cd4 or cd8 easysep mouse t cell isolation kit  (STEMCELL Technologies Inc)

Journal: The Journal of Experimental Medicine

Article Title: A CARMIL2 gain-of-function mutation suffices to trigger most CD28 costimulatory functions in vivo

doi: 10.1084/jem.20250339

Figure Lengend Snippet: Preformed CARMIL2 QE -CARD11 complexes are found in unstimulated Carmil2 QE mouse T cells irrespective of CD28 expression. (A) CD4 + T cells purified from WT, Carmil2 OST , and Carmil2 QE-OST mice were either left untreated (−) or stimulated (+) with anti-CD3 plus anti-CD28 for 2 and 5 min at 37°C. Equal amounts of TL of the specified mouse CD4 + T cells were analyzed by immunoblots and probed with anti-CARMIL2, anti-CARD11, and an anti-ZAP70 loading control. (B) Immunoblot analysis of equal amounts of lysates from mouse CD4 + T cells prepared as in A and subjected to AP on Strep-Tactin Sepharose beads, followed by elution of proteins with D-biotin, and probed with anti-CARMIL2 or anti-CARD11. (C) Quantitation of the immunoblot analysis shown in B. Bars represent normalized CARMIL2-ZAP70 and CARD11-ZAP70 ratios. Data are presented as the mean ± SE in C, F, and I. (D) CD8 + T cells purified from OT-I Carmil 2 WT and OT-I Carmil 2 QE mice were either left untreated (−) or stimulated (+) with pervanadate for 2 and 5 min at 37°C. Equal amounts of TL of the specified mouse CD8 + T cells were analyzed by immunoblots and probed with anti-CARMIL2, anti-CARD11, and an anti-ZAP70 loading control. (E) Immunoblot analysis of equal amounts of lysates from mouse CD8 + T cells activated as in D and from which CARMIL2 or CARMIL2 QE proteins were IP with an anti-CARMIL2 antibody, and subjected to immunoblot analysis with anti-CARMIL2 or CARD11. (F) Quantitation of the immunoblot analysis shown in E. Bars represent normalized CARMIL2-ZAP70 and CARD11-ZAP70 ratios. (G) CD4 + T cells purified from WT, Carmil2 QE , and Carmil2 QE Cd28 −/− mice were either left untreated (−) for 2 min at 37°C or stimulated (+) with pervanadate for 2 min at 37°C. Equal amounts of TL of the specified mouse CD4 + T cells were analyzed by immunoblots and probed with anti-CARMIL2, anti-CARD11, and an anti-ZAP70 loading control. (H) Immunoblot analysis of equal amounts of lysates from mouse CD4 + T cells prepared as in G and from which CARMIL2 or CARMIL2 QE proteins were IP with an anti-CARMIL2 antibody, and subjected to immunoblot analysis with anti-CARMIL2 or CARD11. (I) Quantitation of the immunoblot analysis shown in H. Bars represent normalized CARD11-CARMIL2 units. (J) CD4 + T cells from WT, Carmil2 OST , and Carmil2 QE-OST mice were left untreated (−) or stimulated with either of the anti-CD3 antibodies 2C11 and 17A2 (+) in the presence (+) or absence (−) of the anti-CD28 antibodies MAB4832 for 2 min prior to isolation of whole-cell lysates. Equivalent amounts of lysates were separated by SDS-PAGE and analyzed by immunoblot with an antibody specific for pTyr. Inducible phosphorylation of SLP76 pTyr128 (pSLP76), LAT pTyr171 (pLAT), and ERK1/2 pThr202/Tyr204 (pERK1/2) was also assessed by immunoblotting with phospho-specific antibodies. In A, B, D, E, G, H, and J, molecular weights in kilodaltons are shown in the left margin. Prior to biochemical analysis, comparable levels of TCRβ, CD3, and CD28 were found at the surface of T cells from WT, Carmil2 OST , Carmil2 QE-OST , OT-I Carmil2 , OT-I Carmil2 QE , and Carmil2 QE Cd28 −/− mice . Data in A–J are representative of two independent experiments. AP, affinity purification; TL, total lysates; IP, immunoprecipitated; pTyr, phosphotyrosine. Source data are available for this figure: SourceData F2 .

