Ann. din. Biochem. 13 (1976) 485-487
Cellular Immunology
J. J. T.
OWEN
Department of Anatomy, Medical School, University of Newcastle upon Tyne
to depend on cellular immunity also. Secondly, adoptive transfer of tumour immunity has been obtained by lymphoid suspensions given either systemically- or locally. 3 Indeed, there has been widespread acceptance that a major function of T lymphocytes might be in "immune surveillance", a concept originally proposed by Ehrlich, subsequently rephrased by Thomas, and developed by Burnett.s This theory states that a major driving force in the evolution of the immune response has been the necessity to recognise and eliminate clones of neoplastic cells however they may arise. Although some of the predictions of the immune surveillance theory-for example, that neoplastic cells bear altered cell surface components which can be recognised by the immune system-may hold true.s recognition of these components may not lead to responses which eliminate or suppress neoplastic cells." The role of "blocking" factors in this regard is discussed in a subsequent paper. More strikingly, the prediction that immune deficiency, especially of the T lymphocyte type, should lead to an increased incidence of tumours is not borne out in either immunosuppressed animals or in mice born with congenital thymic deficiency (nude rnice),? But it should be stated that such animals do have a lowered resistance to viral oncogenesis. It does seem, however, that this reflects their lowered immune status to viruses in general rather than a special susceptibility to oncogenesis resulting from their immunosuppressed state. In patients undergoing chronic immunosuppression a higher incidence of tumours has been reported. However, the spectrum of tumours found in these patients is very different from that found in the general population. Up to 40% of these tumours are of mesenchymal origin (mostly lymphomas), and at least two-thirds of the remainder are cancers of the skin, cervix, or lip. These observations may suggest that immunosuppressed patients are especially prone to certain carcinomas, perhaps as a result of their susceptibility to infections. CELLULAR IMMUNITY AND TUMOUR REJECfION A further phenomenon which argues against the The belief that cellular immunity is important in immune surveillance theory has been known for tumour rejection is based firstly on the fact that some time. Very small inocula of highly immunorejection of allogeneic grafts of normal tissue is genic tumours syngeneic or even allogeneic to the primarily T lymphocyte-dependent, and therefore host may grow in experimental animals even though transplantation resistance seen in experimental larger inocula fail under the same circumstances.f animals after ligation or excision of tumours is likely This "sneaking through" phenomenon suggests that
Cellular or cell-mediated immunity has generally been considered to be a major factor in suppression of tumour growth. However, although the term "cellular immunity" is in common usage, it has become increasingly difficult to define. It can be defined as immunity which requires the direct participation of cells at the site of the immune reaction or, more definitively, in experimental animals as immunity which can be adoptively transferred from one individual to another by cells alone. In other words, classical antibody (humoral immunity) is not required. Most recent evidence suggests that cellular immunity defined in this way is mediated by T lymphocytes, which recognise antigen and are activated by it. In turn their activation evokes the participation of other cells (especially macrophages) through the release of a variety of factors or lymphokines. I will not discuss these factors in detail here except to say that most of them have been demonstrated in in-vitro systems, although there is increasing evidence for their role in in-vivo immune responses. Factors such as migration inhibition factor (MIF), macrophage activating factor (MAF), and other chemotactic and mitogenic factors result in the secondary recruitment of cells and the production of delayed hypersensitivity responses. Most of the factors involved are thought to be proteins or glycoproteins with molecular weights ranging from 20 000 to 70 000. 1 However, the borderline between cellular and humoral immunity has become blurred in recent years. T lymphocytes play a regulatory role in antibody production, and recently it has been shown that B lymphocytes are able to produce lymphokines. Furthermore, target cells may be killed in vitro not only by T cells (probably an in-vitro correlate of cellular immunity) but by antibodydependent cellular cytotoxicity or A.D.C.C. This type of cytotoxicity depends upon nonimmune cells (K cells) killing antibody-coated target cells after attaching to the Fe portion of the antibody.
