlling the TCR would restore neonatal CTL responses. We performed adoptive transfers of both nontransgenic and TCR-transgenic OVA(257-264)-specific (OT-I) CD8(+) T cells into influenza-infected hosts, which revealed that naive neonatal and adult OT-I cells expand equally well in neonatal and adult hosts. In contrast, nontransgenic neonatal CD8(+) T cells when transferred into adults failed to expand. We further demonstrate that differences in TCR avidity may contribute to decreased expansion of the endogenous neonatal CTL. These studies highlight the rapid evolution of the neonatal TCR repertoire during the first week of life and show that impaired neonatal CTL immunity results from an immature TCR repertoire, rather than intrinsic signaling defects or a suppressive environment.

A Numerically Subdominant CD8 T Cell Response to Matrix Protein of Respiratory Syncytial Virus Controls Infection with Limited Immunopathology.

CD8 T cells are involved in pathogen clearance and infection-induced pathology in respiratory syncytial virus (RSV) infection. Studying bulk responses masks the contribution of individual CD8 T cell subsets to protective immunity and immunopathology. In particular, the roles of subdominant responses that are potentially beneficial to the host are rarely appreciated when the focus is on magnitude instead of quality of response. Here, by evaluating CD8 T cell responses in CB6F1 hybrid mice, in which multiple epitopes are recognized, we found that a numerically subdominant CD8 T cell response against DbM187 epitope of the virus matrix protein expressed high avidity TCR and enhanced signaling pathways associated with CD8 T cell effector functions. Each DbM187 T effector cell lysed more infected targets on a per cell basis than the numerically dominant KdM282 T cells, and controlled virus replication more efficiently with less pulmonary inflammation and illness than the previously well-characterized KdM282 T cell response. Our data suggest that the clinical outcome of viral infections is determined by the integrated functional properties of a variety of responding CD8 T cells, and that the highest magnitude response may not necessarily be the best in terms of benefit to the host. Understanding how to induce highly efficient and functional T cells would inform strategies for designing vaccines intended to provide T cell-mediated immunity.

The hypervariable region 4 (HV4) and position 93 of the α chain modulate CD1d-glycolipid binding of iNKT TCRs.

TCRs of invariant NKT (iNKT) cells bind α-galactosylceramide (αGC) loaded CD1d in a highly conserved fashion and show a characteristic TCR gene usage: An "invariant" α chain with a canonical AV14/AJ18 rearrangement in mice (AV24/AJ18 in humans) is paired with β chains containing characteristic Vβ segments. In the rat, a multimember AV14 gene family increases the variability within this system. This study characterizes CD1d binding of rat AV14 gene segments in TCR transductants as well as CD1d binding and iNKT TCR expression of expanded polyclonal F344 rat iNKT populations. It defines an important role of position 93 at the V-J transition for TCR avidity and species cross-reactivity of the rat iNKT TCR. Furthermore, for the first time we identified variability within the fourth hypervariable loop (HV4) of the α chain as a modulator of CD1d:αGC binding in rat and mouse. Additionally, we confirmed the importance of the CDR2β for CD1d:αGC binding, but also show that the CDR3β may even have opposite effects on binding depending on the pairing α chain. Altogether, we characterized naturally occurring sources of variability for the iNKT TCR and speculate that they rather level than increase the largely germline encoded differences of iNKT TCR ligand avidity.

Engineered FVIII-specific human CD4 T cells: does TCR avidity modulate T-helper phenotypes? (IRC7P.425)

Abstract We recently demonstrated that expanded human CD4 T cells can be transduced to express TCRs specific for a given epitope. These cells proliferate and secrete cytokines in response to their cognate peptide/MHC. Herein, we examined whether TCR- transduced cells could be skewed to different T-helper subsets, or alternatively if previously skewed T cells could be modulated after transduction. Two HLA DR1-restricted TCRs were cloned from Th2 and Th17/Th1-type CD4+ T-cell clones specific for a coagulation factor VIII peptide (pC2). Engineered CD4+ T cells expressing these TCRs exhibited different proliferation kinetics during titration with pC2. All pC2-stimulated cells expressed IFN-g, IL-4, and IL-2 intracellularly but did not express cytokine signatures characteristic of their parental Th2 and Th17/Th1 clones. CD45RA+ and CD45RA- T cells were skewed to Th1, Th2 or Th17 and then transduced with each of the 2 TCRs. The higher-avidity TCR cells that were skewed to Th2 and then stimulated with low [pC2] maintained a Th2 cytokine signature, whereas the same cells stimulated with higher [pC2] expressed lower levels of IL-4 and GATA3. In contrast, Th1, Th17 and Th0-skewed cells transduced with the same TCR expressed their respective cytokines at all pC2 doses. Differences between the low-avidity TCR-engineered cells were less pronounced. These studies will inform our strategies for designing novel T-helper cellular therapies.

