Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1978 Feb 1;147(2):352–368. doi: 10.1084/jem.147.2.352

H-2-restricted cytotoxic effectors generated in vitro by the addition of trinitrophenyl-conjugated soluble proteins

A Schmitt-Verhulst, CB Pettinelli, PA Henkart, JK Lunney, GM Shearer
PMCID: PMC2184483  PMID: 75237

Abstract

Murine spleen cells from normal donors were cultured in vitro with trinitrobenzene sulfonate (TNBS)-conjugated soluble proteins, i.e., bovine gamma globulin (TNP-BGG) or bovine serum albumin (TNP-BSA). Addition of 100 μg of any of these TNP-proteins to the spleen cell cultures led to the generation of cytotoxic T-cell effectors which were H-2-restricted and TNP- specific. The lytic potential of such effectors was comparable to that generated by sensitization with TNBS-modified syngeneic cells, and was restricted to haplotypes shared at the K or K plus I-A, or the D regions of the H-2 complex. Greater effecter cell activity was generated by addition of TNP-BGG against TNBS-modified targets which shared K plus I-A than against modified targets which shared the D region with the responding cells, which suggests that the same immune response genes are involved when the response is generated by the addition of TNP-conjugated soluble proteins or of TNBS- modified cells. H-2-restricted, TNP-specific effecter cells were generated by culturing mouse spleen cells with syngeneic cells which had been preincubated with TNP- BGG or TNP-BSA for 1.5 h. The addition of unconjugated soluble proteins to the cultures did not result in cytotoxic effectors detectable on H-2-matched targets, whether the targets were prepared by modification with TNBS, or by incubation with either the unconjugated or TNP-conjugated proteins. Depletion of phagocytic cells in the tumor preparation by Sephadex G-10 column fractionation before incubation with TNP-BSA had no effect on their lysis by the relevant effector cells. Immunofluorescent staining of tumor target cells with anti-TNP antibodies indicated that TNP could be detected on the tumor cells within 10 rain of incubation with TNP-BSA. The cytotoxic response generated by addition of the TNP-proteins to spleen cell cultures was found to be T-cell dependent at the effector phase, as shown by the sensitivity of the lytic phase to absorbed RAMB and complement. Furthermore, the response did not appear to be attributable to antibody-dependent cellular cytotoxicity. Three mechanisms were considered which could account for the generation of H-2-restricted, TNP-specific, cytotoxic T-cell effectors by the addition of soluble TNP-proteins. These include covalent linkage of activated TNP groups from the soluble proteins to cell surface components, macrophage processing of the soluble conjugates and presentation to the responding lymphocytes in association with H-2-coded self structures, or hydrophobic interaction of the TNP-proteins to cell surfaces. Results obtained from sodium dodecyl sulfate gel patterns indicating that cell-bound TNP was still linked to BSA, and the observation that phagocytic-depleted cells could interact with the soluble TNP-proteins and function as H-2-restricted targets, appear not to favor the first two proposed mechanisms.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bevan M. J. Interaction antigens detected by cytotoxic T cells with the major histocompatibility complex as modifier. Nature. 1975 Jul 31;256(5516):419–421. doi: 10.1038/256419a0. [DOI] [PubMed] [Google Scholar]
  2. Cline M. J., Lehrer R. I. Phagocytosis by human monocytes. Blood. 1968 Sep;32(3):423–435. [PubMed] [Google Scholar]
  3. Cullen S. E., Freed J. H., Nathenson S. G. Structural and serological properties of murine Ia alloantigens. Transplant Rev. 1976;30:236–270. doi: 10.1111/j.1600-065x.1976.tb00222.x. [DOI] [PubMed] [Google Scholar]
  4. Cullen S. E., Schwartz B. D. An improved method for isolation of H-2 and Ia alloantigens with immunoprecipitation induced by protein A-bearing staphylococci. J Immunol. 1976 Jul;117(1):136–142. [PubMed] [Google Scholar]
  5. Doherty P. C., Blanden R. V., Zinkernagel R. M. Specificity of virus-immune effector T cells for H-2K or H-2D compatible interactions: implications for H-antigen diversity. Transplant Rev. 1976;29:89–124. doi: 10.1111/j.1600-065x.1976.tb00198.x. [DOI] [PubMed] [Google Scholar]
  6. Ellner J. J., Lipsky P. E., Rosenthal A. S. Antigen handling by guinea pig macrophages: further evidence for the sequestration of antigen relevant for activation of primed T lymphocytes. J Immunol. 1977 Jun;118(6):2053–2057. [PubMed] [Google Scholar]
  7. Forman J. On the role of the H-2 histocompatibility complex in determining the specificity of cytotoxic effector cells sensitized against syngeneic trinitrophenyl-modified targets. J Exp Med. 1975 Aug 1;142(2):403–418. doi: 10.1084/jem.142.2.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Forman J., Vitetta E. S., Hart D. A., Klein J. Relationship between trinitrophenyl and H-2 antigens on trinitrophenyl-modified spleen cells. I. H-2 antigens on cells treated with trinitrobenzene sulfonic acid are derivatized. J Immunol. 1977 Mar;118(3):797–802. [PubMed] [Google Scholar]
  9. Forman J., Vitetta E. S., Hart D. A. Relationship between trinitrophenyl and H-2 antigens on trinitrophenyl-modified spleen cells. II. Correlation between derivatization of H-2 antigens with trinitrophenyl and the ability of trinitrophenyl-modified cells to react functionally to the CML assay. J Immunol. 1977 Mar;118(3):803–808. [PubMed] [Google Scholar]
