RAG-dependent peripheral T cell receptor diversification in CD8+ T lymphocytes

Abstract

Rearrangement of T cell receptor (TCR) genes is driven by transient expression of V(D)J recombination-activating genes (RAGs) during lymphocyte development. Immunological dogma holds that T cells irreversibly terminate RAG expression before exiting the thymus, and that all of the progeny arising from mature T cells express the parental TCRs. When single pancreatic islet-derived, NRP-A7 peptide-reactive CD8(+) T cells from nonobese diabetic (NOD) mice were repeatedly stimulated with peptide-pulsed dendritic cells, daughter T cells reexpressed RAGs, lost their ability to bind to NRP-A7K(d) tetramers, ceased to transcribe tetramer-specific TCR genes, and, instead, expressed a vast array of other TCR rearrangements. Pancreatic lymph node (PLN) CD8(+) T cells from animals expressing a transgenic NRP-A7-reactive TCR transcribed and translated RAGs in vivo and displayed endogenous TCRs on their surface. RAG reexpression also occurred in the PLN CD8(+) T cells of wild-type NOD mice and could be induced in the peripheral CD8(+) T cells of nondiabetes-prone TCR-transgenic B10.H2(g7) mice by stimulation with peptide-pulsed dendritic cells. In contrast, reexpression of RAGs could not be induced in the CD8(+) T cells of B6 mice expressing an ovalbumin-specific, K(b)-restricted TCR, or in the CD8(+) T cells of NOD mice expressing a lymphocytic choriomeningitis virus-specific, D(b)-restricted TCR. Extra-thymic reexpression of the V(D)J recombination machinery in certain CD8(+) T cell subpopulations, therefore, enables further diversification of the peripheral T cell repertoire.

Fig 1.

Loss of tetramer reactivity by NRP-A7-specific CD8⁺ clones. (A) Pancreatic islets from nondiabetic NOD mice (n = 16–20 mice per experiment; two experiments) were cultured in the presence of rIL-2 for 7 days, and the islet-derived NRP-A7 tetramer-positive (x) and tetramer-negative (y) CD8⁺ cells were sorted by FACS at one cell per well. (B) Summary of NRP-A7 tetramer reactivity of T cell clones derived from single, originally tetramer-positive cells. Clones were derived from NOD (n = 77, 46, and 21 clones at each restimulation) or rag2^−/− 8.3-NOD islets (n = 15–18 clones per restimulation) upon stimulation with NRP-A7-pulsed DCs. (C) Representative tetramer-staining patterns for clones derived from tetramer-positive cells. (D) TCR levels on clones from NOD or rag2^−/− 8.3-NOD mice (n = 10 and 8, respectively). (E) Representative tetramer/TCRβ-staining patterns. (F) rag2⁺ 8.3-CD8⁺ clones express endogenous TCRs and decrease their avidity for NRP-A7 upon stimulation with NRP-A7-pulsed DCs. (Upper) Images correspond to an islet-derived 8.3-CD8⁺ T cell line before stimulation in vitro. (Lower) Images correspond to 8.3-CD8⁺ clones grown on NRP-A7-pulsed DCs or to splenic 8.3-CD8⁺ T cells. Dead cells were gated out. (G) Expression of RAG mRNAs by 8.3-CD8⁺ MLN cells and rag2⁺ and rag2^−/− 8.3-CD8⁺ clones (harvested 1–2 weeks after the first or second restimulations, respectively) within 3 h of stimulation with NRP-A7-pulsed DCs. T, thymus RNA control.

Fig 2.

TCR repertoire of clones arising from tetramer-positive precursors, as analyzed by RT-PCR. (A) NRP-A7 tetramer-negative clones do not transcribe Vα17-Jα42 mRNA (Upper), but a subset does transcribe at least some Vα17⁺ TCRα rearrangements (Lower). (B) NRP-A7 tetramer-negative clones express multiple Vβ elements. TCRβ cDNAs of clones were amplified with Vβ- and Cβ-specific primers. The Vβ11-binding primer crossreacted with Vβ8 in this experiment.

Fig 3.

Expression of RAGs by NRP-A7-reactive CD8⁺ T cells in vitro and in vivo. (A) Expression of RAG1 mRNA by 8.3-, OT-1-, and LCMV-TCR-transgenic CD8⁺ T cells upon in vitro stimulation with NRP-A7, ova, or gp33-peptide-pulsed DCs (3 h), respectively. Negative control peptides: TUM, gp33, and NRP-A7, respectively. (B) 8.3-CD8⁺ clones from 8.3-B10.H2^g7 mice express endogenous TCRs upon repeated stimulation with NRP-A7-pulsed DCs. Panels correspond to splenic 8.3-CD8⁺ cells from a rag2^−/− 8.3-NOD mouse (control), a Vβ panel⁻ clone from the spleen of a rag2⁺ 8.3-NOD mouse, and Vβ panel⁺ clones from the spleens of rag2⁺ 8.3-NOD or 8.3-B10.H2^g7 mice (experiments independent of those shown in Fig. 1). (C) Proliferation of naïve, CFSE-labeled 8.3-CD8⁺ T cells in the PLNs of NOD but not of B10.H-2^g7 mice. (D) Transcription of RAGs by PLN 8.3-CD8⁺ T cells. CD8⁺ cells were purified from the PLNs or MLNs of 3–10 8.3-NOD mice per experiment. Thymus (T) RNA was prepared from crude thymus lysates. RT-PCR products [5% for the thymus RAG2 RT-PCR shown (T*) and 15% for all other RT-PCR products] were probed with RAG1 and RAG2 cDNAs. (E) Immunoprecipitation Western blot for RAG2. RAG2 was immunoprecipitated from purified 8.3-CD8⁺ T cells or whole thymus (250 μg of protein). (F) RAG transcription in PLN and splenic (S) T cells from NOD mice (3–10 mice per experiment). (G) RSS-specific DNA breaks at Dβ and Jα loci in PLN 8.3-CD8⁺ cells.

Fig 4.

Expression of secondary TCR rearrangements by 8.3-CD8⁺ T cells in vivo. (A and B) RAG-dependent expression of secondary TCRβ chains on PLN 8.3-CD8⁺ cells. Data correspond to PLN and MLN cells from 8.3-NOD (n = 17 and 15, respectively), rag2^−/− 8.3-NOD (n = 5 and 6), 8.3-B10.H2^g7 (n = 5 and 4), OT-1-B6 (n = 3), and LCMV-NOD mice (n = 3; ≥10 weeks of age). Each triangle corresponds to a different sample. (C) Average percentage of tetramer-negative cells within CD4⁻/Vβ panel⁺ and CD4⁻/Vβ8.1/8.2⁺ PLN cells of 8.3-NOD mice (n = 5 mice). (D) Representative tetramer reactivity of CD4⁻/Vβ panel⁺ and CD4⁻/Vβ panel⁻ PLN cells. (E–G) Tetramer-positive/Vβ panel⁺ cells are larger (E) and express higher levels of CD44 (F) and CD69 (G) than tetramer-positive/Vβ panel⁻ cells. Data correspond to eight (E and F) and seven (G) 8.3-NOD mice per group.