Article Snippet: CD4 + and CD8 + T cells were purified by immunomagnetic negative selection using CD4 or CD8 EasySep Mouse T Cell Isolation Kit (STEMCELL Technologies) and then stimulated with plate-bound anti-CD3 (145-2C11; Exbio) and soluble anti-CD28 (37–51; Exbio) antibodies or with phorbol 12-myristate 13-acetate and ionomycin.

Techniques: Expressing, Purification, Western Blot, Control, Quantitation Assay, Isolation, SDS Page, Phospho-proteomics, Affinity Purification, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: A CARMIL2 gain-of-function mutation suffices to trigger most CD28 costimulatory functions in vivo

doi: 10.1084/jem.20250339

Figure Lengend Snippet: Effect of the Carmil2 QE mutation on thymic development including T reg cells and iNKT cells. (A) WT, Cd28 −/− , Carmil2 −/− , Carmil2 QE , and Carmil2 QE Cd28 −/− thymi were analyzed by flow cytometry for the expression of CD4 and CD8. Numbers indicate the percentage of CD4 − CD8 − double-negative, CD4 + CD8 + double-positive, and CD4 + and CD8 + SP cells. (B) Analysis of lineage − (CD25 − , MHCII − , CD11b − , CD161 − ) thymocytes from the specified thymi using TCRβ-CD69 dot plots. They permit to identify TCRβ + CD69 + cells that went through TCR-mediated positive and negative selection . Note that TCRβ hi CD69 − cells were also included in the specified TCRβ + CD69 −/+ gate since they correspond to the most mature SP cells . (C) Analysis of CD4 and CD8 expression on gated TCRβ + CD69 +/− cells showed that they include both DP and SP cells and permit to define CD4 + and CD8 + mature SP cells. (D) Numbers of total cells in thymi isolated from mice of the specified genotypes (see key). (E) Numbers of total TCRβ + CD69 +/− cells in thymi isolated from mice of the specified genotypes (see key in D). (F) Numbers of CD8 + and CD4 + TCRβ + CD69 +/− mature T cells in thymi isolated from mice of the specified genotypes (see key in D). (G) Total CD4 + SP cells from WT thymus were analyzed by flow cytometry using FOXP3 and CD25 expression and the percentage of FOXP3 + CD25 + T reg cells among total CD4 + SP cells defined using the outlined areas. (H) Numbers of FOXP3 + T reg cells found in thymi isolated from mice of the specified genotypes (gating strategy as in G). The difference in FOXP3 + T reg cell numbers between Carmil2 QE and Carmil2 QE Cd28 −/− thymi is not significant. (I) WT thymic iNKT cells were analyzed by flow cytometry using TCRβ expression and binding of α-galactosylceramide–complexed CD1d tetramers (CD11d tet), and their percentage among total thymocytes defined using the outlined area. (J) Numbers of iNKT cells gated as in I in thymi isolated from mice of the specified genotypes. Data in A–J were pooled from four experiments with a total of eight mice per group. The data in D–F, H, and J are shown as box plots with the median, boxed interquartile, and whiskers. Data were analyzed by two-way ANOVA applying the Holm–Sidàk multiple comparison toward the WT group. Only significant values with P ≤ 0.05 are shown. *P < 0.05, **P < 0.01, ***P < 0.001. DP, double positive; SP, single positive.

Article Snippet: CD4 + and CD8 + T cells were purified by immunomagnetic negative selection using CD4 or CD8 EasySep Mouse T Cell Isolation Kit (STEMCELL Technologies) and then stimulated with plate-bound anti-CD3 (145-2C11; Exbio) and soluble anti-CD28 (37–51; Exbio) antibodies or with phorbol 12-myristate 13-acetate and ionomycin.

