Human CD4+CD3- innate-like T cells provide a source of TNF and lymphotoxin-αβ and are elevated in rheumatoid arthritis

Abstract

Innate lymphoid cells encompass a diverse array of lymphocyte subsets with unique phenotype that initiate inflammation and provide host defenses in specific microenvironments. In this study, we identify a rare human CD4(+)CD3(-) innate-like lymphoid population with high TNF expression that is enriched in blood from patients with rheumatoid arthritis. These CD4(+)CD3(-) cells belong to the T cell lineage, but the lack of AgR at the cell surface renders them nonresponsive to TCR-directed stimuli. By developing a culture system that sustains survival, we show that CD4(+)CD3(-) innate-like T cells display IL-7-dependent induction of surface lymphotoxin-αβ, demonstrating their potential to modify tissue microenvironments. Furthermore, expression of CCR6 on the CD4(+)CD3(-) population defines a CD127(high) subset that is highly responsive to IL-7. This CD4(+)CD3(-) population is enriched in the peripheral blood from rheumatoid arthritis patients, suggesting a link to their involvement in chronic inflammatory disease.

Figure 1. Presence of a rare CD4⁺CD3⁻lymphoid population distinct from LTi and ILCs

(A) Peripheral blood lymphocytes enriched for Lin⁻ cells were analyzed by flow cytometry for the indicated surface markers (representative of 15 donors). (B) CD4⁺CD3⁺TCRaP⁺ (1) and CD4⁺CD3- TCRαβ^- (2) cells were sorted on slides and imaged with a fluorescent microscope (representative of two experiments). (C) The number of CD4⁺CD3⁻ and CD117⁺ LTi cells recovered from 200ml of blood. (D) Percentage (%) of CD4⁺CD3⁻ and CD117⁺ LTi cells within the CD127⁺CD56⁻ gate. (E) Tonsil lymphocytes enriched for Lin⁻ cells were analyzed by flow cytometry for the indicated surface markers (representative of five donors; CCR6 staining representative of three donors). In (C) and (D) each symbol represents a donor.

Figure 2. CD4⁺CD3⁻cells are unrelated to NK and γδT and cell

(A) Expression of TCRγδ, NKp46 and NKG2D on CD4⁺CD3⁻ and γδ T and NK cells respectively (representative of three donors). (B)mRNA levels of ID2 in FACS-sorted CD4⁺CD3⁻, CD4⁺ T and NK cells. (C)mRNA levels of PRF1 (perforin) after activation of FACS-sorted CD4⁺CD3⁻ or NK cells with PMA (representative of three experiments). (D) FACS-sorted CD4⁺CD3⁻ or NK cells were cultured on OP-9 stroma for seven days in the presence of IL-2, IL-7 and IL-15 before analyzing for the expression of NKp46 and CD56 (representative of three experiments).

Figure 3. CD4⁺CD3⁻cells are unconventional T cells

(A) Expression of surface CD3 on CD4⁺CD3⁻, LTi and T cells (representative of fifteen donors). (B) Mean fluorescent intensity (MFI) for CD3 expression in CD4⁺CD3⁻, LTi and CD4⁺ T cells. (C) Imaging of CD4⁺CD3⁻ and CD4⁺ T cells in culture for five days with αCD3/αCD28 and IL-2 (representative of three experiments). (D) Expression of CCR6 and CD3 of FACS-sorted CD4⁺CD3⁻ and CD4⁺ T cells that had been co-cultured with OP-9 stromal and cytokines for seven days. (E) Expression of intracellular (ic) CD3 and TCRαβ in CD4⁺CD3⁻, CD117⁺ LTi and CD4⁺ T cells (representative of three experiments).

Figure 4. A culture system to sustain LTi survival and LTαβ expression

(A) Numbers of γ_CR sufficient CD4⁺ LTi cells (Lin^-CD127⁺) before culture (day 0) and two days after culture with or without epithelia (each symbol represents one experiment). (B) Numbers of γ_CR deficient CD4⁺ LTi cells (Lin^-CD127⁺) before culture (day 0) and two days after culture with or without epithelia (each symbol represents one experiment). (C) Expression of Ki67 in CD4⁺ LTi cells cultured for 8, 16 and 24 hours with our without epithelia (control is EL4 cells; representative of two experiments). (D) Expression of LTαβ (LTβR-Fc staining) and CD127 in CD4⁺ LTi cells before culture (day 0) and two days after culture with or without epithelia and IL-7 stimulation (representative of three experiments). (E) Expression of LTαβ (LTβR-Fc staining) and CD127 in γ_CR deficient CD4⁺ LTi cells cultured for two days with epithelia with or without IL-7 (representative of two experiments).

Figure 5. CD127 and CCR6 determine IL-7 responsiveness and LTαβ expression in CD4⁺CD3⁻cells

(A) The numbers of CD4⁺CD3⁻ cells cultured for two days with or without epithelia (each symbol represents one donor). (B) Expression of CD127 and LTαβ (LTβR-Fc staining) on CD4⁺CD3⁻ and CD117⁺ cells that were cultured on an epithelial monolayer in the presence or absence of IL-7 for two days (representative of five donors). (C) Lin⁻ enriched lymphocytes were cultured on an epithelial monolayer in the presence or absence of IL-7 and two days later CD4⁺CD3⁻ cells were sorted and analyzed for the expression of LTA and LTB mRNA by qPCR (mean±sem, n = 3). (D) Expression of CD127 and CCR6 in CD4⁺CD3⁻ cells (representative of twenty donors). (E) Percentage (%) of CCR6⁺ and CCR6⁻ CD4⁺CD3⁻ cells (each symbol represents one donor). (F) Mean fluorescent intensity (MFI) of CD127 expression in CCR6⁺ and CCR6⁻ CD4⁺CD3⁻ cells. (H) Expression of CCR6 and LTαβ (LTβR-Fc staining) on CD4⁺CD3⁻ cells that were cultured on an epithelial monolayer in the presence or absence of IL-7 for two days (representative of three donors).

Figure 6. CD4⁺CD3⁻cells express high levels of TNF and are increased in rheumatoid arthritis

(A)mRNA levels of TNF in CD4⁺CD3⁻, CD4⁺ T cells and monocytes (MNC) directly isolated from blood or MNC-derived macrophages (MΦ) (each symbol represents one donor). (B)mRNA levels of TNF, IFNG, IL17 and IL22 in CD4⁺CD3⁻ cells and MNC or MΦ that had been activated with PMA (each symbol represents one donor). (C) Expression of CD4 and CD3 in Lin⁻-enriched PBMCs from normal or rheumatoid arthritis (RA) patients (representative of ten donors). (D) Frequency of CD4⁺CD3⁻ cells in normal and RA patients. (E) Number of CD4⁺CD3⁻ cells recovered from 2×10⁷ PBMCs of normal or RA patients after Lin⁻-enrichment. (F) Frequency of CD117⁺ LTi cells in normal and RA patients. (G) Number of CD117⁺ LTi cells recovered from 2×10⁷ PBMCs of normal or RA patients after Lin⁻-enrichment. (H) Frequency of CD127⁺Lin^- cells in normal and RA patients. In (D-H) each symbol represents one donor.