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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)
Coyle A.J. et al.(2000). Immunity, 13: 95-105.
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.
Coyle A.J. et al.(2000). Immunity, 13: 95-105.
T cell-dependent Ab production and germinal center formation in T cell-dependent Ab production and germinal center formation in ICOS -/- miceICOS -/- mice
Experimental procedureExperimental procedure
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.
Tafuri A. et al (2001). Nature, 409: 105-109.
ICOS +/+
ICOS +/-
ICOS -/-
Cytokine production after restimulation in vitroCytokine production after restimulation in vitro
Tafuri A. et al (2001). Nature, 409: 105-109.
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
Experimental procedureExperimental procedure
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
Chapoval et al (2001). Nature Immunology, 2(3): 269-274
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
Chapoval et al (2001). Nature Immunology, 2(3): 269-274
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
Primed T cellPrimed T cell
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
Primed T cellPrimed T cell
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.