Functional Role of Kallikrein 6 in Regulating Immune Cell Survival

Kallikrein 6 (KLK6) is a newly identified member of the kallikrein family of secreted serine proteases that prior studies indicate is elevated at sites of central nervous system (CNS) inflammation and which shows regulated expression with T cell activation. Notably, KLK6 is also elevated in the serum of multiple sclerosis (MS) patients however its potential roles in immune function are unknown. Herein we specifically examine whether KLK6 alters immune cell survival and the possible mechanism by which this may occur.Using murine whole splenocyte preparations and the human Jurkat T cell line we demonstrate that KLK6 robustly supports cell survival across a range of cell death paradigms. Recombinant KLK6 was shown to significantly reduce cell death under resting conditions and in response to camptothecin, dexamethasone, staurosporine and Fas-ligand. Moreover, KLK6-over expression in Jurkat T cells was shown to generate parallel pro-survival effects. In mixed splenocyte populations the vigorous immune cell survival promoting effects of KLK6 were shown to include both T and B lymphocytes, to occur with as little as 5 minutes of treatment, and to involve up regulation of the pro-survival protein B-cell lymphoma-extra large (Bcl-XL), and inhibition of the pro-apoptotic protein Bcl-2-interacting mediator of cell death (Bim). The ability of KLK6 to promote survival of splenic T cells was also shown to be absent in cell preparations derived from PAR1 deficient mice.KLK6 promotes lymphocyte survival by a mechanism that depends in part on activation of PAR1. These findings point to a novel molecular mechanism regulating lymphocyte survival that is likely to have relevance to a range of immunological responses that depend on apoptosis for immune clearance and maintenance of homeostasis

Partitioning the Heritability of Tourette Syndrome and Obsessive Compulsive Disorder Reveals Differences in Genetic Architecture

The direct estimation of heritability from genome-wide common variant data as implemented in the program Genome-wide Complex Trait Analysis (GCTA) has provided a means to quantify heritability attributable to all interrogated variants. We have quantified the variance in liability to disease explained

An elite controller of picornavirus infection targets an epitope that is resistant to immune escape.

The emergence of novel viral pathogens can lead to devastating consequences in the infected population. However, on occasion, rare hyper-responsive elite controllers are able to mount a protective primary response to infection and clear the new pathogen. Factors distinguishing elite controllers from other members of the population are not completely understood. We have been using Theiler's murine encephalomyelitis as a model of primary infection in mice and clearance of the virus is limited to one MHC genotype capable of generating a protective response to a single viral peptide VP2121-130. The genetics of host susceptibility to TMEV, a natural mouse pathogen, has been studied extensively and non-protective CD8 responses to other peptides have been documented, however, little is known why the protective response to infection focuses on the VP2121-130 peptide. To study this question, we have generated TMEV mutants that encode for mutations within the VP2121-130 peptide. We find that very few of mutants are able to assemble and infect in vitro. These mutations are not related to virus RNA structure since non-coding mutations do not interfere with assembly. In the rare event when functional VP2121-130 mutant viruses did emerge, they were attenuated to some level or retained the ability to develop an immune response to the wild-type VP2121-130 sequence, demonstrating that the virus is incapable of escaping the protective response. These findings advance our understanding of how characteristics of the host immune response and an infectious agent can interact to lead to the appearance of rare super controllers in a population. Furthermore, the immutable nature of the viral antigen highlights the importance of choosing appropriate vaccine antigens and has implications for the development of agents that are able to generate protective CD8 T-cell responses

Development of TMEV VP2_121-130 mutant viruses.

<p>(A) Real-time RT-PCR expression analysis of viral VP2 and plasmid neomycin phosphotransferase from BHK cells transfected with TMEV VP2_121-130 mutant cDNA. (B) Western blot analysis of whole cell lysates and viral supernatants for TMEV viral proteins from cells transfected with wild-type TMEV-DA cDNA for 3 and 7 days. (C) Western blot of whole cell lysates and supernatants from cells transfected with TMEV VP2_121-130 mutant plasmid cDNA (D) Western blot of supernatants derived from cells transfected with TMEV VP2₁₂₅ mutant plasmid cDNA.</p

VP2_121-130 mutants.

<p>VP2_121-130 mutants.</p

Altered VP2_121-130 codon structure does not influence virus fidelity.

<p>(A) Thirteen silent nucleotide substitutions representing changes to 9 of the 10 codons of VP2_121-130 were introduced into the TMEV-DA plasmid cDNA by site-directed mutagenesis. (B) Sequence verification of codon alternate VP2_121-130 virus recovered from infected BHK cells. (C) Western blot analysis of virus supernatant recovered from cells infected with codon alternate TMEV-VP2_121-130.</p

I-Mutant predicted ΔΔG values for TMEV VP2_121-130 amino acid substitutions.

<p>I-Mutant predicted ΔΔG values for TMEV VP2_121-130 amino acid substitutions.</p

Peptide depletion reveals an overlap in the wild-type VP2_121-130 CD8+ T cell response after infection with S125A and M130L mutant viruses.

<p>(A) Mice were pre-depleted with control E7₄₉, wild-type VP2_121-130, VP2-S125A and VP2-M130L peptide prior to intracranial infection with wild-type virus. CNS infiltrating lymphocytes were analyzed by flow cytometry to determine the percentage and number of CD8+ T-cells specific for the immunodominant epitope after peptide depletion. Data are representative examples of three individual mice per group. (B) Percent and absolute number of H-2D^b/VP2₁₂₁+ CD8 T-cells derived from infected CNS tissue including a no peptide group (* significant by ANOVA).</p

CD8+ T-cell response and virus RNA levels in resistant and susceptible mice after intracranial infection with TMEV-wt, S125A and M130L viruses.

<p>(A) CD45+ cells isolated from the central nervous system of VP2 mutant infected C57BL/6 mice were analyzed for the presence of CD8+ cells. (^a significant compared to M130L by ANOVA, * significant compared to TMEV-wt by ANOVA). Figure is an experiment replicated 3 times with 3-5 animals per group. (B) CNS infiltrating lymphocytes were stained with H-2D^b/VP2₁₂₁ or H-2D^b/E7₄₉ tetramers and analyzed by flow cytometry to determine the percent of the CD8+ T-cell population that is positive for the immunodominant VP2₁₂₁ epitope (* significant compared to S125A and M130L by ANOVA). A representative example of 2 experiments using 3-5 animals per group. (C) Semi-quantitative RT-PCR analysis of RNA isolated by C57BL/6 mice infected with TMEV-wt, S125A or M130L mutants (n = 3/group). No significant differences in viral transcripts were detected between the groups. IgG specific responses to wild-type TMEV in C57BL/6 mice infected for 30 days. No significant differences were detected by ELISA between S125, M130L or pciDA (wild-type TMEV) (D) The same RT-PCR analysis in C on the susceptible strain FVB (* significant compared to TMEV-wild type by ANOVA, n = 5/group). TMEV-specific IgG response and antibody neutralization to wild-type TMEV in FVB mice infected for 30 days. No significant differences were detected by ELISA or neutralization assay between S125, M130L or pciDA (wild-type TMEV).</p