Malaria-specific transgenic CD4+ T cells protect immunodeficient mice from lethal infection and demonstrate requirement for a protective threshold of antibody production for parasite clearance by Robin Stephens, Frank R. Albano, Stuart Quin, Benjamin J. Pascal, Vicky Harrison, Brigitta Stockinger, Dimitris Kioussis, Hans-Ulrich Weltzien, and Jean Langhorne Blood Volume 106(5):1676-1684 September 1, 2005 ©2005 by American Society of Hematology

Intrathymic and peripheral expression of Vα2/Vβ8 Intrathymic and peripheral expression of Vα2/Vβ8.1 Tg TCR chains and a second Vα chain. Intrathymic and peripheral expression of Vα2/Vβ8.1 Tg TCR chains and a second Vα chain. Thymocytes (A) or splenocytes (B) from B5 TCR Tg mice or BALB/c were analyzed by flow cytometry for expression of CD4, CD8, Vβ8.1, and Vα2. Expression of CD4 and CD8 on gated lymphocytes within thymus and spleen is shown as well as expression of the transgenic TCR chains on CD4+ cells and CD8+ cells (thymus) and CD4+ cells (spleen). The numbers indicate the percentage of positive cells within the regions shown. Data are representative of 5 experiments. (C) Thymocytes and splenocytes of the B5 TCR Tg mice on RAG2-/- background were stained for CD4, CD8, Vα2, and Vβ8.1, Vβ8.2. Expression of the transgenic TCR is shown on CD4+CD8+ thymocytes and on CD4+ splenic T cells. CD4+ cells in the spleen were stained for Vα2/Vβ8. (D) Splenocytes of B5 TCR Tg and BALB/c control mice were stained for CD4, Vβ8.1. Expression of the transgenic α chain Vα2, as well as another TCR α, Vα8.3 is shown on gated CD4, Vβ8.1 cells. The numbers indicate the percent of positive gated cells. Arrows indicate gate shown in following plot. The data are representative of 3 independent experiments. Robin Stephens et al. Blood 2005;106:1676-1684 ©2005 by American Society of Hematology

Transgenic CD4+ T cells respond to MSP-1 on the parasite. 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). Robin Stephens et al. Blood 2005;106:1676-1684 ©2005 by American Society of Hematology

MSP1-specific transgenic T cells respond to P chabaudi in vivo after adoptive transfer into BALB/c Vβa mice. MSP1-specific transgenic T cells respond to P chabaudi in vivo after adoptive transfer into BALB/c Vβa mice. CD4+ T cells from B5 TCR Tg (5 × 106) were purified and transferred into Vβa mice (which lack endogenous Vβ8) and infected with 1 × 105P chabaudi parasites. Histogram overlays represent CD69 (A) and CD45RB (B) expression on gated CD4+ Vα2/Vβ8+ splenocytes from uninfected (thin line) and infected (thick line) recipient mice on the indicated days after infection. (C) Expansion and contraction of anti-MSP-1 T cells during infection. The histogram shows the number of anti-MSP-1 B5 CD4+ cells in the spleen, determined from the percentage of CD4+Vα2+Vβ8+ cells and the number of total viable splenocytes. (D) B5 Tg cells make IFN-γ during infection. The mean percentage (of 3 mice) of CD4+Vβ8+Vα2+ cells staining positive for IFN-γ on stimulation. SEMs are less than 10% of the means. Robin Stephens et al. Blood 2005;106:1676-1684 ©2005 by American Society of Hematology

MSP-1 TCR Tg cells protect RAG-/- mice from death and clear P chabaudi parasitemia only when B cells are present. MSP-1 TCR Tg cells protect RAG-/- mice from death and clear P chabaudi parasitemia only when B cells are present. (A) B5 and BALB/c CD4+ cells were purified (> 99%CD4+) by high-speed flow cytometry and transferred into RAG-/- mice, which were then infected with 104P chabaudi parasites. Parasitemia is shown as percent infected RBCs on a logarithmic (log10) scale. (B) Mortality is shown as percent survival. RAG mice survive significantly better than RAG mice with B5 T cells P < .05 (log-rank test). (C) B5 and BALB/c CD4+ cells were purified by high-speed flow cytometry and transferred into nu/nu mice, which were then infected with 104P chabaudi parasites. (D) A total of 5 × 106 anti-OVA TCR Tg, DO11.10, CD4+ cells were purified and transferred into nu/nu mice; 50 μg OVA was given at the same time and the mice were infected with 104P chabaudi. The courses of infection and mortality in BALB/c mice are shown for comparison. The infections shown are the mean parasitemias of 5 to 7 mice and are representative of 2 to 3 independent experiments. Error bars represent SEs of geometric means. (E-F) B5 Tg and BALB/c CD4+ cells were purified (> 99%) by high-speed flow cytometry and transferred into RAG2-/- mice, which were then infected with 104P chabaudi parasites. (F) Some mice were additionally immunized with the T-cell antigen (MSP1900-1507) covalently linked to a protective B-cell antigen (MSP11658-1746 or MSP121) the day after T-cell transfer and 2 days before infection. Parasitemia is shown on a logarithmic (log10) scale, and data are shown as geometric means of 5 to 8 mice and SEMs. Inset graphs in panels E and F represent the same parasitemia curves on a linear scale to day 14, with maximum of 45% iRBC on y-axis. * indicates significance of P < .05 by the Student t test. Robin Stephens et al. Blood 2005;106:1676-1684 ©2005 by American Society of Hematology

B5 T cells make cytokines to parasite infection in RAG mice. B5 T cells make cytokines to parasite infection in RAG mice. B5 and BALB/c CD4+ cells were purified (> 99% CD4+) by high-speed flow cytometry and transferred into RAG-/- mice, which were then infected with 104P chabaudi parasites. Some mice were immunized with the T-cell antigen (MSP1900-1507) covalently linked to a protective B-cell antigen (MSP121). (A-C) On days 7 and 15, splenocytes were analyzed by flow cytometry for surface expression of activation markers CD45RB and CD69 and intracellular IFN-γ, IL-2, and IL-10. Cells shown are lymphocyte gated CD4+Vβ8+ B5 T cells. n.d. indicates not determined. Robin Stephens et al. Blood 2005;106:1676-1684 ©2005 by American Society of Hematology

MSP1-specific TCR Tg T cells help B cells produce quicker and greater levels of malaria-specific antibody. MSP1-specific TCR Tg T cells help B cells produce quicker and greater levels of malaria-specific antibody. (A-F) B5 (▪) and BALB/c CD4+ cells (□) were purified by high-speed flow cytometry and transferred into nu/nu mice, which were then infected with 104P chabaudi parasites. Antibodies specific for (A) MSP11-672, (B) MSP1581-921, (C) MSP1900-1507, (D) MSP11508-1766, (E) whole parasite lysate, and (F) recombinant AMA-1 were measured in the plasma of samples taken weekly during the course of a P chabaudi infection. Specific IgG antibody is expressed as arbitrary units relative to a standard hyperimmune serum (1000 AU). The values shown are the geometric means and SEs of units of antibody measured in plasma of 5 to 7 mice. The asterisk indicates significant differences (P < .05, Student t test). (G) B5 TCR Tg T cells are still specific to MSP1900-1507 after 40 days of exposure to a P chabaudi infection in vivo. Lymph node cells were taken 6 weeks after infection from nu/nu mice reconstituted with B5 and BALB/c T cells and stimulated with 4 MSP-1 recombinant fragments in vitro (from left to right, MSP11-672, MSP1581-921, MSP1900-1507, and MSP11508-1766). Proliferation was determined by 3H-thymidine incorporation for the last 12 hours of culture. Robin Stephens et al. Blood 2005;106:1676-1684 ©2005 by American Society of Hematology

Activated anti-MSP-1 T cells help immune B cells make a faster and greater antibody response, which is critical for clearance. Activated anti-MSP-1 T cells help immune B cells make a faster and greater antibody response, which is critical for clearance. B5 (▪) and BALB/c (□) CD4+ cells were purified by high-speed flow cytometry and transferred into RAG-/- mice, which were then infected with 104P chabaudi (AS) parasites (A-C). Some mice were immunized with the T-cell antigen (MSP1900-1507) covalently linked to the B-cell antigen (MSP121) intraperitoneally 2 days after T-cell transfer and a day before infection (D-F). Antibodies recognizing MSP1900-1507 (A-B), MSP11508-1766 (B,E) and whole parasite lysate (C,F) were measured by ELISA. The results are expressed as arbitrary units of antibody relative to a standard hyperimmune serum. The values shown are the geometric means and SEs of IgG responses of 5 to 7 mice. The asterisk indicates that the differences are significant (P < .05, Student t test). (G) T cells protect from lethal infection but a threshold of antibody is critical in clearance of P chabaudi. A schematic representation of the influence of transferred transgenic CD4+ T cells and immune B cells in the presence and absence of immunization on the rate of clearance of P chabaudi infection (▪), mortality of RAG-/- mice (□), and the speed and magnitude of a malaria-specific antibody response (gray shaded area). Robin Stephens et al. Blood 2005;106:1676-1684 ©2005 by American Society of Hematology