Characterization of CTL Clones Specific for Single Antigen, H60 Minor Histocompatibility Antigen

Abstract

Background: Disparities of Minor H antigens can induce graft rejection after MHC-matched transplantation. H60 has been characterized as a dominant antigen expressed on hematopoietic cells and considered to be an ideal model antigen for study on graft-versus-leukemia effect.

Methods: Splenocytes from C57BL/6 mice immunized with H60 congenic splenocytes were used for establishment of H60-specific CTL clones. Then the clones were characterized for proliferation capacity and cytotoxicity after stimulation with H60. Clone #14, #15, and #23 were tested for the TCR binding avidity to H60-peptide/H-2K(b) and analyzed for TCR sequences.

Results: H60-specific CTL clones showed different levels of proliferation capacity and cytotoxic activity to H60-stimulation. Clones #14, #15, and #23 showed high proliferation activity, high cytotoxicity, and low activities on both aspects, respectively, and have TCRs with different binding avidities to H60-peptide/H-2K(b) with t(1/2) values of 4.87, 6.92, and 13.03 minutes, respectively. The TCR usages were Vα12D-3-01+Jα11-01 and Vβ12-1-01+Dβ1-01+J2-7-01 for clone #14, Vα13D-1-02+Jα34-02 and Vβ13-1-02+Dβ2-01+Jβ2-7-01 for clone #15, and Vα16D+Jα45-01 and Vβ12-1-01+Dβ1-01+Jβ2-5-01 for clone #23.

Conclusion: The results will be useful for modeling GVL and generation TCR transgenic mouse.

Keywords: Avidity; CTL clone; Cytotoxicity; Proliferation; TCR usage.

Figure 1

Establishment and characterization of H60-specific CTL clones. (A) Female C57Bl/6 (B6) mice were immunized with splenocytes from male H60 congenic mice, and, then, peripheral blood lymphocytes (PBLs) from the immunized mice were examined to see whether H60-specific response was induced by staining with H60 tetramer and subsequent flow cytometry on day 10 post-immunization (left). On day 14, splenocytes from the immunized mice were cultured with irradiated splenocytes from H60 congenic mouse and the mixed lymphocyte culture (MLC) was re-stimulated regularly on weekly basis to generate H60-specific CTL line (right). H60-specific CTL clones were derived from the CTL line on passage 5. (B) Proliferation assay of the H60-specific CTL clones. Established H60-specific CTL clones were examined for their proliferation capacity in reaction to the stimulation with H60 congenic cells in the presence of IL-2 at the concentrations of 5 U/ml (left) and 20 U/ml (right). ³H was added to the culture on 48 hr later and, after further induction for 18 hr, cells were harvested and ³H-incorporation was measured. The assay was performed in triplicates and the data represent three independent experiments.

Figure 2

Cytotoxicity of H60-specific CTL clones. CTL clones were incubated with ⁵¹Cr-labeled T2-K^b cells after loading with H60 or VSV peptide at the E:T ratios of 2:1 and 10:1. ⁵¹Cr released into culture supernatant was measured and specific cytotoxicity was calculated in relation to the ⁵¹Cr released in the VSV-peptide-included wells. The assay was performed in triplicates and the mean values were plotted. The data represent three independent experiments.

Figure 3

Biochemical features of the interaction of H60-peptide/H-2K^b-TCR of H60-specific CTL clones (A) CTL clones, #14, #15, and #23, were reacted with H60-tetramer-PE, washed extensively and incubated 37℃ for 1 hr. The dissociation of the tetramer from cells during the chase was monitored by flow cytometry. (B) Cells were also stained with anti-TCRβ antibody post-chase to detect TCR levels of the cells. The MFI values from the flow cytometry (A and B) were plotted. (C) Relative MFIs were MFI values at indicated time points fractionated by MFI value detected at the beginning of the chase. And the t_1/2 was obtained based on this dissociation curve. The data represent two independent experiments.

Figure 4

Sequence analysis of H60-specific CTL clones. (A) Annotation of TCR usages of H60-specific CTL clones, #14, #15, and #23, and (B) sequences of the CDR3 regions were shown.