500 words paper

Kate Pham

W e describe here an analysis o f C D 2 8 and C T L A - 4 sig- naling o n h i g h l y purified T cells, n o t i n g the presence o f B7 o n t he T cells themselves. T h e results indicate that the t w o molecules have o p p o s i n g effects on l y m p h o k i n e p r o d u c t i o n and proliferation, and t h a t the o u t c o m e o f T cell activation is d e t e r m i n e d b y integration o f signals transduced b y these t w o molecules.

Materials and Methods Immunization and Hybn'doma Production. 6-wk-old golden Syrian

hamsters received five footpad injections of 50 #1 (packed volume) heat-killed Staphylococcus A bacteria coated with •100 # g CTLA- 4Ig (27) and suspended in 0.2 ml PBS. 3 d after the final injection, draining lymph nodes were removed, and lymphocytes were iso- lated and fused with the P3X3.Ag8.653 myeloma line using a stan- dard polyethylene glycol fusion technique (28). Hybridoma super- natants were tested for reactivity to CTLA-4 Ig and for a lack of reactivity to CD4 Ig by ELISA (29). Hybridomas from positive wells were repetitively cloned by limiting dilution in the presence of irradiated mouse thymocyte feeder layers. Antibody 9H10 was specific for CTLA-4 by three criteria: (a) Reactivity against CTLA-4 Ig but not CD4 lg; (b) the ability to block CTLA-4 Ig binding to B7 transfectants; (c) the ability to stain activated T ceils but not freshly isolated T cells; and (d) the ability to stain a CTLA-4 trans- fectant but not control transfectants.

Antibodies. Antibodies used include anti-CD3 clone 50(02 (30), anti-CD28 clone 37.51 (31), anti-B7-1 clone 1610A (9), anti-B7-2 (17), anti-Vqr3 clone 536 (32), anti-class II M H C clone 28-16-8s (33), and anti-IA a/b clone BP107 (34). Conjugates of these anti- bodies were prepared in our laboratory. PE, biotin, and FITC con- jugates of anti-CD4 and anti-CD8 were purchased from CALTAG Laboratories (South San Francisco, CA) and PharMingen (San Diego, CA).

T Cell Activation Cultures. Spleens from 4-6-wk-old BALB/c mice were harvested and minced, and suspensions were treated with Geys RBC lysis solution (35). Cells were cultured in RPMI con- taining 10% FCS and soluble anti-CD3 antibody at 10 #g/ml.

Flow Cytometry. 2 x 10 s cells were suspended in 10 #1 ice-cold PBS/1% calf serum/0.05% sodium azide. Antibodies were added for 30 min followed by two 4-ml washes in PBS/calf serumAo- dium azide. Data were acquired on a FACScan | (Becton Dickinson and Co. (Mountain View, CA) and the LYSIS II program was used to electronically gate on relevant populations.

Proliferation Assays. LN cells were isolated from 6-8-wk-old BALB/c mice (Charles River Laboratories, Wilmington, MA). Iso- lated lymphocytes were obtained by mincing and filtration through nylon sieves. Cells were then treated with anti-class II antibodies 28.16.8s and BP107 and a mixture of rabbit and guinea pig com- plement (Accurate Chemical and Scientific Corp., Westbury, NY). Viable cells were isolated over lympholyte 1.119 (Sigma Chemical Co., St. Louis, MO) and residual Ig-positive cells were removed by repetitive panning on rabbit anti-mouse IgG coated tissue cul- ture plates. Typical preparations analyzed by FACS | were typically found to be 99% Thyl.2 + with <0.5% B220-positive cells. Round-bottomed 96-well plates were used for all assays. Where indicated, wells were coated with anti-CD3 at 0.1/xg/ml in 50-t~l vol for 2 h at 37~ then washed extensively and blocked for 30 rain at 37~ with complete RPMI 1640. T cells were added at l0 s per weli in 200/zi complete ILPMI 1640. For soluble assays, anti-CD28 was added at a 1:1,000 dilution of ascites, anti-CTLA-4 was added at 10 #g/ml, and B7 antibodies were added at 2.5 #g/ml

and 10/zg/ml for anti-B7-1 and anti-B7-2, respectively. For cross- linking assays, anti-CD8 was added at 4 #g/ml, anti-CD3 was added at 5 #g/ml, anti-CTLA-4 was added at 20 #g/ml, and anti-Vy3 control antibody F536 was added to produce a constant total anti- body concentration of 30 #g/ml. Polyclonal goat anti-hamster anti- body (Pierce Chemical Co., Rockford, IL) was added at a final con- centration of 20 #g/ml. All cultures were incubated at 37~ for 72 h, then pulsed with 1 #Ci of [3H]thymidine for an additional 16 h before harvesting.

