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The B7/CD28 pathway is much more complicated than expected. Chambers C.A. et al. Immuunol. Rev. 153:27, 1996. B7/CD28/CTLA-4. Activated T cell. AP2. AP2. TCR. CTLA-4. CD28. P. P. peptide/ MHC. B7.1 (CD80). Activated Professional APC. B7.2 (CD86).

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The b7 cd28 pathway is much more complicated than expected

Chambers C.A. et al.

Immuunol. Rev. 153:27, 1996.

The b7 cd28 pathway is much more complicated than expected


Activated T cell










B7.1 (CD80)

Activated Professional APC

B7.2 (CD86)

The b7 cd28 pathway is much more complicated than expected

CTLA-4 is not readily detectable innaïve T cells

but is rapidly upregulated upon T cell activation.

CTLA-4 mRNA can be readily detected within 1 h of

TCR engagementand peaks at about 24–36 h.

However, CTLA-4 is not readilydetectable at the cell surface

until 24–48 h after activation.

An accurate assessment of the kinetics of

CTLA-4protein expression is complicated

by the fact that it is notprimarily expressed at the cell surface.

Surface expression of CTLA-4 is tightly regulated as a result

of the presence of a tyrosine-based intracellular localization

motif in its cytoplasmic tail which allowsan association with

the medium subunit of the clathrin-coated

pit adaptor complex, AP2,thus providing a mechanism for

CTLA-4cellular localization. CTLA-4 expression onthe

T cell surface is stabilized and increased by tyrosine

phosphorylationof the endocytosis motif, which inhibits

AP2 association. Itis noteworthy that the intracellular portion

of CTLA-4 is 100%conserved among many different species

of animals, suggestingthat control of intracellular trafficking

may be extremely importantfor its function. This motif results

in both the rapid endocytosis of CTLA-4 from the cell surface to

endosomalcompartments, as well as the targeting of at

least some CTLA-4to the lysosomes for degradation.

It is unclear whetherendocytosed CTLA-4 can recycle back

to the surface or if thisrepresents a terminal pathway for the


The b7 cd28 pathway is much more complicated than expected

Dynamic integration of TCR, CD28 and CTLA-4

As previously discussed, CD28 is constitutively expressed onT cells, whereas CTLA-4 appears after

activation. Because ofthis, and perhaps as a result of our innate appreciation forsymmetry, the idea arose

that CD28 engagement allowed initiation,while CTLA-4 provided for termination of immune responses.

Surprisingly, the majority of the in vitro data has demonstratedan inhibitory role for CTLA-4 in the early

stages of T cellactivation. IL-2 production, expression of early markers such asCD69 and CD25, and a

number of other aspects of activation areinhibited upon CTLA-4 cross-linking. These events take place

within hours of T cell activation with anti-CD3 and CD28. Infact, the inhibition of the induction of

IL-2 transcriptionwas detected 4 h after stimulation.

This suggests eitherthat there is a physiologically relevant intracellular poolof CTLA-4 present

in naïve T cells or that protein expressionis induced rapidly upon activation.

The possibility that CTLA-4 can inhibit earlystages of T cell activation has led to the

development of modelsthat stress that the dynamic interplay of costimulatory andTCR signals

depends on the activation state of the T cell aswell as the activation state of the antigen-presenting cell.

The b7 cd28 pathway is much more complicated than expected

The threshold model:When B7 levelsare low and TCR signals are weak, the amount of CTLA-4 induced

is low but may be sufficient to minimize costimulation and preventactivation. Under these conditions

CTLA-4 may set a thresholdfor activation. In other words, it plays a role in setting the stimulatory

threshold required for a T cell to progress to full activation.

The b7 cd28 pathway is much more complicated than expected

As in the classicaltwo-signal model,

an encounter of a naïve T cell with acell

expressing appropriate MHC/antigen complex

but lackingB7 does not result in activation of

the T cell owing to lackof costimulation.

The cells receiving a TCR signal in the absence

of CD28-mediated costimulation may be

rendered anergic.

However,engagement of the TCR can lead

to rapid induction and/or mobilizationof

small amounts of CTLA-4. Under conditions

where there isan incompletely activated

APC expressing only low amounts ofB7,

CTLA-4 could, by virtue of its higher affinity,

outcompeteCD28 for B7 and/or deliver

inhibitory signals. This could effectively

raise the threshold of CD28 and/or TCR

signals needed for fullactivation.

Chambers, C.A. et al. Ann.Rev.Immunol. 19:565, 2001

The b7 cd28 pathway is much more complicated than expected

There are two scenarios in which CTLA-4 may play a role in establishinga threshold for

CD28 and/or TCR signals needed for activationof naïve T cells.

