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ified by examination of the C5a receptorknockout mice. C3b on the other hand, hasbeen shown capable of blocking IL-12 produc-tion by its interaction with the αMβ2 integrin(also known as Mac-1 or CR3)7,8, an action per-fectly in keeping with a positive role for C3cleavage in AHR, either via C3a and its recep-tor or through C3b and reduced levels of IL-12.However, although a potential role for IL-12 inthese complement-mediated processes isintriguing, it seems likely that this is not theonly point of intersection between active com-ponents of the complement system and thecytokine network.

Even if IL-12 is a major point of regulation,these observations merely shift responsibilityone step down. One must now ask how IL-12modifies the cytokine mix to prevent, or itsabsence so alters the cytokine mix to promote,the fundamental biochemical and biophysicalprocesses causing airway hyperresponsive-ness. Presumably C3a would be expected toact more directly on these downstream effec-tor pathways—but where? This is still a major

question. Finally, although seemingly self-evident, it may still be important to emphasizeavoidance of the all too common tendency toattribute key roles to any one or two mole-cules in biological phenomenon as complexas asthma, or even the more limited manifes-tation of hyperresponsiveness. The excite-ment in these two studies comes from the newquestions that they raise and the hope for anew understanding of the actual mechanismsunderlying the process of AHR itself. Theyalso clearly raise the possibility that the twomolecules, C3a and C5a—usually thought toact in the same direction—have oppositeeffects. An additional set of important ques-tions might then address the source of the C3or C5 cleavage. Candidate proteases are pre-sent in most inflammatory reactions andwould be expected in asthmatic airways.Bacteria in the upper respiratory tract andsinuses may also contribute to generation ofcomplement fragments that then gain accessto the lower airways9. There is also an increas-ing interest in the possibility that chronic

asthma is associated with the presence ofinfectious agents such as mycoplasma orchlamydia in the lower airways. Whateverrole these might play in the pathogenesis orexacerbation of the human disease, and quiteseparate from the possible source of C3- orC5-cleaving proteases in the murine modelsdiscussed here, complement activation bysuch organisms could certainly add to the mixof bioactive molecules in the airways and, asa consequence, to the balance between normaland altered responses to exposure.

1. Karp, C.L. Nature Immunol. 221–226 (2000).2. Humbles,A.A. Nature, 406 (in the press, 2000).3. Teran, L. M. et al. Clin. Exp.Allergy 27, 396–405 (1997).4. Regal, J. F. & Klos,A. Immunopharmacology 46, 15–28

(2000).5. Irvin, C. G., Berend, N. & Henson, P. M. Am. Rev. Respir. Dis. 134,

777–783 (1986).6. Hopken, U. E., Lu, B., Gerard, N. P. & Gerard, C. Nature 383,

86–89 (1996).7. Sutterwala, F. S., Noel, G. J., Clynes, R. & Mosser, D. M. J. Exp. Med.

185, 1977–1985 (1997).8. Marth,T. & Kelsall, B. L. J. Exp. Med. 185, 1987–1995 (1997).9. Brugman, S. M., Larsen, G. L., Henson, P. M., Honor, J. & Irvin, C. G.

Am Rev Respir Dis 147, 314–320 (1993).

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In contrast to some animal models of autoim-mune disease, such as experimental autoim-mune encephalomyelitis (EAE), that areinduced by immunization with specific self-antigens, diabetes in the nonobese diabetic(NOD) mouse arises spontaneously under theinfluence of more than 18 polymorphicgenes1. The disease goes through definedcheckpoints from peri-insulitis to insulitis andovert diabetes as mice age and the diseaseprogresses2. Also, unlike EAE, in the NODmouse CD8 cells as well as CD4 cells havebeen shown to be pathogenic effectors for theinduction of diabetes3. Antigen recognition iscentral to the disease process, and identifyingthe antigens involved has remained an impor-tant theme in diabetes research. In a recentreport in Nature4, Amrani et al. describe stud-ies that address this question from the per-spective of a specific major histocompatibili-ty complex (MHC) class I–restricted CD8 Tcell response to islet cells. In previous workthe same group exploited the observation that

