Investigations on Autologous T-Cells for Adoptive Immunotherapy of Aids

  • Jan van Lunzen
  • Jörn Schmitz
  • Kathrin Dengler
  • Claudia Kuhlmann
  • Herbert Schmitz
  • Manfred Dietrich
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 374)

Abstract

We report on the preclinical results of an immunotherapeutic approach of AIDS mediated by ex vivo propagated CD4+ and CD8+ T-cells. A mean yield of 6.23 x 109 lymphocytes, containing 1.82 x 109 CD4+, 3.23 x 109 CD8+ T-lymphocytes and 8.39 x 106 CD34+ peripheral blood progenitor cells (PBPC) were be obtained by continuous flow cytapheresis (CFC) in 15 asymptomatic HIV infected patients (CD4-count >350/mm3) The CD4/CD8 ratio (mean: 0.53, SD: ±0.15) in the cell concentrates reflected the distribution of the circulating lymphocyte subsets in vivo. Absolute lymphocyte counts decreased at a mean of 404/µl(25%) immediately after CFC but were replaced from the extravascular pool within one hour. Neither the CD4/CD8 ratio nor p24-antigen and neopterin levels did change significantly after cell separation. No alteration of the number of proviral DNA copies (1/103-1/106) could be detected in peripheral T-helper cells by semiquantitative PCR after lymphapheresis. Cells were cryopreserved in liquid nitrogen without substantial loss of viability or function. Ex vivo propagation of T-cells in a strictly autologous manner in the presence of PHA+IL-2 for 14d resulted in a 50-fold expansion rate (140-fold in healthy controls, p<0.001). Viral replication could be controlled but not completely eliminated by cocultivation with autologous CD8+ T-lymphocytes as measured by limiting dilution nested PCR (NPCR). The expanded cells showed the typical phenotype of highly activated memory type T-lymphocytes (CD3+ CD45RO+ CD25+ HLA-DR+). The distribution of CD4+ and CD8+ T cells did not reveal significant changes before and after culture indicating that both subsets were equally expanded. Functionally important membrane or intracellular epitopes which were found to be decreased in HIV infected subjects (CD7, CD55, CD59) before culture were reconstituted after ex vivo propagation of T-cells. The functional importance of the up-regulation of complement regulating epitopes (CD55, CD59) after culture could be proven by a significant inhibition of cytolysis of T-cells in the presence of autologous complement. The majority (75%) of expanded CD8+ T cells stained positive with mAb TIA-1 which is directed to intracellular granules within cytotoxic T-cells. Furthermore, programmed cell death of expanded T-cells could be prevented by cocultivation with fibroblasts which are believed to secrete a cytokine pattern preventing activated T-cells from apoptosis after withdrawal of IL-2 and other stimuli. Our preclinical data support the idea of reconstituting the immune response of HIV infected patients by adoptive cell mediated immunotherapy in order to an improve prevention and treatment of opportunistic diseases. The methods of cell separation, cryopreservation and ex vivo propagation are established and safe. However, before clinical application and evaluation additional studies are necessary.

