Immunobiol., vol. 185, pp. 268-280 (1992)
Departments of Pathology and Internal Medicine!, University of Iowa, Iowa City, USA
TCR Induced Expression of Fc Receptors on Murine T Cell Subsets in vitro and in vivo MATYAS SANDOR, GEORGEA. COOK!, RANDyE. SACCO, RANJIT MATHEW!, ALEXANDERIBRAGHIMOV,]OEL V. WEINSTOCK!, and RICHARD G.LYNCH
Introduction T cells expressing Fc receptors have the unique potential to use two different cognate systems, TCR and the immunoglobulins bound to Fc receptors. The functional consequences of this dual recognition have not been studied extensively, despite the obvious implications concerning T cell activation and signalling and the interactions of T cells with other cells. Such studies require knowledge about the nature of Fc receptors on T cells. While Fc receptors for each immunoglobulin heavy chain isotype can be expressed on T cells (rev LYNCH and SANDOR, 1990), there is little information on structure, distribution, regulation, and function of these receptors. In contrast, much progress has been made in the study of Fc receptors on other cell types, such as B lymphocytes, macrophages and granulocytes. In this review, we summarize our results on Fc receptor expression on various subpopulations of murine T cells, concentrating on the CD4+ and on the y/b TCR + T cell subsets. Elsewhere in this issue a more detailed review will focus on Fn-:RII(CD23) (LYNCH et aI., 1992), so the emphasis of the present review will be on Fca, Fc!! and Fcy receptors. Detailed studies of Fc receptors on B cells have resulted in the establishment of certain principles about the structure, regulation and function of Fc receptors. Our goal is to examine how these principles might apply to Fc receptors on T cells. Some of our studies used in vitro approaches, and others involved observations in vivo in Schistosoma mansoni-infected mice. Murine schistosomiasis provides an in vivo model to study activated T cells CD4+ T cells play an important role in Schistosoma mansoni induced immune responses GAMES and SHER, 1990). IFN-y produced by Th1 CD4+ T cells in the early phase of infection can provide some degree of resistance. However in the presence of chronic parasite infection, CD4+ T cells that produce a lymphokine pattern characteristic of Th2 CD4+ T cells appear to dominate. The elaboration of IL-4 and IL-S in infected animals results in higher expression of MHC class II antigens, FC!::RII, and IL-2R on splenic
TCR Induced Expression of Fc Receptors on Murine T Cell Subsets . 269
B cells. Adult worms in the portal vein release large numbers of eggs some of which become lodged in the liver and induce the formation of granulomas (WEINSTOCK, 1987). The granulomas contain activated CD4+ T cells which produce the Th2 lymphokines IL-4, IL-5, IL-6 and IL-I0 which are important in granulomas formation. CD4 + T cells are the most numerous T cells in the granulomas, but other T cells, such as y/b T cells, are also present. As a percent of the total T cells, y/b TCR + T cells are enriched in these granulomas compared to lymphoid tissues or blood (SANDOR et ai., 1992). The compartmentalized distribution of different T cell populations in schistomiasis provides a model to examine Fc receptor expression on subsets of resting and activated T cell populations in vivo.
Constitutive expression of Fc receptors Virgin, resting, mature B cells express FcyRII(CD32), FCERII(CD23), FcaR, FCIlR and FcbR constitutively (LYNCH and SANDOR, 1990; LYNCH et ai., 1992). In primary and secondary lymphoid organs 95-99 % of the T cells have a resting phenotype (small size, IL-2R-, TfR-). Using anti-Fc receptor antibodies (2.4G2, b3b4), fluorescence labelled immunoglobulins, and cytofluorometry we found that normal resting T cells isolated from spleen, thymus, lymph nodes and Peyer's patch do not have Fc receptors detected with these methods. When incubated overnight with various doses of cytokines (IL-2, -4, -5, -6, IFN-a and -y, TNF-a, TGF-~) FcRs are not detected on normal spleen T cells. Schistosome-infected mice have enlarged spleens that contain a small fraction of activated T cells which secrete IL-4 and IL-5. The presence of these lymphokines alters the phenotypes of the accompanying B cells as shown by their elevated CD23, class II MHC and IL-2R expression. Overall, our studies indicate that, if Fc receptors are present on resting T cells their level is at least 10-100 times less than on resting B cells. While normal resting T cells express little or no Fc receptors, a panel of T cells clones, lines and lymphomas expressed high levels of FcaR (SANDOR et ai., 1990), and FCIlR (IBRAGHIMOV et ai., in preparation). The T cell lymphoma 549.1 was the first cell from which an FcR, FcyRII~1 (CD32) was cloned (RA VETCH et ai., 1986). These findings led us to investigate the relationship between T cell activation and FcR expression. Intraepithelial T cells seem to be different than T cells of the primary and secondary lymphoid organs, as they express Fc receptors prior to activation. The dendritic epithelial T cells (DEC) isolated from murine skin express a non-variant T cell receptor (Vy3Vbl) and bind 2.4G2 (antiFcyRIIIIII) antibody in the resting state. Many of the DEC clones we examined bound 2.4G2 and using antibodies that distinguished between the cytoplasmic tails of the FcyRII and FcYRIII or PCR primers specific for the intracellular portion of the various FcyRs, it was clear that the DEC clones expressed the FcyRIII (CDI6) isoform and not the FcyRII (CD32) isoform.
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B cells appear to use only one FcyR isoform, the FcyRII~I, while previous results suggest that various T cells use different forms of FcyRs. As the DEC cells represent the first wave of peripheralized T cells in ontogeny, their FcyR expression may be important in the skin of newborns as a defense against microbes. Since childbirth is accompanied by contamination of the child with maternal microbes, maternal IgG might be expected to contain antibodies directed to microbes that contaminate the newborn. Because IgE plays an important role in immune responses in the skin, it is of interest that the freshly-isolated DEC (but not phenotypically activated DEC clones) are CD23+ as shown by their ability to bind b3b4, a monoclonal anti-FcERII-CD23 antibody. It is conceivable that DEC, which have functional properties of cytotoxic cells, are involved in a novel form of IgE-dependent delayed hypersensitivity. Preliminary results suggest that resting y/bTCR + T cells at other epithelial sites are also FcR +. Vaginal y/b TCR+ cells express a nonvariant Vy4Vbl TCR and express FcyR before they are activated (IBRAGHIMOV et aI., unpublished observation). Similarly, a fraction of y/b TCR+ (VyS+ variant TCR+) intraepithelial lymphocytes in the intestinal tract express FcyR prior to activation.
