;Jii;iiiiiiii!i!!iii! Modulation of T-cell function by gliomas Thomas Roszman, Lucinda Elliott and William Brooks Patients with primary intracranial tumors (gliomas) exhibit a profound decrease in immunity, the mechanism of which has, until recently, remained obscure. Here Thomas Roszman, Lucinda Elliott and William Brooks reveal that T cells obtained from these patients exhibit defects in interleukin 2 secretion and in expression of the high-affinity IL-2 receptor and they discuss the role played by immunosuppressive factors produced by gliomas in inducing these defects. The relationship between the central nervous and immune systems is unique because of (1) the sequestration of neural antigens in a 'partial' immunologically privileged site, (2) the antigenic determinants shared by the two systems and (3) the existence of neurally-derived immunoregulatory mediators. Clinical manifestation of this interrelationship is exhibited in patients with gliomas who present with broad suppression of humoral and cellmediated immunity1~. Recently, substantial progress has been made in understanding the immunobiology of patients with gliomas, particularly with regard to the modulation of T-cell function: cellular and biochemical dysfunction appears to be related to the production of immunosuppressive mediators by the glioma. Development of treatment strategies using biologic response modifiers depends on understanding this interaction. Primary malignant gliomas develop and remain within the brain (see Box 1). They are somewhat sequestered from the immune system yet are associated with broad suppression of host immunocompetence, as manifested by T-cell lymphopenia and impaired T-cell responsiveness to a variety of specific and nonspecific stimuli (Table 1). Cumulative data indicate that intrinsic defects, which may be induced by soluble factors secreted by malignant glial cells, exist in the T-cell population obtained from
these patients. Thus, patients bearing these tumors offer an excellent opportunity to investigate how immune function may be modulated by a tumor originating in an immunologically privileged site. Moreover, the immunomodulatory mechanisms inherent in this tumor model may provide insight into the interplay between neoplasms of the central nervous system and mediators of immunity, as well as the mechanisms of neuralimmune interaction. Impaired immune function Numerous studies have demonstrated that peripheral blood lymphocytes (PBLs) obtained from patients with primary iutracranial tumors respond poorly to mitogens and/or antigens 1,5-1°. This proliferative defect resides predominantly in the CD4 ÷ subset 11. Initial characterization of this impairment in T-cell activation revealed that it was not due to an inability of the ceils to bind mitogen and/or anti-CD3 monoclonal antibody 11,12. Nor can the reduced responsiveness be ascribed to T-cell lymphopenia5,6,13 or to trivial explanations attributable to in vitro culturing conditions14. The decreased proliferative capacity of these T cells is ubiquitous and extends to a wide variety of lectins and antigens 1,11. Early investigations of the cellular basis for the suppression of T-cell responsiveness in this group of patients ruled out defects in interleukin 1 (IL-1) production by monocytes15 and enhanced suppressor cell activity 12, suggesting an intrinsic defect within T cells rather than in the accessory cell population. Further studies, showing that there were one-sixth of phytohemagglutinin (PHA)responsive T cells in patients relative to normal subjects, Table 1. Some anomalies in the immune status of patients with glioblastomas
Observation
Refs
Cutaneous anergy 1-3 Decreased antibody response to influenza 4 virus and tetanus toxoid " 5,6,13 Decreased percentage and absolute number of peripheral blood T cells 1,5-10 Decreased peripheral blood lymphocyte reactivity to mitogens and alloantigens © 1991, ElsevierSciencePublishers Lrd, UK. 0167-4919/91/$02.00
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support this hypothesisis. In addition, cytokinetic analysis, using colchicine to block PHA-induced T-cell proliferation, demonstrated that the number of first generation T cells entering the S phase of mitosis was considerably reduced in patients. Although first generation responding cells from patients or normal subjects entered DNA synthesis at the same time, T cells obtained from most patients could not undergo second and subsequent generations. Taken together, these results suggest that T cells obtained from patients cannot undergo clonal expansion after stimulation with mitogen. Defects in IL-2 and IL-2R expression by activated T cells A crucial element in the initiation and maintenance of lymphocyte proliferation is the secretion of IL-2 and expression of the high-affinity IL-2 receptor (IL-2R). The production of IL-2 by lectin-stimulated T cells obtained from glioma-bearing patients is significantly less than that from T cells obtained from normal individuals 15. In addition, cells from patients fail to function as helper cells for immunoglobulin secretion, perhaps indicating a broader failure in cytokine synthesis 16. This defect is predominantly confined to the CD4 + T-cell subpopulation 11. On the basis of these data, it was hypothesized that broad suppression of lymphocyte proliferation resuits from inadequate IL-2 production. This theory was tested by adding exogenous IL-2 in an attempt to restore the lymphocyte proliferative