Cancer Immunol Immunother (1992) 35: 83- 91
ancer mmunology mmunomerapy © Springer-Verlag1992
Induction of interleukin-2 receptor by tumor necrosis factor on cultured ovarian tumor-associated lymphocytes Constantin G. Ioannides 1, Bryan Fisk 1, Barbara Tomasovic 1, Raj Pandita 2, Bharat B. Aggarwal2, and Ralph S. Freedman 1 1Department of Gynecology, and 2 Cytokine Research Laboratory, Department of Clinical Immunology and Biological Therapy, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77 030, USA Received 17 May 1991/Accepted 4 February 1992
Summary. We have recently reported that autologous tumor-specific cytotoxic T lymphocyte (CTL) lines and clones can be developed from lymphocytes infiltrating ovarian malignant ascites (TAL). In this study, we investigated the biological effects of tumor necrosis factor c~ (TNFo9 in the induction, expansion, long-term proliferation and lyric function of CD8 + TAL. TNF~ up-regulated the IL-2 receptor (IL-2R) c~ chain (Tac antigen) on the surface of CD3+ CD8 + CD4- TAL, enhanced the proliferation of antologous tumor-specific CTL, and potentiated their lyric function in long-term cultures. Furthermore, in the induction and expansion phase of CD8 + TAL, the presence of TNF(x was associated with a selective increase in CD8 + IL-2R+ (Tac +) cells, and subsequent decrease in CD4 + IL-2R+ (Tac+) cells. These results suggest that the observed facilitation of the outgrowth of CD8 + cells in TAL cultures may be due, at least in part, to the up-regulation of IL-2R, and indicate the usefulness of TNFc~ in the analysis of signalling in autologous tumor-reactive CTL. Key words: TNFc~ - CD8 + CTL - IL-2R - T1L/TAL
Introduction Lymphocytes infiltrating human tumors (TIL) have been the focus of intense investigation in recent years because of the favorable clinical responses observed when TIL cultured in vitro were administered in vivo to cancer patients [2, 3, 12, 13, 20, 21, 25, 28, 29]. TIL isolated from tumors consist of a mixture of lymphoid cell populations, of which CD3 + CD8 + cells have been demonstrated to mediate preferential killing of autologous tumor in vitro [3, 12]. Culture
Offprint requests to: C. G. Ioannides, M. D. Anderson Cancer Center, Department of Gynecology, Box 67, 1515 Holcombe Boulevard, Houston, Texas 77 030, USA
of TIL with interleukin-2 (IL-2) in the initial presence of autologous tumors has led to enrichment in CD3 + CD8 + cells in certain instances in particular cancers such as melanoma [3, 12, 13]. We focussed our investigation on lymphocytes infiltrating ovarian malignant ascites (TAL) because ovarian cancer frequently expands as an ascitic tumor. We have isolated CTL-TAL lines and clones that mediate preferential killing of autologous tumors [10]. In a large case study (16 TAL samples) we observed that certain TAL expand preferentially as CD3+ CD4+ CD8- lines, whereas others expand as CD3 + CD4- CD8 + lines in the same culture conditions. However, CD8 + CTL have been isolated from predominantly CD3 + CD4+ cultures, indicating that such cells are present and functional [11]. These findings suggested that factors additional to antigen (tumor) and growth-promoting lymphokines (IL-2) are involved in the preferential expansion of CD3 + CD8 + TAL. To date, little is known about the interactions of tumor necrosis factor ~ (TNF~) with other lymphokines in the development of autologous tumor-specific CTL from ovarian TAL. Therefore, to delineate the functional significance of the synergy of IL-2 and TNFo~ in inducing preferential expansion and proliferation of CD3+ CD8 + TAL we focus in this article on the investigation of the expression of the interleukin-2 receptor a chain (p55 Tac antigen) [23] on CD3+ cells cultured with IL-2 plus TNFc~. IL-2-dependent cellular proliferation of activated T lymphocytes is mediated by the high-affinity IL-2 receptors (IL-2R) [6, 18]. Both affinity classes (high and low) of IL-2R contain a 55 kDa glycoprotein (Tac), which upregulates on T cells after antigen/mitogen stimulation. Anti-Tac monoclonal antibodies can block IL-2-dependent cell proliferation [5, 14] and are currently used to recognize IL-2R+ cells. However, an additional protein of 75 kDa (p75, IL-2R ~3chain), when associated with p55, converts the low-affinity to the high-affinity IL-2R, which transduces a proliferative signal to T cells. This study was considered important because the long-term expansion and proliferation of tumor-specific TIL depends on the ability
