Figure 9. Blockade of ICOSL reduces the frequency of particular lymphocyte subsets in the spleen and lymph nodes of MCMV-infected mice.

(A) BALB/c mice (n = 11–12/group) were i.p. inoculated with 2 × 10⁶ PFU of MCMVΔm138 and treated with (α-ICOSL) or without (CTL) 100 mg of anti-ICOSL mAb. At day 14 post-infection mice were sacrificed, and spleens and lymph nodes were isolated and disaggregated. Cell suspensions were analyzed by flow cytometry using anti-mouse CD3, CD4, CD8, CD44, CD62L, CXCR5, and PD1 for T-cell phenotype. For B cell phenotype B220, CD21, CD23, CD95, and GL7 mAbs were used. Percentages of Tfh cells (CD3⁺CD4⁺CXCR5^hiPD1^hi), germinal center (GC) B cells (CD95^hiGL7^hiB220⁺), follicular (FO) B cells (CD21⁺CD23⁺B220⁺), and CD3⁺CD4⁺ or CD3⁺CD8⁺ naive (CD44^lowCD62L^hi) and memory (CD44^hiCD62L^low) T cells from these two organs are shown. (B) Representative immunofluorescence of spleens from mice infected and treated with (panels b and d) or without (panels a and c) the anti-ICOSL mAb, as indicated in A. Two color colocalization with B220 in green and peanut agglutinin (PNA) in red are shown at ×4 and ×10 magnifications. Panels on the right show a graphic representation of follicle and GC relative areas from tissue sections of a representative mouse of each experimental group. In graphs shown in A and B, results are expressed as mean +/-SD. Mice treated with anti-ICOSL mAb are represented as red squares and mice not receiving the mAb are represented as open circles. Two-tailed unpaired t-tests were used to assess statistical differences between experimental groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are pooled from two independent experiments.

Figure 9—figure supplement 1. Effect of ICOSL blockade on the frequency of several lymphocyte subsets in the spleen and lymph nodes of MCMV-infected mice.

Cell suspensions obtained from spleens and lymph nodes of BALB/c mice infected and treated as indicated in Figure 8A were analyzed by flow cytometry using anti-mouse CD3, CD4, CD8, CD5, B220, CD23, CD21, CD138, and CD19 mAbs, and anti-mouse IgM polyclonal Ab. Percentages of total B cells (B220⁺), marginal zone (MZ) B cells (CD21⁺CD23^-B220⁺), B1 B cells (CD19^hiB220^int), B1a (CD19^hiB220^intCD5⁺), and B1b (CD19^hiB220^intCD5^-) subsets, CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells, and plasma cells (CD138^hiB220^lowCD19^-IgM^-) are shown. Results are expressed as mean +/- SD. Mice treated with anti-ICOSL mAb are represented as red squares and mice untreated are represented as open circles. Two-tailed unpaired t-test was used to assess statistical differences between experimental groups.