Nonself-antigens are the cognate specificities of Foxp3+ regulatory T cells

Abstract

The majority of regulatory Foxp3+CD4+ T cells naturally arises in the thymus. It has been proposed that T cell receptors (TCRs) on these cells recognize self-MHC class II-peptide complexes with high or higher affinity and that their specificities mirror specificities of autoreactive T cells. Here, we analyzed hundreds of TCRs derived from regulatory or nonregulatory T cells and found little evidence that the former population preferably recognizes self-antigens as agonists. Instead, these cells recognized foreign MHC-peptide complexes as often as nonregulatory T cells. Our results show that high-affinity, autoreactive TCRs are rare on all CD4+ T cells and suggest that selecting self-peptide is different from the peptide that activates the same regulatory T cells in the periphery.

Figure 1. Overlap of TCRs between naïve T and T_reg populations

(A) The maximum frequency (ϕ_max) of a CDR3α transcript whose sequence did not appear after accumulation of n sequences, with 95% confidence, was computed based on equation 1. Upper and lower limits of the 95% confidence interval for a clone with ϕ_max frequency were calculated based on equation 2. Both equations are shown in Experimental Procedures. (B) Observed frequency of most dominant naive and T_reg clones found in TCR^mini or TCR^miniEp mice. Percentage of overlap was calculated by dividing shared clones by a total number of dominant clones of a given population. Shown amino acid sequences represent fragments of CDR3 regions including amino acids beginning with the third amino acid after the invariant C residue in all TCRAV genes (Y-L/F-C-A-X-1) and spanning the amino acid immediately preceding the TCRAJ motif (2-F/W-G-X-F-G-T). Clones shared between populations are highlighted by grey box. Table below the graphs shows at what frequency dominant clones of one population were found at least once in second population (for both type of mice).

Figure 2. Relative extrathymic proliferation of dominant T cell clones from TCR^mini mice

The proportions of peripheral proliferation of most frequent T cell clones (shown in Fig. 1) was calculated by dividing the peripheral frequency of a given clone by its thymic frequency. CDR3α sequences are aligned from the most expanded to the least proliferated clones. Clones shared between populations are highlighted by grey box.

Figure 3. TCRs on T cell hybridomas derived from expanded T_reg blasts accurately represent TCRs found on T_reg cells

(A) Expression of Foxp3 by T_reg cells isolated from lymph nodes of TCR^miniEp mice and 7−10 days after in vitro culture of these cells. Cells were surface-stained with antibodies against CD4, CD8, and CD25 molecules, followed by intracellular staining with Foxp3 antibody. (B) 2D-F-SSCP revealed extensive (82%) overlap of CDR3 regions of TCRα chains from freshly sorted and expanded T_reg cells. cDNA was synthesized from isolated RNA of both types of cells. Run-off PCR was done with primer specific for TCRVα2, coupled with Cy3 (fluorescent dye). First, fluorescent PCR products were run in the first dimension using capillary electrophoresis in denaturing PAGE. The second dimension was run in a non-denaturing SSCP slab gel. Resulting 2D gels show diversity, distribution, and frequency of dominant CDR3α regions from indicated populations. The intensity of individual dots corresponds to the real frequency of a particular rearrangement in the original sample. Spots migrating with different speed on the second dimension SSCP gel represent different VαJα rearrangements. (C) TCR CDR3α sequences retrieved from T_reg-derived hybridomas (Hyb) were compared with the most abundant CDR3α sequences found in T_reg cells in TCR^miniEp mice (T_reg). (D) Hybridomas derived from naïve T or T_reg cells of TCR^miniEp mice, were co-cultured with splenocytes from C57BL/6 mice. Responding hybridomas were compared for the ability to produce IL-2. Production of IL-2 secreted by activated hybridomas was measured by HT-2 proliferation using MTT assay. Results are shown as arithmetical mean ± SD.

Figure 4. T_reg-derived hybridomas from TCR^miniEp mice frequently recognize foreign but not self MHC-peptide complexes

(A) Hybridomas were co-cultured overnight with splenocytes or bone marrow derived dendritic cells (BMDC's) from C57BL/6 (A^b), Ii-, or A^bEpIi⁻ (AEp) mice. Production of IL-2 secreted by activated hybridomas was measured by HT-2/MTT assay. In each experiment, all hybridomas were separately tested for response to anti-CD3 stimulation in the presence of autologous APCs, to determine the percentages of hybridomas able to respond to activation. The box beneath the bar graph shows number of hybridomas (n) used in each test. (B) Twelve dominant T_reg TCRs identified by single-cell RT-PCR were compared with TCRs found on T cell hybridomas. Based on reactivities of these hybridomas, antigenic specificity was assigned to the particular CDR3α sequence.

Figure 5. Hybridomas derived from naïve T and T_reg cells respond with the same frequency to stimulation with allogeneic APCs but not autologous APCs

(A) Hybridomas derived from naive and T_reg cells from TCR^mini or C57BL/6 mice, were stimulated by splenocytes or BMDC's derived from autologous C57BL/6 (A^b), allogenic CBA Ca/J (A^kE^k) or bm12 (A^bm12) mice. All hybridomas were tested for response to anti-CD3 stimulation in the presence of autologous APCs. Production of IL-2 was measured by HT-2 proliferation using MTT assay. To determine percentages of hybridomas responding to APCs, only hybridomas that responded to the anti-CD3 stimulation were considered. The box beneath the bar graph shows number of hybridomas (n) used in each test. (B) The CDR3α regions of autoreactive TCRs identified in TCR^mini repertoire. TCRs on TCR^miniEp T_reg-derived hybridomas reactive to A^b were compared against database containing CDR3α sequences retrieved by single-cell RT-PCR from T cells in TCR^mini mice. Of the six CDR3 sequences identified so far, five were expressed on Foxp3⁺ T_reg cells and two were found on Foxp3⁻ T cells.

Figure 6. The CD4⁺CD45RB^hi T cells isolated from TCR^mini but not TCR^miniEp mice cause wasting disease upon adoptive transfer into autologous lymphopenic hosts

T cells isolated from lymph nodes were FACS sorted using CD4 and CD45RB antibodies. 4×10⁵ of CD4⁺CD45RB^hi T cells (purity above 99%) from TCR^mini or TCR^miniEp mice were injected intraperitoneally to A^bWT TCRα^−/− or A^bEp TCRα^−/− lymphopenic hosts, respectively. The ratio of the starting weight of recipient mice is shown. Data are representative of two independent experiments with 2 or 3 mice per group. Weight loss was associated with colon inflammation, prolapse, bowel wall thickening, and typical hunched-back appearance (data not shown).