Antibody Incorporation onto Latex Microspheres. Sulfate polysty- rene latex microspheres of 5 +_ 0.1 #M mean diameter were ob- tained from Interracial Dynamics Corporation (Portland, OR). Ap- proximately 1 x 107 beads per ml were suspended in PBS and the indicated antibodies were incubated for 1.5 h at 370C. Anti-CD3 was added at 1 tzg/ml to deliver the antigen-specific signal, and binding solutions were normalized with control antibody 536 to maintain a constant total antibody concentration of 5 #g/rot during binding. Previous data have shown maximal binding is achieved when this concentration is added per 107 particles (36). Beads were washed extensively with PBS and resuspended in I ml RPMI-10% FCS and allowed to block for at least 30 min at room temperature. 1@ cells were incubated in 96-well cultures with 10 s each of the indicated bead preparation.

Results Activation Induces Expression o f C T L A - 4 on Both C D 4 + and

C D 8 + T cells. Antibody 9H10 was used to assess C T L A - 4 expression o n freshly isolated and activated T cells. As shown in Fig. 1 A , C T L A - 4 was undetectable on freshly isolated T cells. C T L A - 4 was readily detected on T cells 48 h after stimulation by addition o f a n t i - C D 3 to splenocytes, and it was returned to resting levels by 72 h. C D 2 8 expression was not greatly altered by stimulation. C T L A - 4 was expressed by b o t h C D 4 § and C D 8 § T cells, w i t h significantly higher levels on the latter.

Blockade o f C T L A - 4 or B 7 - I / 2 Enhances Anti-CD28 Costimu- lation. To determine the functional consequences o f C T L A - 4 e n g a g e m e n t , soluble a n t i - C T L A - 4 o r a n t i - C D 2 8 antibodies were added singly or t o g e t h e r to cultures o f purified T cells exposed to immobilized anti-CD3. As shown in Fig. 2, while anti-CD28 greatly enhances the minimal proliferation induced by C D 3 stimulation, a n t i - C T L A - 4 has no effect. This sug- gests that C T L A - 4 does not function as an alternate costimula- t o r y receptor. Despite its lack o f c o s t i m u l a t o r y activity on its o w n , a n t i - C T L A - 4 markedly increases T cell prolifera- tion w h e n given t o g e t h e r w i t h anti-CD28.

At least t w o mechanisms could account for the potentiating effects o f a n t i - C T L A - 4 : enhancement o f activation signals or removal o f inhibitory signals. Given the observation that a n t i - C T L A - 4 lacked c o s t i m u l a t o r y activity on its own, we favored the latter p o s s i b i l i t y - that blockade o f C T L A - 4 by the a n t i b o d y m i g h t prevent interaction o f C T L A - 4 on the T cell surface with B7-1 and/or B7-2 and thus prevent delivery o f an i n h i b i t o r y signal. T h i s is f u r t h e r suggested b y the ob- servation that a n t i - C T L A - 4 Fab fragments have similar effects as a n t i - C T L A - 4 mAbs in this assay system (data not shown). Since the cells used in o u r experiments were > 9 9 % T h y l + and contained no detectable M H C class-II + or B220 + cells,

460 Opposing Effects of CD28 and CTLA-4 on T Cell Response

C T L A - 4 C D 2 8 aCD3+ a C D ' 2 a a C T L A 4 I R 7 1 + 2

" "~"P++++++++++++++++++++++++++++I-, .4- - i - = -li + + ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

- +

+ + ~iiiiii~i~giiii~::iiiiiii:J:=i!i~iii:=iii~i~iiii..,iii!ii~iii..:i....::~....::=..ii:..!i!i!~::i::::::=:iii=:::::::::N~ 4

++li ; ::::::::::::::::::::::::::::::::::;; ;N; -: +g::;:,i.:+:;;g;;;| + + + :::!:::!:: :::::: : :::~:': : : :::::~:~:::~:::I. " I i i i I 0 ~ I0~ I~000

Figure 2. Influence of anti-CTLA-4 and anti-B7 antibodies on prolifer- ative responses of purified lymph node T cells. 105 BALB/c LN T cells were cultured for 72 h in 96-weU microwells coated with 0.1 #g/ml anti- CD3. Anti-CD28 was used at a 1:1,000 dilution of ascites and B7 anti- bodies were used at 2.5 and 10 #g/ml for anti-B7-1 and anti-B7-2, respec- tively. Anti-CTLA-4 antibody 9H10 was added at 10 ~g/ml.

