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Listeriolysin O is an improved protein carrier for lymphoma immunoglobulin idiotype and provides systemic protection against 38C13 lymphoma

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Abstract

Follicular lymphoma (FL) is a disease that responds to current treatment regimens; however, patients in general relapse with increasingly refractory disease. Idiotype-based vaccines are currently under trial for the treatment of FL. These vaccines comprise the patient’s BCR idiotype (Id) as the tumor antigen conjugated to the protein carrier Keyhole Limpet Hemocyanin (KLH); however, other protein carriers may enhance the immune response to the lymphoma Id. In this study we investigated whether an alternate carrier, Listeriolysin O (LLO), would amplify the immune response to Id protein and provide better protection against challenge by 38C13 murine lymphoma. The Id-LLO vaccine compared favorably against Id-KLH in tumor-protection studies and both vaccines provided systemic immunity against 38C13 lymphoma. However, the immune response to the two conjugates was different in that Id-LLO induced a more powerful Th1 response characterized by high titer IgG2a anti-Id antibodies after one immunization and the presence of CD4 cells secreting IFN-γ. In vivo studies demonstrated that immune serum contributed to the anti-lymphoma efficacy seen following Id-LLO immunization. Interestingly, Id-LLO immunized mice, when challenged twice with 38C13 lymphoma provided better protection against challenge by the BCR loss variant 38C13-V2, suggesting that Id-LLO immunized mice have more potential to develop epitope spreading than Id-KLH. In conclusion, Id-LLO compared favorably against Id-KLH in its anti-lymphoma efficacy. Furthermore, Id-LLO induced a more potent humoral and cell-mediated immune response and promoted epitope spreading after lymphoma challenge. Thus, anti-Id vaccines incorporating LLO may be a better therapeutic option for treatment of B-cell lymphoma.

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Abbreviations

BCR:

B cell receptor

DLN:

Draining lymph node

Id:

Idiotype

KLH:

Keyhole limpet hemocynin

LLO:

Listeriolysin O

NHL:

Non-Hodgkin’s lymphoma

PMA:

Phorbol myristate acetate

NHL:

Non-Hodgkin’s lymphoma

GM-CSF:

Granulocyte macrophage colony stimulating factor

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Acknowledgments

This work was supported by a Leukemia and Lymphoma Society SCOR grant (PI Carl June) and R41CA121765 (Y.P.). We would like to thank Dr Phillip Darcy for critically reading this manuscript. Also, we would like to thank Dr Thorsten Verch for constructive advice on protein-expression induction and affinity chromatography; Dr Paulo Maciag and Dr Reshma Singh for advice on designing the mouse tumor protection experiments and measuring the T cell responses to immunization.

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  1. Paul Neeson

    Present address: Cancer Immunology Program, Peter MacCallum Cancer Institute, Melbourne, Australia

Authors and Affiliations

  1. Microbiology Department, University of Pennsylvania, 323 Johnson Building, 3610 Hamilton Walk, Philadelphia, PA, 19104, USA

    Paul Neeson, Zhen Kun Pan & Yvonne Paterson

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Correspondence to Yvonne Paterson.

Electronic supplementary material

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262_2007_388_MOESM1_ESM.tif

Supplementary Figure 1. Conjugation of 38C13 Idiotype protein to KLH or LLO is complete. Purified KLH or LLO were conjugated to 38C13 lymphoma Id protein using glutaraldehyde. The conjugate proteins were run on a SDS PAGE gel under reducing and non-reducing conditions, stained by Coumassie and the protein bands examined. Results are presented for 2 gels showing the individual plus conjugate proteins for (A) Id-KLH, and (B) Id-LLO. (TIF 797 kb)

262_2007_388_MOESM2_ESM.tif

Supplementary Figure 2. 38Id protein or 38Id conjugates block binding of 0.1ug S1C5 antibody to 38C13 BCR idiotype. FITC-S1C5 antibody (0.1ug) and 38C13 Id protein (A), 38C13 Id-LLO (B), or 38C13 Id-KLH (C) were pre-incubated prior to addition to 38C13 lymphoma cells. The cells were then examined by flow cytometry for the binding of the S1C5 antibody to the BCR Id. Results are depicted as fluorescence histogram overlays where the 38C13 Id protein or the protein conjugates were added at 0 to 20ug to the S1C5 antibody. A shift to the left on the fluorescence histogram indicates that the 38C13 Id protein or 38C13 protein conjugate have the Id protein available for binding by the S1C5 antibody. (TIF 195 kb)