485
Downloaded from acb.sagepub.com by guest on May 21, 2016
486 J. J. T. Owen
small numbers of tumour cells can readily escape the immune response. Indeed, it has been suggested that under certain circumstances the immune response may stimulate growth." In summary, most evidence at the moment argues against the immune surveillance theory. This, however, does not necessarily argue against the role of cellular immunity in restricting the growth of established tumours, and I will consider aspects of this topic in subsequent sections. CELLULAR lMMuNITY IN EXPERIMENTAL TUMOURS
Perhaps the most optimistic model from the point of view of the role of cellular immunity in tumour rejection is found in studies on viral oncogenesis. One of the most intensively investigated tumours from the point ofview of the immunological response is that produced by the murine sarcoma virus (MSV). MSV is a single stranded RNA virus derived from mouse tumours of mixed mesenchymal origin. The pathogenesis of tumours produced by MSV has been studied extensively by Fefer et a/. l D One of the highly interesting features of the tumour is that regression is common if the virus is injected into weanling mice. This regression is dependent on an intact immune system, and this observation together with the fact that the tumour mass itself contains a large number of inflammatory cells argues strongly for the importance of the immune response in the rejection process. An extensive series of in-vitro studies have been carried out in this system. The first studies were performed by Hellstrom and Hellstrom.P who used a microcytotoxicity test to demonstrate the presence of cytotoxic lymphoid cells within the spleens and lymph nodes of animals with growing tumours as well as in those animals whose tumours had regressed. The basis of the microcytotoxicity test is a simple one-s-namely, that target tumour cells are grown in multiwell plates in the presence of lymphoid cell populations. The killing, or growth inhibition, of the tumour cells can then be measured by counting the number of adherent tumour cells remaining at the end of the test. Although simple in principle the test itself is subject to many variables. These variables have been studied in systems in which the tumour cells have been labelled with DNA incorporated l25Iododeoxyuridine. 12 This allows a large number of tests to be performed and all of the variables operating in the test to be studied. Both rnacrophages and T lymphocytes have been found to kill target tumour cells in these assays.13 However, other measures of immunity have suggested the importance of antibody-dependent mechanisms in the rejection of MSV tumours.P Thus, in-
vitro systems have demonstrated a heterogeneity of effector mechanismsw involving not only T and B lymphocytes but also macrophages, It is clearly of considerable importance, therefore, to try to determine to what extent any or all of these mechanisms are operative in vivo. Work along these lines has been performed in the MSV system in adoptive transfer models and the results have suggested firstly that T lymphocytes may play an important part in the rejection of murine sarcomas and also that antibody itself can mediate tumour rejection.t" In-vitro studies have also been performed on rejection of chemically induced tumours. Again the results have shown the complexity of the immune response in tumour rejectionp,18 Recent work, especially in virus models, has indicated that the antigens recognised by T lymphocytes may well be different from those recognised by B lymphocytes and antibody. Indeed there is some evidence that T lymphocytes recognise modified major histocompatibility antigens.P Obviously this work requires further investigation but is clearly of great importance for studies of tumour rejection. CELLULAR IMMUNITY IN HUMAN TUMOURS
There is now an extensive literature on attempts to evaluate cellular immunity in human cancer. 5,2D Much of the work has been aimed at determining aspects of the cell-mediated immune response to tumour antigens. Perhaps the most widely used test has been the microcytotoxicity assay mentioned previously. However, the difficulties in evaluating this assay in human systems are considerable. First, there is the problem of obtaining suitable target tumour cells. Many investigations have used long transplanted lines, but these cells may have lost the antigens in question, or they may have gained new antigens associated with virus infection. In addition to this problem there are problems in determining the specificity of any cytotoxic reactions noted. Cytotoxic reactions by lymphocytes obtained from normal healthy individuals have been widely reported, and therefore it is difficult to know what baseline should be used in evaluating any reaction noted with lymphocytes from cancer patients. 21,22 Until these problems are resolved it seems unlikely that the microcytotoxicity test will be of clinical value. Various other tests have also been extensively investigated. Attempts have been made to obtain lymphocyte stimulation using either fresh or frozen tumour cells,' or membrane extracts of tumour cells.23 Relatively low levels of stimulation have been obtained, and a further problem is that in many of the studies suitable controls have not been
Downloaded from acb.sagepub.com by guest on May 21, 2016
Cellular immunology
included and therefore it is difficult to evaluate the results obtained. Leucocyte migration inhibition has also been used as an assay for Iymphokines released from sensitised T lymphocytes.e Again, there is difficulty in evaluating the data that have been obtained, because in many instances adequate controls were not included in the surveys. Furthermore, in all of these tests there is the possibility that . allogeneic rather than tumour specific reactions are being detected. Some attempts have been made to examine in-vivo reactions to tumour extracts. 25 Some of the results look promising in that they have indicated that patients with tumours are sensitive to antigen preparations obtained from the tumour in question. However, further work is required on the specificity of the effects noted. CONCLUSIONS
I think that it should be clear from this brief account that many of the current assays which are used to assess cellular immunity in cancer patients require much further investigation before they can be used in clinical practice. However, the results from animal experiments are promising, and if immunotherapy is to be of value in the treatment of cancer it is very important to have well evaluated invitro tests to assess the results of treatment. Current progress in studies on the recognition of antigen by T lymphocytes should provide a sound basis for the evaluation of T lymphocyte reactivity to tumour antigens.
