Do human TH1 and TH2 CD4+ clones exist?

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D o human T H I and TH2 CD4 + clones exist ? J.E. de Vries, R. de Waal Malefyt, H. Yssel, M.-G. Roncarolo and H. Spits Department o f Human Immunology, D N A X Research Institute, 901 California Avenue, Paio Alto, CA 94304-1104 (USA)

Introduction Mouse CD4+ helper T cells can be distinguished by their lymphokine production patterns. Two distinct, mutually exclusive lymphokine production profiles were defined. Type 1 T helper (TH1) cells produce IL2, IFN-~ and TNF[3, whereas type 2 T helper (TH2) cells secrete IL4, IL5, IL6 and ILl0 (Mosmann et aL, 1986; Cherwinski et ai., 1987). The existence of THI and TH2 cells was originally demonstrated in vitro (Mosmann et aL, 1986). More recently, THI and TH2 immune responses were also shown to occur in vivo (Mosmann and Coffman, 1989; Street et al., 1990). Immune responses against parasites in particular appear to represent stable TH 1 or TH2 pathways, depending on the mouse strain studied (reviewed in Mosmann and Coffman, 1989). Generally, TH1 responses are preferentially associated with delayed-type hypersensitivity (DTH), whereas TH2 responses are associated with high antibody levels (Mosmann and Coffman, 1989). In addition to T-cell clones that fit in the THI or TH2 group, murine CD4+ T-cell clones have been described that have the capacity to produce virtually every known lymphokine (Street et al., 1990). The latter CD4+ T-cell clones, designated TH0 cells, are similar to the majority of human CD4+ T-cell clones obtained from healthy donors which, as will be

discussed here, cannot be readily divided into TH1 or TH2 cells. Lymphokine production profiles of human T-cell clones obtained from healthy donors The vast majority of CD4 + T-cell clones with well defined specificities, established from healthy donors, including T-cell clones specific for class II MHC antigens, tetanus toxin, Der pI (which is the major allergen in extracts of the house dust mite Dermatophagoides pteronyssimus), and purified protein derivative (PPD) produced IL2, IL4, IL5, IFNI" and GM-CSF, following polyclonal activation (Paliard et al., 1991 ; Bacchetta et aL, 1990; Yssel et ai., 1990). In addition, most clones of a smaller panel expressed mRNA for IL3, IL6, TNF0q TNF[3, G-CSF and M-CSF, indicating that these clones do not fit into the TH1 and TH2 lymphokine production patterns of murine CD4* T cells, but resemble more the "TH0-1ike" pa:tern described for mouse T-cell clones obtained from non-immunized mice (Street et al., 1990). The "TH0-1ike" lymphokine production profile of the human CD4+ T-cell clones seems not to be the result of selection induced by the culture procedures. CD4+ T-cell clones obtained from immunized or n o n - i m m u n i z e d donors via different cloning procedures, using different activation

protocols and feeder cell mixtures, generally fit into this category. The capacity to produce IL2, IL4 and IFN1- was observed not only after polyclonal activation, but also after activation by (allo)antigen, presented by different types of antigen-presenting cells (APC). However, considerable q,-antitative, and occasionally qualitative, differences in lympholdne production could be observed when different p-lyclonal activators or different types of APC are used, indicating that different modes of activation (that were optimal for each situation) can drastically change the ratios of lymphokines produced (de Waal Malefyt et al., 1991a). This may have important biological consequences (see below). One important implication of these findings is that one has to be cautious in claiming defective lymphokine production by certain T-cell clones. In our experience, lack of production of a certain lymphokine following only one, or even two, particular modes of activation does not allow any firm conclusions to be made about the capacity of a T-cell clone to produce certain lymphokines. Human ILl0 is produced by "TH0-1ike" cells Recently, M o s m a n n and Moore (Fiorentino et al., 1989; Moore et al., 1990) characterized a novel cytokine, cytokine-

