T Cells Engaging the Conserved MHC Class Ib Molecule Qa-1b with TAP-Independent Peptides Are Semi-Invariant Lymphocytes

Abstract

The HLA-E homolog in the mouse (Qa-1^b) is a conserved MHC class Ib molecule presenting monomorphic peptides to germline-encoded natural killer receptor CD94/NKG2A. Previously, we demonstrated the replacement of this canonical peptide by a diverse peptidome upon deficiency of the TAP peptide transporter. Analysis of this Qa-1^b-restricted T cell repertoire against these non-mutated neoantigens revealed characteristics of conventional hypervariable CD8⁺ T cells, but also of invariant T cell receptor (TCR)αβ T cells. A shared TCR Vα chain was used by this subset in combination with a variety of Vβ chains. The TCRs target peptide ligands that are conserved between mouse and man, like the identified peptide derived from the transcriptional cofactor Med15. The thymus selection was studied in a TCR-transgenic mouse and emerging naïve CD8⁺ T cells displayed a slightly activated phenotype, as witnessed by higher CD122 and Ly6C expression. Moreover, the Qa-1^b protein was dispensable for thymus selection. Importantly, no self-reactivity was observed as reported for other MHC class Ib-restricted subsets. Naïve Qa-1^b restricted T cells expanded, contracted, and formed memory cells in vivo upon peptide vaccination in a similar manner as conventional CD8⁺ T cells. Based on these data, the Qa-1^b restricted T cell subset might be positioned closest to conventional CD8⁺ T cells of all MHC class Ib populations.

Keywords: CD8+ T cells; Qa-1b; T cell receptor; non-classical MHC; peptide transporter TAP; thymus.

Figure 1

T cell receptor (TCR) usage of Qa-1^b-restricted T cell clones. Three independent Qa-1^b-restricted T cell clones were isolated (Ln12, Ln25, and Ln14). (A) TCRβ usage was determined by flow cytometry. Representative data from three independent experiments with similar results. (B) Amino acid sequence and CDR3 region of the TCRα and TCRβ chains, according to IMGT nomenclature. Complete amino acid sequences are displayed in Figure S1 in Supplementary Material.

Figure 2

Splenocytes and dendritic cells from TAP1^−/− mice present non-mutated neoantigens. T cell clones were tested against titrated numbers of splenocytes (A) and dendritic cells (B) from wild-type B6 or TAP1^−/− mice. The TLR4 ligand LPS was added to target cells to activate them. A twofold titration of target cells was tested, starting with 20,000 cells (splenocytes) or 10,000 cells (DC) per well. IFNγ release by T cells was determined as a measure of reactivity. Data are shown as mean with SD of triplicates from one out of two similar experiments. n.d., not determined.

Figure 3

Qa-1^b-restricted CD8⁺ T cell recognize conserved peptides from human and monkey. T cell clones were tested against human T2 cells and HeLa cells and monkey COS-7 cells. (A) Human TAP-negative T2 lymphoma cells were transduced with the gene for Qa-1^b. (B) Human HeLa cells were transduced with the Qa-1^b gene and the viral TAP-inhibitor BTIP (=UL49.5). (C) TAP-proficient HeLa cells were selected for low or high surface expression of Qa-1^b. (D) Monkey COS-7 cells were rendered Qa-1^b positive by gene transfer. Twofold titration of target cells are depicted, starting with 20,000 cells per well and T cell activation was measure via IFNγ production. Data are representative for at least two independent experiments with comparable results, shown as mean and SD of triplicates.

Figure 4

The Ln12 T cell peptide-epitope Med15 is immunogenic. The peptide derived from the Med15 cofactor for RNA polymerase II with sequence RLIIHFRDI is recognized by Ln12 T cells and immunogenic in C57/BL6 mice. (A) Ln12 T cell clone was tested against the Med15 peptide and other previously reported Qa-1^b-binding peptides. (B) Med15 or Qdm peptides were used for immunization of C57/BL6 mice. Five days after the booster immunization, blood samples were taken and frequencies of CD8⁺ T cell responses were measured by intracellular IFNγ staining after brief ex vivo stimulation with immunized peptide or medium. Shown are means and SEM from eight mice. (C,D) Immunized C57/BL6 mice were challenged with a mix of three different splenocyte populations that were peptide-loaded and differentially labeled with CFSE. Splenocytes were injected at day 5 after booster immunization and harvested at day 7. Specific in vivo killing of these labeled target cells was calculated using the unloaded splenocyte subset as reference. Compiled data (n = 9) of three independent experiments with similar results are shown as mean with SEM. Student’s t-test was used for statistical analyses.

Figure 5

Selection of Ln12 T cell receptor (TCR)-transgenic cells is independent of Qa-1^b. Generation and thymus selection analysis of a TCR-transgenic mouse based on the Ln12 TCR. (A) Splenocytes were retrovirally transduced with cloned alpha and beta (Vβ10) chains of the Ln12 T cell clone. (B) Transduced splenocytes were stimulated with Med15 peptide in vitro and IFNγ release by splenocytes was measured. Data representative of three independent experiments with comparable results and shown as mean with SD of triplicates. (C) Representative flow cytometry plots of thymus and spleen of C57/BL6, Ln12 tg ^WT and Ln12 tg Qa-1^−/− mice. Ln12 tg were both backcrossed to RAG1^−/−. (D,E) Cellularity of thymus and spleen and absolute counts of Vβ10⁺ CD8⁺ T cells in thymus and spleen are depicted. One-way ANOVA was used for statistical analyses.

Figure 6

Normal behavior of peripheral T cell receptor (TCR)-transgenic Ln12 T cells. Mature peripheral T cells of the TCR-transgenic mouse were examined. (A,B) Expression of activation markers on CD8⁺ T cells in spleens of C57BL/6 or Ln12 tg mice was measured by flow cytometry. Combined data from at least eight animals are depicted, shown as mean with SEM of single markers. Student’s t-test was used for statistical analyses. (C,D) In vitro stimulation of purified CD8⁺ T cells from Ln12 tg mice with Med15 or Qdm peptide. Proliferation and IFNγ release were measured. Data are representative of at least two independent experiments. (E) Naïve CFSE-labeled CD8⁺ T cells from Ln12 tg mice were i.v. transferred into C57BL/6 or TAP1^−/−. After 8 days, blood of recipient mice was analyzed for the frequency of Ln12 tg T cells. Data shown from one of the three experiments. Each line represents an individual mouse. (F,G) Ln12 tg CD8⁺ T cells were transferred to C57BL/6 mice and subsequently vaccinated with Med15 or Qdm peptide. Frequency and activation status was followed in blood of recipient mice with the use of a congenic marker. Data shown are mean with SEM of three mice and one of the two comparable experiments is shown. (G) Intracellular IFNγ detection in purified T cells was measured by flow cytometry upon brief ex vivo incubation with dendritic cells (DC) or peptides.