capacity; however, the addition of various concentrations of delectinated or recombinant IL-2 failed to improve the proliferative abilities of lectin-stimulated T cells obtained from patients 17. The inability of IL-2 to restore the responsiveness of T cells obtained from glioma patients led to the suggestion that these lymphocytes have defective IL-2R expression, hence they cannot bind IL-2. Studies with lymphocytes obtained from patients revealed that lectin-stimulated T cells do not express the high-affinity IL-2R, as evidenced by ligand-binding studies with I2SI-labeledIL-2 (Ref. 18). The high-affinity IL-2R is composed of a p55 chain (e~or Tac protein) and a p75 chain (~). Both p55 and p75 bind to IL-2; the former with low affinity and the latter with intermediate affinity. Although p75 is expressed constitutively on T cells, p55 is induced only after antigen or mitogen stimulation and, subsequently, noncovalently associates with p75 to form a heterodimer that binds IL-2 with high-affinity IL-2R 19. p55 is expressed in excess of p75 on activated T cells, presumably to ensure that p75 is always occupied, thus resulting in the formation of the physiologic high-affinity IL-2R. The failure to assemble the high-affinity IL-2R subsequent to appropriate stimulation suggests a defect in the expression of the p55 and/or p75 chains. T cells obtained from patients with gliomas express significantly less of the p55 chain at 24-72 h after lectin stimulation when compared with normal subjects 18, while p75 is expressed normally. The explanation for the decreased expression of p55 after mitogen stimulation of these T cells remains to be determined. Decreased expression could occur because there is a defect in the regulation of gene transcription or
Immunology Today
translation. However, northern blot analysis of total RNA extracted from mitogen-stimulated T cells obtained from patients with malignant primary brain tumors indicated that these cells express normal steady-state levels of mRNA for p55. In these studies, the two predominant species of mRNA (1.5 and 3.5 kb) 18 were detected, and the ratio of 3.5:1.5 kb mRNA was similar for T cells obtained from patients and normal subjects. The question of whether translation of the mRNA for p55 occurs normally in mitogen-activated T cells obtained from patients has also been examined by labeling activated T cells with [3sS]-methionine and performing a pulse chase experiment. The primary product of the mRNA for p55 is a 34 kDa (p34) peptide which undergoes extensive post-translational alterations, including N-linked and O-linked glycosylation, to yield the final p55 product2°. The results demonstrated that appropriate translational and post-translational modifications occur in T cells of these patients, resulting in the formation of mature p55 (Ref. 18). The molecular basis of the failure of p55 expression remains to be determined. Several possibilities exist: although the mRNA for the p55 component appears to be produced in normal amounts in steady-state studies, the rate of synthesis and stability of this mRNA in these lymphocytes is unknown. Similarly, post-translational processing of p55 appears to be intact, but further kinetics studies will be necessary to evaluate the possibility that the protein is sequestered and degraded. In addition to translational defects leading to altered p55 expression, it is possible that the receptor is shed, abnormally, from the T-cell membrane. Interestingly, p55, with a molecular mass of 45-50 kDa, is shed in appreciable concentrations after mitogen stimulation of normal T cells21,22. Thus, it is possible that this apparently normal regulatory process could be upregulated in T cells obtained from patients, resulting in a marked decrease in membrane p55. Finally, the p55 could be masked or folded in such a way that it cannot be detected by specific antibody and these alterations could also result in it being unable to associate with p75 to form a high-affinity IL-2R. Glioblastoma-derived T-ceU suppressor factors An adequate explanation for the generalized impairment of host immunity observed in patients remains to be established. Previous findings 1,7, indicating that patients' sera can suppress both autologous and homologous normal lymphocyte function, suggest that immunosuppressive mediators secreted by the tumor may induce lymphocyte hyporesponsiveness. Support for this proposition is provided by the reports from a number of laboratories that human glioblastomas produce and secrete factors that inhibit lymphocyte function. Thus, addition of culture supernatants obtained from either cloned human glioblastoma cell lines or fresh, surgically removed glioblastomas inhibit a variety of lymphocyte functions including mitogen- and antigen-induced T-cell proliferation23,24, cytotoxic T-cell generation23, IL-2induced T-cell proliferation23, the production of tumorinfiltrating lymphocytes (TILs)25 and the generation of lymphokine-activated killer (LAK) cells26. Although there is general agreement on the observation that glioblastomas secrete immunosuppressive substances, there
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Glioblastoma
G-TSF/TGF-J] 2
T cell
Mitogen/antigen activation
I L-2
IL-2R
Decreased expression of membrane p55
y
Decreased IL-2 secretion
Impaired humoral and cell-mediated immunity Fig. 1. A possible scheme is shown that explains the broad suppression of humoral and cell-mediated immunity observed in patients with glioblastomas.
