A central role of CD40 ligand in the regulation of CD4+ T-cell responses

IMMUNOL(

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TODAY

A central ro regulation 0 Many of the cosfimuiafovy molecules thought to be essential for T-cell activation have now been identified. The CD4U-CD40 ligand he study of T-cell activation reendeavor, even though some of the molecular details of T-cell actimCCns an exciting

interacfion is one such receptor/counter-recepfor

pair that

hns been shown to be importanf in

The importance

of CD4OL expression

in vivo was revealed by the studies of hyper-

IgM syndrome

(HIGMl) patients, in whom

high levels of IgM but severely reduced levels of other Ig isotypes were observeds. HIGMI is a human X-linked immunodeficiency,

vation have become apparent. A number of B- and T-cell cognate interactions. molecules (receptor/counter-receptor pairs) Here Iqbal Grewal and usually caused by mutations in CD40L that thought to be crucial for the function of result in a lack of functional expression of Richard Flavell examine recent data T celis have been identified. These include CD4OL on the surface of activated T cells. costimulatory molecules such as CD24, from studies using gene knockout HIGMl patients suffer from recurrent CD28, CD54 and CD58 which are widely techniques that have helped provide upper-respiratory-tract infections, and cerdistributed on many tissues, and CD80 (87-l) tain opportunistic infections such as cryptoand CD86 (B7-2) molecules which are rea better understanding of CD4OL’s sporidial diarrhea and Pnewnocystis curinii stricted to antigen-presenting cells (AI’Cs). role in vivo, in regulation of the pneumonias. B cells in HIGMl patients proHowever, levels of CD80 and CD86 on immune response. duce normal levels of IgM upon challenge resting APCs are generally low, becoming with T-cell dependent (TD) antigens, but are upregulated following AK activation. unable to switch to produce other classes of Ig and to establish A further receptor/counter-receptor pair, CD40-CD40 ligand B-cell memory. Although patients show abnormal antibody refCD4OL),has been identified to be involved in B- and T-cell cognate interactions: an association that has been shown to be important for sponses, their B cells are capable of producing normal antibodies in vitro when co-cultured with normal T helper (Th) cells, indicating humoral immunity. Recently, CD40L-knockout mice (CD4OL-/-) have provided significant clues towards understanding the in viva that the defect lies in the inability of T cells to activate B cells. In functions of CD4OL in immune responses. This article focuses on common with HIGMl patients CD40L- and CD40-knockout mice have defective humoral immunity, establishing that CD4O-CD4OL the irt uivo role of CDIOL in regulation of immune responses, in particular its involvement

in the activation of antigen-specific

naive T

cells. Contrary to prior belief, recent data suggest that CD4O-CD40L interactions are not only required for B-cell activation events but are also essential for the initiation of antigen-specific T-cell responses.

CD40L and its counteweceptor CD4OL is a type II membrane protein of 33 kDa, a member of the tumor necrosis factor (TNF) gene family and is preferentially expressed on activated CD4’ T cells and mast cells’. Expression of CD4OL on T cells was defined both in humans2 and in mice3 by specific antibodies, and human and murine CD4OL has been cloned4,i. The counter-receptor for CD40L is CD40 (40 kDa), a member of the TNF-receptor (TNFRI family, which is found on APCs including B cells, dendritic cells (DCs), activated macrophages and follicular dendritic cells (FDCs). Extensive studies using monoclonal antibodies to CD4OL, as well as soluble CD40L-immunoglobulin Fc fusion proteins (sCD40L-Ig) and CD4OL-transfected cells, have established the role of CD40-CD40L interactions in B-cell activation and differentiation, and in immunoglobulin (Ig) class switching6m9.

interactions are crucial for B-cell responses to TD antigens’&**.

egulation of costimulatary activity on

cells by

CD40_CD40L interaction A reciprocal dialogue between inducible molecules both on T cells and APCs provides regulated activation of T cells, which is important for amplification of the in vivo immune response. The current understanding of T-cell activation is that naive T cells require two distinct signals for activation’3. The first signal is provided by the engagement of the T-cell receptor (TCR) with major histocompatibility complex (MHC)-peptide ligand on the surface of the AK, thus ensuring antigenic specificity. The second signal is delivered to T cells by costimulatory molecules such as CD80 and CD86 on the surface of APC via their interaction with CD28/CTLA-4 on the surface of T cells. However, APCs such as B cells posses antigenspecific Ig receptors and MHC class II molecules but do not normally express the necessary costimulatory molecules unless they are first activated. Recognition of the MHC-peptide complex by the TCR leads to the induction of CWOL on T cells, whereas simultaneous engagement of MHC-peptide by the TCR, and CD40 by

