Concise Review International Journal of Cell Cloning 9:438-450(1991)

Transforming Growth Factor-0: An Important Mediator of Immunoregulation John H . Kehrl Laboratory of Immunoregulation, National Institute of Allergy and Xnfectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

Key Words. "GF-6 Monocytes

- Immunosuppression

Jinmunoregulation

Lymphocytes

a

Abstract. Transforming growth factor-beta ('IGF-8) is synthesized and secreted by a wide variety of cells, including cells of the immune system. Lymphocytes and monocytes possess high affinity TGF-/3 receptors and the addition of TGF-@to in vitro cell cultures results in significant modulation of immune function. E F - 8 inhibits the proliferation of thymocytes, T cells, B cells, and natural killer cells. Additionally, it inhibits certain differentiative functions of lymphocytes including a marked inhibition of immunoglobulin production by human B lymphocytes. TUF-6 has dichotomous actions on monocytes. It is a potent chemoattractant for monocytes and induces interleukin 1 mRNA expression while inhibiting generation of reactive oxygen intermediates and monocyte killing, Evidence is accumulating that TGF-6 regulatesimmune function in vivo and that overpduction of E F - 6 may be associated with immunosuppression.

Historical Aspects of Transforming Growth Factor-p (TGF-0)

n?F-p is a 25,000 Dalton homodimericprotein which was on@y described by its ability to induce a transformed phenotype in soft agar of normal fibroblast indicator cells [I, 21. It was purified from human placenta and platelets, and bovine kidney [3-51.Partial amino acid (aa) sequencing of the purified protein led to the isolation of cDNA clones. The predicted amino acid sequence indicated that the TGF-0 monomer was encoded in the 122 amino acid carboxy terminus of a 390 aa precursor molecule [6].Mature or active "GF-8 is a homodimer composed of two chains of the carboxy terminal monomer. However, most cell types including lymphocytesand monocytes synthesize and secrete mF-8in a biologically inactive or latent form [7-91.Latent TGF-6 is a complex of one molecule of the dimeric mature form of E F - P associated with 2 molecules of the cleaved Correspondence: Dr. John H. Kehrl, National Institutes of Allergy and Infectious Diseases, National Institutes &Health, Bldg. 10,Room llB-l3, Bethesda, MD 20892, USA. Received May 10, 1991;accepted for publication May 10, B91. 0737-14541911$2.00/0@AlphaMedPress

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N-terminus of the TGF-P precursor and a third 135 kDa protein [lo, ll]. While latent TGF-P is readily activated in vitro by transient acidification or exposure to heat, alkali, or chaotropic agents, the precise mechanism by which E F - p is activated in vivo is unknown [lo, 121. Active TGF-P, but not the latent complex, is capable of binding to the TGF-/3 receptor. Several proteins highly related to E F - p have been isolated and the E F - P family of proteins now includes five distinct, yet highly homologous members. TGF-pl, 2, and 3 are each more than 99% conserved between species as diverse as chicken and man, whereas homology between the various isofonns ranges from 62%to 84%at the amino acid level [13-15]. The TGF-p4 and TGF-fl5 isoforms have been isolated from chicken [16] and frogs [17]; however, they have not yet been detected in any mammal. While much of the original interest in mF-0was related to its transforming properties, there is now considerable excitement concerning its role as a growth inhibitor for epithelial cells, as a mediator of inflammation and tissue repair, and as an important cytokine in the regulation of immunological function. The biochemistry and major biologic effects of E F - p have been previously reviewed [18-201. This review will focus on the effects of TGF-6 on immune function, the likely role of TGF-P as an endogenousregulator of the immune system, and the potential importance of TGF-p in the pathogenesis of immunosuppression accompanying infection with either the adult T cell leukemia (ATL) virus or the human immunodeficiency virus (HIV). Effects of Exogenous TGF-/3 on Immunocompetent Cells

