The current study investigated the correlation between various genotypes of two Granzyme B SNPs (Q-55R and A-295G)) and the frequency of Granzyme B precursor cells in PBMC of normal healthy controls. In order to establish the prevalence of these Granzyme B SNPs, we have examined a large and racially diverse pediatric heart transplant cohort.
Recently, McIlroy et al. (12
) identified a mutated Granzyme B allele responsible for the substitution of three amino acids (R48
) that encodes for a stable protein. A prevalence of 25–30% of RAH allele was reported in European, African and Asian populations (12
). Concordant with previous reports, in this large cohort of pediatric heart recipients, we also found a similar prevalence of the R allele. Furthermore, we report a significant difference in the distribution of -55 R/R genotype among various racial/ethnic groups. In addition, the pediatric group was genotyped for Granzyme B A–295G SNP and the racial/ethnic distribution of this SNP was reported. Black non-Hispanics were identified as being more likely to be homozygous for the A allele compared to White non-Hispanic and Hispanic recipients.
The impact of Q-55R SNP on cytolytic function of Granzyme B is still controversial. One report showed that R48
allele was incapable of apoptosis in vivo
), while other authors showed that this allele retained the biochemical and cytotoxic function of the wild type variant (13
Our investigation focused on establishing a correlation between the Granzyme B SNPs and protein expression following non-specific mitogen stimulation of fresh, unmanipulated cells from normal individuals. Granzyme B ELISPOT assay was preformed to evaluate the frequency of Granzyme B precursor cells. We did not have the opportunity to measure the frequency of Granzyme B precursor cells in the pediatric heart transplant group. Furthermore, donor blood and/or tissue was not available for cytotoxic assays. However, these results were likely to be impaired by different immunosuppression regimens used across the various centers.
Our results on normal controls clearly showed a significant difference among Q-55R genotypes: carriers of Q/Q genotype exhibited a higher frequency of Granzyme B precursor cells as compared to R/R genotype. In contrast to our findings, other investigators have reported similar expression of Granzyme B among the two genotypes. These discrepancies might be explained by the source of the cells, different cohorts and the method of detection. We examined the frequency of Granzyme B precursor cells in freshly isolated PBMC from normal healthy controls, as Granzyme B might be produced by several types of cells in PBMC including NK cells, CD8+ and CD4+ T cells (11
). In contrast, the other investigators limited their observations to a subset of cells, CD8 positive T cells, from frozen samples of HIV positive donors. Furthermore, we analyzed the frequency of Granzyme B by release of protein and capture at single cell level with the Granzyme B ELISPOT assay, whereas previous investigators applied a less sensitive assay of intracellular staining for Granzyme B in CD8 positive T cells. Although PMA and ionomycin calcium induced a stronger stimulation than PHA, following both modalities of activation we detected a similar pattern. In contrast, for the other Granzyme B SNP A-295G we did not find a functional correlation as assessed by ELISPOT assay.
Using ELISPOT assay, immune cell frequencies can be measured at the single cell level without elaborate expansion or manipulation of cell populations (20
). ELISPOT assay may be preferable to assess the functional expression of certain molecules due to its capacity to detect low-level responses, while flow cytometry allows more for phenotypic discrimination of responding cells upon stimulation (21
). Furthermore, Granzyme B ELISPOT assay measures the release of a cytolytic protein (14
) and was documented to correlate with the frequency of cytotoxic T lymphocytes after HLA-identical living-related kidney transplantation (15
) and to measure ex vivo antigen-specific cytotoxicity of PBMC in clinical trials for cancer vaccines (16
). Moreover, ex vivo ELISPOT measurements of Granzyme B within 24 hours after antigen challenge also allowed for discrimination of active memory CD8+ cells from resting memory cells (22
Common genetic variations (genetic polymorphisms) in transplant recipients (and possible donors) may influence post-transplant outcomes (23
). In our prior studies, we have focused on the impact of various cytokine and growth factor gene polymorphisms on post-transplant outcomes of PHTx recipients (26
). We have also suggested that the racial/ethnic distribution of certain genetic polymorphisms might explain some of the observed variation in post-transplant outcomes among different racial groups (28
). In the current study, we turn our attention to effector molecules, which are key components of the host response to the allograft and mediate cell death (11
). Although Black recipients exhibited a higher frequency of the Granzyme B R/R genotype in comparison with Whites, the overall frequency of this genotype/phenotype is low and we do not expect a significant change in their rejection profile based only on this parameter. In order to evaluate the risk of rejection for a certain race/ethnic group, multiple parameters (e.g. age at the time of transplant, donor/recipient race, HLA compatibility) need to be considered, including additional cytokine and/or growth factor genetic polymorphisms that may also affect the clinical outcome (26
). Furthermore, the information that we have obtained on the association between specific polymorphisms and the function/phenotype of a given mediator is very useful for designing future studies that correlate clinical outcomes with various genetic variations.
We have shown that the prevalence of two Granzyme B SNPs was also influenced by race/ethnicity. In addition, we determined a significant correlation between the frequency of Granzyme B precursor cells and certain genetic polymorphisms. Further analysis will focus on the clinical impact of Granzyme B polymorphisms in pediatric heart transplant recipients.