84 of IL-2 to promote proliferation of effector cells, which comprise activated C T L [27]. W e found that addition of TNFcx to T A L cultures at both the induction and expansion phase increased the proportion of CD8 ÷ cells in the cultures and m a i n l y the percentage of CD8 + I L - 2 R + (Tac +) cells b u t not of CD4 + IL-2R + (Tac +) cells. Interestingly, the increase in the CD8 + cells in T A L cultures supplemented with IL-2 and TNFcz demonstrated an inverse relationship with the concentrations of IL-2. Furthermore, TNFoc was f o u n d to induce the expression of Tac antigen directly in a CD8 ÷ C T L - T A L line, which was followed by increased C T L proliferation and stable lytic function over time compared with the same C T L line culture in IL-2 alone. These results demonstrate that TNFcz m a y be important for expansion of ovarian T A L used for i m m u n o t h e r a p y trials.
Materials and methods TAL. TAL were isolated from ovarian malignant ascites by centrifugation over 75%- 100% Ficoll/Hypaque gradients as previousy described [10]. Ovarian malignant ascites were collected from patients in the Gynecology Clinic under institutionally (M. D. Anderson) approved protocols. TAL were separated 85%-95% free of tumor cells. In most TAL samples tumor samples represented less than 10% of the total amount cells at the initiation of the cultures.
Lymphokines. Recombinant IL-2 (IL-2) and recombinant TNF(z (TNFc~) were generous gifts from Cetus Corporation (Emeryville, Calif.). The specific activities of IL-2 and TNFcc are 3.87
ancer mmunology mmunomerapy © Springer-Verlag1992
Induction of interleukin-2 receptor by tumor necrosis factor on cultured ovarian tumor-associated lymphocytes Constantin G. Ioannides 1, Bryan Fisk 1, Barbara Tomasovic 1, Raj Pandita 2, Bharat B. Aggarwal2, and Ralph S. Freedman 1 1Department of Gynecology, and 2 Cytokine Research Laboratory, Department of Clinical Immunology and Biological Therapy, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77 030, USA Received 17 May 1991/Accepted 4 February 1992
Summary. We have recently reported that autologous tumor-specific cytotoxic T lymphocyte (CTL) lines and clones can be developed from lymphocytes infiltrating ovarian malignant ascites (TAL). In this study, we investigated the biological effects of tumor necrosis factor c~ (TNFo9 in the induction, expansion, long-term proliferation and lyric function of CD8 + TAL. TNF~ up-regulated the IL-2 receptor (IL-2R) c~ chain (Tac antigen) on the surface of CD3+ CD8 + CD4- TAL, enhanced the proliferation of antologous tumor-specific CTL, and potentiated their lyric function in long-term cultures. Furthermore, in the induction and expansion phase of CD8 + TAL, the presence of TNF(x was associated with a selective increase in CD8 + IL-2R+ (Tac +) cells, and subsequent decrease in CD4 + IL-2R+ (Tac+) cells. These results suggest that the observed facilitation of the outgrowth of CD8 + cells in TAL cultures may be due, at least in part, to the up-regulation of IL-2R, and indicate the usefulness of TNFc~ in the analysis of signalling in autologous tumor-reactive CTL. Key words: TNFc~ - CD8 + CTL - IL-2R - T1L/TAL
Introduction Lymphocytes infiltrating human tumors (TIL) have been the focus of intense investigation in recent years because of the favorable clinical responses observed when TIL cultured in vitro were administered in vivo to cancer patients [2, 3, 12, 13, 20, 21, 25, 28, 29]. TIL isolated from tumors consist of a mixture of lymphoid cell populations, of which CD3 + CD8 + cells have been demonstrated to mediate preferential killing of autologous tumor in vitro [3, 12]. Culture
