Calcium-Modulating Cyclophilin Ligand Is Essential for the Survival of Activated T Cells and for Adaptive Immunity.

Calcium-modulating cyclophilin ligand (CAML) is an endoplasmic reticulum resident protein that is widely expressed. Although it has been demonstrated to participate in the tail-anchored protein insertion pathway, its physiological role in the mature immune system is unknown. In this work, we show that mature, peripheral T cells require CAML for survival specifically following TCR-induced activation. In this study, we examined mature T cells from spleen and lymph nodes of tamoxifen-inducible CAML knockout mice (tCAML(-/-)). Whereas CAML-deficient T cells were able to express the early activation markers CD25 and CD69, and produce IL-2 normally upon stimulation, deficient cells proliferated less and died. Cells did not require CAML for entry into the S phase of the cell cycle, thus implicating its survival function at a relatively late step in the T cell activation sequence. In addition, CAML was required for homeostatic proliferation and for Ag-dependent cell killing in vivo. These results demonstrate that CAML critically supports T cell survival and cell division downstream of T cell activation.

Essential role for CAML in follicular B cell survival and homeostasis.

Calcium-modulating cyclophilin ligand (CAML) is a ubiquitously expressed protein that is important during thymopoiesis. However, whether it serves a function in mature lymphocytes is unknown. In this article, we show that CAML is essential for survival of peripheral follicular (Fo) B cells. Conditional deletion of CAML in CD19-Cre transgenic mice caused a significant reduction in Fo cell numbers and increased rates of homeostatic proliferation. CAML-deficient Fo cells showed increased cellular turnover and normal proliferative ability. Although CAML-deficient Fo cells responded to AgR stimulation and to B cell activating factor, they displayed decreased survival and increased apoptosis following stimulation with LPS and IL-4 in vitro. Failure to survive was not due to aberrant B cell development in the absence of CAML, because induced deletion of the gene in mature cells resulted in a similar phenotype. These data establish an essential and ongoing role for CAML in the long-term survival of mature B cells.

CAML mediates survival of Myc-induced lymphoma cells independent of tail-anchored protein insertion.

Calcium-modulating cyclophilin ligand (CAML) is an endoplasmic reticulum (ER) protein that functions, along with WRB and TRC40, to mediate tail-anchored (TA) protein insertion into the ER membrane. Physiologic roles for CAML include endocytic trafficking, intracellular calcium signaling, and the survival and proliferation of specialized immune cells, recently attributed to its requirement for TA protein insertion. To identify a possible role for CAML in cancer cells, we generated Eµ-Myc transgenic mice that carry a tamoxifen-inducible deletion allele of Caml. In multiple B-cell lymphoma cell lines derived from these mice, homozygous loss of Caml activated apoptosis. Cell death was blocked by Bcl-2/Bcl-xL overexpression; however, rescue from apoptosis was insufficient to restore proliferation. Tumors established from an Eµ-Myc lymphoma cell line completely regressed after tamoxifen administration, suggesting that CAML is also required for these cancer cells to survive and grow in vivo. Cell cycle analyses of Caml-deleted lymphoma cells revealed an arrest in G2/M, accompanied by low expression of the mitotic marker, phospho-histone H3 (Ser10). Surprisingly, lymphoma cell viability did not depend on the domain of CAML required for its interaction with TRC40. Furthermore, a small protein fragment consisting of the C-terminal 111 amino acid residues of CAML, encompassing the WRB-binding domain, was sufficient to rescue growth and survival of Caml-deleted lymphoma cells. Critically, this minimal region of CAML did not restore TA protein insertion in knockout cells. Taken together, these data reveal an essential role for CAML in supporting survival and mitotic progression in Myc-driven lymphomas that is independent of its TA protein insertion function.

CAML loss causes anaphase failure and chromosome missegregation.

Calcium modulating cyclophilin ligand (CAML) is a ubiquitously expressed cytoplasmic protein that is implicated in the EGFR and LCK signaling pathways and required for early embryonic and thymocyte development. To further define the critical biological functions of CAML at the cellular level, we generated CAML-deleted mouse embryonic fibroblasts (MEFs) using an in vitro Cre-loxP mediated conditional knockout system. We found that CAML(-/-) MEFs have severely impaired proliferation and a strong reduction of normal anaphases. The primary mitotic defect of CAML(-/-) MEFs is that duplicated chromosomes fail to segregate in anaphase, resulting in nuclear bisection by the cleavage furrow as cells decondense their DNA and exit mitosis, highly reminiscent of the "cut" phenotype in fission yeast. This phenotype is due to spindle dysfunction rather than inability to resolve physical connections between sister chromatids. Furthermore, CAML(-/-) MEFs display defects often seen in cells with mitotic checkpoint gene deficiencies, including lagging and misaligned chromosomes and chromatin bridges. Consistent with this, we found that CAML(-/-) MEFs have a modestly weakened spindle assembly checkpoint (SAC) and increased aneuploidy. Thus, our data identify CAML as a novel chromosomal instability gene and suggest that CAML protein acts as a key regulator of mitotic spindle function and a modulator of SAC maintenance.