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.

Trial registration: ClinicalTrials.gov NCT02964494.

Keywords: Bone marrow transplantation; Cell Biology; Immunology; T cells.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. COPII-dependent ER-to-Golgi pathway is engaged upon T cell activation.

(A) Transmission electron micrographs of WT murine T cells that received no stimulation or stimulation with αCD3 and αCD28 for 3 days. Measurements of rough ER widths were taken from 3 different ERs from 5 cells in each group (n = 15). Scale bars: 100 nm. (B) Real-time quantitative PCR analysis of ER stress–associated genes in WT murine T cells that received no stimulation or 24 hours of stimulation with αCD3 and αCD28 (n = 3). (C) Western blots showing protein levels of SEC23B, SEC23A, and β-actin in Sec23bfl/– Cd4Cre (KO) T cells relative to Sec23bfl/– (FL) controls. Densitometric analysis on the right plot indicates normalized levels of SEC23A and SEC23B relative to β-actin expression (n = 4). (D) TEM highlighting an increase in rough ER dilation in Sec23bfl/– Cd4Cre T cells when compared with Sec23bfl/– controls following activation with αCD3 and αCD28 for 3 days (n = 13). Scale bars: 100 nm. (A and B) Data represent mean ± SEM, with **P < 0.01 and ***P < 0.001 (2-tailed unpaired Student’s t test). (D) Statistical significance was determined by 1-way ANOVA and post hoc Tukey’s test; ****P < 0.0001.

Figure 2. SEC23B deficiency does not affect mature T cell development but regulates its secretome.

(A) Percentages and absolute numbers of Sec23bfl/– (FL) and Sec23bfl/– Cd4Cre (KO) CD3+ T cells in whole thymus (n = 3), spleen (n = 5), and lymph node (n = 5) homogenate samples (left), and those of CD4+ and CD8+ T cell subsets gated on CD3+ cells (right). (B) Volcano plot representing 3 biological replicates of SEC23B-deficient T cell secretomes after 24 hours of stimulation compared with WT. Log2 ratios were calculated based on P values. (C) Luminex panel showing mean cytokine levels derived from Sec23bfl/– (FL) and Sec23bfl/– Cd4Cre (KO) T cell supernatants following 3 days of stimulation with αCD3 and αCD28. (A and C) Data represent mean ± SEM, with *P < 0.05 (2-tailed unpaired Student’s t test).

Figure 3. Deficiency of SEC23B-dependent COPII leads to accumulation of secreted proteins.

(A) Histograms based on flow cytometry of surface TCR-β on Sec23bfl/– and Sec23bfl/– Cd4Cre T cells relative to isotype levels present on naive unstimulated WT T cells. (B) Flow cytometry of phosphorylated ZAP70 and ERK1/2 molecules in Sec23bfl/– and Sec23bfl/– Cd4Cre T cells that received no stimulation or stimulation with αCD3 and αCD28 for 30 minutes. (C) Flow cytometry of surface CD69 present on Sec23bfl/– and Sec23bfl/– Cd4Cre T cells stimulated with αCD3 and αCD28 for 6 h compared with isotype levels present on naive unstimulated WT T cells. Flow cytometric data are representative of 3 replicate experiments. (D) qRT-PCR analysis of IL-2 in SEC23B-deficient T cells compared with WT following stimulation with αCD3 and αCD28 for 4 days (n = 5/group). (E) Flow cytometry measuring intracellular IL-2 levels in T cells after stimulation by αCD3 and αCD28 for 3 days followed by 5 hours of stimulation with PMA and ionomycin in the presence or absence of BFA (n = 6/group). (F) Immunofluorescence confocal micrographs of SEC23B (green) and IL-2 (pink) in WT or SEC23B-deficient T cells after 3 days of αCD3 and αCD28 stimulation, and 5 hours of PMA and ionomycin stimulation with or without BFA. (D) Data represent mean ± SEM, and significance was determined by 2-tailed unpaired Student’s t test. (E) Statistical significance was determined by 1-way ANOVA and post hoc Tukey’s test; *P < 0.05, ***P < 0.001. Scale bars: 10 μM.