REFERENCES
lYaldimarsson, H. Effector mechanisms in cellular immunity, p, 179, in The Immune System, ed. M. I. Hobart and I. McConnell. Oxford, Blackwell (1975). "Mitchison, N. A. Proc, roy. Soc. Ser. B. 142 (1954) 72.
487
·Klein, G. Sjogren, H. O. Cancer Res. 20 (1960) 452. 'Burnett, F. M. Progr. Exp. Tumour Res. 13 (1970) 1. 'Currie, G. A. Cancer and the immune response, in Current Topics in Immunology, No.2, ed, I. Turk. London, E. Arnold (1974). "Hellstrom, K. E., Hellstrom, I. Adv. Cancer Res. 12 (1969) 167. 7Rygaard, I., Povlsen, C. O. Transplant. Rev. 28 (1976) 43. "Bonmasser, E., Melconi, E., Goldin, A., Cudkowicz, G. J. nat. Cancer Inst, 53 (1974) 475. ·Prehn, R. T. Transplant. Rev. 28 (1976) 34. lOFefer, A., McCoy, I. L., Perk, K., Glynn, I. P. Cancer Res. 28 (1968) 1577. llHellstrom, I., Hellstrom, K. E. Int. J. Cancer 5 (1970) 195. lISeeger, R. C., Rayner, S. A., Owen, I. I. T. Int. J. Cancer 13 (1974) 697. 13Seeger, R. c., Owen, I. I. T. Nature (Lond.) 252 (1974) 420. "Lamon, E. W., Andersson, B., Wigzell, H., Fenyo, E. M., Klein, E. Int. J. Cancer 13 (1974) 91. 1&Plata, F., Gomard, E., Leclerc, I. c., Levy, I. P. J. Immunol. 112 (1974) 1477. uPearson, G. R., Redmon, L. W., Bass, L. R. Cancer Res. 33 (1973) 171. 17Kearney, R., Basten, A., Nelson, D. S. Int. J. Cancer 15 (1975) 438. uBaldwin, R. W. Transplant. Rev. 28 (1976) 62. lOChesebro, B., Wehrly, K. J. expo Med. 143 (1976) 85. IOAlexander, P. Tumour immunology, in The Immune System, p. 296, ed. M. I. Hobart and I. McConnell. Oxford, Blackwell (1975). 'lHeppner, G., Henry, E., Stolbach, L., Cummings, F., McDonough, E., Calabresi, P. Cancer Res. 35 (1975) 1931. .ISulit, H. L., Golub, S. H., Irie, R. F., Gupta, R. K., Grooms, G. A., Mortan, D. L. Int. J. Cancer 17 (1976) 461. '·Stjernsward, I., Vanky, F. Nat. Cancer Inst, Monogr. 35 (1972) 237. "Churchill, W. H. Nat. Cancer Inst, Monogr. 37 (1973) 135. "Oren, M. E., Herberman, R. B. (1971). Clin. expo Immunol. 9 (1971) 45.
