T cell receptors in an IL-10-secreting amino acid copolymer-specific regulatory T cell line that mediates bystander immunosuppression

Abstract

The T cell receptors from the regulatory IL-10-secreting T cell line induced by the random amino acid copolymers poly(F,Y,A,K,)n in SJL mice (H-2(s)) have been characterized, cloned, sequenced and expressed both in 293T cells and in 2 different TCR alpha(-)/beta(-) T cell hybridomas. The usage of TCR alpha and beta V regions in the cell line was oligoclonal. Four TCR alpha/beta pairs cloned from single cells of the T cell line were inserted into a retrovirus vector linked by an oligonucleotide encoding the 2A peptide that spontaneously cleaves in vivo. After cotransfection of this vector with a CD3 vector into the 293T cells, the TCR were surface expressed. Moreover, after transduction into the 2 T cell hybridomas, all 4 were functional as evidenced by their response to stimulation by poly(F,Y,A,K)n. All 4 pairs were Valpha3.2(3.5)/Vbeta14, a prominent clonotype found in the poly(F,Y,A,K)n-specific T cell line. These V regions are identical to those recently found in a regulatory T cell line that secretes both IL-4 and IL-10 induced in B10.PL mice with a different MHC hapotype (H-2(u)) by a small peptide obtained from an autoimmune TCR of that strain. These data lead to a hypothesis regarding the origin of the epigenetic modifications that lead to selective cytokine secretion in T cells.

Fig. 1.

Predominance of the TCR Vβ4 and Vβ14 usage in FYAK-specific CD4⁺ T cell lines. (A) FACS analysis of Vβ segments. Frequencies of each segment are shown as means of at least 3 experiments. Error bars show SD values. (B) Staining of FYAK-specific T cell line with antibodies against CD4 and Vβ4 or Vβ14. (C) Sorted CD4⁺Vβ4⁺ and CD4⁺Vβ14⁺ populations were restimulated with FYAK, GA or PLP139–151 for 72 h. Proliferation of unsorted and sorted populations to the different antigens was evaluated by CFSE-labeling. (D) In the last 4 h of restimulation, unsorted or Vβ sorted FYAK-specific T cell populations were incubated with PMA, ionomycin, and BFA; then, IL-10 expression was analyzed by intracellular staining. (E) IL-4 and IL-10 expression on one FYAK-specific T cell line was analyzed by intracellular staining.

Fig. 2.

TCR Vα repertoire analysis. RNA was isolated from T cells and reverse-transcribed into cDNA for qPCR with specific 5′ Vα primers and a 3′ Cα primer. (A and B) Expression of Vα1 was designated as 100%, and relative expression of each Vα segment on established unsorted FYAK-specific T cell line (FYAK-TCL) (A) or Vβ14-expressing sorted FYAK-TCL (B) are shown. (C) The top 5 Vα segments with the highest expression in both A and B were compared by qPCR among different T cell populations. They are (from left to right) Vβ14⁺ FYAK-TCL, Vβ4⁺ FYAK-TCL, unsorted FYAK-TCL, Vβ14⁺ PLP-TCL, Vβ4⁺ PLP-TCL, and unsorted PLP-TCL. The expression of each Vα segment on unsorted PLP-TCL was designated as 1. All of the qPCR data are shown as means of triplicates. (D) Flow cytometry showing the expression of Vα3.2 on different Vβ sorted T cell populations.

Fig. 3.

CDR3 analysis of TCR Vα3.2 and Vβ14 fragments. (A) The CD4⁺Vα3.2⁺Vβ14⁺ T cells from FYAK-TCL were sorted at 1 cell per well into 96-well PCR plate for specific amplification of TCR fragments by single-cell RT-PCR. The PCR products from 29 single cells are shown: 14 cells for the Vα3.2 fragment, 14 cells for the Vβ14 fragment, and 1 cell for the β-actin gene (C1). RNA from a pool of FYAK-specific T cells was used as a positive control for the PCR amplification (C2). NC, negative control with no cells. (B) PCR products from single cells (7 for Vα3.2; 8 for Vβ14) were subjected to sequencing. Alignments of the translated protein sequences are shown. Red, V and C regions; green, J region; yellow, those amino acids that differ from the most frequent residues appearing at the same position. The N-Jα or N-Dβ-N region is not colored.

Fig. 4.

TCR Cloning. (A) Every TCR pair including the full-length α chain and the full-length of β chain was PCR amplified from the same single CD4⁺Vα3.2⁺Vβ14⁺ FYAK-specific T cell. PCR products from 32 single cells are shown. Only those pairs with good amplification of both α and β chain were used for further subcloning. (B) CDR3 amino acid sequences for Vα3.2 and Vβ14 TCR from 18 individual cells. The conserved residues CAXS or CAWS flanking the 5′ CDR3 of Vα3.2 and Vβ14 respectively, or FGXG flanking the 3′ CDR3 is not shown. Residues found in the J region are indicated in green. The amino acids different from the most frequent residues of the J region are indicated in yellow. The asterisk denotes the 4 TCR αβ pairs used for subcloning. (C) Structure of retroviral TCR construct. Amplified TCR α and β chain cDNA sequences were linked with the 2A peptide sequence of porcine teschovirus-1 (P2A) by recombinant PCR and then subcloned into a cassette vector, MSCV-IRES-GFP (pMIG), for retrovirus-mediated expression. (D) 293T cells cotransfected with TCR construct and CD3 vector (pMIG-CD3δγεζ) were stained with mAbs against TCR Vα3.2 and Vβ14. Flow cytometry showing the expression of cloned TCR on the surface of transfected 293T cells.

Fig. 5.

Expression and function of retroviral TCR in T-hybridoma cells. Supernatant from the retrovirus-producing GP+E86 cells were collected for infection of the TCR-deficient BW-1 and 4G4.CD4 T-hybridoma cells. (A) BW-1 cells were stained with TCRβ antibody. Flow cytometry shows the expression of all 4 TCR pairs (αβ1, αβ7, αβ11, and αβ16) on infected (GFP-positive) cells. No TCR expression was detected on BW-1 cells infected by vector only. (B and C) The TCR-bearing and vector-only infected T-hybridoma cells were sorted for GFP⁺TCRβ⁺ (groups αβ1, -7, -11, and -16) or GFP⁺ (vector) populations and stimulated with different antigens presented by APC. Supernatant from stimulated BW-1 (B) or 4G4.CD4 (C) cells were harvested at 48 h, and IL-2 production was measured by ELISA.