Techniques: Mutagenesis, Flow Cytometry, Expressing, Selection, Isolation, Binding Assay, Comparison

Journal: The Journal of Experimental Medicine

Article Title: A CARMIL2 gain-of-function mutation suffices to trigger most CD28 costimulatory functions in vivo

doi: 10.1084/jem.20250339

Figure Lengend Snippet: Effect of the Carmil2 QE mutation on peripheral T cell homeostasis. (A) Total T cells from LN of WT, Cd28 −/− , Carmil2 −/− , Carmil2 QE , and Carmil2 QE Cd28 −/− mice were analyzed by flow cytometry for the expression of TCRβ and CD5. Numbers indicate the percentage of cells in the specified quadrants. (B) TCRβ + cells from LN of the specified mice (gated as in A) were analyzed by flow cytometry for the expression of CD4 and CD8. Numbers indicate the percentage of cells in the CD4 + CD8 − and CD8 + CD4 − quadrants. (C) Numbers of TCRβ + CD4 + and TCRβ + CD8 + T cells in LN of mice of the specified genotypes (see key). (D) MFI of CD5 and TCRβ expression on CD4 + T conv and CD8 + T cells isolated from the LN of the specified mice (see key in C). (E) CD4 + T conv (CD4 + conv) cells and CD8 + T cells from LN of WT, Cd28 −/− , Carmil2 −/− , Carmil2 QE , and Carmil2 QE Cd28 −/− mice were analyzed by flow cytometry for the expression of CD44 and CD62L. It allows to segregate CD4 + T cells into naive (CD44 lo CD62L hi ) and effector-memory (CD44 hi CD62L lo ) cells, and CD8 + T cells into naive (CD44 lo CD62L hi ) and antigen-experienced CD44 hi , which comprise central memory (CD62L hi ) and effector-memory (CD62L lo ) CD8 + T cells. Numbers indicate the percentage of cells in the specified quadrants. (F) Numbers of central memory (cm) and of effector-memory (em) CD8 + and CD4 + T conv cells in the LN of the specified mouse (see key). (G) Numbers of CD161 + NK cells in the spleen of the specified mouse (see key in F). Data in A–G were pooled from four experiments with a total of eight mice per group. Data in C, E, and F were analyzed by one- or two-way ANOVA applying the Holm–Sidàk multiple comparison toward the WT group. Only significant values with P ≤ 0.05 are shown in black, and values comparing Carmil2 QE and Carmil2 QE Cd28 −/− mice are shown in red. *P < 0.05, **P < 0.01, ***P < 0.001. MFI, mean fluorescence intensity.

Article Snippet: CD4 + and CD8 + T cells were purified by immunomagnetic negative selection using CD4 or CD8 EasySep Mouse T Cell Isolation Kit (STEMCELL Technologies) and then stimulated with plate-bound anti-CD3 (145-2C11; Exbio) and soluble anti-CD28 (37–51; Exbio) antibodies or with phorbol 12-myristate 13-acetate and ionomycin.

Techniques: Mutagenesis, Flow Cytometry, Expressing, Isolation, Comparison, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: A CARMIL2 gain-of-function mutation suffices to trigger most CD28 costimulatory functions in vivo

