The B7/CD28 pathway is much more complicated than expected
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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

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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)


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

protein.


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 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.


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


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 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 attenuation model: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.


Under conditions where the APC is activated

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


CTLA-4 would prevent this high-affinity population fromdominating 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.


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).


Attenuation model: a

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).


CTLA- regulates cell cycle progression, not cell death

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

machinery.


CTLA-4 induces inhibitory cytokines?

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.


CTLA-4 and regulatory T cells?

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.


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


No treatment

Depletion of Treg

Vacc. + CTLA-4 blocking

Vacc. + depletion of Treg

Vacc.

+ 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


In conclusion, the data presented in this paper reveal thatcombination 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.


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

leads to tumor rejection but also induces autoimmune responses

(depigmentation).


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