Selecting killers: the linebetween life and deathLINDSAY B. NICHOLSON AND VIJAY K. KUCHROO

How do T cells that are specific for pancreaticislet cell antigens cause diabetes? A recent paperin Nature provides evidence from NOD micethat the killer T cells responsible increase theiravidity as the disease progresses—removing thehigh avidity clones prevents disease.

a majority of the CD8 T cells that infiltrate theislets of the pancreas in diabetes bear arestricted set of T cell receptor (TCR) αchains, paired with a diverse repertoire of Vβ

chains. With a representative TCR from theseCD8+ T cells, they generated the 8.3 TCRtransgenic mouse whose CD8+ T cells caninduce diabetes, although diabetes is inducedmore efficiently when CD4 cells are also pre-sent5. The antigenic epitopes that these CD8+

T cells recognize in the islets is not known.However, screening a peptide library yieldedtwo peptides, NRP (KYNKANWFL) andNRP-A7 (KYNKANAFL), that activate thetransgenic 8.3 TCR–bearing T cells in thecontext of H-2Kd (ref. 6). Neither of thesepeptides is a known autoantigen from pancre-atic islets. In these activation assays, it wasclear that whereas the NRP peptide had ago-nist properties, an alanine mutant of the pep-tide called NRP-A7 acted as a superagonistpeptide for this TCR.

They have now taken the next step and

addressed the role of the T cells whose TCRsbind these peptides in the pancreas of normalNOD mice, by using NRP and NRP-A7–Kd

tetramers to identify them. To do this theyexpanded islet-derived T cells from nondia-betic mice in the presence of interleukin 2(IL-2) for a week before analyzing them,which may explain why the frequency oftetramers recognizing an insulin peptide–Kd

complex is lower than previously reported7.They have come to the arresting conclusionthat the average avidity of the population of Tcells that recognizes NRP-A7 increases asdisease progresses and that elimination of thehigh avidity cells prevents diabetes.

The study raises several interesting ques-tions for immunologists investigating thebiology of autoimmune diseases. The firstissue is why should increases in avidity cor-relate with pathogenicity and disease pro-gression? The second is why should elimina-tion of high avidity T cells of just one clono-type result in almost complete prevention of

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diabetes? TCRs are known for the weaknature of their ligand interaction. Moreover,activation is often seen as a threshold phe-nomenon on one side of which T cells aredormant, and on the other side, fully activat-ed. Consistent with the observations of P.Allen, A. Sette and R. Germain, the findingspresented in this study lend support to mod-els of T cell activation in which differenteffector functions are induced at incremen-tally higher activation signals. Therefore,perhaps higher avidity cells are more patho-genic because their killing functions areinduced more efficiently. On the other hand,the monoclonal 8.3 TCR transgenic mousestill develops diabetes soon after developinginsulitis5. Therefore avidity maturation maynot only be critical for pathology to develop,but also for selectively expanding cells froma diverse pool of infiltrating cells of vary-ing avidity, which can overcome the check-points to disease and mediate diabetes. Tounderstand whether this is the case, weneed to understand what drives the processof avidity maturation in immunization,infection and autoimmune disease.

For antibodies, affinity maturation is dri-ven by limiting the amounts of antigen.One might have expected that in the pan-creatic islets, where a large pool of islet tis-sue antigen is available, even low avidity Tcells could expand and contribute to theprogression of disease. There are severalreasons why avidity maturation (or moreprecisely selection of high avidity T cells)may be necessary for the development ofpathogenic CD8+ effector cells in autoim-mune diabetes. Cytolytic CD8+ cells arebelieved to lyse pancreatic islet cells direct-ly, even though these cells lack expressionof costimulatory molecules (B7s andCD40) and generally express few class Imolecules. By the time diabetes develops,the majority of the β-cells in the islets havebeen destroyed and so the availability ofthe pancreatic antigen recognized by theNRP-A7–reactive cells in the tissue may belimiting. The limited availability of both theligand for the TCR (class I MHC and antigen)and a lack of expression of costimulatorymolecules on the pancreatic islet cells may becritical factors leading to the expansion ofcells of high avidity that have a lowerrequirement for costimulation8, and whichovercome the regulatory checkpoints andcause tissue destruction (Fig. 1). There mayalso be other fundamental mechanisms dri-ving the selection of a single or a few clonesfrom the whole population of cells that canrespond to a particular antigen, as eventually