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synthesis-inhibiting factor (CSIF) or ILl0. This cytokine is produced by TH2 cells in the mouse and cross-regulates TH 1 cell functions. ILl0 inhibits proliferation and cytokine production of THI cells, induced by activation via antigen and using macrophages as APC. Interestingly, human ILl0 is produced by CD4+ T-cell clones that do not belong to the TH2 type (Vieira et al., 1990), but reflect the general "TH0-1ike" CD4+ T-cell population (Street et al., 1990). A limited panel of CD4+ T-ceU clones, including clones specific for class II MHC allo-antigens, the Derpl allergen, or tetanus toxin, all produced IL2, IFN?, IL4, and expressed ILl0 mRNA, following polyclonal or antigenic stimulation (de Waal Malefyt et ai., 1991b). For example, the human CD4 + tetanus-toxin-specific T-cell clone from which an IL10-encoding eDNA was isolated is well characterized and produces, in addition to ILl0, IL2, IL4, IL5, IFNy and GM-CSF, and expresses IL3, IL6, TNF~, TNF[3, M-CSF and GCSF mRNA (Roncarolo et al., 1988). Sub~tq o f C D 4 + T-cell clones in human disease Despite the fact that the vast majority of CD4+ T-cell clones from healthy donors produce IL2, IFNy, IL4, and IL5, exceptional subpopulations have been reported. Bacchetta et al. (1990), showed that a series of host-reactive CD4 + T-cell clones isolated from a transplanted severe combined immunodeficiency (SCID) patient had a selective defect in their capacity to produce IL4. The lack of IL4 synthesis was firmly established by using different modes of activation and PCR analysis. These host-reactive CD4 + cells produced levels of IL2, ILS, IFNy and GM-CSF that were in the normal range. In addition, they expressed IL3, IL6, TNFa and G-CSF mRNA following activation. Thus, subsets of CD4+

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T-cell clones exist that have a selective defect in IL4 synthesis, but these are rare in our experiments. It is well established that IL4 induces IgE synthesis both in vitro and in vivo (Coffman et al., 1986; Finkelman et al., 1986; P~ne et ai., 1988). This induction of IgE synthesis by IL4 is suppressed by IFN T ( C o f f m a n and Carty, 1986; Finkelman et al., 1986; P6ne et al., 1988; Finkelman et al., 1988; King et ai., 1989). Because of its capacity to induce IgE switching in B cells (Lebman and Coffman, 1988; Rothman et al., 1990; Gauchat et al., 1990), we hypothesized that excessive production of IL4 by allergen-specific CD4+ T cells could be responsible for enhanced IgE synthesis in atopic patients. We showed that CD4+ T-cell clones specific for DerpI established from atopic patients allergic to house-dust mite produced excessive levels of IL4, but normal levels of IFN T and IL2 (Yssel et al., 1991). The extremely high levels of IL4 production were observed not only after activation by allergen and APC, but also after polyclonal activation or activation via the CD2 pathway, indicating that high IL4 production is an intrinsic property of these Derpi-specific T cells obtained from atopic patients. In contrast, non-DerpI-specific CD4+ T-cell clones established from the same patients and DerpIspecific T-cell clones obtained from a non-atopic donor produced normal levels of IL4, IFN~. and IL2. Despite their normal level~ of IFN T production, only the highIL4-producing T-cell clones or their supernatants induced IgE synthesis by normal B cells. Although these T-cell clones still fit the "TH0-1ike" phenotype, they are, because of their aberrant IL4/IFNy production ratios, functionally different. Although it remains to be established, it is tempting to speculate that excessive IL4 production by allergenspecific T-cell clones is responsible for the enhanced total, and