Keywords

Agarose Citrat Electrophoresis Iodide Capron 

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References

  1. 1.
    Phillips AN, Lee CA, Elford J, Janossy G, Timms A, Bofill M, KemoffPB: Serial CD4 lymphocyte count and development of AIDS. Lancet 337:389–392, 1991PubMedCrossRefGoogle Scholar
  2. 2.
    Barre-Sinoussi F, Chermann JC, Rey F., Nugeyre MT, Chamaret S, Gruest J, Dauguet C, Axler-Blin C, Vezinet-Brun F, Rouzioux C, Rozenbaum W, Montagnier L: Isolation of a T-lymphotropic from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220: 868–871, 1983PubMedCrossRefGoogle Scholar
  3. 3.
    Gallo RC, Salahuddin SZ, Popovic M, Shearer GM, Kaplan M, Haynes BF, Palker TJ, Redfield R, Oleske J, Safai B: Frequent detection and isolation of cytopathic retroviruses (HTLV III) from patients with AIDS and at risk for AIDS. Science 224: 500–503Google Scholar
  4. 4.
    Schnittman SM, Psallidopoulos MC, Lane HC, Thompson L, Baseler M, Massari F, Fox CH, Salzman NP, Fauci AS: The reservoir for HIV-1 in human peripheral blood is a T-cell that maintains expression of CD4. Science 245: 305–308, 1989PubMedCrossRefGoogle Scholar
  5. 5.
    Miedema F, Petit AJC, Terpstra FG, Eeftinck Schattenkerk JKM, de Wolf F, Al BJM, Roos M, Lange JMA, Danner SA, Goudsmit J, Schellenkens PTA. Immunological abnormalities in HIV-infected asymptomatic homosexual men J Clin. Invest. 82: 1908–1914, 1988PubMedCrossRefGoogle Scholar
  6. 6.
    Schnittman SM, Lane HC, Greenhouse J, Justement JS, Baseler M, Fauci AS: Preferential infection of CD4+ memory T cells by human immunodeficiency virus type 1: evidence for a role in the selective T-cell functional defects observed in infected individuals. Proc Natl Acad Sci 97: 6058–62, 1990CrossRefGoogle Scholar
  7. 7.
    Ameisen JC, Capron A: Cell dysfunction and depletion in AIDS: The programmed cell death hypothesis. Immunol Today 12: 102–105, 1991PubMedCrossRefGoogle Scholar
  8. 8.
    Groux H, Torpier G, Monte D, Mouton Y, Capron A, Ameisen JC: Activation-induced Death by apoptosis in CD4+ T cells from human immunodeficiency virus-infected asymptomatic individuals. J Exp Med 175: 331–340, 1992PubMedCrossRefGoogle Scholar
  9. 9.
    Tyler DS, Stanley SD, Nastala CA, Austin AA, Bartlett JA, Stine KC, Lyerly HK, Bolognesi DP, Weinhold KJ: Allterations in antibody-dependant cellular cytotoxicity during the course of HIV-1 infection. J Immunol 144: 3375–84, 1990PubMedGoogle Scholar
  10. 10.
    Walker BD, Flexner C, Birch-Limberger K, Fisher L, Paradis TJ, Aldovini A, Young R, Moss B, Schooley RT: Long-term culture and fine specifity of human cytotoxic T-lymphocyte clones reactive with human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA, 86: 9514–9518, 1989PubMedCrossRefGoogle Scholar
  11. 11.
    Clerici M, Shearer GM: A TH1–TH2 switch is a critical step in the etiology of HIV infection. Immunology Today, 14: 107–111, 1993PubMedCrossRefGoogle Scholar
  12. 12.
    Scott-Algara D, Vuillier F, Marasescu M, de Saint Martin J, Dighiero G: Serum levels of II-2, Il-1 alpha, TNF-alpha, and soluble receptor of Il-2 in HIV-1 infected patients. AIDS Res. Hum. Retroviruses, 7: 381–386,1991PubMedCrossRefGoogle Scholar
  13. 13.
    Gluckman JC, Klatzmann D, Cavaille-Coll M, Brisson E, Messiah A, LacHIVer D, Rozenbaum W: Is there correlation of T-cell proliferative functions and surface marker phenotypes in patients with AIDS or LAS? Clin. Exp. Immunology, 60: 8–16, 1985Google Scholar
  14. 14.
    Clerici M, Stocks NI, Zajac RA, Boswell RN, Lucey DR, Via CS, Shearer GM: Detection of three distinct patterns of T-helper cell dysfunction in asymptomatic, human immunodeficiency virus-seropositive patients. J. Clin. Invest. 84: 1892–1899, 1989PubMedCrossRefGoogle Scholar
  15. 15.
    Hufert FT, v. Laer MD, Schramm C, Tarnok A, Schmitz H: Detection of HIV-1 DNA in different subsets of human peripheral blood mononuclear cells using the polymerase chain reaction. Arch Virol 106: 341–345,1989PubMedCrossRefGoogle Scholar
  16. 16.
    Schnittman SM, Greenhouse JJ, Psallidopoulos MC, Baseler M, Salzman NP, Fauci AS, Lane HC: Increasing viral burden in CD4+ T-cells from patients with HIV-infection reflects rapidly progressive immunosuppression and clinical disease. Ann. Intern. Med. 113: 438–443, 1990PubMedGoogle Scholar
  17. 17.
    Hufert FT, v. Laer D, Fenner TE, Schwander S, Kern P, Schmitz H: Progression of HIV-1 infection. Monitoring of HIV-1 DNA in peripheral blood mononuclear cells by PCR. Arch Virol 120: 233–240, 1991PubMedCrossRefGoogle Scholar
  18. 18.
