Titration of CD45 expression differentially regulates AgR signaling in T and B cells. (A) CD19⁺, CD4⁺, and CD8⁺ LN cells from an allelic series of mice generated by crossing CD45 WT, lightning (L), and null alleles, and CD45 ‘H’ Tg were stained for surface CD45 expression and assessed by flow cytometry. Representative histograms are plotted. (Genotypes in this and all subsequent figures: −/− = CD45^−/−; WT or +/+ = CD45^+/+; L/L = two lightning alleles; L/− = one lightning allele and one CD45 null allele; H/H = two H Tg in a CD45^+/+ background; H/− = one H Tg in a CD45^+/+ background.) CD45^−/− mice are omitted from peripheral T-cell histograms and in B and C, because T-cell development is completely blocked in the thymus. (B) Allelic series LN cells were stimulated with α-IgM for 2.5 min and then fixed, permeabilized, and stained for phospho-Erk. Cells were costained for CD23 and B220 to identify mature follicular B cells. Data were collected by flow cytometry. The graph quantifies phospho-Erk⁺ CD23⁺ LN B cells (as depicted in E). Values are mean ± SEM of three independent experiments. (C and D) Allelic series LN cells were stimulated with α-CD3ε for 2.5 min and then fixed, permeabilized, and stained for phospho-Erk. Cells were costained for CD44, CD8, and CD4 to identify naïve T-cell subsets. Data were collected by flow cytometry. The graph quantifies phospho-Erk⁺ naïve CD44^lo CD4 (C) or CD44^lo CD8 (D) LN T cells (as depicted in E). Values are mean ± SEM of three independent experiments. (E) Lymphocytes were stimulated and stained as described in B–D. Histograms depict intracellular phospho-Erk expression in CD23⁺ B, CD4 T, or CD8 T LN cells from WT mice (gray-shaded histogram) and H/H mice (black line). (F and G) Intracellular calcium increase was assessed by flow cytometry in Fluo-3-loaded LN B cells (F) or LN CD4 T cells (G) from WT mice (gray) or H/H mice (black) after either AgR or ionomycin stimulation. Data in A, E, F, and G are representative of at least three independent experiments.

CD45 differentially regulates Src family kinases in T and B cells. (A) Whole-cell lysates of resting allelic series LN CD4 T cells were blotted with Ab to the inhibitory and activating tyrosines of Lck (Lck505/Src416). Src416 Ab binds activating tyrosines of all SFKs. The lower band represents p56 Lck; the upper band, p59 Fyn. Total Lck is detected as a loading control. Quantification was performed by densitometry on single Lck505 bands and lower Src416 bands (representing Lck). Values are normalized to total Lck and adjusted relative to WT values of 1. Data are representative of two independent experiments. (B and C) Whole-cell lysates of resting allelic series splenic (B) or LN (C) B cells were blotted with Ab to the inhibitory tyrosine of Lyn (Lyn507) and activating tyrosines of the SFKs (Src416). Total Lyn was detected as a loading control using an Ab from cell signaling (CS) that recognizes one Lyn isoform or anti-serum from Clifford Lowell's laboratory (AS) that recognizes both Lyn isoforms. Quantification was performed by densitometry on two lower Lyn507 and Src416 bands (representing Lyn). Values are normalized to total Lyn and adjusted relative to WT values of 1. Data in B are representative of three independent experiments; data in C, of two independent experiments.

High CD45 expression negatively regulates follicular B-cell development. (A) Graph showing total splenic cells from allelic series mice. Values are mean ± SEM of between four and six biological replicates. (B) Graph presenting total splenic CD19+ B-cells from allelic series mice. Values are mean +/− SEM of 5–12 biological replicates. (C) Representative plots of CD19⁺ BM B cells from allelic series mice stained for IgM and IgD to identify developmental subsets as gated in SI Appendix, Fig. S1A. (D) Representative plots of CD19⁺ splenic B cells from allelic series mice stained for AA4.1 and CD23 to identify developmental subsets as gated in SI Appendix, Fig. S1C. Data in C and D are representative of at least five independent experiments. (E) Graph quantifying T1, T2, and FM splenic B-cells from allelic series mice as gated in D. Values are mean ± SEM of between five and seven biological replicates. (F) Competitive chimeras were generated with a 1:1 mix of H/H and WT donor BM. BM and splenic B cells from chimeras were stained to identify B-cell developmental subsets (as gated in SI Appendix, Fig. S1 A and C). The bar graph represents relative contribution of 45.1⁺ H/H and 45.2⁺ WT B cells to each developmental compartment (gating as in SI Appendix, Fig. S3 F–I). Data are normalized to a 1:1 ratio of 45.1:45.2 cells at the IgM⁻ BM B-cell stage of development. Values are mean ± SEM of three biological replicates. (G) BM cells from allelic series mice were surface stained for CD19, IgM, and IgD expression to identify immature BM B cells (IgM⁺IgD⁻), and then fixed, permeabilized, and stained with antibodies to either Igκ or Igλ. The graph quantifes the percentage of λ⁺ cells in the immature BM B-cell compartment (gating as in SI Appendix, Fig. S1A). Values are mean ± SEM of three biological replicates. In this and subsequent figures, pairwise comparisons of data as in A, B, and F were performed using the unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (i.e, P > 0.05). Linear regression analysis was performed as described in Materials and Methods. In all figures where linear regression is performed (as in A, B, E, and G), r² represents goodness of fit, and P values were determined using Fisher's exact test. In E, each B-cell compartment (T1, T2, and FM) was independently subjected to linear regression analysis.