is much less accord as to their mode of action. Primarily, two issues contribute to this discord. First, these studies have been performed with crude culture supernatants obtained from glioblastoma cell lines and with several different factors isolated from culture supernatants, making direct and strict comparisons difficult. Second, most studies have used mouse rather than human lymphocytes, further complicating matters. Early descriptions of a glial-derived immunosuppressive factor 23 demonstrated that culture supernatants obtained from these cells, but not from a variety of other tumor cell lines, exhibited a broad range of effects on mouse lymphocyte function, including inhibition of concanavalin A (ConA)-induced thymocyte proliferation, of antigen- and IL-2-induced proliferation byan antigenspecific T-cell clone, and of the generation of cytotoxic Immunology Today
T cells. Interestingly, the culture supernatant did not affect the production of IL-2 by mitogen-stimulated spleen cells but did inhibit the proliferation of several neuroblast, but not fibroblast, cell lines. The general conclusion drawn from these data was that the suppressor factor present was acting on the late events involved in T-cell activation, possibly the IL-2-induced events. Recently, we have investigated the immunomodulatory effects of culture supernatants obtained from cloned human glioblastoma cell lines, as well as those obtained from fresh, surgically removed glioblastomas 24, on human PBLs. PHA- and anti-CD3 monoclonalantibody-induced responses of normal lymphocytes are inhibited in a dose-dependent fashion and maximal suppression observed only if the glioma-derived culture supernatants are added during the first 24 h of culture. This suggests that the major effects occur during the early events of lymphocyte activation. Inhibition occurs after only a 2 h preincubation with the tumor culture supernatants. In contrast to the observations of Fontana et al. 23, we found that addition of glioma-derived culture supernatants to PHA-stimulated PBLs also results in a significant decrease in IL-2 production and the subsequent diminished T-cell proliferation is refractory to the addition of exogenous IL-2. Another difference between these two studies is that culture supernatants from our glioblastoma cell lines do not inhibit the IL-2induced proliferation of a T-cell clone. The failure of exogenous IL-2 to restore the mitogen responsiveness of normal lymphocytes previously incubated in the presence of these culture supernatants suggests a defect in the expression of the high-affinity IL-2R. Additional studies have confirmed this: PHA-stimulated lymphocytes incubated with glioma-derived supernatant fail to express high-affinity IL-2R, as measured by ligand-binding studies (authors' unpublished observations). The dichotomy between these studies and those of Fontana et al. 23 may relate to differences in the experimental systems used, that is mouse versus human lymphocytes, different suppressive factors and/or their relative concentration in the culture supernatants. The latter may be of particular importance: the well-known heterogeneity of glioblastomas 27 could result in clones of differing genetic potential. None the less, collectively these results establish that human glioblastomas secrete a factor(s) the immunosuppressive mode of action of which, on normal human lymphocytes, is similar to that observed with lymphocytes obtained from patients with glioblastomas. This concept is further supported by the findings that serum obtained from patients with these tumors is immunosuppressive when added to normal human lymphocytes undergoing mitogen or antigen stimulation 1,7and that the immunosuppression observed in patients is partially relieved by surgical removal of the tumor 28. However, it remains to be established whether this suppressive factor is directly or indirectly responsible for the immunodeficiencies observed in these patients. Isolation and characterization of glioblastoma-derived T-ceU suppressor factors Attempts have been made to isolate and characterize the suppressor factor(s) from culture supernatants of human glioblastoma cell lines. Initial studies a9 demon-
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strated that the suppressor factor is a heat- and acidstable 97 kDa protein, which was termed glioblastomaderived T-cell suppressor factor (G-TSF). In general, this factor appears to be similar in its immunosuppressive capabilities to that of the original culture supernatant from which it was obtained. However, in more recent studies the same group isolated a 12.5 kDa protein, also termed G-TSF, with amino acid homology to transforming growth factor [3 (TGF-~3)3°. Subsequently, a cDNA was obtained from a human glioblastoma cell line using oligonucleotide probes deduced from the partial amino acid sequence of G-TSF31. By deducing the amino acid sequence from this cDNA, it appears that G-TSF and TGF-[32 are identical. The TGF-[3s are multifunctional peptides composed of two disulfide-linked 25 kDa homodimers that can modulate cell growth and differentiation of lymphocytes32. Studies comparing the effects of G-TSF and TGF-[32 on lymphocyte functions reveal that their effects are identical and that the actions of TGF-[32 can be neutralized by antibody specific to TGF-[3233. Furthermore, although mRNA for TGF-[31 and -[32 can be detected in human glioblastoma cell lines and fresh, surgically-removed glioblastomas, only TGF-f32 is secreted34. Recently, the examination of a large number of glioblastoma cell lines as well as surgically-obtained glioblastomas and astrocytomas revealed mRNA for TGF-131 or -~2 in almost all cases3~. In addition, there is evidence that some human glioblastoma cell lines can also secrete TGF-~ 36. Collectively, these results provide a link between the impaired immunity observed in patients with glioblastomas and secretion of G-TSF (Fig. 1). This correlation is strengthened by the observations that a suppressor factor is present in the sera of these patients 1,7, and that surgical removal of the tumor partially ameliorates suppression 28. Although the precise mechanism of action of G-TSF on T-cell function remains to be established, these findings raise the possibility of using a novel form of adjunctive therapy that could be used to inhibit the production and/or secretion of G-TSF, potentially resulting in improvement in the immune status of patients.
