"Phytohemagglutinin-Activated Autochthonous Lymphocytes for Systemic Immunotherapy of Human Neoplasms".
John H. Frenster
Department of Medicine
Stanford University School of Medicine
Stanford, California 94305
Patients with disseminated neoplasms often display high concentrations of circulating tumor antigens (1,2) or antigen fragments (3) in their peripheral blood. These tumor antigens are apparently shed from the large number of neoplastic cells in these patients (1, 4), and although useful for diagnostic purposes (1, 5), such tumor antigens may be deleterious to the immune reactions mounted by the patient against the neoplastic cells (6-8). Tissue tumor antigens have been shown at high concentrations to inhibit the activity of immune lymphocytes (9-12) and are thought to play a role in sensitizing antigen-reactive lymphocytes to the action of macrophages (13, 14). As a consequence, any attempt to develop an immunotherapeutic approach toward disseminated human neoplasms must devise methods for overcoming such lymphocyte-blocking or lymphocyte-sensitizing activities of free tumor antigen.
Studies of lymphocyte activity in-vivo in intreated patients with Hodgkin's Disease have suggested approaches by which these deleterious effects of free tumor antigens on immune lymphocytes may be overcome (15).
The numbers of lymphocytes present within the involved lymph nodes of untreated patients with Hodgkin's Disease correlate directly with a favorable prognosis of such patients after radiotherapy (16). Such lymphocytes are often observed to be tightly apposed to the neoplastic cells (15), a condition found in-vitro during lymphocyte-mediated destruction of target cells (17). The in-vivo correlate of such tight apposition by lymphocytes is a corresponding severe cytotoxicity within the neoplastic cells (15). The lymphocytes closest to the neoplastic cells have been shown by ultrastructural molecular probe techniques to be the most active (18), accompanied by a clustering of macrophages close to the neoplastic cells (19). The majority of lymphocytes apposed to neoplastic cells in Hodgkin's Disease are positive for acid phosphatase activity in their Golgi zone (20), a condition characteristic of thymus-dependent T-lymphocytes (21). Further direct immunologic study has revealed these apposed lymphocytes in Hodgkin's Disease to be T-lymphocytes and the neoplastic Reed-Sternberg cells to be immunoglobulin-positive non-T cells (22).
Electron microscopic studies have revealed frequent phagocytosis in-vivo of such T-lymphocytes by active macrophages within Hodgkin's Disease lymph nodes (13), and recent studies have suggested that such macrophage attack is mediated after uptake by lymphocytes of free tumor antigen (13, 14). Although macrophages are also active against tumor cells in-vivo (13), stimulation of macrophages by Corynebacterium Parvum may suppress lymphocyte-mediated tumor immunity (14) and may require immunotherapy methods more selective for lymphocytes than for macrophages or tumor antigens per se (Fig. 1):
T-lymphocytes are important in mediating cellular immune reactions against a variety of systemic neoplasms in-vivo (23). Such T-lymphocytes can be activated by tumor antigens at low concentration (10) but appear to be inhibited by the same tumor antigens at higher concentrations (10). It is significant in this regard that T-lymphocytes can additionally be activated by the plant mitogen Phytohemagglutinin (PHA), which has a marked selectivity for T-lymphocytes as compared to B-lymphocytes or other mononuclear cells (24). In addition, the plasma membrane binding sites for PHA appear to be distinct from those for tumor antigens on T-lymphocytes (25) so that, even when T-lymphocytes have been inhibited by high concentrations of tumor antigens, these lymphocytes can still be activated by PHA to express their cytotoxicity against tumor cells (26).
In addition, although macrophages are important in mediating the response of T-lymphocytes to defined antigens (27), macrophages are less important in the response of T-lymphocytes to PHA (28). Because macrophages can apparently misread antigen-bearing T-lymphocytes as tumor cells (13, 14), it is desirable to employ conditions of lymphocyte activation in which the role of macrophage interaction with T-lymphocytes is minimized, a situation characteristic of PHA activation (28).
