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Presidential Address to The American Association of Immunologists

Stimulating Naive T Cells¹

Department of Immunology, IMM4, The Scripps Research Institute, La Jolla, CA 92037

	Abstract

Top Abstract Introduction T cell stimulation: the... Transfected Drosophila cells as... Requirements for TCR down... Intensifying signal 1 by... A model for costimulation CD28-dependent absorption of B7... Discussion References

 

	Introduction

Top Abstract Introduction T cell stimulation: the... Transfected Drosophila cells as... Requirements for TCR down... Intensifying signal 1 by... A model for costimulation CD28-dependent absorption of B7... Discussion References

 
Being introduced for this address by Jacques Miller, my Ph.D. supervisor 30 years ago, gives me particular pleasure because Jacques has always been a hero of mine, and I consider myself very fortunate to have had the opportunity to train in the lab of one of the great figures in immunology.

View larger version (122K): [in this window] [in a new window]   Jonathan Sprent

There is one AAI issue I would like to mention briefly, namely membership of the AAI. Table I shows membership numbers for all of the immunology societies affiliated with the International Union of Immunological Societies (IUIS). Not surprisingly, the United States is at the top with 5500 members, but notice that the United Kingdom and Japan are not far behind. In fact, if total population numbers are factored in, the United States is actually last on this list of five western countries: 200/10⁷ compared with about 800/10⁷ for Scandinavia, the Netherlands and Britain. So, assuming the per capita representation of immunologists is the same here as in these European countries, then instead of 5500 AAI members we could have 20,000 members. Therefore, the implication is that a very sizeable proportion of people doing immunology in this country are not AAI members. Although there could be multiple explanations for this poor representation, to my mind the most likely—and worrying—possibility is that, for junior immunologists, the profile of the AAI is just too low to attract their membership. If so, we have a fundamental problem on our hands. The Council debated this issue at some length at a recent meeting, and any input from the AAI membership at large would be gratefully received. Now for some science.

	T cell stimulation: the signal 1/signal 2 hypothesis

Top Abstract Introduction T cell stimulation: the... Transfected Drosophila cells as... Requirements for TCR down... Intensifying signal 1 by... A model for costimulation CD28-dependent absorption of B7... Discussion References

How signals 1 and 2 converge to induce T cell proliferation and differentiation is still controversial. For costimulation via CD28, the prevailing view is that signal 2 is primarily important for inducing T cells to synthesize growth-promoting cytokines such as IL-2; however, which particular intracellular signaling pathways are involved is still unclear, although signal 2 may involve activation of Jun kinase and possibly PI3 kinase (7, 8, 9).

For signal 1, TCR ligation via contact with peptide/MHC complexes is thought to initiate a chain of phosphorylation-driven events involving initial activation of p56^lck and p59^fyn followed by tyrosine phosphorylation of CD3 and -chains (5). Recruitment of ZAP-70 then leads to signaling via several downstream pathways, including the PI/Ca and mitogen-activating protein kinase pathways, and eventual activation of transcription factors such as NF-AT, AP-1, and NF-B. Although the signaling events elicited by TCR ligation are now understood in some detail, it should be emphasized that the data are based largely on studies with cell lines exposed to cross-linked anti-TCR mAbs. Hence the relevance of the data to physiological T cell activation induced following exposure of naive T cells to Ag on normal APC remains questionable.

Determining the effects of a "physiological" dose of signal 1 on naive T cells is obviously difficult. The key problem is in devising an experimental system where T cells can interact with peptide/MHC complexes in the absence of costimulation. A priori, the obvious approach is to expose T cells to purified peptide/MHC complexes bound to an inert surface, e.g., to beads or plastic. However, there is the objection that the degree of TCR cross-linking induced by a ligand attached to a solid matrix may be far higher than occurs with presentation via the lipid bilayer of a normal APC. The alternative is to use APC that are devoid of costimulatory molecules. But here there is the problem that virtually all cell lines express a wide spectrum of cell-surface molecules, some of which may be capable of providing significant costimulation for T cells.

