Cell activation-based screening of natively paired human T cell receptor repertoires

Adoptive immune therapies based on the transfer of antigen-specific T cells have been used successfully to treat various cancers and viral infections, but improved techniques are needed to identify optimally protective human T cell receptors (TCRs). Here we present a high-throughput approach to the identification of natively paired human TCRα and TCRβ (TCRα:β) genes encoding heterodimeric TCRs that recognize specific peptide antigens bound to major histocompatibility complex molecules (pMHCs). We first captured and cloned TCRα:β genes from individual cells, ensuring fidelity using a suppression PCR. We then screened TCRα:β libraries expressed in an immortalized cell line using peptide-pulsed antigen-presenting cells and sequenced activated clones to identify the cognate TCRs. Our results validated an experimental pipeline that allows large-scale repertoire datasets to be annotated with functional specificity information, facilitating the discovery of therapeutically relevant TCRs

Modulation of CD40 ligand (CD40L) expression by polypyrimidine tract-binding protein

CD40 ligand (CD40L or CD154) is a protein expressed on activated CD4+ T cells, which is crucial for antibody-dependent and cell-mediated immunity. The expression of CD40L is tightly regulated at multiple levels throughout a time course of T cell activation. At the post-transcriptional level the CD40L message is rapidly degraded at early time points of activation followed by a significant increase in message stability at later times of activation (24-48 hr). Previous work from our lab revealed that a cytoplasmic polypyrimidine tract binding protein (PTB)-containing-complex binds to the CD40L 3'UTR at later times of T cell activation. To understand the direct relationship between PTB and CD40L mRNA stability and subsequently CD40L expression, we used viral RNA interference against PTB and scrambled shRNA (Control) sequence in model CD40L mRNA stability T cell line, Jurkat/D1.1. Downregulation of PTB resulted in dramatic decrease in half-life of the CD40L mRNA. The downregulation of PTB did not significantly change the percentage of CD40L+ cells, but caused an approximate 2-fold decrease in the mean fluorescence (MFI) of CD40L. Cellular fractionation of CD40L mRNA from shCTRL- and shPTB-infected cells revealed a novel role for nuclear PTB in retaining the CD40L mRNA in the nucleus. In addition, cytoplasmic PTB is important for optimal association of CD40L message with translating polysomes. 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 cytoplasmic PTB 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 post-translational modification of cytoplasmic PTB that appears to correlate with a change in phosphorylation status of PTB. Confocal microscopy analysis of CD4+ T cells with activation-induced CD40L mRNA stability revealed co-localization of PTB and CD40L mRNA at distinct foci, suggesting a role of PTB in the localization of the stable CD40L message during T cell activation.M.S.Includes abstractIncludes bibliographical referencesby Rodrigo A. Matus Nicodemo

The Mechanism for HIV Infection and Persistence in Resting Memory CD4 T cells

HIV remains one of the most challenging viruses in modern times. There are no effective vaccines for it, and HIV+ individuals must be on antiretroviral therapy (ART) for the entirety of their lives. ART is lifesaving but does not prevent or eliminate the HIV reservoir that persists in resting CD4 T cells during therapy. The mechanism for the establishment of this resting HIV reservoir is a central question in the field. Here, I tested the hypothesis that HIV, as a lentivirus, establishes a reservoir by direct infection of resting CD4 T cells. I designed a replication-competent HIV that expressed the fluorescent protein, GFP, from the HIV Nef gene. I used this HIVgfp to infect highly pure primary resting CD4 T cells from HIV-negative blood donors and investigated the mechanism for the infection. I found HIV directly infected resting memory CD4 T cells. Resting CD4 T cells had high levels of the CCR5 co-receptor for entry and sufficient dNTPs for reverse transcription (RT) due to a cell cycle-independent dNTP synthesis pathway. I found evidence that the non-canonical nucleotide deoxyuridine (dU) was incorporated into the provirus during RT. The dU-containing proviruses integrated into the host chromatin at regions with open DNA. HIV-infected resting CD4 T cells expressed HIV mRNAs encoding for Nef, VpuEnv, Vpr, and Vif. These four expressed HIV accessory proteins primed the infected resting cell to evade the host immune system and efficiently replicate HIV after T cell receptor (TCR) activation. TCR activation triggered HIV replication that correlated with the expression of the HIV Tat and Rev regulatory proteins. Additionally, I did a preliminary study of TCR recognition of HIV-specific CD8 T cells of HIV peptides presented by MHC class I molecules (pMHCs). I found in this study that TCR recognition was the primary determinant for controlling HIV infection. Together, this work provided a clear picture of why HIV can establish a persistent reservoir in resting CD4 T cells. Lastly, my work further demonstrates why HIV is so difficult to find a cure and/or an effective vaccine

The RNA-Binding Protein, Polypyrimidine Tract-Binding Protein 1 (PTBP1) Is a Key Regulator of CD4 T Cell Activation

<div><p>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.</p></div

PTB is critical for expression of multiple activation markers.

<p>(A) pLV-shCTRL- or pLV-shPTB-infected CD4 T cells were expanded for 13 days and either left untreated or activated with anti-CD3/anti-CD28 beads for 2 h or 48 h. Following activation, cells were stained with antibodies to selected cell surface markers and analyzed for expression by flow cytometry. Results showing the median fluorescence intensity (top graph) and the percent positive (lower graph) are presented and the boxed bars indicate observed changes in absolute number of positive cells. (B) Expanded CD4 T cells were left untreated or activated with 1 ng/ml PMA and 1 μg/ml Ionomycin for 5 h or anti-CD3/anti-CD28 mAb-bound beads for 48 h and analyzed by intracellular staining with specific antibodies to IL-2, TNFα and IFNγ. Data shown represent the mean and SEM of three independent experiments with *p≤0.05, **p ≤ 0.01, and ***p≤0.005.</p

PTBP1 is required for optimal expansion of CD4 T cells.

