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Immunodominant peptides derived from the heavy constant region of IgG1 stimulate natural regulatory T cells: identification of pan-
HLA binders for clinical translation
Alessandra Franco MD PhDUCSD School of MedicineDepartment of Pediatrics
Division of Allergy Immunology and Rheumatology
• Immune regulation is key in the immune homeostasis
• Immune regulation is key in the response to therapy in a large variety of immune-mediated diseases
Two main human Treg lineages have been described
1) natural (n)Treg are derived from the thymus during fetal life and recognize self peptides.
2) peripherally-induced (i)Treg recognize not-self, arise from:a) naïve T cells under unique repeated stimulatory conditions (i.e.
transforming grow factor (TGF-b)
b) pro-inflammatory T cells (Th17, Th1, CD8+ cytotoxic T cells) following repeated antigenic stimulation (iTreg are specific for the pathogen)
Regulatory T cells phenotypically CD4+ CD25 high
Kawasaki Disease: a disease model where immune regulation is key in the response to therapy
• Kawasaki Disease (KD) is a self-limited T cell-mediated pediatric vasculitis of the coronary arteries.
• In San Diego County, the average incidence is 25/100,000 children <5 yrs. of age and approximately 80-90 new cases are diagnosed and treated each year by the KD Team at Rady Children's Hospital San Diego
• If untreated, 25% of children will develop aneurysms of the coronary arteries.
• Treatment with high dose intravenous immunoglobulin (IVIG) reduces the rate of aneurysms to 5%
T helper 17 cells recruit CD8+ T cells (CTL) in the arterial walls
Human Pathology 2012
α-SMA α-SMA αSMA and IL17
Understanding IVIG therapy and its success in Kawasaki disease: Fc-specific Treg?
Fc-specific Treg expand only in sub-acute KD subjects with normal arteries after IVIG
% C
D4+
CD
25hi
ghT
cells
subject 1 subject 2 subject 3 subject 4 subject 5
subject 6 subject 7 subject 8 subject 9 subject 10
subject 11 subject 12 subject 13 subject 14 subject 20
Fc µg/ml
0.01.53.0
No
Ag 1 10 100
0.01.53.0
No
Ag 1 10 100
0.01.53.0
No
Ag 1 10 100
0.01.53.0
No
Ag 1 10 100
0.01.53.0
No
Ag 1 10 100
0.01.53.0
No
Ag 1 10 100
0.01.53.0
No
Ag 1 10 100
0.01.53.0
No
Ag 1 10 100
0.01.53.0
No
Ag 1 10 100
0.01.53.0
No
Ag 1 10 100
0.01.53.0
No
Ag 1 10 100
0.01.53.0
No
Ag 1 10 100
0.01.53.0
No
Ag 1 10 100
0.01.53.0
No
Ag 1 10 100
Dilated arteries or aneurysm
CD4
CD
25
Control 1µg/ml 10µg/ml10µg/ml1µg/mlFc F(ab)2100µg/ml 100µg/ml
0.01.53.0
No
Ag 1 10 100
Subj
ect
7
Autoimmunity 2014
Autoimmunity 2014
Fc-specific nTreg circulate in healthy donors but not adult KD patients
Hypothesis for the lack of Fc-specific nTreg
• Clonal deletion (mutated maternal Fc?)• Clonal anergy• HLA type/HLA binding affinity of the relevant Fc
peptides• Antigen processing
The T cell receptor (TCR) recognizes a complex represented by MHC molecules and a peptide bound to the MHC binding groove
Antigen Processing and Presentation to MHC class II-restricted T Cells (CD4+): the data suggest that relevant Fc peptides are not presented to nTreg in KD patients that develop arterial complications
Fine specificity of nTreg: 64 peptides 15 amino acid long, 10 amino acid overlap
Method to define nTreg specificity
PBMC separation
Culture 4 days PBMC with 20µg/ml Fc peptide (97% pure) without exogenous IL-2
Read outs:1. IL-10 measured in culture supernatants2. CD4+ CD25 high T cell expansion by flow cytometry
Study population – KD subjects after IVIG
5 6 8
IL10 responses to 6 immunodominant Fc peptides recognized by nTreg in KD subjects after IVIG
Fc 51-65(Rank 1, 4/8, 50%)
Fc 181-195 (Rank 2, 3/8, 37.5%)
Fc 61-75 (Rank 2, 3/8, 37.5%)
Fc 21-35 (Rank 3, 2/8, 25%)
Fc 271-285 (Rank 3, 2/8, 25%)
Fc 306-320 (Rank 3, 2/8, 25%)
5 6 8
5 6 8
5 6 8
5 6 8
5 6 8
Fc position Sequence21-35 TAALGCLVKDYFPEP
26-40 CLVKDYFPEPVTVSW
31-45 YFPEPVTVSWNSGAL
36-50 VTVSWNSGALTSGVH
51-65 TFPAVLQSSGLYSLS
56-70 LQSSGLYSLSSVVTV
61-75 LYSLSSVVTVPSSSL
66-80 SVVTVPSSSLGTQTY
121-135 SVFLF PPKPKDTLMI
126-140 PPKPKDTLMISRTPE
181-195 TYRVVSVLTVLHQDW
