Characterisation of mice lacking all functional isoforms of the pro-survival BCL-2 family member A1 reveals minor defects in the haematopoietic compartment

Abstract

The pro-survival proteins of the BCL-2 family regulate the survival of all cells, and genetic deletion models for these proteins have revealed which specific BCL-2 family member(s) is/are critical for the survival of particular cell types. A1 is a pro-survival BCL-2-like protein that is expressed predominantly in haematopoietic cells, and here we describe the characterisation of a novel mouse strain that lacks all three functional isoforms of A1 (A1-a, A1-b and A1-d). Surprisingly, complete loss of A1 caused only minor defects, with significant, although relatively small, decreases in γδTCR T cells, antigen-experienced conventional as well as regulatory CD4 T cells and conventional dendritic cells (cDCs). When examining these cell types in tissue culture, only cDC survival was significantly impaired by the loss of A1. Therefore, A1 appears to be a surprisingly redundant pro-survival protein in the haematopoietic system and other tissues, suggesting that its targeting in cancer may be readily tolerated. Cell Death and Differentiation (2017) 24, 534-545; doi:10.1038/cdd.2016.156; published online 13 Janaury 2017The pro-survival proteins of the BCL-2 family prevent apoptosis 1 and studies using gene-targeted mice have revealed which cell types rely on which pro-survival protein for their survival. For example, Bcl-2 −/− mice exhibit thymic and splenic atrophy, a loss of fur pigment and die~30 days post birth from polycystic kidney disease, attributable to excess lymphocyte, melanocyte and renal epithelial cell apoptosis, respectively. 2-4 Bcl-X −/− mice die before E14.5 of embryonic development because of aberrant death of erythroid and neuronal cells. 5 The generation of chimaeric or tissue-specific Bcl-X −/− revealed a critical role for BCL-XL in the survival of developing lymphocytes 5 and platelets. 6,7 Mcl-1 −/− embryos die before implantation (E3.5), 8 but conditional Mcl-1 deletion models have demonstrated an essential role for MCL-1 in the survival of haematopoietic stem cells, lymphocytes, neurons and cardiomyocytes. 9-15 Bcl-W −/− mice have impaired spermatogenesis. 16,17 A1 remains the only pro-survival BCL-2 family member for which a knockout mouse strain has not been developed. A1 was first discovered as a GM-CSF-inducible gene with significant sequence similarity to BCL-2 and MCL-1, 18 and its human homologue BFL-1 was later identified in fetal liver. 19 Overexpression of A1 protected an IL-3-dependent cell line from growth factor deprivation-induced apoptosis, thus demonstrating its pro-survival function. 20 In mice, A1 expression is restricted to the haematopoietic compartment. 18 Human BFL-1 expression is more widespread, but also predominantly haematopoietic. 21 A1 can be upregulated by NF-κB signalling, and it has been proposed that A1/BFL-1 is important for the survival of several activated immune cell subsets through stimulation of antigen or cytokine receptors. [22][23][24][25] Studies of A1 in mice are complicated by the presence of multiple isoforms that are the result of gene duplication ev...

Differential effects of Vav‐promoter‐driven overexpression of BCLX and BFL1 on lymphocyte survival and B cell lymphomagenesis

“…In a broad variety of immune cells, including neutrophils, granulocytes, B and T cells, A1 is rapidly inducible in response to diverse stimuli, including antigen receptor stimulation, GM‐CSF, BAFF receptor or CD40‐ligation 40, 41, predictive of crucial roles in inflammation and immunity. Nevertheless, complete deficiency of A1 does not impair the normal development or function of the immune system nor does it influence the normal behaviour and life span of mice 9, 10.…”

The BCL-2 pro-survival protein A1 is dispensable for T cell homeostasis on viral infection

“…The lack of an obvious phenotype was unexpected, as the levels of A1 mRNA and protein were both rapidly increased in T cells on TCR/CD3 ligation or stimulation with mitogens 17,18,23 A1 induction has been defined as a central step in rewiring the cell survival machinery in T cells during the transition from a resting to an activated state. This is associated with a change from a cytokine receptor (mainly IL-7R) to a TCR-driven survival programme and includes the induction of BCL-XL and A1, accompanied by a downregulation of BCL-2.…”

“…In a separate study, we found that unchallenged A1 −/− mice have slightly reduced numbers of memory T cells compared with their wild-type counterparts. 17 Therefore, we analysed the impact of A1-deficiency in memory T cell formation. Hence, mice were infected intra-nasally with influenza HKx31 virus and memory T cells were enumerated 41 days after infection.…”

“…17 These observations suggested a role for A1 as a survival factor for long-lived memory T cells that are still present after the termination of an anti-viral immune response. Although it has been shown that the BIM/BCL-2 axis is a main regulator of the formation and maintenance of memory T cells, 26,27 NOXA has also been proposed to have a role in regulating the size and clonal diversity of memory T cell populations.…”

“…To unambiguously determine the functions of A1, we developed an A1 knockout mouse strain in which all three functional paralogues of A1 (A1-a, A1-b and A1-d) had been deleted. 17 Interestingly, these A1-deficient mice show little to no abnormalities under steady state conditions. However, these observations might not be totally surprising, given that A1 protein levels are very low in unstimulated cells.…”