Clinical implications Conventional treatment by surgery, radiation and/or chemotherapy is rarely curative and has done little to improve the poor prognosis of patients with malignant glioma. The observation that mononuclear infiltration of these tumors may correlate with survival 14,37 suggests that immunobiological response modifiers might be useful in treatment. Several treatment protocols, using a variety of response modifiers, have been reported yet none has proved superior to currently available conventional therapy38~3. Despite these failures, the potential usefulness of immune response modifiers remains, strengthened by the findings that glioblastoma-derived TILs may be expanded and induced to kill human glioblastoma cells in vitro after incubation with IL-2 (Refs 44,45) and that LAK cells obtained from patients and normal individuals can kill glioblastoma cells in vitro 45,46. These observations form the basis of current adoptive immunotherapy, with IL-2 and intratumoral implantation of autologous LAK cells being evaluated in Immunology Today
several clinics. Unfortunately, the results reported so far have not been encouraging4°. The poor clinical response to IL-2 and LAK cell infusion, despite the in vitro studies, almost certainly results from the basic immunobiologic relationship between the neoplasm and host. For example, these patients have significantly reduced numbers of mononuclear cells capable of IL-2 LAK cell induction47 and manifest intrinsic deficiencies in IL-2 secretion and IL-2R assembly as previously discussed. More importantly, secretion of immunosuppressive substances such as TGF-[32 (Ref. 26) and prostaglandin E2 (Ref. 48) by glioblastomas reduce the effectiveness of adoptively-transferred cells. Further insights into the intrinsic mechanisms of host immunosuppression, the relationship of malignant and normal glial cells or immunomodulatory factors secreted by both normal and neoplastic glial cells will, therefore, be required before immunobiologic response modifiers can be used effectively.
Perspective The immunologic consequences of primary malignant brain tumors clearly reveal a functional link between T cells, soluble mediators secreted by gliomas, and loss of function through tumor infiltration with possible destruction of specific areas in the brain that are associated with modulation of responsiveness 49. The number and mode of action of immunosuppressive factors secreted by glioblastomas remains to be determined. Of equal importance, the precise molecular defect responsible for the inability of T cells obtained from patients to secrete normal amounts of IL-2 and express the p55 chain of IL-2R must be identified. Only when these tasks are accomplished can immunobiologic response modifiers be rationally developed and clinically evaluated. Nevertheless, patients with gliomas provide a naturally-occurring model with well-defined immunologic anomalies with which to investigate tumor immunity in relationship to neural-immune interactions.
Thomas Roszman, Lucinda Elliott and William Brooks are at the Dept of Microbiology and Immunology, University of Kentucky Medical Center, Lexington, KY 40536, USA.
References 1 Brooks,W.H, Netsky, M.G., Horwitz, D.A. and Normansell, D.E. (1972)J. Exp. Med. 136, 1631-1747 2 Brooks,W.H., Caldwell, H.D. and Mortara, R.H. (1974) Surg. Neurol. 2, 419-423 3 Albright,L., Seab,J.A. and Ommaya,A.K. (1977) Cancer 39, 1331-1336 4 Mahaley,M.8., Brooks, W.H., Roszman,T.L. et al. (1977) J. Neurosurg. 46, 467-476 5 Brooks,W.H., Roszman,T.L. and Rogers, A.S. (1976) Cancer 37, 1869-1973 6 Brooks,W.H., Roszman,T.L., Mahaley, M.S. and Woosley, R. (1977) Clin. Exp. Immunol. 29, 61-66 7 Young,H.R., Saka|a, R. and Kaplan, A.M. (•986) Surg. Neurol. 5, 19-23 8 Roszman,T.L. and Brooks, W.H. (1980) Clin. Exp. Immunol. 39, 395-402 9 Braun, O.P., Penn, R.O., Flannery,A.M. and Harris, J.E. (1982) Neurosurgery 10, 203-209
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10 Wood, G.W. and Morantz, R.A. (1982) J. Natl Cancer
E M B O J . 6, 1633-1636
Inst. 68, 27-33
31 de Martin, R., Haendler, B., Hofer-Warbinek, R. et al.