<p>(A) Negatively selected CD4 T cells were infected with either pLV-shCTRL or pLV-shPTB lentivirus, expanded for 10 days in IL-2 and removed from IL-2 for one day. At day 11, GFPposCD4pos cells were analyzed for PTBP1 expression using intracellular immunostaining with anti-PTBP1 mAb or isotype control. Median fluorescence intensity (MFI) of 5 independent experiments is indicated in table below. (B) CD4 T cells infected with either pLV-shCTRL or pLV-shPTB were expanded for 13 days and activated for two days with anti-CD3 + anti-CD28 antibodies. The GFPpos population in each panel is presented as a percentage of the total CD4pos population. (C) Experiments were carried out as in “B” and data compiled from a minimum of three independent experiments. Comparisons between pLV-shCTRL or pLV-shPTB infected cultures were carried out at 1–3 and 10–13 days post infection and again 2 days post activation. (D) Analysis of phospho-STAT5 after a 10 day IL-2-dependent expansion, followed by either 2 days with no IL-2 (grey bar), or 1 day with no IL-2 and either a 20 min stimulation with 250 U of IL-2 (black line) or 250 U IL-2 plus anti-CD3 + anti-CD28 beads (red line).</p

Steady-state RNA expression of different marker genes varies between early and late times of activation.

<p>(A) 5 X 10⁶ CD4 T cells were purified from total blood and cultured for 2 h or 48 h with anti-CD3 + anti-CD28 antibodies. Total RNA was reverse transcribed with random primers and analyzed using real time quantitative RT-qPCR. Shown is the fold change in expression between 2 h and 48 h of activation. (B) 2 h and 48 h stimulated CD4 T cells were incubated with DRB (50 μg/ml) for 15, 30, and 60 min. Analysis of RNA levels of the indicated targets was carried out following reverse transcription with poly(A) and RT-qPCR normalized to 18S RNA in each sample. Results represent the average and the SEM of three independent experiments.</p

CD40L, CD38 and IL-2 messages are less stable in cells expressing shPTB.

<p>(A) Sorted GFPposCD4pos T cells expressing either pLV-shCTRL or pLV-shPTB were incubated with anti-CD3 +anti-CD28 antibodies for 48 h followed by treatment with 50 μg/ml DRB for 15 min. mRNA decay was analyzed by RT-qPCR normalizing against 18S as an internal control. (B) Total RNA was extracted from CD4 T cells expressing either shCTRL or shPTB and reverse transcribed using poly(dT) as a primer. Following incubation with anti-CD3 + anti-CD28 antibody-coated beads for 48 h RNA was quantified using real time qPCR using 18S as an internal control. Results are presented as the fold difference of values obtained in shPTB-expressing cells over those from shCTRL-expressing cells. (C) Cytoplasmic extracts from CD4 T cells activated for 48 h with anti-CD3 + anti-CD28 antibodies were immunoprecipitated with anti-PTBP1 antibodies. RNA was isolated and analyzed by RT-qPCR for enrichment of transcripts in the bound fraction relative to the transcript representation in total cytoplasm.</p

PTBP1 regulates ERK1/2 and NF-κB signaling.

<p>(A) CD4 T cells infected with either pLV-shCTRL or pLV-shPTB lentivirus were activated with either anti-CD3/anti-CD28 beads or 1 ng/ml PMA and 1 μg/ml Io between 2 and 20 min (depending on the optimal response of individual signals). Phosphflow analysis was carried out by fixing cells in paraformaldehyde and permeablizing them for intracellular staining with antibodies to the indicated targets. (B) Histograms showing total (left panel) and phosphorylated ERK1/2 (right panel) in PMA/Io stimulated GFPposCD4pos T cells. (C) Histograms showing ERK1/2 signaling in cells expressing shCTRL and shPTB over 10 min of stimulation with PMA/ionomycin. (D) CD4 T cells infected with either pLV-shCTRL or pLV-shPTB lentivirus were activated with 1 ng/ml PMA and 1 μg/ml Ionomycin for 10 min and analyzed for PLCγ1, PKCθ, and IκBα activity (mean values (+/- SEM) with a *p≤0.05).</p

Reduced PTBP1 inhibits CD4 T cell Proliferation but not viability.

<p>(A) Analysis of cell viability in pLV-shCTRL or pLV-shPTB infected cells was carried out by staining 2×10⁵ cells with Annexin-V and analyzing GFPpos Annexin-Vpos cells prior to and after 48 h activation with anti-CD3 and anti-CD28 antibodies. Numbers shown are the percentage of PE-Anexin-Vpos cells in the total (GFPpos and GFPneg) population and comparisons were made between the pLV-shCTRL and pLV-shPTB infected cells as well as the infected and uninfected cells in the same population. (B) Compiled data from three independent experiments showing the extent of cell death in uninfected and pLV-shCTRL- and pLV-shPTB-infected T cells before and 48 h after stimulation with anti-CD3 and anti-CD28 beads. (C) 5 X 10⁶ pLV-shCTRL- and pLV-shPTB-infected T cells were incubated at day13 post-infection with 10 μM cell proliferation dye eFluor670 concurrently with the addition of anti-CD3/anti-CD28 conjugated beads for activation. Uninfected (GFPneg) and infected (GFPpos) populations were analyzed by flow cytometry at day 0 and day 2. (D) Division Index, Percent Divided and Proliferation Index were determined using FloJo software from three independent experiments (mean values (+/- SEM) with a *p≤0.05).</p