CDIOL leads to the upregulation

of CD80 and CDS6 on B cells. This

costimulatory activity induced on I3 cells then acts to amplify the response of the T cells. Several pieces of evidence support the idea that CD4O-CD4OL interactions are required for the induction of B-cell-mediated costimulatory activity for T-cell activation: (1) monoclonal antibodies against CD4OL or CD40 inhibit activated T-cells from inducing CD80 and CD86 on B cells*4,15;(2) recombinant CD4OL induces costimulatory activity on B cells?; and (3) it has recently been demonstrated that CDBOLinduces a novel costimulatory activity on B cells”. These studies strongly suggest that CD40CD40L interactions control costimulatory activity on B cells by inducing expression of CDSO, CDS6 and other molecules, and therefore might play a key role in T-cell activation and development of effector functions.

ale of Although

HIGMl patients are susceptible to Pncu!nocystij and infections, which are typically associated with severe

Cryptosporidiu

T-cell immunodeficiency, a possible role of CD4OL in initiation and/or maintenance of T-cell immunity has not been resolved. However, several clues pointing to the involvement of CD4O-CD40L interactions in T-cell immunity are apparent in the literature. For example, CD40 is expressed on conventional APCs, i.e. DCs, activated macrophages, FDCs and l3 cells. Moreover, CD4OL is preferentially expressed on T cells, indicating that CD4&CD40L interactions might play a crucial role in T-cell activation by APCs (Ref. 1). In addition, monoclonal antibodies against CD4OL prevent the activation of antigen-specific T cells14.

T cell functions in CD4(DL-‘-

mice

CD4OL-/- mice are severely impaired in primary T-cell responses to protein antigens Is. The defective T-cell responses of these mice were mapped to an inability of CD40Gdeficient T cells to undergo effective clonal expansion in viva and to enter the cell cycle. An adoptive-transfer system was used to monitor the expansion of a limiting number of T cells from TCR transgenic mice following their transfer into normal recipients that were challenged with antigen. T cells lacking CD40L failed to expand in vivo, whereas wild-type cells expanded normally, indicating that CD40CD40L interaction is required for effective in vivo priming of CD4+ T cells. The impact of a lack of CD4O-CD4OL interaction has been further studied in the context of the development of the T-cellmediated autoimmune disease, experimental allergic encephalomyelitis (EAE), a murine model for multiple sclerosisi9. When the CD4OL mutation was bred onto myelin basic protein (MBP)-specific

the induction of ill tGo costimu!atory activity on ApCs, r\hicIl is required for activation and priming of T cell5 and to evoke E;\E (Ref. 41). The inability of CD4OL-deficient T cells to undergo clonat erpansion and enter the cell cycle’” suggests a defect at the early stage of T-cell activation. Since DCs are implicated in early T-cell priming events and as EAE cannot be induced in CD40L / mice following immunization with short encephalitogenic peptide’“, the hypothesis is proposed that CD40-CD4OL interactions are required for the activation of DCs. Although DCs constitutively express low levels of CD86, they do not generally express costimulatory activity2’. Furthermore, this low level expression of CD86 on DCs in the thymus was not enough to rescue deletion of T cells in the thymus”?. Moreover, of APCs other than B cells in T-cell priming is .
the potential role ported by several to keyhole !impet immunodeficiency tuted only with mice2”, and by

deficient mice IC.A. Janeway Jr, unpublished). CD4O-CD40L interactions play a crucial role in the development of effector functions, as well as the newly described role in T-cell priming: CD40L is required for B-cell activation and differentiation (see above), and to activate macrophages and monocytes to produce TNF-(Y,interleukin 1 (IL-l), IL-12 , IFN-y and nitric oxide (NO)‘i-‘K. CD4OL has also been shown to rescue circulating monocytes from apoptotic death, thus prolonging their survival at the site of inflammation (J. Suttles et nl., unpublished). These studies indicate that CD4OL plays a significant role in mediating inflammation by the induction of cytokine secretion by monocytes and macrophages, and prolongation of their survival: CD4U-CD40L interaction during T-cell activation by antigen-presentin, n macrophages results in IL-12 production, and IL-12 is shown to play a critical role in development of Thl cellsr9”o.Thus it can be concluded that CD4O-CD4OLinteractions between T cells and macrophages play a role in maintenance of Thl type cellular responses and mediation of inflammatory responses. Consequently, CD40L-‘- mice have been tested for their ability to resist Leishrtzania ~rnz~zone~rsis infection, which is believed to be contained by T-cell activated macrophages”. In response to parasite challenge, wild-type mice were susceptible to Lrishtr~nftininfection, and developed progressive ulcerative lesions. However, CD4OL “

mice developed a more severe form of infection with higher oarasite burden than wild-type mice. The enhanced susceptibility of CD4OL-/- mice was associated with low levels of IFN-y, Iymphotoxin (LT)/TNF and NO production by macrophages. In addition, these mice failed to generate a protective immune response after immunization. These results clearly indicate the importance of