only a single clone may be able to occupy aspecific niche. This idea of competitiveexclusion has been considered theoreticallyin the context of T cell repertoires9, but thereis little experimental data to assess its validi-ty. In the future it will also be interesting todetermine whether there is a need for patho-genic CD4+ effector cells to also undergoavidity maturation before they induce dis-ease, since their expansion will be driven byprofessional antigen presenting cells (APCs)that express many class II and costimulatorymolecules.

In their analysis of this system, the authorshave concluded that the cells that bind NRPtetramer are a subset of the whole populationof cells that bind the NRP-A7 tetramer,because the affinity of the 8.3 TCR is higherfor the NRP-A7 tetramer than for the NRP

tetramer. This seems inconsistent with theresults presented in the paper for two reasons.First the NRP-A7 tetramer binds more cellsthan NRP tetramer because it binds the lowavidity cells that the NRP tetramer cannot.Therefore if the avidity of the population isincreasing, then the fraction of cells that bindsNRP tetramer should increase, but in fact itdecreases. Second, in cells derived from theislets of 5-week-old mice, the percentage andnumber of NRP-A7 tetramer–binding cells islower than the percentage and number of NRP

tetramer–binding cells. This cannot be thecase if all the NRP tetramer–binding cells arecontained within the set of cells that bindNRP-A7 tetramer. Therefore a more likelyinterpretation of the data is that cells that bindNRP-A7 tetramer and NRP tetramer are qual-itatively distinct but overlapping populations,rather than one population being a subset ofthe other. This interpretation makes the rela-tive ineffectiveness of NRP inhibition of dis-ease compared with NRP-A7 much easier tounderstand because the NRP-A7–Kd tetramermay recognize qualitatively distinct cells andnot just cells with higher avidity compared tothe NRP-Kd–binding cells.

The inhibition of disease by administrationof NRP-A7 peptide is very striking, and thefact that it is accompanied by an expansion oflow avidity cells that recognize NRP-A7 at

the expense of high avidity cells raises sev-eral important questions for regulation ofimmunity and autoimmunity. One questionis why is the effect is so profound? Simpledeletion of one pathogenic clonotype can-not easily explain this result. In fact, thenumber of high avidity cells may be rela-tively unchanged, if low avidity cells areexpanding (Fig. 1). Although it is possiblethat all the NRP-A7–recognizing CD8+ Tcells that are high affinity and cause dis-ease could have been eliminated, wouldthis lead to such a profound reduction indisease in normal NOD mice? There areseveral other pathogenic CD4 and CD8clonotypes available7,10, and these clono-types can induce profound diabetes: theyshould therefore be able to take over andcause disease. Blocking reactivity to oneantigen would only be expected to halt dis-ease progression if there was a sequentialrecognition of different antigens, andblocking recognition to one preventsprogress to the next. It should also stop thedevelopment of insulitis, which is not seenin these experiments.

