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Gating strategy, Shannon entropy, PD-1 expression, additional co-culture data, primers sequence, Frequency of the top 10 CD8+PD-1+ TCRB in bulk unsorted TIL and sorted TIL subsets, Retrospective analysis of pairing the most frequent TCRB in the CD8+PD-1+ TIL subset with the most frequent TCRA from the same subset, Method of identification of TCR pairs, TCR expression on genetically engineered PBL.

Adoptive transfer of T cells with engineered T-cell receptor (TCR) genes that target tumor-specific antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. To test this hypothesis, we developed a multistep strategy that involved TCRB deep sequencing of the CD8+PD-1+ T-cell subset, matching of TCRA–TCRB pairs by pairSEQ and single-cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes, which included reactivity against neoantigens. These data show the feasibility of developing a rapid, personalized TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity. Cancer Immunol Res; 4(9); 734–43. ©2016 AACR.

Our previous work found that the RNA binding protein polypyrimidine tract-binding protein (PTBP1) is critical for regulating multiple events in T cell activation including changes in proliferation, and expression of activation markers and cytokines. These changes corresponded to the regulation of the ERK1/2 and NF-κB pathways as well as through changes in steady-state RNA levels. Because proliferation is critical for driving T cell activation, it was unclear whether PTBP1 was required for optimal activationper seor whether changes were secondary to a requirement for initiating/sustaining proliferation. To address this question, the human T cell lymphoma cell line, Jurkat, which recapitulates many of the molecular events of TCR-induced activation, was used to understand how PTBP1 impacts early events in T cell activation with ongoing proliferation. Using two phenotypically distinct Jurkat subclones (D1.1 and B2.7), we first profiled global RNA expression patterns using RNAseq analysis and found marked differences between the two cell lines with the D1.1 line giving a more antigen-experienced phenotype. Reducing PTBP1 by shPTB expression, to 60% WT levels resulted in no significant decrease in proliferation in the two subclones. However, we observed that PTBP1 was required for both optimal expression of activation markers, CD25, CD38, CD69, and CD40L, and signaling through the ERK1/2, P38 and AKT pathways. Importantly, limiting PTBP1 had different effects on the activation signals for each cell line suggesting that the differentiation state of the cell is a critical factor in understanding the role of PTBP1 in T cell activation. This was further reinforced by our finding that PTBP1 regulated distinct groups of genes specific for each line. Together, our findings suggest that PTBP1 regulates specific T cell activation responses independent of its role in proliferation and that the initial phenotype of the T cell plays an essential role in the dependency of the cell on PTBP1 for driving these changes.

Functional analyses of the T cell receptor (TCR) landscape can reveal critical information about protection from disease and molecular responses to vaccines. However, it has proven difficult to combine advanced next-generation sequencing technologies with methods to decode the peptide-major histocompatibility complex (pMHC) specificity of individual TCRs. We developed a new high-throughput approach to enable repertoire-scale functional evaluations of natively paired TCRs. In particular, we leveraged the immortalized nature of physically linked TCRα:β amplicon libraries to analyze binding against multiple recombinant pMHCs on a repertoire scale, and to exemplify the utility of this approach, we also performed affinity-based functional mapping in conjunction with quantitative next-generation sequencing to track antigen-specific TCRs. These data successfully validated a new immortalization and screening platform to facilitate detailed molecular analyses of disease-relevant antigen interactions with human TCRs.

Although many HIV cure strategies seek to expand HIV-specific CD8+ T cells to control the virus, all are likely to fail if cellular exhaustion is not prevented. A loss in stem-like memory properties (i.e., the ability to proliferate and generate secondary effector cells) is a key feature of exhaustion; little is known, however, about how these properties are regulated in human virus-specific CD8+ T cells. We found that virus-specific CD8+ T cells from humans and nonhuman primates naturally controlling HIV/SIV infection express more of the transcription factor TCF-1 than noncontrollers. HIV-specific CD8+ T cell TCF-1 expression correlated with memory marker expression and expansion capacity and declined with antigenic stimulation. CRISPR-Cas9 editing of TCF-1 in human primary T cells demonstrated a direct role in regulating expansion capacity. Collectively, these data suggest that TCF-1 contributes to the regulation of the stem-like memory property of secondary expansion capacity of HIV-specific CD8+ T cells, and they provide a rationale for exploring the enhancement of this pathway in T cell-based therapeutic strategies for HIV.

