Application of B cell immortalization for the isolation of antibodies and B cell clones from vaccine and infection settings

The isolation and characterization of neutralizing antibodies from infection and vaccine settings informs future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL and transform primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. However, application of this methodology to B cell subsets from different tissues and B cells from chronically infected individuals has not been well characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In healthy individuals, naïve and memory B cell subsets from PBMCs and tonsil tissue transformed with similar efficiencies, and displayed similar characteristics with respect to their longevity and immunoglobulin secretion. In HIV-1-viremic individuals or in individuals with recent malaria infections, the exhausted CD27-CD21- memory B cells transformed with lower efficiency, but the transformed B cells expanded and secreted IgG with similar efficiency. Importantly, we show that this methodology can be used to isolate broadly neutralizing antibodies from HIV-infected individuals. Overall, we demonstrate that Bcl-6/Bcl-xL B cell immortalization can be used to isolate antibodies and generate B cell clones from different B cell populations, albeit with varying efficiencies

A Second SNARE Role for Exocytic SNAP25 in Endosome Fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play key roles in membrane fusion, but their sorting to specific membranes is poorly understood. Moreover, individual SNARE proteins can function in multiple membrane fusion events dependent upon their trafficking itinerary. Synaptosome-associated protein of 25 kDa (SNAP25) is a plasma membrane Q (containing glutamate)-SNARE essential for Ca(2+)-dependent secretory vesicle–plasma membrane fusion in neuroendocrine cells. However, a substantial intracellular pool of SNAP25 is maintained by endocytosis. To assess the role of endosomal SNAP25, we expressed botulinum neurotoxin E (BoNT E) light chain in PC12 cells, which specifically cleaves SNAP25. BoNT E expression altered the intracellular distribution of SNAP25, shifting it from a perinuclear recycling endosome to sorting endosomes, which indicates that SNAP25 is required for its own endocytic trafficking. The trafficking of syntaxin 13 and endocytosed cargo was similarly disrupted by BoNT E expression as was an endosomal SNARE complex comprised of SNAP25/syntaxin 13/vesicle-associated membrane protein 2. The small-interfering RNA-mediated down-regulation of SNAP25 exerted effects similar to those of BoNT E expression. Our results indicate that SNAP25 has a second function as an endosomal Q-SNARE in trafficking from the sorting endosome to the recycling endosome and that BoNT E has effects linked to disruption of the endosome recycling pathway

Selective Loss of Early Differentiated, Highly Functional PD1high CD4 T Cells with HIV Progression.

The role of PD-1 expression on CD4 T cells during HIV infection is not well understood. Here, we describe the differential expression of PD-1 in CD127high CD4 T cells within the early/intermediate differentiated (EI) (CD27highCD45RAlow) T cell population among uninfected and HIV-infected subjects, with higher expression associated with decreased viral replication (HIV-1 viral load). A significant loss of circulating PD-1highCTLA-4low CD4 T cells was found specifically in the CD127highCD27highCD45RAlow compartment, while initiation of antiretroviral treatment, particularly in subjects with advanced disease, reversed these dynamics. Increased HIV-1 Gag DNA was also found in PD-1high compared to PD-1low ED CD4 T cells. In line with an increased susceptibility to HIV infection, PD-1 expression in this CD4 T cell subset was associated with increased activation and expression of the HIV co-receptor, CCR5. Rather than exhaustion, this population produced more IFN-g, MIP1-a, IL-4, IL-10, and IL-17a compared to PD-1low EI CD4 T cells. In line with our previous findings, PD-1high EI CD4 T cells were also characterized by a high expression of CCR7, CXCR5 and CCR6, a phenotype associated with increased in vitro B cell help. Our data show that expression of PD-1 on early-differentiated CD4 T cells may represent a population that is highly functional, more susceptible to HIV infection and selectively lost in chronic HIV infection

Rhesus macaque Bcl-6/Bcl-xL B cell immortalization: Discovery of HIV-1 neutralizing antibodies from lymph node

Many HIV-1 vaccines are designed to elicit neutralizing antibodies, and pre-clinical testing is often carried out in rhesus macaques (RMs). We have therefore adapted a method of B cell immortalization for use with RM B cells. In this system, RM B cells are activated with CD40 ligand and RM IL-21 before transduction with a retroviral vector encoding Bcl-6, Bcl-xL, and green fluorescent protein. Importantly, RM B cells from lymph nodes are more effectively immortalized by this method than B cells from PBMC, a difference not seen in humans. We suggest the discrepancy between these two tissues is due to increased expression of CD40 on RM lymph node B cells. Immortalized RM B cells expand long-term, undergo minimal somatic hypermutation, express surface B cell receptor, and secrete antibodies into culture. This allows for the identification of cells based on antigen specificity and/or functional assays. Here, we show the characterization of this system and its application for the isolation of HIV-1 neutralizing antibodies from a SHIV.CH505-infected animal, both with and without antigen probe. Taken together, we show that Bcl-6/xL immortalization is a valuable and flexible tool for antibody discovery in RMs, but with important distinctions from application of the system in human cells

Interaction dynamics between innate and adaptive immune cells responding to SARS-CoV-2 vaccination in non-human primates