Both presume that low levels of CTLA-4pre-exist or can be rapidly induced in naïve T cells upon

engagement of the TCR and CD28.

The first scenario deals with the regulation of the responseof T cells to tonic signaling by

self-peptide/MHC interactions.

Continuous TCR interactions with self-peptide/MHCprovide important signals for the survival of

peripheral T cells.

Some of these tonic interactions, under conditions of lowlevels of CD28/B7 interaction, might be

sufficiently stimulatoryto lead to the activation of CD4+ T cells and the inductionand/or mobilization of

CTLA-4. Based on the analysis of theCTLA-4-/- mice, it is speculated that CTLA-4might prevent the

signals generated by these interactions fromleading to full activation of CD4+ T cells.

Thus, CTLA-4may be involved in maintaining naïve CD4+ T cells and previouslyactivated T cells

in a resting state.

This model is supported by the phenotype of CTLA-4-/- mice. The expansion of T cells that occurs in these

miceis polyclonal. Thissuggests that the expansion is not the result of a failureto terminate responses

to few environmental pathogens. Thus, the phenotype of CTLA-4-/- mice results from the activation and

expansion of T cells reactive to low-affinity self-ligands due to a decreased activation threshold.

The b7 cd28 pathway is much more complicated than expected

The second scenario suggests a role for CTLA-4 in maintainingperipheral tolerance of T cells with

specificity for tissue-specificantigens that are not expressed in the thymus and have not beendeleted as

a consequence of negative selection. CTLA-4 may provide an additional level of regulation toensure

peripheral tolerance by preventing activation when aT cell encounters a normal self-antigen in the

context of lowB7 expression.

This scenario may explain the observationthat CTLA-4 blockade or deficiency accelerates the onset and

severity of insulitis and diabetes in nonobese diabetic (NOD)mice expressing a transgenic ß TCR cloned

from anislet-ß-cell-specific CD4+ T cell clone isolated froma NOD mouse. T cells bearing this

TCR are efficientlyselected, rather than deleted in the thymus, demonstrating thatcentral tolerance is not

effective for T cells with this specificity.The observation that blockade or loss of CTLA-4 dramatically

accelerates disease in this model system indicates a role for CTLA-4 in maintaining an activation threshold

for autoreactive T cells bearingTCRs specific to autoantigens.

The b7 cd28 pathway is much more complicated than expected

The attenuation model maintaining:When B7 levelsare high and TCR signals are strong, the higher levels of CTLA-4

induced after activationmay be able to attenuate the response of activated cells. This model suggest that

after activation and subsequent entry into the cell cycle, CTLA-4 can limit the capacity of a T cell

to divide.

The b7 cd28 pathway is much more complicated than expected

Under conditions where the APC is activated maintaining

and expressing high levels of B7,

CD28 costimulation may dominate and

activation proceeds. However, this results in

induction and/or mobilization of CTLA-4

that may be proportional to the strength of the

TCR signal, resulting in differential inhibition.

CTLA-4 preferentially attenuatesthe expansion

of T cells that have been strongly activated.

This notion is supported by the effect of

CTLA-4-blockade on the proliferative capacity,

or averagenumber of daughter cells per responder,

for T cells primed withagonist or weak agonist

peptides in adjuvant.

Blockade significantlyincreased the proliferative

capacity of T cells primed withthe agonist ligand

but had a minimal effect on the cells responding

to the weak agonist peptide..

Chambers, C.A. et al. Ann.Rev.Immunol. 19:565, 2001

The b7 cd28 pathway is much more complicated than expected

CTLA-4 would prevent this high-affinity population from maintainingdominating the primed pool by restricting

proliferation earlyin the response. Engagement of CTLA-4 would then serve to broadenthe pool of

T cells by limiting clonal representation of thehigh-affinity population, thus allowing more equal

representationof the cells bearing lower affinity TCRs in the early stagesof the clonal evolution of

the response.

It appears thatincreased CTLA-4 expression upon activation modulates T cellresponses

differentially and might serve to limit the burstsize of responding T cells. Overall, the results suggest

thatthe quality of the TCR signal is critical to determining ifand/or how dramatically CTLA-4 regulates

the proliferative capacityof any antigen-specific clone selected from the T cell repertoire.

While the TCR and CD28 might primarily determine the range ofT cells responding to antigen,

CTLA-4 would limit clonal representationof T cells with high-affinity TCRs.

The b7 cd28 pathway is much more complicated than expected

Role played by CTLA-4 in regulating antigen-specific T cell responses. (a)

The threshold and attenuation models make different predictions about the

function of CTLA-4, depending on the stimulatory conditions encountered during

activation. Shown are graphical representations that relate the effect of CTLA-4–

mediated inhibition on T cell expansion to the degree of stimulation (TCR +

CD28 signals). Threshold model: T cells that are stimulated below a given

activation threshold (dashed lines) will not commit to entering the cell cycle.

