Figure 2.
Immunological and functional phenotypes in CD137-deficient patients. (A) T- and B-cell features in our patients: immunophenotyping revealed decreased frequencies of class-switched (CD27+ immunoglobulin D–negative [IgD−]) B cells and TFH (CD45RO+CXCR5+) cells in patient 1 (P1) compared with an HD, as measured by flow cytometry. (B) T-cell proliferation with carboxyfluorescein succinimidyl ester (CFSE) fluorescence cell incorporation assay 4 days poststimulation exhibiting reduced CD3+ T-cell proliferation in response to anti-CD3 and anti-CD3 in combination with CD137L in patients 1, 2 (P2), and 4 (P4) with partial and complete restoration upon OX40 and CD28 costimulation, respectively (****P < .0001; 2-way analysis of variance [ANOVA]). (C) Representative surface expression of CD25 on T cells as measured by flow cytometry 4 days poststimulation in patient 2 compared with an HD, demonstrating a T-cell activation defect with a compensatory effect upon CD28 costimulation. (D) Rescue of T-cell proliferation and activation via CD25 expression in patient 3 (P3) by exogenous expression of wild-type CD137. (E) Analysis of T-cell receptor γ (TRG) repertoire diversity with a tree-map representation for patients 1, 2, and 3, and age-matched healthy controls. Each colored square represents a unique clone and its size reflects its productive frequency within the repertoire. Simpson's D diversity index and Shannon's H index quantify repertoire clonality. (F) Flow cytometric expression of Tregs, displaying reduced Treg rates in patients 1 and 2 compared with an HD. (G) Top, Flow cytometric expression of CD86+ and CD25+ of CD19+CD3− cells 1 day poststimulation with CD40L in combination with IL4 showed impaired activation in patients 2 and 3. Bottom, quantification of B-cell activation, showing significantly lower B-cell activation in patient cells compared with HDs (*P < .05; ***P < .001; 2-way ANOVA). (H) Top, class-switched IgG+ and IgA+ of CD19+ cells upon various stimulations, displayed impaired class switch recombination in patient 3 in response to T-cell–dependent and –independent stimuli. Bottom, Quantification of class-switched (CD27+IgD−) CD19+ cell rates showing significantly lower frequencies in the patients (**P < .01; ***P < .001; 2-way ANOVA). (I) Top, B-cell proliferation measured by violet proliferation dye (VPD450) 4 days poststimulation showing reduced proliferating B cells in patient 3 in response to T-cell–dependent and –independent stimuli. Bottom, quantification of CD19+ proliferating cells, showing decreased rates in patients (*P <.05; 2-way ANOVA). All error bars indicate plus or minus SEM. CDR, complementarity-determining region; CpG, cytosine guanine dinucleotide; IGH, immunoglobulin heavy; Unstim, unstimulated.

Immunological and functional phenotypes in CD137-deficient patients. (A) T- and B-cell features in our patients: immunophenotyping revealed decreased frequencies of class-switched (CD27+ immunoglobulin D–negative [IgD−]) B cells and TFH (CD45RO+CXCR5+) cells in patient 1 (P1) compared with an HD, as measured by flow cytometry. (B) T-cell proliferation with carboxyfluorescein succinimidyl ester (CFSE) fluorescence cell incorporation assay 4 days poststimulation exhibiting reduced CD3+ T-cell proliferation in response to anti-CD3 and anti-CD3 in combination with CD137L in patients 1, 2 (P2), and 4 (P4) with partial and complete restoration upon OX40 and CD28 costimulation, respectively (****P < .0001; 2-way analysis of variance [ANOVA]). (C) Representative surface expression of CD25 on T cells as measured by flow cytometry 4 days poststimulation in patient 2 compared with an HD, demonstrating a T-cell activation defect with a compensatory effect upon CD28 costimulation. (D) Rescue of T-cell proliferation and activation via CD25 expression in patient 3 (P3) by exogenous expression of wild-type CD137. (E) Analysis of T-cell receptor γ (TRG) repertoire diversity with a tree-map representation for patients 1, 2, and 3, and age-matched healthy controls. Each colored square represents a unique clone and its size reflects its productive frequency within the repertoire. Simpson's D diversity index and Shannon's H index quantify repertoire clonality. (F) Flow cytometric expression of Tregs, displaying reduced Treg rates in patients 1 and 2 compared with an HD. (G) Top, Flow cytometric expression of CD86+ and CD25+ of CD19+CD3− cells 1 day poststimulation with CD40L in combination with IL4 showed impaired activation in patients 2 and 3. Bottom, quantification of B-cell activation, showing significantly lower B-cell activation in patient cells compared with HDs (*P < .05; ***P < .001; 2-way ANOVA). (H) Top, class-switched IgG+ and IgA+ of CD19+ cells upon various stimulations, displayed impaired class switch recombination in patient 3 in response to T-cell–dependent and –independent stimuli. Bottom, Quantification of class-switched (CD27+IgD−) CD19+ cell rates showing significantly lower frequencies in the patients (**P < .01; ***P < .001; 2-way ANOVA). (I) Top, B-cell proliferation measured by violet proliferation dye (VPD450) 4 days poststimulation showing reduced proliferating B cells in patient 3 in response to T-cell–dependent and –independent stimuli. Bottom, quantification of CD19+ proliferating cells, showing decreased rates in patients (*P <.05; 2-way ANOVA). All error bars indicate plus or minus SEM. CDR, complementarity-determining region; CpG, cytosine guanine dinucleotide; IGH, immunoglobulin heavy; Unstim, unstimulated.

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