b

I . . . . . '1 . . . . . . . . I ' ' ' " ~ ' J

F i g u r e 1. Expression of CTLA-4 and CD28 on resting and activated splenic T cells. BALB/c splenic cell suspensions (2 x 10S/ml) were stimu- lated in vitro with 10/~g/ml soluble anti-CD3. (a) Cells were double stained with Thyl.2PE and either hamster Ig-control FITC, anti-CTLA-4 FITC, or anti-CD28 FITC. Data were electronically gated for Thyl.2-positive cells; CTLA-4 and CD28 expressions are shown on freshly explanted cells and after 48- and 72-h incubations with anti-CD3. (b) 48-h cultures were stained for anti-CD4 biotin or anti-CD8 biotin followed by avidin tri- color and FITCylated irrelevant or CTLA-4 antibodies. Subpopulation- gated data show modestly higher CTLA-4 expression on CD8 populations.

this possibility would require that the T cells themselves pro- vide a source of ligand. As shown in Fig. 3, flow cytometric analysis revealed that the freshly isolated T cells did indeed express significant levels of B7-2 and trace levels of B7-1.

To determine the functional consequence of B7 expression by T cells in our assay system, we determined the effects of anti-B7 antibodies on CD28-mediated costimulation. As shown in Fig. 2, anti-B7 antibodies by themselves had no significant effect on anti-CD3-induced T cell proliferation. The addition of anti-B7 antibodies to cultures containing anti- CD28 resulted in a threefold increase in proliferation over that obtained w i t h anti-CD28 alone. A similar increase in

proliferation was obtained when chimeric CTLA-4 Ig instead of anti-B7 antibodies was added to block B7 interactions (data not shown). The magnitude of the increase was similar to that obtained when anti-CTLA-4 is added to CD28-treated cells in the absence o f B7 blockade. The addition o f anti- CTLA-4 to cultures in which anti-B7 antibodies are present results in no further increase in CD28 costimulation; indeed, a slight but reproducible decrease is observed.

Together, these results suggest that T cells express B7 at levels that are insufficient to provide costimulation via CD28 engagement in the assay system used. However, perhaps be- cause of the fact that CTLA-4 has a much higher affinity than CD28 for B7 binding, these levels are sufficient to generate a signal that at least partially inhibits activation. Blockade of the C T L A - 4 / B 7 interaction with either anti-CTLA-4 or antioB7 antibodies removes the inhibitory signal, resulting in an increase of the costimulatory effect of CD28 ligation.

Cross-linking of CTLA-4 with the TCR and CD28 Inhibits T Cell Proliferation and IL-2 Production. The results shown

7 ? -~ ,27. t

Figure 3. Expression of B7-2 and B7-1 on purified, freshly isolated lymph node T cells. Cells that were >99% Thyl.2 + were isolated and stained with GL1 (anti-B7-2), 1610A (anti-B7-1), or irrelevant antibodies.

461 Krummel and Allison

in Fig. 2 suggested that soluble, bivalent anti-CTLA-4 anti- body was effective in blocking B7-mediated signals, but was inefficient in providing signals. We next examined the effects of using anti-hamster Ig to cross-link CD3, CD28, and C T L A - 4 singly or together. As shown in Fig. 4, no prolifer- ation was obtained when CD3, CD28, or CTLA-4 were cross- linked individually. As expected, cross-linking of CD3 to- gether with CD28 resulted in potent costimulation, while cross-linking of CD3 and C T L A-4 had no effect. Co-cross- linking o f CTLA-4 together wit h C D3 and CD28 consis- tently resulted in a 5- to 10-fold reduction in proliferation. This inhibition was largely reversed by the addition of IL-2 to the cultures, suggesting that the effect is not caused by toxicity. Finally, cross-linking of C T L A-4 wi th CD3 and CD28 also resulted in a profound decrease in IL-2 produc- tion in the cultures (Fig. 4 B). These results demonstrate that CTLA-4 can deliver signals that inhibit T cell responses to T C R ligation, and that the effects observed in the experi-

m A b X - l i n k e d

C~rl t aCD3,Ctrl 1

aCD2S,Ctrl 1 aCTLA-4,Ctrl 1

aCD3,aCD28.Ctrl-[~ !! ! ~:.~:.~:.! !~:. i:.i:.~:.i iii ~il ![[~:.i:. i:.i ;.i:.~i~:. i:.i:.i:.i :.iii!i:. i :.i:. ~ I - - I

ac~D3aca~A-4aCTL:~ ~

aCD3,aCD28 ,aCTLA-4JL2 '-~ i!i!ii!iiii::!!ii~i!:i!i!i!::~i~i!::~i~i!::!::!~ ] -I I I I I i

