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. 2011 Dec 22;118(26):6824-35.
doi: 10.1182/blood-2011-06-362533. Epub 2011 Oct 28.

IL-21 is the primary common γ chain-binding cytokine required for human B-cell differentiation in vivo

Mike Recher  1 , Lucinda J Berglund, Danielle T Avery, Morton J Cowan, Andrew R Gennery, Joanne Smart, Jane Peake, Melanie Wong, Sung-Yun Pai, Sachin Baxi, Jolan E Walter, Umaimainthan Palendira, Gillian A Tangye, Michael Rice, Shannon Brothers, Waleed Al-Herz, Hans Oettgen, Hermann Eibel, Jennifer M Puck, Federica Cattaneo, John B Ziegler, Silvia Giliani, Stuart G Tangye, Luigi D Notarangelo
Affiliations

IL-21 is the primary common γ chain-binding cytokine required for human B-cell differentiation in vivo

Mike Recher et al. Blood. .

Abstract

SCID resulting from mutations in IL2RG or JAK3 is characterized by lack of T and natural killer cells; B cells are present in normal number, but antibody responses are defective. Hematopoietic cell transplantation (HCT) is curative for SCID. However, B-cell dysfunction persists in a substantial proportion of patients. We hypothesized that impaired B-cell responses after HCT in IL2RG/JAK3 deficiency results from poor donor B-cell engraftment and defective γc-dependent cytokine signaling in host B cells. To test this, and to identify which γc cytokine(s) is critical for humoral immunity, we studied 28 transplanted patients with IL2RG/JAK3 deficiency. Lack of donor B-cell engraftment associated with persistent humoral dysfunction and significantly reduced memory B cells. B-cell proliferation induced by CD40L alone or together with CpG, anti-Ig, IL-4, IL-10, or IL-13 was comparable in healthy controls and in post-HCT SCID patients, irrespective of their chimerism status. However, in vitro stimulation with CD40L/IL-21 induced B-cell proliferation, plasmablast differentiation, and antibody secretion in patients with donor B cells, but not in patients with autologous B cells. These data imply that IL-21-mediated signaling is critical for long-lived humoral immunity and to restore antibody responses in IL2RG/JAK3-deficient patients after HCT. Furthermore, in vitro stimulation with CD40L/IL-21 can predict in vivo B-cell immunity in IL2RG/JAK3 SCID after transplantation.

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Figures

Figure 1
Figure 1
Impaired humoral immune responses in X-SCID/JAK3–deficient patients correlates with reduced generation of total and class-switched memory B cells. (A-B) PBMCs from age-matched controls (n = 11 [total B cells]; n = 18 [memory B cells]) or transplanted X-SCID/JAK3–deficient patients with either donor-derived B cells (group 1; n = 13) or autologous host B cells (n = 15 [total B cells]; n = 11 [memory B cells]; group 2) were labeled with mAb against CD19 (or CD20) and CD27. The frequencies of total B cells (A) or memory B cells (B) were then determined by flow cytometry. The plots are from representative donors and patients, with the values representing the mean of each group. The graphs show data points for all donors and patients examined, with the horizontal line representing the mean. ns indicates not significant (*P < .05; **P < .01). (C-D) Memory (CD20+CD27+) B cells from age-matched controls (n = 11) or group 1 (n = 6) or group 2 (n = 8) X-SCID/JAK3–deficient patients were labeled with mAb specific for IgM, IgG, or IgA. The frequency of memory B cells that were IgM+, IgG+, or IgA+ was then determined. (C) Histogram plots show IgG and IgA expression on memory B cells from representative patients corresponding to group 1 (gray-filled histogram) or group 2 (dashed black histogram). (D) Data points for all donors and patients examined, with the horizontal line representing the mean (*P < .05; ***P < .005).
Figure 2
Figure 2
Diminished plasmablast formation and Ig secretion in response to CD40L/IL-21 in SCID patients with poor donor-derived B-cell reconstitution. PBMCs of the indicated patient-groups were either unstimulated, or cultured with CD40L/IL-21 (A,C) or CpG (B,D). After 4 days, the frequency of B cells that differentiated into plasmablasts, as determined by acquisition of a CD38hiCD27hi phenotype (A-B), and secretion of IgM and IgG (C-D) were determined by flow cytometry and ELISA, respectively. Representative FACS plots derived from healthy donors are presented in panels A and B. Statistical analysis was performed using 1-way ANOVA and Bonferroni posttest analysis (*P < .05; **P < .01; ***P < .001). NS indicates nonsignificant changes.
Figure 3
Figure 3
B cells from Group 2 X-SCID/JAK3-deficient patients fail to proliferate in response to CD40L/IL-21 stimulation. (A-C) PBMCs were labeled with CFSE and were either unstimulated or stimulated with CD40L/IL-21 or CpG. After 4 days, proliferation of B cells was assessed by incubating the cultured cells with anti-CD19 mAb and determining dilution of CFSE by CD19+ cells by flow cytometric analysis. (A-B) Percentage of CFSElo cells in cultures of PBMCs from the indicated donor groups after stimulation with CD40L/IL-21 (A) or CpG (B). (C) Representative FACS plots of CFSE dilution in PBMCs isolated from the indicated donor groups after stimulation in the absence or presence of CD40L/IL-21. Statistical analysis was performed using 1-way ANOVA and Bonferroni posttest analysis (**P < .01; ***P < .001).
Figure 4
Figure 4
IL2RG mutant naive B cells fail to proliferate in response to IL-21. (A) Naive B cells from control donors (n = 15) or transplanted X-SCID/JAK3–deficient patients with either donor-derived (group 1; n = 6) or autologous (group 2; n = 7) B cells were stimulated with CD40L alone or together with anti-Ig, CpG, IL-4, IL-10, IL-13, IL-15, or IL-21. Proliferation was determined after 5 days by measuring incorporation of [3H]thymidine during the final 18 hours of culture. The results are expressed as fold-change in proliferation relative to CD40L alone. [3H]Thymidine uptake (mean cpm ± SEM) by naive B cells isolated from healthy donors (n = 15), group 1 (n = 6), or group 2 (n = 7) transplanted X-SCID/JAK3–deficient patients who were stimulated with CD40L alone was 393 ± 63, 419 ± 104, and 442 ± 71, respectively. (B-C) Naive B cells from control donors or transplanted group 1 (B) or group 2 (C) X-SCID/JAK3–deficient patients were labeled with CFSE and then cultured with CD40L alone or together with IL-4 or IL-21. Proliferation, as determined by dilution of CFSE, was measured after 5 days of culture. Data from experiments that examined responses of naive B cells from 3 different group 2 X-SCID patients is presented in panel C.
Figure 5
Figure 5
IL2RG mutant naive B cells can express AICDA and differentiate into Ig-secreting cells in response to diverse stimuli, but not IL-21. Naive B cells from control donors (A; n = 5; B-E; n = 14) or transplanted X-SCID/JAK3–deficient patients with either donor-derived (group 1; A, n = 2; B-E, n = 6) or autologous (group 2; A, n = 3; B-E, n = 7) B cells were stimulated with CD40L alone or together with IL-4, IL-10, CpG, or IL-21. (A) Expression of AICDA was determined by quantitative PCR after 5 days. The levels of secreted IgM (B-D), IgG (E), and IgA (F) were determined by ELISA after 12 days. Each symbol represents AICDA expression of Ig secretion by naive B cells from an individual normal donor or patient (*P < .05).
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