Resting lymphocytes are induced to undergo clonal expansion via the interaction of antigen or processed antigen with either the immunoglobulin (Is) receptors on B lymphocytes or the T cell receptors on T lymphocytes. These interactions lead to induction of growth factor receptors and, in the presence of the appropriate growth factors, to cell division [reviewed 21-23]. While much is known about the mechanismsof cell activation and proliferation, includingthe lymphoid growth factors and their receptors, the mechanisms by which cell growth is limited and eventually terminated are less well understood. While there is assuredly a role for decreased availability of growth factor and downregulation of growth factor receptors, there are likely other regulatory mechanisms which lead to a cessation of proliferation and depression of immune activation. ll3F-P is a candidate molecule to perform such a function. The addition of exogenousE F - 0 to in vitro lymphocyte cultures inhibits the proliferation of B cells, mature T cells as well as thymocytes, natural killer cells (NK),and lymphocyte-activatedkiller cells [8, 24-27]. The anti-proliferative effects of TGF-P on immune cells are summarized in Table I. Several experiments have shown that the decrease in proliferation is not related to an inhibition of lymphocyte activation but rather to a direct inhibition of prolikration. For example, TGF-@does not inhibit the early proto-oncogene expression induced by mitogens, and the inhibitory effects of TGF-p added at the

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initiation of cell cultures are equivalent to the addition of TGF-P at 36 hours after mitogen stimulation [28,29]. These findmgs suggest that EF-/3 inhibits the transition of G,to S phase of the cell cycle. The mechanism by which E F - P inhibits the proliferation of lymphoid cells is unknown; however, studies with epithelial cells have suggested that the addition of TGF-P in mid- to late G,phase of the cell cycle prevents phosphorylation of the retinoblastoma (RB) protein and arrests epithelial cells in late G,[30]. The RB protein is a ubiquitously expressed 105 kDa phosphoprotein whose state of phosphorylation has been linked to certain phases of the cell cycle. The underphosphorylated form is the predominant species during G, and the hyperphosphorylated form is predominant during S and G2/M [31, 321. Whether E F - P similarly regulates the RB protein in lymphocytes is under investigation. The c-myc gene has also been implicated as a target of TGF-@[33]. Treatment of keratinocytes with E F - 6 inhibits c-myc transcription, which may be secondary to the induction of a negative regulatory transcription factor which suppresses c-myc transcription [33]. While this data suggests that the inhibition of c-myc is important in the inhibition of keratinocyte proliferation, there is little data to suggest that inhibition of c-myc transcription is important in the anti-proliferative effects of EF-/3 on primary lymphoid cells. Studies with human B cells activated with a B cell mitogen and interleukin 2 (IL-2) have shown that the presence of TGF-/3 in culture decreases levels of another transcription factor, AP-1 [34]. This decrease in AP-1 levels was not due to a decrease in the transcription of the genes which encode the two major proteins in the AP-1 complex, c-jun and c-fos, but presumably is related to a decrease in binding of AP-1 to its cognate sequence [34]. Whether this decrease in AP-1 levels is in part responsible for the inhibition in lymphocyte prolikration or secondary to the growth inhibitionby TGF-P per se remains to be determined. Besides limiting lymphocyte proliferation, TGF-0 also inhibits certain differentiative functions of lymphocytes (Table I>, The cytolytic activity of cytotoxic T cells, NK cells, and LAK cells [26,27, 351; the acquisition of the x light chain by murine pre-B cells [36]; and the secretion of Ig by activated B cells [24] are all inhibited by TCF-P. In contrast, TGF-8 has been shown to promote B cell isotype switching to IgA. Murine B cells stimulated with lipopolysaccharide (LPS) in the presence of TGF-P undergo switching to IgA; however, the continued presence of E F - P inhibits IgA secretion [37]. The effects of EF-/3 on B cell Ig secretion in vitro have been studied in some detail [34]. The addition of EF-/3 to activated human B cells dramatically inhibits Ig secretion as well as reduces Ig membrane expression and Ig synthesis. an apNorthern blot analysis revealed an 80% reduction in light chain -As, proximately 50%reduction in b heavy chain mRNA, and a marked reduction in the switch from the membrane forms to the secreted hrms of p and y heavy chains. The decrease in switching from the membrane form to the secreted form of Ig observed in the presence of TGF-P likely accounts for the dramatic reduction in Ig secretion. Nuclear run-ons are consistent with a reduction in x and X light chain mRNA transcription [34]. Evaluation of some of the factors known to be impor-