doi: 10.1084/jem.20250339

Figure Lengend Snippet: Effect of the Carmil2 QE mutation on peripheral T reg cell homeostasis and suppressive function. (A) Total CD4 + T cells from LN of the specified mice were analyzed by flow cytometry for the expression of CD25 and FOXP3. The FOXP3 + CD25 lo to high gate corresponds to T reg cells, and their percentages are shown. (B) Numbers of T reg cells in the spleen and LN of the specified mouse (see key). Data were pooled from four experiments with a total of 14 mice per group. Data are shown as box plots with the median, boxed interquartile, and whiskers. (C) Sorted T reg cells from WT, Carmil2 QE , and Carmil2 QE Cd28 −/− spleens were cultured at the indicated ratio with CTV-labeled CD4 + CD25 − T conv cells from WT mice in the presence of anti-CD3-CD28-coated beads, and the percentage of WT CD4 + CD25 − T conv cells that have divided was evaluated after 72 h of culture (see Materials and methods). Data were pooled from four experiments with a total of six mice per group. Percent suppression was calculated using the following formula: ( proliferation of T conv cells alone – proliferation of T conv cells treated with T reg cells ) / ( proliferation of T conv cells alone ) × 100 . Mean value ± SEM are represented. Data in B and C were analyzed by one- or two-way ANOVA applying the Holm–Sidàk multiple comparison toward the WT group. Only significant values with P ≤ 0.05 are shown in black. *P < 0.05, ***P < 0.001.

Article Snippet: CD4 + and CD8 + T cells were purified by immunomagnetic negative selection using CD4 or CD8 EasySep Mouse T Cell Isolation Kit (STEMCELL Technologies) and then stimulated with plate-bound anti-CD3 (145-2C11; Exbio) and soluble anti-CD28 (37–51; Exbio) antibodies or with phorbol 12-myristate 13-acetate and ionomycin.

Techniques: Mutagenesis, Flow Cytometry, Expressing, Cell Culture, Labeling, Comparison

Journal: The Journal of Experimental Medicine

Article Title: A CARMIL2 gain-of-function mutation suffices to trigger most CD28 costimulatory functions in vivo

doi: 10.1084/jem.20250339

Figure Lengend Snippet: CARMIL2-CARD11-dependent signals maximize TCR-induced proliferation, cytokine production, and expression of activation markers. (A) Naive CD4 + and CD8 + T cells purified from the spleen and LN of WT, Carmil2 QE , and Carmil2 QE Cd28 −/− mice were activated in vitro with the specified concentrations of plate-bound anti-CD3 in the presence or absence of a fixed concentration (1 μg/ml) of soluble anti-CD28. CD4 + and CD8 + T cell proliferation was measured by luminescence after 48 h. Stimulation of Carmil2 QE CD8 + T cells with anti-CD3 resulted in twofold increased proliferation as compared to anti-CD3–stimulated Carmil2 QE Cd28 −/− CD8 + T cells, the reason for which remains to be elucidated. Data were analyzed by one-way ANOVA applying the Holm–Sidàk multiple comparison toward the specified groups. *P < 0.05, **P < 0.01, ***P < 0.001. (B) Naive CD4 + T cells from the specified mice (see key in C) were activated as in A with 3 μg/ml of anti-CD3 in the presence or absence of 1 μg/ml anti-CD28 and the content of IL-2 present in the supernatant of 40-h-long coculture assessed. Also shown is the IL-2 produced upon stimulation with PMA and ionomycin. (C) Naive CD8 + T cells from the specified mice were activated as in A with 3 μg/ml of anti-CD3 in the presence or absence of 1 μg/ml anti-CD28 and the content of IFN-γ present in the supernatant of 40-h-long coculture assessed (see key). Data in A–C were pooled from two experiments out of four, each with a total of four mice per group. Mean value ± SEM are represented; ND indicates nondetectable IL-2 level. Data in B and C were analyzed by one-way ANOVA applying the Holm–Sidàk multiple comparison toward the specified groups. Only significant values with P ≤ 0.05 are shown. **P < 0.01, ***P < 0.001. (D) Naive CD4 + and CD8 + T cells purified from the spleen and LN of the specified mice (see key) were activated in vitro using anti-CD3 (3 μg/ml) plus anti-CD28 (1 μg/ml) cross-linkage, and the levels of CD278 (ICOS), CD71, CD91, CD272 (BTLA), and CD279 (PD-1) were determined by flow cytometry. The ratio of the MFI at t 48h to the MFI at t 0 h is shown for each of the analyzed molecules. Data in D are shown as box plots with the median, boxed interquartile, and whiskers, and pooled from two experiments, each with a total of six mice per group. Data in D were analyzed by two-way ANOVA applying the Holm–Sidàk multiple comparison toward the specified groups. Only significant values with P ≤ 0.05 are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Histograms corresponding to the levels of CD278 (ICOS), CD71, CD91, CD272 (BTLA), and CD279 (PD-1) on CD4 + and CD8 + T cells from WT, Carmil2 OST , and Carmil2 QE-OST mice prior to and after 48 h of activation are shown in Fig. S2 B . MFI, mean fluorescence intensity.

