Transgenic CD4⁺ T cells respond to MSP-1 on the parasite. (A-D) In vitro stimulation of anti-MSP-1 transgenic T cells with peptide, recombinant protein, and parasites. (A) A diagrammatic representation of MSP-1 showing its proteolytic cleavage into 6 fragments during invasion of RBCs. The fragments shed on invasion of the RBC are indicated by the arrow. The 4 recombinant fragments, which cover the full length of the protein, are shown below. The location of the B5 peptide, to which the Tg TCR is specific, and a control peptide, B7, are indicated by black bars. (B-F) CD4⁺ T cells from B5 TCR transgenic mice. Results are shown as counts per minute of ³H-thymidine incorporation measured for proliferation of CTLL-2 cells incubated with supernatant of various combinations of B5 CD4⁺ cells with antigen-presenting cells. B5 Tg and BALB/c T cells were incubated with (B) bone marrow dendritic cells (BMDCs) and specific MSP-1 B5 peptide or nonspecific B7 peptide (1024 nM), (C) irradiated splenocytes and recombinant fragments of MSP1 (44 pmol), and (D) BMDCs and parasitized RBCs at the schizont stage (50:1 RBC/DC). The B5 hybridoma was also incubated with the protein or parasite as a positive control and BALB/c CD4⁺ T cells are shown as a negative control. (E) BMDCs and B5 or control B7 peptide (100 nM) were added to CD4⁺ B5 or littermate control T cells. Forty-eight hours later, the concentration of IFN-γ in the supernatant was measured by ELISA. Means and SEM of triplicate wells are shown. These data are representative of at least 5 independent experiments. (F) Proliferation and activation of B5 TCR Tg cells is shown after 5 days in culture. Collagenase-treated B5 spleens were labeled with CFSE and incubated with B5 peptide for 5 days. The cells were then stained for activation markers CD25, CD44, CD45RB, and L-selectin (CD62L).