Inhibition of follicular T-helper cells by CD8(+) regulatory T cells is essential for self tolerance

Abstract

The ability to produce vigorous immune responses that spare self tissues and organs depends on the elimination of autoreactive T and B cells. However, purging of immature and mature self-reactive T and B cells is incomplete and may also require the involvement of cells programmed to suppress immune responses. Regulatory T cells (T(reg)) belonging to the CD4(+) T-cell subset may have a role in preventing untoward inflammatory responses, but T-cell subsets programmed to inhibit the development of autoantibody formation and systemic-lupus-erythematosus-like disease have not yet been defined. Here we delineate a CD8(+) regulatory T-cell lineage that is essential for the maintenance of self tolerance and prevention of murine autoimmune disease. Genetic disruption of the inhibitory interaction between these CD8(+) T cells and their target Qa-1(+) follicular T-helper cells results in the development of a lethal systemic-lupus-erythematosus-like autoimmune disease. These findings define a sublineage of CD8 T cells programmed to suppress rather than activate immunity that represents an essential regulatory element of the immune response and a guarantor of self tolerance.

Figure 1. B6.Qa-1(D227K) mice develop an autoimmune phenotype

a) Serum IgG levels of B6.Qa-1(WT) and B6.Qa-1(D227K) mice (n=6), b) Kidney sections from 2 and 6 month old WT and D227K (n=4) mice stained with anti-mouse IgG antibody and quantified. c) Dilated capillary loops of glomeruli in kidney of 6 month old D227K mice and quantification are shown. d) ANA generation in WT and D227K (n=9) mice in 6–7 month old mice. e) Qa-1 expression on T_FH cells (ICOS+CXCR5+) in steady state. f) Analysis of surface markers on T_FH cells from 6 month old WT and D227K mice. g) Germinal centers in spleen and quantification of GC area (n=4/group). h) Isotype switched GC B cells (B220⁺Fas⁺IgM^-) from 6 month old WT and D227K mice. Error bars represent mean ± SEM.

Figure 2. Germinal Center formation and antibody response in B6.Qa-1(WT) and B6.Qa-1(D227K) mice challenged with protein antigen or virus

a) GC formation in spleen of WT or D227K mice (6––8 wk old) upon KLH/CFA immunization and quantification of GL-7⁺ area. b) Autoantibody generation and c) immunopathology in D227K mice upon multiple immunization (at day 0, 4 and 10) with antigen KLH/CFA. d) Autoantibody generation, e) histopathological analysis (day 28) in D227K mice after LCMV infection. f) IgG deposition in glomeruli from WT and D227K mice (day 28) after LCMV-Armstrong infection. 3–6 mice/group were used. Data shown represent mean ± SEM (20–30 kidney areas/group).

Figure 3. CD44⁺ ICOSL⁺ CD8⁺ cell population targets T_FH to block generation of high affinity antibodies

a) CD44⁺ICOSL⁺CXCR5⁺ CD8 cells 10 days after KLH/CFA immunization of WT mice (6–8 wk old). b, c) Dose dependent suppression of total and high affinity NP-specific antibodies. Rag2^−/− mice were transferred with 2×10⁶ naïve WT B cells along with 1×10⁶ nasve WT (●) or D227K (○) CD4 cells with or without ICOSL⁺ enriched CD8 cells. Recipients were immunized i.p. with NP₁₉-KLH/CFA and reimmunized i.p. with NP₁₉-KLH/IFA at day 10. Serum was analyzed at day 7 for primary (b) and at day 18 for secondary (c) Ab responses. Data are representative of three independent experiments. d) Suppression of high affinity Ab responses by in vitro enriched ICOSL⁺ CD8⁺ T cells. Rag2^−/− mice were transferred with B, CD4 and CD8 cells and immunized as described above. ICOSL⁺ CD8 cells were enriched in vitro. Secondary Ab response is shown at day 28 after transfer. Data represent one of two independent experiments. e, f) Ag-specific Ab and autoantibody generation in secondary hosts infused with WT (●) or D227K (○) CD4 cells. g) Qa-1-dependent suppression of monoclonal T_FH cells by CD8 Treg (see legend to Supplementary Figure 10). (h) Number of recovered OT-II cells (V_β5+CD4+) in spleen of Rag2^−/− recipients. 3–6 mice/group were used. Error bars denote mean ± SEM.

Figure 4. Mechanism of Qa-1 restricted suppression by CD8 Treg

a) Rag2^−/− hosts were transferred with CD8 cells isolated from KLH/CFA immunized WT or IL-15^−/− donor. Total and high affinity Ab was measured at day 18 (secondary response) after immunization. (b) IL-15-dependent and IL-10-independent suppression of Ab response by CD8 Treg: Rag2^−/− recipients transferred with B, CD4 and CD8 Treg were immunized and reimmunized with NP-KLH/CFA and NP-KLH/IFA. Upon initial immunization and every 3 days thereafter, antibodies were injected i.v. (100 μg/mouse). (c) Transfer of high affinity Ab responses into normal mice: B6 mice were transferred with in vitro differentiated WT or D227K OT-II T_FH cells. B6 recipients were immunized i.p. either with 100 μg NP₁₃-OVA or NP₁₉-KLH in CFA. NP specific total (c) and high affinity (d) Ab response was measured at day 7 after immunization. (e) Qa-1-dependent suppression of transferred T_FH cells: In vitro differentiated WT or D227K OT-II TFH cells (CD45.2⁺; see skewing conditions in Additional Methods) were transferred into syngeneic CD45.1⁺ B6 recipients and immunized with 100 μg NP₁₃-OVA/CFA. CD45.2⁺ OT-II T_FH cells were analyzed 10 days later by FACS (e) and enumerated (f). 3–6 mice/group were used. Data shown represent mean ± SEM.