	Transfected Drosophila cells as APC

Top Abstract Introduction T cell stimulation: the... Transfected Drosophila cells as... Requirements for TCR down... Intensifying signal 1 by... A model for costimulation CD28-dependent absorption of B7... Discussion References

 
The approach that we have adopted is to bypass the use of normal cells and instead try to create artificial APC by transfecting genes for MHC and known costimulatory/adhesion molecules into Drosophila cells. The theoretical advantage of this system is that the endogenous cell-membrane components of nonmammalian cells are presumably only very distantly related to mammalian molecules and therefore are unlikely to be recognized by the cohort of costimulatory/adhesion molecules on T cells. Hence, after transfection with MHC molecules, Drosophila cells presenting peptide/MHC complexes to T cells would be expected to deliver a "pure" source of signal 1 in the absence of signal 2. Our results on the effects of culturing TCR transgenic CD8⁺ cells with MHC class I-transfected Drosophila cells are summarized below (10, 11, 12). The experiments involved a close collaboration between my lab at Scripps and the group of Per Peterson, Michael Jackson, and Anders Brunmark who recently moved from Scripps to the R. W. Johnson Pharmaceutical Research Institute (RWJPRI). The bulk of the work was done in my lab by a very talented young investigator, Zeling Cai, who is now at RWJPRI. In parallel studies, my colleague Sue Webb at Scripps has been doing comparable experiments with MHC class II-transfected Drosophila cells as APC for naive CD4⁺ cells (13).

We cotransfected a Drosophila cell line (Schneider SC2 cells) with cDNA encoding murine MHC class I molecules and ß₂-microglobulin under the control of the metallothionein promoter (10, 14); the molecules are expressed following overnight exposure to CuS0₄. Because insect cells lack TAP peptide transporters, the class I molecules expressed in Drosophila cells fail to bind endogenous peptides and hence are unstable (14). This is an advantage because incubating the cells with appropriate exogenous peptides leads to the expression of a high density of stable peptide/MHC complexes on the cell surface. In the experiments discussed below we used L^d-transfected Drosophila cells as APC for naive CD8⁺ cells from the 2C TCR transgenic line. This line (15) has specificity for two L^d-associated peptides, p2Ca and QL9 (16, 17, 18, 19). Both peptides are derived from a natural endogenous Ag (a Krebs cycle enzyme) and have a near-identical sequence, the only difference being that QL9 (a 9-mer) has one extra amino acid than p2Ca. This subtle difference is highly significant, however, because QL9 is a much stronger peptide for 2C cells than p2Ca (even though the immunogenicity of p2Ca is well above average).

The effects of exposing naive 2C CD8⁺ cells to L^d-transfected Drosophila cells (L^d Dros) preincubated with p2Ca or QL9 peptides are shown in Fig. 1. As controls, we used L^d Dros cells cotransfected with either B7 (B7-1) molecules (L^d.B7) or ICAM-1 molecules (L^d.ICAM) or both (L^d.B7.ICAM). The key finding is that, as manifested by up-regulation of CD69 and CD25 (IL-2R), even high concentrations of p2Ca peptide (10 µM) presented by L^d Dros APC have no detectable capacity to activate naive 2C cells. Likewise, p2Ca fails to induce T proliferative responses even when 2C cells are supplemented with exogenous IL-2 (Fig. 2). With the much stronger QL9 peptide, high concentrations of this peptide cause low-level CD69 and CD25 expression on 2C cells (Figs. 1 and 3) and lead to significant proliferative responses, though only in cultures supplemented with IL-2 (Fig. 2). With lower concentrations of QL9 (0.1 µM), signs of T cell activation are totally absent. In marked contrast to these findings with Drosophila cells expressing L^d alone, conspicuous activation of 2C cells occurs when either p2Ca or QL9 peptides are presented by L^d.B7 or L^d.ICAM APC (Figs. 1 and 3 and see below).