Article Snippet: CD4 + and CD8 + T cells were purified by immunomagnetic negative selection using CD4 or CD8 EasySep Mouse T Cell Isolation Kit (STEMCELL Technologies) and then stimulated with plate-bound anti-CD3 (145-2C11; Exbio) and soluble anti-CD28 (37–51; Exbio) antibodies or with phorbol 12-myristate 13-acetate and ionomycin.

Techniques: Expressing, Activation Assay, Purification, In Vitro, Concentration Assay, Comparison, Produced, Flow Cytometry, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: A CARMIL2 gain-of-function mutation suffices to trigger most CD28 costimulatory functions in vivo

doi: 10.1084/jem.20250339

Figure Lengend Snippet: Comparative expression of cell surface markers on CD4 + and CD8 + T cells of WT, Carmil2 OST , Carmil2 QE-OST , OT-I Carmil2 , OT-I Carmil2 QE , and Carmil2 QE Cd28 −/− mice. (A) Related to . Naive CD4 + and CD8 + T cells purified from the spleen and LN of WT, Carmil2 OST , Carmil2 QE-OST , OT-I Carmil2 , OT-I Carmil2 QE , and Carmil2 QE Cd28 −/− mice were analyzed by flow cytometry for the levels of TCR, CD3, and CD28 prior to biochemical analysis (shaded curves). Dashed line curves correspond to isotype-matched control antibodies (negative controls), and data are representative of two independent experiments. (B) Related to Fig. 6 . Naive CD4 + and CD8 + T cells purified from the spleen and LN of WT, Cd28 −/− , Carmil2 −/− , Carmil2 QE , and Carmil2 QE Cd28 –/– mice (see key) were activated in vitro using anti-CD3 plus anti-CD28 cross-linkage, and the levels of ICOS (CD278), CD71, CD98, BTLA (CD272), and PD-1 (CD279) were measured by flow cytometry prior to activation (t 0 , solid line curves) or after 48 h of activation (t 48 , shaded curves). Data are representative of two experiments, each with a total of six mice per genotype.

Article Snippet: CD4 + and CD8 + T cells were purified by immunomagnetic negative selection using CD4 or CD8 EasySep Mouse T Cell Isolation Kit (STEMCELL Technologies) and then stimulated with plate-bound anti-CD3 (145-2C11; Exbio) and soluble anti-CD28 (37–51; Exbio) antibodies or with phorbol 12-myristate 13-acetate and ionomycin.

Techniques: Expressing, Purification, Flow Cytometry, Control, In Vitro, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: A CARMIL2 gain-of-function mutation suffices to trigger most CD28 costimulatory functions in vivo