Adoptive transfer of T cells with engineered T-cell receptor (TCR) genes that target tumor-specific antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. To test this hypothesis, we developed a multistep strategy that involved TCRB deep sequencing of the CD8+PD-1+ T-cell subset, matching of TCRA–TCRB pairs by pairSEQ and single-cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes, which included reactivity against neoantigens. These data show the feasibility of developing a rapid, personalized TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity. Cancer Immunol Res; 4(9); 734–43. ©2016 AACR.

Prior studies of T-cell responses to KSHV have included relatively few participants and focused on relatively few KSHV antigens. To provide a more comprehensive analysis, we investigated T-cell responses to the whole KSHV proteome using IFN-γ ELISpot. Using ∼7,500 overlapping 15mer peptides we generated one to three peptide pools for each of the 82 KSHV ORFs. IFN-γ ELISpot analysis of PBMCs from 19 patients with a history of KSHV-associated disease and 24 healthy donors (11 KSHV seropositive) detected widely varied responses. Fifty six of the 82 ORFs were recognized by at least one individual but there was little overlap between participants. Responses to at least one ORF pool were observed in all 19 patients and in 7 seropositive donors. Four seropositive donors and 10 seronegative donors had no detectable responses while 3 seronegative donors had weak responses to one ORF. Patients recognised more ORFs than the donors (p=0.04) but the response intensity (spot forming units: SFU per million cells) was similar in the two groups. In four of the responding donors, individual peptides eliciting the predominant responses were identified: three donors responded to only one peptide per ORF, while one recognized five. Using intracellular cytokine staining in four participant samples, we detected peptide-induced IFN-γ, MIP1-β, and TNF-α as well as CD107a degranulation, consistent with multifunctional effector responses in CD8+ and CD4+ T cells. Sequence analysis of TCRs present in peptide specific T-cell clones generated from two participants showed both mono- and multi-clonotypic responses. Finally, we molecularly cloned the KSHV specific TCRs and incorporated the sequences into retroviral vectors to transfer the specificities to fresh donor cells for additional studies. This study suggests that KSHV infected individuals respond to diverse KSHV antigens, consistent with a lack of shared immunodominance and establishes useful tools to facilitate KSHV immunology studies.

CD40L (CD154) is regulated at the posttranscriptional level by an activation-induced process that results in a highly stable transcript at extended times of T cell activation. Transcript stability is mediated by polypyrimidine tract-binding protein (PTB)-containing complexes (complex I and II) that bind to three adjacent CU-rich sequences within the 3′ untranslated region. To assess the role of PTB in the expression and distribution of CD40L mRNA, PTB was targeted using short hairpin RNA in both primary T cells and a T cell line that recapitulates the stability phase of regulated CD40L mRNA decay. PTB knockdown resulted in a marked decrease in the mRNA stability that resulted in lowered CD40L surface expression. PTB was also critical for appropriate distribution of CD40L mRNA between the nucleus and cytoplasm and in the cytoplasm between the cytosol and the translating polysomes. The activation-induced formation of PTB-specific ribonucleoprotein complexes was observed only with cytoplasmic and not nuclear PTB indicating functional differences in the protein defined by cellular localization. Finally, we observed that cytoplasmic and nuclear PTB isoforms were differentially modified relative to each other and that the changes in cytoplasmic PTB were consistent with activation-induced phosphorylation. Together this work suggests that differentially modified PTB regulates CD40L expression at multiple steps by 1) retaining CD40L mRNA in the nucleus, 2) directly regulating mRNA stability at late times of activation, and 3) forming a ribonuclear complex that preferentially associates with translating ribosomes thus leading to an enhanced level of CD40L protein.

