HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2

Anti-CD20 therapy depletes activated myelin-specific CD8+ T cells in multiple sclerosis.

CD8+ T cells are believed to play an important role in multiple sclerosis (MS), yet their role in MS pathogenesis remains poorly defined. Although myelin proteins are considered potential autoantigenic targets, prior studies of myelin-reactive CD8+ T cells in MS have relied on in vitro stimulation, thereby limiting accurate measurement of their ex vivo precursor frequencies and phenotypes. Peptide:MHC I tetramers were used to identify and validate 5 myelin CD8+ T cell epitopes, including 2 newly described determinants in humans. The validated tetramers were used to measure the ex vivo precursor frequencies and phenotypes of myelin-specific CD8+ T cells in the peripheral blood of untreated MS patients and HLA allele-matched healthy controls. In parallel, CD8+ T cell responses against immunodominant influenza epitopes were also measured. There were no differences in ex vivo frequencies of tetramer-positive myelin-specific CD8+ T cells between MS patients and control subjects. An increased proportion of myelin-specific CD8+ T cells in MS patients exhibited a memory phenotype and expressed CD20 compared to control subjects, while there were no phenotypic differences observed among influenza-specific CD8+ T cells. Longitudinal assessments were also measured in a subset of MS patients subsequently treated with anti-CD20 monoclonal antibody therapy. The proportion of memory and CD20+ CD8+ T cells specific for certain myelin but not influenza epitopes was significantly reduced following anti-CD20 treatment. This study, representing a characterization of unmanipulated myelin-reactive CD8+ T cells in MS, indicates these cells may be attractive targets in MS therapy

Increased TCR Avidity after T Cell Activation A Mechanism for Sensing Low-Density Antigen

AbstractWhile activated T cells are known to have enhanced biological responses to antigen stimulation, the biophysical basis of this increased sensitivity remains unknown. Here, we show that, on activated T cells, the TCR avidity for peptide-MHC complexes is 20- to 50-fold higher than the TCR avidity of naive T cells. This increased avidity for peptide-MHC depends on TCR reorganization and is sensitive to the cholesterol content of the T cell membrane. Analysis of the binding data indicates the enhanced avidity is due to increases in cross-linking of TCR on activated T cells. Activation-induced membrane (AIM) changes in TCR avidity represent a previously unrecognized means of increasing the sensitivity of activated T cells to small amounts of antigen in the periphery

Enhanced Antigen-Specific Antitumor Immunity with Altered Peptide Ligands that Stabilize the MHC-Peptide-TCR Complex

AbstractT cell responsiveness to an epitope is affected both by its affinity for the presenting MHC molecule and the affinity of the MHC-peptide complex for TCR. One limitation of cancer immunotherapy is that natural tumor antigens elicit relatively weak T cell responses, in part because high-affinity T cells are rendered tolerant to these antigens. We report here that amino acid substitutions in a natural MHC class I–restricted tumor antigen that increase the stability of the MHC-peptide-TCR complex are significantly more potent as tumor vaccines. The improved immunity results from enhanced in vivo expansion of T cells specific for the natural tumor epitope. These results indicate peptides that stabilize the MHC-peptide-TCR complex may provide superior antitumor immunity through enhanced stimulation of specific T cells

T-Cell Memory Responses Elicited by Yellow Fever Vaccine are Targeted to Overlapping Epitopes Containing Multiple HLA-I and -II Binding Motifs