11 Elliott, L.H., Brooks, W.H. and Roszman, T.L. (1987) J. Neurosurg. 67, 231-236 12 Roszman, T.L., Brooks, W.H. and Elliott, L.H. (1982) Cancer 50, 1273-1279 13 Apzzo, M.L.J. and Mitchell, M.S. (1981)J. Neurosurg. 55, 1-18 14 Roszman, T.L. and Brooks, W.H. (1980) Clin. Exp. Immunol. 39, 395-402 15 Elliott, L.H., Roszman, T.L. and Brooks, W.H. (1984) J. tmmunol. 132, 1208-1215 16 Roszman, T.L., Brooks, W.H., Steele, C. and Elliott, L.H. (1985) J. Immunol. 134, 1545-1550 17 Elliott, L., Brooks, W. and Roszman, T. (1987) J. Natl Cancer Inst. 78, 919-922 18 Elliott, C.H., Brooks, W.H. and Roszman, T.L. (1990) J. Clin. Invest. 86, 80-86 19 Smith, K.A. (1988) Adv. Immunol. 42, 165-179 20 Leonard, W.J., Depper, J.M., Kronke, M. et al. (1985) J. Biol. Chem. 260, i872-1880 21 Rubin, L.A., Kurman, C.C., Fritz, M.E. et al. (1985) J. Immunol. 135, 3172-3177 22 Nelson, D.L., Rubin, L.A., Kurman, C.C. et al. (1986) J. Clin. Immunol. 6, 114-120 23 Fontana, A., Hengarmer, H., DeTribolet, N. and Weber, E. (1984)J. Immunol. 132, 1837-1844 24 Elliott, L.H., Brooks, W.H. and Roszman, T.L. (1987) J. Neurosurg. 67, 231-236 25 Kuppner, M.C., Hamou, M.F., Sawamura, Y. et al. (1989) J. Neurosurg. 71,211-217 26 Kuppner, M.C., Hamou, M.F., Bodmer, S. et al. (1988) Int. J. Cancer 42, 562-567 27 Rubinstein, L.J. (1976) Adv. Neurol. 15, 1-26 28 Brooks, W.H., Latta, R.B., Mahaley, M.S. et al. (1981) J. Neurosurg. 54, 331-337 29 Schuryzer, M. and Fontana, A. (1985) J. Immunol. 134, 1003-1009 30 Wrann, M., Bodmer, S., de Martin, R. et al. (1987)
lmmunology Today
(1987) EMBO J. 6, 3673-3677 32 Sporn, M.B., Roberts, A.B., Wakefield, L.M. and Assoian, R.K. (1986) Science 233,532-534 33 Siepl, C., Bodmer, S., Frei, K. et al. (1988) Eur. J. Immunol. 18,593-600 34 Bodmer, S., Strommer, K., Frei, K. et al. (1989) J. Immunol. 143, 3222-3229 35 Mapstone, T., McMichael, M. and Goldthwait, D. (1991) Neurosurgery 28,216-222 36 Rutha, J.T., Rosenblum, M.L., Stern, R. et al. (1989) J. Neurosurg. 71, 875-883 37 Brooks, W.H., Markesbery, W.R., Gupta, G.D. and Roszman, T.L. (1978) Ann. Neurol. 4, 219-224 38 Mahaley, M.S., Bigner, D.D., Dudka, L.F. et al. (1983) J. Neurosurg. 59, 201-207 39 Duff, T.A., Borden, E., Bay, J. et al. (1986) J. Neurosurg. 64, 408-413 40 Lillehei, K.O., Mitchell, D.H., Johnson, S.P. et al. (1991) Neurosurgery 28, 16-23 41 Jacobs, S.K., Wilson, D.J., Melin, G. and Parkham, C.W. (1986) Neurol. Res. 8, 81-87 42 Barba, D., Saris, S.C., Holder, C. et al. (1989) J. Neurosurg. 70, 175-182 43 Mahaley, M.S., Bertsch, L., Cush, S. and Gillespie, G.Y. (1988) J. Neurosurg. 69, 826-829 44 Kuppner, M.C., Hamou, M.F. and DeTribolet, N. (1988) Cancer Res. 48, 6926-6932 45 Miescher, S., Whiteside, T.L., DeTribolet, N. and Von Fliedner, V. (1988) J. Neurosurg. 68,438-448 46 Jacobs, S.K., Wilson, D.J., Kornblith, P.L. and Grimm, E.A. (1986)J. Neurosurg 64, 114-117 47 Bosnes, V. and Hirschberg, H. (1988) J. Neurosurg. 69, 234-238 48 Kuppner, M.C., Sawamura, Y., Hamou, M.F. and DeTribolet, N. (1990) J. Neurosurg. 72, 619-625 49 Roszman, T.L. and Brooks, W.H. (1985) J. Neuroimmunol. 10, 59-69
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these patients. Thus, patients bearing these tumors offer an excellent opportunity to investigate how immune function may be modulated by a tumor originating in an immunologically privileged site. Moreover, the immunomodulatory mechanisms inherent in this tumor model may provide insight into the interplay between neoplasms of the central nervous system and mediators of immunity, as well as the mechanisms of neuralimmune interaction. Impaired immune function Numerous studies have demonstrated that peripheral blood lymphocytes (PBLs) obtained from patients with primary iutracranial tumors respond poorly to mitogens and/or antigens 1,5-1°. This proliferative defect resides predominantly in the CD4 ÷ subset 11. Initial characterization of this impairment in T-cell activation revealed that it was not due to an inability of the ceils to bind mitogen and/or anti-CD3 monoclonal antibody 11,12. Nor can the reduced responsiveness be ascribed to T-cell lymphopenia5,6,13 or to trivial explanations attributable to in vitro culturing conditions14. The decreased proliferative capacity of these T cells is ubiquitous and extends to a wide variety of lectins and antigens 1,11. Early investigations of the cellular basis for the suppression of T-cell responsiveness in this group of patients ruled out defects in interleukin 1 (IL-1) production by monocytes15 and enhanced suppressor cell activity 12, suggesting an intrinsic defect within T cells rather than in the accessory cell population. Further studies, showing that there were one-sixth of phytohemagglutinin (PHA)responsive T cells in patients relative to normal subjects, Table 1. Some anomalies in the immune status of patients with glioblastomas
Observation
Refs
Cutaneous anergy 1-3 Decreased antibody response to influenza 4 virus and tetanus toxoid " 5,6,13 Decreased percentage and absolute number of peripheral blood T cells 1,5-10 Decreased peripheral blood lymphocyte reactivity to mitogens and alloantigens © 1991, ElsevierSciencePublishers Lrd, UK. 0167-4919/91/$02.00
Immunology Today 370
Vol.12 No. 10 1991