allergen-specific IgE synthesis observed in atopic patients which results from activation o f "bystander-" and antigen-specific B cells, respectively (Hirohata et al., 1988; Julius et al., 1988; Whalen et al., 1988). Thus, allergen-specific CD4+ T cells that have the intrinsic capacity to produce excessive amounts of IL4 are selected in atopic patients with elevated serum IgE levels. Murine studies have indicated that selection of TH1 or TH2 cells can be directed by the class II MHC haplotype (Murray et al., 1989). However, selection of high IL4 producers in atopic patients seems not to be determined by particular peptide class II MHC haplotype associations, since the DerpI-specific T-cell clones obtained from a healthy donor, which produced normal levels of IL-4, recognized the same peptide in the same restriction element as did the high-IL4-producer T-cell clones obtained from one of the patients Cgssel et al., 1991). Romagnani et al. (see this volume) reported relatively large subsets of human CD4+ T-cell clones that produced IL4 and IL2, ing polyclonal activation (Maggi et al., 1988; reviewed in Romagnani, 1990). These CD4+ T-cell clones with unknown specificities were isolated from polyclonally activated peripheral blood mononuclear cells from patients suffering from parasite infections and from other patients with elevated serum IgE levels. More recently, Wierenga et al. (1990) described allergenspecific CD4+ T-cell clones isolated from atopic patients which also belonged to this subset of IL4- and IL2-producing T-cell clones that fail to synthesize IFNT. Since these latter authors used low numbers of T cells and only antigen-specific, not polyclonal stimulation protocols, we do not know whether defective IFNT production is an intrinsic property of their clones, or whether it is associated with activation by specific antigen. In ad-

CD4 + T-CELL SUBSETS: DIFFERENTIATION AND FUNCTION

dition, both laboratories reported large proportions of CD4 + T-cell clones generated from healthy donors that failed to produce detectable levels of IL4 after antigenic or polyclonal activation (Maggi et al., 1988; Romagnani, 1990; Wierenga et al., 1990). These latter data are at variance with our results which indicated that the vast majority of human CD4 + T-eell clones have the capacity to produce IL4, provided they are optimally activated. Therefore, at this stage, it is not clear whether CD4+ T-cell clones obtained from healthy donors that fail to produce IL4 represent a particular subset, or that the lack of IL4 production is related to the mode of activation used. This point needs to be investigated more thoroughly. The information gained thus far tends to indicate that, particularly in disease situations, subsets of human CD4+ T cells seem to exist with lymphokine production patterns that are different from the "TH0-1ike" cells. This notion was further supported by analysing CD4 + T-cell clones specific for the 65-kDa heat shock protein (HSP) of Mycobacterium leprae, isolated from patients with tuberculoid leprosy. This form of leprosy is characterized by high cellular immunity to M . ieprae and immunopathology due to delayed hypersensitivity. Most of these clones produced excessive levels of IFNT and approximately half of them failed to produce detectable levels of IL4 and IL5, as compared to CD4+ T-cell clones specific for antigens different from the 65-kDa HSP, obtained from the same patients and as compared to other control CD4 + T-cell clones (Haanen et al., 1990). The levels of IL2 production by the 65-kDa HSPspecific CD4+ T-cell clones were in the normal range. This "THl-like" lymphokine production profile of some of these clones was a stable property of these cells, because excessive IFN,f and reduced or absent IL4 and IL5 synthesis were also observed after ac-

tivation by ConA. However, preliminary data indicated that many 65-kDa HSP-specific CD4 ÷ T-cell clones isolated from peripheral blood mononuclear cells of healthy individuals also failed to produce detectable IL4 and IL5 suggesting that this cytokine production profile is an intrinsic property of 65-kDa HSPspecific T cells and is not associated with tuberculoid leprosy.

Conclusions The vast majority of human CD4 + T-cell clones obtained from b o t h immunized and nonimmunized healthy donors produce IL2, IFNT, IL4 and IL5, and do not fit in the T H I or TH2 T-cell subsets described for murine CD4+ T helper ceils. The capacity of human CD4+ T-cell clones to produce a whole array of lymphokines indicates that these cells resemble more the mouse TH0 subset that has been isolated from non-immunized mice. It is of interest to note that ILl0 (which is a TH2- and not THl-cell product in the mouse) is also produced by "TH0-1ike" cells in man. Although most human CD4 + T-cell clones have the intrinsic capacity to produce IL2, IFNT, IL4, IL5 and ILl0 (which has been less extensively tested), major quantitative, and occasionally qualitative, differences in lymphokine production levels were observed alLOt d , fer~m modes of activation. This implies that careful analysis of supernatants followiag stimulation, using different actieation protocols, is required before any conclusions can be drawn regarding the exact lymphokine production phenotype of human T-cell clones. Nevertheless, subsets of human CD4÷ T-cell clones based on differences in lymphokine production were isolated from patients in different disease situations. A series of host-reactive CD4 + T-cell clones that selectively failed