Downloaded from acb.sagepub.com by guest on May 21, 2016
Cellular Immunology
J. J. T.
OWEN
Department of Anatomy, Medical School, University of Newcastle upon Tyne
to depend on cellular immunity also. Secondly, adoptive transfer of tumour immunity has been obtained by lymphoid suspensions given either systemically- or locally. 3 Indeed, there has been widespread acceptance that a major function of T lymphocytes might be in "immune surveillance", a concept originally proposed by Ehrlich, subsequently rephrased by Thomas, and developed by Burnett.s This theory states that a major driving force in the evolution of the immune response has been the necessity to recognise and eliminate clones of neoplastic cells however they may arise. Although some of the predictions of the immune surveillance theory-for example, that neoplastic cells bear altered cell surface components which can be recognised by the immune system-may hold true.s recognition of these components may not lead to responses which eliminate or suppress neoplastic cells." The role of "blocking" factors in this regard is discussed in a subsequent paper. More strikingly, the prediction that immune deficiency, especially of the T lymphocyte type, should lead to an increased incidence of tumours is not borne out in either immunosuppressed animals or in mice born with congenital thymic deficiency (nude rnice),? But it should be stated that such animals do have a lowered resistance to viral oncogenesis. It does seem, however, that this reflects their lowered immune status to viruses in general rather than a special susceptibility to oncogenesis resulting from their immunosuppressed state. In patients undergoing chronic immunosuppression a higher incidence of tumours has been reported. However, the spectrum of tumours found in these patients is very different from that found in the general population. Up to 40% of these tumours are of mesenchymal origin (mostly lymphomas), and at least two-thirds of the remainder are cancers of the skin, cervix, or lip. These observations may suggest that immunosuppressed patients are especially prone to certain carcinomas, perhaps as a result of their susceptibility to infections. CELLULAR IMMUNITY AND TUMOUR REJECfION A further phenomenon which argues against the The belief that cellular immunity is important in immune surveillance theory has been known for tumour rejection is based firstly on the fact that some time. Very small inocula of highly immunorejection of allogeneic grafts of normal tissue is genic tumours syngeneic or even allogeneic to the primarily T lymphocyte-dependent, and therefore host may grow in experimental animals even though transplantation resistance seen in experimental larger inocula fail under the same circumstances.f animals after ligation or excision of tumours is likely This "sneaking through" phenomenon suggests that
Cellular or cell-mediated immunity has generally been considered to be a major factor in suppression of tumour growth. However, although the term "cellular immunity" is in common usage, it has become increasingly difficult to define. It can be defined as immunity which requires the direct participation of cells at the site of the immune reaction or, more definitively, in experimental animals as immunity which can be adoptively transferred from one individual to another by cells alone. In other words, classical antibody (humoral immunity) is not required. Most recent evidence suggests that cellular immunity defined in this way is mediated by T lymphocytes, which recognise antigen and are activated by it. In turn their activation evokes the participation of other cells (especially macrophages) through the release of a variety of factors or lymphokines. I will not discuss these factors in detail here except to say that most of them have been demonstrated in in-vitro systems, although there is increasing evidence for their role in in-vivo immune responses. Factors such as migration inhibition factor (MIF), macrophage activating factor (MAF), and other chemotactic and mitogenic factors result in the secondary recruitment of cells and the production of delayed hypersensitivity responses. Most of the factors involved are thought to be proteins or glycoproteins with molecular weights ranging from 20 000 to 70 000. 1 However, the borderline between cellular and humoral immunity has become blurred in recent years. T lymphocytes play a regulatory role in antibody production, and recently it has been shown that B lymphocytes are able to produce lymphokines. Furthermore, target cells may be killed in vitro not only by T cells (probably an in-vitro correlate of cellular immunity) but by antibodydependent cellular cytotoxicity or A.D.C.C. This type of cytotoxicity depends upon nonimmune cells (K cells) killing antibody-coated target cells after attaching to the Fe portion of the antibody.