Abstract As SARS-CoV-2 variants continue evolving, testing updated vaccines in non-human primates remains important for guiding human clinical practice. To date, such studies have focused on antibody titers and antigen-specific B and T cell frequencies. Here, we extend our understanding by integrating innate and adaptive immune responses to mRNA-1273 vaccination in rhesus macaques. We sorted innate immune cells from a pre-vaccine time point, as well as innate immune cells and antigen-specific peripheral B and T cells two weeks after each of two vaccine doses and used single-cell sequencing to assess the transcriptomes and adaptive immune receptors of each cell. We show that a subset of S-specific T cells expresses cytokines critical for activating innate responses, with a concomitant increase in CCR5-expressing intermediate monocytes and a shift of natural killer cells to a more cytotoxic phenotype. The second vaccine dose, administered 4 weeks after the first, elicits an increase in circulating germinal center-like B cells 2 weeks later, which are more clonally expanded and enriched for epitopes in the receptor binding domain. Both doses stimulate inflammatory response genes associated with elevated antibody production. Overall, we provide a comprehensive picture of bidirectional signaling between innate and adaptive components of the immune system and suggest potential mechanisms for the enhanced response to secondary exposure

The frequency of less differentiated PD-1^high CD127^high CD4 T cells is reduced compared with more differentiated subsets in advanced HIV infection.

<p><b>(A)</b> Gating strategy to define differentiation status of CD127, PD-1 and CTLA-4 expression by CD4 T cells. Differentiation was defined by gating on CD27 and CD45RA with CD27^highCD45RA^high (referred to as <b>Naïve</b>), CD27^highCD45RA^low (<b>Early/Intermediate</b>), and CD27^lowCD45RA^low (<b>Late</b>). <b>(B)</b> Distribution plots from HIV- infected subjects compared to HIV-uninfected (open circles, n = 15) from two cohorts with HIV infection: Cohort 1 (median CD4 count 525 cells/μl, filled circles, n = 31); and Cohort 2 with more advanced infection (median CD4 count 148 cells/μl, filled squares, n = 14) of PD-1 and PD-1/CTLA-4 expression by differentiation status and CD127 (IL-7Ra) staining demonstrating an altered/reduced frequency of PD-1^high CTLA-4^high/low CD127^high CD4 T cells of early/intermediate differentiation compared to more differentiated subsets which show increased PD-1 expression with more advanced HIV infection. Plots include median and interquartile range, *p< 0.05, **p< 0.001, ***p< 0.0001 by Kruskal-Wallis or Mann-Whitney test.</p

Seletctive loss of PD-1^highCTLA-4^low/highCD127^high Early/Intermediate CD4 T cells occurs with higher plasma HIV-1 viral RNA levels and higher cell-associated viral DNA.

<p><b>(A)</b> Scatter plots of HIV-1 viral RNA and fitted regression lines for total (naïve and memory) CD8 and CD4 T cells demonstrating increased PD-1 expression with higher viral replication. However, for CD4 T cells of Early/Intermediate differentiation expressing CD127 and PD-1 or PD-1/CTLA-4 there is a negative association compared with more differentated (CD127^low) CD4 T cells. Spearman rank correlation coefficients and associated p-values are shown. <b>(B)</b> Donors (n = 14, five from Cohort 1 and nine from Cohort 3) with HIV Gag-specific CD4 T-cell responses are more differentiated (CD127^low) and co-express both PD-1 and CTLA-4. <b>(C)</b> Cell-associated HIV-1 <i>gag</i> DNA (no. copies/cell) for sorted T cell populations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144767#pone.0144767.s003" target="_blank">S3 Fig</a> for gating strategy). Individual differences between differentiation subsets (shown for each individual by a connecting line) are statistically significant (p = 0.031 by Friedman test). <b>(D)</b> PD-1^highCTLA-4^lowCD127^high Early/Intermediate CD4 T cells are increased after antiretroviral therapy. Relative frequencies of bulk CD4 populations before and after initiation of combination antiretroviral therapy (cART). Connected symbols represent pre-cART and 48 weeks post-cART (Cohort 2, n = 14, Wilcoxon matched-pairs test, p-values shown in figure). The PD-1^highCTLA-4^low CD127^high group is analyzed separately for subjects who started cART with a CD4 count less than 200.</p

CD4 T follicular helper cell dynamics during SIV infection

CD4 T follicular helper (TFH) cells interact with and stimulate the generation of antigen-specific B cells. TFH cell interaction with B cells correlates with production of SIV-specific immunoglobulins. However, the fate of TFH cells and their participation in SIV-induced antibody production is not well understood. We investigated the phenotype, function, location, and molecular signature of TFH cells in rhesus macaques. Similar to their human counterparts, TFH cells in rhesus macaques represented a heterogeneous population with respect to cytokine function. In a highly differentiated subpopulation of TFH cells, characterized by CD150lo expression, production of Th1 cytokines was compromised while IL-4 production was augmented, and cells exhibited decreased survival, cycling, and trafficking capacity. TFH cells exhibited a distinct gene profile that was markedly altered by SIV infection. TFH cells were infected by SIV; yet, in some animals, these cells actually accumulated during chronic SIV infection. Generalized immune activation and increased IL-6 production helped drive TFH differentiation during SIV infection. Accumulation of TFH cells was associated with increased frequency of activated germinal center B cells and SIV-specific antibodies. Therefore, chronic SIV does not disturb the ability of TFH cells to help B cell maturation and production of SIV-specific immunoglobulins