In vitro activation through the antigen receptor modifies Fc receptor expreSSIon The crosslinking of surface immunoglobulins on B cells induces a transient upregulation of FcyRII-CD32 (AMIGO RENA et aI., 1989) and FCERIICD23 (GORDON and GUY, 1987). These changes occur early in the cell cycle, during the transition from GO to G 1 (CD23) and in early G 1 (CD32). Antigen-activated cells of the CD4+ Th2 clone DI0, which recognizes conalbumin in an H-2k context, express IgA, IgM and IgD receptors, but IgE and IgG FcRs are not detected by monoclonal antibodies. The gradual withdrawal of IL-2 from the medium results in a change in the phenotype of DIO cells. They become smaller and acridine orange staining indicates that they are in the resting phase of the cell cycle. The level of FcR expression was much lower on these DI0 cells than on activated DI0 cells. The resting DI0 cells re-enter the cell cycle after co culture with antigenprimed, irradiated spleen cells and the level of Fc receptor on the cells starts to rise by 6 h and reaches maximum 12 h after activation. Simultaneous acridine orange staining and FcR analysis has established that the induction of Fc receptors on D 10 cells is linked to the early G 1 phase of the cell cycle. The role of TCR in the induction of FcR was further emphasized by experiments where antibodies reacting with the TCR-complex had the same effect on FcR expression as did activation with antigen-pulsed APC. The close relationship between T cell activation and Fc receptor expression was shown in experiments where repeated cycles of TCR-mediated activation were followed by periods of resting the cells in IL-2 depleted media. This resulted in a concomitant fluctuation in FcR expression wherein activation
TCR Induced Expression of Fc Receptors on Murine T Cell Subsets . 271
up regulated receptor expression. It is important to note that there are CD4 + T cell clones and CD4+ T cells which do not express Fc receptors even after activation. This issue will be discussed later.
In vivo activation modifies Fc receptor expression Schistosoma mansoni-infected mice have developed numerous liver granulomas by 8 weeks after infection. These granulomas contain S-8 % CD4 + T cells which actively secrete lymphokines (including IL-4, -S, -6 and -10). The schistosome activated CD4 + T cells, like the activated CD4 + T cell clones examined in vitro, express high levels of IgA and IgM receptors. The resting CD4+ T cells in the spleens of schistosome-infected mice were found not to express FcR. These results provide an in vivo model verifying the in vitro findings which link Fc receptor expression to TCRmediated activation. Similar experiments were carried out to investigate the relationship of TCR-induced activation and FcR expression on y/o TCR+ cells. y/o TCR+ cells were prepared either from y/o TCR transgenic mice (DENT et al., 1990) or from cultures where polyclonal splenic or intestinal y/o TCR + cells had been selectively expanded and enriched by the addition of a mitogenic antiy/o TCR antibody (UC7-13DS). In a series of in vitro experiments we found that fresh, resting y/o T cR+ cells or postactivated, resting cells did not express Fc receptors. However the y/o cells expressed IgA, IgM and to a lesser extent IgG receptors after primary and secondary culture with a mitogenic anti-CD3£ antibody (14S-2CII). These experiments identified a relationship between the TCR-induced activation and FcR expression on y/o TCR + cells similar to that previously observed in studies of alB TCR+ CD4+ cells. The time dependence of FcR induction has been studied using spleen cells prepared from y/o TCR transgenic mice (Fig. I). Fresh spleen cells from transgenic mice are more heterogeneous in cell cycle position than the semisynchronized resting cells of the DIO clone. After 24 h of stimulation with mitogenic anti-CD3£ antibody, a portion of the y/o TCR+ spleen cells are still small, resting cells. These small y/o TCR + cells bind very little immunoglobulin, while the large, activated cells bind IgA, IgM and IgG. Cell enlargement is a step that relates to the entry into the G I phase of the cell cycle, and our finding with alB TCR+ and y/o TCR+ cells indicate that the induction of Fc receptors on T cells is an early activation event. Liver granulomas in Schistosoma-infected mice contain O.S-4 % y/o TCR + cells. These large, y1.1 TCR positive, transferrin receptor positive cells bind IgA, IgM and the 2.4G2 antibody. Resting, splenic y/o TCR+ cells from the same mice do not display FcR. Before leaving this section that deals with the relation of TCR-induced activation and FcR expression, some interesting findings should be discussed that indicate a somewhat more complex situation than has been
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Figure 1. Effect of anti-CD3E antibody on IgA binding by y/6 TCR + cells. Spleen cells isolated from y/o TCR transgeneic mice contain a high portion of y/oTCR+ cells (first left panel). Spleen cells were cultured in the presence of 10 ug/ml of mitogeneic anti-CD3E antibody. At various time points specified as h at the right upper corner of the panels, cells were double stained with anti-y/o TCR antibody (GL3) and IgA-FITC.
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TCR Induced Expression of Fc Receptors on Murine T Cell Subsets . 273
suggested. First, there are CD4 + and y/6 TCR + cells which do not express Fc receptors following TCR-mediated activation, a finding that will be discussed shortly. In addition, TCR-mediated signals in some cases are necessary but not sufficient to induce FcR expression. For example, primary activation of splenic y/6 TCR + cells induces Fcy R expression, but after additional in vitro culture, resting and then reactivation, these cells do not re-express FcR. In contrast, in vivo reactivation results in the reexpression of FcyR. Thus repeated, in vivo administration of mitogenic anti-TCR antibody induces the expression of FCERII on some CD4+ cells. Changing the activation protocol by doing the secondary stimulation in vitro results in the appearance of FeaR, and FCflR+, but not FCERII on CD4+ T cells. Thus, factors present in vivo, but missing in vitro are required for the induction of FCERII on CD4+ T cells. Another element of complexity is demonstrated by the observation that some y/6 TCR+ DEC express FCERII and FcyRIII while resting, but when activated with anti-CD3E antibody the FCERII is completely downregulated and FeaR and FCflR are induced. The loss of FCERII is exceptional since TCR-induced activation, when it influences FcR expression, typically brings about an increase in the level of FcR on the T cell.
