The impact of DM on MHC class II–restricted antigen presentation can be altered by manipulation of MHC–peptide kinetic stability

DM edits the peptide repertoire presented by major histocompatibility complex class II molecules by professional antigen-presenting cells (APCs), favoring presentation of some peptides over others. Despite considerable research by many laboratories, there is still significant uncertainty regarding the biochemical attributes of class II–peptide complexes that govern their susceptibility to DM editing. Here, using APCs that either do or do not express DM and a set of unrelated antigens, we found that the intrinsic kinetic stability of class II–peptide complexes is tightly correlated with the effects of DM editing within APCs. Furthermore, through the use of kinetic stability variants of three independent peptides, we demonstrate that increasing or decreasing the kinetic stability of class II–peptide complexes causes a corresponding alteration in DM editing. Finally, we show that the spontaneous kinetic stability of class II complexes correlates directly with the efficiency of presentation by DM+ APCs and the immunodominance of that class II–peptide complex during an immune response. Collectively, these results suggest that the pattern of DM editing in APCs can be intentionally changed by modifying class II–peptide interactions, leading to the desired hierarchy of presentation on APCs, thereby promoting recruitment of CD4 T cells specific for the preferred peptides during an immune response

Identification of new cytokine combinations for antigen-specific T-cell therapy products via a high-throughput multi-parameter assay.

Infusion of viral-specific T cells (VSTs) is an effective treatment for viral infection after stem cell transplant. Current manufacturing approaches are rapid, but growth conditions can still be further improved. To optimize VST cell products, we designed a high-throughput flow cytometry-based assay using 40 cytokine combinations in a 96 well plate to fully characterize T cell viability, function, growth, and differentiation. Peripheral blood mononuclear cells (PBMC) from six consenting donors were seeded at 100,000 cells/well with pools of CMV peptides from IE-1 and pp65, and combinations of IL15, IL6, IL21, IFNα, IL12, IL18, IL4, and IL7. Ten-day cultures were tested by 13 color flow cytometry to evaluate viable cell count, lymphocyte phenotype, memory markers, and IFNγ and TNFα expression. Combinations of IL15/IL6 and IL4/IL7 were optimal for the expansion of viral-specific CD3+ T cells, (18-fold and 14-fold respectively compared with unstimulated controls). CD8+ T cells expanded 24-fold in IL15/IL6, and 9-fold in IL4/IL7 cultures (p< 0.0001). CD4+ T cells expanded 27-fold in IL4/IL7 and 15-fold in IL15/IL6 (p< 0.0001). CD45RO+ CCR7- effector memory T cells were the preponderant cells (76.8% and 72.3% in IL15/IL6 and IL15/IL7 cultures, respectively). Cells cultured in both cytokine conditions were potent, with 19.4% of CD3+ cells cultured in IL15/IL6 producing IFNγ (7.6% producing both TNFα and IFNγ), and 18.5% of CD3+ cells grown in IL4/IL7 (9% producing both TNFα and IFNγ). This study shows the utility of this single plate assay to rapidly identify optimal growth conditions for VST manufacture using only 10(7) PBMC

Direct Ex Vivo Analyses of HLA-DR1 Transgenic Mice Reveal an Exceptionally Broad Pattern of Immunodominance in the Primary HLA-DR1-Restricted CD4 T-Cell Response to Influenza Virus Hemagglutinin▿

The recent threat of an avian influenza pandemic has generated significant interest in enhancing our understanding of the events that dictate protective immunity to influenza and in generating vaccines that can induce heterosubtypic immunity. Although antigen-specific CD4 T cells are known to play a key role in protective immunity to influenza through the provision of help to B cells and CD8 T cells, little is known about the specificity and diversity of CD4 T cells elicited after infection, particularly those elicited in humans. In this study, we used HLA-DR transgenic mice to directly and comprehensively identify the specificities of hemagglutinin (HA)-specific CD4 T cells restricted to a human class II molecule that were elicited following intranasal infection with a strain of influenza virus that has been endemic in U.S. human populations for the last decade. Our results reveal a surprising degree of diversity among influenza virus-specific CD4 T cells. As many as 30 different peptides, spanning the entire HA protein, were recognized by CD4 T cells, including epitopes genetically conserved among H1, H2, and H5 influenza A viruses. We also compared three widely used major histocompatibility class II algorithms to predict HLA-DR binding peptides and found these as yet inadequate for identifying influenza virus-derived epitopes. The results of these studies offer key insights into the spectrum of peptides recognized by HLA-DR-restricted CD4 T cells that may be the focus of immune responses to infection or to experimental or clinical vaccines in humans