doi: 10.1084/jem.20250339

Figure Lengend Snippet: OT-I T cells develop normally in the presence of CARMIL2 QE molecules. (A) Total cells from OT-I and OT-I Carmil2 QE thymi were analyzed by flow cytometry for the expression of CD4 and CD8. Numbers indicate the percentage of CD4 − CD8 − double-negative, CD4 + CD8 + double-positive, and CD4 + and CD8 + SP cells. Also shown on the right is the cellularity of OT-I and OT-1 Carmil2 QE thymi. (B) Total cells from OT-I and OT-1 Carmil2 QE thymi were analyzed for the expression of TCRβ and CD24. It permits to distinguish mature CD8 + T cells (TCRβ + CD24) and immature SP CD8 + T cells (TCRβ − CD24 + ), the percentages of which are indicated by the number adjacent to outlined areas. Also shown on the right is the quantification of the numbers of TCRβ + CD24 – mature CD8 + T cells found in OT-I and OT-1 Carmil2 QE thymi. (C) T cells from OT-I and OT-1 Carmil2 QE LN were analyzed by flow cytometry for the presence of CD4 + and CD8 + T cells, the percentages of which are indicated by the number adjacent to outlined areas. Also shown on the right is the quantification of the numbers of T cells in OT-I and OT-1 Carmil2 QE LN. (D) Gated CD8 + T cells from LN of OT-I and OT-1 Carmil2 QE mice (see C) were analyzed using CD44 and CD62L expression. The percentages of naive (CD44 lo CD62L hi ), effector-memory (CD44 hi CD62L lo ), and central memory (CD44 hi CD62L hi ) CD8 + T cells are indicated by the number adjacent to outlined areas. Also shown on the right is the quantification of the numbers of CD8 + effector-memory T cells in OT-I and OT-1 Carmil2 QE LN. Data in A–D were pooled from three independent experiments with a total of six mice per group. Data quantification is shown as box plots with the median, boxed interquartile, and whiskers. Data were analyzed by two-way ANOVA applying the Holm–Sidàk multiple comparison toward the OT-I group. *P < 0.05, ns, nonsignificant. SP, single positive.

Article Snippet: CD4 + and CD8 + T cells were purified by immunomagnetic negative selection using CD4 or CD8 EasySep Mouse T Cell Isolation Kit (STEMCELL Technologies) and then stimulated with plate-bound anti-CD3 (145-2C11; Exbio) and soluble anti-CD28 (37–51; Exbio) antibodies or with phorbol 12-myristate 13-acetate and ionomycin.

Techniques: Flow Cytometry, Expressing, Comparison

Journal: The Journal of Experimental Medicine

Article Title: A CARMIL2 gain-of-function mutation suffices to trigger most CD28 costimulatory functions in vivo

doi: 10.1084/jem.20250339

Figure Lengend Snippet: Expression of CARMIL2 QE molecules substitutes for CD28 engagement during responses to solid tumors. (A) Cohorts of 10-wk-old, WT, Cd28 −/− , Carmil2 −/− , Carmil2 QE , and Carmil2 QE Cd28 −/− C57BL/6 mice were injected subcutaneously into the flank with 1 × 10 5 cells of the BRAF V600E Ptgs −/− syngeneic melanoma tumor and monitored for tumor growth using tumor size and weight. The mean and SEM are shown for the tumor size values, and the weight panel corresponds to box plots with the median, boxed interquartile, and whiskers. Data were pooled from three independent experiments with a total of 12–14 mice per group. (B) Immune cell infiltrate analysis of BRAF V600E Ptgs1/Ptgs2 −/− tumors 11 days after implantation in WT, Cd28 −/− , Carmil2 −/− , Carmil2 QE , and Carmil2 QE Cd28 –/– mice. The percentages of intratumoral TCRβ + cells, CD4 + T cells, CD8 + T cells, T reg cells, and NK cells among CD45 + cells are shown (see key in A). Box plots with the median, boxed interquartile, and whiskers are shown, and data were pooled from three independent experiments, each with a total of nine mice per group. (C) Tumor growth analysis in WT mice injected subcutaneously with MC38-OVA carcinoma cells and treated with isotype control antibody, anti-PD-1 antibody, OT-I T cells, or Carmil2 Q538E OT-I T cells 6 days after tumor inoculation. The lines indicate tumor volume over time in individual mice up to the time they had to be euthanized. Tumor growth was monitored three times a week. Data are representative of two independent experiments each involving 10 mice per condition. (D) Results in C were expressed as mean tumor volume (mm 3 ± SEM) and P values shown for day 24. (E) Analysis of immune cell infiltrates of MC38-OVA tumors 11 days after implantation in mice that have been infused with OT-I T cells and Carmil2 QE OT-I T cells. The percentages of Vα2 + Vβ5 + OT-I T cells, Vα2 + Vβ5 + Carmil2 QE OT-I T cells, NK cells, and T reg cells among intratumoral CD45 + cells are shown. Data in A, B, D, and E were analyzed by one-way or two-way ANOVA applying the Holm–Sidàk multiple comparison toward the specified groups. Only significant values with P ≤ 0.05 are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: CD4 + and CD8 + T cells were purified by immunomagnetic negative selection using CD4 or CD8 EasySep Mouse T Cell Isolation Kit (STEMCELL Technologies) and then stimulated with plate-bound anti-CD3 (145-2C11; Exbio) and soluble anti-CD28 (37–51; Exbio) antibodies or with phorbol 12-myristate 13-acetate and ionomycin.