0 50000 100000 150000 20~C00 250000 cpm

M A b ) [ - l i n k e d

aCD28~"CU'[ - ~

aCTLA-4-',Ctrl -

aCD3+Ctrl -

aCD3+aCTLA-4-t(2trt - ~

aCD3+aCD28+aCTLA4 - T

0 5 10 15 20 25 IL2 (ng/ml)

Figure 4. Cross-linked anti-CTLA-4 can diminish both proliferation and lymphokine production by purified LN T cells. 10s BALB/c LN T cells were cultured with the indicated hamster antibodies together with control hamster antibodies. Anti-hamster Ig antibody was added at 20 /~g/ml to cross-link. Where indicated, anti-CD3 was added at 5 ~tg/ml, anti-CD28 was added at 4 ~g/ml, anti-CTLA-4 was added at 20 #g/ ml, and control was added to normalize antibody concentration at 30 p.g/ml. (a) Calls were cultured for 72 h, pulsed with 1 ~Ci [3H]thymidine, and harvested after an additional 16 h. (b) Supernatants were removed and ana- lyzed for II.-2 production at 48 h using an ELISA detection system.

ment presented in Fig. 2 most likely result from perturba- tion of B 7 / C T L A - 4 interactions.

CD28 and C T L A - 4 Deliver Quantitatively Opposing Sig- nals. The preceding data indicate that CTLA-4 cross-linking in the presence of CD28 signaling can inhibit IL-2 secretion and proliferation. We next sought to determine whether sig- naling above the threshold for CTLA-4 inhibition is inde- pendent of the magnitude of CD28 costimulation, or whether the threshold increases as CD28 signaling increases. To ad- dress this issue, T cells were stimulated by incubation with polystyrene beads coated with a constant amount of anti- CD3 and varying amounts of anti-CD28 and anti-CTLA-4. As shown in Fig. 5, costimulation with increasing amounts of anti-CD28 in the absence of anti-CTLA-4 resulted in a gradual increase in proliferation, reaching at the highest dose a 1,500-fold increase over that obtained with anti-CD3 alone. The addition of increasing amounts of anti-CTLA-4 reduced that proliferation in a stepwise manner at all doses of anti- CD28. These results suggest that T cells integrate signals from CD28 with signals from CTLA-4, and the balance o f these signals regulates the magnitude of the response to T C R ligation.

Discussion

The results presented here clearly demonstrate that CTLA-4 does not serve as a functional alternative to CD28 in providing costimulatory signals to T cells. This finding is in agreement with earlier studies showing that CTLA- 4 did not replace CD28 function in CD28 mutant mice (37). The finding that anti-CTLA-4 increases proliferation of T cells activated by anti-CD3 and anti-CD28 is in agreement with the results of Linsley et al. (24). However, the fact that a similar result is obtained when blocking antibodies to B7 are included sug- gests that this apparent cooperativity of CTLA-4 is in fact a result o f removal of preexisting inhibitory B 7 - C T L A - 4 in-

Figure 5. Proliferation in response to a constant CD3 signal is affected by the relative concentrations of CD28 and CTLA-4 signals. 107 5 ~tM microspheres were coated with 1 ~tg/ml of anti-CD3, the indicated con- centrations of anti-CD28 and anti-CTLA-4, and control hamster Ig con- stituting a total antibody concentration of 5 pg/ml. 10 s coated spheres were incubated in 96-well cultures with 10 s purified LN T calls for 72 h, pulsed with 1 ~tCi [3H]thymidine, and harvested after an additional 16 h.

462 Opposing Effects of CD28 and CTLA-4 on T Cell Response

teractions by the soluble CTLA-4 antibodies rather than a synergism between the two antibody-generated signals. The ability of CTLA-4 to directly signal is supported by the fact that cross-linking of anti-CTLA-4, either with second anti- body or by presentation immobilized on beads, results in in- hibition of both IL-2 production and proliferation. This di- rect demonstration of signaling by CTLA-4 supports the report of Walunas et al. (26) that CTLA-4 is a negative regu- lator of T cell activation.

