Replication Study: Melanoma genome sequencing reveals frequent PREX2 mutations.

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Chroscinski et al., 2014) that described how we intended to replicate selected experiments from the paper "Melanoma genome sequencing reveals frequent PREX2 mutations" (Berger et al., 2012). Here we report the results of those experiments. We regenerated cells stably expressing ectopic wild-type and mutant phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2 (PREX2) using the same immortalized human NRASG12D melanocytes as the original study. Evaluation of PREX2 expression in these newly generated stable cells revealed varying levels of expression among the PREX2 isoforms, which was also observed in the stable cells made in the original study (Figure S6A; Berger et al., 2012). Additionally, ectopically expressed PREX2 was found to be at least 5 times above endogenous PREX2 expression. The monitoring of tumor formation of these stable cells in vivo resulted in no statistically significant difference in tumor-free survival driven by PREX2 variants, whereas the original study reported that these PREX2 mutations increased the rate of tumor incidence compared to controls (Figure 3B and S6B; Berger et al., 2012). Surprisingly, the median tumor-free survival was 1 week in this replication attempt, while 70% of the control mice were reported to be tumor-free after 9 weeks in the original study. The rapid tumor onset observed in this replication attempt, compared to the original study, makes the detection of accelerated tumor growth in PREX2 expressing NRASG12D melanocytes extremely difficult. Finally, we report meta-analyses for each result.

Role of anti-glycoproteins D (anti-gD) and K (anti-gK) IgGs in pathology of herpes stromal keratitis in humans.

PURPOSE: To assess the relative impact of antibodies specific for HSV-1 glycoproteins on eye disease in response to HSV-1 infection, the composition of antibodies specific for 10 of the viral glycoproteins, and the effect of anti-glycoprotein (g)D and anti-gK antibodies on antibody-dependent enhancement (ADE). METHODS: In a prospective case-control study, sera from patients with a history of herpes stromal keratitis (HSK) were compared with sera from nonocular HSV-1-seropositive and HSV-seronegative control subjects. HSV-1 neutralizing antibody titer and type-specific IgG and IgM were measured. In addition, the presence of anti-HSV-1 gD and gK antibodies in the sera of all patients also was determined by ELISA using gD and gK antigens. Finally, the role of anti-gD- and gK-specific antibodies to ADE was investigated. RESULTS: Average neutralizing antibody titers and levels of HSV-1 IgG were similar between HSK- and non-HSK-seropositive patients. However, the contribution of gD to the neutralizing antibody titer in HSK sera was significantly lower than that in non-HSK-seropositive patients, despite higher anti-gD ELISA titers. Overall, sera from patients with HSK had higher anti-gK antibody titers and induced ADE in vitro compared with non-HSK or seronegative sera. The ADE response in HSK sera was attributed to anti-gK antibody. CONCLUSIONS: These results suggest that sera from HSK patients had higher anti-gD and -gK antibody titers than sera from seropositive patients who had no history of HSK despite similar levels of neutralizing antibody titers and HSV-1 IgG, that HSK sera induced ADE whereas sera from non-HSK patients did not induce ADE, and that anti-gD antibody in sera of HSK patients contributed less to the HSV-1 neutralization antibody titer than did sera from non-HSK patients.

Treatment of mice with anti-CD86 mAb reduces CD8+ T cell-mediated CTL activity and enhances ocular viral replication in HSV-1-infected mice.

PURPOSE: To determine the relative impact of the CD86 (B7-2) costimulatory molecule in protection against ocular HSV-1 infection. METHODS: BALB/c mice were depleted of CD86 by antibody and depleted mice were examined for their ability to withstand HSV-1 ocular infection. Depleted mice were tested for the presence of virus replication, T-cell activation, survival, and eye disease. RESULTS: Mice that had been depleted of CD86 had significantly higher titers of HSV-1 in their eyes compared to mock-depleted infected mice. However, the levels of corneal scarring between the two groups of mice were similar. Following ocular infection, the levels of class I MHC-restricted cytotoxic T lymphocytes (CTL) were significantly higher in mock-depleted mice than in CD86-depleted mice. Finally, adoptive transfer of primed CD8(+) T cells but not CD4(+) T cells to CD86-depleted mice resulted in a decrease in peak virus titers in the eyes, such that HSV-1 titers were similar to that of their mock-depleted counterparts. CONCLUSIONS: These data demonstrate an important role for CD86 in the development of CTL and reduction of virus replication in the eyes of HSV-1-infected mice.

Involvement of CD8+ T-cells in exacerbation of corneal scarring in mice.

PURPOSE: To determine the specific immune responses involved in the exacerbation of corneal scarring induced by HSV-1 in gK vaccinated mice. MATERIALS AND METHODS: BALB/c mice were vaccinated with HSV-1 glycoprotein K (gK) and ocularly challenged with HSV-1. Infiltration into the cornea of T cells and macrophages was monitored by immunocytochemistry, and the effect of depletion of CD4+ T-cells, CD8+ T-cells, or macrophages on corneal scarring was determined. RESULTS: Following ocular challenge, CD4+ and CD8+ T-cells and macrophages were more abundant in the corneas of gK-vaccinated mice than in the corneas of mock vaccinated mice. Depletion of CD8+ T-cells, but not of CD4+ T-cells or macrophages, reduced the severity of corneal scarring in gK-vaccinated mice. CONCLUSIONS: We have shown that gK vaccination causes an overall increase in T cells and macrophages in the cornea after ocular HSV-1 challenge. The immunopathology induced by gK vaccination appears to be related to CD8+ T-cell activity, as depletion of these cells, but not other immune cells, reduced corneal scarring.