TCR transgenic mice, a T-cell priming deficiency became apparent: EAE could not be provoked in these mice by immunizing with

CD4OCD4OL interactions in the development

encephalitogenic peptide, whereas the same peptide acutely induceL EAE ln CD40L+/+ MBP-TCR transgenic mice. However, T cells in CD40-/- MBP-TCR transgenic mice could be primed to produce

results were reported

by others,

Leishfnnnin

investigated

IFN-1 and mediate EAE by providing them with B7-l+ APCs by adoptive transfer. These findings suggest that CD4OL is required for

of cellular immune responses for killing LeiS/V?lrrlli~ nrn020iWHSis.Furthermore, similar major

were

where

immune

in CD40L ’

response>

miceZ”3, Challenge of these mice with Leishrmrir~ mjw ulcerative

rcdtd

in

lesion, and failure to mount a ThI type inlIn CD4OL-” mice, the impairment of Thl tvl’c

cutaneous

mune response.

to

and CDdt)

IMtiiJNOLOGY

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&primed

APC

(b) Step 2

-

Activation

Primed APC Siglial Fig. I. The role of CD40-CD4OL in T-cel1octivntiorr. Two-step ~norlelojncfiz~tio~z of T cells. (n) Step I: iirducfiorr of CD4OLon T cells. Ajfiigetls me iden up by unprimed nnive DC5 (Zmgerlmns cells Dz tissues), a~d these DCs migrate fo the LN zcdierr flzq prmwf T cells in the fom

of MC-pepfide

conzylexes, zuhich deliver the arrfigenic sigmd fsigml

late CD4OLon their surfnce. (b) Step 2: inducfion of cosfimhfory

processed mfigetz to rraive

1) zh the TCA to nnivc T cells. As n result, T cells upregu-

ncfiuify 011APCs. CD4OL OIIt/w swfncc of T crlls induces cosfinzlrl.!fory ncfbify

on DCs via rlre CD40-CD40L internctiotr. This prinrerl APC mm expressing cosfimtlnfq tnolrcules serzds a srrord cosfimlofoy sigrmf to T cells nlong with sigtml 1 for full acfivnfion of T cells to produce cytokines nrzd to perform effpcfor jkrcfiotzs. Abbrecinfiorzs: AK. nilfigfIz-~Jrl,srtzfilzg cell; DC, dendrific cell; IL-1 2, inter/e&i!? 12; LN, fy?npli node; MI-K, major histoconzpfibility cotnpkx; TCR. T-cell rewpfo):

immune responses was shown to be the result of the inability of macrophages to produce IL-U; however, this defect was corrected by administration of recombinant IL-12 to infected mice. Furthermore, administration of soluble CDBOL was shown to offer partial protection from development of Leishmnnimis. Further studies of CD40-‘- mice show that T cells from Leishmania-infected

rect signal delivered to T cells via triggering or crosslinking of CD4OL is required in addition to the conventional signals 1 and 2. In support of the second hypothesis, a recent report by van Essen ef RI. indicates that CD40-‘- mice are defective in the formation of germinal centers in response to TD antigens, but this defect can be corrected

with administration

of sCD4OL-Fcyl fusion protein3”.

CDIO-I- mice produce lower amounts of IFN-1 and IL-12 mRNA from draining lymph nodes than those from wild-type mice. Thus,

These hypotheses are not mutually exclusive. The fact that IL-12 can reconstitute the CD40L-deficiency

these studies, using two different strains of Leishmanilr and two different model systems, strongly suggest that CD4O-CD4OL interactions play an important role in two distinct phases of the cell-mediated immune response to Leishunin infection through macrophage activation: (1) the generation of the Thl response required for macrophage activation; and (2) the effector functions of macrophages in-

resistance to Leishnmnin mjoP? and overcome blockade of colitis by anti-CDBOL antibodiesA’ suggests that the induction of IL-12 by CD40L is important in T-cell activation as well as T-cell-

cluding othe* mediators of inflammation contain Leish?nnnininfection.

which are required to

Based on the above discussion, two likely hypotheses can be considered to explain the defective T-cell responses in CD4OL-/mice. The first hypothesis (described in the next section) is that CD4O-CD40L interactions are required for the activation/priming of APCs. The second hypothesis considers the possibility that a di-

SEPTEMBER

1996

in

macrophage interaction. One possibility that seems likely is that CD40L stimulation of DCs leads to IL-12 secretion which, in conjunction with the costimulatory effect described above, drives CD4+ T cells to differentiate into Thl effector celP.

