Cytotoxic herpes simplex type 2-specific, DQ0602-restricted CD4 T+-cell clones show alloreactivity to DQ0601

Abstract

Alloreactivity is one of the most serious problems in organ transplantation. It has been hypothesized that pre-existing alloreactive T cells are actually cross-reacting cells that have been primed by the autologous major histocompatibility complex (MHC) and a specific peptide. CD8+ cytotoxic T lymphocytes that are alloreactive and recognize a virus-peptide that is presented by the autologous MHC have been reported. Here we demonstrate a cross-reactivity that exists between DQ0602 restricted, herpes simplex type 2 VP16 40-50 specific CD4+ T-cell clones, which can be alloreactive to DQ0601. Though most of the DQ0602 restricted T-cell clones we isolated from two different donors were not alloreactive, weakly cross-reacting T-cell clones could be isolated from both donors. Two strongly cross-reacting T-cell clones with high affinity interaction of their T-cell receptor (TCR) with both DQ0602/VP16 40-50 and DQ0601 could be isolated from one donor. DNA sequencing of the a fragment of the Vbeta gene used in their TCR confirmed that these two T cells indeed are two independent clones. These clones are cytotoxic and produce cytokines of a T helper 2-like pattern. Possible implications in a DR-matched transplantation setting are discussed.

Figure 1

Proliferation of clone 44 in response to different concentrations of its cognate peptide HSV-2 VP16 40–50 presented by different HLA-DQ6 molecules. There is no proliferation seen with DQ0603 and DQ0604, a peptide dosage dependent proliferation with DQ0602, and VP16-peptide independent proliferation with DQ0601 as restriction element.

Figure 2

Tetramer staining of stimulated bulk PBMC cultures from HSV-2 positive patients. (a) VP16 40–50 stimulated culture. The rectangle in the upper right quadrant shows the approximate location of the sort rectangle that was used in the single cell sorts. (b) DQ0601 allo-stimulated culture. The difference in the number of tetramer-positive cells compared to (a) is clearly visible.

Figure 3

DQ0602/VP16 40–50 tetramer staining. The cells were gated on viable CD4⁺ cells. The left image shows a tetramer-negative, the right image a tetramer-positive clone.

Figure 4

PE-DQ0602/VP16 40–50 tetramer staining of the two tetramer-positive and strongly DQ0601 alloreactive clones.

Figure 5

Identical amino acids are symbolized by an asterisk in the clone 11f6 sequence. Dashes mark a deletion in the clone 44 sequence.

Figure 6

Granzyme B assays with T-cell clones 44 and 11f6 were done in triplicate. BLS-1 DQ0602 and TAB 089 were used as APCs with or without peptide, respectively.

Figure 7

Cytotoxicity of clone 44. Cytotoxicity of clone 44 was evaluated by using a flow cytometry assay based on cleavage of a flurogenic caspase substrate within the target cells. T cells were incubated with dye-loaded BLS-1 DQ0602 or TAB089 cells at a 1 : 1 ratio in the presence and absence of 10 µg/ml VP16 40–50 peptide. Apoptotic target cells are shown as percentage of total dye loaded target cells.

Figure 8

Cytokines were measured form the supernatant of a proliferation assay using cytometric bead arrays for human Th1/Th2 cytokines. Black and white patterned bars are from clone 44, black and grey from clone 11f6. Similarly to the proliferation results, no cytokine expression was detected with DQ0602 as restriction element in the absence of VP16 40–50 peptide, while cytokine production was independent of externally added peptide with DQ0601 as restriction element.