CD10 delineates a subset of human IL-4 producing follicular helper T cells involved in the survival of follicular lymphoma B cells

Abstract

In follicular lymphoma (FL), follicular helper T cells (TFH) have been depicted as one of the main components of the malignant B-cell niche and a promising therapeutic target. Although defined by their capacity to sustain FL B-cell growth together with specific gene expression and cytokine secretion profiles, FL-TFH constitute a heterogeneous cell population. However, specific markers reflecting such functional heterogeneity are still lacking. In this study, we demonstrate that CD10 identifies a subset of fully functional germinal center TFH in normal secondary lymphoid organs. Importantly, this subset is amplified in the FL context, unlike in other B-cell lymphomas with a follicular growth pattern. Furthermore, whereas FL-TFH produce high levels of interleukin (IL)-21 and low levels of IL-17 irrespectively of their CD10 expression, CD10(pos) FL-TFH specifically exhibit an IL-4(hi)IFN-γ(lo)TNF-α(hi) cytokine profile associated with a high capacity to sustain directly and indirectly malignant B-cell survival. Altogether, our results highlight the important role of this novel functional subset in the FL cell niche.

Figure 1

Characterization of CD10^pos T cells in B-cell lymphomas. (A) Double staining revealed the presence of scattered strong CD10^pos cells within FL neoplastic follicles. Numerous CD10^pos (brown) PAX5^neg (red) lymphocytes are evidenced (arrows). Original magnification: objective ×20 (left) and ×40 (right). (B) Representative characterization of ICOS, CD10, CXCR5, and PD-1 staining by flow cytometry on CD3^posCD4^pos viable cells from FL LNs. (C) In situ characterization of CD10^pos T cells in RLH including reactive LNs and tonsils, and B-cell lymphomas with follicular growth pattern (MCL, NLPHL, and FL). For each sample, the numbers of CD10^posPAX5^neg and PD-1^pos cells found inside GCs were counted in 10 high power fields (×40), and the median of these 10 values was calculated. Data are expressed as the median [range] of the different samples tested. ND, not done. (D) Expression of MME/CD10 in CD3^posCD4^pos CXCR5^highICOS^highCD25^neg T_FH purified from 7 reactive tonsils and 7 FL samples (203435_s_at probeset of the GeneChip HG-U133 Plus 2.0 oligonucleotide arrays). **P < .01. (E) Frequency of CD10^pos T_FH among CD4^pos T cells in 15 FL LNs and 25 reactive tonsil samples, evaluated by flow cytometry. ***P < .001.

Figure 2

Functional characterization of CD10^pos and CD10^neg FL-T_FH. (A) FL LN or reactive tonsil (Tons) cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin for 6 hours, and with brefeldin A for the last 4 hours of stimulation before intracytoplasmic detection of cytokines. The percentage of singlet viable CD10^neg (gray) or CD10^pos (white) viable T_FH producing IL-21, IL-17A, IL-4, and IFN-γ was determined. *P < .05, **P < .01. (B) Representative plots of IFN-γ/IL-4 and CD10/TNF-α staining on FL-T_FH. (C) Purified malignant FL B cells were cultured alone (Ø), with an activation cocktail (recombinant human CD40 ligand, IL-2, and IL-4), or in the presence of autologous CD10^neg or CD10^pos T_FH at a 1:1 ratio for 48 hours. Representative plots of active caspase-3 staining gated on CD20^pos B cells (n = 3).