The yellow fever vaccines (YF-17D-204 and 17DD) are considered to be among the safest vaccines and the presence of neutralizing antibodies is correlated with protection, although other immune effector mechanisms are known to be involved. T-cell responses are known to play an important role modulating antibody production and the killing of infected cells. However, little is known about the repertoire of T-cell responses elicited by the YF-17DD vaccine in humans. In this report, a library of 653 partially overlapping 15-mer peptides covering the envelope (Env) and nonstructural (NS) proteins 1 to 5 of the vaccine was utilized to perform a comprehensive analysis of the virus-specific CD4+ and CD8+ T-cell responses. The T-cell responses were screened ex-vivo by IFN-γ ELISPOT assays using blood samples from 220 YF-17DD vaccinees collected two months to four years after immunization. Each peptide was tested in 75 to 208 separate individuals of the cohort. The screening identified sixteen immunodominant antigens that elicited activation of circulating memory T-cells in 10% to 33% of the individuals. Biochemical in-vitro binding assays and immunogenetic and immunogenicity studies indicated that each of the sixteen immunogenic 15-mer peptides contained two or more partially overlapping epitopes that could bind with high affinity to molecules of different HLAs. The prevalence of the immunogenicity of a peptide in the cohort was correlated with the diversity of HLA-II alleles that they could bind. These findings suggest that overlapping of HLA binding motifs within a peptide enhances its T-cell immunogenicity and the prevalence of the response in the population. In summary, the results suggests that in addition to factors of the innate immunity, "promiscuous" T-cell antigens might contribute to the high efficacy of the yellow fever vaccines. © 2013 de Melo et al

Tumor and Microenvironment Evolution during Immunotherapy with Nivolumab.

The mechanisms by which immune checkpoint blockade modulates tumor evolution during therapy are unclear. We assessed genomic changes in tumors from 68 patients with advanced melanoma, who progressed on ipilimumab or were ipilimumab-naive, before and after nivolumab initiation (CA209-038 study). Tumors were analyzed by whole-exome, transcriptome, and/or T cell receptor (TCR) sequencing. In responding patients, mutation and neoantigen load were reduced from baseline, and analysis of intratumoral heterogeneity during therapy demonstrated differential clonal evolution within tumors and putative selection against neoantigenic mutations on-therapy. Transcriptome analyses before and during nivolumab therapy revealed increases in distinct immune cell subsets, activation of specific transcriptional networks, and upregulation of immune checkpoint genes that were more pronounced in patients with response. Temporal changes in intratumoral TCR repertoire revealed expansion of T cell clones in the setting of neoantigen loss. Comprehensive genomic profiling data in this study provide insight into nivolumab\u27s mechanism of action

Fas-Mediated Apoptosis Regulates the Composition of Peripheral αβ T Cell Repertoire by Constitutively Purging Out Double Negative T Cells

BACKGROUND: The Fas pathway is a major regulator of T cell homeostasis, however, the T cell population that is controlled by the Fas pathway in vivo is poorly defined. Although CD4 and CD8 single positive (SP) T cells are the two major T cell subsets in the periphery of wild type mice, the repertoire of mice bearing loss-of-function mutation in either Fas (lpr mice) or Fas ligand (gld mice) is predominated by CD4(-)CD8(-) double negative alphabeta T cells that also express B220 and generally referred to as B220+DN T cells. Despite extensive analysis, the basis of B220+DN T cell lymphoproliferation remains poorly understood. In this study we re-examined the issue of why T cell lymphoproliferation caused by gld mutation is predominated by B220+DN T cells. METHODOLOGY AND PRINCIPAL FINDINGS: We combined the following approaches to study this question: Gene transcript profiling, BrdU labeling, and apoptosis assays. Our results show that B220+DN T cells are proliferating and dying at exceptionally high rates than SP T cells in the steady state. The high proliferation rate is restricted to B220+DN T cells found in the gut epithelium whereas the high apoptosis rate occurred both in the gut epithelium and periphery. However, only in the periphery, apoptosis of B220+DN T cell is Fas-dependent. When the Fas pathway is genetically impaired, apoptosis of peripheral B220+DN T cells was reduced to a baseline level similar to that of SP T cells. Under these conditions of normalized apoptosis, B220+DN T cells progressively accumulate in the periphery, eventually resulting in B220+DN T cell lymphoproliferation. CONCLUSIONS/SIGNIFICANCE: The Fas pathway plays a critical role in regulating the tissue distribution of DN T cells through targeting and elimination of DN T cells from the periphery in the steady state. The results provide new insight into pathogenesis of DN T cell lymphoproliferation