Various lymphocyte subpopulations differ in their Fc receptor expreSSiOn Mature virgin murine CDS-(B2Iineage) B cells express FcRs for each of the five Ig isotypes while under the same conditions, CDS+ (B1Iineage) cells do not express FCERII -CD23 (W ALDSCHMlDT et al., 1991). The pattern of expressed FcRs also changes during different stages of B cell development (LYNCH et al., 1992). Upon activation, CD4+ T cells produce a series of lymphokines and the pattern of the secreted lymphokines defines the functions of these cells. Recent research has shown that CD4 + T cell populations differ in their lymphokine production (MOSMANN and COFFMAN, 1989). Virgin CD4+ T cells, when activated, produce large amounts of IL-2 but low levels of other lymphokines. Most activated CD4+ memory T cells secrete IL-2, IFN-y, IL-4, IL-S, IL-6, IL-10 and have been designated as Tho CD4+ cells. In the chronic presence of antigens, such as in parasite infections, the frequency of two types of CD4+ T cells increases. One of these subsets of CD4+ T cells is known as Th1 cells and they secrete IL-2 and IFN-y, but not IL-4, IL-S, IL-6 or IL-10. Th 1 cells are the effector cells of the DTH reaction. The other subset of CD4+ T cells, Th2 cells, secrete IL-4, IL-S, IL-6, IL-IO and one of their functions is to deliver «help» for B cells that induces their proliferation and differentiation. We have demonstrated Fc receptor expression on the various CD4 + T cell populations using fresh splenic cells as a source of virgin T cells and 22 characterized, long term
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CD4+ T cell clones as representatives of the ThO, Th1 or Th2 subsets. The TCR -activated Th2 and ThO clones were found to express a high level of multiple types of FcRs. IgA, IgM and IgD binding was the most common, but some clones also bound IgG or IgE. In contrast, Th1 clones consistently expressed very low levels of FcRs. Considering that each clone was activated by its respective antigen, these findings show that activation through the TCR induces FcR expression on Th2 and ThO cells, but in the case of CD4+ Th1 cells, activation either does not induce FcR expression or induces a much lower level of expression (SANDOR et al., 1990). The increasing evidence that the biochemical pathways of TCR-mediated signalling are different in Th1 and Th2 clones (GAJEWSKI et al., 1989) might be important in understanding the differences observed in Fc receptor expression on these T cells when activated. In the supernatants of normal spleen cells activated with mitogenic anti-CD3E antibody there are only trace amounts of the Th2 cytokines IL-4 and IL-S, and these activated CD4+ T cells bind only low quantities of IgA and IgM. However, the supernatants of anti-CD3E activated spleen cells from Schistosoma mansoni-infected mice contain 40-fold higher levels of IL-4 and soo-fold higher levels of IL5, and there is a S-10-fold increase in IgA and IgM binding by these CD4+ T cells. In the liver granulomas of Schistosoma-infected mice there are activated CD4+ cells which produce high levels of Th2 cytokines and avidly bind IgA and IgM (SANDOR et al., 1992). The in vivo injection of T cell activating antibodies (anti-CD3E) resulted in data similar to the findings in Schistosoma-infected mice in that the appearance of Th2 lymphokineproducing CD4+ cells coincides with the presence of CD4+ T cells expressing high levels of Fc receptors. An interesting feature of y/b TCR+ cells is their heterogeneity. Some y/b T cells are located in the epithelial layers of organs and are sessile, others that are found in lymphoid organs, in the circulation and at sites of inflammation are mobile cells. The TCR usage by y/b T cells at various anatomical sites is different. The y/b TCR + cells in the skin (DEC) express Vy3b1, the y/b TCR+ cells in the epithelium of the reproductive organs, the urinary tract and the tongue express Vy4bl, and the expression of these families of TCR genes by y/b T cells at these sites is invariant. At other anatomical sites the y/b TCR gene usage is quite variable. VyS TCR+ cells are located in the epithelium of the intestine (IEL), and Vyl.1, Vy1.2, and Vy2 TCR + cells are present in lymphoid organs and in the circulation. We are in the process of characterizing FcR expression of these cells and the results obtained to date clearly show that on various y/b TCR + cell populations different classes of FcRs are present. The availability of y/b TCR transgenic mice made it possible to investigate the resting y/b TCR+ cells in lymphoid organs (spleen, thymus, lymph nodes and Peyer's patches) and in blood. These cells do not express Fc receptors. The splenic y/b TCR+ cells activated with anti-CD3E in vitro and the yl.1 + cells activated in vivo in the granulomas expressed cell surface IgA, IgM and IgG Fc receptors.
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A fraction of fresh IELs express FcyR. The in vitro enrichment of IELs that followed TCR-mediated stimulation or restimulation resulted in two populations. One population had no detectable FeR expression but the other population bound IgA and IgM. It will be interesting to learn whether FcR expression relates to the functional subsets previously described for y/6 TCR+ IELs (LEFRANCOIS and GOODMAN, 1989). DEC freshly isolated from murine skin react with monoclonal antibodies specific for FcyR and FCERII. Activation of these cells with anti-CD3E antibody results in the loss of FcRdI and the appearance of IgA and IgM Fe receptors on a fraction of the cells. As previously found with other intraepithelial y/6 TCR+ cells, we have recently found that y/6 TCR+ cells in the vagina express FcyR (IBRAGHIMOV and LYNCH, unpublished). While the characterization of FcR distribution on various T cell subsets is not yet complete, it is already clear that the pattern of FeR expression on various T cell subpopulations is different.
Physical association between the antigen receptors and Fc receptors The crosslinking of the antigen receptors on B cells induces the cocapping of FcyRs (DICKLER, 1982) and Fq.lRs (SCHAIFF and LYNCH, 1992).
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Figure 2. Cocapping of FcaR with TCR on DID cells. Laser confocal microscopy images show binding of IgA- FITC and anti-CD3E antibody. Cells were stained at 4°C with anti-TCR antibody. Staining was visualized using biotinylated anti-hamster IgG and Avidin-Texas red. Avidin-Texas red was used at 4 °C or 37 °C. Conditions that induced capping of the TCR resulted in cocapping of IgA FeR.
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LEE and CONRAD (1985) precipitated the murine FCERII from murine B cells previously treated with an extracellular crosslinker. The presence of membrane IgM and IgD in the FCERII precipitates suggested that the antigen receptors were associated with the FCERII on the surface membrane of the B cell. To assess the relationship between Fc receptors and antigen receptors on T cells, co-capping experiments have been carried out using the CD4 + Th2 clone D10. According to the images generated by laser confocal fluorescence microscopy, IgA and IgM bind to these cells with an evenly distributed pattern. The crosslinking of TCR with anti- V~8 antibody (F23.1) or anti-CD3E antibody (145-2C11) under conditions that allow for capping of the TCR induced the co-capping of the IgA and the IgM binding sites on these cells. The co-localization of the IgA receptors and the TCR is demonstrated in Figure 2. The association of these molecules is also detected on other T cells. The crosslinking of y/o TCR by GL3 antibody on mitogen-activated splenic y/o TCR+ cells induces the co-capping of IgA and IgM receptors (SANDOR et aI., 1992). The precise nature of the physical association of these molecules is not known. However, a direct association can not be excluded since in some cases FcRs and TCRs have been shown to form complexes. FcyRIII (CD16) appears to share a subunit (y) with the TCR complex (RAVETCH and KINET, 1991). A tyrosine kinase (fyn) has been shown to be associated both with the TCR-complex and with FCERII (SUGIE et aI., 1991). Elements of the TCR complex and some FcRs contain immunoglobulin-like domains, which in some molecules are shown to interact with each other. Such domain-domain interactIon might be involved in the TCR-FcR association. A less direct interaction can be suggested through TCR induced modification of the cytoskeletal system. An association of FcRs with the elements of the cytoskeletal apparatus has been reported (UHER et aI., 1982; GREENBERG, 1990). Immunoprecipitations of the crosslinked TCRs after using bifunctional crosslinkers might provide insight into the interaction of various FcRs and TCRs. TCR crosslinking is an important part of the T cell induction by APC, and of the cellular interactions that occur when effector functions such as help or killing are delivered. The accumulation of Fc receptors at these interfaces might enhance the interactions between the T cell FcRs and immunoglobulins.
Antigen receptor induced responses are modified by the Fc receptors It is well established, that under certain conditions crosslinking of the antigen receptors on B lymphocytes induces cellular differentiation and immunoglobulin production. This response can be modified to a large extent by ligands that bind to Fc receptors on the B lymphocytes.