support this hypothesisis. In addition, cytokinetic analysis, using colchicine to block PHA-induced T-cell proliferation, demonstrated that the number of first generation T cells entering the S phase of mitosis was considerably reduced in patients. Although first generation responding cells from patients or normal subjects entered DNA synthesis at the same time, T cells obtained from most patients could not undergo second and subsequent generations. Taken together, these results suggest that T cells obtained from patients cannot undergo clonal expansion after stimulation with mitogen. Defects in IL-2 and IL-2R expression by activated T cells A crucial element in the initiation and maintenance of lymphocyte proliferation is the secretion of IL-2 and expression of the high-affinity IL-2 receptor (IL-2R). The production of IL-2 by lectin-stimulated T cells obtained from glioma-bearing patients is significantly less than that from T cells obtained from normal individuals 15. In addition, cells from patients fail to function as helper cells for immunoglobulin secretion, perhaps indicating a broader failure in cytokine synthesis 16. This defect is predominantly confined to the CD4 + T-cell subpopulation 11. On the basis of these data, it was hypothesized that broad suppression of lymphocyte proliferation resuits from inadequate IL-2 production. This theory was tested by adding exogenous IL-2 in an attempt to restore the lymphocyte proliferative capacity; however, the addition of various concentrations of delectinated or recombinant IL-2 failed to improve the proliferative abilities of lectin-stimulated T cells obtained from patients 17. The inability of IL-2 to restore the responsiveness of T cells obtained from glioma patients led to the suggestion that these lymphocytes have defective IL-2R expression, hence they cannot bind IL-2. Studies with lymphocytes obtained from patients revealed that lectin-stimulated T cells do not express the high-affinity IL-2R, as evidenced by ligand-binding studies with I2SI-labeledIL-2 (Ref. 18). The high-affinity IL-2R is composed of a p55 chain (e~or Tac protein) and a p75 chain (~). Both p55 and p75 bind to IL-2; the former with low affinity and the latter with intermediate affinity. Although p75 is expressed constitutively on T cells, p55 is induced only after antigen or mitogen stimulation and, subsequently, noncovalently associates with p75 to form a heterodimer that binds IL-2 with high-affinity IL-2R 19. p55 is expressed in excess of p75 on activated T cells, presumably to ensure that p75 is always occupied, thus resulting in the formation of the physiologic high-affinity IL-2R. The failure to assemble the high-affinity IL-2R subsequent to appropriate stimulation suggests a defect in the expression of the p55 and/or p75 chains. T cells obtained from patients with gliomas express significantly less of the p55 chain at 24-72 h after lectin stimulation when compared with normal subjects 18, while p75 is expressed normally. The explanation for the decreased expression of p55 after mitogen stimulation of these T cells remains to be determined. Decreased expression could occur because there is a defect in the regulation of gene transcription or
Immunology Today
translation. However, northern blot analysis of total RNA extracted from mitogen-stimulated T cells obtained from patients with malignant primary brain tumors indicated that these cells express normal steady-state levels of mRNA for p55. In these studies, the two predominant species of mRNA (1.5 and 3.5 kb) 18 were detected, and the ratio of 3.5:1.5 kb mRNA was similar for T cells obtained from patients and normal subjects. The question of whether translation of the mRNA for p55 occurs normally in mitogen-activated T cells obtained from patients has also been examined by labeling activated T cells with [3sS]-methionine and performing a pulse chase experiment. The primary product of the mRNA for p55 is a 34 kDa (p34) peptide which undergoes extensive post-translational alterations, including N-linked and O-linked glycosylation, to yield the final p55 product2°. The results demonstrated that appropriate translational and post-translational modifications occur in T cells of these patients, resulting in the formation of mature p55 (Ref. 18). The molecular basis of the failure of p55 expression remains to be determined. Several possibilities exist: although the mRNA for the p55 component appears to be produced in normal amounts in steady-state studies, the rate of synthesis and stability of this mRNA in these lymphocytes is unknown. Similarly, post-translational processing of p55 appears to be intact, but further kinetics studies will be necessary to evaluate the possibility that the protein is sequestered and degraded. In addition to translational defects leading to altered p55 expression, it is possible that the receptor is shed, abnormally, from the T-cell membrane. Interestingly, p55, with a molecular mass of 45-50 kDa, is shed in appreciable concentrations after mitogen stimulation of normal T cells21,22. Thus, it is possible that this apparently normal regulatory process could be upregulated in T cells obtained from patients, resulting in a marked decrease in membrane p55. Finally, the p55 could be masked or folded in such a way that it cannot be detected by specific antibody and these alterations could also result in it being unable to associate with p75 to form a high-affinity IL-2R. Glioblastoma-derived T-ceU suppressor factors An adequate explanation for the generalized impairment of host immunity observed in patients remains to be established. Previous findings 1,7, indicating that patients' sera can suppress both autologous