Blood, Vol. 114, Issue 18, 3880-3889, October 29, 2009

Myeloma cell line–derived, pooled heat shock proteins as a universal vaccine for immunotherapy of multiple myeloma
Blood Qian et al. 114: 3880

Supplemental materials for: Qian et al

Files in this Data Supplement:

• Figure S1. Schematic presentation of mouse vaccination experiments (JPG, 277 KB) -
  (A) Vaccinations as myeloma prophylaxis; (B) Vaccinations and other therapies as treatments for established myeloma.
  
  
  
  
  
  
• Figure S2. Protective effects of gp96 vaccines against myeloma development in tumor-B model (JPG, 217 KB) -
  Balb/c mice (10 per group) were s.c. vaccinated twice with either gp96N, gp96A, gp96B, gp96C, gp96AC, or gp96ACD, followed by challenge with tumor-B cells. Tumor burdens were measured twice every wk. Mice were euthanized when s.c. tumors reached 225 mm² or when mice became moribund. Results shown are measurements of tumor burdens and survival of mice received different treatments. *P P
  
  
  
  
  
• Figure S3. Protective effects of gp96 vaccines against myeloma development in tumor-D model (JPG, 226 KB) -
  Balb/c mice (10 per group) were s.c. vaccinated twice with either gp96N, gp96A, gp96B, gp96D, gp96AB, or gp96ABC, followed by challenge with tumor-D cells. Tumor burdens were measured twice every wk. Mice were euthanized when s.c. tumors reached 225 mm² or when mice became moribund. Results shown are measurements of tumor burdens and survival of mice that received different treatments. *P P
  
  
  
  
  
• Figure S4. Protective effects of gp96 vaccines against myeloma development in tumor-C model (JPG, 205 KB) -
  Balb/c mice (10 per group) were s.c. vaccinated twice with either gp96N, gp96A, gp96B, gp96C, gp96AB, or gp96ABD followed by challenge with tumor-C cells. Tumor burdens were measured twice every wk. Mice were euthanized when s.c. tumors reached 225 mm² or when mice became moribund. Results shown are measurements of tumor burdens and survival of mice that received different treatments. *P P
  
  
  
  
  
• Figure S5. Protective effects of gp96 vaccines against myeloma development in tumor E and F models (JPG, 172 KB) -
  (A) C57BL/KaLwRij mice (5 per group) were s.c. vaccinated twice with either gp96N, gp96E, and gp96BCD, followed by i.v. challenge with tumor-E cells. (B) C3H mice (5 per group) were s.c. vaccinated twice with gp96N, gp96F and gp96BCD, followed by s.c. challenge with tumor-F cells. Tumor burdens were measured twice every wk. Mice were euthanized when s.c. tumors reached 225 mm² or when mice became moribund. Results shown are measurements of tumor burdens and survival of mice that received different treatments. * P P
  
  
  
  
  
• Figure S6. gp96 vaccines induce CD4⁺ and CD8⁺ tumor-specific T-cell responses (JPG, 568 KB) -
  Balb/c mice (3 per group) were s.c. vaccinated twice with either normal g96N, gp96B, gp96AC, gp96ACD, followed by challenge with tumor-B cells. One wk later, splenocytes were isolated, pooled, and restimulated with irradiated tumor-B cells for 5 d. Shown are the results of T cells from mice vaccinated with different gp96 vaccines: (A) T-cell proliferation measured by CFSE dilution assay on gated CD4⁺ and CD8⁺ T cells; (B) Intracellular staining for IFN-γ and IL-4 expression on gated CD4⁺ and CD8⁺ T cells; and (C) Cytotoxicity of CTLs against tumor-B cells. (D) Longevity of gp96 vaccine-induced immune responses. Balb/c mice were s.c. vaccinated twice with either normal g96N, gp96A, and gp96BCD. At mo 1, 2, and 3 from the second vaccination, mice (3 per group) were sacrificed and splenocytes were isolated, labeled with CFSE, and restimulated for 5 d with irradiated tumor A in vitro. T-cell proliferation was measured on gated CD8⁺ T cells by CFSE dilution. Shown are percentages of proliferating CD8⁺ T cells from mice vaccinated with either gp96N, gp96A, or gp96BCD before vaccination or 1, 2, or 3 mo after the second vaccination. Representative results of one out of two independent experiments are shown.
  
  
  
  
  
  
• Figure S7. Importance of T cells, NK cells, and IFN-γ in gp96 vaccine-induced tumor protection (JPG, 201 KB) -
  Mice (5 per group) were s.c. vaccinated twice with pooled heterologous gp96ACD and then depleted of CD4⁺ or CD8⁺ T cells or NK cells by i.p. injections of specific mAbs before tumor-B challenge, or IFNγ^−∕− mice were s.c. vaccinated twice with gp96ACD and followed by tumor-B challenge. Tumor burdens were measured twice every wk. Results shown are measurements of tumor burdens and survival of mice that received different treatments. * P P