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The simultaneous crosslin king of FcyRII and antigen receptors generates negative signals for the B cells (SINCLAIR, 1969). In addition, it has been suggested that the antigen receptor-FeaR interaction has a similar effect. In contrast, the crosslinking of Fc£RII and antigen receptors has an enhancing effect on antigen receptor-mediated induction (CAMPBELL et aI., 1991; WALDSCHMIDT et aI., 1991). In cognate T cell and target cell cytoconjugation the presented antigen crosslinks the antigen receptors. Conceivably, Fe receptors might colocalize with the TCRs at the interfaces of these cellular interactions. Surface immunoglobulins on B ceUs are concentrated at the T -B cell cognate interaction site (NOELLE et aI., 1990), and were the FcRs to colocalize they could interact with the surface immunoglobulins and provide additional signal transduction. To study the influence of Fc receptors on TCRmediated signals, we coated plates with anti-CD3£ antibody and TNPI0BSA, and measured cytokine mRNA levels in FeaR+, Fc~R+ DI0 Th2 cells in the presence or absence of anti-TNP IgA (MOPC 315) or anti-TNP IgM
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IL-4 IL-5 IL-6 Figure 3. The effect of IgA and IgM binding to their respective Fe receptors on the TCRinduced increase of IL-4, IL-5 and IL-6 mRNA in DIO cells. Tissue culture plates were coated with 10 ug/ml anti-CD3t:: antibody and then with 200 ug/ml TNPIO-BSA. The washed plates were further incubated with medium, 300 ug/ml anti-TNP IgA (MOPC 315) or 40 ug/ml antiTNP IgM (F5D6). DIO cells (I x 106 cell/ml) were cultured for 24 hours on the different plates and then RNA was precipitated from the harvested cells. After electrophoresis the blotted RNA was consecutively probed with lL-4, IL-5 and IL-6 eDNA.
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(F5D6). The interleukins IL-4, IL-5, IL-6 were selected for study because of their important role in B cell «help» by this clone. The Northern blots (Fig. 3) show a decrease in the level of IL-4 and IL-6 mRNA in the presence of IgA or IgM. The effect of simultaneous signalling through Fc receptors changed the TCR induced response, but the influence was not as dramatic as it was in the case of the BCR signal change that followed costimulation of BCR and CD23 or FcyRIIB1 (CAMPBELL et aI., 1991; WALDSCHMIDT et aI., 1991 ).
Concluding remarks Recent progress indicates an interesting relationship between antigen receptors and Fc receptors on T cells. The TCR complex and Fc receptors (FcERI, FcyRIII) have similar subunits which can be utilized by either type of molecule (RAVETCH and KINET, 1991). This is especially interesting in the case of those y/b TCR + T cells which express both TCR and FcyRIII, an occurrence that permits for common usage of the subunits. The association of the tyrosine kinase fyn (p59) with the TCR and the FCERII (SUGIE et aI., 1991) is another example of the association of FcR with molecules known to be important in cellular recognition and activation. In this paper, we summarized data which address other aspects of the interrelationships of these receptors. An important finding is that antigen receptor-mediated signals induce the expression of various Fc receptors on certain T cell subsets, both in vitro and in vivo. In cognate interactions, there is a concentration of the crosslinked TCRs at the interface of the interacting cells. Our results suggest that Fc receptors are co-localized with the TCR in the surface membrane complex that mediates cell-cell interactions, since capping of TCRs brings about the co-capping of the Fc receptors. When Fc receptor expression is compared on murine Band T cells, the striking difference is that resting B cells constitutively express multiple types of Fc receptors, while resting T cells either do not express FcR or express very low levels of Fc receptors. However after TCR-induced T cell activation, T cells express multiple classes of Fc receptors and these receptors share a series of characteristics with Fc receptors expressed on B cells. The level of FcR expression is dependent on the activation status of the B or T cells. In addition, various subpopulations differ in the extent and the pattern of Fc receptors expressed. Fc receptors are associated with crosslinked antigen receptors on Band T cells. Fc receptors play an important role in the physiology of B cells by their ability to alter the antigen receptor-induced B cell response. Further studies will be needed to determine whether the Fc receptors present on activated T cells can playa similar regulatory function. It is likely that the rapid progress being made in Fc receptor research will have a significant impact on our understanding of T cell biology.
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References AMICORENA, S., C. BONNEROT, D. CHOQUET, W. H. FRIDMAN, and J. L. TEILLAUD. 1989. FcyRII expression in resting and activated B lymphocytes. Eur. J. Immunol. 19: 1379. CAMPBELL, K. A., A. LEES, F. D. FINKELMAN, and D. H. CONRAD. 1991. Crosslinking FCERII and surface IgD enhances anti-IgE mediated B cell activation. FASEB J. 5: A609. DENT, A. L., L. A. MATIS, F. HOOSMAND, S. M. WIDACKI, J. A. BLUESTONE, and S. M. HEDRICK. 1990. Self reactive y/o T cells are eliminated in the thymus. Nature 343: 714. DICKLER, H. B. 1982. Interactions between receptors for antigen and receptors for antibody a review. Molec. Immunol. 19: 1301. GAJEWSKI, T. F., S. R. SHELL, G. NAU, and F. W. FITCH. 1989. Regulation ofT cell activation: Differences among T cell subsets. Immunol. Rev. 111: 79. GORDON, J. and G. R. GUY. 1987. The molecules controlling B lymphocytes. Immunol. Today 8: 339. GREENBERG, S., K. BURRIDGE, and S. C. SILVERSTEIN. 1990. Co localization of F-actin and talin during Fc receptor-mediated phagocytosis in mouse macrophages. J. Exp. Med. 172: 1853. JAMES, S. L. and A. SHER. 1990. Cell-mediated immune response to Schistosomiasis. Curf. Top. Microbiol. Immunol. 155: 21. LEE, W. T. and D. H. CONRAD. 1985. The murine lymphocyte receptor for IgE. III. Use of chemical crosslinking reagents to further characterize the B lymphocyte FeE receptor. J. Immunol. 134: 518. LEFRANCOIS, L. and T. GOODMAN. 1989. In vivo modulation of cytolytic activity and Thy expression in y/o + intraepitheliallymphocytes. Science 243: 1716. LYNCH, R. G., M. SANDOR, T. J. WALDSCHMIDT, A. MATHUR, W. T. SCHAIFF, D. J. BERG, K. SNAPP, A. MUELLER, M. G. ROBINSON, N. NOBEN, and M. G. ROSENBERG.1990. Lymphocyte Fc receptors: Expression, regulation and function. Molec. Immunol. 27: 1167. LYNCH, R. G., M. SANDOR, A. MATHUR, M. HAGEN, T. J. WALDSCHMIDT, B. VAN NESS, K. NELMS, N. NOBEN, A. IBRAGHIMOV, D. MORDUE, R. SACCO, P. TEERARATKUL, T. W. SCHAIH, and L. IAKOUBOV. 1992. Lymphocyte Fc receptors: The immunbiology and pathology of CD23. Immunobiol. 185: 235. MOSMANN, T. R. and R. L. COFFMAN. 1989. Thl and Th2 cells: different patterns of Iymphokine secretion lead to different functional properties. Ann. Rev. Immunol. 7: 145. NOELLE, R. J. and E. C. SNOW. 1990. Cognate interaction between helper T cells and B cells. Immunol. Today 11: 361. RAVETCH, J. V. and J. P. KINET. 1991. Fe receptors. Ann. Rev. Immunol. 9: 457. RAVETCH,j. V., A. D. LUSTER, R. WEINSHANK,j. KOCHAN, A. PAVLOVEC, Y. C. E. PAN, and J. C. UNKELESS. 1986. Structural heterogeneity and functional domains of murine immunoglobulin G Fc receptors. Science 234: 718. SANDOR, M., T. GAJEWSKI, j. THORSON, J. D. KEMP, F. W. FITCH, and R. G. LYNCH. 1990a. CD4+ murine T cell clones that express high level of immunoglobulin binding belong to the IL-4 producing Th2 subset. J. Exp. Med. 171: 2171. SANDOR, M., T. J. WALDSCHMIDT, K. R. WILLIAMS, and R. G. LYNCH 1990b. IgA-induced avidity maturation of IgA Fc receptors on murine T lymphocytes J. Immunol. 144: 4562. SANDOR, M., B. HOULDEN, J. A. Bl.UESTONE, S. M. HEDRICK, J. V. WEINSTOCK, and R. G. LYNCH. 1992. In vitro and in vivo activation of murine y/o T cells induces the expression of IgA, IgM and IgG Fc receptors. J. Immunol., 148: 2363. SCHAII'F, W. T. and R. G. LYNCH. 1992. Functional association of surface membrane immunoglobulins and MHC class II molecules with overt and cryptic pools of IgM receptors on normal murine B-Iymphocytes, submitted for publication. SINCLAIR, N. R. ST C. 1969. Regulation of the immune response. 1. Reduction in ability of specific antibody to inhibit long lasting IgG immunological priming after removal of the Fc fragment. J. Exp. Med. 129: 1183. SUCIE, K., T. KAWAKAMI, Y. MAEDA, T. KAWABE, A. UCHIDA, and J. YODO!. 1991. Fyn tyrosine kinase associated with FCERII/CD23: Possible multiple roles in lymphocyte activation. Proc. Nat!. Acad. Sci. USA 88: 9132.