View larger version (32K): [in this window] [in a new window]   FIGURE 1. Expression of CD69 and CD25 on CD8+ 2C cells stimulated with 10 µM peptides presented by transfected Drosophila cells. Purified CD8+ 2C cells were incubated with transfected Drosophila cells plus p2Ca or QL9 peptides (10 µM) in bulk (2 ml) culture for 12 h and then stained for the markers shown. The data show staining of gated CD8+ cells; staining of noncultured 2C cells (top) is shown as a control. Data are from Ref. 10 .

View larger version (30K): [in this window] [in a new window]   FIGURE 2. IL-2-dependent proliferative responses of CD8+ 2C cells to peptides presented by Drosophila cells transfected with Ld only. Responses to p2Ca (a) and QL9 (b) peptides were measured by culturing 5 x 104 purified CD8+ 2C cells with 2 x 105 Drosophila cells in the presence or absence of the indicated concentrations of peptides for 3 days. [3H]Thymidine was added during the last 8 h of culture; rIL-2 was added at a final concentration of 20 U/ml. The data refer to the mean of triplicate cultures. Data are from Ref. 10 .

View larger version (35K): [in this window] [in a new window]   FIGURE 3. Influence of peptide dose on expression of CD25 on CD8+ 2C cells. Purified CD8+ 2C cells were cultured with the indicated concentration of peptides presented by Drosophila cell APC for 12 h and then stained for CD25 expression. The data show staining on gated CD8+ cells. Data are from Ref. 10 .

Because CD28/B7 interaction provides a quintessential source of signal 2, it comes as no surprise that presentation of QL9 and p2Ca peptides by L^d.B7 Dros cells leads to strong T cell activation as measured by CD69 and CD25 expression (Figs. 1 and 3); L^d.B7 Dros cells also induce high T proliferative responses, though only for QL9 and not p2Ca (Fig. 4, Table II). The unexpected finding is the strong APC function displayed by L^d.ICAM APC. Indeed, in terms of costimulating proliferative responses (Fig. 4) and inducing CD69 and CD25 up-regulation (Fig. 3), L^d.ICAM APC are just as efficient as L^d.B7 APC. Thus, even though ICAM-1, the ligand for LFA-1, is often viewed simply as an adhesion molecule, ICAM-1 clearly displays costimulatory function for 2C cells.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Features of proliferative responses of CD8+ 2C cells to peptides presented by transfected Drosophila cells. a, Influence of peptide concentration on the day 3 response of CD8+ 2C cells to different Drosophila APC. b, Kinetics of the response of CD8+ 2C cells to 10 µM QL9 peptide presented by different Drosophila APC. c, Influence of peptide dose on the day 5 response of CD8+ 2C cells with different Drosophila APC. CD8+ cells (5 x 104) were cultured with 2 x 105 Drosophila APC. The data show the mean of triplicate cultures. Data are from Ref. 10 .

It should be noted that, as with bacterial DNA (23), the unmethylated CpG motifs of Dros DNA are able to activate B cells and other APC and thereby provide a form of bystander costimulation for T cells (11). However, this is only a problem when the responding T cell population is contaminated with non-T cells. With highly purified T cells, which we use routinely, Dros DNA has no effect on the T cell response to Ags. This is apparent from the finding that 2C cells are unresponsive to peptides presented by L^d Dros APC.