Restricting the BCR repertoire to eliminate clonal deletion does not rescue B-cell survival in H/H mice. (A) Graph quantifying splenic B-cell number in CD45^−/−, ^+/+, and H/H mice expressing the IgHEL Tg in the absence of sHEL ligand. Values are mean ± SEM of between three and six biological replicates. (B) Representative plots of CD19⁺ splenocytes from mice in A stained for IgM and IgD expression to identify T1 (IgM^hiIgD^int) and T2/FM (IgD^hi) subsets. (C) Representative plots of CD19+ splenocytes from mice in A stained for CD23 and AA4.1 to identify T1, T2, and FM subsets (as gated in SI Appendix, Fig. S1C). (D and E) Representative plots of CD19⁺ splenocytes from mice in A stained for CD21 and CD23 (D) or IgM and CD1d (E) to identify CD21^hi, CD1d^hi, and IgM^hi MZ B cells. Data in B–E are representative of at least three mice per genotype.

Supraphysiological CD45 expression transforms anergy into deletion in the context of a restricted BCR repertoire. (A) Graph quantifying splenic B-cell number in CD45^+/+ and H/− mice expressing IgHEL Tg with or without sHEL expression. Mice were generated genetically rather than through chimeras. Values are mean ± SEM of between three and six biological replicates. (B) Graph representing MFI of surface IgM in mice described in A. Values are mean ± SEM of three biological replicates. (C) Representative plots of total splenocytes from CD45^+/+ and H/− mice with or without restricted BCR repertoire (IgHEL) in the presence or absence of autoantigen (sHEL) stained for IgM and IgD to identify phenotypic distribution of B cells. (D) Intracellular calcium increase upon α-IgM stimulation assessed by flow cytometry in fluo-3–loaded B lymphocytes from IgHEL⁺ CD45^+/+ and IgHEL H/− mice with or without sHEL antigen. Data are representative of three biological replicates. (E) Graph representing MFI of CD86 expression on B lymphocytes from IgHEL⁺ CD45^+/+ and IgHEL⁺ H/− mice with or without sHEL antigen expression stimulated overnight with either PMA/ionomycin or α-IgM (μg/mL). Values are mean ± SEM of three biological replicates. The unpaired t-test was used to compare genotypes stimulated with 1 μg, 4 μg, and PMA/ionomycin stimuli independently. The lowest P value of each independent pairwise comparison is plotted for ease of interpretation. (F) Graph representing MFI of CD86 expression on LN B cells from CD45^+/+ and H/H mice with an unrestricted repertoire stimulated as in E. Values are mean ± SEM of three biological replicates.

High CD45 expression renders B cells refractory to BAFF. (A) L/−, CD45^+/+, and H/H LN B cells were incubated in media with or without 20 ng/mL of BAFF for 5 d. Viability of allotype-marked CD23⁺ follicular LN B cells was assessed at d0, d1, d3, and d5 by flow cytometry. The graph represents viable B cells of each genotype normalized to starting viability. Values are mean ± SEM of three biological replicates. (B) CD45^+/+ and H/H LN B cells from mixed chimeric mice were assayed for survival as described in A to isolate cell intrinsic contributions to this assay. Values are mean ± SEM of three biological replicates. (C) Histograms representing BAFFR expression on CD23⁺ (T2 and FM) splenic B cells from allelic series mice. Gray shading represents background staining of non–B-cell splenocytes. BAFFR MFI of distinct histograms is plotted on the right. Data are representative of three independent experiments. (D) CD45.1 (H/H) and CD45.2 (WT) splenic B cells from mixed chimeras stained for allotype markers and expression of BAFFR, CD23, and AA4.1 to identify the cell intrinsic contribution of each genotype to BAFFR expression at sequential B-cell developmental stages. The graph shows MFI of BAFFR surface expression on T1, T2, and FM subsets (gated as in SI Appendix, Fig. S1C) in H/H (45.1) and WT (45.2) splenocytes from mixed chimeras. Background staining is defined by BAFFR staining of non–B-cell splenocytes and is plotted for reference. Values are mean ± SEM of three biological replicates. (E) Graph representing MFI of BAFFR surface expression in CD23⁺ splenocytes from IgHEL⁺ CD45^+/+ and IgHEL⁺ H/− mice with or without autoantigen (sHEL) expression. Values are mean ± SEM of three biological replicates.

CD45 differentially regulates Src family kinase phosphorylation in T and B cells. Increasing the expression of the signaling molecule CD45 leads to progressive dephosphorylation of the inhibitory tyrosine of the Src family kinases in both T and B cells. However, only in T cells does increasing CD45 expression result in dephosphorylation of the tyrosine in the activation loop of the T cell-specific Src family kinase (Lck). This, in turn, confers a dual positive and negative regulatory role on CD45 during T-cell antigen receptor signaling. In contrast, CD45 plays a purely positive regulatory role during B-cell antigen receptor signaling.