Affinity for self antigen selects Treg cells with distinct functional properties

Abstract

The manner in which regulatory T cells (Treg cells) control lymphocyte homeostasis is not fully understood. We identified two Treg cell populations with differing degrees of self-reactivity and distinct regulatory functions. We found that GITR(hi)PD-1(hi)CD25(hi) (Triple(hi)) Treg cells were highly self-reactive and controlled lympho-proliferation in peripheral lymph nodes. GITR(lo)PD-1(lo)CD25(lo) (Triple(lo)) Treg cells were less self-reactive and limited the development of colitis by promoting the conversion of CD4(+) Tconv cells into induced Treg cells (iTreg cells). Although Foxp3-deficient (Scurfy) mice lacked Treg cells, they contained Triple(hi)-like and Triple(lo)-like CD4(+) T cells zsuper> T cells infiltrated the skin, whereas Scurfy Triple(lo)CD4(+) T cells induced colitis and wasting disease. These findings indicate that the affinity of the T cell antigen receptor for self antigen drives the differentiation of Treg cells into distinct subsets with non-overlapping regulatory activities.

Figure 1

Characterisation of Triple^hi and Triple^lo T_reg cells. (a) Flow cytometry analyzing the expression of Foxp3, GITR and PD-1 and CD25 by CD4⁺ LN cells from 6-12 week old B6 mice. Numbers adjacent to outlined areas indicate percent of Foxp3⁺CD4⁺ cells (left; green) and frequencies of Triple^hiT_reg cells (GITR^hiPD-1^hiCD25^hi, middle, red) and Triple^loT_reg cells (GITR^loPD-1^loCD25^lo, middle, brown) among Foxp3⁺CD4⁺ cells (n= 6 mice). Middle left panel shows quantification of those results. Histogram (right) shows CD25 expression on Triple^hi (red line) or Triple^lo(brown, line) T_reg cells (n=4 mice) and quantification of median fluorescence intensity (MFI) of those results. (b,c) Flow cytometry analyzing the expression of (b) CD44, CD62L, CD103, CCR7, ICOS and (c) Helios and Nrp-1 by Foxp3⁺CD4⁺ Triple^hi (red lines and bars) and Triple^lo (brown lines and bars) T_reg cells from LNs of 6-12 week old B6 mice (n= 4 mice) and quantification of those results. Each symbol represents an individual mouse; bars graphs indicate the mean (± s.e.m.) NS = not significant (P> 0.05), *P≤ 0.01, **P≤ 0.001, ***P≤0.0001 (unpaired, two-tailed t-test). Data are from three (a) or two (b,c) independent experiments.

Figure 2

Analysis of Triple^hi and Triple^lo T_reg cell origin (a) Flow cytometry analyzing the expression of GITR and PD-1 on Foxp3⁺CD4 single positive (SP) thymocytes of 6-12 week old B6 mice. Numbers adjacent to outlined areas (left) indicate frequencies of Triple^hi (red) and Triple^lo (brown) T_reg cells among Foxp3⁺CD4SP thymocytes. Bar graph (right) shows quantification of those results. (n=10 mice) (b) Flow cytometry analyzing expression of Rag-GFP reporter, GITR and PD-1 in Foxp3⁺CD4SP thymocytes from Foxp3-RFP Rag2-GFP dual-reporter mice. Numbers near bracketed lines indicate percent Rag-GFP^– (left) and Rag-GPF⁺ (right) among Foxp3⁺CD4SP thymocytes. Numbers adjacent to outlined areas indicate frequencies of Triple^hi (red) and Triple^lo (brown) T_reg cells among recirculating (Rag-GPF^–, middle) and de novo generated thymic (Rag-GPF⁺, right) T_reg cells. Bar graphs (middle right and far right) show quantification of those results. (n= 3 mice, data taken from 1 experiment) (c) Flow cytometry analyzing Foxp3, CD4, GITR and PD-1 expression in LN cells from specific pathogen free (SPF), germ free (GF) and antigen free (AF) B6 mice. Numbers adjacent to outlined areas indicates frequencies of Foxp3⁺CD4⁺ T cells (top row, gray) and Triple^hi (red) and Triple^lo (brown) T_reg cells in these mice (bottom row). Bar graphs (right) show quantification of those results. (n=2 mice per group) Each symbol represents an individual mouse; bar graphs indicate the mean (± s.e.m.) NS = P> 0.05 (Kruskal-Wallis Test). Data are from five independent (a) or one (b,c) experiments.