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to produce IL4 were isolated from a transplanted SCID patient. Such clones may be rare or even absent in healthy individuals, but their frequency in this patient were high. Why these clones fail to produce IL4 is unknown, but it may be associated with their particular biological function. The allergenspecific CD4+ T cells isolated from atopic donors still fit in the "TH0-1ike" subset, but because of their excessive levels o f IL4 production they were, in contrast to control T-cell clones, able to induce IgE synthesis, indicating that they were functionally different. The 65-kDa H S P - s p e c i f i c CD4 + T-cell clones isolated from patients with the tuberculoid form of leprosy resembled the murine TH 1 subset most. These clones all produced high levels of IFNT and half of the clones failed to produce detectable levels of IL4 and IL5, whereas the other half secreted reduced levels of IIA and IL5. It has been proposed that murine TH0 cells upon stimulation by strong immunogens, differentiate to THI or TH2 cells in vivo (Street et al., 1990). Therefore, it is possible that the low IL4 and IL5 producers represent intermediate stages ,vhich, following chronic antigenic stimulation, can differentiate into " T H l-like" cells that lost their capacity to produce IL4 and IL5. Similarly, the allergen-specific T-cell clones isolated from atopic donors that produce excessive amounts of IL4, but normal levels of IFN~, and IL2, may also represent different intermediate stages that finally differentiate into subtypes of T cells that lose the capacity to produce IFNT. The mechanisms by which these subsets of CD4 + T cells are selected remain to be determined, but chronic exposure to allergen or activation of T cells by particular allergen-derived peptide class II M H C haplotype combinations seem not to play a role in the selection process in atopic patients, since allergen-specific CD4 + T-

62 cell clones isolated from a nonatopic healthy donor, who was also chronically exposed to allergen and recognized the allergen in the same class II M H C restriction element, as did the T-cell clones o f one of the patients, produced norreal levels o f IL4. In contrast, preliminary data obtained with 65-kDa HSP-specific CD4 + T-cell clones isolated from a healthy individual indicated that the lack o f IL4 and IL5 production is a specific property o f T-cell clones recognizing 65-kDa H S P and is not associated with the disease. More extensive studies on CD4 + T-cell clones derived from atopic patients or patients suffering from infectious diseases are required to determine whether selection o f subsets o f CD4+ T cells with stable, aberrant lymphokine production phenotypes does indeed occur frequently in vivo. Determination o f the factors that set the stage for the development of TH1, TH2, as well as other CD4+ T helper subsets in vivo is a major question which presently needs to be addressed. Acknowledgements The DNAX Research Institute of Molecularand CellularBiologyis supported by Schedng-PloughCorporation. References Bacchetta, R., de Waal Malefyt, R., Yssel, H., Abrams, J., de Vries, J.E., Spits, H. & Roncarolo, M.-G. (1990), Host-reactive CD4 + and CD8 + T-cell clones isolated from a human chimera produce IL-5, IL-2, IFNT, and GM-CSF, but not IL-4. J. Immunol., 144, 902-909. Cherwinski, H.M., Schumacher, J.H., Brown, K.D. & Mosmann, T.R. (1987), Two types of mouse helper T cell clone. - - III. Further differences in lymphokine synthesis between THI and TH2 clones revealed by RNA hybridization, fuactional monospecific bioassays and monoclonal antibodies. J. exp. Med., 166, 129-1244.