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Table I. Summary of the immunosuppressiveeffects of EF-@on in vitm immune function'

Effect

Cell type B cells

T cells

NK cells

Monocytes

~~~

1. Suppress proliferation 2. Impair antibody production 3. Impair cytolytic activity 4. Impair cytokine production

5. Enhance chemotaxis 6. Deactivate oxidative burst 7. Enhance IL-1production

+++ +++

+++ +++ ++

+++ +++

++ ++++ ++++ ++

'References included in text.

tant in Ig gene transcription [reviewed 381 revealed no obvious changes in the levels of nuclear factor-xB, the octamer-binding protein Oct-2, or xE2 in nuclear extracts prepared from cells treated with TGF-0 compared to controls [34]. However, preliminary evidence suggests that TGF-0 inhibits the activity of another transcription factor Pu.1 (ClaireThevenin, unpublished observation). A Pu.1 site located in the 3 ' kappa enhancer has been recently shown to be important for the transcriptional activation of this enhancer in B lymphocytes [39,40]. Similar to the regulation of the RB protein by TGF-0, a potentialmechanismby which TGF-0 might regulate Pu.1 is by altering its state of phosphorylation. TGF-P also significantly modulates monocyte and macrophage function. Exposure of peripheral blood monocytes (PBM) to a femtomolar concentration gradient of TGF-0 is a potent chemotaxic signal [41]. The release of E F - / 3 by degranulathg platelets in inflammatory foci, for example, may be an early recruitment signal for monocytes. Higher concentrationsof TGF-0 in the picomolar range enhances monocyte cytokine production. TGF-0 increases IL-1,platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and tumor necrosis factoralpha (TNF-a)mRNA levelsin monocytes [41,42]. In contrast, exposureof macrophages to "GF-0 results in a decrease in hydrogen peroxide production and ablation of the respiratory burst [43]. TGF-P does not impair the phagocytic capacity of the macrophages [43]. Thus, the initial production of TGF-0 is likely to recruit and activate monocytes, but as monocytes differentiate into macrophages their ability to kill via the respiratory burst is inhibited. The deactivation of macrophages by TGF-P is reversed by exposure of the macrophages to activating agents such as y-interferon or TNF-a! [43]. This reversibility of the inhibitory effects of TGF-8 by stimulatory cytokines has been consistently observed with both lymphoid cells and monocytes suggesting that a balance between stimulatory cytokinesand 7GF-0 may play a significant role in immunoregulation [8, 351.

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Lymphocytes and Monocytes Possess High Affinity TGF-8 Receptors Scatchard analysis of 12sI-TGF-/3binding assays has revealed low numbers of high affinity TGF-P receptors on resting T cells, resting B cells, and monocytes [8,24,44]. The differentiation of monocytes to macrophages is accompanied by a decrease in TGF-/3 receptors [44]; conversely, T and B cell activation results in a tenfold increase in the number of TGF-P receptors [8, 241. Using receptor cross-linking and competitive binding assays, the major species that make up the ‘IGF-Pl receptor complex on human B and T cells have been identified. Two crosslinked species of 65 and 90 kDa were found under reducing conditions, which would correspond to two proteins of 52 and 77 kDa cross-linked to one chain of the homodimeric TGF-01 [45]. Although TGF-Pl was effective in competitively inhibiting the detection of these cross-linked species, TGF-P2 did so poorly. A 125T-EF-pl competitivebinding assay revealed that TGF-01, but not TGF-D, competitively inhibited the binding of iodinated TGF-P1 to its high-affinity receptor, again suggesting a difference between the two receptor types despite the similar biologic responses to the two TGF-0s [45]. The precise nature of the TGF-/3 receptors of lymphoid and other cell types awaits