280 . M. SANDOR et al. UHER, F., A. JANCSO, M. SANDOR, K. PINTER, E. N. A. BIRO, and J. GERGELY. 1981. Interaction between actomyosin complexes and Fc receptors of human peripheral mononuclear cells. Immunol. Lett. 2: 213. WALDSCHMIDT, T. J., F. G. M. KROESE, L. T. TYGRETT, D. H. CONRAD, and R. G. LYNCH. 1991. The expression of B cell surface receptors. III. The murine low affinity IgE Fc receptor is not expressed on Ly 1 or «Ly I-like» B cells. Internat. Immunol. 3: 305. WALDSCHMIDT, T. J. and L. T. TYGRETf. 1991. Crosslinking surface Ig and the low affinity IgE Fc receptor induces B cells to enter cell cycle. FASEB J. 5: A609. WEINSTOCK, J. R. 1987. Immunoregulation of granulomatous inflammation in the liver and intestines. In: Immunopathology of small intestine, ed. M. N. MARS. J. Whiley & Sons Ltd., p.73. Dr. MATYAS SANDOR, Dept. of Pathology, University of Iowa, Iowa City, IA 52242, USA
Departments of Pathology and Internal Medicine!, University of Iowa, Iowa City, USA
TCR Induced Expression of Fc Receptors on Murine T Cell Subsets in vitro and in vivo MATYAS SANDOR, GEORGEA. COOK!, RANDyE. SACCO, RANJIT MATHEW!, ALEXANDERIBRAGHIMOV,]OEL V. WEINSTOCK!, and RICHARD G.LYNCH
Introduction T cells expressing Fc receptors have the unique potential to use two different cognate systems, TCR and the immunoglobulins bound to Fc receptors. The functional consequences of this dual recognition have not been studied extensively, despite the obvious implications concerning T cell activation and signalling and the interactions of T cells with other cells. Such studies require knowledge about the nature of Fc receptors on T cells. While Fc receptors for each immunoglobulin heavy chain isotype can be expressed on T cells (rev LYNCH and SANDOR, 1990), there is little information on structure, distribution, regulation, and function of these receptors. In contrast, much progress has been made in the study of Fc receptors on other cell types, such as B lymphocytes, macrophages and granulocytes. In this review, we summarize our results on Fc receptor expression on various subpopulations of murine T cells, concentrating on the CD4+ and on the y/b TCR + T cell subsets. Elsewhere in this issue a more detailed review will focus on Fn-:RII(CD23) (LYNCH et aI., 1992), so the emphasis of the present review will be on Fca, Fc!! and Fcy receptors. Detailed studies of Fc receptors on B cells have resulted in the establishment of certain principles about the structure, regulation and function of Fc receptors. Our goal is to examine how these principles might apply to Fc receptors on T cells. Some of our studies used in vitro approaches, and others involved observations in vivo in Schistosoma mansoni-infected mice. Murine schistosomiasis provides an in vivo model to study activated T cells CD4+ T cells play an important role in Schistosoma mansoni induced immune responses GAMES and SHER, 1990). IFN-y produced by Th1 CD4+ T cells in the early phase of infection can provide some degree of resistance. However in the presence of chronic parasite infection, CD4+ T cells that produce a lymphokine pattern characteristic of Th2 CD4+ T cells appear to dominate. The elaboration of IL-4 and IL-S in infected animals results in higher expression of MHC class II antigens, FC!::RII, and IL-2R on splenic
TCR Induced Expression of Fc Receptors on Murine T Cell Subsets . 269
B cells. Adult worms in the portal vein release large numbers of eggs some of which become lodged in the liver and induce the formation of granulomas (WEINSTOCK, 1987). The granulomas contain activated CD4+ T cells which produce the Th2 lymphokines IL-4, IL-5, IL-6 and IL-I0 which are important in granulomas formation. CD4 + T cells are the most numerous T cells in the granulomas, but other T cells, such as y/b T cells, are also present. As a percent of the total T cells, y/b TCR + T cells are enriched in these granulomas compared to lymphoid tissues or blood (SANDOR et ai., 1992). The compartmentalized distribution of different T cell populations in schistomiasis provides a model to examine Fc receptor expression on subsets of resting and activated T cell populations in vivo.