459. CAR-Transduced T Cells Demonstrate Increased CTL Function

Infection with HIV-1 results in CD4+ T cell depletion and the subsequent loss of immune function results in AIDS. Although HAART lowers plasma viremia, it requires life-long drug therapy. However, elite controllers are able to exert control over viral replication in the absence of cART therapy by maintaining polyfunctional HIV-specific T cells with high avidity TCR binding that target CTL epitopes where escape mutants have reduced fitness.To mimic increase immunosurveillance of elite controllers and prevent viral replication in a non-controlled setting, we will transduce T cells with CD4-CAR vectors to bolster and redirect the “designer T cells” (dTc) towards an HIV-specific target. Chimeric antigen receptors (CARs) are fusion proteins of an extracellular antigenbinding domain linked to intracellular T cell signaling domains. In CD4-CARs, the CD4 extracellular domain binds directly to diverse HIV envelopes and redirects the CTL activity. Env/CD4 binding is independent of both the MHC restriction and Nef down regulation. As in elite controllers, CD4-CARs targets an essential viral function where mutations would lower viral fitness and make viral escape difficult to achieve. The first-generation CD4-CAR vector, with only the T-cell receptor zeta (TCRζ) intracellular domain, killed Env+ cells in vitro but failed to control viremia in clinical trials. Recent studies with anti-tumor CARs show that additional co-stimulatory signals (CD28 and 4-1BB) improved CTL function in vitro, in murine models, and showed objective tumor regression in clinical trials.We hypothesize that genetically modified T cells designed to increase CTL killing of HIV+ cells, to persist in vivo, and to prevent viral infection will provide durable viral suppression in vivo and eliminate the need for life-long drug therapy as modeled by elite controllers. To determine what signaling domains would be necessary to mimic this immune protection, we developed a series of CD4-CAR vectors with various intracellular signaling domains. To protect dTc from infection, we co-transduced cells with the membrane-associated C46 (maC46), a fusion inhibitor tethered to dTc, that binds HIV and blocks viral replication thereby providing a strong selective advantage to transduced cells. Therefore, we stimulated PBMCs with αCD3αCD28 and co-transduced T cells with the series of CD4-CAR and maC46 vectors. To evaluate the functional activity of the dTc, we measured 1) HIV Env-specific cytotoxicity, 2) cytokine production using IFN-γ ELIspot, and 3) CAR-specific proliferation of dTc. CD4-CAR T cells demonstrated Env+ specific killing, increased frequency of IFN-γ spot-forming cells, and CAR-specific proliferation.Our long-term goal, to adoptively transfer dTc with increased CTL function (cytokine production, proliferative capacity, viral suppression). The control of viremia in the absence of HAART would revolutionize HIV treatments for patients with AIDS.

Defects in pro-apoptotic genes, Bim or Bax/Bak, cause accumulation of TCRβhi DN4 cells in thymus (HEM4P.241)

Abstract Regulated induction of apoptosis during thymocyte development is critical to prevent the generation of self-reactive T cells whilst maintaining a repertoire of T cells that can promote protective immunity. Although the pro-apoptotic Bcl-2 family member Bim has been shown to promote apoptotic negative selection of self-reactive thymocytes, the role of Bim in this process is controversial. Here, we have investigated the role of Bim and downstream mediators Bax and Bak on thymocyte selection. Surprisingly, the absence of Bim results in a striking accumulation of DN4 cells with surface TCRβhi feature in the thymus. Similar results were observed in mice with a T cell specific loss of Bax in a Bak knock out background. DN4 cells from Bim-/- or LckCreBaxfl/flBak-/- mice cultured with OP9-DL1 cells largely failed to generate DP cells relative to WT DN4 cells. Further, using a dLckCre system, in which Cre is expressed late in the DP stage, revealed that conditional knock out of Bim or Bax did not result in an accumulation of DN4-TCRβhi cells. Combined, these data suggest that accumulation of DN4-TCRβhi cells in either Bim-/- or LckCreBaxfl/flBak-/- are the result of cells having failed negative selection that downregulate CD4 and CD8 coreceptors. Such a strategy would inherently limit their TCR avidity and reduce their potential for autoreactivity. Our current studies are examining the persistence and location of DN-TCRβhi cells in the periphery of Bim-/- or LckCreBaxfl/flBak-/- mice.

The Influenza Virus–Specific CTL Immunodominance Hierarchy in Mice Is Determined by the Relative Frequency of High-Avidity T Cells

Virus-specific CTL responses typically fall into reproducible hierarchies with particular epitopes eliciting either immunodominant or subdominant responses after viral challenge. The recently acquired capacity to directly enumerate naive CTL precursors (CTLps) in both mice and humans has implicated CTLp frequency as a key predictor of immune response magnitude after Ag challenge. However, recent studies have indicated that naive CTLp frequencies do not necessarily predict the size of the Ag-driven response, indicating an important role for differential CTLp recruitment and/or expansion. This study characterizes the early emergence of various influenza epitope-specific CTL responses at multiple sites in C57BL/6 mice, and probes the role of Ag dose and TCR avidity in dictating immune response hierarchies. Despite large naive CTLp numbers, subdominance was found to arise largely as a consequence of the abrupt and premature cessation of CTL proliferation, at least for one epitope specificity. Investigation into the possible drivers of the poor proliferation observed for subdominant specificities showed that the immunodominance hierarchy endured irrespective of epitope abundance, and correlated with the prevalence of high-avidity T cells in both the naive and immune compartments. Our study strongly indicates that the quality, and not simply the quantity, of antiviral CTLs dictate response magnitude.

