Fig 2: 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-a, NF-?B and phosphorylated CD3-? chain (pCD3-?), ß-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-a, NF-?B, and pCD3-? 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 3: CD3 is expressed on RAW cells. (A) RNA of human T cells and monocyte-derived macrophages (MDM) and RAW cells were obtained to assess relative expression (R.E) of CD3-S by qPCR. Graphic shows murine and human data; however, each one was normalized to its corresponding endogenous control. (B) Immunoblot analysis of CD3-S identification in RAW cells, human monocytes (CD14+), MDM, human CD4+ T cells, murine splenocytes (S), and murine bone marrow cells (BM). ß-actin was used as a loading control. (C) Representative dot plot showing analysis strategy to identify intra- and extracellular CD3 expression in RAW cells by flow cytometry. (D) Frequency of RAW cells positive for F4/80 and CD3-S obtained by cell surface staining and evaluated by flow cytometry. (E) Frequency of RAW cells positive for intracellular or extracellular CD3 or both. (F) Representative immunoblot analysis of samples immunoprecipitated with a system of protein G coupled to magnetic beads and monoclonal anti-CD3. n = 5 independent experiments (for flow cytometry), n = 3 independent experiments by duplicate (for qPCR), (B,F), a representative image of 3 independent experiments. Bars represent the mean of means ± SEM (to (A)) or the mean value ± SD (to (D,E)). One-way ANOVA followed by the Bonferroni test were used for qPCR analyses. ** p < 0.01.

Fig 4: CD3 is endocytosed on RAW macrophages. (A) Representative dot plot showing analysis strategy to identify by flow cytometry the extracellular CD3 expression at different times after adding anti-CD3. (B) Frequency of CD3+ RAW at different indicated times. (C) Mean fluorescence intensity (MFI) of CD3 in RAW cells at indicated time points. (D) RNA was obtained from the RAW cells after anti-CD3 addition at different times to assess the relative expression (R.E) of CD3-S by qPCR. n = 5 independent experiments (for flow cytometry), n = 3 independent experiments performed by duplicate (for qPCR). Bars represent the mean value ± SD (to (B,C)) or the mean of means ± SEM (to (D)). Differences between times were analyzed using the Kruskal–Wallis and corrected for the Dunn test for flow cytometry data and one-way ANOVA followed by the Bonferroni test for qPCR. * p < 0.05, ** p < 0.01.

Fig 5: Gating strategy for identification of respiratory lymphocyte subsets and Linnegleukocytes. Respiratory B and T lymphocytes were identified according to low side scatter characteristics, CD19 and CD3e surface expression. This gating strategy resulted also in exclusion of autofluorescent respiratory macrophages. Out of the CD3e-positive population CD4+ T helper, CD4+CD25+ T helper and CD8 T cells were gated according to staining with the respective mAbs and ?d T cells were identified by using a ?d TCR mAb. Please note that the CD4+CD25+ T cells are not CD25high. A more detailed gating strategy of Treg subsets including isotpye controls is shown in Figure 8 . Natural killer cells were identified according to CD49b+Cd3eneg expression. Respiratory lineage-negative leukocytes were identified by absence of CD3e, CD8a, CD11b, CD11c, CD19, B220, TER-119, FceR1 (Lineage cocktail) and CD4 expression and subsequently separated into CD90+ and CD90neg cells.