Bi-specific MHC heterodimers for characterization of cross-reactive T cells

Abstract

T cell cross-reactivity describes the phenomenon whereby a single T cell can recognize two or more different peptide antigens presented in complex with MHC proteins. Cross-reactive T cells have previously been characterized at the population level by cytokine secretion and MHC tetramer staining assays, but single-cell analysis is difficult or impossible using these methods. In this study, we describe development of a novel peptide-MHC heterodimer specific for cross-reactive T cells. MHC-peptide monomers were independently conjugated to hydrazide or aldehyde-containing cross-linkers using thiol-maleimide coupling at cysteine residues introduced into recombinant MHC heavy chain proteins. Hydrazone formation provided bi-specific MHC heterodimers carrying two different peptides. Using this approach we prepared heterodimers of the murine class I MHC protein H-2K(b) carrying peptides from lymphocytic choriomeningitis virus and vaccinia virus, and used these to identify cross-reactive CD8+ T cells recognizing both lymphocytic choriomeningitis virus and vaccinia virus antigens. A similar strategy could be used to develop reagents to analyze cross-reactive T cell responses in humans.

FIGURE 1.

CD8+ T cells specific for VV-A11R and LCMV-GP34. A, CD8+ T cell response against VV-A11R and LCMV-GP34 was measured by intracellular IFNγ (y axis) and TNFα (x axis) staining in response to 1 μm of the indicated peptides. T cells from an LCMV immune mouse were isolated and expanded in vitro using LCMV-GP34 peptide-pulsed targets, and then analyzed for cytokine secretion in response to K^b-expressing cells pulsed with VV-A11R or LCMV-GP34 peptides, using an intracellular cytokine staining assay, with the results presented as dot plots. B, visualization of CD8+ T cell response against VV-A11R and LCMV-GP34 by peptide-K^b tetramer staining using 300 nm tetramer. The T cell line from panel A was used. T cells were stained with R-PE or APC-labeled streptavidin-based K^b tetramers folded with VV-A11R, LCMV-GP34, LCMV-GP118, or control SIY peptides. The indicated tetramer stain (blue traces) was overlaid with the control-SIY tetramer stain (red traces), with results presented as histograms.

FIGURE 2.

Cross-reactive T cells engage cognate pMHC tetramers in a manner that reveals that there exists cross-reactive TCR on these cells. Visualization of VV-A11R- and LCMV-GP34-specific T cells by double MHC tetramer staining at 300 nm. Double MHC tetramer staining experiments were set up using a PE-labeled peptide-K^b tetramer and a APC-labeled peptide-K^b tetramer as follows. A, A11R tetramer (PE) and GP118 tetramer (APC); B, A11R tetramer (PE) and SIY tetramer (APC); C, GP118 tetramer (PE) and GP34 tetramer (APC); D, SIY tetramer (PE) and GP118 tetramer (APC); and E, A11R tetramer (PE) and GP34 tetramer (APC).

FIGURE 3.

Chemical reaction scheme of heterobifunctional cross-linkers MHPH and MTFB. Peptide-K^b monomers (R₁ and R₂) containing a free thiol at the C terminus of the class I heavy chain (position 282) were reacted with heterobifunctional linkers, MTFB or MHPH through their maleimide moieties. The hydrazine on the MHPH-modified peptide-K^b monomer subsequently cross-links with the benzaldehyde on the MTFB-modified peptide-K^b monomer (when mixed in a 1:1 molar ratio) to generate a hydrazone cross-linked peptide-K^b dimer.

FIGURE 4.

Heterobifunctional cross-linking of peptide-K^b monomers. K^b monomers modified with MTFB (GP34 MTFB) or MHPH (A11R MHPH), cross-linked K^b heterodimer reaction mixture (GP34-K^b MTFB and A11R-K^b MHPH), and Alexa 647-labeled cross-linked K^b heterodimer (A647-labeled A11R-GP34 dimer) were analyzed by size exclusion chromatography and 12% SDS-PAGE. The MTFB-MHPH chemistry is specific because the A11R MHPH K^b monomer and GP34 MTFB K^b monomers do not form cross-linked K^b dimers independently. Additionally, Alexa 647 succinimidyl ester labeling does not alter the hydrodynamic radius or purity of the cross-linked K^b dimer. A, in the A11R-GP34 dimer reaction mixture, cross-linked K^b dimer with a retention volume ∼14 ml was purified from modified K^b monomers (retention volume ∼15.5 ml) by size exclusion chromatography using a Superdex 200 column. The molecular weight standards are indicated along with the approximate retention volumes. B, reducing SDS-PAGE confirms the purity of the modified K^b monomers and cross-linked K^b dimers. Proteolysis is not observed after modification (GP34 MTFB, A11R MHPH) or cross-linking (A11R-GP34 dimer reaction mixture). Molecular mass markers are indicated on the left.

FIGURE 5.

MHC tetramer and MHC monomer staining of cross-reactive T cells. A, T cells previously characterized by tetramer staining as specific for both VV-A11R and LCMV-GP34 maintained their specificity after additional restimulation with LCMV-GP34 peptide-pulsed targets. After restimulation and passage in vitro, T cells were stained with the indicated streptavidin-based K^b tetramers at 300 nm (blue trace) shown overlaid with an unstained control (red trace). Positive staining with the VV-A11R tetramer and the LCMV-GP34 tetramer shows that the specificity of the T cells is maintained after passage. B, MHC monomers do not have sufficient affinity required for T cell staining. The T cell culture from panel A was stained with peptide-K^b monomers at 2 μm as indicated. The indicated monomer stain (blue trace) was overlaid with the unstained control (red trace) in each histogram. Positive staining of T cells is not observed with any of the monomers.

FIGURE 6.

MHC heterodimer staining of cross-reactive T cells. The T cell culture shown in Fig. 5 was stained with the indicated K^b-homodimers (A) or K^b-heterodimers (B) at 1 μm. A, MHC homodimer staining demonstrates that the novel hydrazone linkage does not disturb MHC engagement of TCR and confirms that the T cells are cross-reactive for LCMV-GP34 and VV-A11R as seen in the tetramer staining experiment. B, bi-specific MHC heterodimer (A11R-GP34-K^b) staining shows that cross-reactive T cells can be readily identified. All of the indicated MHC homodimers and heterodimers (blue trace) were overlaid with an unstained control (red trace). The MFI of the positive staining population is indicated in the upper right in each histogram.