Summary We previously identified a cis-acting element within the 3′ untranslated region of CD40 ligand messenger RNA (mRNA) that is composed of three complex binding sites and acts to increase mRNA stability in both in vitro and in vivo systems. We now demonstrate the functional consequences of the three binding sites with respect to increasing both luciferase activity and mRNA stability in a heterologous transcript expressed in a T-cell line. The internal region B was shown to be a bona fide stability element because its presence increased luciferase activity fourfold over the unmodified transcript and its removal from the XbaI–HaeIII region resulted in rapid degradation of the transcript. Region A contained both a binding site for a polypyrimidine-tract-binding protein (PTB)-mediated complex (Complex I) and an upstream, adjacent sequence that was a negative regulator of mRNA stability. Region C bound Complex II, which contained both PTB and heterogeneous nuclear ribonucleoproteinL (hnRNPL), and was less effective as a stability element on its own compared to region B. Our findings demonstrate differential levels of activity for the three binding sites relative to the turnover of CD40 ligand mRNA, suggesting that the lack of binding of Complex I/II during the early stages of T-cell activation contributes to the rapid degradation of the CD40 ligand mRNA transcript.

We have previously shown that the RNA binding protein, polypyrimidine tract-binding protein (PTBP1) plays a critical role in regulating the expression of CD40L in activated CD4 T cells. This is achieved mechanistically through message stabilization at late times of activation as well as by altered distribution of CD40L mRNA within distinct cellular compartments. PTBP1 has been implicated in many different processes, however whether PTBP1 plays a broader role in CD4 T cell activation is not known. To examine this question, experiments were designed to introduce shRNA into primary human CD4 T cells to achieve decreased, but not complete ablation of PTBP1 expression. Analyses of shPTB-expressing CD4 T cells revealed multiple processes including cell proliferation, activation-induced cell death and expression of activation markers and cytokines that were regulated in part by PTBP1 expression. Although there was an overall decrease in the steady-state level of several activation genes, only IL-2 and CD40L appeared to be regulated by PTBP1 at the level of RNA decay suggesting that PTBP1 is critical at different regulatory steps of expression that is gene-specific. Importantly, even though the IL-2 protein levels were reduced in cells with lowered PTBP1, the steady-state level of IL-2 mRNA was significantly higher in these cells suggesting a block at the translational level. Evaluation of T cell activation in shPTB-expressing T cells revealed that PTBP1 was linked primarily to the activation of the PLCγ1/ERK1/2 and the NF-κB pathways. Overall, our results reveal the importance of this critical RNA binding protein in multiple steps of T cell activation.

Broadly neutralizing antibodies (bNAbs) protect against HIV-1 infection, yet how they are generated during chronic infection remains unclear. It is known that T follicular helper (TFH) cells are needed to promote affinity maturation of B cells during an immune response; however, the role of TFH during HIV-1 infection is undefined within lymph node germinal centers (GCs). We use nonhuman primates to investigate the relationship in the early stage of chronic SHIVAD8 (simian-human immunodeficiency virus AD8) infection between envelope (Env)-specific TFH cells, Env-specific B cells, virus, and the generation of bNAbs during later infection. We found that both the frequency and quality of Env-specific TFH cells were associated with an expansion of Env-specific immunoglobulin G-positive GC B cells and broader neutralization across HIV clades. We also found a correlation between breadth of neutralization and the degree of somatic hypermutation in Env-specific memory B cells. Finally, we observed high viral loads and greater diversity of Env sequences in rhesus macaques that developed cross-reactive neutralization as compared to those that did not. These studies highlight the importance of boosting high-quality TFH populations as part of a robust vaccine regimen aimed at eliciting bNabs.