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Coffman, R.L. & Carty, J. (1986), A T cell activity that enhances polyclonal IgE production and its inhibition by IFN T. J. Immunoi., 136, 949-954. Coffman, R.L., Ohara, J., Bond, M.W., Carty, J., Zlotnik, A. & Paul, W.E. (1986), B cell stimulatory factor-I enhances the IgE response of lipopolysaccharideactivated B cells. J. bnmunol., 136, 4538-4541. De Waal Malefyt, R., Yssel, H., Roncarolo, M.-G., Spits, H. & de Vries, J.E. (1991a), Quantitative and qualitative differences in lymphokine production profiles by human CD4 + T cell clones following different modes of activation (submitted). De Waal Malefyt, R., Yssel, H., Roncarolo, M.-G., Vieira, P., Moore, K.W., Spits, H. & de Vries, J.E. (1991b), IL-10 mRNA expression in human (allo) antigen specific CD4 + non-THl, non-TH2 cells (manuscript in preparation). Finkelman, F.D., Katona, I.M., Urban, J., Snapper, C.M., Ohara, J. & Paul, W.E. (1986), Suppression of in vivo polyclonal IgE responses by monoclonal antibody to lymphokine BSF-1. Proc. nat. Acad. Sci. (Wash.), 83, 9675-9678. Finkelman, F.D., Katona, I.M., Mosmann, T.R. & Coffman, R.L. (i988), IFNT regulates the isotypes of Ig secreted during in vivo humoral immune responses. J. Immunol., 140, 10221027. Fiorentino, D.F., Bond, M. & Mosmann, T.R. (1989), Two types of mouse helper cells. - - IV. TH2 cells secrete a factor that inhibits cytokine production by THI clones. J. exp. Med., 170, 2081-2095. Gauchat, J.F., Lebman, D.A., Coffman, R.L., Gascan, H. & de Vries, J.E. (1990), Structure and expression of germline ¢ transcripts in human B cells, induced by interleukin 4 to switch to IgE production. J. exp. Med., 172, 463-473. Haanen, J., de Waal Malefyt, R., de Vries, R.R.P. & Spits, H. (1991), Type l-like human CD4 + T cell clones specific for the 65 kD hsp of Mycobacterium lepra (manuscript in preparation). Hirohata, S., Jelinek, D.F. & Lipsky, P.E. (1988), T-cell-dependent ac-

tivation of B cell proliferation and differentiation by immobilized monoclonal antibodies to CD3. J. Immunoi., 140, 37263744. Julius, M.H., Rammensee, H.G., Ratcliffe, M.J., Lamers, M.C., Langhorne, J. & Kohler, G. (1988), The molecular interactions with helper T cells which limit antigen specific B cell differentiation. Europ. J. Immunol., 18, 381-386. King, C.L., Galiin, J.I., Malech, H.L., Abramson, S.L. & Nutman, T.B. (1989), Regulation of immunoglobulin production in hyperimmunoglobuUn ~ recurrent-infection syndrome by interferon T. Proc. nat. Acad. Sci. (Wash.), 86, 10085-10089. Lebman, D.A. & Coffman, R.L. (1988), Interleukin-4 causes isotype switching to IgE in T-cellstimulated clonal B cell cultures. J. exp. Med., 168, 853-862. Maggi, E., Del Prete, G., Macchia, D., Parronchi, A., Tiri, I., Chr6tien, I., Ricci, M. & Romagnani, S. (1988), Profilesof lymphokine activity and helper functions for IgE in human T cell clones. Europ. J. lmmunoL, 18, 10451054. Moore, K.W., Vieira, P., Fiorentino, D.F., Trounstein, M.L., Khan, T.A. & Mosmann, T.R. (1990), Homology of cytokine synthesis inhibit factor (iL-i0) to the Epstein-Barr virus gene BCRFI. Science, 248, 1230-1234. Mosmann, T.R., Cherwinski, H., Bond, M.W., Giedlin, M.A. & Coffman, R.L. (1986), Two types of murine helper T cell clones. - I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol., 136, 2348-2357. Mosmann, T.R. & Coffman, R.L. (1989), THI and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Ann. Rev. lmmunol., 7, 145-173. Murray, J.S., Madri, J., Tire, J.~ Carding, S.R. & Bottomly, K. (1989), MHC control of CD4 + T cell subset activation. J. exp. Med., 170, 2135-2140. Paliard, X., de Waal Malefyt, R., Yssel, H., Blanchard, D., Chr6tien, I., Abrams, J., de Vries, J.E. & Spits, H. (1990), Simultaneous production of IL-2, IL-4 and IFN-T by human CD4 + and