	Requirements for TCR down-regulation

Top Abstract Introduction T cell stimulation: the... Transfected Drosophila cells as... Requirements for TCR down... Intensifying signal 1 by... A model for costimulation CD28-dependent absorption of B7... Discussion References

 
Returning to the effects of signal 1 alone, the above data on Drosophila APC indicate that TCR contact with peptide/MHC complexes in the absence of costimulation is nonimmunogenic and leads to little or no detectable sign of T cell activation. Others have argued that exposure to signal 1 alone causes T cells to become anergic (24). However, this is clearly not the case for naive 2C CD8⁺ cells. Thus, in our hands, culturing 2C cells for 2 days with QL9 peptide presented by Drosophila APC expressing L^d alone fails to impair the capacity of the cells to subsequently respond to QL9 plus L^d.B7.ICAM APC (I. Hwang and J. Sprent, unpublished data). In fact, the cultured T cells behave identically to naive cells.

An obvious question is whether 2C cells can even "see" QL9/L^d complexes on APC lacking costimulatory molecules. In fact, these complexes are clearly recognized because exposing 2C cells to QL9 on L^d Dros APC causes marked TCR down-regulation (Ref. 12 and Fig. 5). Moreover, with limiting concentrations of peptide, TCR down-regulation is as prominent with L^d APC as with L^d.B7 APC; TCR down-regulation is slightly enhanced with ICAM-1⁺ APC (Fig. 5), perhaps reflecting the increased cell adhesion provided by LFA-1/ICAM-1 interaction. These findings indicate that, in the absence of costimulation, peptide/MHC complexes on APC are readily recognized by the TCR but lead only to TCR down-regulation and not to T cell activation.

View larger version (26K): [in this window] [in a new window]   FIGURE 5. Influence of B7-1 and ICAM-1 expression on peptide-induced 2C TCR down-regulation. A total of 5 x 105 purified CD8+ 2C cells were cultured with 1 x 106 Drosophila APC in the presence of a titrated concentration of QL9 peptides for 12 h. The cells were harvested and stained with FITC-conjugated 1B2 anti-clonotypic mAb. The data show TCR expression as defined by mean fluorescence intensity (MFI) on gated CD8+ 2C cells. Data are from Ref. 12 .

	Intensifying signal 1 by coupling peptide-MHC complexes to solid matrices

Top Abstract Introduction T cell stimulation: the... Transfected Drosophila cells as... Requirements for TCR down... Intensifying signal 1 by... A model for costimulation CD28-dependent absorption of B7... Discussion References

View larger version (22K): [in this window] [in a new window]   FIGURE 6. Activation of 2C CD8+ T cells by peptides presented by Ld.beads. A, Purified 2C CD8+ cells (5 x 105) were cultured with 10 µM peptide-loaded Ld.beads (1 x 106) for 12 h; the cells were then harvested and stained for TCR, CD25 (not shown), and CD69 expression. For proliferation, 2C CD8+ cells (5 x 104/well) were cultured with peptide-loaded APC (2 x 105/well) for 2 days; [3H]TdR was added during the last 8 h of culture. B, Proliferative responses by various doses of 2C CD8+ cells were measured on the days shown; Ld.beads loaded with 10 µM QL9 peptide were used as APC. Data are from Ref. 25 .

To investigate this latter idea, we attached L^d molecules to the surface of mouse RBC (mRBC) using the avidin/biotin procedure (25). As shown in Fig. 7, the APC function of mRBC is the same as for Drosophila cells. Thus, unless accompanied by costimulation (provided by anti-CD28 mAb), culturing 2C cells with L^d.mRBC plus QL9 peptide induces only minimal CD69 up-regulation, despite complete TCR down-regulation (Fig. 7A); likewise, proliferative responses are undetectable (Fig. 7, A and B).