Constitutive expression of Fc receptors Virgin, resting, mature B cells express FcyRII(CD32), FCERII(CD23), FcaR, FCIlR and FcbR constitutively (LYNCH and SANDOR, 1990; LYNCH et ai., 1992). In primary and secondary lymphoid organs 95-99 % of the T cells have a resting phenotype (small size, IL-2R-, TfR-). Using anti-Fc receptor antibodies (2.4G2, b3b4), fluorescence labelled immunoglobulins, and cytofluorometry we found that normal resting T cells isolated from spleen, thymus, lymph nodes and Peyer's patch do not have Fc receptors detected with these methods. When incubated overnight with various doses of cytokines (IL-2, -4, -5, -6, IFN-a and -y, TNF-a, TGF-~) FcRs are not detected on normal spleen T cells. Schistosome-infected mice have enlarged spleens that contain a small fraction of activated T cells which secrete IL-4 and IL-5. The presence of these lymphokines alters the phenotypes of the accompanying B cells as shown by their elevated CD23, class II MHC and IL-2R expression. Overall, our studies indicate that, if Fc receptors are present on resting T cells their level is at least 10-100 times less than on resting B cells. While normal resting T cells express little or no Fc receptors, a panel of T cells clones, lines and lymphomas expressed high levels of FcaR (SANDOR et ai., 1990), and FCIlR (IBRAGHIMOV et ai., in preparation). The T cell lymphoma 549.1 was the first cell from which an FcR, FcyRII~1 (CD32) was cloned (RA VETCH et ai., 1986). These findings led us to investigate the relationship between T cell activation and FcR expression. Intraepithelial T cells seem to be different than T cells of the primary and secondary lymphoid organs, as they express Fc receptors prior to activation. The dendritic epithelial T cells (DEC) isolated from murine skin express a non-variant T cell receptor (Vy3Vbl) and bind 2.4G2 (antiFcyRIIIIII) antibody in the resting state. Many of the DEC clones we examined bound 2.4G2 and using antibodies that distinguished between the cytoplasmic tails of the FcyRII and FcYRIII or PCR primers specific for the intracellular portion of the various FcyRs, it was clear that the DEC clones expressed the FcyRIII (CDI6) isoform and not the FcyRII (CD32) isoform.
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B cells appear to use only one FcyR isoform, the FcyRII~I, while previous results suggest that various T cells use different forms of FcyRs. As the DEC cells represent the first wave of peripheralized T cells in ontogeny, their FcyR expression may be important in the skin of newborns as a defense against microbes. Since childbirth is accompanied by contamination of the child with maternal microbes, maternal IgG might be expected to contain antibodies directed to microbes that contaminate the newborn. Because IgE plays an important role in immune responses in the skin, it is of interest that the freshly-isolated DEC (but not phenotypically activated DEC clones) are CD23+ as shown by their ability to bind b3b4, a monoclonal anti-FcERII-CD23 antibody. It is conceivable that DEC, which have functional properties of cytotoxic cells, are involved in a novel form of IgE-dependent delayed hypersensitivity. Preliminary results suggest that resting y/bTCR + T cells at other epithelial sites are also FcR +. Vaginal y/b TCR+ cells express a nonvariant Vy4Vbl TCR and express FcyR before they are activated (IBRAGHIMOV et aI., unpublished observation). Similarly, a fraction of y/b TCR+ (VyS+ variant TCR+) intraepithelial lymphocytes in the intestinal tract express FcyR prior to activation.
In vitro activation through the antigen receptor modifies Fc receptor expreSSIon The crosslinking of surface immunoglobulins on B cells induces a transient upregulation of FcyRII-CD32 (AMIGO RENA et aI., 1989) and FCERIICD23 (GORDON and GUY, 1987). These changes occur early in the cell cycle, during the transition from GO to G 1 (CD23) and in early G 1 (CD32). Antigen-activated cells of the CD4+ Th2 clone DI0, which recognizes conalbumin in an H-2k context, express IgA, IgM and IgD receptors, but IgE and IgG FcRs are not detected by monoclonal antibodies. The gradual withdrawal of IL-2 from the medium results in a change in the phenotype of DIO cells. They become smaller and acridine orange staining indicates that they are in the resting phase of the cell cycle. The level of FcR expression was much lower on these DI0 cells than on activated DI0 cells. The resting DI0 cells re-enter the cell cycle after co culture with antigenprimed, irradiated spleen cells and the level of Fc receptor on the cells starts to rise by 6 h and reaches maximum 12 h after activation. Simultaneous acridine orange staining and FcR analysis has established that the induction of Fc receptors on D 10 cells is linked to the early G 1 phase of the cell cycle. The role of TCR in the induction of FcR was further emphasized by experiments where antibodies reacting with the TCR-complex had the same effect on FcR expression as did activation with antigen-pulsed APC. The close relationship between T cell activation and Fc receptor expression was shown in experiments where repeated cycles of TCR-mediated activation were followed by periods of resting the cells in IL-2 depleted media. This resulted in a concomitant fluctuation in FcR expression wherein activation
TCR Induced Expression of Fc Receptors on Murine T Cell Subsets . 271
up regulated receptor expression. It is important to note that there are CD4 + T cell clones and CD4+ T cells which do not express Fc receptors even after activation. This issue will be discussed later.
In vivo activation modifies Fc receptor expression Schistosoma mansoni-infected mice have developed numerous liver granulomas by 8 weeks after infection. These granulomas contain S-8 % CD4 + T cells which actively secrete lymphokines (including IL-4, -S, -6 and -10). The schistosome activated CD4 + T cells, like the activated CD4 + T cell clones examined in vitro, express high levels of IgA and IgM receptors. The resting CD4+ T cells in the spleens of schistosome-infected mice were found not to express FcR. These results provide an in vivo model verifying the in vitro findings which link Fc receptor expression to TCRmediated activation. Similar experiments were carried out to investigate the relationship of TCR-induced activation and FcR expression on y/o TCR+ cells. y/o TCR+ cells were prepared either from y/o TCR transgenic mice (DENT et al., 1990) or from cultures where polyclonal splenic or intestinal y/o TCR + cells had been selectively expanded and enriched by the addition of a mitogenic antiy/o TCR antibody (UC7-13DS). In a series of in vitro experiments we found that fresh, resting y/o T cR+ cells or postactivated, resting cells did not express Fc receptors. However the y/o cells expressed IgA, IgM and to a lesser extent IgG receptors after primary and secondary culture with a mitogenic anti-CD3£ antibody (14S-2CII). These experiments identified a relationship between the TCR-induced activation and FcR expression on y/o TCR + cells similar to that previously observed in studies of alB TCR+ CD4+ cells. The time dependence of FcR induction has been studied using spleen cells prepared from y/o TCR transgenic mice (Fig. I). Fresh spleen cells from transgenic mice are more heterogeneous in cell cycle position than the semisynchronized resting cells of the DIO clone. After 24 h of stimulation with mitogenic anti-CD3£ antibody, a portion of the y/o TCR+ spleen cells are still small, resting cells. These small y/o TCR + cells bind very little immunoglobulin, while the large, activated cells bind IgA, IgM and IgG. Cell enlargement is a step that relates to the entry into the G I phase of the cell cycle, and our finding with alB TCR+ and y/o TCR+ cells indicate that the induction of Fc receptors on T cells is an early activation event. Liver granulomas in Schistosoma-infected mice contain O.S-4 % y/o TCR + cells. These large, y1.1 TCR positive, transferrin receptor positive cells bind IgA, IgM and the 2.4G2 antibody. Resting, splenic y/o TCR+ cells from the same mice do not display FcR. Before leaving this section that deals with the relation of TCR-induced activation and FcR expression, some interesting findings should be discussed that indicate a somewhat more complex situation than has been
24
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Figure 1. Effect of anti-CD3E antibody on IgA binding by y/6 TCR + cells. Spleen cells isolated from y/o TCR transgeneic mice contain a high portion of y/oTCR+ cells (first left panel). Spleen cells were cultured in the presence of 10 ug/ml of mitogeneic anti-CD3E antibody. At various time points specified as h at the right upper corner of the panels, cells were double stained with anti-y/o TCR antibody (GL3) and IgA-FITC.