and homologous normal lymphocyte function, suggest that immunosuppressive mediators secreted by the tumor may induce lymphocyte hyporesponsiveness. Support for this proposition is provided by the reports from a number of laboratories that human glioblastomas produce and secrete factors that inhibit lymphocyte function. Thus, addition of culture supernatants obtained from either cloned human glioblastoma cell lines or fresh, surgically removed glioblastomas inhibit a variety of lymphocyte functions including mitogen- and antigen-induced T-cell proliferation23,24, cytotoxic T-cell generation23, IL-2induced T-cell proliferation23, the production of tumorinfiltrating lymphocytes (TILs)25 and the generation of lymphokine-activated killer (LAK) cells26. Although there is general agreement on the observation that glioblastomas secrete immunosuppressive substances, there
371
vo~12 No. lO 1991
Glioblastoma
G-TSF/TGF-J] 2
T cell
Mitogen/antigen activation
I L-2
IL-2R
Decreased expression of membrane p55
y
Decreased IL-2 secretion
Impaired humoral and cell-mediated immunity Fig. 1. A possible scheme is shown that explains the broad suppression of humoral and cell-mediated immunity observed in patients with glioblastomas.
is much less accord as to their mode of action. Primarily, two issues contribute to this discord. First, these studies have been performed with crude culture supernatants obtained from glioblastoma cell lines and with several different factors isolated from culture supernatants, making direct and strict comparisons difficult. Second, most studies have used mouse rather than human lymphocytes, further complicating matters. Early descriptions of a glial-derived immunosuppressive factor 23 demonstrated that culture supernatants obtained from these cells, but not from a variety of other tumor cell lines, exhibited a broad range of effects on mouse lymphocyte function, including inhibition of concanavalin A (ConA)-induced thymocyte proliferation, of antigen- and IL-2-induced proliferation byan antigenspecific T-cell clone, and of the generation of cytotoxic Immunology Today
T cells. Interestingly, the culture supernatant did not affect the production of IL-2 by mitogen-stimulated spleen cells but did inhibit the proliferation of several neuroblast, but not fibroblast, cell lines. The general conclusion drawn from these data was that the suppressor factor present was acting on the late events involved in T-cell activation, possibly the IL-2-induced events. Recently, we have investigated the immunomodulatory effects of culture supernatants obtained from cloned human glioblastoma cell lines, as well as those obtained from fresh, surgically removed glioblastomas 24, on human PBLs. PHA- and anti-CD3 monoclonalantibody-induced responses of normal lymphocytes are inhibited in a dose-dependent fashion and maximal suppression observed only if the glioma-derived culture supernatants are added during the first 24 h of culture. This suggests that the major effects occur during the early events of lymphocyte activation. Inhibition occurs after only a 2 h preincubation with the tumor culture supernatants. In contrast to the observations of Fontana et al. 23, we found that addition of glioma-derived culture supernatants to PHA-stimulated PBLs also results in a significant decrease in IL-2 production and the subsequent diminished T-cell proliferation is refractory to the addition of exogenous IL-2. Another difference between these two studies is that culture supernatants from our glioblastoma cell lines do not inhibit the IL-2induced proliferation of a T-cell clone. The failure of exogenous IL-2 to restore the mitogen responsiveness of normal lymphocytes previously incubated in the presence of these culture supernatants suggests a defect in the expression of the high-affinity IL-2R. Additional studies have confirmed this: PHA-stimulated lymphocytes incubated with glioma-derived supernatant fail to express high-affinity IL-2R, as measured by ligand-binding studies (authors' unpublished observations). The dichotomy between these studies and those of Fontana et al. 23 may relate to differences in the experimental systems used, that is mouse versus human lymphocytes, different suppressive factors and/or their relative concentration in the culture supernatants. The latter may be of particular importance: the well-known heterogeneity of glioblastomas 27 could result in clones of differing genetic potential. None the less, collectively these results establish that human glioblastomas secrete a factor(s) the immunosuppressive mode of action of which, on normal human lymphocytes, is similar to that observed with lymphocytes obtained from patients with glioblastomas. This concept is further supported by the findings that serum obtained from patients with these tumors is immunosuppressive when added to normal human lymphocytes undergoing mitogen or antigen stimulation 1,7and that the immunosuppression observed in patients is partially relieved by surgical removal of the tumor 28. However, it remains to be established whether this suppressive factor is directly or indirectly responsible for the immunodeficiencies observed in these patients. Isolation and characterization of glioblastoma-derived T-ceU suppressor factors Attempts have been made to isolate and characterize the suppressor factor(s) from culture supernatants of human glioblastoma cell lines. Initial studies a9 demon-