Developing predictive animal models to assess how candidate vaccines and infection influence the ontogenies of Envelope (Env)-specific antibodies is critical for the development of an HIV vaccine. Here we use two nonhuman primate models to compare the roles of antigen persistence, diversity and innate immunity. We perform longitudinal analyses of HIV Env-specific B-cell receptor responses to SHIVAD8 infection and Env protein vaccination with eight different adjuvants. A subset of the SHIVAD8-infected animals with higher viral loads and greater Env diversity show increased neutralization associated with increasing somatic hypermutation (SHM) levels over time. The use of adjuvants results in increased ELISA titres but does not affect the mean SHM levels or CDR H3 lengths. Our study shows how the ontogeny of Env-specific B cells can be tracked, and provides insights into the requirements for developing neutralizing antibodies that should facilitate translation to human vaccine studies., HIV vaccine development will be facilitated by having animal models that are predictive for translation to humans. Here, the authors use two nonhuman primate models to compare the effects of natural infection and different adjuvants on antigen persistence, diversity and humoral immunity.

Because of significant viral diversity, vaccines that elicit durable and broad protection against influenza have been elusive. Recent research has focused on the potential of highly conserved regions of the viral hemagglutinin (HA) as targets for broadly neutralizing Ab responses. Abs that bind the highly conserved stem or stalk of HA can be elicited by vaccination in humans and animal models and neutralize diverse influenza strains. However, the frequency and phenotype of HA stem–specific B cells in vivo remain unclear. In this article, we characterize HA stem–specific B cell responses following H5N1 vaccination and describe the re-expansion of a pre-existing population of memory B cells specific for stem epitopes. This population uses primarily, but not exclusively, IGHV1-69–based Igs for HA recognition. However, within some subjects, allelic polymorphism at the ighv1-69 locus can limit IGHV1-69 immunodominance and may reduce circulating frequencies of stem-reactive B cells in vivo. The accurate definition of allelic selection, recombination requirements, and ontogeny of neutralizing Ab responses to influenza will aid rational influenza vaccine design.

OA30.05 Background: To assess the ability of immunogens to elicit broadly neutralizing antibodies (bNAbs) against HIV-1, an appropriate animal model is necessary. It has been previously established that the CCR5-tropic SHIV-AD8-EO molecular clone is a pathogenic and mucosally transmissible virus in Rhesus macaques. Furthermore, some macaques infected with this SHIV develop bNAbs; however the mechanism of eliciting bNabs in this model remains unclear. Methods: One macaque, JG7, slowly developed breadth on a multi-clade 21-virus panel from weeks 47–131 post-infection. Frequency of antigen-specific germinal center B cells and T-follicular helper (TFH) cells were evaluated at the early wk47 time-point as well as somatic hypermutation in these B cell receptors (BCRs). Monoclonal antibodies were isolated and characterized by ELISA binding and neutralization profile. Results: By wk47, JG7 had high viral load, and high levels of antigen-specific TFH and IgG+B cells in the germinal center. A fingerprint analysis of sera neutralization data suggested a CD4-induced (CD4i) specificity at the early wk47 time-point. BCR sequencing revealed a large clonal family distinguished by high V-gene mutation frequency and long heavy chain CDR3s of 31 amino acids. Four BCRs were cloned and found to recapitulate most of the serum breadth at this time. Furthermore, these monoclonals competed with 17b for binding to gp120 and had enhanced neutralization activity in the presence of soluble CD4, confirming the CD4i prediction. Conclusions: Our findings support the hypothesis that in the presence of high antigen levels and T cell help, Rhesus macaques can develop cross-neutralizing anti-HIV antibodies with high levels of mutation and long CDRH3s akin to bNAbs isolated from HIV infected humans. These data highlight the utility of the SHIV model for studying development of bNAbs in Rhesus macaques.

Adoptive transfer of T-cell receptor (TCR) gene engineered T-cells targeting tumor-associated antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T-cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. We thus developed a multi-step strategy that involved CDR3-TCRB deep sequencing of tumor infiltrating lymphocytes and matching of TCRA-TCRB pairs by pairSEQ and single cell RT-PCR. CDR3 TCRA-TCRB pairs identified with this approach, were then reconstructed to full length based on IMGT database [http://www.imgt.org], synthetized and cloned into non-viral and retroviral expression vectors. T-cells were gene-engineered with these TCRs and tested against tumor cell lines and antigen presenting cells expressing tandem minigenes encoding mutated tumor neoantigens and/or pulsed with corresponding mutated peptides. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes including reactivity against neoantigens. These data demonstrate the feasibility of developing a rapid approach for the identification of tumor-reactive TCRs that can be used for personalized TCR-gene therapy.