View larger version (23K): [in this window] [in a new window]   FIGURE 7. Activation of 2C cells by Ld.mRBC as APC only occurs with fixed APC. A, To measure TCR and CD69 expression, 2C CD8+ cells (5 x 105) were cultured with unfixed mRBC that had been coupled with Ld alone or with Ld and anti-CD28 mAb; QL9 or PIA peptide were added at 10 µM and cells were stained at 12 h. Proliferative responses were measured at day 2 with 5 x 104 2C cells and 2 x 105 APC. B, Proliferative responses and IL-2 production by 2C CD8+ cells (1 x 105) elicited by Ld.beads and fixed (F) vs unfixed mRBC (2.5 x 105) plus 10 µM QL9 peptide. Treatment with 1% glutaraldehyde was used for fixation; Ld expression on fixed and unfixed cells was comparable. a, 2C CD8+ cells were cultured with titrated numbers of Ld.beads, Ld.mRBC, or Ld. F.mRBC in the presence of 10 µM QL9 peptide. Proliferation was measured on day 2. b, 2C CD8+ cells were cultured with APC for 2, 3, and 4 days. [3H]TdR was added during the last 8 h of culture. c, 2C CD8+ cells were cultured with titrated numbers of APC for 24 h. Culture supernatants were measured for IL-2 activity using the CTLL-2 cell line. Data are from Ref. 25 .

	A model for costimulation

Top Abstract Introduction T cell stimulation: the... Transfected Drosophila cells as... Requirements for TCR down... Intensifying signal 1 by... A model for costimulation CD28-dependent absorption of B7... Discussion References

 
The above data indicate that, in the absence of costimulation, peptide/MHC complexes displayed on a solid matrix or on fixed APC are strongly immunogenic (though only for proliferation and not for IL-2 production). Why then are peptide/MHC complexes nonimmunogenic when presented on normal APC? As discussed earlier, the lack of immunogenicity may be a reflection of poor TCR cross-linking. Here, the simplest idea is that peptide/MHC complexes on the surface of normal cells have a tendency to move laterally in the lipid bilayer, thus limiting their capacity to induce TCR cross-linking. In favor of this idea, MHC molecules with truncated intracytoplasmic tails, which presumably have an enhanced tendency to move laterally in the cell membrane, are much less immunogenic than normal MHC molecules (27).

This line of reasoning begs the question of how costimulatory molecules enhance T cell activation. Thus, if peptide/MHC complexes on normal APC are themselves incapable of causing TCR cross-linking (signal 1), how does concomitant T cell interaction with costimulatory molecules on APC induce T cell activation?

First, in keeping with current dogma, costimulatory molecules may elicit a discrete signal (signal 2) which synergizes with the TCR signal (signal 1). Here, the underlying assumption is that costimulatory molecules function solely as signaling molecules: these molecules operate by boosting the effects of signal 1 but do not intensify signal 1 itself (which is very weak). This idea fits well with the evidence on cytokine production. Thus, in the absence of costimulation, IL-2 is barely detectable even when peptide/MHC complexes are presented in cross-linked form (see above). However, the data on CD69/25 up-regulation and early T proliferative responses are less easy to explain. Here, the point to emphasize is that, by these parameters, T cell activation becomes independent of costimulatory molecules when peptide/MHC complexes are artificially cross-linked. This is difficult to explain by the signal 1/signal 2 concept.

Second, for the early stages of T cell activation, costimulatory molecules may function not by inducing a discrete signaling pathway but by enhancing TCR cross-linking, thereby boosting the intensity of signal 1; costimulatory molecules also induce unique signaling (signal 2) but this is only important for cytokine production and not for initial T cell activation.

The notion that costimulatory molecules can enhance TCR cross-linking rests on the assumption that costimulatory molecules have significant binding affinity for their respective ligands on APC. This is clearly the case for LFA-1/ICAM-1 interaction, but whether CD28 acts as an adhesion molecule is less clear (but see Refs. 28, 29). Evidence in favor of this possibility is discussed below.

