Inter-strain tissue-infiltrating T cell responses to minor histocompatibility antigens involved in graft-versus-host disease as determined by Vbeta spectratype analysis

Abstract

Lethal graft-vs-host disease (GVHD) can be induced between MHC-matched murine strains expressing multiple minor histocompatibility Ag differences. In the B6->BALB.B model, both CD4(+) and CD8(+) donor T cells can mediate lethal GVHD, whereas in the B6->CXB-2 model, only CD8(+) T cells are lethal. TCR Vbeta CDR3-size spectratyping was previously used to analyze CD8(+) and CD4(+) T cell responses in lethally irradiated BALB.B and CXB-2 recipients, which showed significant overlap in the reacting repertoires. However, CD4(+) T cells exhibited unique skewing of the Vbeta2 and 11 families in only BALB.B recipients. These Vbeta family reactivities were confirmed by immunohistochemical staining of lingual epithelial infiltrates, and by positive and negative selection Vbeta family transfer experiments for GVHD induction in BALB.B recipients. We have now extended these studies to examine the T cell repertoire responses involved in target tissue damage. Infiltrating B6 host-presensitized CD8(+) and CD4(+) T cells were isolated 8-10 days post-transplant from the spleens, intestines and livers of CXB-2 and BALB.B transplant recipients. For both T cell subsets, the results indicated overlapping tissue skewings between the recipients, also between the tissues sampled within the respective recipients as well as tissue specific responses unique to both the BALB.B and CXB-2 infiltrates. Most notably, the CD4(+) Vbeta 11(+) family was skewed in the intestines of BALB.B but not CXB-2 recipients. Taken together, these data suggest that there are likely to be target tissue-related anti-multiple minor histocompatibility Ag-specific responses in each of the strain recipients, which may also differ from those found in peripheral lymphoid organs.

FIGURE 1

Representative CDR3-size spectratype analysis of tissue-infiltrating B6 CD4⁺Vβ11⁺ T cells. BALB.B and CXB-2 mice were lethally irradiated and injected with 2 × 10⁷ CD8⁺ or CD4⁺ enriched T cells from host-presensitized B6 mice. The liver, spleen, and the distal ileum were excised for further processing. The livers were dissociated by passage through a stainless steel mesh and the spleens were ground with a tissue grinder. Sections of the small intestine were flushed with PBS, cut into small pieces and processed to collect infiltrating lymphocytes before re-suspension in Ultraspec. Total cellular RNA, RT-PCR, and CDR3-size spectratype analysis was performed as previously described in the Materials and Methods section. A band was considered skewed if the mean area under the peak was greater than the mean ± 3× the SD of the corresponding control peak (from naive B6 CD4⁺ or CD8⁺ T cells). Arrows indicate the presence of a skewed CDR3-size length.

FIGURE 2

GVHD-associated CD4⁺Vβ11⁺ infiltration and injury of intestinal epithelium. In brief, 4.6 × 10⁵ eGFP⁺ CD4⁺Vβ11⁺ cells were injected into lethally irradiated BALB.B and CXB-2 recipients. Greater numbers of eGFP⁺ T cells (in green) and TUNEL⁺ cells (in red) were observed in the intestine of BALB.B mice (A) compared with that of CXB-2 (B) recipients. C, Quantitative comparison of infiltrated eGFP⁺CD4⁺Vβ11⁺ T cells per tissue area (pixel²) (p ≤ 0.01). D, TUNEL-positive cells per tissue area (pixel²) (p ≤ 0.02).