CD4 + T-CELL SUBSETS: DIFFERENTIATION AND FUNCTION

CD8 + T cell clones. J. lmmunol., 144, 849-855. P~ne, J., Rousset, F., Bri~re, F., Chr~tien, I., Bonnefoy, J.Y., Spits, H., Yokota, T., Arai, N., Arai, K.I., Banchereau, J. & de Vries, J.E. (1988), IgE production by human B cells is induced by IL-4 and suppressed by interferons y, and prostaglandin E2. Proc. nat. Acad. Sci. (Wash.), 85, 6880-6884. Romagnani, S. 0990), Regulation and deregulation of human IgE synthesis. Immunol. Today, ll, 316-321. Roncarolo, M.G., Yssel, H., Touraine, J.L., Bacchetta, R., Gebuhrer, L., de Vries, J.E. & Spits, H. (1988), Antigen recognition by MHC-incompatible cells isolated from a human mismatched chimera. J. exp. Med., 168, 2139-2152. Rothman, P., Chen, Y.Y., Lutzker,

S., Li, S.C., Stewert, V., Coffman, R. & Alt, F.W. (1990), Structure and expression of germline Ig heavy chain ~ transcripts: IL-4 plus LPS-directed switching to C~. Mol. Cell. Biol., 10, 1672-1680. Street, N.E., Schumacher, J.H., Fong, A.T., Bass, H., Fiorentino, D.F., Leverah, J.A. & Mosmann, T. (1990), Heterogeneity of mouse helper T cells. Evidence from bulk cultures and limiting dilution cloning for precursors of THI and TH2 cells. J. Immunoi., 144, 1629-1639. Vieira, P., de Waal Malefyt, R., Dang, M.W., Johnson, K.E., Kastelein, R., Fiorentino, D.F., de Vries, J.E., Roncarolo, M.-G., Mosmann, T.R. & Moore, K.W. (1990), Isolation and expression of human cytokine synthesis inhibitory factor (CSIF/IL-10) cDNA clones: homology to

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Epstein-Barr virus open reading frame BCRFI. Proc. nat. Acad. Sci. (Wash.) (in press). Whalen, B.J., Tony, H.-P. & Parker, D.C. (1988), Characterization of the effector mechanism of help for antigen-presenting and bystander resting B cell growth mediated by I-A-restricted Th2 helper T cell lines. J. Immunoi., 141, 2230-2239. Wierenga, E.A., Snoek, M., de Groot, C., Chr6tien, I., Bos, J.D., Jansen, H.M. & Kapsenberg, M.L. (1990), Evidence for compartmentalizatioa of functional subsets of CD4 + T lymphocytes in atopic patients. J. Immunol., 144, 4651-4656. Yssel, H., Gascan, H., Schneider, P., Spits, H. & de Vries, J.E. (1991), Excessive IL-4 production by allergen specific T cell clones from atopic patients is associated with enhanced IgE synthesis(submitted).

S. R o m a g n a n i , E. Maggi, P. Parronchi, D. Macchia a n d M . - P . Piccinni Department o f Clinical Immunology and Allergology, University o f Florence. Istituto di Clinica medica 3, Policlinico di Careggi, 50134 Florence (Italy)

Introduction The interaction between environmental allergens and the immune system is critical to the development of specific human allergy. This interaction is presumably initiated by uptake and presentation of allergens by classII-MHC-positive accessory cells to allergen-specific helper T lymphocytes. Activated helper T cells then induce B lymphocytes to produce

allergen specific antibodies mainly belonging to the IgE class. However, in individuals genetically determined to recognize allergenic epitopes (Marsh et al., 1982; Ansari et al., 1989), the origin of the preferential IgE antibody production is still unclear. In the last few years, a pathway of lgE regulation, essentially based on the reciprocal activity of interleukin-4 (IL4) and g a m m a - i n t e r f e r o n

(IFNy), has been discovered in mice (Coffman and Carty, 1986; Coffman et al., 1986; Finkelman et al., 1986). Further research has provided substantial information also on the mechanisms that regulate human IgE synthesis and has allowed significant advances towards the knowledge of alterations responsible for its deregulation in allergic disorders (Romagnani, 1990).