	CD28-dependent absorption of B7 molecules from APC

Top Abstract Introduction T cell stimulation: the... Transfected Drosophila cells as... Requirements for TCR down... Intensifying signal 1 by... A model for costimulation CD28-dependent absorption of B7... Discussion References

 
Inkyu Hwang in my lab has made the surprising observation that culturing T cells with APC leads to rapid transfer of various cell-surface molecules from APC to T cells. Such transfer involves several cell-surface molecules, including B7, ICAM-1, and MHC, and is easily visualized by FACS analysis. Interestingly, at least for B7, the absorbed molecules are first bound to the surface of T cells and are then internalized. Parallel experiments by Zeling Cai and Jing-Feng Huang at RWJPRI have shown that T cell internalization of APC-derived molecules also applies to MHC (L^d) molecules and is followed by rapid localization in lysosomes, presumably indicating degradation.

Although the notion that T cells can absorb molecules from APC has a long history (30, 31, 32, 33, 34, 35), which particular receptor/ligand interactions control this process has been unclear. To date, our studies indicate that T cell uptake of molecules from APC involves two sets of molecules, namely CD28/B7 and TCR/peptide/MHC. For CD28, the notable finding is that T cell uptake of molecules from APC can occur in the absence of specific Ag but applies only to CD28⁺ and not to CD28^-/- T cells. In this situation, short-term (1 h) culture of CD28⁺ T cells with APC leads to absorption of B7 molecules and coabsorption of other molecules (e.g., ICAM-1 and MHC class I), presumably indicating that the APC-derived molecules become bound to CD28 as membrane fragments or vesicles. CD28-mediated absorption is apparent with resting T cells, but is much more prominent with activated T cells.

These data suggest that the affinity of CD28/B7 interaction is sufficiently strong to enable T cells to absorb (and internalize) B7 and other molecules from APC. Operationally, CD28 thus displays conspicuous adhesive function. Interestingly, adhesion of LFA-1 to ICAM-1 seems to be much weaker. Thus, we have failed to see T cell uptake of ICAM-1 from APC unless the T cells are CD28⁺ and the APC are B7⁺. However, indirect uptake of ICAM-1 by T cells through CD28/B7 interaction can be partially inhibited by anti-LFA-1 mAb. This finding suggests that, though weak, the affinity of LFA-1/ICAM-1 interaction is sufficient to stabilize the absorption of molecules mediated by CD28/B7 interaction.

The above data refer to Ag-independent absorption. Essentially similar absorption can occur via TCR/peptide/MHC interaction. This is best exemplified with CD28^-/- TCR transgenic T cells; i.e., where CD28-mediated absorption is avoided. Thus, culturing CD28^-/- CD8⁺ cells with L^d+ APC plus QL9 peptide leads to marked absorption of L^d (and B7) molecules from the APC; in the absence of peptide, absorption is low or undetectable.

	Discussion

Top Abstract Introduction T cell stimulation: the... Transfected Drosophila cells as... Requirements for TCR down... Intensifying signal 1 by... A model for costimulation CD28-dependent absorption of B7... Discussion References

 
The above findings indicate that CD28, a prototypic costimulatory molecule, can also act as an adhesion molecule. The adhesive function of CD28/B7 interaction is sufficient to cause B7 (and other) molecules on APC to be transferred to the surface of T cells and then be internalized.

These findings suggest that CD28/B7 interaction may make a significant contribution to the overall avidity of T/APC interaction. On this point, it is notable that Ag-dependent T/APC interactions lead to the formation of supramolecular activation clusters (SMACs) at the contact site of T/APC interaction (36, 37, 38, 39, 40). SMAC formation is associated with local clustering of a number of different cell-surface molecules, including TCR, B7, and LFA-1; comparable SMAC formation probably also occurs on APC. The point to emphasize is that accumulation of mutually interacting sets of molecules at the contact site of T/APC interaction presumably serves to promote and stabilize TCR/peptide/MHC interaction. This could explain costimulation. Thus, during the initial stages of T/APC interaction, molecules such as CD28 and LFA-1 may serve a sheep-dog role by corralling TCR molecules into SMACs and keeping these molecules in tight proximity (Fig. 8). Perhaps by associating with the cytoskeleton, the various molecules in the corral (SMACs) may have little capacity for lateral movement: the cell membrane in this site becomes relatively rigid, with the result that TCR/peptide/MHC interaction now leads to strong TCR cross-linking. The latter leads to a strong and/or prolonged dose of signal 1 and the T cells become activated.

