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. 2011 Jul 22;35(1):109-22.
doi: 10.1016/j.immuni.2011.03.029. Epub 2011 Jun 30.

A requisite role for induced regulatory T cells in tolerance based on expanding antigen receptor diversity

Dipica Haribhai  1 , Jason B Williams, Shuang Jia, Derek Nickerson, Erica G Schmitt, Brandon Edwards, Jennifer Ziegelbauer, Maryam Yassai, Shun-Hwa Li, Lance M Relland, Petra M Wise, Andrew Chen, Yu-Qian Zheng, Pippa M Simpson, Jack Gorski, Nita H Salzman, Martin J Hessner, Talal A Chatila, Calvin B Williams
Affiliations

A requisite role for induced regulatory T cells in tolerance based on expanding antigen receptor diversity

Dipica Haribhai et al. Immunity. .

Abstract

Although both natural and induced regulatory T (nTreg and iTreg) cells can enforce tolerance, the mechanisms underlying their synergistic actions have not been established. We examined the functions of nTreg and iTreg cells by adoptive transfer immunotherapy of newborn Foxp3-deficient mice. As monotherapy, only nTreg cells prevented disease lethality, but did not suppress chronic inflammation and autoimmunity. Provision of Foxp3-sufficient conventional T cells with nTreg cells reconstituted the iTreg pool and established tolerance. In turn, acute depletion of iTreg cells in rescued mice resulted in weight loss and inflammation. Whereas the transcriptional signatures of nTreg and in vivo-derived iTreg cells were closely matched, there was minimal overlap in their T cell receptor (TCR) repertoires. Thus, iTreg cells are an essential nonredundant regulatory subset that supplements nTreg cells, in part by expanding TCR diversity within regulatory responses.

