mhcii ko mice  (Jackson Laboratory)

Journal: Cell reports

Article Title: CD4 T cell help prevents CD8 T cell exhaustion and promotes control of Mycobacterium tuberculosis infection

doi: 10.1016/j.celrep.2021.109696

Figure Lengend Snippet: Purified polyclonal CD8 T cells from C57BL/6J mice were transferred to TCRα KO mice with or without Ag85-specific CD4 T cells (P25) (i.e., helped and helpless, respectively) and then infected with aerosolized Mtb. (A) The survival of helped, helpless, TCRα KO mice that received only P25 cells and TCRα KO mice that did not receive any cells was monitored. Data were combined from three experiments using a total of 17–19 transferred mice per group and 5 TCRα KO mice that received no cells. The difference between the groups was statistically significant (p < 0.0001)as determined using the log-rank test for trend. (B) Determination of synergy between P25 and CD8 T cells as described in the text. The theoretical additive benefit is calculated by the “independent effects” function, while the “synergy” group is the actual survival observed after transfer of P25 and CD8 T cells (i.e., from A). These two scenarios differed statistically (p < 0.0001) by the log-rank test. (C) Lungs from TCRα KO mice that received P25 and CD8 T cells (helped) versus only CD8 T cells (helpless) were analyzed at 4 and 7 wpi by flow cytometry to determine the frequencies of TB10.4 4-11 -specific CD8T cells. (D) The proportion of lung KLRG1 + CD127 − (SLEC) and KLRG1 − CD127 + (MPEC) CD8 T cells was determined 7 wpi. Quadrant numbers represent percentages. (E) The frequencies of lung KLRG1 + CD127 − (SLEC) among total CD8 T cells was determined 4 and 7 wpi by flow cytometry and analyzed statistically. (C and E) Bars, mean ± SD. Data are representative of three independent experiments, 3–5 mice/group. Statistical significance was analyzed by unpaired t test. *p < 0.01. ns, no significant differences. See also - .

Article Snippet: P25 TCRtg, MHCII KO, and TCRα KO mice were purchased from Jackson Lab and bred locally.

Techniques: Purification, Infection, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Roles of Alum and Monophosphoryl Lipid A Adjuvants in Overcoming CD4 + T Cell Deficiency to Induce Isotype-Switched IgG Antibody Responses and Protection by T-Dependent Influenza Vaccine

doi: 10.4049/jimmunol.1600173

Figure Lengend Snippet: (A) Pro-inflammatory cytokine production of BMDCs after adjuvant treatment. (B) Activation marker expression on adjuvant-treated BMDCs. Fold increase of marker expression were determined by mean fluorescence index of each groups compared to that of control group. BMDCs were generated from BM cells of WT mice. BMDCs were cultured with media (control), or adjuvants for 48 h. (C) Cell viability of BM derived DCs (BMDCs) treated with MPL+Alum, MPL or alum. Cell viability was determined by an MTT assay after 2 days culture with adjuvants. (D) In vitro antibody production by adjuvant-treated BMDCs. Spleen cells containing naïve B cells were harvested from naïve CD4KO mice and cultured with MPL+Alum, MPL+Alum pre-treated mature DCs (mDC), or mDC plus mDC culture supernatants (sup, a source of cytokines). The mDCs were prepared by pre-treating with adjuvants for 2 days. After 7 days’ culture, total IgG levels in culture supernatants were determined by ELISA. (E) Percentages of proliferated double negative (DN, CD4−CD8− in CD3+ T cells) T cells after 5 days co-culture with or without BMDCs. w/o DCs; CD4KO mouse splenocytes containing DN T cells were cultured without BMDCs and proliferation of CFSE-labeled DN T cells was analyzed by flow cytometry. imDCs; DN T cells cultured with untreated control BMDCs. mDCs; DN T cells cultured with MPL+Alum pre-treated BMDCs. mDCs+MHCII Ab; DN T cells cultured with MPL+Alum and anti-mouse MHCII (clone M5/114.15.2) antibody (1 µg/ml) pre-treated BMDCs. (F) Percentages of proliferated double negative (DN, CD4−CD8− in CD3+ T cells) T cells after 5 days co-culture with different antigen-treated BMDCs. w/o DCs; CD4KO mouse splenocytes containing DN T cells were cultured without BMDCs. OVA DCs; DN T cells were cultured with ovalbumin pre-treated BMDCs. iPhil DCs; DN T cells were cultured with inactivated A/Philippine H3N2 virus pre-treated BMDCs. iCal DCs; DN T cells were cultured with inactivated A/California H1N1 virus vaccine strain pre-treated BMDCs. All data were shown as mean ± SEM. Statistical significance were calculated by 1-way ANOVA and Tukey’s multiple comparison test. *; p<0.05, **; p<0.01 and ***; p<0.001 as indicated among the groups. nd; not detected.

Article Snippet: Animals and reagents Female and male 6 to 8-week old C57BL/6, CD4 knock out (CD4KO, B6.129S6-Cd4 tm1Mak /J), and major histocompatibility complex class II (MHCII) KO (I-Aβ −/− ) mice were purchased from Jackson Laboratory and maintained in the animal facility at Georgia State University (GSU).

Techniques: Adjuvant, Activation Assay, Marker, Expressing, Fluorescence, Control, Generated, Cell Culture, Derivative Assay, MTT Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Labeling, Flow Cytometry, Virus, Comparison