O u r demonstration o f a dynamic competition between CD28 and CTLA-4 indicates that in addition to CD3 and CD28 signal integration, there exists an integration point for CTLA-4-derived signals. At present there is little knowl- edge of the biochemical events that follow CD28 or CTLA-4 ligation. There have been reports that CD28 stimulation results in induction of protein tyrosine kinase activity (38), and re- cent evidence suggest the Tec family kinase ITK represents one associated protein kinase (39). In addition, it has been demonstrated that phosphoinositides accumulate in T cells stimulated by ligation of CD28 with B7-1, suggesting an involvement of phosphatidylinositol 3'-kinase (PI3K) 1 with CD28 (40). In this regard, it is of interest that the cytoplasmic domains of both CD28 and CTLA-4 contain the sequence Y M / V X M , a motif found in several growth factor receptors that associate with PI3K (41, 42). Several recent reports have documented a stimulation-induced association between CD28 and PI3K, and it has been reported that mutation of the PI3K- binding motif destroys the costimulatory activity of CD28 (43-47). These findings strongly suggest that binding of PI3K plays an important role in CD28 signaling. W i t h respect to CTLA-4, however, there have been contradictory findings. Whereas a chimeric protein containing the cytoplasmic do- main of CTLA-4 was unable to bind PI3K (46), another study

1 Abbreviation used in this paper: PI3K, phosphatidylinositol 3'-kinase.

reported the coprecipitation of PI3K activity with CTLA-4 (48). In any event, these findings raise the possibility that CD28 and CTLA-4 might compete for PI3K and affect its role in subsequent signal transduction.

It is also possible that CD28 and CTLA-4 signals might intersect at later stages in the pathway. It has been demon- strated that the CD28 and CD3 pathways intersect at the level of the MAP kinase J N K (49). CTLA-4 might in someway interfere with this coupling, thus preventing costimulation. Finally, it is possible that CTLA-4 signals might interfere with those of CD28 even further downstream by interfering with IL-2 transcriptions or m R N A stabilization (46, 50).

O u r results further suggest that regulation of the outcome of T cell stimulation is a complex process with regard to events at the cell surface. It is clear that in the absence of costimula- tory signals provided by the B7 family, T cells do not proliferate. It appears that even small amounts of B7, such as those present on T cells themselves, are ineffectual in sup- porting CD28-mediated costimulation of anti-CD3 responses. This appears to be less a consequence of the absence of CD28 signal being delivered, but rather a result of an inhibitory signal delivered through CTLA-4. This implies that either CTLA-4 is quickly expressed after activation and aborts the response, or that CTLA-4 is expressed at functionally significant levels on resting T cells. At higher levels of B7 expression, as might be encountered on activated dendritic cells and activated B cells, CTLA-4 expression on the T cells might become limiting, and the costimulation provided by CD28 becomes dominant. As expression of CTLA-4 rises after activation, the signals generated through CTLA-4 might become dominant and terminate the response. Decay of CTLA-4 expression with time would allow the T cell to re- turn to a state where the CD28 costimulatory signal would predominate. In any event, accumulating evidence suggests that in addition to antigen receptor and CD28-mediated signals, a third signal, provided by CTLA-4, is important in determining the outcome of T cell activation.

We are grateful to Drs. P. Lane and K. Karljialeinen for providing CTLA-4Ig and CD4Ig fusion chimeras. We thank David Raulet for critical reading of the manuscript.

This work was supported by National Institutes of Health grants CA40041 and CA09179.

Address correspondence to James P. Allison, Dept. of Molecular and Cell Biology and Cancer Research Laboratory, University of California, Berkeley, CA 94720.

Received for publication 8 December 1994 and in revised form 6 March 1995.

References 1. June, C.H., J.A. Bluestone, L.M. Nadler, and C.B. Thompson.

1994. The B7 and CD28 receptor families. IrnmunoL Today. 15:321-331.

2. Jenkins, M.K. 1994. The ups and downs ofcostimulation. Im- munity. 1:443-446.

3. Linsley, ES., andJ.A. Ledbetter. 1993. The role of the CD28 receptor during T cell responses to antigen. Annu. Rev. Im- munot. 11:191-212.

4. Harding, F., J.G. McArthur, J.A. Gross, D.H. Raulet, and J.P. Allison. 1994. CD28 mediated signalling costimulates murine

463 Krummel and Allison

T cells and prevents the induction of anergy in T cell clones. Nature (Lond.). 356:607-609.