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strated that the suppressor factor is a heat- and acidstable 97 kDa protein, which was termed glioblastomaderived T-cell suppressor factor (G-TSF). In general, this factor appears to be similar in its immunosuppressive capabilities to that of the original culture supernatant from which it was obtained. However, in more recent studies the same group isolated a 12.5 kDa protein, also termed G-TSF, with amino acid homology to transforming growth factor [3 (TGF-~3)3°. Subsequently, a cDNA was obtained from a human glioblastoma cell line using oligonucleotide probes deduced from the partial amino acid sequence of G-TSF31. By deducing the amino acid sequence from this cDNA, it appears that G-TSF and TGF-[32 are identical. The TGF-[3s are multifunctional peptides composed of two disulfide-linked 25 kDa homodimers that can modulate cell growth and differentiation of lymphocytes32. Studies comparing the effects of G-TSF and TGF-[32 on lymphocyte functions reveal that their effects are identical and that the actions of TGF-[32 can be neutralized by antibody specific to TGF-[3233. Furthermore, although mRNA for TGF-[31 and -[32 can be detected in human glioblastoma cell lines and fresh, surgically-removed glioblastomas, only TGF-f32 is secreted34. Recently, the examination of a large number of glioblastoma cell lines as well as surgically-obtained glioblastomas and astrocytomas revealed mRNA for TGF-131 or -~2 in almost all cases3~. In addition, there is evidence that some human glioblastoma cell lines can also secrete TGF-~ 36. Collectively, these results provide a link between the impaired immunity observed in patients with glioblastomas and secretion of G-TSF (Fig. 1). This correlation is strengthened by the observations that a suppressor factor is present in the sera of these patients 1,7, and that surgical removal of the tumor partially ameliorates suppression 28. Although the precise mechanism of action of G-TSF on T-cell function remains to be established, these findings raise the possibility of using a novel form of adjunctive therapy that could be used to inhibit the production and/or secretion of G-TSF, potentially resulting in improvement in the immune status of patients.
Clinical implications Conventional treatment by surgery, radiation and/or chemotherapy is rarely curative and has done little to improve the poor prognosis of patients with malignant glioma. The observation that mononuclear infiltration of these tumors may correlate with survival 14,37 suggests that immunobiological response modifiers might be useful in treatment. Several treatment protocols, using a variety of response modifiers, have been reported yet none has proved superior to currently available conventional therapy38~3. Despite these failures, the potential usefulness of immune response modifiers remains, strengthened by the findings that glioblastoma-derived TILs may be expanded and induced to kill human glioblastoma cells in vitro after incubation with IL-2 (Refs 44,45) and that LAK cells obtained from patients and normal individuals can kill glioblastoma cells in vitro 45,46. These observations form the basis of current adoptive immunotherapy, with IL-2 and intratumoral implantation of autologous LAK cells being evaluated in Immunology Today
several clinics. Unfortunately, the results reported so far have not been encouraging4°. The poor clinical response to IL-2 and LAK cell infusion, despite the in vitro studies, almost certainly results from the basic immunobiologic relationship between the neoplasm and host. For example, these patients have significantly reduced numbers of mononuclear cells capable of IL-2 LAK cell induction47 and manifest intrinsic deficiencies in IL-2 secretion and IL-2R assembly as previously discussed. More importantly, secretion of immunosuppressive substances such as TGF-[32 (Ref. 26) and prostaglandin E2 (Ref. 48) by glioblastomas reduce the effectiveness of adoptively-transferred cells. Further insights into the intrinsic mechanisms of host immunosuppression, the relationship of malignant and normal glial cells or immunomodulatory factors secreted by both normal and neoplastic glial cells will, therefore, be required before immunobiologic response modifiers can be used effectively.
Perspective The immunologic consequences of primary malignant brain tumors clearly reveal a functional link between T cells, soluble mediators secreted by gliomas, and loss of function through tumor infiltration with possible destruction of specific areas in the brain that are associated with modulation of responsiveness 49. The number and mode of action of immunosuppressive factors secreted by glioblastomas remains to be determined. Of equal importance, the precise molecular defect responsible for the inability of T cells obtained from patients to secrete normal amounts of IL-2 and express the p55 chain of IL-2R must be identified. Only when these tasks are accomplished can immunobiologic response modifiers be rationally developed and clinically evaluated. Nevertheless, patients with gliomas provide a naturally-occurring model with well-defined immunologic anomalies with which to investigate tumor immunity in relationship to neural-immune interactions.
Thomas Roszman, Lucinda Elliott and William Brooks are at the Dept of Microbiology and Immunology, University of Kentucky Medical Center, Lexington, KY 40536, USA.
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