further biochemical and molecular characterization of these receptor proteins. Certain lymphoblastoid B cell lines are refractory to the inhibitory effects of TGF-/3 and the lack of growth inhibition by TGF-P has been considered to be of possible importance in the outgrowth of B cell tumors [45, 461. The failure of these B cell lines to respond to TGF-P is not related to the lack of TGF-/3 receptors since receptor numbers and affinities are similar to those of activated B cells [45]. Additionally, the cross-linked receptor species are similar when responsive and nonresponsiveB cell lines are compared [45]. Thus, the refractorinessof these lymphoblastoid cell lines to growth inhibition by TGF-P remains unexplained, although it is likely secondary to a failure of the TGF-/3 receptor signalling or to an altered response to the TGF-/3 growth inhibition signal.

Lymphocytes and Monocytes Synthesize and Secrete TGF-P Lymphocytes and monocytes not only respond to TGF-P, but in addition, following cell activation, they synthesize and secrete TGF-P. Unactivated T or B cells and unstimulated monocytes secrete very low levels of IGF-P, however, following mitogen activation of lymphocytes or lipopolysaccharideactivation of monocytes these cells synthesize increased amounts of TGF-Pl mRNA and secrete significant quantities of TGF-Pl into culture [8,9,24]. The TGF-P secreted by lymphocytes and monocytes is almost exclusively TGF-fl with very little to no TGF-02 detected. Whether lymphoid cells or monocytes can synthesize and secrete TGF-03 is under investigation. As with other cell types, lymphoid cells stimulated with mitogens secrete TGF-@predominantly in a latent form, although

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low levels of active TGF-0 are present in lymphoid cultures as assessed by the use of anti-TGF-0 antibodies [45, 471. LPS-stimulated monocytes also secrete TGF-0 in a latent form. A recent report suggests that under certain circumstances lymphocytes may secrete active TGF-0. Resting murine T lymphocytes stimulated with IL-la! were found to secrete TGF-/3 predominantly in an active form [48]. The production of active TGF-0 by any cell type would likely have profound effects on neighboring lymphocytes and monocytes. Since TGF-0 is likely to be an important mediator in inflammatory foci, a mechanism for activation of latent TGF-/3 in these sites is to be expected. Both environmental conditions and enzymes have been proposed to be responsible for activation of TGF-0 [lo, 121. Whether stimulated macrophages or macrophage products might be able to activate TGF-0 is an interesting but unknown possibility. Thus,while the mechanism by which TGF-p is activated in vivo remains obscure, it is clear that the production of TGF-0 in a largely latent form protects lymphocytes from its immunosuppressive effects and provides an important control mechanism governing the biologic activity of TGF-0.

The Effects of Exogenous TGF-/3 Administration on In Vivo Immune Function There are now several examples where the administrationof TGF-0 to rodents markedly depresses inflammatory and immunological responses (Table II). For example, TGF-pl has been shown to prolong the time to rejection of ectopic cardiac transplants [49]. In both a rat and mouse model of arthritis the prior injection of TGF-01 protects the animals from the development of arthritis following the injection of either collagen or streptococcal cell wall ( S C W ) extracts [50,51]. In addition, the prior administration of TGF-0 delays the onset of relapsing experimental allergic encephalomyelitis(an animal model of multiple sclerosis) and continued administrationof TGF-0 prevents the Occurrence of relapses [51]. The mechanisms by which TGF-0 inhibits these immunological and inflammatory responses in vivo are unknown, but based on the in vitro studies, the suppressive effects of TGF-0 are likely mediated