5. Holsti, M.A., J. McArthur, J.P. Allison, and D.H. Raulet. 1994. Role of I1,-6, I L l , and CD28 signaling in responses of mouse CD4 § T cells to immobilized anti-TCR monoclonal antibody. J. Immunot. 152:1618-1624.

6. Caux, C., B. Vanvervliet, C. Massacrier, M. Azuma, K. Okumura, L.L. Lanier, and J. Banchereau. 1994. B70/B7-2 is identical to CD86 and is the major functional ligand for CD28 expressed on human dendritic cells, j . Exp. Med. 180:1841-1847.

7. Linsley, P.S., E.A. Clark, andJ.A. Ledbetter. 1990. The T cell antigen, CD28, mediates adhesion with B cells by interacting with the activation antigen, B7/BB-1. Proc. Natl. Acad. Sci. USA. 87:5031-5035.

8. Azuma, M., D. Ito, H. Yagita, K. Okumura, J.H. Phillips, L. Lanier, and C. Somoza. 1993. B70 antigen is a second ligand for CTLA-4 and CD28. Nature (Lond.). 366:76-79.

9. Razi-Wolf, Z., G.J. Freeman, F. Galvin, B. Benacerraf, L. Nad- let, and H. Reiser. 1992. Expression and function o f the mu- fine B7 antigen, the major costimulatory molecule expressed by peritoneal exudate cells. Proc. Natl. Acad. Sci. USA. 89:4210-4214.

10. Reiser, H., G.J. Freeman, Z. Razi-Wolf, C.D. Gimmi, B. Benacerraf, and N.M. Nadler. 1992. Murine B7 antigen pro- vides a costimulatory signal for activation of murine T lym- phocytes via the T cell receptor/CD3 complex. Proa Natl. Acad. Sci. USA. 89:271-275.

11. Linsley, P.S., W. Brady, L. Grosmaire, A. Aruffo, N.K. Damle, and J.A. Ledbetter. 1991. Binding of the B cell activation an- tigen B7 to CD28 costimulates T cell proliferation and inter- leukin 2 m R N A accumulation. J. Exp. Ailed. 173:721-730.

12. Gimmi, C.D., G.J. Freeman, J.G. Gribben, K. Sugita, A.S. Freedman, C. Morimoto, and L.M. Nadler. 1991. B-cell sur- face antigen B7 provides a costimulatory signal that induces T cells to proliferate and secret interleukin 2. Proc. Natl. Acad. Sci. USA. 88:6575-6579.

13. Reiser, H., G.J. Freeman, Z. Razi-Wolf, C.D. Gimmi, B. Benacerraf, and L.M. Nadler. 1992. Murine B7 antigen pro- vides an efficient costimulatory signal for activation of routine T lymphocytes via the T-cell receptor/CD3 complex. Proa Natl. Acad. Sci. USA. 89:271-275.

14. Murphy, E.E., G. Terres, S.E. Macatonia, C.S. Hsieh, J. Mattson, L.L. Lanier, M. Wysocka, G. Trinchieri, K. Murphy, and A. O'Garra. 1994. B7 and interleukin-12 cooperate for proliferation and I F N - g production by mouse T h l clones that are unresponsive to B7 costimulation. J. Exp. Med. In press.

15. Inaba, K., M. Witmer-Pack, M. Inaba, K.S Hathcock, H. Sakuta, M. Azuma, H . Yagita, K. Okumura, P.S. Linsley, S. Ikehara, et al. 1994. The tissue distribution of the B7-2 costimu- lator in mice: Abundant expression on dendritic cells in situ and during maturation in vitro.J. Exp. Med. 180:1849-1860.

16. Linsley, P.S., W. Brady, M. Urnes, L.S. Grosmaire, N.K. Damle, andJ.A. Ledbetter. 1991. CTLA-4 is a second receptor for the B cell activation antigen B7. J. Exp. Med. 174:561-569.

17. Freeman, G.J., F. Borriello, R.J. Hodes, H. Reiser, K.S. Hath- cock, G. Laszlo, A.J. McKnight, J. Kim, L. Ku, D.B. Lom- bard, et al. 1993. Uncovering of functional alternative CTLA-4 counter-receptor in B7-deficient mice. Science (Wash. DC). 262:907-909.

18. Lenschow, D.J., G.H.-T. Su, L.A. Zuckerman, N. Nabavi, C.L. Jellis, G.S. Gray, J. Miller, and J.A. Bluestone. 1993. Expres- sion and functional significance o f an additional ligand for

CTLA-4. Proc. Natl. Acad. Sci. USA. 90:11054-11058. 19. Linsley, P.S., P.M. Wallace, J. Johnson, M.G. Gibson, J.L.