A model of costimulation/TceH activation Interactions between T cells and APCs must be caref-ally regulated such that activation of quiescent self-reactive or bystander T cells is avoided. The presence of CD40 and MHC molecules on resting

IMMUNOLOGY

TODAY

APCs, in conjuction with TCR and CD28 on resting T cells, is insufficient to trigger an immune response; in constrast, it has been shown that resting 6 ceils tolerize resting T cells to specific antigens37J8. A reciprocal dialogue between AK and T cell can only follow if either AK or T cell is activated. One possibility is that cognate interaction followed by CD40-CD40L interaction is necessary for the activation of costimulatory activity on all APCs, including B cells, macrophages and DCs. However, it is DCs which are implicated in the initiation of the immune response13. A recent study supports the possibility of CD40CD40L interactions

“‘-1

I

0 cell

Proliferation and differentiation lg class switch CDBO/CD86 Other costimulatorj activity

inducing costimulatory activity on DCs, by showing that CD40+ DCs, generated from

IL-l IL-12 NO CDBOICDFE TNF-U Kill intracellular pathogens

CD80/CD86 TNF-u IL-12

human cord blood CD34’ progenitor cells, upregulate CD80 and CD86 when activated by mouse L cells that are stably expressing CD4OL (Ref. 39). How can a T cell activate its AK before the T cell is itself activated? Interestingly, it has also been shown that the upregulation of CD40L requires stimulation through theantigenreceptor (signal 1) but that costimulation (signal 2) is not required’*,‘O. We therefore propose a two-step model for the activation of T cells in the initiation of the immune response (Fig. 1). In the first step, DCs take up antigens at the site of injury or infection and migrate to the lymph node, where they present antigens to naive T cells. The T cell receives Ihe antigenic signal (signal l!, which causes the upregulation of CD4OL. This in turn activates the expression of the costimulatory

activity of the AK.

of the immune response and might provide information to develop selective immunotherapies and to treat T-cell immunodeficiencies and auloimniune

disease.

In the second

phase, the costimulatory signal is received by the T cell (for example via CD28) which drives the cell to enter into the cell cycle and become fully active. These activated T cells can now enter into secondary cognate CD40CD40L-dependent effector recognition: with B cell?,

We thank E.E. Eynon for valuahlr suggestions and F. Manzo for help ure artwork. I.%.

in fig-

is an Associate, R.A.F. is an Investigator of the Howard

Hughes Medical Institute.

to promote Ig class switch; with macrophages to produce cytokines; and with DCs to upregulate costimulatory activity (Fig. 2).

Conclusions Overall, the data suggest that CD4OL is a master regulator of the immune system, with strong influence on both B-and T-cell activation,

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ita

i

The balance of CD4’ and CD8+ T cells in humans is controlled by a nzajor autosomnl gene. Here, Afherto Amadori and colleagues espite

intense

pathogenetic

fects

directly

immunodeficiency discernible

from

are still unclear;

exerted

by

virus the

that result in depletion

the

in AIDS in hu-

involved mans

efforts,

mechanisms

the

ef-

human

(HIV) itself1,2 are

indirect

phenomena

of noninfected

CD4’

T cells, and thus amplify the damage orig-

inally caused by the virus (reviewed in Ref.

propose that in view of tlze /zig/z CD4+ cell turnover durizzg HIV infection, individuals genetically predisposed to a high CD4:CDB ratio can withstand HIV-associated CD4+ cell losses better than tllose predisposed to tz low ratio.

3). Overall, HIV infection is perceived as a titanic struggle between the virus an? the immune systemti. Huge numbers of CD4+ T cells are destroyed daily and, since in most individuals the decline in CD4’ T-cell number over time is gradual, an almost equivalent number must be gener&d to reconstitute the peripheral pool.

In an analogy provided by Ho et a1.4,the seropositive patient can be compared to a

bathtub, where a drain (the HIV) causes a steady loss of water (the CD4’ T cells), while a tap (the thymus or post-thymic involution and other sites of T-cell generation) steadily strives to maintain the water level; a similar idea has recently been formalized in a schematic view by Heeney7. This notion

undoubtedly summarizes an important piece of the puzzle, but a universal view that could explain different disease progression in different individuals is not forthcoming. In this regard, an area of intense research concerns the category of HIV-infected individuals termed long-term non-progressors (LTNPP9. This definition identifies asymptomatic, untreated