by inhibition of lymphocyte and monocyte function. Another study more directly examined the efkcts of TGF-p on a specific immune function, cytolytic T cell function. Simultaneous injection of TGF-01 and infection with either lymphocytic choriomeningitis virus (LCMV) or vaccinia virus suppressed the generation of virus-specific cytotoxic T cells [52]. In addition, the local swelling reaction normally noted after injection into the mouse foot pad was reduced. These data provide further evidence suggesting that TGF-0 may be an important mediator in the regulation of immune function in vivo.

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Table II. Studies examining the in vivo effects of exogenous TGF-pon immune function'

1. Suppression of cytolytlc T cells responses to LCMV and vaccinia. 2. Delayed onset of rejection of ectopic cardiac transplantation. 3. Suppression of acute and chronic arthritis. 4. Suppression of acute and chronic experimental allergic encephalomyelitis.

'Reference included in the text.

The Potential Role of Endogenous TGF-0Production on In vivo Immune Function While it is clear that TGF-0 can suppress immune function in vitro and that there is increasing evidence that the injection of exogenous TGF-p inhibits immune reactivity, it is still unresolved whether endogenous TGF-0 has a regulatory role in limiting immune function during either physiologic or pathologic activation of the immune system (Table III). There are several pathologic conditions which are characterized by immune suppression and associated with elevated levels of TGF-0. High levels of TGF-0 have been found in the synovial fluid derived from the joints of patients with rheumatoid arthritis [53,54].The presence of TGF-0 in the synovial fluid may explain some of the functional and phenotypic changes found with synovial lymphocytes, including decreased mitogen responsiveness [%I. Not dissimilarly, in a mouse model of chronic arthritis high levels of TGF-fl have been found in macrophages isolated from mice injected with S C W extracts and splenic lymphocytes from these mice are poorly responsive to mitogens [55]. Cultured macrophages from the SC!W extract-treated mice secreted a soluble molecule which suppressed mitogenesis and was neutralized with anti-TGF-01 antibodies [55]. Another example of immune suppression associated with overproduction of TGF-0 is the immune suppression noted in patients with glioblastomas [56, 571. These patients often have marked impairment in cell-mediated immunity and their serum can inhibit the mitogen and antigen responsivenessof T cells. Culture media conditioned by glioblastoma tumor cells suppresses thymocyte proliferation, and purification of the responsible agent revealed it to be TGF-02 [56,57]. Surgical removal of the tumor and the source of the excessive TGF-02 production results in reversal of the immune suppression. Peripheral blood and leukemic cells isolated from patients with the adult T cell leukemia (ATL) also produce excessive amounts of TGF-0 in in vitro cultures [58,59]. The human T lymphotrophic virus type l (HTLV-l) is the causative agent of ATL and clinical features include suppression of both cellular and humoral immunity [60].Freshly isolated ATL cells secrete 16- to 80-fold more TGF-01 than normal control mononuclear cells. No significantamount of T3F-m was detected in media conditioned by either normal cells or ATL cells [59]. Ele-

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Table III. Human diseases associated with immunosuppressionand overproduction of TGF-0' 1. Glioblastoma is associated with overproduction of TGF-02. 2. Adult T cell leukemia is associated with overproduction of TGF-pl. 3. The acquired immunodeficiency syndrome is associated with overproduction of TGF-01.

'References included in the text.