We have previously shown that the RNA binding protein, polypyrimidine tract-binding protein (PTB) plays a crucial role in regulating the expression of CD40L in activated CD4 T cells. Regulation occurs through stabilizing the message at late times of activation as well as changing the distribution of CD40L mRNA within distinct cellular compartments. Further experiments were designed to assess how PTB affects global CD4 T cell activation by using shRNA introduced into primary human CD4 T cells. Specifically, we found that PTB is critical for the proliferation and viability of activated T cells. The propensity to undergo cell death was evident in both infected and uninfected CD4 T cells within the same population suggesting that either soluble and/or cognate interactions were being affected by decreased PTB. Accordingly, PTB was found to be required for optimal expression of CD25, CD69, TNF-α and IL-2 but not CD38 or IFN-γ. When the decay rates were assessed in anti-CD3/anti-CD28-stimulated CD4 T cells, the pattern of IL-2, TNF-α and CD69 mRNA decay did not change over a time course of activation whereas CD25 showed a pattern of mRNA decay that was highly similar to CD40L. Evaluation of signaling pathways using phosphoflow analysis revealed PTB-specific changes in the NF-κB and MAPK-ERK pathways. Together, these findings support a role for PTB in the control of CD4 T cell activation through processes that are both dependent on and independent of PTB-regulated mRNA stability.

The expression of the CD40 ligand (CD40L) is controlled at the posttranscriptional level by an activation-induced program of regulated mRNA stability whereby at early times of activation the mRNA is highly unstable and at later times (24-48 hr) the transcript becomes significantly stabilized. Regulated stability is dependent on the formation of a cytoplasmic polypyrimidine tract binding protein (PTB)-containing-complex (Complex I) that binds to the 3’UTR of the CD40L mRNA. Here we report that the broad-spectrum PKC inhibitor H7 destabilizes the CD40L mRNA in both a Jurkat subclone (Jurkat/D1.1) that recapitulates the “stability” phase of the CD40L regulated decay program and in primary human CD4+ T cells activated with PMA and ionomycin. In both cell populations, treatment with H7 also resulted in a corresponding decrease in surface CD40L expression. Analysis of RNA binding complexes by electrophoretic mobility shift assays (R-EMSA) following H7 treatment of Jurkat/D1.1 cells corresponded to an increase in Complex I as well as the formation of a novel PTB-independent complex that bound to the CD40L stability element. These results demonstrate the relationship between PKC-regulated pathways and the control of CD40L expression through mRNA stability. Future work is underway to identify the protein component(s) of this new complex, as well as to identify PKC isoforms that are involved in regulating PTB-mediated decay.

The expression of CD40L is tightly regulated at the posttranscriptional level throughout a time course of T cell activation, which corresponds to a significant increase in message stability at late times of activation (24-48 hr). Previous work revealed a cytoplasmic polypyrimidine tract binding protein (PTB)-containing-complex binds to the CD40L 3’UTR at late times of T cell activation. We used RNA interference to downregulate PTB in D1.1 Jurkat T cell line to address the functional roles of PTB in CD154 mRNA stability and surface expression. Real-time qPCR measurement of the CD154 mRNA decay revealed that downregulating PTB caused a > 2-fold decrease in the CD40L mRNA half-life. The downregulation of PTB also caused an approximate 2-fold decrease in the mean fluorescence (MFI) of CD40L. Analysis of PTB cellular distribution during a time course of CD4+ T cell activation revealed cytoplasmic and nuclear localization in all resting and activated cells. However, there was an increase in expression at late times of activation. Binding studies revealed that CD40L mRNA is bound by nuclear PTB at all times of activation indicating that the requirements for binding of CD40L message by nuclear versus cytoplasmic PTB is highly distinct. Finally, the binding of CD40L message corresponded to a change in phosphorylation status of cytoplasmic PTB. These findings support a model whereby activation-induced phosphorylation of PTB is required for cytoplasmic binding to CD40L mRNA.