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Knutson, K.L., M.L. Disis

IL-12 enhances the generation of tumour antigen-specific Th1 CD4 + T-cells during ex vivo expansion. Knutson, K.L., M.L. Disis. Interleukin-12 (IL-12). IL-12 is a cytokine produced primarily by activated macrophages.

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Knutson, K.L., M.L. Disis

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  1. IL-12 enhances the generation of tumour antigen-specific Th1 CD4+ T-cells during ex vivo expansion Knutson, K.L., M.L. Disis

  2. Interleukin-12 (IL-12) • IL-12 is a cytokine produced primarily by activated macrophages. • IL-12 is a potent inducer of Interferon-Gamma (INF-g) and Tumor Necrosis Factor Alpha (TNF-a) to a lesser extent. • IL-12 directs naïve T helper cells to a Th1 pattern of cytokine release. • IL-12 only works on activated T-Cells.

  3. IFN-g • IFN-g is a cytokine that promotes proliferation of activated human B cells. • Natural Killer (NK) cell activity is boosted by IFN-g. • IFN-g combats tumors by its stimulating effect on the cell-mediated immune system, but can also take a direct action against the tumor cell.

  4. TNF-a • TNF-a has a growth inhibiting effect and growth stimulatory effects on a wide variety of cell types. • TNF-a has a growth inhibiting effect on normal cells. • TNF-a has a growth stimulatory effect on lymphocyte cells.

  5. Origins of Th1 Response Naïve T Cell Macrophages, Dendritic Cells IL-4 IL-12 Th2 Cell Th1 Cell

  6. CD4 T-Cells • CD4 T cells fall into the Th1 subset. • CD4 is a signaling and adhesion coreceptor • CD4 responds mainly to class II MHC molecules.

  7. MHC II Pathway

  8. Immunotherapy • It has been known that the immune system has a response to cancer antigens like HER-2/neu, which is used in this experiment. • Generally, the Th1 subset of T Cells is most effective at combating tumors. • However, systemic application of cytokines has proven toxic.

  9. Immunotherapy • Adoptive Immunotherapy = The in vitro proliferation of antigen specific T Cells to be later reintroduced to patient. • IL-12 boosts the rate and extent of proliferation in the presence of the traditionally used cytokine, IL-2

  10. IL-12 enhances the generation of tumour antigen specific Th1 CD4 T cells during ex vivo expansion • Article by K.L Knutson and M. L. Disis. • Varying levels of IL-12 added to culture containing peripheral blood mononuclear cells of cancer patients. • Enzyme linked immunosorbent spot (ELIspot assay) used to count peptide specific T lymphocytes and TNF-a. • Flow cytometry was used to count and identify T cells.

  11. Purpose • Systemic application of IL-12, although successful in mice, has been proven to produce toxic results in humans. • IL-12 has been known to have augmenting effects on CD4+ T cell proliferation and specificity. • How can IL-12 be manipulated to produce antigen specific T cell production in patients suffering from cancer without toxic side effects?

  12. Materials and Methods • T Cell proliferation was measured by 3-H Thymidine Incorporation assays. • Trypan Blue Exclusion Method was used to test the number of viable human cells after in vitro stimulation. • Enzyme-linked immunosorbent spot (ELIspot) assay used to measure frequencies of peptide-specific t-lymphocytes.

  13. Materials and Methods • Peripheral Blood Mononuclear Cells (PBMC) from HER-2/neu cancer patients was isolated by density gradient centrigugation. • PBMC was stimulated in vitro with peptide. • Viable cell count was determined. • T-Lymphocyte proliferation was determined. • Cytokine secretion was determined.

  14. 3-H Thymidine Incorporation Assay • 3-H Thymidine is incorporated into the DNA of dividing cells. • Number of proliferating cells can thus be determined by the amount of 3-H thymidine used. • The comparison of 3-H Thymidine yeilded in response to an antigen is compared to incorporation in control wells to provide the stimulation index (SI) of proliferation.

  15. Generation of Immunity to HER-2/neu Peptide • The image to the right displays stimulation index of peptide-specific immunities. • ICD represents a protein derived from HER-2/neu. • P776-790 is the HER-2/neu immunizing peptide. P42-56 is the non-immunizing peptide.

  16. Generation of Immunity to HER-2/neu Peptide and Protein: Before (pre) and after (post) IL-12

  17. Generation of Immunity to HER-2/neu Peptide and Protein: Before (pre) and after (post) IL-12 HER-2/neu antigen peptide

  18. Trypan Blue Dye Exclusion following IVS • Trypan Blue is a common dye used in cell assays. • Trypan Blue only penetrates those cells that are dead because of lack of membrane integrity. • Thus, by the method of Trypan Blue Exlusion, the amount of viable cells can be counted.

  19. Absolute Viable Cell Number Recovery from Culture • In Vitro Stimulation (IVS) to HER-2/neu p776-790 helper peptide • Overall number of viable cells was not altered by inclusion of IL-12.

  20. Absolute Viable Cell Number

  21. Cells that Stained positive for CD3, CD4, or CD8 Protein • This image displays counts of T Cells that employ either CD3, CD4, or CD8. • There was no significant increase in CD4+ T cells overall after the inclusion of IL-12.

  22. Cells that Stained positive for CD3, CD4, or CD8 Protein

  23. Stimulation Index (SI) at Varying Levels of IL-12 with IL-2. • SI levels above 2 indicate a significant change. • Demonstrates a dose-dependant increase of SI with IL-12.

  24. Stimulation Index (SI) at Varying Levels of IL-12 with IL-2. These numbers indicate amount of IL-12.

  25. Stimulation Index (SI) at Varying Levels of IL-12 with IL-2 in Other Patient Very Significant Change

  26. ElIspot Analysis for Peptide Specific CD4+ Cells • Proteins are added to a well in buffer. • Those proteins that do not attach are washed away. • Enzyme labeled antibodies are added and the antibodies that do not bind are washed away. • Substrate with chromagenic dye solution is added. Substrate interacts with die solution and gives a color reaction. • The color reaction is the interpreted data.

  27. Proliferation Assessed in Counts Per Minute (CPM) with Varying levels of IL-12 • Addition of IL-12 raises the number of proliferating immune cells in a dose-dependant manner.

  28. Correlation of Peptide Specific T-Cells Proliferation to IL-12 Concentration • White bar represents proliferation of peptide specific T-Cells with 10 U/mL of IL-2 alone. • Black bar represents proliferation of peptide specific T-Cells Containing10 U/mL of IL-2 and 10 U/mL of IL-12

  29. Correlation of Peptide Specific T-Cells Proliferation to IL-12 Concentration NON-IMMUNIZING PEPTIDE

  30. Correlation of Peptide Specific T-Cells Proliferation to IL-12 Concentration

  31. IL-12 Increases Peptide Specific TNF-a Release by T Cells

  32. IL-12 Increases Peptide Specific TNF-a Release by T Cells • This graph displays the amount of Tumor Necrosis Factor Alpha released by T-Cells at varying levels of IL-12. • The amount of TNF-a increased from 63 +/- 7 pg/ml to 390 +/- 33 pg/ml.

  33. IL-12 Increases Peptide Specific TNF-a Release by T Cells No antigen Non-immunizingpeptide

  34. Conclusions • Animal studies with adoptive immunotherapies have been shown to have positive affects in tumor treatment. • IL-12 may be a big help, since it is now possible to make even more antigen specific T cells and introduce them into the patient. • Hopefully the results of this paper will lead to clinical studies of adoptive immunotherapy with IL-12 as a major compononent of the T cell proliferation process.

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