262_2007_388_MOESM3_ESM.tif

Supplementary Figure 3. Gating strategy for CFSE proliferation assay and intracellular cytokine detection. Draining lymph nodes (DLNs) were harvested 14 days after the last immunization for each vaccine group. DLN cells were stained with CFSE and incubated in triplicate wells with purified protein. After five days, PMA/Ionomycin and Brefelden A were added to each well for 5 hours. The cells were harvested, surface antigens stained and then fixed, permeabilized and stained for intracellular cytokines. Cells were then run on a BD FACS Calibur flow cytometer and analyzed by FlowJo software. A series of dot plots are provided which depict the gating strategy used to define CD4 versus CD8 T cells, the cytokines secreted by these cells and, lastly, CD4 T cell proliferation. PMA/Ionomycin treatment is known to decrease surface levels of CD4 and CD8, nonetheless these two T cell subsets were still clearly defined at the end of the in vitro re-stimulation step. (TIF 299 kb)

262_2007_388_MOESM4_ESM.tif

Supplementary Figure 4. Mice vaccinated with 38Id conjugated to either LLO or KLH are protected from 38C13 lymphoma growth. C3H mice were immunized with the 38Id protein, 38Id-LLO or 38Id-KLH protein conjugates, and challenged with 38C13 lymphoma (as per the Methods section). Individual mice were then monitored for formation of s.c. tumors. The results have been plotted as the mean tumor diameter versus time post-inoculation for individual mice from each vaccine group. Graphs shown are for PBS control (A), 38Id only (B), 38Id-KLH (C) and 38Id-LLO. (TIF 182 kb)

262_2007_388_MOESM5_ESM.tif

Supplementary Figure 5. Anti-idioypic antibodies are present in mouse serum after immunization and tumor challenges. These figures depict the binding characteristics of induced anti-Id antibodies (in mouse serum) to the idiotype present in its native state on the 38C13 lymphoma cell. The binding characteristics of S1C5 to 38C13 and not 38C13-V2 cells was shown (A). The ability of anti-Id antibodies to inhibit S1C5 binding to 38C13 cells was also examined (B), this was performed using sera collected pre-immune (red), post-1st immune (brown), post-2nd immune (pink), post-38C13 challenge (pale blue). The binding of S1C5-FITC to 38C13 cells was compared in the presence or the absence of immunized mouse sera. A decrease in log fluorescence indicated the presence of anti-Id antibodies after immunization. (TIF 351 kb)

262_2007_388_MOESM6_ESM.tif

Supplementary Figure 6. Representative dot plots of CD4 CFSE proliferation assay. DLN cells were harvested 14 days after the last immunization, Cells were recovered and stained with CFSE. After 5 days of re-stimulation, the cells were stained for surface (CD4) markers and examined by flow cytometry for dilution of CFSE fluorescence as an indicator of CD4 T cell proliferation. Results presented are representative dot plots showing CD4 versus CFSE fluorescence for three vaccine groups following re-stimulation with either PBS (negative control), ConA (positive control), 38C13 Id protein, KLH or LLO. (TIF 313 kb)

262_2007_388_MOESM7_ESM.tif

Supplementary Figure 7. Mouse serum IgG binds to 38C13 but not 38C13-V2 after Id-LLO immunization and post-lymphoma challenges. This study examines whether the humoral response to immunization (and subsequent 38C13 lymphoma challenge) induces epitope spreading. The 38C13 or 38C13-V2 (BCR loss variant) cell lines were incubated with serum (collected at various time points during immunization or after 38C13 lymphoma challenge) and the binding of mouse IgG to the 38C13 lymphoma cells detected by indirect staining. The stained 38C13 lymphoma cells were run on a FACSCalibur and the data analyzed by FlowJo software. The specificity of the mouse antibody binding to the lymphoma cells was determined by performing blocking studies in the presence of purified 38C13 Id protein. Data is presented as a series of overlay histograms depicting mouse antibody binding to 38C13 or 38C13-V2 cells in the presence or absence of Id protein. (TIF 235 kb)

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Neeson, P., Pan, Z.K. & Paterson, Y. Listeriolysin O is an improved protein carrier for lymphoma immunoglobulin idiotype and provides systemic protection against 38C13 lymphoma. Cancer Immunol Immunother 57, 493–505 (2008). https://doi.org/10.1007/s00262-007-0388-y

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