Greene, J.A. Ledbetter, C. Singh, and M.A. Tepper. 1992. Im- munosuppression in vivo by a soluble form of the CTLA-4 T cell activation molecule. Science (Wash. DC). 257:792-795.

20. Lenschow, D.J., Y. Zeng, J.Ik. Thistlewaite, A. Montag, W. Brady, M.G. Gibson, P.S. Linsley, and J.A. Bluestone. 1992. Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4Ig. Science (Wash. DC). 257:789-792.

21. Brunet, J.F., E Denizot, M.F. Luciani, M. Roux-Dosseto, M. Suzan, M.F. Mattei, and P. Golstein. 1987. A new member of the immunoglobulin superfamily CTLA-4. Nature (Lond.). 328:267-270.

22. Harper, K., C. Balzano, E. Rouvier, M.G. Mattei, M.F. Lu- ciani, and P. Golstein. 1991. CTLA-4 and CD28 activated lym- phocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chro- mosomal location. J. Immunol. 147:1037-1044.

23. Lindsten, T., K.P. Lee, E.S. Harris, B. Petryniak, N. Craig- head, P.J. Reynolds, D.B. Lombard, G.J. Freeman, L.M. Nad- let, G.S. Gray, et al. 1993. Characterization of CTLA-4 struc- ture and expression on human T cells.J. Fmmunol. 151:3489- 3499.

24. Linsley, P.S., J.L. Greene, P. Tan, J. Bradshaw, J.A. Ledbetter, C. Anasetti, and N.K. Damle. 1992. Coexpression and func- tional cooperativity of CTLA-4 and CD28 on activated T lym- phocytes. J. Exp. Med. 176:1595-1604.

25. Damle, N.K., K. Klussman, G. Leytze, S. Myrdal, A. Aruffo, J.A. Ledbetter, and P.S. Linsley. 1994. Costimulation of T lym- phocytes with integrin ligands intercellular adhesion molecule-1 or vascular cell adhesion molecule-1 induces functional expres- sion of CTLA-4, a second receptor for B7. J. lmmunol. 152:2686-2697.

26. Walunas, T.L., D.J. Lenschow, C.Y. Bakker, P.S. Linsley, G.J. Freeman, J.M. Green, C.B. Thompson, and J.A. Bluestone. 1994. CTLA-4 can function as a negative regulator of T cell activation. Immunity. 1:405-413.

27. Lane, P., W. Gerhard, S. Hubele, A. Lanzavecchia, and F. McConnell. 1993. Expression and functional properties of mouse BB1/B7 using a fusion protein between mouse CTLA-4 and human gl. Immunology. 80:56-61.

28. McKearn, T.J., F.W. Fitch, D.E. Smilek, M. Sarmiento, and F.P. Stuart. 1979. Properties of rat anti-MHC antibodies pro- duced by cloned rat-mouse hybridomas. Immunol. Rev. 47:91-98.

29. Jitsukawa, T., S. Nakajima, I. Sugawara, and H. Watanabe. 1989. Increased coating efficiency of antigens and preservation of original antigenic structures after coating in ELISA. J. Im- munol. Methods. 116:251-256.

30. Allison, J.P., W.L. Havran, M. Poenie, J. Kimura, L. Degraffenreid, S. Ajami, G. Duwe, A. Weiss, and R. Tsien. 1987. Expression and function of CD3 on murine thymocytes. In The T Cell Receptor, U C L A Symposia on Molecular and Cellular Biology, New Series. J. Kappler, and M. Davis, editors. Alan R. Liss, Inc., New York. pp. 33-45.

31. Gross, J.A., E. Callas, and J.P. Allison. 1992. Identification and distribution of the costimulatory receptor CD28 in the mouse. J. Immunol. 149:380-388.

32. Havran, W.L., S.C. Grell, G. Duwe, J. Kimura, A. Wilson, A.M. Kruisbeek, 1K.L. O'Brien, W. Born, R.E. Tigelaar, and J.P. Allison. 1989. Limited diversity o f T C R g chain expres- sion of routine Thy-1 * dendritic epidermal cells revealed by Vg3-specific monoclonal antibody. Proc. Natl. Acad. &i. USA. 86:4185-4189.

