Other Clinical Diagnoses

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1 Donor Case ID 14 year old fe npod year old npod year old fe npod year old fe npod69 22 year old fe npod year old T1D.6 27 year old T1D.7 30 year old T1D.8 22 year old T1D.9 Duration of T1D (years) Other Clinical Diagnoses Supplemental Table 1. Summary of characteristics and tally of CD4+ and CD8+ lines and clones derived from islets of donors with T1D. The number of ex vivo CD4 or CD8 islet infiltrating T cells detected by flow cytometry is shown (4 th column from the left) and the number of CD4 and CD8 T cell lines grown from individual islets is shown (5 th column from the left). The T cells clones that were sorted and grown are included in this tally. The number of T cell lines grown from individual islets that contained mixtures of CD4 and CD8 T cells is shown (5 th column from the left as mixed); examples of lines grown from individual islets that contained both CD4 and CD8 T cells is shown in Supplemental Figure 1. No. T cells detected by flow cytometry No. T cells lines grown No. of lines grown as mixtures of CD4 and CD8 2 none reported 34/105 25/10 10 mixed 2 bipolar disorder, depression 1178/83 14/3 3 hypothyroidism ND 7/3 10 mixed 3 spinal muscular 104/569 31/0 atrophy type I, chronic respiratory failure, gastroesophageal reflux disease, seizures 6 depression/ 0/20 10/1 anxiety 12 mixed 7 diabetic seizures ND 17/4 17 none reported 10/0 5/0 20 diabetic seizures, unspecified psychiatric disorder 20 none reported 23 mixed 0/150 0/2 ND 40 mixed 2/0 7 mixed

2 Donor Case ID Clinical diagnoses HLA No. T cell detected by flow cytometry No. T cell lines grown 3 month old Is.1 not reported A1, A1 DR17, DR7 3 year old Is.2 not reported A2, A24 DR7, DR11 DQ2, DQ7 5 year old Is.3 untreated asthma A2, A24 DR7, DR11 DQ2, DQ7 ND no lines grown 6 year old Is.4 not reported A23, A30 DR12, DR17 7 year old Is.5 seasonal allergies, asthma A4, A65 DR-, DR44 ND no lines grown 8 year old fe Is.6 allergies not available ND no lines grown 11 year old Is.7 appendectomy at age 9 A2, A11 DR13, DR15 DQ6, DQ98 3/31 5/0 25 year old fe Is.8 type 2 diabetes for 2 years, alopecia, thyroiditis, migraines A2, A68 DR13, DR17 DQ2,7 51 yr old Is.9 type 2 diabetes, hypertension, seizure disorder A2, A2 DR4, DR8 DQ4, DQ8 Supplemental Table 2. Summary of characteristics of normal donors (Is) without T1D and lack of islet infiltrating T cells. CD4 and CD8 islet infiltrating T cells of unknown reactivity were detected in one case, Is.7. Causes of death were accidental (cases Is.2, Is.3, Is.4, Is.5, and Is.7,) self-inflicted (Is.8), anoxia secondary to intracranial hemorrhage (Is.1), cerebrovascular accident secondary to intracranial hemorrhage (Is.6), and intracerebral hemorrhage (Is.9). The number of CD4 and CD8 T cells detected ex vivo by flow cytometryis shown in the 4 th column from the left and the tally of T cells grown directly from individual islets or clones ex vivo sorted from dispersed islets and grown is shown in the 5 th column from the left.

3 Age of donor (years) T1D duration (years) Total Islet IEQ isolated npod ,000 npod ,750 T1D ,000 T1D ,000 T1D ,500 T1D ,000 Mean IEQ +/- SD 35,883 +/- 32,757 Supplemental Table 3. Islet equivalents (IEQ) recovered from six of the islet isolations from donors with T1D. The mean and standard deviaton of the IEQ recovered from six of the donors with T1D is shown.

4 Supplemental Figure 1. One hundred and two T cells lines grown from individual islets from donors with T1D were mixtures of CD4 and CD8 T cells. Islets were cultured on a gel matrix with media supplemented with factors for T cell stimulation and with cytokines for 5-10 days as described in the Methods. Cells observed growing from an individual islet were collected separately under a disssectng microscope and expanded as individual lines. An aliquot of each line was stained for CD4 and CD8β expression and analyzed by flow cytometry. One hundred and eleven lines from 8/9 islet donors with T1D contained T cells that stained only for CD4. Twenty three lines from 6/9 islet donors with T1D contained T cells stained only for CD8β. One hundred and two lines from 6/9 islet donors with T1D contained mixtures of T cells staining for CD4 or CD8β (designated mixed, Supplemental Table 1). Four examples of lines designated as mixed are shown above.