Electron microscopic studies of the localization of 3H-labeled purified octamer of PHA have revealed that PHA first attaches to the plasma membranes of both lymphocytes and monocytes during incubation with buffy coat fractions from the peripheral blood of normal human subjects (29). Within 15 min. of such incubation, a significant amount of PHA is found localized within the nucleus of the lymphocytes but is confined to the cytoplasm of the monocytes (29). After 24 hr. of such incubation, the majority of the PHA found bound to the lymphocytes is localized in the cell nucleus of the lymphocytes, perhaps accounting for the nuclear activation and mitogenic response observed within such lymphocytes (30). The lack of such nuclear localization in the incubated monocytes may account for the lack of a mitogenic response in these cells, even though a regular conversion of incubated monocytes to macrophages is observed during PHA incubation (29). Because of these dual effects on lymphocytes and monocytes during incubation with PHA, it is probably desirable to separate the nonadherent T-lymphocytes from the adherent monocytes and macrophages (31) before incubation with PHA to avoid the inclusion of activated monocytes and macrophages within therapeutic infusions of T-lymphocytes during immunotherapy trials (32).
It has been possible to infuse intravenously up to 109 activated autochthonous peripheral blood lymphocytes into patients after 48-hr. incubations in-vitro of buffy coat fractions with purified octamer form of PHA (32). Such activated lymphocytes have been washed free of the externally adherent highly purified octamer of PHA utilized for such in-vitro activation (33) and have been well tolerated by these patients, with only mild febrile reactions, which probably represent responses to the distintegrating granulocytes and macrophages in these cell infusions.
Five patients with widespread disseminated neoplasms have received 109 lymphocytes per infusion for 5 consecutive days per month for at least 2 consecutive months without significant hematologic, allergic, or neoplastic complications (34). Patients were hospitalized on the General Clinical Research Center Service at Stanford University Hospital for only 7 days each month and were able to return to their jobs or school immediately after discharge.
Each of the study patients had multile pulmonary metastases evident on conventional chest radiography (Table 1). Each patient had failed to respond to prior chemotherapy, which had been discontinued at least 3 months before the immunotherapy trial. The response of each patient was assessed by serial chest radiography during the reinfusion of activated autochthonous lymphocytes and for 3 months thereafter. Three of five study patients displayed a decrease in the size of pulmonary metastases of more than 50% in the product of two diameters of each nodule for more than 2 months duration, with no other form of concurrent therapy (Table 1).
| Patient | Histology | Visceral Metastases | Immunotherapy Response |
| O.A. | Malignant Melanoma | Pulmonary, Hepatic | Pulmonary Regression |
| P.D. | Malignant Melanoma | Pulmonary, Hepatic | None |
| R.F. | Ewing's Sarcoma | Pulmonary | Pulmonary Regression |
| R.G. | Embryonal Cell Carcinoma | Pulmonary, Hepatic | Pulmonary Regression |
| P.C. | Teratocarcinoma | Pulmonary | None |
It is noteworthy that two patients who had concurrent liver metastases displayed progression of these liver metastases while their pulmonary metastases were regressing, which suggests that the number of lymphocytes infused was insufficient to deal with the pulmonary and hepatic metastases simultaneously (34).
Current Immunotherapy Protocols:
Because of such numerical insufficiency, larger numbers of lymphocytes are now being collected by means of the IBM blood cell separator, which yields up to 1012 lymphocytes per 4-hr. collection period (35). In addition, macrophages, monocytes, and B lymphocytes are being removed from the buffy coat preparations prior to PHA activation (36) to reduce their interference with T-lymphocytes that attack target neoplastic cells in-vivo (13, 14).
Finally, continuous-flow plasmapheresis by means of the IBM blood cell separator (35) is being utilized to reduce the titer of free circulating tumor antigens in the peripheral blood of study patients to test the possibility of reducing the inhibitory effects of such tumor antigens (10) on T-lymphocytes that attack neoplastic cells in-vivo (15, 37, 38).
These recent and current clinical protocols that utilize larger numbers of T-lymphocytes freed of contaminating macrophages, monocytes, B-lymphocytes, and circulating tumor antigens promise to provide more critical tests of the potential of activated T-lymphocytes for the systemic immunotherapy of disseminated human neoplasms.