  10. Freedman R. B., Radda G. K. The reaction of 2,4,6-trinitrobenzenesulphonic acid with amino acids, Peptides and proteins. Biochem J. 1968 Jul;108(3):383–391. doi: 10.1042/bj1080383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gardner I. D., Bowern N. A., Blanden R. V. Cell-medicated cytotoxicity against ectromelia virus-infected target cells. III. Role of the H-2 gene complex. Eur J Immunol. 1975 Feb;5(2):122–127. doi: 10.1002/eji.1830050210. [DOI] [PubMed] [Google Scholar]
  12. Gomard E., Duprez V., Henin Y., Levy J. P. H-2 region product as determinant in immune cytolysis of syngeneic tumour cells by anti-MSV T lymphocytes. Nature. 1976 Apr 22;260(5553):707–709. doi: 10.1038/260707a0. [DOI] [PubMed] [Google Scholar]
  13. Gordon R. D., Simpson E., Samelson L. E. In vitro cell-mediated immune responses to the male specific(H-Y) antigen in mice. J Exp Med. 1975 Nov 1;142(5):1108–1120. doi: 10.1084/jem.142.5.1108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Henkart P. A., Schmitt-Verhulst M., Shearer G. M. Specificity of cytotoxic effector cells directed against trinitrobenzene sulfonate-modified syngeneic cells. Failure to recognize cell surface-bound trinitrophenyl dextran. J Exp Med. 1977 Oct 1;146(4):1068–1078. doi: 10.1084/jem.146.4.1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hodes R. J., Handwerger B. S., Terry W. D. Synergy between subpopulations of mouse spleen cells in the in vitro generation of cell-mediated cytotoxicity: evidence for the involvement of a non-T cell. J Exp Med. 1974 Dec 1;140(6):1646–1659. doi: 10.1084/jem.140.6.1646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Koszinowski U., Thomssen R. Target cell-dependent T cell-mediated lysis of vaccinia virus-infected cells. Eur J Immunol. 1975 Apr;5(4):245–251. doi: 10.1002/eji.1830050405. [DOI] [PubMed] [Google Scholar]
  17. Ly I. A., Mishell R. I. Separation of mouse spleen cells by passage through columns of sephadex G-10. J Immunol Methods. 1974 Aug;5(3):239–247. doi: 10.1016/0022-1759(74)90108-2. [DOI] [PubMed] [Google Scholar]
  18. Rehn T. G., Inman J. K., Shearer G. M. Cell-mediated lympholysis to H-2-matched target cells modified with a series of nitrophenyl compounds. J Exp Med. 1976 Oct 1;144(4):1134–1140. doi: 10.1084/jem.144.4.1134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rehn T. G., Shearer G. M., Koren H. S., Inman J. K. Cell-mediated lympholysis of N-(3-nitro-4-hydroxy-5-iodophenylacetyl)-beta-anaylglycylglycyl-modified autologous lymphocytes. Effector cell specificity to modified cell surface components controlled by the H-2K and H-2D serological regions of the murine major histocompatibility complex. J Exp Med. 1976 Jan 1;143(1):127–142. doi: 10.1084/jem.143.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schirrmacher V., Rubin B., Golstein P., Wigzell H., Andersson B. Cytotoxic immune cells with specificity for defined soluble antigens. 3. Separation from helper cells and from antibody-forming cell precursors. Transplant Proc. 1973 Dec;5(4):1447–1450. [PubMed] [Google Scholar]
  21. Schirrmacher V., Rubin B., Pross H., Wigzell H. Cytotoxic immune cells with specificity for defined soluble antigens. IV. Antibody as mediator of specific cytotoxicity. J Exp Med. 1974 Jan 1;139(1):93–107. doi: 10.1084/jem.139.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schmitt-Verhulst A. M., Sachs D. H., Shearer G. M. Cell-mediated lympholysis of trinitrophenyl-modified autologous lymphocytes. Confirmation of genetic control of response to trinitrophenyl-modified H-2 antigens by the use of anti-H-2 and anti-Ia antibodies. J Exp Med. 1976 Jan 1;143(1):211–217. doi: 10.1084/jem.143.1.211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schmitt-Verhulst A. M., Shearer G. M. Bifunctional major histocompatibility-linked genetic regulation of cell-mediated lympholysis to trinitrophenyl-modified autologous lymphocytes. J Exp Med. 1975 Oct 1;142(4):914–927. doi: 10.1084/jem.142.4.914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schmitt-Verhulst A. M., Shearer G. M. Multiple H-2 linked immune response gene control of H-2 D-associated T-cell-mediated lympholysis to trinitrophenyl-modified autologous cells: Ir-like genes mapping to the left of I-A and within the I region. J Exp Med. 1976 Dec 1;144(6):1701–1706. doi: 10.1084/jem.144.6.1701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schrader J. W., Edelman G. M. Joint recognition by cytotoxic T cells of inactivated Sendai virus and products of the major histocompatibility complex. J Exp Med. 1977 Mar 1;145(3):523–539. doi: 10.1084/jem.145.3.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shearer G. M. Cell-mediated cytotoxicity to trinitrophenyl-modified syngeneic lymphocytes. Eur J Immunol. 1974 Aug;4(8):527–533. doi: 10.1002/eji.1830040802. [DOI] [PubMed] [Google Scholar]
  27. Vitetta E. S., Baur S., Uhr J. W. Cell surface immunoglobulin. II. Isolation and characterization of immunoglobulin from mouse splenic lymphocytes. J Exp Med. 1971 Jul 1;134(1):242–264. doi: 10.1084/jem.134.1.242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zinkernagel R. M., Doherty P. C. Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature. 1974 Apr 19;248(5450):701–702. doi: 10.1038/248701a0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES