Other B7 molecules

In spite of the considerable body of evidence that CD28

provides a critical costimulatory signal for T cell responses,

not all T cell-mediated responses are CD28-dependent.

Kunding T.M. et al. Immunity 5:41, 1996

VSV does not replicate extensively in vivo, in contrast to LCMV.

Continued presence of signal 1 alone, either through prolonged viral replication or repeated injection of peptide, generates a functional T cell response in vivo in absence of CD28.

The duration of TCR stimulation determines the costimulatory requirement of T cells.

Schweitzer, A.N. and Sharpe A.H. J. Immunol. 61:2762, 1998.

T cells + WT APC pulsed with peptide

T cells4 days

London C.A. et al. J. Immunol. 164:265, 2000.

TCR Trsg T cells + WT APC pulsed with peptide

4 days

T cells Transferred into mice

6-7months

Memory T cells Stimulated in vitro with

WT APCB7 KO APC

In the presence of strong or prolonged signal 1, T cells don’t need costimulation.

Costimulation is not required for effector and memory T cells.

Now,

In the presence of strong signal 1, T cells don’t need CD28-mediatedcostimulation.

CD28-mediated costimulation is not required for effector and memory T cells.

These responses may involve additional costimulatory pathways.

The B7 family and its receptorsThe B7 family and its receptors

B7 family members Receptors

B7.1B7.2

CD28CTLA-4

B7RP-1 ICOS

PD-L1PDL-2

PD-1

B7-H3 Unknown- Expressed byactivated T cells

ICOS expression:ICOS is not constitutively expressed on naïve T cells but is induced on CD4+ and CD8+ T cellsfollowing stimulation through the TCR and is further enhanced by CD28-mediated costimulation.

A new molecule with structural characteristic similar to the B7 molecules was identify in 1999,and was named B7h (B7-related protein 1; also GL-50 or B7RP-1 or ICOS-L).

B7h does not bind to CD28 or CTLA-4, but bind to ICOS (inducible costimulatory molecule).ICOS shares 30-40% sequence similarity with CD28 and CTLA-4.

McAdam A.J. et al. J. Immunol. 165:5035, 2000.

FIGURE 2. Expression of ICOS on activated T cells. Dissociated splenocytes from wild-type or B7-1/2-/- 129/SvS4Jae mice were incubated with anti-CD3, anti-CD3 and CD28, or no Ab. The thick line shows ICOS expression on T cells from wild-type splenocyte cultures, the dotted line shows ICOS expression on T cells from B7-1/2-/- splenocyte cultures, and the thin line represents a negative staining control (rat IgG-FITC).

B7h/ICOS costimulatory pathway

ICOS is expressed on memory T cells (CD45RO+) and on T cells in germinal centers.

More highly expressed on Th2 than Th1 clones. ICOS expression decreases during the differentiation of Th0 cells to Th1.

Coyle A.J. et al. Immunity 13:95, 2000. McAdam A.J. et al. J. Immunol. 165:5035, 2000.

Hutloff A. et al. Nature 397:263, 1999.

b, Localization of ICOS+ cells in the apical light zone of germinal centres, as determined from histochemical stains of frozen human tonsillar sections with monoclonal antibody F44 using the APAAP technique30. LZ, light zone; DZ, dark zone; MZ, mantle zone (original magnification 69).

• Strongly expressed in the B-cell area of the lymph nodes and spleen and in germinal centers.• Induced on DCs and monocytes• Induced on non-lymphoid cells (kidney, lungs and testis) by inflammatory stimuli

Yoshinaga S.K. et al. Nature 402:827, 1999.

The expression of B7RP-1 is strong in the lymph nodesfrom both normal and sensitized mice (Fig. 2a), particularly in the cortex (a B-cell area) and in both primary and secondary follicles. In other lymphoid tissues, B7RP-1expression is primarily localized in B-cell areas of the spleen, the follicles in the Peyer's patchesand the medulla of the thymus (Fig. 2b).

B7h expression

Roles for the B7h/ICOS costimulatory pathway

Activation of naive and recently activated CD4Activation of naive and recently activated CD4++TcellsTcellsExperimental procedureExperimental procedure

+

CD4+ OVA-specific TcR Tg

APC

OVA peptide

CTLA-4 Ig ICOS Ig hIg

Proliferation

IL-2 production (ELISA)

Naive T cells Recently activated T cells

1- First activation of CD4+ T cells

with APC-peptide

Coyle A.J. et al.(2000). Immunity, 13: 95-105.

+

Cytokine production (ELISA)

2- Second activation

+

CD4+ OVA-specific TcR Tg

APC

OVA peptide

IgCTLA-4 ICOS Ig hIg

+

Activation of naive and recently activated CD4Activation of naive and recently activated CD4++Tcells Tcells in vitroin vitro

• ICOS is not implicated in the activation of naive CD4ICOS is not implicated in the activation of naive CD4++ T cells and production of IL-2. T cells and production of IL-2. • ICOS pathway is important for cytokine production from recently activated CD4ICOS pathway is important for cytokine production from recently activated CD4++ T cells. T cells.

ICOS-Ig

CTLA-4Ig

hIg

ICOS-Ig

CTLA-4Ig

Cytokine production by fully differentiated Th1 and Th2 effectors cellsCytokine production by fully differentiated Th1 and Th2 effectors cells

Experimental procedureExperimental procedure

+Three rounds of

repetitive stimulations with

OVA peptide

CD4CD4+ + T cellsT cells APCAPC

+

hIg

CTLA-4CTLA-4 IgIg

ICOSICOS IgIg

Th1Th1 Th2Th2

Cytokine production Cytokine production (ELISA)(ELISA)


Cytokine production by Th1 and Th2 effectors cellsCytokine production by Th1 and Th2 effectors cells

B7RP-1/ ICOS pathway contributes to the cytokine production from Th2 but not Th1 cells in vitro.B7RP-1/ ICOS pathway contributes to the cytokine production from Th2 but not Th1 cells in vitro.


T cell-dependent Ab production and germinal center formation in T cell-dependent Ab production and germinal center formation in ICOS -/- miceICOS -/- mice


Primary Immunization

with KLH/ CFA

ICOS +/+ ICOS +/- ICOS -/-

Antibody response (ELISA)

GC formation (Immunohistochemistry)

Second Immunization

with KLH/ CFA

Cytokine production (Immunostaining and cytofluorimetry)

Tafuri A. et al (2001). Nature, 409: 105-109.

Antibody response and germinal center formation in ICOS -/- miceAntibody response and germinal center formation in ICOS -/- mice

ICOS is required for antibody responses and GC formation. ICOS is required for antibody responses and GC formation.


ICOS +/+

ICOS +/-

ICOS -/-

Cytokine production after restimulation in vitroCytokine production after restimulation in vitro


ICOS is required for Th2 differentiation.ICOS is required for Th2 differentiation.

ICOS +/+ T cells

ICOS -/- T cells

Additional in vivo evidences that Th2 responses are primarily regulated by B7h/ICOS pathway

Mucosal model of inflammation in the lung (Th2-mediated) Gonzalo JA et al (2001). Nature Immunol, 2: 597-604.

Blocking B7h/ICOS during priming: no effectBlocking B7/CD28 during priming: inflammation

Blocking B7h/ICOS during effector phase: inflammationBlocking B7/CD28 during effector phase: no effect

EAE (Th1-mediated disease)

EAE in ICOS deficient animal : Exacerbation of the disease Dong C et al (2001). Nature, 409: 97-101.

Blocking B7h/ICOS during antigen priming : Increased disease severityRottman JB et al (2001). Nature Immunol, 2: 605-611. augmentation of Th1 polarization

by inhibition of Th2 polarization

In vivo evidences that B7h/ICOS pathway also regulatesTh1 responses

Th1 cells express lower levels of ICOS compared to Th2 cells. In vitro studies have indicatedthat ICOS is not relevant in Th1 polarized T cells. However, in vivo studies indicate that expression of ICOS by Th1 cells appears to be relevant

EAE (Th1-mediated disease) Rottman JB et al (2001). Nature Immunol, 2: 605-611.

Blocking B7h/ICOS during antigen priming : Increased disease severityaugmentation of Th1 polarizationby inhibition of Th2 polarization

Blocking B7h/ICOS during effector phase: Limits disease progression byinhibiting Th1 functions (IFNg).

Th1-mediated cardiac allograft rejection:Ozkaaaynak E et al (2001). Nature Immunol, 2: 591-596.

Blocking ICOS prolongs allograft survival by preventing both acute and chronic rejection.

Thus, these results show that ICOS is also important in regulating Th1 responses in vivo.

Role of B7h/ICOS in CD8 T cell responses

• B7h/ICOS interactions are not relevant in CD8 T cell responses to LCMVKopf M et al (2000)J. Exp. Med 192: 53-62.

• New studies indicate that ICOS may be relevant for effective CD8 T cell mediated anti-tumor responses:Wallin JJ et al (2001) J. Immunol. 167: 132-139.Liu X et al (2001)J. Exp. Med 194: 1339-1348.

In both studies, expression of B7h on tumor cells results in efficient CD8 mediated tumor rejection. (Delayed kinetic compared with rejection of B7.2-expressing tumors).

PD-L1, PD-L2 and their receptor PD-1PD-L1, PD-L2 and their receptor PD-1

• PD-L1 (programmed death 1 ligand),PD-L1 (programmed death 1 ligand), PD-L2 (programmed death 2 ligand) and PD-L2 (programmed death 2 ligand) and do notdo not bind to CD28, ICOS, or CTLA-4, but bind to PD-1 (programmed death bind to CD28, ICOS, or CTLA-4, but bind to PD-1 (programmed death 1 molecule). 1 molecule).

• PD-1 shows 24 % of sequence similarity to CD28 and CTLA-4.PD-1 shows 24 % of sequence similarity to CD28 and CTLA-4.

• An ITIM motif (An ITIM motif (IImmunoreceptor mmunoreceptor TTyrosine-based yrosine-based IInhibitory nhibitory MMotif) is present otif) is present in the cytoplasmic tail of PD-1. The ITIM motif is known to be present in in the cytoplasmic tail of PD-1. The ITIM motif is known to be present in receptors that have an inhibitory function on lymphocyte responses.receptors that have an inhibitory function on lymphocyte responses.

Expression patterns of PD-L1, PD-L2 Expression patterns of PD-L1, PD-L2

Coyle A.J. et al (2001). Nature Immunology, 2(3): 203-209

- PD-L1 and PD-L2 are expressed on DCs - PD-L1 and PD-L2 are expressed on DCs and monocytes.and monocytes.

- They are constitutively expressed on - They are constitutively expressed on several non-lymphoid tissues.several non-lymphoid tissues.Dong H. et al (1999). Nature Med, 5(12): 1365-1369 Latchman et al. (2001). Nature Immunol., 2(3):261-268

Expression pattern of PD-1 Expression pattern of PD-1

• PD-1 is constitutively expressed on T cells following activationPD-1 is constitutively expressed on T cells following activation ..

(Chambers, C. (2001) Trends Immunol., 22(4): 217-223).

• C57Bl/6 PD-1 -/- mice develop lupus-like arthritis and glomerulonephritis with C57Bl/6 PD-1 -/- mice develop lupus-like arthritis and glomerulonephritis with an increase in serum IgG3 and B cell proliferation.an increase in serum IgG3 and B cell proliferation.(Nishimura, H. el al (1999). Immunity, 11: 141-151).

• BALB/c PD-1 -/- develop an autoimmune dilated cardiomyopathy, with IgG BALB/c PD-1 -/- develop an autoimmune dilated cardiomyopathy, with IgG deposites on cardiomyocytes and an increase of IgG in blood.deposites on cardiomyocytes and an increase of IgG in blood.(Nishimura, H. el al (2001). Science, 291: 319-322).

• CTLA-4 has a stronger negative effect than PD-1 on T cell activation. All Ig CTLA-4 has a stronger negative effect than PD-1 on T cell activation. All Ig isotypes are elevated in CTLA-4 -/- mice independently of the genetic background. isotypes are elevated in CTLA-4 -/- mice independently of the genetic background. The lymphoproliferation is more severe, and the onset of the phenotype and the The lymphoproliferation is more severe, and the onset of the phenotype and the lethality occur faster in CTLA-4 -/- mice. lethality occur faster in CTLA-4 -/- mice.

(Chambers, C. (2001) Trends Immunol., 22(4): 217-223).

CD4+ T cellsCD4+ T cells

+

CD3

hIgPD-L1-Ig

CD3

ProliferationProliferation

Cytokine production (ELISA)Cytokine production (ELISA)

T cell proliferation and cytokine production after PD1- T cell proliferation and cytokine production after PD1- PD-L1PD-L1 engagement engagement


Freeman et al (2000). J. Exp. Med, 192 (7): 1027-1034.

T T cell proliferation and cytokine production after PD-1/ cell proliferation and cytokine production after PD-1/ PD-L1PD-L1 engagement engagement

PD-L1/ PD-1 interactions can inhibit TcR-mediated proliferation PD-L1/ PD-1 interactions can inhibit TcR-mediated proliferation and cytokine production.and cytokine production.

Freeman et al (2000). J. Exp. Med, 192 (7): 1027-1034

Proliferation and cytokine production after Proliferation and cytokine production after PD-L2PD-L2/ PD-1 engagement / PD-1 engagement Experimental procedureExperimental procedure

APCAPC

OVA-specific TcR TgOVA-specific TcR Tg

CD4+ T cellsCD4+ T cells

OVA OVA peptide peptide ++

First activation

CHO.I-ACHO.I-Add CHO.I-ACHO.I-Add.PD-L2.PD-L2Second activation

Proliferation

Cytokine production (ELISA)

Latchman et al. (2001). Nature Immunol., 2(3):261-268

OVA OVA peptide peptide ++

Cytokine production after Cytokine production after PD-L2PD-L2/ PD-1 engagement/ PD-1 engagement

PD-L2/ PD-1 interactions can inhibit TcR-mediated PD-L2/ PD-1 interactions can inhibit TcR-mediated proliferation and cytokine production.proliferation and cytokine production.

Latchman et al. (2001). Nature Immunol., 2(3):261-268

Contradicting Results as to the function of the PD-L2/PD-L1/PD-1 pathwayContradicting Results as to the function of the PD-L2/PD-L1/PD-1 pathway

Costimulation of T cells by PD-L2Costimulation of T cells by PD-L2

Tseng, SY et al. (2001). J. Exp. Med., 193 (7): 839-845.

PD-L2 costimulates :PD-L2 costimulates :

• a T cell proliferative response to a greater level than B7.1.a T cell proliferative response to a greater level than B7.1.

• mainly CD4+ T cells. mainly CD4+ T cells.

• greater levels of IFN-greater levels of IFN- than B7-1, and fails to promote IL-4 or IL-10 production. than B7-1, and fails to promote IL-4 or IL-10 production. • PD-L2 appears to drive Th1 responses.PD-L2 appears to drive Th1 responses.

Contradicting resultsContradicting results

• T cell proliferation and IL-10 secretion increased with PD-L1 stimulation.T cell proliferation and IL-10 secretion increased with PD-L1 stimulation.

Dong et al (1999). Nature Medecine, 5(12): 1365-1369).

Tamura et al (2001). Blood, 97(6): 1809-1816.

• It is unclear whether the PD-L1/L2 costimulation reported by these groups is PD-1 It is unclear whether the PD-L1/L2 costimulation reported by these groups is PD-1 dependent or dependent or whether it could be mediated by an alternative receptor for PD-L1whether it could be mediated by an alternative receptor for PD-L1: Parallel : Parallel of CD28/ CTLA-4 receptors- B7.1/ B7.2 ligands. of CD28/ CTLA-4 receptors- B7.1/ B7.2 ligands. Freeman et al (2000). J. Exp. Med, 192 (7): 1027-1034

B7-H3 B7-H3

• B7-H3 (B7 homolog 3)B7-H3 (B7 homolog 3) binds to an unknown receptor on activated T cells that is binds to an unknown receptor on activated T cells that is

distinct from CD28, CTLA-4, ICOS or PD-1.distinct from CD28, CTLA-4, ICOS or PD-1.

• Expression pattern:Expression pattern:

Chapoval et al (2001). Nature Immunology, 2(3): 269-274

B7-H3

Ctrl serum

B7-H3 Ig

Ctrl Ig

B7-H3 is an inducible B7-H3 is an inducible molecule on the surface of molecule on the surface of DCs and monocytes. DCs and monocytes.

Not restricted to lymphoid Not restricted to lymphoid tissue but also observed in tissue but also observed in heart, kidney, testis, colon and heart, kidney, testis, colon and human tumor lineshuman tumor lines

Transient expression of Transient expression of the unknown receptor of the unknown receptor of B7-H3 on activated T B7-H3 on activated T cells.cells.

Costimulating T cell responses by B7-H3Costimulating T cell responses by B7-H3Experimental procedureExperimental procedure

T cells T cells

Coated Coated CD3 CD3

ImmobilizedImmobilizedB7-H3 Ig B7-B7-H3 Ig B7-1 Ig control 1 Ig control IgIg

Proliferation Proliferation

T cellsT cells

+

Melanoma cell Melanoma cell line transfected line transfected with B7-H3 or with B7-H3 or

CT vectorCT vector

CTL activityCTL activity


Cytokine productionCytokine production

Costimulating T cell responses by B7-H3Costimulating T cell responses by B7-H3

B7-H3:B7-H3:

• increases T cell proliferation but is less potent than B7.1. increases T cell proliferation but is less potent than B7.1.

• enhances proliferation of both CD4+ and CD8+ T cells. enhances proliferation of both CD4+ and CD8+ T cells.

• B7-H3- transfected cells induces CTL activity.B7-H3- transfected cells induces CTL activity.

• selectively enhances IFN selectively enhances IFN production with modest effects on TNFa and IL-8 production with modest effects on TNFa and IL-8


SummarySummary

B7-H3B7-H3

Primed T cellPrimed T cell

?? ++ Th1 (INFTh1 (INF) and CTLs. ) and CTLs.

PD-L1PD-L1

PD-L2PD-L2Primed T cellPrimed T cell

PD-1PD-1 -- Negative regulation of T cell Negative regulation of T cell activation.activation.

DC, MDC, M

DC, MDC, M

B7RP-1B7RP-1


ICOSICOS ++ T cell activation in 2T cell activation in 2ndnd responses, differentiation in responses, differentiation in

Th2 Th2

APC (B)APC (B)

B7.1B7.1

B7.2B7.2

Resting T cellResting T cell

CD28CD28


CTLA-4CTLA-4

+ + Initiation of T cell activation. Initiation of T cell activation.

Inhibition of T cell activation Inhibition of T cell activation

--

APCAPC

Th1 responses (INF Th1 responses (INF ))..++?

ConclusionConclusion

• Regulation of T cell activation by costimulation is more complex than originally Regulation of T cell activation by costimulation is more complex than originally envisioned.envisioned.

• None of the newly discovered pathways appears to be completely redundant with CD28 None of the newly discovered pathways appears to be completely redundant with CD28 in terms of naïve T cell activation but rather regulate the fate of primed and memory T in terms of naïve T cell activation but rather regulate the fate of primed and memory T cells.cells.

• The spirit of the two-signal model of T-cell activation remains intact, but a second The spirit of the two-signal model of T-cell activation remains intact, but a second activation step process called « step two of signal 2» can be add to this model. activation step process called « step two of signal 2» can be add to this model.

Coyle A.J. et al (2001). Nature Immunology, 2(3): 203-209

• Complementation in functions: Complementation in functions: ICOS/B7h: ICOS/B7h: Th2Th2

?/B7-H3:?/B7-H3:Th1 and CTLTh1 and CTL

• Spatial differences in expression: The novel B7 family members have aSpatial differences in expression: The novel B7 family members have a

broad distribution in non-lymphoid tissues and are therefor uniquely broad distribution in non-lymphoid tissues and are therefor uniquely position to regulate antigen-specific T cell functions at sites of position to regulate antigen-specific T cell functions at sites of inflammation.inflammation.

Other B7 molecules

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