Targeting Sec23b in COPII Vesicles Regulates T Cell Immunity
Authors: Kim Stephanie , Khoriaty Rami , Valanparambil Rajesh , Wu Julia , Taylor Austin , Fujiwara Hideaki , Peltier Dan , Sun Yaping , Henig Israel , Zajac Cynthia , Oravecz-Wilson Katherine , Brabbs Stuart , Ahmed Rafi , Ginsburg David , Reddy Pavan 
Published: October 2019

Abstract
T cells play critical roles in both protective and pathogenic adaptive immune responses. The release of cytokines by T cells is critical for their development, differentiation, and effector functions. However, significant gaps remain in our understanding of the fundamental cellular and molecular pathways that are critical for T cell cytokine release. Coat Protein Complex II (COPII) enables protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. It consists of an outer layer that facilitates the budding of vesicles from the ER membrane, and an inner layer responsible for selecting cargo proteins that comprises the protein SEC23B. The critical ER-Golgi pathway in T cell cytokine release is not known.
Therefore, to explore the role of COPII secretory pathways, we generated novel Sec23bfl/-Cd4Cre mice that will lack Sec23b in T cells. We hypothesized that SEC23B-mediated COPII deficiency will lead to abnormalities in T cell development given the critical roles of T cell-derived cytokines in their ontogeny. Contrary to our hypothesis, SEC23B-deficient T cells developed normally, and were present in similar numbers in the thymus (p=0.20), lymph nodes (p=0.69), and spleen (p=0.10) as WT T cells. They also showed normal levels of surface phenotypic markers (TCR, CD25, CD44, CD62L, CD28). To rule out potential Cre-recombinase driven artifacts we next generated Sec23bfl/-Vav1Cre mice and found that T cell development was not affected. We explored the functions of mature fully developed T cells by stimulating them with aCD3/aCD28 for 3 days. Compared to WT T cells, SEC23B-deficient T cells demonstrated decreased release of cytokines such as IL-2, IL-7, and IFNg (all p<0.01), but not others, such as IL-17 and IL-10. This selective reduction in cytokine secretion was not due to defective T cell signaling events (such as phosphorylation of ZAP-70, LCK, AKT, ERK1/2) or production of cytokines at the mRNA level (including IL-2, IFNg, IL-10). However, the altered cytokine levels in the supernatant was associated with decreased proliferation by SEC23B-deficient T cells compared to WT when measured by CFSE (p=0.0003).
We therefore hypothesized that SEC23B-deficient T cells were able to produce cytokines, but unable to release them selectively. To test this, we used Brefeldin A (BFA), an inhibitor of anterograde transport from the ER to the Golgi apparatus. In the presence of BFA, intracellular levels of IL-2 and IL-17 were similar in WT and SEC23B-deficient T cells (p=0.86). Collectively, these data suggest SEC23B-deficient T cells exhibit secretory defects of select cytokines upon stimulation compared to WT T cells.
We next explored the in vivo relevance of SEC23B-dependent COPII vesicle formation in two clinically relevant models. We first tested the impact of disrupting Sec23b on in vivo protective functions of T cells in a model of viral immunity by utilizing the well-characterized LCMV Armstrong model of acute infection. Sec23b WTor Sec23bfl/-Cd4Cre mice were infected with 2x105 p.f.u. of LCMV Armstrong i.p., and T cells were analyzed on day 8 post-infection. In contrast to WT mice, Sec23bfl/-Cd4Cre mice demonstrated impaired ability to clear the virus by day 8 (9.1x107 p.f.u/gm vs N.D., p=0.0008), reduced expansion of viral antigen-specific cells (p=0.0001 for gp33, gp66, p=0.0006 for gp276), and reduced numbers of IFNg (p=0.0004) and TNFa (p=0.0003) producing T cells on day 8 post-infection.
We next determined the pathogenic capacity of SEC23B-deficient T cells by utilizing a well-established MHC-mismatched C57BL/6->BALB/c model of Graft-versus-Host Disease (GVHD). BALB/c and C57BL/6 recipients were lethally irradiated (8.5 Gy) and transplanted with 5x106 allogeneic C57BL/6 WT bone marrow cells, and either 2x106 C57BL/6 WT or SEC23B-deficient T cells. All syngeneic recipients survived. By contrast, 100% of the recipients of allogeneic WT T cells died by day 61 due to GVHD. However, only 6% of allogeneic recipients of SEC23B-deficient T cells died (n=18/group, p<0.0001) and all recipients showed lower GVHD clinical scores compared to recipients of WT T cells (p<0.01 to 0.001 over 6 weeks).
Collectively, our data demonstrate a heretofore unrecognized but critical role for SEC23B-dependent COPII in T cell protective and pathogenic effector functions and thus provide fundamental insights into T cell-mediated immunity.

Disclosures
Ginsburg: Portola pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Shire plc: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.