Extended Data Fig. 2: Characterization of mice devoid of XBP1 in T cells.

a, Deletion efficiency was analysed by qPCR using a primer set that specifically detects the exon 2 region of Xbp1. Data were normalized to endogenous expression of Actb and presented as relative expression compared with wild-type littermates (n = 8). b, Absolute cell numbers in the thymus, spleen and lymph nodes. c, FACS-based phenotyping of double-negative (CD4⁻CD8⁻), double-positive (CD4⁺CD8⁺) or single-positive (CD4⁺ or CD8⁺) thymocytes. d–g, Frequency of TCRβ⁺ cells (d, f) and CD4⁺ or CD8⁺ cells (gated on TCRβ⁺ cells) (e, g) in lymph nodes or spleen. h, i, Expression of CD44 and CD62L on both CD4⁺ (h) and CD8⁺ (i) TCRβ⁺ subsets in the spleen. j, Frequency of splenic TCRβ⁺CD4⁺FoxP3⁺ T cells. k, l, Frequency of non-T-cell populations among total live cells in spleen (k) and lymph nodes (l). b–g, n = 5; h–j, n = 3; k, l, Xbp1^f/f (n = 4), Xbp1^f/fCd4^cre (n = 5). m, Reconstitution efficiency of CD4⁺ and CD8⁺ T cells in bone marrow and spleen from mixed bone marrow chimaeras (n = 3 per chimaera type). Chimaeras were generated with a mixture of wild-type bone marrow (CD45.1⁺) plus either Xbp1^f/f or Xbp1^f/fVav1^cre bone marrow (CD45.2⁺). n, Flow cytometry assessing cell proliferation of CD4⁺ T cells stained with the division-tracking dye (Cell Trace Violet). Cells were left unstimulated or stimulated for 72 and 96 h with plate-bound anti-CD3 (5 μg ml⁻¹) and soluble anti-CD28 (1 μg ml⁻¹). Histograms (left) and proliferation index (right) are shown (n = 4). o, Cell-cycle analysis of CD4⁺ T cells activated for 72 h by staining with propidium iodide. Representative plots from two experiments. p, Transmission electron microscopy of in vitro activated wild-type versus XBP1-deficient CD4⁺ T cells. Naive CD4⁺ T cells isolated from three biologically independent mice were activated with plate-bound anti-CD3 and soluble anti-CD28 antibodies for 48 h. White arrowheads indicate the ER; M, mitochondria; magnification 12,000× (left); 50,000× (right). Average mitochondrial area of independent cells was estimated using ImageJ software. Xbp1^f/f (n = 19); Xbp1^f/fCd4^cre (n = 29). q, Histogram (left) and quantification (right) for mitochondrial staining (Mitotracker) in in vitro activated CD4⁺ T cells (n = 2 from two independent experiments). r, Activated wild-type versus XBP1-deficient CD4⁺ T cells were incubated in glucose-containing, glucose-depleted or 2-deoxyglucose (2-DG, 10 mM)-treated medium for 6 h and PGC1α expression was analysed by immunoblot. β-Actin was used as loading control. Representative plots from two independent experiments. Data are shown as mean ± s.e.m. (a–n, p). n values represent biologically independent samples (a–n, p, q). Two-tailed Student’s t-tests (b–l, n, p); *P < 0.05; NS, not significant.

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