186-200 SVLTVLHQDWLNGKE
271-285 NNYKTTPPVLDSDGS
276-290 TPPVLDSDGSFFLYS
301-315 QGNVFSCSVMHEALH
306-320 SCSVMHEALHNHYTQAutoimmunity 2015
Study population – healthy adult donors
Fc 306-320 (Rank 1, 18/36, 50.0%)
Fc 181-195 (Rank 2, 16/36, 44.4%)
Fc 271-285 (Rank 3, 15/36, 41.7%)
Fc 21-35 (Rank 4, 14/36, 38.9%)
Fc 61-75 (Rank 7, 11/36, 30.6%)
Fc 51-65 (Rank 9, 9/36, 25%)
IL10 responses to 6 immunodominant Fc peptides recognized by nTreg in healthy adult donors
DR alleles DQ alleles DP alleles
No binding
Fc 181-195
No binding
Fc 61-75
Fc 51-65
HLA bindingFc 306-320
Fc 21-35
Fc 271-285
Binding affinity of 6 immunodominant Fc peptides to different DR, DQ and DP alleles of MHC class II
Study population – RA subjects
Fc 181-195 (Rank 1, 6/14, 42.9%)
Fc 21-35 (Rank 2, 5/14, 35.7%)
Fc 306-320 (Rank 2, 5/14, 35.7%)
Fc 61-75 (Rank 3, 3/14, 21.4%)
Fc 51-65 (Rank 3, 3/14, 21.4%)
Fc 271-285 (Rank 4, 2/14, 14.3%)
IL10 responses to 6 immunodominant Fc peptides recognized by nTreg in RA subjects
Promiscuous HLA class II binder Fc 181-195 Promiscuous HLA class II binder Fc 61-75 Promiscuous HLA class II binder Fc 51-65
Fc 21-35 (binds DRB1*12:01) Fc 306-320 (binds DQB1*06:02) Fc 271-285 (binds DRB1*07:01 and DRB1*04:05 )
21-35 61-75 181-195 306-320
nTreg expansion in response to peptide stimulation
Healthyadult
donors
RA subjects In progress
Culture supernatant of a Fc 21-35 specific nTreg line down-regulate IFNγ secretion by autologous pro-inflammatory T cells
P = 0.0013, Mann-Whitney test
nTreg responses to the Fc are different in healthy donors and RA patients
The phenotype of Fc-specific Treg clones indicate that these T cells are natural Treg(nTreg)
CD4
CD
25
CCR7CCR6IL-15rIL-7r CD45RAFOXP3 CCR4
Treg clonesIL-10 IL-4 TGFβCTLA-4
Treg clonesIL-17GITR PDCD1
Treg expansion IL-10
IL-4B cell expansion?
TCR
Treg
HLAClass II
IgG+ B cells and their role in expanding Fc-specific nTreg
A new model of B cell-T cell cooperation?
Fc-specific nTreg recognize autologous IgG+ B cells
Treg clones
IL-4
pg/
ml
Live autologous IgG+ B cells
Fixed autologous IgG+ B cellsIL
-10
pg/m
l
Treg clones
Live autologous IgG+ B cells
Fixed autologous IgG+ B cells
IgG molecules on B cells need to be processed and presented by HLA molecules to stimulate Fc-specific Treg clones
live versus fixed IgG+ autologous B cells as antigen presenting cells
Anti-CD4102 103 104 105
102
103
104
105
Ant
i-CD
25 71.7
Anti-CXCR5102 103 104 105
42.3
Even
ts
Anti-CCR7
Even
ts
52.9
102 103 104 105
Chemokine receptors suggest homing to the lymph nodes and proximity to the germinal center: representative Fc 181-195 specific Treg line
Conclusions
We identified the fine specificity of an important nTreg lineage involved in immune homeostasis in health and diseases: 3-5 immunodominant Fc peptides could serve in the clinic as the first approach to induce immune regulation
CCR7 expression and B cell antigen presentation suggest that Fc-specific nTreg operates in the lymph nodes
IgG+ B cells can activate Fc-specific nTreg that reside in the germinal centers (CXCR5+)
Fc-specific nTreg secrete IL-4 in addition to IL-10 and promotes B cell survival/expansion
This is the main mechanism of IVIG in some clinical settings: Fc peptides by-pass the need for antigen processing that is affected in the two disease models studied (KD and RA): Fc peptides offer an optimized approach to expand Fc-specific nTreg in vivo
Application of the technology
Autoimmunity
Vascular inflammation
Prevention of miscarriages in autoimmune women
Neurological disorders successfully treated with IVIG
Kawasaki disease and other diseases where the IVIG mechanism resides in Immune regulation
Acknowledgments
Li-En HisiehNegar BenhamfarRanim Touma
Gary FiresteinDavid BoyleAndre Matti
Jane C BurnsAdriana H TremouletChisato ShimizuJoan PancheriDeeAnna Sherrer
UCSD School of MedicineDepartment of Medicine RAI division
UCSD School of MedicineDepartment of Pediatrics AIR Division
La Jolla Institute for Allergy and Immunology
Alessandro SetteJohn Sidney
Kawasaki Disease Center
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