Supported in part by Research Grants CA-10174 and CA-13524 from the National Cancer Institute, by Research Grant IC-45 from the American Cancer Society, and by a Research Scholar Award from the Leukemia Society.
1. Thomson DMP, Krupey J, Freedman SO, and Gold P, 1969, "The radio-immunoassay of circulating carcinoembryonic antigen of the human digestive system", Proc. Natl. Acad. Sci. USA 64: 161-167.
2. Currie GA, and Basham C, 1972, "Serum-mediated inhibition of the immunological reactions of the patient to his own tumor: possible role for circulating antigen", Brit. J. Cancer 26: 427-438.
3. Currie GA, 1973, "The role of circulating antigen as an inhibitor of tumor immunity in man", Brit. J. Cancer 28 (Suppl. 1): 153-161.
4. Currie GA, and Alexander P, 1974, "Spontaneous shedding of TSTA by viable sarcoma cells: its possible role in facilitating metastatic spread", Brit. J. Cancer 29: 72-75.
5. Gold P, and Freedman SO, 1975, "Tests for carcinoembryonic antigen. Role in diagnosis and management of cancer", J. Am. Med. Assn. 234: 190-192.
6. Hellstrom I, Sjogren HO, Warner GA, and Hellstrom KE, 1971, "Blocking of cell-mediated tumor immunity by sera from patients with growing neoplasms", Int. J. Cancer 7: 226-237.
7. Baldwin RW, Embleton MJ, Price MR, and Robins A, 1974, "Immunity in the tumor-bearing host and its modification by serum factors", Cancer 34: 1452-1460.
8. Hellstrom I, and Hellstrom KE, 1974, "Cell-mediated immune reactions to tumor antigens with particular emphasis on immunity to human neoplasms", Cancer 34: 1461-1468.
9. Sjogren HO, Hellstrom I, Bansal SC, and Hellstrom KE, 1971, "Suggestive evidence that the blocking antibodies of tumor-bearing individuals may be antigen-antibody complexes", Proc. Natl. Acad. Sci. USA 68: 1372-1375.
10. Mavligit GM, Ambus U, Gutterman JU, Hersh EM, and McBride CM, 1973, "Antigen solubilized from human solid tumors: lymphocyte stimulation and delayed hypersensitivity", Nature (New Biol.) 243: 188-190.
11. Blair PB, Lane MA, and Yagi MJ, 1975, "Blocking of spleen cell activity against target mammary tumor cells by viral antigens", J. Immunol. 115: 190-194.
12. Chism S, Burton R, and Warner NL, 1975, "Lymphocyte activation in-vitro to onco-fetal antigens", Nature 257: 594-596.
13. Masek MA, Rhoades DJ, and Frenster JH, 1973, "In-vivo macrophage interactions with lymphocytes in Hodgkin's Disease", Proc. Am. Assoc. Cancer Res. 14: 8-9.
14. Kirchner H, Glaser M, and Herberman RB, 1975, "Suppression of cell-mediated tumor immunity by Corynebacterium Parvum", Nature 257: 396-398.
15. Archibald RB, and Frenster JH, 1973, "Quantitative ultrastructural analysis of in-vivo lymphocyte Reed-Sternberg cell interactions in Hodgkin's Disease", Natl. Cancer Inst. Monogr. 36: 239-245.
16. Keller AR, Kaplan HS, Lukes RJ, and Rappaport H, 1968, "Correlation of histopathology with other prognostic indicators in Hodgkin's Disease", Cancer 22: 487-499.
17. Biberfeld P, and Johansson A, 1975, "Contact areas of cytotoxic lymphocytes and target cells", Exp. Cell Res. 94: 79-87.
18. Masek MA, Landrum S, Wilson LS, and Frenster JH, 1974, "Spatial patterns of lymphocyte activity within Hodgkin's Disease lymph nodes", Proc. Am. Assoc. Cancer Res. 15: 2-3.