5 a b Supplemental Figure 2. DQ8 restriction and proinflammatory cytokine secretion of a CD4+ T cell line reactive with hybrid peptide heggg:np-y (GQVELGGG:SSPETLI). A CD4+ T cell line grown from an islet from donor T1D.6 was previously reported to be reactive with the hybrid peptide 28 (Delong, T., et al. Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion. Science 351, (2016)). In a, Priess B cells (HLA Class II: DRB1*04 +/+, DRB4*101, DQB1*03 +/+, DR53) were pulsed with the hybrid peptide heggg:np-y and blocking anti-dr or anti-dq antibodies were added with the CD4+ T cell line. After 48 hours, secretion of IFNγ was measured by ELISA. In b, cytokine secretion was measured by Luminex after 48 hours culture of Priess cells, CD4+ T cell line +/- peptide at 50 µg/ml. p values are shown in the figure. One of three representative experiments is shown.

6 Supplemental Figure 3. Isletderived autoreactive T cells are proinflammatory. After 1-3 rounds of polyclonal stimulation, T cell lines and clones were stimulated with and without peptide with the B cells described below for each line or clone. Supernatants were collected 48 hours later and cytokine secretion determined by Luminex. The donor designation and T cell line identify is shown at the top of each set of apnels along with the HLA restriction in italics, if determined. a, a CD4+ T cell line grown from an islet from donor T1D.6 recognized GAD in the context of a bare lymphocyte syndrome (BLS) B cell expressing only HLA-DR1*0101/DRB1*0401 by secreting IL-2, IFNγ, TNFα, IL-9, CCL20/MIP3α and GM-CSF in response to the peptide. b, a CD4+ T cell line grown from an islet from donor npod69 recognized proinsulin in the context of a bare lymphocyte syndrome (BLS) B cell expressing only HLA- DR1*0101/DRB1*0401 by secreting IIFNγ and TNFα in response to the peptide. c, a CD4+ T cell line grown from an islet from donor npod6323 responded to HLA-matched EBV transformed B cells pulsed with hybrid insulin peptide (insulin C-peptide and IAPP, heggg:iapp1 (GQVELGGG:TPIESHQ) with IFNγ and TNFα secretion. d, a CD4+ T cell line grown from an islet from donor npod6323 responded to HLA-matched EBV transformed B cells pulsed with hybrid insulin peptide (insulin C-peptide and IAPP, heggg:iapp2 (GQVELGGG:NAVEVLK) with IL-2, IFNγ and TNFα secretion. No IL-4 or IL- 5 was detected from any of the lines though 3 lines did secrete IL-13 in a non-peptide specific manner. The mean values +/- standard deviation (error bars) is shown for triplicates of cell culture wells. Representative experiments are shown. p values are shown in the figures. ns - no significant difference.

7 Supplemental Figure 4. Autoreactive T cell clones recognize HLA-matched B cells transduced with autoantigen. Priess EBV-transformed B cells (HLA Class II: DRB1*04, DRB4*101, DQB1*03, DR53) transduced with lentiviral vectors expressing OFR clones of the genes for autoantigens or control antigens were washed, irradiated and plated at the cell number/well indicated with 2.5x10 4 human T cell clone cells for 48 hours and then supernatants were collected and assayed for IFNγ by ELISA. A, T cell clone 164 (reactive with glutamic acid ecarboxylase 65 peptide, GAD in the context of DRB1*0401 secreted IFNγ when co-cultured with Priess B cells expressing the ORF for GAD2, but not when cultured with the Priess B cells expressing the ORF for myelin oligodendrocyte glycoprotein (MOG). B, T cell clone Ob.1A12 secreted IFNγ when co-cultured with Priess B cells expressing the ORF for myelin basic protein (MBP), but not when cultured with the Priess B cells expressing the MOG ORF. This clone responds to MBP in the context of DRB1*15:01 and DRB1*04. One of three representative experiments is shown.