Fig 2: Maternal CD8+ T Cell-Blunted Responsiveness to Fetal Antigen Re-stimulation during Secondary Pregnancy(A) C57BL/6 (H-2b) females 21 days following allogeneic mating with Act-OVA male mice on the BALB/c background were compared to pregnant mice mid-gestation during secondary pregnancy sired by Act-OVA or non-transgenic male mice on the BALB/c background.(B) Number of H-2Kb:OVA257–264 CD8+ T cells in the spleen and pooled peripheral lymph nodes of mice described in (A).(C) Percentage of IFN-?-, TNF-a-, or IL-2-producing H-2Kb:OVA257–264 CD8+ T cells following PMA-ionomycin stimulation for each group of mice described in (A).(D) H-2Kb:OVA257_264 tetramer staining intensity and CD44 expression levels by H-2Kb:OVA257_264 CD8+ T cells for each group of mice described in (A).(E) PD-1, LAG-3, CTLA-4, and TIM-3 expression levels by H-2Kb:OVA257_264 CD8+ T cells for each group of mice described in (A).(F) Relative expression of PD-1, LAG-3, CTLA-4, and TIM-3 among H-2Kb:OVA257–264 CD8+ T cells mid-gestation during primary or secondary pregnancy, each sired by Act-OVA male mice on the BALB/c background.Data are from at least 3 independent experiments, each with similar results, with each point representing data from an individual mouse.Bar, mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001.

Fig 3: EV-TßRII mediates the exhaustion of CD8+ T cells by TEVs.a Experimental analysis in vivo: BALB/c mice were nipple injectied with 4T1 cells (2 × 105 cells per mouse) expressing control shRNA or doxycycline-inducible shRNA targeting TßRII (shTßRII) and tumors were grown for 3 weeks (n = 6 mice per group), followed by the administration of doxycycline (Dox) as in Fig. 6a (left panel). Frequency of CD8+ T cells expressing TNFa, IFN?, PD1, TIM3, LAG-3, and Ki-67 in tumor-infiltrating lymphocytes (TIL) populations from mice (right panel). b qRT-PCR analysis of CD8 T cells in TILs from mice and the results shown as a heatmap. c Confocal microscopy (left panel) and FACS analysis (right panel) of purified CD8+ T cells pre-treated with control EVs or EVs (40 µg) derived from MDA-MB-231 cells expressing TßRII-GFP for 48 h. Scale bar, 5 µm. d–f Experimental analysis in vivo: BALB/c mice were tail vein-injected of control EVs, TßRII-GFP+ EVs, or TßRII- EVs for 3 weeks (n = 6 mice per group); then the percentage of GFP positive CD8+ T cells of mice blood were quantified d. The percentage of CD8+ T cells in lym-node or spllen and TIM3+, PD1+, IFN?+, IFN?+&TNFa+, LAG3+ of CD8+ T cells in TIL were analyzed by FACS (n = 6 mice per group) e. The cellular proliferation of CD8+ T cells were analyzed by CFSE dilution f. g Experimental analysis in vivo: OT-I TCR transgenic mice were subcutaneous injected with B16 melanoma cells expressing MHC class I specific epitope of OVA (B16-OVA), followed by tail vein-injection of EVs derived from control (TßRII+) or TßRII knock-out (TßRII-) 4T1 cell (50 µg per mouse every other day) for 3 weeks (n = 5 mice per group) (left); Quantification of the percentage of PD1+, LAG3+, IFN?+, TNFa+, Ki67+, T-bet+, Eomes+ of CD8+ T cells and qPCR analysis of TOX mRNA from tumor-infiltrating lymphocyte (TIL). *p < 0.05 (two-tailed Student’s t-test a, d, e, g). Data are analyzed of three independent experiments and shown as mean + SD a, d, e, g. Source data are provided as a Source Data file.

Fig 4: PD-1 and LAG-3 Restrict Re-expansion of Fetal-Specific CD8+ T Cells Selectively during Secondary Pregnancy(A) Schematic showing the administration of aPD~L1 and/or aLAG-3 antibodies mid-gestation (E10.5) during primary or secondary pregnancies sired by Act-OVA male mice on the BALB/c background.(B) Percentage of fetal wastage 3 days after the administration of aPD-L1 and/or aLAG-3 antibodies for mice described in (A).(C) Number of H-2Kb:OVA257–264 CD8+ T cells in the spleen and pooled peripheral lymph nodes for each group of mice described in (A).(D) Fetal wastage 3 days after the administration of aPD-L1/LAG-3 antibodies with or without co-administration of aCD8- and/or aCD4-depleting antibody during secondary pregnancy sired by Act-OVA male mice on the BALB/c background.(E) Schematic showing the administration of aPD-L1 and/or LAG-3 antibodies mid-gestation (E10.5) during allogeneic compared with syngeneic secondary pregnancies.(F) Percentage of fetal wastage 3 days following aPD-L1/LAG-3 antibody treatment mid-gestation for groups of mice described in (E).Data are from at least 3 independent experiments, each with similar results, with each point representing data from an individual mouse.Bar, mean ± SEM. **p < 0.01 and ****p < 0.0001.

Fig 5: AFAP1L2 is upregulated in chronically activated T cells and blunts their survival(A) FACS-purified naive CD8+ T cells from four healthy donors were either analyzed directly by LC-MS or after increasing times of activation with antibodies to CD3 and CD28. Heatmap shows the abundance of proteins that were significantly upregulated in tumor-infiltrating CD8+ T cells with a high percentage of PD-1+ cells (see Figure 4). Each column represents a different donor.(B) Schematic of the activation conditions of naive CD8+ T cells and flow cytometry plots showing the expression of PD-1, TIM-3, CD39, and LAG-3. See also Figure S4B for quantifications.(C) Naive CD8+ T cells were activated as indicated in the schematic in (B) and then analyzed by LC-MS. Heatmap shows the abundance of indicated proteins, where each column represents a different donor.(D) Plot shows AFAP1L2 protein copy numbers at different time points following activation. n = 4 for resting T cells, and n = 3 for other time points.(E) AFAP1L2 protein copy numbers in CD8+ T cells following different activation schemes. n = 3. Two-tailed t test.(F) Same as in (E) but T cells were analyzed by RNA-seq, and AFAP1L2 mRNA counts are shown.(G) NTC and AFAP1L2-edited CD8+ T cells were labeled with CTV and then stimulated with ImmunoCult CD3/CD28/CD2 Activator (1:333). Proliferation (CTV intensity) was followed by flow cytometry over the time course of 5 days. n = 9 from three different donors.(H) NTC and AFAP1L2-edited CD8+ T cells were stimulated with plate-bound anti-CD3/CD28 antibodies for 8 and 12 days, and the number of surviving T cells was quantified by flow cytometry. n = 5 from five donors. Two-tailed t test.