According to the threshold model, CTLA-4 may participate in setting a threshold

for activation, thereby keeping cells that receive low amounts of stimulation from

responding (red lines). In the absence of CTLA-4 signaling, the T cell activation

threshold would be shifted, so that cells that receive weaker amounts of stimulation

could now become activated and proliferate (green lines).

The b7 cd28 pathway is much more complicated than expected

Attenuation model: a responses.

certain amount of T cell stimulation may be required to up-regulate and/or mobilize

CTLA-4, thereby allowing it to participate in the regulation of a T cell response.

Thus, according to the attenuation model, CTLA-4 does not affect the threshold of

stimulation required for a T cell to enter the cell cycle; rather, it affects the extent of

subsequent expansion. Based on its expression and localization patterns, CTLA-4

may most significantly restrict the expansion of T cells that receive stronger amounts

of stimulation. (b) A diverse population of cells may respond to a given antigen, but

due to the process of antigen-driven selection, a greater number of T cells bearing

TCRs of higher affinity will be present in this population. By preferentially restricting

the expansion of cells that receive stronger TCR signals, CTLA-4 would prevent T

cells bearing higher affinity TCRs from dominating the response (red line). Thus,

in the absence of CTLA-4, the distribution of cells within the responding population

would shift toward higher affinity TCRs, resulting in a polyclonal response of reduced

diversity (green line).

The b7 cd28 pathway is much more complicated than expected

CTLA- regulates cell cycle progression, not cell death responses.

The process by which CTLA-4-induced negative regulation occurs is clearly distinctfrom activation-induced

cell death. There is no evidence that CTLA-4 ligationin conjunction with TCR and CD28 cross-linking on

resting murineT cells induces apoptosis. Further, CTLA-4ligation does not alter the CD28-mediated

upregulation of survivalfactor bcl-2, arguing against apoptosis induction beingthe major mechanism of

CTLA-4 inhibition.

The fact that CTLA-4 deficiency is lethal relatively early inlife and that the Fas pathway is intact

indicates that CTLA-4can play a critical role in limiting T cell expansion.

CTLA-4has effects on at least two aspects of activation that havecritical relevance to proliferation.

The first is on IL-2 production.CD28 costimulation enhances IL-2 production both at the levelof

transcription and mRNA stabilization. Extensiveligation of CTLA-4 under suboptimal conditions of

stimulationby TCR plus CD28 can result in inhibition of IL-2 production,probably at the level of

transcription. CTLA-4ligation inhibits TCR-induced productionof cdk4, cdk6, and cyclin D3, all of which

are required forG0/G1 progression.

Thus CTLA-4 can limit expansion notonly by reducing production of an important growth factor,

butalso by inhibiting TCR-mediated induction and assembly of essentialcomponents of the cell cycle


The b7 cd28 pathway is much more complicated than expected

CTLA-4 induces inhibitory cytokines? responses.

It has been reported that cross-linking of CTLA-4 may enhanceproduction of TGFß by activated T cells.

This raises the possibility that CTLA-4 does not directly inhibitT cell activation but does so by the active

induction of thisinhibitory cytokine. One observation taken as evidence for anindirect role for CTLA-4

in the inhibition of T cell responseswas the failure of mixed bone marrow chimeric mice generatedwith

CTLA-4-/- and wild-type bone marrow to develop a phenotypeequivalent to the ctla-4 null mice.

It was proposed thatthere is no primary defect in CTLA-4-/- T cells but that theCTLA-4-/- phenotype is

due to a failure of T cells to secreteinhibitory cytokines such as TGFß.

Recent studies showed that CTLA-4 cross-linking resulted inthe inhibition of proliferation of T cells

from TGFß-/- mice or of T cells from mice lacking Smad3, a critical downstreamsignaling molecule in the

TGFß pathway. This suggests that neither TGFßproduction nor its signaling pathway is required for

CTLA-4-mediatedinhibition of T cell responses. These studies also failed toshow a role for

CTLA-4 in regulating TGFß production,since it was produced by CTLA-4-/- T cells.

Finally, CTLA-4ligation failed to induce production of TGFß by normalnaïve T cells.

The b7 cd28 pathway is much more complicated than expected

CTLA-4 and regulatory T cells? responses.

Over the last several years, a considerable literature has documenteda role for CD25+CD45RBlowCD4+

regulatory T cells (Treg) in themaintenance of peripheral tolerance to organ-specific self-antigens.

