Fig 2: RAW cells increase CD3 and TNF at the transcriptional level after in vitro BCG infection. (A) Representative dot plot showing analysis strategy to identify, by flow cytometry, the CD3 and transmembrane TNF (tmTNF) expression in RAW cells uninfected (Uninf) and infected in vitro with BCG MOI 0.1 and 1 during 5 and 24 h. Controls of unstained and uninfected cells are shown down to the respective molecule. Percentage of CD3+ and tmTNF+ RAW cells evaluated by flow cytometry ((B,C), respectively). (D) Immunoblot evaluation of CD3-Σ in lysates of uninfected and infected RAW cells. GAPDH was used as a loading control. (E) Relative units (R.U) of CD3-ε and TNF expression evaluated by immunoblot, and relative expression (R.E) of CD3-ε and TNF genes assessed by qPCR in uninfected and infected RAW cells ((F,G), respectively). Quantification of soluble TNF and IL-6 by ELISA in culture supernatants from uninfected and infected RAW cells ((H,I), respectively). n = 7 independent experiments (for flow cytometry), n = 5 independent experiments (for immunoblot), and n = 7 independent experiments performed in triplicate (for qPCR and ELISA). Bars represent the mean value ± SD (B,C,E) or the mean of means ± SEM (F–I). Kruskal–Wallis and corrected for the Dunn test were used for flow cytometry and immunoblot analysis, and one-way ANOVA followed by Bonferroni test for qPCR and ELISA data. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig 3: Anti-CD3 stimulation induces the production of TNF and IL-6 in CD3+ RAW cells. (A) Sorted RAW cells were used to evaluate NFAT, phosphorylated C-Jun (pC-Jun), and IKK-α by immunoblot. β-Tubulin was used as a loading control. CD3+ and CD3- RAW cells were cultured for 24 h under the following conditions: RPMI, anti-CD3, and isotype control (I.C). (B) Relative units (R.U) of NFAT-1, (C) pC-Jun, and (D) IKK-α in RAW CD3+ and CD3- cell lysates. TNF and IL-6 levels ((E,F), respectively) were measured in supernatants collected from CD3+ and CD3- RAW cells by ELISA. n = 3 independent experiments (for immunoblot) and n = 3 independent experiments performed in triplicate (for ELISA). Bars represent the mean value ± SD (B–D) or the mean of means ± SEM (E,F). Kruskal–Wallis and corrected for the Dunn test were used for immunoblot data and one-way ANOVA followed by Bonferroni test for ELISA. * p < 0.05, ** p < 0.01.

Fig 4: The anti-CD3 monoclonal antibody does not induce activation in total RAW cells. (A) Immunoblot evaluation of proteins involved in the CD3 signaling pathway; NFAT, phosphorylated C-Jun (pC-Jun), IKK-α, NF-κB and phosphorylated CD3-ζ chain (pCD3-ζ), β-Tubulin as loading control in lysates of RAW cells cultured for 24 h under the following conditions: RPMI (control), anti-CD3 (stimulus), and isotype control. Relative units (R.U) of NFAT, pC-Jun, IKK-α, NF-κB, and pCD3-ζ in RAW cell lysates ((B–F), respectively). Levels of TNF and IL-6 were measured in supernatants collected from total RAW cells by ELISA sandwich assay ((G,H), respectively). n = 5 independent experiments (immunoblot) and n = 5 independent experiments performed in triplicate (ELISA). Bars represent the mean value ± SD (B–F) or the mean of means ± SEM (G,H). Kruskal–Wallis and corrected for the Dunn test were used for immunoblot data and one-way ANOVA followed by Bonferroni test for ELISA. * p < 0.05.

Fig 5: Induction of tolerance to a hybrid insulin peptide restrains functional effector differentiation of graft-infiltrating, antigen-specific T cells.a Method for generating peptide-coupled PLGA nanoparticles (NPs) by covalent conjugation of the 2.5HIP (treatment) or HEL (control) peptide. b Islet transplant and NP treatment experimental design where mice are treated IV on day -7, +1, and +7 relative to the day of transplant with 2.5 mg of HEL- or HIP-conjugated NPs. c Random blood glucose readings and survival curve of transplanted mice treated with 2.5 mg of HEL NP 3x, 2.5HIP NP 3x, or 2.5HIP NP 9x (final treatment 67 d). Survival was compared between groups using Kaplan-Meier analysis with a log-rank test and Bonferroni correction for multiple comparisons. * HEL NP 3x vs HIP NP 3x, p = 0.0303; HEL NP 3x vs HIP NP 9x, p = 0.0303; HIP NP 3x vs HIP NP 9x, p = 0.0201. d Representative MHC-tetramer staining with 2.5HIP-loaded I-Ag7 or IGRP-loaded H-2kd in islet grafts. Proportion (%) and absolute number (#) of tetramer-positive 2.5HIPCD4 or IGRPCD8 T cells in islet grafts. e Representative staining of TCF1 and percentage of TCF1hi on graft-infiltrating 2.5HIPCD4 or IGRPCD8 T cells. CD44loCD62Lhi bulk, splenic CD8 T cells used as naïve controls. f Absolute number (#) of TCF1lo and TCFhi 2.5HIPCD4 or IGRPCD8 T cells in islet grafts. g, h Representative staining of inhibitory receptors TIM3 and PD1 and proportion of each IR on TCF1lo and TCF1hi, 2.5HIPCD4 and IGRPCD8 T cells. Representative staining of TCF1 by TIM3 on (i) 2.5HIPCD4 and (k) IGRPCD8 T cells. Representative cytokine staining (IFNγ, TNF) on (1) TCF1hiTIM3lo, (2) TCF1loTIM3lo, and (3) TCF1loTIM3hi T cells i 2.5HIPCD4 and (k) IGRPCD8 T cells. Proportion (%) of IFNγ+TNF+ double-positive j 2.5HIPCD4 and l IGRPCD8 T cells. In d,e,f, data are depicted as the mean ± s.e.m., with p-values obtained from a two-sided unpaired Student’s t-test. In (f–l), data are depicted as the mean ± s.e.m. with p-values obtained from one-way ANOVA with Šídák’s multiple comparison test. In (d), n = 4 mice per group for HEL NP and HIP NP. In (e), n = 3 for HEL NP 2.5HIPCD4 TCF1hi% and n = 4 for HIP NP 2.5HIPCD4 TCF1hi%, while n = 4 for HEL NP IGRPCD8 TCF1hi% and n = 5 for HIP NP IGRPCD8 TCF1hi%. In (f), n = 3 for HEL NP 2.5HIPCD4 TCF1lo/hi# and n = 4 for HIP NP 2.5HIPCD4 TCF1lo/hi#, with n = 4 for HEL NP IGRPCD8 TCF1lo/hi# and n = 5 for HIP NP IGRPCD8 TCF1lo/hi #. In (g), n = 4 mice per group for 2.5HIPCD4 TIM3% and PD1% in HEL NP and HIP NP treated mice. In (h), n = 4 mice per group for IGRPCD8 TIM3% and PD1% in HEL NP and HIP NP treated mice. In (i), n = 3 mice per group for HEL NP and HIP NP. In (i), n = 3 for HEL NP and HIP NP. In (d–f), the data are from 6 independent experiments; in (g, h, j, l), the data are from 4 independent experiments. Grey shading indicates TCF1lo T cells.