19. Frenster JH, 1975, "Analysis of queueing and renewal systems in Hodgkin's Disease", Proc. Am. Assoc. Cancer Res. 16: 223-224.
20. Rowan RA, Masek MA, Thompson JM, and Frenster JH, 1975, "Electron microscopic localization of acid phosphatase activity within Hodgkin's Disease lymph nodes", Proc. Am. Assoc. Cancer Res. 16: 10-11.
21. Catovsky D, 1975, "T-cell origin of acid-phosphatase-positive lymphoblasts", lancet 2: 327-328.
22. Kadin ME, Newcom SR, Gold SB, and Stites DP, 1974, "Origin of Hodgkin's cell", Lancet 2: 167-168.
23. Weston BJ, Carter RL, Easty GC, Connell DI, and Davies AJS, 1974, "The growth and metastasis of an allografted lymphoma in normal, deprived, and reconstituted mice", Int. J. Cancer 14: 176-185.
24. Ozato K, Ebert JD, and Adler WH, 1975, "Pretreatment of murine thymocytes by PHA inhibits the binding of 3H-concanavalin-A" J. Immunol. 115: 339-344.
25. Henkart PA, and Fisher RI, 1975, "Characterization of the lymphocyte surface receptors for Con-A and PHA", J. Immunol. 114: 710-714.
26. Bevan MJ, and Cohn M, 1975, "Cytotoxic effects of antigen- and mitogen-induced T-cells on various targets", 114: 559-565.
27. Rosenthal AS, Blake JT, and Lipsky PE, 1975, "Inhibition of macrophage-lymphocyte interaction by cytochalasin-B during antigen recognition by T-lymphocytes", J. Immunol. 115: 1135-1139.
28. Wedner HJ, Dankner R, and Parker CW, 1975, "Cyclic GMP and lectin-induced lymphocyte activation", J. Immunol. 115: 1682-1687.
29. Stanley DA, Frenster JH, and Rigas DA, 1971, "Localization of 3H-PHA within human lymphocytes and monocytes", in: Proceedings of the Fourth Leukocyte Culture Conference", McIntyre OR, ed., pp. 1-11, Appleton-Century-Crofts, New York.
30. Sasaki MS, and Norman A, 1966, "Proliferation of human lymphocytes in culture", Nature 210: 913-914.
31. Ben-Sasson SZ, Paul WE, Shevach EM, and Green I, 1975, "In-vitro selection and extended culture of antigen-specific T-lymphocytes", J. Immunol. 115: 1723-1731.
32. Frenster JH, and Rogoway WM, 1968, " In-vitro activation and re-infusion of autologous human lymphocytes", Lancet 2: 979-980.
33. Rigas DA, and Head C, 1968, "the dissociation of PHA of Phaseolus Vulgaris by 8.0 M urea and the separation of the mitogenic from the erythro-agglutinating activity", Biochem. Biophys. Res. Comm. 34: 633-639.
34. Frenster JH, and Rogoway WM, 1970, "Immunotherapy of human neoplasms with autologous lymphocytes activated in-vitro", in: Proceedings of the Fifth Leukocyte Culture Conference, Harris JE, ed., pp. 359-373, Academic Press, Inc. New York.
35. Lowenthal RM, Buskard NA, Goldman JM, Spiers ASD, Bergier N, Graubner M, and Galton DAG, 1975, "Intensive leukopheresis as initial therapy for chronic granulocytic leukemia", Blood 46: 835-844.
36. Peter HH, Pavie-Fisher J, Fridman WH, Aubert C, Cesarini JP, Roubin R, and Kourilsky FM, 1975, "Cell-mediated cytotoxicity in-vitro of human lymphocytes against a tissue culture melanoma cell line (IGR3)", J. Immunol. 115: 539-548.
37. Cheema RA, and Hersh EM, 1972, "Local tumor immunotherapy with in-vitro activated autochthonous lymphocytes", Cancer 29: 982-986.