View larger version (24K): [in this window] [in a new window]   FIGURE 8. A model for early costimulation. In the absence of costimulatory/adhesion molecules, peptide/MHC complexes on APC can move laterally in the cell membrane and thus cause poor TCR cross-linking and only a low dose of signal 1. Costimulatory/adhesion molecules may serve to immobilize TCR/peptide/MHC complexes at the site of T/APC interaction. Aided by local recruitment of tyrosine kinases, such immobilization induces optimal TCR cross-linking, thus resulting in a much higher dose of signal 1. By preventing lateral movement of peptide/MHC complexes on APC, costimulatory/adhesion molecules may create the situation that occurs when T cells are exposed to peptide/MHC complexes immobilized on a solid matrix, or B cells encounter cross-linked epitopes expressed on TI-2 Ags such as TNP-Ficoll. Here, ligand immobilization causes strong TCR/Ig cross-linking and the cells are activated without a requirement for costimulatory adhesion molecules. For T cells, the model proposes that costimulatory/adhesion molecules such as CD28 and LFA-1 have a dual role, functioning first as adhesion molecules and only later as signaling molecules.

The essential feature of the above model is that the initial role of costimulatory molecules is not to provide T cells with signal 2 but to intensify signal 1, i.e., by augmenting TCR cross-linking. The strong dose of signal 1 is adequate to cause T cell activation and early proliferation, but not to induce cytokine production. Lack of cytokines is not important initially, but growth-promoting cytokines such as IL-2 become crucial for sustaining the later stages of the proliferative response. At this time, by controlling cytokine production, the unique signaling function of costimulatory molecules (signal 2) is essential.

This model for costimulation is undoubtedly an oversimplification in at least two respects. First, in addition to enhancing TCR cross-linking, the adhesive function of costimulatory molecules may also promote local accumulation of tyrosine kinases such as p56^lck in the vicinity of the TCR, thus augmenting TCR-mediated signaling (signal 1) (39, 42). Second, the model does not easily explain the marked synergy observed for CD28/B7 and LFA-1/ICAM-1 interactions. Thus, it is puzzling that these molecules act synergistically not only for IL-2 production (where signal 2 is mandatory) but also for CD69/CD25 expression. Yet, based on the studies with peptide/MHC complexes on a solid matrix (Fig. 6), CD69/CD25 up-regulation seems to depend only on TCR cross-linking and not on costimulation. One possibility is that CD28/LFA-1 synergy reflects the different distribution of these molecules in SMACs (36, 37, 38, 39, 40); thus, CD28 and TCR occupy the central region of SMACs whereas LFA-1 has a more peripheral distribution. How this difference in location results in synergy, however, is obscure.

	Acknowledgments

 
I thank Barbara Marchand for typing the manuscript.

	Footnotes

 
¹ This work was supported by Grants CA38355, CA25803, AI21487, AI32068 and AG01743 from the U.S. Public Health Service. This is publication no. 12699-IMM from the Scripps Research Institute.

² Address correspondence and reprint requests to Dr. Jonathan Sprent, Department of Immunology, IMM4, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address:

³ Abbreviations used in this paper: AAI, American Association of Immunologists; RWJPRI, R. W. Johnson Pharmaceutical Research Institute; mRBC, mouse RBC; SMAC, supramolecular activation cluster.

Received for publication September 8, 1999. Accepted for publication September 8, 1999.

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Top Abstract Introduction T cell stimulation: the... Transfected Drosophila cells as... Requirements for TCR down... Intensifying signal 1 by... A model for costimulation CD28-dependent absorption of B7... Discussion References

 

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