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TCR Induced Expression of Fc Receptors on Murine T Cell Subsets . 273
suggested. First, there are CD4 + and y/6 TCR + cells which do not express Fc receptors following TCR-mediated activation, a finding that will be discussed shortly. In addition, TCR-mediated signals in some cases are necessary but not sufficient to induce FcR expression. For example, primary activation of splenic y/6 TCR + cells induces Fcy R expression, but after additional in vitro culture, resting and then reactivation, these cells do not re-express FcR. In contrast, in vivo reactivation results in the reexpression of FcyR. Thus repeated, in vivo administration of mitogenic anti-TCR antibody induces the expression of FCERII on some CD4+ cells. Changing the activation protocol by doing the secondary stimulation in vitro results in the appearance of FeaR, and FCflR+, but not FCERII on CD4+ T cells. Thus, factors present in vivo, but missing in vitro are required for the induction of FCERII on CD4+ T cells. Another element of complexity is demonstrated by the observation that some y/6 TCR+ DEC express FCERII and FcyRIII while resting, but when activated with anti-CD3E antibody the FCERII is completely downregulated and FeaR and FCflR are induced. The loss of FCERII is exceptional since TCR-induced activation, when it influences FcR expression, typically brings about an increase in the level of FcR on the T cell.
Various lymphocyte subpopulations differ in their Fc receptor expreSSiOn Mature virgin murine CDS-(B2Iineage) B cells express FcRs for each of the five Ig isotypes while under the same conditions, CDS+ (B1Iineage) cells do not express FCERII -CD23 (W ALDSCHMlDT et al., 1991). The pattern of expressed FcRs also changes during different stages of B cell development (LYNCH et al., 1992). Upon activation, CD4+ T cells produce a series of lymphokines and the pattern of the secreted lymphokines defines the functions of these cells. Recent research has shown that CD4 + T cell populations differ in their lymphokine production (MOSMANN and COFFMAN, 1989). Virgin CD4+ T cells, when activated, produce large amounts of IL-2 but low levels of other lymphokines. Most activated CD4+ memory T cells secrete IL-2, IFN-y, IL-4, IL-S, IL-6, IL-10 and have been designated as Tho CD4+ cells. In the chronic presence of antigens, such as in parasite infections, the frequency of two types of CD4+ T cells increases. One of these subsets of CD4+ T cells is known as Th1 cells and they secrete IL-2 and IFN-y, but not IL-4, IL-S, IL-6 or IL-10. Th 1 cells are the effector cells of the DTH reaction. The other subset of CD4+ T cells, Th2 cells, secrete IL-4, IL-S, IL-6, IL-IO and one of their functions is to deliver «help» for B cells that induces their proliferation and differentiation. We have demonstrated Fc receptor expression on the various CD4 + T cell populations using fresh splenic cells as a source of virgin T cells and 22 characterized, long term
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CD4+ T cell clones as representatives of the ThO, Th1 or Th2 subsets. The TCR -activated Th2 and ThO clones were found to express a high level of multiple types of FcRs. IgA, IgM and IgD binding was the most common, but some clones also bound IgG or IgE. In contrast, Th1 clones consistently expressed very low levels of FcRs. Considering that each clone was activated by its respective antigen, these findings show that activation through the TCR induces FcR expression on Th2 and ThO cells, but in the case of CD4+ Th1 cells, activation either does not induce FcR expression or induces a much lower level of expression (SANDOR et al., 1990). The increasing evidence that the biochemical pathways of TCR-mediated signalling are different in Th1 and Th2 clones (GAJEWSKI et al., 1989) might be important in understanding the differences observed in Fc receptor expression on these T cells when activated. In the supernatants of normal spleen cells activated with mitogenic anti-CD3E antibody there are only trace amounts of the Th2 cytokines IL-4 and IL-S, and these activated CD4+ T cells bind only low quantities of IgA and IgM. However, the supernatants of anti-CD3E activated spleen cells from Schistosoma mansoni-infected mice contain 40-fold higher levels of IL-4 and soo-fold higher levels of IL5, and there is a S-10-fold increase in IgA and IgM binding by these CD4+ T cells. In the liver granulomas of Schistosoma-infected mice there are activated CD4+ cells which produce high levels of Th2 cytokines and avidly bind IgA and IgM (SANDOR et al., 1992). The in vivo injection of T cell activating antibodies (anti-CD3E) resulted in data similar to the findings in Schistosoma-infected mice in that the appearance of Th2 lymphokineproducing CD4+ cells coincides with the presence of CD4+ T cells expressing high levels of Fc receptors. An interesting feature of y/b TCR+ cells is their heterogeneity. Some y/b T cells are located in the epithelial layers of organs and are sessile, others that are found in lymphoid organs, in the circulation and at sites of inflammation are mobile cells. The TCR usage by y/b T cells at various anatomical sites is different. The y/b TCR + cells in the skin (DEC) express Vy3b1, the y/b TCR+ cells in the epithelium of the reproductive organs, the urinary tract and the tongue express Vy4bl, and the expression of these families of TCR genes by y/b T cells at these sites is invariant. At other anatomical sites the y/b TCR gene usage is quite variable. VyS TCR+ cells are located in the epithelium of the intestine (IEL), and Vyl.1, Vy1.2, and Vy2 TCR + cells are present in lymphoid organs and in the circulation. We are in the process of characterizing FcR expression of these cells and the results obtained to date clearly show that on various y/b TCR + cell populations different classes of FcRs are present. The availability of y/b TCR transgenic mice made it possible to investigate the resting y/b TCR+ cells in lymphoid organs (spleen, thymus, lymph nodes and Peyer's patches) and in blood. These cells do not express Fc receptors. The splenic y/b TCR+ cells activated with anti-CD3E in vitro and the yl.1 + cells activated in vivo in the granulomas expressed cell surface IgA, IgM and IgG Fc receptors.
TCR Induced Expression of Fc Receptors on Murine T Cell Subsets . 275
A fraction of fresh IELs express FcyR. The in vitro enrichment of IELs that followed TCR-mediated stimulation or restimulation resulted in two populations. One population had no detectable FeR expression but the other population bound IgA and IgM. It will be interesting to learn whether FcR expression relates to the functional subsets previously described for y/6 TCR+ IELs (LEFRANCOIS and GOODMAN, 1989). DEC freshly isolated from murine skin react with monoclonal antibodies specific for FcyR and FCERII. Activation of these cells with anti-CD3E antibody results in the loss of FcRdI and the appearance of IgA and IgM Fe receptors on a fraction of the cells. As previously found with other intraepithelial y/6 TCR+ cells, we have recently found that y/6 TCR+ cells in the vagina express FcyR (IBRAGHIMOV and LYNCH, unpublished). While the characterization of FcR distribution on various T cell subsets is not yet complete, it is already clear that the pattern of FeR expression on various T cell subpopulations is different.
Physical association between the antigen receptors and Fc receptors The crosslinking of the antigen receptors on B cells induces the cocapping of FcyRs (DICKLER, 1982) and Fq.lRs (SCHAIFF and LYNCH, 1992).