IL-4 Attenuates Th1-Associated Chemokine Expression and Th1 Trafficking to Inflamed Tissues and Limits Pathogen Clearance

<div><p>Interleukin 4 (IL-4) plays a central role in the orchestration of Type 2 immunity. During T cell activation in the lymph node, IL-4 promotes Th2 differentiation and inhibits Th1 generation. In the inflamed tissue, IL-4 signals promote innate and adaptive Type-2 immune recruitment and effector function, positively amplifying the local Th2 response. In this study, we identify an additional negative regulatory role for IL-4 in limiting the recruitment of Th1 cells to inflamed tissues. To test IL-4 effects on inflammation subsequent to Th2 differentiation, we transiently blocked IL-4 during ongoing dermal inflammation (using anti-IL-4 mAb) and analyzed changes in gene expression. Neutralization of IL-4 led to the upregulation of a number of genes linked to Th1 trafficking, including CXCR3 chemokines, CCL5 and CCR5 and an associated increase in IFNγ, Tbet and TNFα genes. These gene expression changes correlated with increased numbers of IFNγ-producing CD4+ T cells in the inflamed dermis. Moreover, using an adoptive transfer approach to directly test the role of IL-4 in T cell trafficking to the inflamed tissues, we found IL-4 neutralization led to an early increase in Th1 cell recruitment to the inflamed dermis. These data support a model whereby IL-4 dampens Th1-chemokines at the site of inflammation limiting Th1 recruitment. To determine biological significance, we infected mice with <i>Leishmania major</i>, as pathogen clearance is highly dependent on IFNγ-producing CD4+ T cells at the infection site. Short-term IL-4 blockade in established <i>L. major</i> infection led to a significant increase in the number of IFNγ-producing CD4+ T cells in the infected ear dermis, with no change in the draining LN. Increased lymphocyte influx into the infected tissue correlated with a significant decrease in parasite number. Thus, independent of IL-4's role in the generation of immune effectors, IL-4 attenuates lymphocyte recruitment to the inflamed/infected dermis and limits pathogen clearance.</p></div

Short-term IL-4 blockade modulates the inflamed tissue environment.

<p><b>A</b>) Antigen-specific IL-4 and IFNγ production by ELISPOT, day 7 following OVA/CFA immunization. <b>B</b>) Heat map of TLDA array genes (n = 94 genes examined, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071949#pone.0071949.s001" target="_blank">Table S1</a>) expressed in the ear dermis of control and anti-IL-4 treated OVA/CFA immunized mice. Anti-IL-4 treatment (or PBS) was administered on day 7 and 10 post-immunization and RNA extracted on day 14. 29 genes (rows) were identified based on the treatment p-value (<0.05) after a 2-way ANOVA. The columns correspond to 18 samples. The colored circles beneath the heat map indicate which of the four experiments the sample was processed in. Color in the heat map corresponds to the z-score of relative expression data. For each gene, the z-score was computed separately in each of the four experiments rather than using all 18 samples together. Genes were clustered using correlation and average linkage.</p

Increased T cell infiltration and improved <i>L. major</i> parasite clearance following short-term IL-4 blockade.

<p><b>A</b>) Increased immune cell accumulation after anti-IL-4 treatment in the <i>L. major</i> infected dermis: number of CD4+CD3+ T cells, representative data from one of three experiments. Anti-IL-4 treatment was administered on day 7 and 10 post-infection and cells analyzed on day 14. <b>B</b>) Anti-<i>Leishmania</i> IFNγ and IL-4 producing T cells by ELISPOT in infected ear (top) and draining LN (bottom) after anti-IL-4 treatment or PBS as in A). Data from 4 independent experiments. Statistics comparing PBS and 11B11 by ANOVA. <b>C</b>) Parasite load in ear dermis after <i>L. major</i> infection and anti-IL-4 treatment on day 7 and day 10, infected tissue harvested on day 21. A) and C) Statistics by Mann Whitney: *p<0.05. ***p<0.001. <b>D</b>) Relative gene expression in the <i>L. major</i>-infected dermis after 11B11 or PBS treatment (as in A); 8 mice per group, from two independent experiments. Statistics by one-tailed T test, * <0.05.</p