Very low avidity endogenous memory CD8+ T cells control tumor growth (TUM2P.893)

Abstract In this study, we describe mice bearing a very low avidity TCR transgene (TCRvlo) specific for a melanoma antigen. Remarkably, a majority of peripheral TCRvlo CD8+ T cells display a phenotype consistent with the expression pattern for endogenous memory (EM) T cells: CD44hi, CXCR3hi, Gr1hi, GITRhi, CD69lo, CD25lo, CD49dlo. This phenotype was not observed among thymic CD8+CD4- TCRvlo T cells. EM-T cells are proposed to arise from naïve T cells in the absence of activation by foreign antigen and are a very rare population of cells which may contribute to primary immune responses, but have not been studied in cancer models. We hypothesize that EM-T cells may provide a unique reservoir of cells that can control tumor growth. Our previous observations indicate that T cells bearing a high avidity TCR with identical antigenic specificity initially control tumor growth but are prone to tumor-induced tolerance. In our preliminary studies, compared to wildtype animals, TCRvlo transgenic mice displayed delayed subcutaneous B16 melanoma growth. In an adoptive transfer setting, TCRvlo T cells also delayed B16 tumor growth compared to untreated animals. Ongoing studies are focused on characterizing the functional responses of the TCRvlo CD8+ T cells to antigen and cytokines as well as determining whether TCRvlo T cells resist tolerization in the tumor microenvironment. Results from these studies may enable us to use EM-T cells as a resource for generating more durable tumor immunity.

High Avidity CD8+ T Cells Efficiently Eliminate Motile HIV-Infected Targets and Execute a Locally Focused Program of Anti-Viral Function

The dissemination of HIV from an initial site of infection is facilitated by motile HIV-infected CD4+ T-cells. However, the impact of infected target cell migration on antigen recognition by HIV-specific CD8+ T-cells is unclear. Using a 3D in vitro model of tissue, we visualized dynamic interactions between HIV-infected or peptide-pulsed CD4+ T-cells and HIV-specific CD8+ T-cells. CTLs engaged motile HIV-infected targets, but ∼50% of targets broke contact and escaped. In contrast, immobilized target cells were readily killed, indicating target motility directly inhibits CD8+ T-cell function. Strong calcium signals occurred in CTLs killing a motile target but calcium signaling was weak or absent in CTLs which permitted target escape. Neutralization of adhesion receptors LFA-1 and CD58 inhibited CD8+ T-cell function within the 3D matrix, demonstrating that efficient motile target lysis as dependent on adhesive engagement of targets. Antigen sensitivity (a convolution of antigen density, TCR avidity and CD8 coreceptor binding) is also critical for target recognition. We modulated this parameter (known as functional avidity but referred to here as “avidity” for the sake of simplicity) by exploiting common HIV escape mutations and measured their impact on CTL function at the single-cell level. Targets pulsed with low avidity mutant antigens frequently escaped while CTLs killed targets bearing high avidity antigen with near-perfect efficiency. CTLs engaged, arrested, and killed an initial target bearing high avidity antigen within minutes, but serial killing was surprisingly rare. CD8 cells remained committed to their initial dead target for hours, accumulating TCR signals that sustained secretion of soluble antiviral factors. These data indicate that high-avidity CD8+ T-cells execute an antiviral program in the precise location where antigen has been sensed: CTL effector functions are spatiotemporally coordinated with an early lytic phase followed by a sustained stationary secretory phase to control local viral infection.

Differential selection of Th1 effector and memory pools depends on the qualitative nature of TCR-driven CD4+ T cell activation (P1175)

Abstract Not all clones that participate in the effector Th1 response to intracellular viral or bacterial infection can give rise to memory T cells. In this study, we generated a mouse with a fixed TCRα chain of known specificity in order to characterize the evolution of effector and memory TCRβ repertoires following LCMV or Listeria infection by deep sequencing. The repertoire diversity of antigen-specific Th1 cells significantly decreased in the transition from effector to memory, and hierarchical cluster analysis revealed that the TCR repertoires of memory T cells, regardless of infectious model, were more similar to each other than to the effector Th1 populations from which they arose. We further generated a large panel of TCRs derived from our sequencing results that were cloned into retroviral expression vectors and used to generate TCR “retrogenic” bone marrow chimeras. Individual clonal T cell populations harvested from these mice were analyzed in adoptive transfer experiments to allow for their longitudinal analysis following infection. We found that the individual memory potential of each clone did not correspond to overall TCR avidity, as measured in tetramer equilibrium binding assays, but to the acquisition of high functional avidity and slow off-rates, as determined by tetramer decay. Our findings support a model in which differential selection of the Th1 effector and memory pools depends at least in part on the qualitative nature of TCR-driven CD4+ T cell activation.