38. Frenster JH, 1965, "Analysis of queueing and renewal within human systems", Nature 207: 1139-1140.
0. Electron Microscopy of Human Lymphocytes before and after Activation by PHA (Busch H, 1974).
1. Childs R, et al, "Regression of Metastatic Renal-Cell Carcinoma after Non-Myeloablative Allogeneic Peripheral-Blood Stem-Cell Transplantation", New Eng. J. Med. 343: 750-758 (Sept. 14, 2000).
2. Hellstrom I, et al, "CD3-Mediated Activation of Tumor-Reactive Lymphocytes from Patients with Advanced Cancer", Proc. Natl. Acad. Sci. USA, 98: 6783-6788 (June 5, 2001).
3. Fong L, et al, "Altered Peptic Ligand Vaccination with Flt3 Ligand Expanded Dendritic Cells for Tumor Immunotherapy", Proc. Natl. Acad. Sci. USA, 98: 8809-8814 (July 17, 2001).
4. Stanley DA, Frenster JH, and Rigas DA, "Subnuclear Localization of Tritiated Phytohemagglutinin during Gene De-repression within Human Lymphocytes", J. Cell Biol. vol. 39, part 2, p. 129a (1968).
5. Chapman ALN, Rickinson AB, Thomas WA, Jarrett RF, Crocker J, and Lee SP, "Epstein-Barr Virus-Specific Cytotoxic T Lymphocyte Responses in the Blood and Tumor Site of Hodgkin's Disease Patients: Implications for a T-Cell-based Therapy", Cancer Res. vol. 61, no. 16, pp. 6219-6226 (August 15, 2001).
6. Chen JJW, Lin Y-C, Yao P-L, Yuan A, Chen H-Y, Shun C-T, Tsai M-F,
Chen C-H, Yang P-C, "Tumor-Associated Macrophages: The Double-Edged Sword
in Cancer Progression", Journal
of Clinical Oncology, vol. 23, no. 5, pp. 953-964 (February 10, 2005).
Dr. J. V. Schlosser (New Orleans, LA): I'd like to reinforce Dr. Frenster's paper by mentioning several patients in whom we used a similar technique. The first patient had undifferentiated carcinoma of the parotid with diffuse spread to subcutaneous tissues and axillary lymph nodes, and we observed about a 75% reduction in the tumor masses. The second patient (in whom we used freeze-thawed and irradiated autologous lymphocytes) had embryonal cell carcinoma of the testes with pulmonary metastases. No other treatment was used. In about 6 weeks, he rejected the pulmonary metastases and is now free of disease 4 years later. In our series of about 40 patients, we also observed other regressions, which were transitory and less striking than these two, but it does indicate that there is something in this system. Why should phytohemagglutinin-stimulated autologous lymphocytes do anything to a patient's tumor, when we are putting back virtually the same thing that we took out ? I think our colleagues in basic research might look into why this mechanism works at all.
Dr. Frenster: That is a very interesting comment, Dr. Schlosser. Were your thawed lymphocytes viable when re-infused into your patients ?
Dr. Schlosser: The viability of the irradiated cells would depend, of course, on the radiation dosage. We used a range of doses. I forget exactly what dose we used in this particular man, but I think that there probably were dead cells after irradiation. The freeze-thawed cells were certainly dead, because we froze and thawed them two or three times.
Dr. Frenster: We may be dealing with lymphokines or similar materials that are formed during phytohemagglutinin activation. I agree that these cells are the same ones that were removed from the patients. However, they are quite different in their physiologic activity, and I suspect that they remain in the activated state for at least 1-2 days in patients, because the label is intranuclear and persists in these cells as they are re-infused.
Dr. Schlosser: Yes, they are blastoid, of course, and this may make the big difference.
Dr. I. Siegel (Mt. Sinai Hospital, New York, NY): I was interested in your observation that the macrophage was a phagocytized T-lymphocyte. Some of our work (J. Immunol. 1970) may throw some light on this matter. Working with guinea pig cells, we found that at a certain stage of macrophage differentiation, phagocytosis of thymocytes in the presence of autologous serum was a natural phenomenon.
Dr. Frenster: I think that we're beginning to understand some relationships between macrophages and lymphocytes, and it may be that they have both adversary and cooperative roles.