vated levels of TGF-j31 mRNA were found with both ATL cell lines and primary leukemic T cells. A potential mechanism for the overproduction of TGF-j31 was found. The TGF-01 promoter was found to be transactivated by the HTLV-1 p4Ox (Tax) protein which is known to transactivate a variety of cellular genes [59]. Another disease caused by a human retrovirus, the acquired immunodeficiency syndrome, is also associated with excessive production of TGF-j3 [61]. Peripheral blood mononuclear cells (PBMC) isolated from HIV-positiveindividuals secrete excessive amounts of TGF-j3. Impairment of T and B cell function was correlated with the amount of TGF-/3 produced by the PBMC [61,62]. Furthermore, HIV-infected monocytes have been shown to secrete significantly more EF-j3 than control cells, implicatingthem as a potential source of excessiveTGF-j3 production [63]. Besides being implicated in impairment of the immune response in HIV-infected individuals, TGF-j3 has also been shown to modify the production of HIV by infected cells. Exposure of monocytes to TGF-P in vitro prior to HIV infection enhances viral production while concurrent exposure of monocytes to H N and TGF-j3 inhibits subsequent viral production [MI.In addition, TGF-j3 blocks the induction of virus production by a chronically infected promonocytic cell line treated with IL-6 [MI. Thus, the overproductionof TGF-j3 in HTV-infected' individuals may have different regulatory effects on virus production and immune function. depending on the stage of the illness. These examples of immunosuppression associated with overproduction of TGF-j3 in vivo implicate TGF-P as the potential mediator of the immune suppression and suggest that overproduction of lI3F-p or, alternatively, aberrant activation of latent TGF-j3 may have important consequences for the function of the immune system. Finally, there is at least one example where E F - P production by immune cells has been shown to be important in the normal physiologic regulation of immunity. The clinical manifestations and histologic evidence of experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by immunization with myelin basic protein (MBP) can be suppressedby the oral administrationof MBP either prior to or following the immunization [65]. Transkr of CDS-positiveT cells from the orally tolerized rats to naive rats blocks the induction of EAE in the naive rats. Supernatantsconditioned by the same CDS-positive T cells stimulated with MBP can suppress in vitro T cell proliferative responses to MBP. The suppres-

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sive activity in the supernatantsis neutralized by antibodies against TGF-/3 [66]. Thus, while the induction of the suppressor T cells is antigen specific, i.e., MBP specific, the effector phase appears to be mediated by a nonspecific immunosuppressant, TGF-0. This observation also implies that the antigen-activatedT cells are secreting active rather than latent TGF-6. Conclusion TGF-0 is a potent inhibitor of lymphocyte function in vitro. Evidence is accumulating that TGF-0 is important in normal immune regulation and that overproduction of TGF-@is associated with immune dysfunction. In vitro studies have shown that TGF-0 impairs the proliferation of lymphoid cells and inhibits certain differentiated functions, including antibody production by B cells and the cytolytic activity of cytotoxic cells. TGF-/3 is a potent chemotaxic molecule for monocytes and increases IL-1 gene transcription. Both lymphocytes and monocytes have high-affinity TGF-/3 receptors which are differentially regulated following cell activation. Receptor cross-linking has identified two major cross-linked species of 65 and 90 kDa. Lymphocytes and monocytes synthesize and secrete TGF-/3 following cell activation predominantly in a latent form, although under certain circumstancesthey may secrete active TGF-0. That TGF-0 is an important molecule in immune regulation and inflammation is supported by studies where