The observation that these cells constitutively expressCTLA-4 has raised the possibility that CTLA-4

may be directlyinvolved in their function. Moreover, some studies indicate that someCTLA-4 functions

may not be necessarily T cell autonomous:(cf. bone marrow chimera with CTLA-4 -/- bone marrow cells or a

mixture of CTLA-4 -/- and CTLA-4 +/+ bone marrow cells.)

Administrationof either anti-CTLA-4 antibodies or anti-TGFß reversedthe inhibitory effects of transferred

CD25+ Treg cells on theinduction of colitis by transferred CD4+ CD25- cells in SCIDmice. This was taken

as evidence for a blocking of thesuppressive effects of Treg cells by preventing CTLA-4-mediatedinduction

of TGFß production. However, it does not exclude the possibility that theeffect of anti-CTLA-4 was a

result of enhancement of the effectorT cells. Other investigators have shown that CTLA-4 does not have a role

In the function of Treg cells. CD25+CD4+ T cells from CTLA-4-/- mice retainedinhibitory activity in in vitro

inhibition assays.

One interestingcharacteristic of Treg cells is a failure to secrete IL-2 orto proliferate in response to ligation of

the TCR and CD28. The hyporesponsiveness might be attributed to the inhibitoryproperties of CTLA-4.

However, to date, attempts to releasethe block on proliferation in response to TCR engagement by

CTLA-4 blockade have not been successful.

The b7 cd28 pathway is much more complicated than expected

T cell–mediated immunotherapy represents a promising treatmentfor human malignancies.

In cancer patients, the absence of efficienttumor-specific immunity can be related to inadequate

APC functionor to T cell tolerance/ignorance towards tumor antigens.

Mice were injected intravenouslywith 5 x 104 or 105 B16-F10 melanoma cells.

Treatment using irradiatedF10/g cells (GM-CSF transfected B16-F10 cells; Presumably,GM-CSF

production at the site of vaccination might attract hostAPCs and enhance their function in vivo.) and

antibodies was started after 24 h.

These results indicatethat CTLA-4 blockade and GM-CSF–producing

vaccines actsynergistically to cause rejection of poorly immunogenic tumors.

van Elsas A. J. Exp. Med. 190:355, 1999

The b7 cd28 pathway is much more complicated than expected

No treatment treatment

Depletion of Treg

Vacc. + CTLA-4 blocking

Vacc. + depletion of Treg


+ CTLA-4 blocking

+ depletion of Treg

Figure 3. CD25+ T cell depletion before vaccination enhances efficacy of treatment.

(A) Survival data of mice challenged subcutaneously with 2.5 x 103 B16-BL6 tumor cells.

Mice received either no treatment (n = 6, ), or depleting anti-CD25 on day -4 (n = 6, ) or vaccination with

GM-CSF–producing B16 on days 0, 3, and 6. The vaccinated mice were divided over three groups that

received the following Ab: CTLA-4 blocking Ab on days 0, 3, and 6 (n = 8, •); depleting anti-CD25 Ab on

day -4 (n = 8, x); or depleting anti-CD25 Ab on day -4 plus CTLA-4–blocking Ab on days 0, 3, and 6 (n = 8, ).

(B) Survival data of mice challenged subsutaneously (day 0) with 5 x 103 B16-BL6 tumor cells. Mice received

either depleting anti-CD25 Ab on day -4 (n = 6, ) or were vaccinated on days 0, 3, and 6 with GM-CSF–

producing B16. The vaccinated mice were divided over two groups that received the following Ab:

blocking anti–CTLA-4 Ab on days 0, 3, and 6 (n = 9, •); or depleting anti-CD25 Ab on day -4 combined with

blocking anti-CTLA-4 Ab on days 0, 3, and 6 (n = 9, )..

Sutmuller, R.P. et al. J. Exp. Med. 194:823, 2001

The b7 cd28 pathway is much more complicated than expected

In conclusion, the data presented in this paper reveal that treatmentcombination of CTLA-4 blockade

and elimination of CD25+ Tregcells can result in more effective therapeuticantitumor immunity than

when these intervention strategies areapplied separately.

These findings support the notion that CD25+ Treg cells and CTLA-4 represent two alternative pathways

forsuppression of autoreactive T cell immunity, but do not excludethat functional overlap between these

pathways exists.

Simultaneous intervention with both regulatorymechanisms appears to be a highly promising strategy

for theinduction of T cell immunity against tumor-associated autoantigensin the immunotherapy of cancer.

The b7 cd28 pathway is much more complicated than expected

Therapy with anti-CTLA-4 and GM-CSF B16 melanoma vaccine treatment

leads to tumor rejection but also induces autoimmune responses