Lindquist, Lonn D.

Abstract Effective T cell function protects us from pathogens as well as cancer. However when T cell activation is dysfunctional, it has detrimental effects. If T cell activation cannot be shut down, it may cause autoimmunity. Therefore, it is crucial for us to study how T cell activation is regulated. Calcium modulating cyclophilin ligand (CAML) is a ubiquitously expressed endoplasmic reticulum protein. Using a transgenic conditional knock-out mouse model to delete CAML, we previously demonstrated that CAML is required for early embryonic development and thymopoiesis. Additionally, cells lacking CAML do not respond to EGFR stimulation due to a receptor recycling defect. However, the role of CAML in mature, peripheral T cells is not well understood. Here we provide preliminary data showing that loss of CAML in peripheral T cells leads to defective T cell activation and proliferation upon TCR stimulation, and inducing cell death through Fas ligand engagement. We hypothesize that CAML regulates components that are required to sustain optimum TCR signaling and function. T cell responsiveness to TCR stimulation is critical for T cells to deliver effector functions during antigen recognition and activation. Therefore understanding the regulation of TCR signaling by CAML will provide insight into novel mechanisms of immune regulation. These studies may provide potential treatments of immunodeficiency, autoimmunity or other inflammation associated diseases.

Abstract Calcium-modulating cyclophilin ligand (CAML) is an endoplasmic reticulum (ER) protein that functions in a complex to mediate tail-anchored (TA) protein insertion into the ER membrane. CAML has also been implicated as a regulator of endocytic trafficking, modulator of intracellular calcium signaling, and essential factor for the survival and proliferation of specialized immune cells. To investigate the role of CAML in cell biology and cancer, we generated Eμ-Myc transgenic mice that carry a conditional deletion allele of CAML. From these mice, we derived B cell lymphoma cell lines driven by c-Myc and induced deletion of the CAML gene by treatment with tamoxifen to activate Cre-ERT2 in vitro. Homozygous loss of CAML in multiple independent B cell lymphoma lines activated apoptosis, while heterozygous deletion led to reduced cell proliferation. Apoptosis was inhibited by caspase inhibitor Q-VD-OPh or Bcl-2/xL overexpression; however, blocking apoptosis did not restore cell division. Loss of CAML did not activate the unfolded protein response, but did induce c-Jun and JNK phosphorylation. Structure-function studies indicated that lymphoma cell viability did not depend on interaction of CAML with TRC40, another component of TA protein insertion machinery. Conversely, expression of a minimal region of CAML that interacts with the TA insertion co-factor WRB was sufficient to rescue growth of CAML-deleted cells. In summary, our study demonstrates an essential pro-survival role for CAML in c-Myc-driven lymphomas. Future investigations will focus on the underlying mechanism of apoptosis in this system to advance our understanding of the biological functions of CAML, and consider CAML-dependent survival as a potential novel target for anticancer therapy. Citation Format: Jennifer C. Shing, Lonn D. Lindquist, Richard J. Bram. c-Myc-driven lymphomas require calcium-modulating cyclophilin ligand (CAML) for survival and proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4412.

Abstract 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.

Abstract 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.