Offprint requests to: C. G. Ioannides, M. D. Anderson Cancer Center, Department of Gynecology, Box 67, 1515 Holcombe Boulevard, Houston, Texas 77 030, USA
of TIL with interleukin-2 (IL-2) in the initial presence of autologous tumors has led to enrichment in CD3 + CD8 + cells in certain instances in particular cancers such as melanoma [3, 12, 13]. We focussed our investigation on lymphocytes infiltrating ovarian malignant ascites (TAL) because ovarian cancer frequently expands as an ascitic tumor. We have isolated CTL-TAL lines and clones that mediate preferential killing of autologous tumors [10]. In a large case study (16 TAL samples) we observed that certain TAL expand preferentially as CD3+ CD4+ CD8- lines, whereas others expand as CD3 + CD4- CD8 + lines in the same culture conditions. However, CD8 + CTL have been isolated from predominantly CD3 + CD4+ cultures, indicating that such cells are present and functional [11]. These findings suggested that factors additional to antigen (tumor) and growth-promoting lymphokines (IL-2) are involved in the preferential expansion of CD3 + CD8 + TAL. To date, little is known about the interactions of tumor necrosis factor ~ (TNF~) with other lymphokines in the development of autologous tumor-specific CTL from ovarian TAL. Therefore, to delineate the functional significance of the synergy of IL-2 and TNFo~ in inducing preferential expansion and proliferation of CD3+ CD8 + TAL we focus in this article on the investigation of the expression of the interleukin-2 receptor a chain (p55 Tac antigen) [23] on CD3+ cells cultured with IL-2 plus TNFc~. IL-2-dependent cellular proliferation of activated T lymphocytes is mediated by the high-affinity IL-2 receptors (IL-2R) [6, 18]. Both affinity classes (high and low) of IL-2R contain a 55 kDa glycoprotein (Tac), which upregulates on T cells after antigen/mitogen stimulation. Anti-Tac monoclonal antibodies can block IL-2-dependent cell proliferation [5, 14] and are currently used to recognize IL-2R+ cells. However, an additional protein of 75 kDa (p75, IL-2R ~3chain), when associated with p55, converts the low-affinity to the high-affinity IL-2R, which transduces a proliferative signal to T cells. This study was considered important because the long-term expansion and proliferation of tumor-specific TIL depends on the ability
84 of IL-2 to promote proliferation of effector cells, which comprise activated C T L [27]. W e found that addition of TNFcx to T A L cultures at both the induction and expansion phase increased the proportion of CD8 ÷ cells in the cultures and m a i n l y the percentage of CD8 + I L - 2 R + (Tac +) cells b u t not of CD4 + IL-2R + (Tac +) cells. Interestingly, the increase in the CD8 + cells in T A L cultures supplemented with IL-2 and TNFcz demonstrated an inverse relationship with the concentrations of IL-2. Furthermore, TNFoc was f o u n d to induce the expression of Tac antigen directly in a CD8 ÷ C T L - T A L line, which was followed by increased C T L proliferation and stable lytic function over time compared with the same C T L line culture in IL-2 alone. These results demonstrate that TNFcz m a y be important for expansion of ovarian T A L used for i m m u n o t h e r a p y trials.
Materials and methods TAL. TAL were isolated from ovarian malignant ascites by centrifugation over 75%- 100% Ficoll/Hypaque gradients as previousy described [10]. Ovarian malignant ascites were collected from patients in the Gynecology Clinic under institutionally (M. D. Anderson) approved protocols. TAL were separated 85%-95% free of tumor cells. In most TAL samples tumor samples represented less than 10% of the total amount cells at the initiation of the cultures.
Lymphokines. Recombinant IL-2 (IL-2) and recombinant TNF(z (TNFc~) were generous gifts from Cetus Corporation (Emeryville, Calif.). The specific activities of IL-2 and TNFcc are 3.87