Techniques: Expressing, Injection, Control, Comparison

Journal: The Journal of Experimental Medicine

Article Title: A CARMIL2 gain-of-function mutation suffices to trigger most CD28 costimulatory functions in vivo

doi: 10.1084/jem.20250339

Figure Lengend Snippet: Composition, dynamics, and stoichiometry of the CARMIL2 and CARMIL2 QE interactomes of primary mouse CD4 + T cells. (A) Plots showing the interaction stoichiometry (in log 10 scale) of CARMIL2 OST and CARMIL2 QE-OST molecules with the CAPZB-, CD28-, CARD11-, and CK1-α–interacting proteins in CD4 + T cells before (NS = not stimulated) and after 2, 5, and 10 min of activation via pervanadate treatment. The CARMIL2 QE-OST -CK1-α bait–prey interaction was the sole to show a temporal pattern of interaction stoichiometry similar to that of CARMIL2 QE-OST -CARD11. Data are representative of three independent experiments each involving three replicates (mean ± SEM; n = 9 for each time point). (B) Stoichiometry plots of the CARMIL2 OST and CARMIL2 QE-OST interactome in CD4 + T cells prior to activation (NS) and after 2 min of activation (see ). The CARMIL2 OST (yellow dots) and CARMIL2 QE-OST (orange dots) proteins (corresponding to the two “baits”), and the CD28 (green dots)-, CARD11 (pink dots)-, PKC-θ (purple dots)–, and CK1-α (blue dots)–interacting proteins (corresponding to select high-confidence “preys”) are highlighted, whereas the remaining high-confidence preys are shown as gray dots. For each of the bait–prey interactions, the ratio of prey to bait cellular abundance (abundance ratio in the log 10 scale) was plotted as a function of the interaction stoichiometry of the considered bait–prey interaction (interaction stoichiometry in the log 10 scale). As already noted in the case of the TCR signaling network ( Voisinne et al., 2019 ), substoichiometric bait–prey interactions play a central role in the organization of the CARMIL2 OST and CARMIL2 QE-OST interactome. The two exceptions corresponded to the almost stoichiometric bait–prey interactions involving CARMIL2 OST and CARMIL2 QE-OST with CAPZB and CAPZA2 and to the maximal interaction stoichiometry reached after 2 min of activation by the CARMIL2 QE-OST -CARD11 bait–prey interaction, a condition in which 12% of the available CARMIL QE-OST molecules are complexed to CARD11. The area corresponding to bait–prey interaction involving >10% of the available prey molecules is indicated in light gray and includes CAPZA2 and CAPZB in all the analyzed conditions, and CARD11 in the case of the CARMIL2 QE-OST interactome after 2 min of stimulation.

