MHCII restriction demonstrates B cells have very limited capacity to activate tumour-specific CD4⁺ T cells in vivo

Figures & data

Figure 1. Naïve tumour-specific B cells are poor activators of naïve tumour-specific CD4⁺ T cells in vivo despite a lymphopenic environment.

(a) Experimental set-up. I-E⁺ or I-E⁻ RAG^−/− host mice were s.c. injected in the flank with 1x10⁶ B16.mHELMCC tumour cells. Mice received I-E⁺ or I-E⁻ SW_HEL B cells and CFSE labelled 5C.C7 CD4⁺ T cells i.v. 7 days later. (b) Representative flow cytometry plots depicting CD4⁺ T cell proliferation (CFSE dilution). (c) Frequency and (d) absolute number of 5C.C7 CD4⁺ T cells in the spleen 7 days after transfer. (e) Representative plots of 5C.C7 CD4⁺ T cell CFSE dilution and expression of CD62L and CD44. Frequency of (f) effector memory (CD44+CD62L-) and (g) central memory (CD44+CD62L+) 5C.C7 CD4⁺ T cells in the spleen 7 days post transfer. (h) Representative plots of splenic HEL⁺ B cells 7 days after transfer. (i) Frequency and (j) absolute number of HEL⁺ B cells. n=3/group, 12 mice total for experiment. Representative of two independent experiments. ns = not significant, * = p < .05, ** = p < .01,*** = p < .001, **** = p < .0001.

Figure 2. CD40 activated tumour-specific B cells have a limited capacity to activate naïve tumour-specific CD4 T cells.

(a) Experimental set-up. I-E⁻ RAG^−/− mice were s.c. injected in the flank with 1x10⁶ B16.mHELMCC tumour cells and after 7 days, naive 5C.C7 CD4⁺ T cells and activated SW_HEL B cells were co-transferred. To generate activated SW_HEL B cells, trangenic SW_HEL mice were injected10 days prior to transfer with s.c. B16.mHELMCC tumour cells in the flank followed by 2 i.p. injections of anti-CD40 agonistic antibody on days 3 and 6. (b) Representative flow cytometry plots depicting CD4⁺ T cell proliferation. (c) Frequency and (d) absolute number of 5C.C7 CD4⁺ T cells in the spleen 7 days after cell transfer. (e) Representative flow cytometric plots of HEL⁺ B cells in the spleens 7 days after cell transfer. (f) Frequency and (g), absolute number of HEL⁺ B cells. (h): Mean Fluorescence Intensity (MFI) of MHCII I-E expression on naïve and activated B cells 7 days after adoptive transfer. n = > 4 mice/group, 9 mice total for experiment. ns = not significant, * = p < .05.

Figure 3. Naïve tumour-specific B cells have a limited capacity to activate effector/memory tumour-specific CD4 T cells.

(a) Experimental set-up as for Figure 1. except that 5C.C7 T cells were harvested from donor mice immunised with antigen emulsified in CFA 21 days prior. (b) Representative flow cytometry plots depicting CD4⁺ T cell proliferation. (c) Frequency and (d) absolute number of 5C.C7 CD4⁺ T cells in the spleen 7 days after cell transfer. (e) Representative flow cytometric plots of HEL⁺ B cells in the spleens 7 days after cell transfer. (f) Frequency and (g), absolute number of HEL⁺ B cells. n = 3 mice/group, 12 mice total for experiment. Representative of two independent experiments. ns = not significant, * = p < .05, ** = p < .01, **** = p < .0001ss

Data availability statement

Raw data were generated at the Centenary Institute. Derived data supporting the findings of this study are available from the corresponding author BF on request.