A more provocative interpretation of thedata would be that the treatment of NOD

mice by soluble NRP-A7 can itself inhibit theprogression of disease. A possible mechanismcould be deletion of pathogenic NRP-A7–reactive CD8+ cells and simultaneousinduction and/or expansion of cells that regu-late diabetes by producing anti-inflammatorycytokines, such as IL-10 and transforminggrowth factor β (TGF-β)11, or by other lesswell understood mechanisms12. Thus it ismore plausible that the mechanism by whichNRP-A7 peptide inhibits disease is by induc-ing regulatory T cells that can mediatebystander suppression and inhibit the patho-

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Figure 1. Preventing clinical disease. In prediabeticmice a mixture of low and high avidity autoantigen-reactiveT cells accumulate outside the pancreatic islet. Under con-ditions of limiting antigen or costimulation high avidity cellsare selected and expand to become pathogenic effectorsthat kill β-cells. Treatment with soluble NRP-A7 peptidefavors the development of low avidity cells. Insulitis stilldevelops but with much reduced clinical disease.

Prediabetic Clinical disease

Protection from disease

Islet with peri-insulitis or insulitis Insulitis andb-cell death

Insulitis withoutclinical disease

Limited antigen/costimulation

Aviditymaturationand diseaseprogression

NRP-A7treatment

Pancreatic islet

High avidity autoantigenreactive T cell

Low avidity autoantigenreactive T cell

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NEWS AND VIEWS

genic effects of T cells of other specificities,rather than by deleting one among many path-ogenic cell specificities.

It is clear from other data that there is a bal-ance in vivo of pathogenic and protectiverepertoires with specificity for the sameautoantigenic epitopes13,14. Treatment withaltered peptide ligands and soluble peptidemay shift this balance and alter the course ofdisease15. This study supports the possibilitythat one important characteristic of pathogenicversus protective repertoires may be theiraffinity for the peptide-MHC complex.Therefore, changes in the balance of differentTCRs in the repertoire, which differ in their

avidity for autoantigen, may differentiate pathogenic clonotypes from protective ones. Ifexperiments directed at testing this hypothesisare informative, then we will have data notonly for the role of high avidity interactions inthe pathogenesis of autoimmune disease butalso for the role of low avidity interactions inits regulation.

1. Wicker, L. S.,Todd, J.A. & Peterson, L. B. Annu. Rev. Immunol. 13,179–200 (1995).

2. Andre, I., Gonzalez,A.,Wang, B., Katz, J., Benoist, C. & Mathis, D.Proc. Natl Acad. Sci. USA 93, 2260–2263 (1996).

3. Wong, F. S. & Janeway, C.A., Jr J. Autoimm. 13, 290–295 (1999).4. Amrani,A., J.Verdaguer, P. Serra, S.Taturo, R.Tan & Santamaria, P.

Nature 406, 739–742 (2000).5. Verdaguer, J., Schmidt, D.,Amrani,A.,Anderson, B.,Averill, N. &

Santamaria, P. J. Exp. Med. 186, 1663–1676 (1997).

6. Anderson, B., Park, B. J.,Verdaguer, J.,Amrani,A. & Santamaria, P.Proc. Natl Acad. Sci. USA 96, 9311–9316 (1999).

7. Wong, F. S. et al. Nature Medicine 5, 1026–1031 (1999).8. Nicholson, L. B.,Waldner, H.-P., Carrizosa,A., Sette,A., Collins,

M., & Kuchroo,V. K. Proc. Natl Acad. Sci. USA 95, 264–269 (1998).9. De Boer, R. J. & Perelson,A. S. J.Theor. Biol. 169, 375–390

(1994).10. Katz, J. D.,Wang, B., Haskins, K., Benoist, C., & Mathis, D. Cell

74, 1089–1100 (1993).11. Nicholson, L. B., Greer, J. M., Sobel, R.A., Lees, M.A. & Kuchroo,

V. K. Immunity 3, 397–405 (1995).12. Thornton,A. M. & Shevach, E. M. J. Exp. Med. 188, 287–296

(1998).13. Mason, D. & Powrie, F. Cur. Opin. Immunol. 10, 649–655 (1998).14. Prabhu Das, M., Nicholson, L. B., Greer, J. M. & Kuchroo,V. K. J.

Exp. Med. 186, 867–876 (1997).15. Nicholson, L. B., Carrizosa,A. M. & Kuchroo,V.K. Immunologist 6,

151–157 (1998).