Figure 3

Sequence similarity analysis between Triple^hi T_reg, Triple^lo T_reg and CD4⁺T_conv TCRs. V_α2⁺ TCRα sequences generated from Triple^hi T_reg, Triple^lo T_reg and CD4⁺T_conv LN cells from Yae62 Vβ8.2 (single TCRβ chain), TCRα^+/KO Foxp3-GFP^KI mice. Sequences from each subset within each group of mice were individually compared to all subsets from all groups of mice. (a) Frequency of V_α2 expressing cells among CD4⁺ T_conv cells (blue), Triple^hiT_reg cells (red) and Triple^loT_reg cells (brown). Evaluation of TCR sequence similarity based on the Morisita-Horn similarity index (MHI) (b,c,d) MHI comparison of V_α2⁺ TCR sequences (b) CD4⁺ T_conv clonotypes (blue) were compared to each other and to Triple^lo (brown) T_reg and Triple^hi (red) T_reg clonotypes (c) Triple^loT_reg clonotypes (brown) were compared to each other and to CD4⁺T_conv (blue) and Triple^hiT_reg (red) clonotypes and (d) Triple^hi T_reg clonotypes (red) were compared to each other and to CD4 T_conv (blue) and Triple^lo T_reg (brown) clonotypes (see Methods for full description). Evaluation of TCR sequences diversity based on Shannon Entropy (e) and Simpson Diversity (f) scores (see Methods for full description). Each symbol represents the value from a group of mice (a) or sequences (e,f); in (b,c,d) each symbol represents a MHI comparison between clonotypes from two individual groups; small horizontal lines indicate the mean (± s.e.m.) NS = not significant (P> 0.058), *P=0.058, *P≤ 0.01, ***P≤0.001 (Mann-Whittney U test, b-d and unpaired, two-tailed t-test, e,f). Data are from one experiment with three independent groups of TCRβ chain mice (2 mice per group).

Figure 4

Self-reactivity of Triple^hi and Triple^lo T_reg cells. (a) Flow cytometry analyzing Triple^hi (red) and Triple^lo (brown) LN T_reg cells from 6-10 week old B6 mice for CD5 (n=4 mice) and Nur77-GFP (n=2 mice) expression and quantification of MFI of those results. (b) Proliferation of LN-derived Triple^hi T_reg (red), Triple^lo T_reg (brown) and T_conv (blue) cells in vivo after intraperitoneal injection of BrdU into B6 mice (n=4 mice). Frequencies of BrdU incorporating (BrdU⁺) cells are shown. (c) Flow cytometry analyzing in vitro proliferation (CFSE dilution) of LN-derived Triple^hi T_reg (red) Triple^lo T_reg (brown) and CD4⁺T_conv (blue) cells from purified CFSE labeled CD4⁺LN-derived T cells cultured on B6 or B6.MHCII KO BMDCs. Representative histogram (left) and quantification of those results (numbers of proliferated cells, right) are shown (n= 5 biological replicates). (d) Flow cytometry analyzing GITR, PD-1, Foxp3 and CD4 expression by 3BK508TCR-tg CD4SP (top and middle row) or GITR and PD-1 expression by 3BK508TCR-tg Foxp3⁺CD4SP thymocytes (bottom row) after 48h of culture in the presence of P-1A, P2A, 3K or no peptide, mature B6 BMDCs as APCs, IL-2 and TGF-β (e) Flow cytometry analyzing expression of Foxp3, CD4 on OTII CD4SP thymocytes (left) and GITR and PD-1 on Foxp3⁺OTII CD4SP thymocytes obtained from chimeras generated by reconstitution of RIP-OVA (top) or B6 (bottom) hosts with a mixture of bone marrow cells from OTII Rag2^–/– and B6 mice (n=4 mice each group). Numbers in quadrants indicate percent cells; numbers adjacent to outlined areas indicate (d, middle) percent Foxp3⁺ among 3BK508TCR-tg CD4SP and (e, left) percent of Foxp3⁺ among OTII CD4SP. Each symbol represents an individual chimera (a,b). Bar graphs indicate the mean (± s.e.m.). NS = not significant (P>0.054), *P≤ 0.054, **P≤ 0.01, ***P≤0.0001 (unpaired, two-tailed t-test). Data are from one (a, Nur77), two (a, CD5; b,c) independent experiments or from one representative experiment from at total of three (d) or two (e) independent experiments with similar results.

Figure 5

Triple^hi but not Triple^lo T_reg cells suppress in vivo lymphoproliferation. Analysis of in vivo suppressive function of sorted Ly5.2 Triple^hi T_reg, Ly5.2 Triple^lo T_reg cells or total Ly5.2 T_reg cells transferred into 6-10 week old Ly5.1 Foxp3^DTR mice treated every other day with diphtheria toxin (DTx). (a) Expansion of endogenous CD4⁺-(left), CD8⁺-(middle) or B- (right) cells of DTx-treated Ly5.1 Foxp3^DTR mice, previously injected (intravenously) with Triple^lo T_reg (brown, n=4 mice), Triple^hi T_reg (red, n=6 mice) or total T_reg (green, n=3 mice) cells was analyzed at d11-13 after cell transfer. DTx-treated Ly5.1 Foxp3^DTR mice receiving no cells (black, n=10 mice) or DTx-treated B6 mice (gray, n=4 mice) were used as controls. (b) Representative flow cytometry analyzing CD44 and CD62L expression on endogenous CD4⁺ T cells, isolated from LNs of DTx treated Ly5.1 Foxp3^DTR or B6 mice (described in a) (c) quantification of endogenous naive (CD44^– CD62L⁺) CD4⁺ T_conv cells of the results in (b). Numbers in quadrants indicate percent cells in each throughout. Bar graphs indicate the mean (± s.e.m.). NS = not significant (P>0.05), *P≤ 0.05, **P≤ 0.001 (unpaired, two-tailed t-test). Data are from 2-4 independent experiments (a,b).