exogenous TGF-/3 has been shown to suppress experimental autoimmune diseases in rodents and to impair immune reactivity. In one particular instance TGF-@appears to mediate the physiologic immunosuppressioninduced following oral tolerance. Finally, two human diseases caused by the human retroviruses HIV and HTLV-1 are characterized by viral infection of lymphoid cells and immune suppression, and in both instances there is an apparent overproduction of X F - p . Acknowledgments The author would like to thank Drs. Michael Sporn and Anita Roberts for their help on the initial TGF-/3 studies; Dr. Anthony Fauci for his encouragement and assistance over the years; and Ms.Mary Rust for her excellent editorial assistance. References 1 Roberts AB, Frolik CA,Anzano MA, Sporn MB. Transforming growth factors from

neoplastic and non-neoplastic tissues. Fed Proc 1983;42:2621. 2 Anzano MA, Roberts AB,Smith JM, Sporn MB, DeLarco JE. Sarcoma growth factor from conditioned medium of virally transformed cells is composed of both type CY and type P transforming growth factors. Proc Natl Acad Sci USA 1983;80:6264-6267.

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3 Assoian RK, Komoriya A, Meyers CA,Smith DM, Sporn MB. Transforming growth factor-@in human platelets: identification of a major storage site, purification, and characterization. J Biol Chem 1983;258:7155-7160. 4 Frolik CA,Dar LL, Meyers CA,Smith DM, Sporn MB. Purification and initial characterization of a type @transforminggrowth factor from human placenta. Prcx Natl Acad Sci USA 1983;80:3676-3680. 5 Roberts A, Anzano M, Meyers C, et al. Purification and properties of a type 0 transforming growth factor from bovine kidney. Biochemistry 1983;22:5692-5698. 6 Derynck RR, Jarret JA, Chen EY, et al. Human transforming growth factor-@complementary DNA sequence and expression in normal and transformed cells. Nature 1985;316:701-705. 7 Kryceve-Martinerie C, Lawrence DA, Crochet J, Jullien P, Vigier P. Further study of @-TGF release by virally transformed and non-transformed cells. Int J Cancer 1985;35:553-557. 8 Kehrl JH, Wakefield LM, Roberts AB, et al. The production of TGF-@by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 1986;16311037-1050. 9 Assoian RK, Fleurdelys BE, Stevenson HC, et al. Expression and secretion of type p transforming growth factor by activated human macrophages. Proc Natl Acad Sci USA l987;84:6020-6023. 10 Wakefield LM, Smith DM, Flanders KC, Sporn MB. Latent transforming growth factor-@from human platelets. J Biol Chem 1988:263:7646-7654. 11 Kanzaki T, Olofosson A, Moren A, et al. TGF-@binding protein: a component of the large latent complex of TGF-@with multiple repeat sequences. Cell 1990;61: 1051-1061. 12 Lyons RM, Keski-Oja J, Moses HL. Proteolytic activation of latent transforrnig growth factor-@from fibroblast-conditionedmedium. J Cell Biol 1987106;1659-1665. 13 Seyedin SM, Segarini PR, Rosen DM, Thompson AY, Bentz H, Graycar J. Cartilageinducing factor-@is a unique protein structurally and functionally related to transforming growth factor-beta. J Biol Chem l987;262:1946-1949. 14 Derynck R, Lindquist PB, Lee A, et al. A new type of transforming growth factor-@, [email protected] J l988;73737-3743. 15 Jakowlew SB, Dillard PJ, Kondaiah P, Sporn MB, Roberts AB.Complementary deoxyribonucleic acid cloning of a novel transforming growth factor-@messenger ribonucleic acid from chick embryo chondrocytes. Mol Endocrinol 1988;2:747-755. 16 Jakowlew SB, Dillard PJ, Sporn MB, Roberts AB. Complementary deoxyribonucleic acid cloning of an mRNA encoding transforming growth factor-@4 from chicken embryo chondrocytes. Mol Endocrinol 1988;2:1186-1195. 17 Kondaiah P, Sands MJ, Smith JM, et al. Identification of a novel transforming growth factor-@(TGF-@5)&A in Xenopus luevis. J Biol Chem 1990;265:1089-1093. 18 Lyons RM, Moses HL. Transforming growth factors and the regulation of cell proliferation. Eur J Biochem 1990;187:467-473. 19 Massague J. The transforming growth factor-@family. Annu Rev Cell Biol 1990;6: 597-641.