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The two-signal or costimulation model of Tcell activation was first proposed by Laffertyand colleagues1,2 to explain why transplants offoreign tissue are not always rejected. Theypostulated that full activation of naïve T cellsrequires engagement of an antigen receptor byforeign antigen (signal one) as well as engage-ment of a “costimulatory” receptor by a solu-ble or cell surface ligand provided by the anti-gen presenting cell (signal two). This two-sig-nal model is consistent with a considerablebody of experimental data and is now widelyaccepted. Variants of the model, for which theevidence is less compelling, propose thatdelivery of signal one alone inactivates T cellsby killing them3 or rendering them unrespon-sive (anergic)4. In this issue of NatureImmunology Gett and Hodgkin investigatecostimulation using modeling5.

When the two-signal model was first pro-posed neither the T cell antigen receptor(TCR) nor any candidate costimulatory recep-tor–ligand interaction had been identified.Subsequently the CD28 molecule and its lig-ands B7-1 and B7-2 were shown to have therequisite properties6, and for a time the termsCD28 and costimulatory receptor were consid-ered by some to be synonymous. However, thedemonstration that T cell antigen recognitionoccurs in mice deficient in CD28 showed thatother receptor-ligand systems must contributeto signal two7,8. To date a large number of

Modeling costimulationP. ANTON VAN DER MERWE

T cell activation usually requires TCRengagement by antigen and another,costimulatory, signal.Modeling studies nowindicate that this second signal may only slightlyenhance TCR signaling,but nevertheless resultsin an exponential increase in cell numbers.

receptor-ligand interactions have been shownto enhance or inhibit T cell antigen recogni-tion, through several different mechanisms9.Furthermore it is evident that signal one canvary because slightly different peptides caninduce different T cell responses followingpeptide-MHC recognition10. These results sug-gest that it is necessary to update the two-sig-nal model along the lines outlined in Fig. 1a.Instead of “costimulatory receptor” the moregeneral term “accessory receptor” is used.This term refers to all T cell surface receptors,other than the TCR itself, that inhibit orenhance T cell antigen recognition upon bind-ing to their soluble or cell-surface ligands. Animportant feature of the updated model is thatthe outcome of T cell antigen recognition isdetermined by the nature the TCR ligand.

Increasingly sophisticated experimentalapproaches are needed to study the complexset of accessory receptors that modulate T cellresponses to antigen. The study by Gett andHodgkin in this issue5 is a good example ofsuch an analysis. They used a simplified sys-tem in which T cells are stimulated by animmobilized monoclonal antibody (mAb) toCD3, and the CD28 and IL-4 receptors areengaged with soluble mAb to CD28 and inter-leukin 4 (IL-4), respectively. Instead of usingthe usual, rather crude, measures of cell prolif-eration, Gett and Hodgkin labeled the cellswith the fluorescent dye CFSE and used flow

cytometry to monitor cell division directly.They found that naïve CD4 T cells given anidentical stimulus vary considerably in thedelay time to the first division, but that thesubsequent division rate was the same for allcells. By measuring the response to mAb toCD3 with or without anti-CD28 at differenttime points they showed that CD28 engage-ment shortened the time to the first divisionbut had no effect on the subsequent divisionrate. In contrast, addition of IL-4 both short-ened the time to the first division andincreased the division rate. Furthermore, com-bining CD28 and IL-4 receptor engagementresulted in strictly additive effects on time tofirst division and division rate, suggestingindependent mechanisms of action.

Using a simple mathematical model of pro-liferation, Gett and Hodgkin go on to showthat the cell division parameters they mea-sured experimentally accurately predicted theobserved cell division profiles, but substantial-ly overestimated the cell numbers. When themodel was modified to take into account thespontaneous cell death that occurs in T cellcultures it accurately predicted cell numbersuntil ∼ 90 h, after which time cell numberswere again overestimated, probably becausethe model did not account for activation-induced cell-death. This model was then usedto perform a “what if” experiment, examiningthe effect of varying parameters such as the

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