464 Opposing Effects of CD28 and CTLA-4 on T Cell Response

33. Ozato, K., and D.H. Sachs. 1981. Monoclonal antibodies to mouse MHC antigens. J. Immunol. 126:317-323.

34. Symington, F., and J. Sprent. 1981. A monoclonal antibody detecting an Ia specificity mapping in the I-A or I-E subregion. Immunogenetics. 14:53-61.

35. MisheU, B., and S. Shiigi. 1980. Selected Methods in Cellular Immunology. W.H. Freeman Co., San Francisco. pp. 23-24.

36. Mescher, M.F. 1992. Surface contact requirements for activa- tion of cytotoxic T lymphocytes.J. Immunol. 149:2402-2405.

37. Green, J.M., P.J. Noel, A.I. Sperling, T.L. Walunas, G.S. Gray, J.A. Bluestone, and C.B. Thompson. 1994. Absence of B7- dependent responses in CD28-deficient mice. Immunity. 1:501-508.

38. Lu, Y., A. Granelli-Piperno, J.M. Bjorndahl, C.A. Phillips, and J.M. Trevillyan. 1992. CD28-induced T cell activation. Evidence for a protein-tyrosine kinase signal transduction pathway. J. Immunol. 149:24-29.

39. August, A., S. Gibson, Y. Kawakami, G.B. Mills, and B. Dupont. 1994. CD28 is associated with and induces the im- mediate tyrosine phosphorylation and activation of the Tec family kinase ITK/EMT in the human Jurkat leukemic T-cell line. Proc. Natl. Acad. Sci. USA. 91:9347-9351.

40. Ward, S.G., J. Westwick, N.D. Hall, and D.M. Sansom. 1993. Ligation of CD28 receptor by B7 induces formation of D-3 phosphotides in T lymphocytes independently of T cell receptor activation. Eur. J. Immunol. 23:2572-2577.

41. Songyang, Z., S.E. Shoelson, J. McGlade, P. Olivier, T. Pawson, X.R. Bustelo, M. Barbacid, H. Sabe, H. Hanafusa, T. Yi, et al. 1994. Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GKB-2, HCP, SHC, Syk, Vav. MoI. Ceil. Biol. 14:2777-2785.

42. Songyang, Z., S.E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W.G. Haser, F. King, T. Roberts, S. Ratnofsky, R.J.

Lechleider, et al. 1993. SH2 domains recognize specific phos- phopeptide sequences. Cell. 72:767:778.

43. August, A., and B. Dupont. 1993. CD28 of T lymphocytes associates with phosphatidylinositol 3-kinase. Int. Immunol. 6:769-774.

44. Prasad, K.V., Y.C. Cai, M. Raab, B. Duckworth, L. Cantley, S.E. Shoelson, and C.E. Rudd. 1994. T-cell antigen CD28 in- teracts with the lipid kinase phosphatidylinositol 3-kinase by a cytoplasmic Tyr(P)-Met-Xaa-Met motif. Proc Natl. Acad. Sci. USA. 91:2834-2838.

45. Truitt, K.E., C.M. Hicks, and J.B. Imboden. 1994. Stimula- tion of CD28 triggers an association between CD28 and phos- phatidylinositol Y-kinase in Jurkat T cells. J. Exp. Med. 179:1071-1076.

46. Stein, P.H., J.D. Fraser, and A. Weiss. 1994. The cytoplasmic domain of CD28 is both necessary and sufficient for costimu- lation of interleukin-2 secretion and association with phospha- tidylinositol Y-kinase. Mol. Cell. Biol. 14:3392-3402.

47. Pages, F., M. Ragueneau, K. Rottapel, A. Truneh, J. Nunes, J. Imbert, and D. Olive. 1994. Binding of phophatidyl-inositol- 3-OH kinase to CD28 is required for T-cell signalling. Nature (Lond.). 369:327-329.

48. Schneider, H., V.S. Prasad, S.E. Shoelson, and C.E. Rudd. 1995. CTLA-4 binding to the lipid kinase phosphatidylinositol 3-kinase in T cells. J. Exp. Med. 181:351-355.

49. Su, B., E. Jacinto, M. Hibi, T. Kallunki, M. Karin, and Y. Ben-Neriah. 1994. JNK is involved in signal integration during costimulation of T lymphocytes. Cell. 77:727-736.

50. Lindsten, T., C.H. June, J.A. Ledbetter, G. Stella, and C.B. Thompson. 1989. Regulation oflymphokine messenger RNA stability by a surface-mediated T cell activation pathway. Science (Wash. DC). 244:339-343.

465 Kmmmel and Allison