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Figures

Figure 1
Figure 1
Adoptive transfer immunotherapy of newborn Foxp3-deficient mice. (A) Kaplan-Meier survival curves of Foxp3K276X mice bred onto the BALB/c (n=68) and C57BL/6 (n=19) backgrounds. (B) Kaplan-Meier survival curves of BALB/c Foxp3K276X mice rescued by neonatal intraperitoneal adoptive transfer with 60×106 unfractionated splenocytes (n=194) or purified 5×106 CD4+ T cells (n=45). (C) Representative flow cytometry analysis of lymphocytes from the indicated mice stained for naïve (CD62Lhi CD44lo) and effector or memory (CD62Llo CD44hi) CD4+ T cells. For these and all subsequent representative FACS plots, numbers indicate the mean percent of cells in the quadrant. Groups included untreated Foxp3K276X mice, n=4; Foxp3K276X mice rescued (R) with unfractionated splenocytes, n=4; 6–8 week old Foxp3EGFP control mice, n=16. The CD4+ Thy1.2+ gate identified host T cells in the rescued mice. (D) The serum concentration of pro-inflammatory cytokines from the indicated mice (n=10 for all groups). *p<0.05, **p<0.005, ***p<0.0005, ns-not significant.
Figure 2
Figure 2
The effect of nTreg cell monotherapy on therapeutic outcome. (A) Linear regression analysis of weight change over time following intraperitoneal adoptive transfer of the indicated number of nTreg cells into newborn Foxp3K276X mice. Mice treated with 0.125×106 nTreg cells did not survive to weaning and are not shown. (B) Representative flow cytometry analysis of lymph node cells gated on CD4+ T cells. Thy1.1 marks the transferred nTreg cell population. (C) Frequency of nTreg cells (CD4+ gate) in the lymph nodes of treated mice. (D, E) Bar graphs depicting the average sum (per mouse) of nTreg cells (D), unfractionated lymphocytes and CD4+ T cells (E) recovered from the spleen, axillary, brachial, superficial inguinal and mesenteric lymph nodes of treated mice and healthy 6–8 week old control mice. Where indicated, dashed horizontal lines mark the mean values for healthy 6–8 week old control mice (n=16). This control value does not apply to mice treated with 0.125 ×106 nTreg cells, which did not survive beyond 22 days. (F) Representative flow cytometry analysis of host CD4+ Tconv cells stained for CD44 and CD62L. (G) Frequency of CD44+CD62L− host T cells. (H) Serum concentration of TNF-α, IFN-γ, and IL-17A in treated mice. Colored dots represent individual mice and the horizontal bars indicate mean values. Treatment dose key: 0.125 ×106 nTreg cells (green, n=16); 0.25×106 nTreg cells (orange, n=9); 0.5×106 nTreg cells (blue, n= 27); 1.0×106 nTreg cells (red, n=10); control (black, n=9); 3–5 experiments per nTreg cell dose. ns - not significant, *p<0.05, **p<0.005, ***p<0.0005.
Figure 3
Figure 3
In vivo derived iTreg cells contribute to tolerance. (A) Linear regression analysis of weight change over time following intraperitoneal adoptive transfer of the indicated number of nTreg and Tconv cell populations into newborn Foxp3K276X mice (red − 1.0×106 nTreg cells, n=10; green − 1×106 nTreg cells + 4×106 Tconv cells, n=29; orange − 1×106 nTreg cells + 4×106 ΔTconv cells from Foxp3ΔEGFP mice, n=8; blue − 1×106 nTreg cells + 1×106 iTreg cells generated in vitro, n=6; 3–7 experiments per treatment group. (B) Representative FACS analysis of lymph node cells from the indicated groups. Analysis is gated on CD4+ T cells. Thy1.1 marks different transferred populations: nTreg cells in 1st and 3rd panels, Tconv cells in the 2nd panel, or in vitro produced iTreg cells in the 4th panel. (C) Frequency of nTreg and iTreg cells. (D, E) Bar graphs depicting the average sum of nTreg cells (D), CD4+ T cells or CD19+ B cells (E) recovered from the spleen, axillary, brachial, superficial inguinal and mesenteric lymph nodes of treated mice. Dashed horizontal lines mark the mean values for healthy 6–8 week old control mice (n=16), and iTreg cells are shown in pink. (F) Representative FACS analysis of host CD4+ Tconv cells stained for CD44 and CD62L. (G) Frequency of CD44+CD62L− host T cells. (H) Representative sections from the colons of the indicated mice stained with hematoxylin and eosin. (I) Colitis scores from treated mice. Colored dots represent individual mice and the horizontal bars indicate mean values. (J) Serum concentration of TNF-α, IFN-γ, and IL-17A in treated mice. Data from 1×106 nTreg cell treatment group shown in Figure 2 are repeated here to facilitate direct comparison. *p<0.05, **p<0.005, ***p<0.0005.
Figure 4
Figure 4
Selective iTreg cell depletion from treated C57BL/6 Foxp3ΔEGFP mice. (A) Weight change over time following intraperitoneal adoptive transfer of 1×106 Foxp3EGFP nTreg cells + 4×106 Foxp3EGFP Tconv cells (blue, n=3) or 1×106 Foxp3EGFP nTreg cells + 4×106 Foxp3EGFP/DTR+ Tconv cells (red, n=3) into newborn Foxp3-deficient mice. Diphtheria toxin was injected daily for 15 days beginning at 50 days of age (arrow) and analyzed 5 days later. Two experiments were performed. (B) Representative flow cytometric analysis of lymph node cells gated on CD4+ T cells. CD45.1 marks the transferred Foxp3EGFP and Foxp3EGFP/DTR+ Tconv cell populations. (C) Frequency of nTreg and iTreg cells following DT treatment. (D, E) Average numbers (±S.E.M.) of nTreg cells (D), total lymphocytes and CD4+ T cells (E) isolated from peripheral lymphoid tissues of Foxp3EGFP and Foxp3EGFP/DTR+ mice post DT treatment. (F, G) FACS analysis of CD44 and CD62L expression (F) and frequency of CD44+CD62L− cells (G) in host (Foxp3ΔEGFP) CD4+ Tconv. (H, I) Representative FACS analysis (H) and cell frequencies (I) of host CD4+ Tconv cells stained for IFNγ pre- and post-DT treatment. (J–K) Representative sections from the lung (J), and colon, liver, and small intestine (K) of the indicated mice stained with hematoxylin and eosin.
Figure 5
Figure 5
Comparison of iTreg and nTreg cells from treated mice. (A) Representative cell surface marker analysis of CD4+EGFP+ lymph node iTreg (red) or nTreg (blue) cells stained as indicated. (B) Hierarchical clustering of probe sets significantly regulated in iTreg cells derived in vivo (top), n Treg cells from the same mice (middle), and in vitro derived iTreg cells 72 hours after Foxp3 induction (bottom), versus naïve Tconv cells from healthy mice. The scale represents fold change relative to Tconv cells (−3 fold to +3 fold). (C) Venn diagram depicting the number of commonly and uniquely regulated probe sets in the in vivo derived iTreg and nTreg cell transcriptional signatures. Bar graphs compare the fold change in expression of select prototypical genes found within the canonical Treg cell transcriptosome. (D) Venn diagram depicting the number of commonly and uniquely regulated probe sets in the in vivo derived iTreg and in vitro derived iTreg cell genetic signatures. Bar graphs compare the fold change in expression of the same genes shown in Figure 6c. (E) Methylation status of the CpG motifs in the Treg cell-specific demethylation region (TSDR) of conserved non-coding sequence 2 (CNS2) in Foxp3. Residue numbers represent individual CpG motifs in reference to the transcription initiation site of Foxp3.
Figure 6
Figure 6
TCR repertoire analysis of iTreg and nTreg cells from treated mice. (A) Representative iTreg and nTreg cell spectratype analysis using Vβ2 or Vβ8.3 5’ PCR primers. (B) Bar graphs comparing the utilization of Jβ segments by Vβ2 and Vβ8.3-containing iTreg and nTreg cell clones. (C) Venn diagrams showing the distribution of unique and overlapping iTreg and nTreg cell CDR3 amino acid sequences of Vβ2 and Vβ8.3-containing clones isolated from individual mice. The number of sequences in each category and the Morsita-Horn Similarity Index (MHI) for the comparison are shown. Ln-rank versus ln-rank frequency plots for each repertoire are shown below the corresponding Venn diagram.
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