Article Snippet: CD4 + and CD8 + T cells were purified by immunomagnetic negative selection using CD4 or CD8 EasySep Mouse T Cell Isolation Kit (STEMCELL Technologies) and then stimulated with plate-bound anti-CD3 (145-2C11; Exbio) and soluble anti-CD28 (37–51; Exbio) antibodies or with phorbol 12-myristate 13-acetate and ionomycin.

Techniques: Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: A CARMIL2 gain-of-function mutation suffices to trigger most CD28 costimulatory functions in vivo

doi: 10.1084/jem.20250339

Figure Lengend Snippet: Thymus and LN of WT, Carmil2 QE , and Carmil2 QE-OST mice have similar phenotypes. (A) WT, Carmil2 QE , and Carmil2 QE-OST thymi were analyzed by flow cytometry for the expression of CD4 and CD8. Numbers indicate the percentage of CD4 − CD8 − double-negative, CD4 + CD8 + double-positive, and CD4 + and CD8 + single-positive cells. Also shown on the right is the cellularity of WT, Carmil2 QE , and Carmil2 QE-OST thymi. (B) Total cells from WT, Carmil2 QE , and Carmil2 QE-OST LN were analyzed by flow cytometry for the expression of TCRβ and TCRγδ, and the corresponding percentages of TCRβ + and TCRγδ + cells were indicated by the number adjacent to outlined areas. Also shown on the right is the number of TCRβ + and TCRγδ + cells in WT, Carmil2 QE , and Carmil2 QE-OST LN (see key in A). (C) TCRβ + cells from WT, Carmil2 QE , and Carmil2 QE-OST LN (see gating strategy in B) were analyzed for CD4 and CD8 expression and the percentages of CD4 + and CD8 + cells indicated by the number adjacent to outlined areas. Also shown on the right is the number of TCRαβ + CD4 + and CD8 + Τ cells in WT, Carmil2 QE , and Carmil2 QE-OST LN (see key in A). (D) FOXP3 + T reg cells present among total CD4 + T cells isolated from WT, Carmil2 QE , and Carmil2 QE-OST LN were analyzed by flow cytometry using FOXP3 and CD25 expression, and their percentages were indicated by the number adjacent to outlined areas. Also shown on the right is the number of FOXP3 + T reg cells in WT, Carmil2 QE , and Carmil2 QE-OST LN (see key in A). (E) CD4 + T conv cells found in the LN of WT, Carmil2 QE , and Carmil2 QE-OST mice (gated as in D) were analyzed by flow cytometry using CD44 and CD62L expression, and the percentages of naive (CD44 lo CD62L hi ) and effector-memory (CD44 hi CD62L lo ) cells were indicated by the number adjacent to outlined areas. Also shown on the right is the number of effector-memory CD4 + T cells in WT, Carmil2 QE , and Carmil2 QE-OST LN (see key in A). (F) CD8 + T cells found in the LN of WT, Carmil2 QE , and Carmil2 QE-OST mice (gated as in C) were analyzed by flow cytometry using CD44 and CD62L expression, and the percentages of naive (CD44 lo CD62L hi ) and central plus effector-memory (CD44 hi CD62L low to high ) cells were indicated by the number adjacent to outlined areas. Also shown on the right is the number of central plus effector-memory CD8 + T cells in WT, Carmil2 QE , and Carmil2 QE-OST LN (see key in A). Data in A–D are representative of two independent experiments, with two mice per genotype.

Article Snippet: CD4 + and CD8 + T cells were purified by immunomagnetic negative selection using CD4 or CD8 EasySep Mouse T Cell Isolation Kit (STEMCELL Technologies) and then stimulated with plate-bound anti-CD3 (145-2C11; Exbio) and soluble anti-CD28 (37–51; Exbio) antibodies or with phorbol 12-myristate 13-acetate and ionomycin.

Techniques: Flow Cytometry, Expressing, Isolation