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 PhD UCSD School of Medicine Department 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
Regulatory T cells phenotypically CD4+ CD25 high 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)
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 α-sma α-sma αsma and IL17 Human Pathology 2012
Understanding IVIG therapy and its success in Kawasaki disease: Fc-specific Treg?
Autoimmunity 2014 Fc-specific Treg expand only in sub-acute KD subjects with normal arteries after IVIG Subject 7 CD25 % CD4+CD25 high T cells Control 10µg/ml 100µg/ml 1µg/ml 10µg/ml CD4 3.0 1.5 0.0 3.0 1.5 0.0 subject 1 subject 2 subject 3 subject 4 subject 5 No Ag 1 10 100 3.0 1.5 0.0 Fc µg/ml No Ag 1 10 100 3.0 1.5 0.0 No Ag 1 10 100 3.0 1.5 0.0 No Ag 1 10 100 3.0 1.5 0.0 No Ag 1 10 100 subject 6 subject 7 subject 8 subject 9 subject 10 3.0 3.0 3.0 3.0 1.5 1.5 1.5 1.5 0.0 0.0 0.0 0.0 No Ag 1 10 100 No Ag 1 10 100 No Ag 1 10 100 No Ag 1 10 100 No Ag 1 10 100 No Ag 1 10 100 subject 11 subject 12 subject 13 subject 14 subject 20 3.0 3.0 3.0 3.0 3.0 1.5 1.5 1.5 1.5 1.5 0.0 0.0 0.0 0.0 0.0 No Ag 1 10 100 1µg/ml Fc F(ab) 2 Dilated arteries or aneurysm No Ag 1 10 100 No Ag 1 10 100 No Ag 1 10 100 100µg/ml
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 supernatants 2. CD4+ CD25 high T cell expansion by flow cytometry
Study population KD subjects after IVIG
IL10 responses to 6 immunodominant Fc peptides recognized by ntreg in KD subjects after IVIG Fc 51-65(Rank 1, 4/8, 50%) Fc 21-35 (Rank 3, 2/8, 25%) Fc 181-195 (Rank 2, 3/8, 37.5%) Fc 271-285 (Rank 3, 2/8, 25%) Fc 61-75 (Rank 2, 3/8, 37.5%) Fc 306-320 (Rank 3, 2/8, 25%) 5 6 8 5 6 8 5 6 8 5 6 8 5 6 8 5 6 8
Fc position Sequence 21-35 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 SCSVMHEALHNHYTQ Autoimmunity 2015
Study population healthy adult donors
IL10 responses to 6 immunodominant Fc peptides recognized by ntreg in healthy adult donors Fc 306-320 (Rank 1, 18/36, 50.0%) Fc 21-35 (Rank 4, 14/36, 38.9%) Fc 181-195 (Rank 2, 16/36, 44.4%) Fc 61-75 (Rank 7, 11/36, 30.6%) Fc 271-285 (Rank 3, 15/36, 41.7%) Fc 51-65 (Rank 9, 9/36, 25%)
Binding affinity of 6 immunodominant Fc peptides to different DR, DQ and DP alleles of MHC class II DR alleles DQ alleles DP alleles Fc 181-195 HLA binding Fc 306-320 No binding Fc 61-75 Fc 21-35 No binding Fc 51-65 Fc 271-285
Study population RA subjects
IL10 responses to 6 immunodominant Fc peptides recognized by ntreg in RA subjects Fc 181-195 (Rank 1, 6/14, 42.9%) Fc 61-75 (Rank 3, 3/14, 21.4%) Fc 21-35 (Rank 2, 5/14, 35.7%) Fc 51-65 (Rank 3, 3/14, 21.4%) Fc 306-320 (Rank 2, 5/14, 35.7%) Fc 271-285 (Rank 4, 2/14, 14.3%)
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 )
ntreg expansion in response to peptide stimulation 21-35 61-75 181-195 306-320 Healthy adult 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
ntreg responses to the Fc are different in healthy donors and RA patients P = 0.0013, Mann-Whitney test
The phenotype of Fc-specific Treg clones indicate that these T cells are natural Treg (ntreg) Treg clones CTLA-4 GITR PDCD1 IL-10 IL-4 TGFβ Treg clones IL-17 FOXP3 CD45RA IL-7r IL-15r CCR6 CCR7 CCR4 CD25 CD4
IgG+ B cells and their role in expanding Fc-specific ntreg Treg expansion IL-10 B cell expansion? IL-4 HLA Class II TCR Treg A new model of B cell-t cell cooperation?
Fc-specific ntreg recognize autologous IgG+ B cells
live versus fixed IgG+ autologous B cells as antigen presenting cells Live autologous IgG+ B cells Fixed autologous IgG+ B cells Live autologous IgG+ B cells Fixed autologous IgG+ B cells IL-10 pg/ml IL-4 pg/ml Treg clones Treg clones IgG molecules on B cells need to be processed and presented by HLA molecules to stimulate Fc-specific Treg clones
Chemokine receptors suggest homing to the lymph nodes and proximity to the germinal center: representative Fc 181-195 specific Treg line Anti-CD25 10 5 10 4 10 3 10 2 71.7 Events 42.3 Events 52.9 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 Anti-CD4 Anti-CXCR5 Anti-CCR7
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
UCSD School of Medicine Department of Pediatrics AIR Division Li-En Hisieh Negar Benhamfar Ranim Touma Acknowledgments UCSD School of Medicine Department of Medicine RAI division Gary Firestein David Boyle Andre Matti La Jolla Institute for Allergy and Immunology Alessandro Sette John Sidney Kawasaki Disease Center Jane C Burns Adriana H Tremoulet Chisato Shimizu Joan Pancheri DeeAnna Sherrer