D 10 Th2 clone
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Figure 2. Cocapping of FcaR with TCR on DID cells. Laser confocal microscopy images show binding of IgA- FITC and anti-CD3E antibody. Cells were stained at 4°C with anti-TCR antibody. Staining was visualized using biotinylated anti-hamster IgG and Avidin-Texas red. Avidin-Texas red was used at 4 °C or 37 °C. Conditions that induced capping of the TCR resulted in cocapping of IgA FeR.
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LEE and CONRAD (1985) precipitated the murine FCERII from murine B cells previously treated with an extracellular crosslinker. The presence of membrane IgM and IgD in the FCERII precipitates suggested that the antigen receptors were associated with the FCERII on the surface membrane of the B cell. To assess the relationship between Fc receptors and antigen receptors on T cells, co-capping experiments have been carried out using the CD4 + Th2 clone D10. According to the images generated by laser confocal fluorescence microscopy, IgA and IgM bind to these cells with an evenly distributed pattern. The crosslinking of TCR with anti- V~8 antibody (F23.1) or anti-CD3E antibody (145-2C11) under conditions that allow for capping of the TCR induced the co-capping of the IgA and the IgM binding sites on these cells. The co-localization of the IgA receptors and the TCR is demonstrated in Figure 2. The association of these molecules is also detected on other T cells. The crosslinking of y/o TCR by GL3 antibody on mitogen-activated splenic y/o TCR+ cells induces the co-capping of IgA and IgM receptors (SANDOR et aI., 1992). The precise nature of the physical association of these molecules is not known. However, a direct association can not be excluded since in some cases FcRs and TCRs have been shown to form complexes. FcyRIII (CD16) appears to share a subunit (y) with the TCR complex (RAVETCH and KINET, 1991). A tyrosine kinase (fyn) has been shown to be associated both with the TCR-complex and with FCERII (SUGIE et aI., 1991). Elements of the TCR complex and some FcRs contain immunoglobulin-like domains, which in some molecules are shown to interact with each other. Such domain-domain interactIon might be involved in the TCR-FcR association. A less direct interaction can be suggested through TCR induced modification of the cytoskeletal system. An association of FcRs with the elements of the cytoskeletal apparatus has been reported (UHER et aI., 1982; GREENBERG, 1990). Immunoprecipitations of the crosslinked TCRs after using bifunctional crosslinkers might provide insight into the interaction of various FcRs and TCRs. TCR crosslinking is an important part of the T cell induction by APC, and of the cellular interactions that occur when effector functions such as help or killing are delivered. The accumulation of Fc receptors at these interfaces might enhance the interactions between the T cell FcRs and immunoglobulins.
Antigen receptor induced responses are modified by the Fc receptors It is well established, that under certain conditions crosslinking of the antigen receptors on B lymphocytes induces cellular differentiation and immunoglobulin production. This response can be modified to a large extent by ligands that bind to Fc receptors on the B lymphocytes.
TCR Induced Expression of Fc Receptors on Murine T Cell Subsets . 277
The simultaneous crosslin king of FcyRII and antigen receptors generates negative signals for the B cells (SINCLAIR, 1969). In addition, it has been suggested that the antigen receptor-FeaR interaction has a similar effect. In contrast, the crosslinking of Fc£RII and antigen receptors has an enhancing effect on antigen receptor-mediated induction (CAMPBELL et aI., 1991; WALDSCHMIDT et aI., 1991). In cognate T cell and target cell cytoconjugation the presented antigen crosslinks the antigen receptors. Conceivably, Fe receptors might colocalize with the TCRs at the interfaces of these cellular interactions. Surface immunoglobulins on B ceUs are concentrated at the T -B cell cognate interaction site (NOELLE et aI., 1990), and were the FcRs to colocalize they could interact with the surface immunoglobulins and provide additional signal transduction. To study the influence of Fc receptors on TCRmediated signals, we coated plates with anti-CD3£ antibody and TNPI0BSA, and measured cytokine mRNA levels in FeaR+, Fc~R+ DI0 Th2 cells in the presence or absence of anti-TNP IgA (MOPC 315) or anti-TNP IgM
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IL-4 IL-5 IL-6 Figure 3. The effect of IgA and IgM binding to their respective Fe receptors on the TCRinduced increase of IL-4, IL-5 and IL-6 mRNA in DIO cells. Tissue culture plates were coated with 10 ug/ml anti-CD3t:: antibody and then with 200 ug/ml TNPIO-BSA. The washed plates were further incubated with medium, 300 ug/ml anti-TNP IgA (MOPC 315) or 40 ug/ml antiTNP IgM (F5D6). DIO cells (I x 106 cell/ml) were cultured for 24 hours on the different plates and then RNA was precipitated from the harvested cells. After electrophoresis the blotted RNA was consecutively probed with lL-4, IL-5 and IL-6 eDNA.
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et al.
(F5D6). The interleukins IL-4, IL-5, IL-6 were selected for study because of their important role in B cell «help» by this clone. The Northern blots (Fig. 3) show a decrease in the level of IL-4 and IL-6 mRNA in the presence of IgA or IgM. The effect of simultaneous signalling through Fc receptors changed the TCR induced response, but the influence was not as dramatic as it was in the case of the BCR signal change that followed costimulation of BCR and CD23 or FcyRIIB1 (CAMPBELL et aI., 1991; WALDSCHMIDT et aI., 1991 ).
Concluding remarks Recent progress indicates an interesting relationship between antigen receptors and Fc receptors on T cells. The TCR complex and Fc receptors (FcERI, FcyRIII) have similar subunits which can be utilized by either type of molecule (RAVETCH and KINET, 1991). This is especially interesting in the case of those y/b TCR + T cells which express both TCR and FcyRIII, an occurrence that permits for common usage of the subunits. The association of the tyrosine kinase fyn (p59) with the TCR and the FCERII (SUGIE et aI., 1991) is another example of the association of FcR with molecules known to be important in cellular recognition and activation. In this paper, we summarized data which address other aspects of the interrelationships of these receptors. An important finding is that antigen receptor-mediated signals induce the expression of various Fc receptors on certain T cell subsets, both in vitro and in vivo. In cognate interactions, there is a concentration of the crosslinked TCRs at the interface of the interacting cells. Our results suggest that Fc receptors are co-localized with the TCR in the surface membrane complex that mediates cell-cell interactions, since capping of TCRs brings about the co-capping of the Fc receptors. When Fc receptor expression is compared on murine Band T cells, the striking difference is that resting B cells constitutively express multiple types of Fc receptors, while resting T cells either do not express FcR or express very low levels of Fc receptors. However after TCR-induced T cell activation, T cells express multiple classes of Fc receptors and these receptors share a series of characteristics with Fc receptors expressed on B cells. The level of FcR expression is dependent on the activation status of the B or T cells. In addition, various subpopulations differ in the extent and the pattern of Fc receptors expressed. Fc receptors are associated with crosslinked antigen receptors on Band T cells. Fc receptors play an important role in the physiology of B cells by their ability to alter the antigen receptor-induced B cell response. Further studies will be needed to determine whether the Fc receptors present on activated T cells can playa similar regulatory function. It is likely that the rapid progress being made in Fc receptor research will have a significant impact on our understanding of T cell biology.
TCR Induced Expression of Fe Receptors on Murine T Cell Subsets . 279
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