485
Downloaded from acb.sagepub.com by guest on May 21, 2016
486 J. J. T. Owen
small numbers of tumour cells can readily escape the immune response. Indeed, it has been suggested that under certain circumstances the immune response may stimulate growth." In summary, most evidence at the moment argues against the immune surveillance theory. This, however, does not necessarily argue against the role of cellular immunity in restricting the growth of established tumours, and I will consider aspects of this topic in subsequent sections. CELLULAR lMMuNITY IN EXPERIMENTAL TUMOURS
Perhaps the most optimistic model from the point of view of the role of cellular immunity in tumour rejection is found in studies on viral oncogenesis. One of the most intensively investigated tumours from the point ofview of the immunological response is that produced by the murine sarcoma virus (MSV). MSV is a single stranded RNA virus derived from mouse tumours of mixed mesenchymal origin. The pathogenesis of tumours produced by MSV has been studied extensively by Fefer et a/. l D One of the highly interesting features of the tumour is that regression is common if the virus is injected into weanling mice. This regression is dependent on an intact immune system, and this observation together with the fact that the tumour mass itself contains a large number of inflammatory cells argues strongly for the importance of the immune response in the rejection process. An extensive series of in-vitro studies have been carried out in this system. The first studies were performed by Hellstrom and Hellstrom.P who used a microcytotoxicity test to demonstrate the presence of cytotoxic lymphoid cells within the spleens and lymph nodes of animals with growing tumours as well as in those animals whose tumours had regressed. The basis of the microcytotoxicity test is a simple one-s-namely, that target tumour cells are grown in multiwell plates in the presence of lymphoid cell populations. The killing, or growth inhibition, of the tumour cells can then be measured by counting the number of adherent tumour cells remaining at the end of the test. Although simple in principle the test itself is subject to many variables. These variables have been studied in systems in which the tumour cells have been labelled with DNA incorporated l25Iododeoxyuridine. 12 This allows a large number of tests to be performed and all of the variables operating in the test to be studied. Both rnacrophages and T lymphocytes have been found to kill target tumour cells in these assays.13 However, other measures of immunity have suggested the importance of antibody-dependent mechanisms in the rejection of MSV tumours.P Thus, in-
vitro systems have demonstrated a heterogeneity of effector mechanismsw involving not only T and B lymphocytes but also macrophages, It is clearly of considerable importance, therefore, to try to determine to what extent any or all of these mechanisms are operative in vivo. Work along these lines has been performed in the MSV system in adoptive transfer models and the results have suggested firstly that T lymphocytes may play an important part in the rejection of murine sarcomas and also that antibody itself can mediate tumour rejection.t" In-vitro studies have also been performed on rejection of chemically induced tumours. Again the results have shown the complexity of the immune response in tumour rejectionp,18 Recent work, especially in virus models, has indicated that the antigens recognised by T lymphocytes may well be different from those recognised by B lymphocytes and antibody. Indeed there is some evidence that T lymphocytes recognise modified major histocompatibility antigens.P Obviously this work requires further investigation but is clearly of great importance for studies of tumour rejection. CELLULAR IMMUNITY IN HUMAN TUMOURS
There is now an extensive literature on attempts to evaluate cellular immunity in human cancer. 5,2D Much of the work has been aimed at determining aspects of the cell-mediated immune response to tumour antigens. Perhaps the most widely used test has been the microcytotoxicity assay mentioned previously. However, the difficulties in evaluating this assay in human systems are considerable. First, there is the problem of obtaining suitable target tumour cells. Many investigations have used long transplanted lines, but these cells may have lost the antigens in question, or they may have gained new antigens associated with virus infection. In addition to this problem there are problems in determining the specificity of any cytotoxic reactions noted. Cytotoxic reactions by lymphocytes obtained from normal healthy individuals have been widely reported, and therefore it is difficult to know what baseline should be used in evaluating any reaction noted with lymphocytes from cancer patients. 21,22 Until these problems are resolved it seems unlikely that the microcytotoxicity test will be of clinical value. Various other tests have also been extensively investigated. Attempts have been made to obtain lymphocyte stimulation using either fresh or frozen tumour cells,' or membrane extracts of tumour cells.23 Relatively low levels of stimulation have been obtained, and a further problem is that in many of the studies suitable controls have not been
Downloaded from acb.sagepub.com by guest on May 21, 2016
Cellular immunology
included and therefore it is difficult to evaluate the results obtained. Leucocyte migration inhibition has also been used as an assay for Iymphokines released from sensitised T lymphocytes.e Again, there is difficulty in evaluating the data that have been obtained, because in many instances adequate controls were not included in the surveys. Furthermore, in all of these tests there is the possibility that . allogeneic rather than tumour specific reactions are being detected. Some attempts have been made to examine in-vivo reactions to tumour extracts. 25 Some of the results look promising in that they have indicated that patients with tumours are sensitive to antigen preparations obtained from the tumour in question. However, further work is required on the specificity of the effects noted. CONCLUSIONS
I think that it should be clear from this brief account that many of the current assays which are used to assess cellular immunity in cancer patients require much further investigation before they can be used in clinical practice. However, the results from animal experiments are promising, and if immunotherapy is to be of value in the treatment of cancer it is very important to have well evaluated invitro tests to assess the results of treatment. Current progress in studies on the recognition of antigen by T lymphocytes should provide a sound basis for the evaluation of T lymphocyte reactivity to tumour antigens.
