ER-to-Golgi transport and SEC23-dependent COPII vesicles regulate T cell alloimmunity
Stephanie Kim, … , David Ginsburg, Pavan Reddy
Stephanie Kim, … , David Ginsburg, Pavan Reddy
Published January 19, 2021
Citation Information: J Clin Invest. 2021;131(2):e136574. https://doi.org/10.1172/JCI136574.
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Research Article Cell biology Immunology

ER-to-Golgi transport and SEC23-dependent COPII vesicles regulate T cell alloimmunity

Abstract

T cell–mediated responses are dependent on their secretion of key effector molecules. However, the critical molecular determinants of the secretion of these proteins are largely undefined. Here, we demonstrate that T cell activation increases trafficking via the ER-to-Golgi pathway. To study the functional role of this pathway, we generated mice with a T cell–specific deletion in SEC23B, a core subunit of coat protein complex II (COPII). We found that SEC23B critically regulated the T cell secretome following activation. SEC23B-deficient T cells exhibited a proliferative defect and reduced effector functions in vitro, as well as in experimental models of allogeneic and xenogeneic hematopoietic cell transplantation in vivo. However, T cells derived from 3 patients with congenital dyserythropoietic anemia II (CDAII), which results from Sec23b mutation, did not exhibit a similar phenotype. Mechanistic studies demonstrated that unlike murine KO T cells, T cells from patients with CDAII harbor increased levels of the closely related paralog, SEC23A. In vivo rescue of murine KO by expression of Sec23a from the Sec23b genomic locus restored T cell functions. Together, our data demonstrate a critical role for the COPII pathway, with evidence for functional overlap in vivo between SEC23 paralogs in the regulation of T cell immunity in both mice and humans.

Authors

Stephanie Kim, Rami Khoriaty, Lu Li, Madison McClune, Theodosia A. Kalfa, Julia Wu, Daniel Peltier, Hideaki Fujiwara, Yaping Sun, Katherine Oravecz-Wilson, Richard A. King, David Ginsburg, Pavan Reddy

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Figure 6

Characterizing the role of SEC23 paralogs in human T cells.

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Characterizing the role of SEC23 paralogs in human T cells. (A) Normaliz...
(A) Normalized expression levels of SEC23B and SEC23A in naive T cells isolated from healthy humans relative to β-actin by Western blot (n = 4/group). (B) Experimental steps in CRISPR/Cas9-mediated KO by Cas9/RNP nucleofection in healthy human T cells. (C) qRT-PCR and Western blot analysis of knock-out efficiency of T cells that underwent CRISPR/Cas9-mediated Sec23b KO. T cells were analyzed 3 days following nucleofection with control or Sec23b-targeting crRNA-tracrRNA duplexes complexed with Cas9 (n = 5/group). (D) ELISA measuring IL-2 secreted by control or KO T cells (n = 5/group). (C and D) Data represent mean ± SEM, with *P < 0.05 and *P < 0.01 (2-tailed unpaired Student’s t test). (E) Representative flow cytometric plots demonstrating intracellular IL-2 in healthy T cells that underwent CRISPR/Cas9-based Sec23b KO compared with those that received control guide RNA. (F) Survival curve and GVHD scores of NSG mice that received control or CRISPR/Cas9-mediated SEC23B KO T cells (n = 5/group). (G) Proliferative capacity of T cells derived from a patient with CDAII compared with healthy controls, as measured by CFSE dilutions following stimulation in vitro with αCD3 and αCD28 for 3 days (n = 5/controls, n = 1/CDAII). (H) Flow cytometry measuring levels of intracellular IL-2, TNF-α, and IFN-γ in T cells from a patient with CDAII and healthy controls, following stimulation in vitro with αCD3 and αCD28 for 3 days, and an additional 5 hours with PMA and ionomycin in the presence or absence of BFA (n = 5/controls, n = 1/CDAII). (I) Quantification of surface CD69 and CD25 expression as measured by flow cytometry in T cells from healthy controls or from a patient with CDAII on day 3 after no stimulation or stimulation with αCD3 and αCD28 (n = 6/controls, n = 1/CDAII). (J) Western blots showing expression of SEC23B and SEC23A relative to β-actin in naive unmanipulated T cells from healthy human donors or patients with CDAII (n = 4/controls, n = 3/CDAII).