Figure 4. SEC23B-dependent COPII regulates naive T cell functions in vitro and in vivo.

(A) Overlay of histograms based on flow cytometry plots indicating expression of intracellular T-bet, GATA3, and RORγt in WT and SEC23B-deficient naive CD4+ T cells that were cultured in media containing Th1-, Th2-, and Th17-polarizing cytokines, respectively (n = 3). (B) Proliferative capacity of WT and SEC23B-deficient T cells as measured by CFSE dilutions in vitro (n = 6/group). Partial rescue of proliferation upon addition of exogenous murine IL-2 in T cells stimulated with αCD3 and αCD28 for 3 days (n = 6/group). (C) Cell death as measured by Annexin V and 7AAD staining (n = 3/group) in SEC23B-deficient T cells compared with WT following stimulation by αCD3 and αCD28 for 3 days. (D) In vivo proliferative capacity of SEC23B-deficient T cells compared with WT as measured by CFSE dilutions following their transfer into allogeneic BALB/c recipient mice (n = 3/group). (E) 51Cr release assay using WT or SEC23B-deficient T cells primed with allogeneic splenic cells and used as effector cells against either syngeneic C57BL/6J or allogeneic BALB/c BMDC target cells (n = 3/group). (A, C and D) Statistical significance was determined by 2-tailed unpaired Student’s t test; ***P < 0.001. (B) Statistical significance was determined by 1-way ANOVA and post hoc Tukey’s test; **P < 0.01, ***P < 0.001. (F and G) Survival and GVHD scores of mice that received syngeneic T cell–depleted bone marrow, and T cells from either syngeneic donors, allogeneic WT C57BL/6J donors, or allogeneic Sec23bfl/– Cd4Cre C57BL/6J donors. Allogeneic recipients were BALB/c (n = 12/group) (F) or C3H.SW (n =6/group) (G) strain. Significance was determined by 1-way ANOVA and post hoc Tukey’s test for GVHD score and Mantel-Cox log rank test for survival; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5. Mechanisms of COPII-dependent regulation of T cell functions.

(A) Representative Western blot showing SEC23B and SEC23A expression in purified T cells from Sec23bfl/–, Sec23bfl/– Cd4Cre, Sec23afl/– Cd4Cre, and Sec23b-a/b-a mice. Normalized levels of each protein relative to β-actin are plotted on the right (n = 4/group). (B) ELISA measuring IL-2 secreted by WT and SEC23A-deficient T cells over 3 days in culture with αCD3- and αCD28-stimulating antibodies (n = 5/group). (C) Flow cytometry measuring levels of intracellular IL-2 in WT and SEC23A-deficient T cells following their stimulation in vitro for 3 days with αCD3 and αCD28, and 5 hours with PMA and ionomycin in the presence or absence of BFA (n = 3/group). (D) Proliferation of isolated T cells from the indicated mice stimulated in vitro with αCD3 and αCD28, as measured by CFSE dilutions (n = 6/group). (E) Schematic diagram of Sec23b endogenous genomic locus in Sec23b-a/b-a mice, which contains the Sec23a coding sequence (from C367 to A2298) followed by a poly(A) termination signal in lieu of the full Sec23b sequence as previously described (26). (F) Survival and composite GVHD scores of mice (n = 12/group) that received syngeneic T cell–depleted bone marrow and T cells from either syngeneic WT C57BL/6J donors or allogeneic WT C57BL/6J, Sec23afl/– Cd4Cre C57BL/6J, or Sec23b-a/b-a C57BL/6J donors. (B and C) Significance was determined by 2-tailed unpaired Student’s t test. (D) Significance was determined by 1-way ANOVA and post hoc Tukey’s tests; **P < 0.01. (F) Significance was determined by Mantel-Cox log rank test for survival and 1-way ANOVA and post hoc Tukey’s tests for GVHD score; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. Characterizing the role of SEC23 paralogs in human T cells.

(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).