    Schmitz J, van Lunzen J, Tenner-Racz K, Großschupff G, Racz P, Schmitz H, Dietrich M, Hufert FT: Follicular dendritic cells retain HIV-1 on their plasma membrane but are not productively infected in asymptomatic patients with follicular hyperplasia. J. Immunol. 153: 1352–1359, 1994PubMedGoogle Scholar
  19. 19.
    Embretson J, Zupancic M, Ribas JL, Burke A, Racz P, Tenner-Racz K, Haase AT: Massive covert infection of helper T-lymphocytes and macrophages by HIV during the incubation period of AIDS. Nature 362: 359–362,1993PubMedCrossRefGoogle Scholar
  20. 20.
    Pantaleo G, Graziosi C, Demarest JF, Butini L, Montroni M, Fox CH, Orenstein JM, Kotler DP, Fauci AS: HIV infection is active and progressive in lymphoid tissue during the clinical latent stage of disease. Nature 362: 355–359, 1993PubMedCrossRefGoogle Scholar
  21. 21.
    Walker CM, Moody DJ, Stites DP, Levy JA: CD8+ lymphocytes can control HIV infection in vitro by supressing virus replication. Science 234: 1563–1566, 1986PubMedCrossRefGoogle Scholar
  22. 22.
    Whiteside TL, Elder EM, Moody D, Armstrong J, Ho M, Rinaldo C, Huang X, Torpey D, Gupta P, McMahon D, Okarma T, Herberman RB: Generation and characterization of ex vivo propagated autologous CD8+ cells used for adoptive immunotherapy of patients infected with human immunodeficiency virus. Blood 81: 2085–2092, 1993PubMedGoogle Scholar
  23. 23.
    Ho M, Armstrong J, MacMahon D, Pazin G, Huang X, Rinaldo C, Whiteside TL, Tripoli C, Levine G, Moody D, Okarma T, Elder E, Gupta P, Tauxe N, Torpey D, Herberman RB: A phase 1 study of adoptive transfer of autologous CD8+ T lymphocytes in patients with AIDS-related complex or AIDS. Blood 81: 2093–2101, 1993PubMedGoogle Scholar
  24. 24.
    Klimas N, Patarca R, Walling J, Garcia R, Mayer V, Moody D, Okarma T, Fletcher MA: Clinical and immunological changes in AIDS patients following adoptive therapy with activated autologous CD8 T cells and interleukin-2 infusion. AIDS 8: 1073–1081, 1994PubMedCrossRefGoogle Scholar
  25. 25.
    Tenner-Racz K, Racz P, Thome C, Meyer CG, Anderson PJ, Schlossman SF, Letvin NL: Cytotoxic effector cell granules recognized by the monoclonal antibody TIA-1 are prsent in CD8+ lymphocytes in lymph nodes of HIV-1 infected patients. Am J Pathol 124: 1750–1758, 1993Google Scholar
  26. 26.
    Wolfs TFW, Zwart G, Bakker M, Valk M, Kuiken CL, Goudsmit J: Naturally occorring mutations within HIV-1 V3 genomic RNA lead to antigenic variation dependant on a single amino acid substitution. Virology 185: 195–205, 1991PubMedCrossRefGoogle Scholar
  27. 27.
    Folks TM, Clouse KA, Justement J, Rabson A, Duh E, Kehrl JA, Fauci AS: Tumor necrosis factor alpha induces expression of HIV in a chronically infected T cell clone. Proc Natl Acad Sci (USA) 86: 2365–2368, 1989CrossRefGoogle Scholar
  28. 28.
    van Lunzen J, Schmitz J, Dengler K, Schmidt 1, Schmitz H, Dietrich M: Recovery of T-lymphocytes for adoptive immunotherapy by lympapheresis of HIV-infected patients without alterations of virological, immunological or clinical parameters. Br J Hematol 88: 46–51, 1994CrossRefGoogle Scholar
  29. 29.
    Westermann J, Pabst R: Lymphocyte subsets in the blood: A diagnostic window of the lymphoid system? Immunology Today 11: 406–410, 1990PubMedCrossRefGoogle Scholar
  30. 30.
    Dreger P, Haferlach T, Eckstein V, Jacobs S, Suttorp M, Löffler H, Müller-Ruchholtz W, Schmitz N: G-CSF mobilized peripheral blood progenitor cells for allogeneic transplantation: safety, kinetics of mobilization, and composition of the graft. Br J Haematol 87: 609–613, 1994PubMedCrossRefGoogle Scholar
  31. 31.
    Akbar AN, Borthwick N, Salmon M, Gombert W, Bofill M, Shamsadeen N, Pilling D, Pett S, Grundy JE, Janossy G: The significance of low bc1–2 expression by CD45RO T cells in normal individuals and patients with acute viral infections. The role of apoptosis in T cell memory. J. Exp. Med. 178: 427–438, 1993PubMedCrossRefGoogle Scholar
  32. 32.
    Scott S, Pandolfi F, Kurnick JT: Fibroblasts mediate T cell survival: A proposed mechanism for retention of primed T cells. J. Exp. Med. 172: 1873–1876, 1990PubMedCrossRefGoogle Scholar
  33. 33.
    Pandolfi F, Oliva A, Sacco G, Polidori V, Liberatore D, Mezzaroma I, Giovanetti A, Kurnick JT, Aiuti F: Fibroblast-derived factors preserve viability in vitro of mononuclear cells isolated from subjects with HIV-1 infection. AIDS 7: 323–330, 1993PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • Jan van Lunzen
    • 1
  • Jörn Schmitz
    • 1
  • Kathrin Dengler
    • 1
  • Claudia Kuhlmann
    • 1
  • Herbert Schmitz
    • 1
  • Manfred Dietrich
    • 1
  1. 1.Clinical Medicine Section and Department of VirologyBernhard-Nocht-Institute for Tropical MedicineHamburgGermany

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