Therapeutic effect of idiotype-specific CD4+ T cells against B-cell lymphoma in the absence of anti-idiotypic antibodies by Katrin U. Lundin, Peter O. Hofgaard, Hilde Omholt, Ludvig A. Munthe, Alexandre Corthay, and Bjarne Bogen Blood Volume 102(2):605-612 July 15, 2003 ©2003 by American Society of Hematology

Id-specific TCR-transgenic SCID mice are protected against subcutaneous challenges with Id+ B lymphomas. Id-specific TCR-transgenic SCID mice are protected against subcutaneous challenges with Id+ B lymphomas. (A) TCR-transgenic SCID mice and SCID littermates were challenged with the indicated titrated amounts of either A20 cells transfected with the λ2315 Ig L chain gene (F9) or A20 cells transfected with the empty pSV2neo vector alone (F55). A palpable tumor of 3 mm was scored as a tumor take. The lowest number of injected F9 cells, 1.25 × 10,3 was not tumorigenic. (B) Id+ λ2315 Ig in sera of mice injected 12 days earlier with 1.25 × 106 cells. Results were statistically significant (P < .0001), Mann-Whitney test. Katrin U. Lundin et al. Blood 2003;102:605-612 ©2003 by American Society of Hematology

Nonleaky Id-specific TCR-transgenic Rag2-/- mice are protected against subcutaneous challenges with Id+ B lymphomas. Nonleaky Id-specific TCR-transgenic Rag2-/- mice are protected against subcutaneous challenges with Id+ B lymphomas. TCR-transgenic Rag2-/- mice and Rag2-/- littermates were challenged with either (A) Id+ F9 or (B) Id- F55 cells (1.25 × 105). Katrin U. Lundin et al. Blood 2003;102:605-612 ©2003 by American Society of Hematology

Transfer of Id-specific CD4+ T cells confers tumor protection. Transfer of Id-specific CD4+ T cells confers tumor protection. (A) TCR-transgenic CD4+ cells, but not CD4- cells, protect. SCID mice were reconstituted with purified TCR-transgenic CD4+ or CD4- cells, or BALB/c CD4+ T cells, injected intravenously (2 × 106). Three days later, the mice were challenged with 1.25 × 105 Id+ F9 cells. TCR-transgenic CD4+ cells versus TCR-transgenic CD4- cells: P = .018. TCR-transgenic CD4+ cells versus BALB/c CD4+ cells: P = .0014. TCR transgenic CD4- versus BALB/c: P = 0.59. (B) Number of Id-specific CD4+ T cells required for transfer of protection. Groups of Rag2-/- mice were injected intravenously with the indicated titrated amounts of purified TCR-transgenic CD4+ T cells, or left nonreconstituted, and challenged 3 days later with 1.25 × 105 F9 cells. (C) Id-specific CD4+ T cells purified from recombination deficient TCR-transgenic SCID mice confer protection. Groups of SCID mice were reconstituted with 2.5 × 105 CD4+ cells purified from either TCR-transgenic SCID or BALB/c mice, or left nonreconstituted. Three days later, the mice were challenged with 1.25 × 105 F9 cells. TCR-transgenic SCID CD4+ cells versus BALB/c CD4+ cells: P = .014. BALB/c CD4+ versus nonreconstituted mice: P = .44. Katrin U. Lundin et al. Blood 2003;102:605-612 ©2003 by American Society of Hematology

Curative potential of Id-specific CD4+ T cells. Curative potential of Id-specific CD4+ T cells. Groups of SCID mice (n = 6-7) were injected with 1.25 × 105 F9 cells at day 0 in all graphs. At the indicated time points subsequent to tumor cell injection, mice received 106 CD4+ T cells purified from TCR-transgenic mice. Percent tumor avoidance (short dashes; tumor ≥ 3 mm was defined as tumor take), % survival (long dashes), and tumor size in diameter (solid lines; mm) were recorded (split y-axis). * indicates late emergence of tumors (survival ≥ 60 days). P value in each graph represents comparison with nonreconstituted mice for survival. Katrin U. Lundin et al. Blood 2003;102:605-612 ©2003 by American Society of Hematology

Id-specific CD4+ T cells accumulate and become activated in Id+ B-lymphoma tumors. Id-specific CD4+ T cells accumulate and become activated in Id+ B-lymphoma tumors. SCID mice were injected with 1.25 × 105 F9 or F55 cells and 13 days later received 106 Id-specific CD4+ cells purified from TCR-transgenic mice. At this time point, none of the mice had tumors. Five days later, single-cell suspensions from 2 F55 tumors (∼2 mm) and 2 F9 tumors (8-10 mm) were analyzed by flow cytometry. (A) Number of Id-specific (GB113+) CD4+ T cells per 106 F9 and F55 tumor cells. (B) Number of activated (CD69+ GB113+) CD4+ cells per 106 F9 and F55 tumor cells. (C) Gated GB113+ CD4+ T cells in F9 and F55 tumor cell suspensions were analyzed for CD69 expression and size (forward scatter [FSC]). Katrin U. Lundin et al. Blood 2003;102:605-612 ©2003 by American Society of Hematology

Histologic analysis of inflamed F9 tumors after injection of Id-specific T cells. Histologic analysis of inflamed F9 tumors after injection of Id-specific T cells. SCID mice with barely palpable subcutaneous F9 or F55 tumors were injected intravenously with purified Id-specific CD4+ T cells (1 × 106) and analyzed 4 to 5 days later. (A) Hematoxylin and eosin–stained section of paraffin-embedded acutely inflamed F9 (15 mm diameter) tumor. V indicates vessel; and T, tumor. Arrows mark some of the neutrophils infiltrating the perivascular edematous connective tissue. (B-C) Immunohistochemistry on cryosections of tumor tissue. Ly-6G (Gr-1) is shown in red, IgG in green, and nuclei (DAPI) in blue. Lymphoma cells express surface IgG2a and are therefore stained green. (B) F9 tumor in mice injected with T cells. (C) F9 tumor in mice not injected with T cells. (D) F55 tumor in mice injected with T cells. (E) Same tumor as in panel B, but stained for detection of Id-specific T cells (GB113, clonotype-specific, red) and IgG+ F9 cells. Original magnification, × 60. Katrin U. Lundin et al. Blood 2003;102:605-612 ©2003 by American Society of Hematology

Memory but not naive Id-specific T cells kill Id+ B-lymphoma cells by apoptosis. Memory but not naive Id-specific T cells kill Id+ B-lymphoma cells by apoptosis. Id+ F9 and Id- F55 cells (104) were tested in a 6- or 18-hour 3H-Td release assay (JAM assay) for susceptibility to killing by unstimulated naive or Th1- or Th2-polarized Id-specific TCR-transgenic SCID splenocytes that had been stimulated in vitro for one (× 1) or 2 (× 2) 10-day cycles. TCR-transgenic lymph node cells served as a source of unstimulated naive CD4+ T cells. Katrin U. Lundin et al. Blood 2003;102:605-612 ©2003 by American Society of Hematology

Id+ B-lymphoma cells become annexin V+ when exposed to cloned Id-specific Th1 cells. Id+ B-lymphoma cells become annexin V+ when exposed to cloned Id-specific Th1 cells. Id+ F9 and Id- F55 B-lymphoma cells were incubated for 4 hours together with either an Id-specific Th1 cell clone (7A10B2), or magnetic bead–purified naive CD4+ T cells from a TCR-transgenic mouse (effector-target ratio [E/T] 2:1), followed by staining. Annexin-V staining of gated B220+ propidium iodide–negative lymphoma cells is shown as shaded histograms, open histograms represent annexin-V staining of lymphoma cells in the absence of T cells. Katrin U. Lundin et al. Blood 2003;102:605-612 ©2003 by American Society of Hematology