20 Roberts AB, Sporn MB. The transforming growth [email protected]: Sporn MB, Roberts AB, Heidelberg FRG,eds. Handbook of Experimental Pharmacology. Peptide Growth Factors and Their Receptors. 1990:4l9-472. 21 Smith KA. Interleukin-2: inception, impact, and implications. Science 1986;240: ll69-1176. 22 Kehrl JH, Muraguchi A, Butler JL, Falkoff RJM, Fauci AS. Human B cell activation, proliferation and differentiation. Immunological Reviews 1984;78:78-96.

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23 Kishimoto T, Hirano T. Molecular regulation of B lymphocyte response. Ann Rev Immunol 1988;6:485-512. 24 Kehrl JH, Roberts AB,Wakefield LM, Jakowlew S, Sporn MB, Fauci AS. Transforming growth factor P is an important immunomodulatory protein for human B lymphocytes. J Immunol 1986;137:3855-3860. 25 Wahl SM, Hunt DA, Song HL, et al. Transforming growth factor-p is a potent immunosuppressive agent that inhibits IL-I dependent lymphocyte proliferation. J Immunol 1988;140:3026-3032, 26 Rook AH, Kehrl JH, Wakefield LM, et al. Effects of transforming growth factor-p on the functions of natural killer cells: depressed cytolytic activity and blunting of interferon responsiveness. J Immunol 1986;136:3916-3920. 27 Kuppner MC, Hamou MF, Bodmer S, Fontana A, De Tribolet N. The glioblastomaderived suppressor factor/transforming growth factor inhibits the generation of lymphokine activated killer (LAK) cells. Int J Cancer 1988;42:562-567. 28 Smeland EB, Blomhoff HK, Holte H, et al. Transforming growth factor type 6 (TGF-p) inhibits G1 to S transition, but not activation of human B lymphocytes. Exp Cell Res 1987;171:213-222. 29 Kehrl JH, Taylor A, Kim SJ, Fauci AS. Transforming growth factor-6 is apotent negative regulator of human lymphocytes. Ann NY Acad Sci 1991;628:345-353. 30 Laiho M, DeCaprio JA, Ludlow JW,Livingston DM, Massaque J. Growth inhibition by V3F-p linked to suppression of retinoblastoma protein phosphorylation. Cell 1990;62:175-185. 31 DeCaprio, JA, Ludlow JW,Lynch D, et al. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell 1989;58: 1085-1095. 32 Buchkovich K, Duffy LA, Harlow E. The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell 1989;58:1097-ll05. 33 Pietenpol JA, Holt JT, Stein RW, Moses HL. Transforming growth factor 01 suppression of c-myc gene transcription: role in inhibition of keratinocyte proliferation. Proc Natl Acad Sci USA 1990;87:3758-3762. 34 Kehrl JH, Thevenin C, Rieckmann P, Fauci AS. Transforming growth factor-p suppresses human B lymphocyte immunoglobulin production by inhibiting synthesis and the switch from the membrane form to the secreted form of immunoglobulin mRNA. J Immunol 1991;146:4016-4022. 35 Ranges GE, Figari IS, Espevik T, Palladino MA. Inhibition of cytotoxic T cell development by transforming growth factor-6and reversal by recombinant tumor necrosis factor-a. J Exp Med 1987;166:991-998. 36 Lee G, Ellingsworth LR, Gillis S, Wall R, Kincade PK. 6 transforming growth factors are potential regulators of B lymphopoiesis. J Exp Med l987;166:1290-1299. 37 Coffman RL, Lebman DA, Shrader B. Transforming growth-@specifically enhances IgG production by lipopolysaccharide-stimulatedmurine B lymphocytes. J Exp Med 1989;170:1039-1044. 38 h f m c G, Lefranc MP. Regulation ofthe immunoglobulingene transcription. Biochimie 1990;72:7-17. 39 Klemsz MJ, McKercher SR, Celada A, Van Beveren C, Maki RA. The macrophage and B cell-specific transcription factor PU.1 is related to the efs oncogene. Cell 1990;61:113-l24. 40 Fbngubala JMR, Atchison ML. Functional characterization of the developmentally controlled immunoglobulin kappa 3 'enhancer: regulation by Id, a repressor of helixloophelix transcription factors. Mol Cell Biol 1991;ll:1040-1047.

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