Figure 6

Triple^lo but not Triple^hi T_reg cells suppress colitis. Analysis of in vivo suppressive function of sorted Ly5.2 Triple^hi T_reg and Ly5.2 Triple^lo T_reg cells co-transferred with sorted, naive (CD4⁺CD25^–) CD4⁺ T_conv cells from B6 Ly5.1 (a,b,c,d CD4⁺ B6 T_conv cells) or Ly5.1 Foxp3^DTR mice (b,c,d,e CD4⁺ Foxp3^DTR T_conv cells) into 6-10 week old T cell-deficient CD3ε^–/– mice. (a) Weight change in CD3ε^–/– mice following intravenous adoptive transfer of CD4⁺ T_conv cells alone (blue, n=9 mice), CD4⁺ B6 T_conv + Triple^loT_reg cells (brown, n=9 mice) or CD4⁺ B6 T_conv + Triple^hiT_reg (red, n=6 mice) cells. CD3ε^–/– mice injected with no cells (black, n=5) were used as controls. b) Hematoxylin-and-eosin staining of colon sections from CD3ε^–/– mice adoptively transferred with cell populations indicated in a and e. Scale bar, 100μm. c) Flow cytometry analyzing of Foxp3 and CD4 expression by CD4⁺ B6 T_conv cells or CD4⁺ Foxp3^DTR T_conv cells isolated from LNs and mesenteric LNs (mLN) from mice described in a and e six weeks after transfer. Numbers adjacent to outlined areas indicate frequencies of Foxp3⁺CD4⁺ (iT_reg cells) among those cells and d) quantification of those results. e) Weight change in CD3ε^–/– mice following intravenous adoptive transfer of CD4⁺ FoxP3^DTR T_conv cells alone (dashed blue, n=3 mice) or CD4⁺ FoxP3^DTR T_conv + Triple^loT_reg cells (dashed brown, n=3 mice). CD3ε^–/– mice not receiving cells (black, n=5) were used as controls. All mice were treated with DTx. NS = not significant (P>0.068), *P=0.068, **P≤ 0.05, ***P≤ 0.01, ****P≤ 0.001(unpaired, two-tailed t-test). Data are from 2-4 independent experiments. Each symbol represents an individual mouse (d); mean ± s.e.m in (a,d,e).

Figure 7

Foxp3-deficient (scurfy) mice contain B6 T_reg cell-like cells with distinct pathogenicities. a) Flow cytometry analyzing the expression of GITR and CD25 on LN CD4⁺ T cells from Foxp3-deficient (scurfy) mice (top) and PD-1, GITR, CD25 (middle), Helios, CD5 and CD62L (bottom) expression by Scurfy Triple^hi (PD-1^hiGITR^hiCD25^hi; orange solid line) CD4⁺T cells, Scurfy Triple^lo (PD-1^negGITR^negCD25^neg; purple solid line) CD4⁺ T cells, B6 CD4⁺ Triple^hi T_reg cells (dotted red line) and B6 CD4⁺ T_conv cells (dotted blue line) from 2-3 week old mice (n=4 mice each group). (b-d) Analysis of in vivo pathogenicity induced by sorted Scurfy Triple^hi and Scurfy Triple^lo CD4⁺ T cells isolated from sick, 2-3 week old FoxP3-deficient mice and adoptively transferred into 6-10 week old CD3ε^–/– mice. (b) Weight change over time following intravenous adoptive transfer of Scurfy Triple^hi (orange) or Scurfy Triple^lo (purple) CD4⁺T cells into 6-10 week old CD3ε^–/– mice (n= 14 mice each group). CD3ε^–/– mice receiving no cells (black, n=7 mice) were used as controls. (c) Representative hematoxylin-and-eosin staining of colon and tail skin sections from CD3ε^–/– mice adoptively transferred with cell populations indicated in (b) and B6 control mice. Scale bar, 100μm. (d) Numbers of Scurfy Triple^hi (orange) and Scurfy Triple^lo (purple) CD4⁺ T cells recovered from peripheral LNs and mLNs six weeks after cell transfer (n=10 mice each group). NS = not significant (P>0.05), *P≤ 0.05, **P≤ 0.001 (unpaired, two-tailed t-test). Data are from seven (b) or five (d) independent experiments or one experiment representative of two (a) or five (c) independent experiments with similar results; mean ± s.e.m in (b).