REFERENCES
lYaldimarsson, H. Effector mechanisms in cellular immunity, p, 179, in The Immune System, ed. M. I. Hobart and I. McConnell. Oxford, Blackwell (1975). "Mitchison, N. A. Proc, roy. Soc. Ser. B. 142 (1954) 72.
487
·Klein, G. Sjogren, H. O. Cancer Res. 20 (1960) 452. 'Burnett, F. M. Progr. Exp. Tumour Res. 13 (1970) 1. 'Currie, G. A. Cancer and the immune response, in Current Topics in Immunology, No.2, ed, I. Turk. London, E. Arnold (1974). "Hellstrom, K. E., Hellstrom, I. Adv. Cancer Res. 12 (1969) 167. 7Rygaard, I., Povlsen, C. O. Transplant. Rev. 28 (1976) 43. "Bonmasser, E., Melconi, E., Goldin, A., Cudkowicz, G. J. nat. Cancer Inst, 53 (1974) 475. ·Prehn, R. T. Transplant. Rev. 28 (1976) 34. lOFefer, A., McCoy, I. L., Perk, K., Glynn, I. P. Cancer Res. 28 (1968) 1577. llHellstrom, I., Hellstrom, K. E. Int. J. Cancer 5 (1970) 195. lISeeger, R. C., Rayner, S. A., Owen, I. I. T. Int. J. Cancer 13 (1974) 697. 13Seeger, R. c., Owen, I. I. T. Nature (Lond.) 252 (1974) 420. "Lamon, E. W., Andersson, B., Wigzell, H., Fenyo, E. M., Klein, E. Int. J. Cancer 13 (1974) 91. 1&Plata, F., Gomard, E., Leclerc, I. c., Levy, I. P. J. Immunol. 112 (1974) 1477. uPearson, G. R., Redmon, L. W., Bass, L. R. Cancer Res. 33 (1973) 171. 17Kearney, R., Basten, A., Nelson, D. S. Int. J. Cancer 15 (1975) 438. uBaldwin, R. W. Transplant. Rev. 28 (1976) 62. lOChesebro, B., Wehrly, K. J. expo Med. 143 (1976) 85. IOAlexander, P. Tumour immunology, in The Immune System, p. 296, ed. M. I. Hobart and I. McConnell. Oxford, Blackwell (1975). 'lHeppner, G., Henry, E., Stolbach, L., Cummings, F., McDonough, E., Calabresi, P. Cancer Res. 35 (1975) 1931. .ISulit, H. L., Golub, S. H., Irie, R. F., Gupta, R. K., Grooms, G. A., Mortan, D. L. Int. J. Cancer 17 (1976) 461. '·Stjernsward, I., Vanky, F. Nat. Cancer Inst, Monogr. 35 (1972) 237. "Churchill, W. H. Nat. Cancer Inst, Monogr. 37 (1973) 135. "Oren, M. E., Herberman, R. B. (1971). Clin. expo Immunol. 9 (1971) 45.
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