Regulatory Vs. Inflammatory Cytokine T-Cell Responses to Mutated Insulin Peptides in Healthy and Type 1 Diabetic Subjects

Regulatory vs. inflammatory cytokine T-cell responses to mutated insulin peptides in healthy and type 1 diabetic subjects

Maki Nakayamaa,b, Kristen McDaniela, Lisa Fitzgerald-Millera, Carol Kiekhaefera, Janet K. Snell-Bergeona, Howard W. Davidsona,b, Marian Rewersa, Liping Yua, Peter Gottlieba, John W. Kapplerb,c,d,1, and Aaron Michelsa,1

aBarbara Davis Center for Childhood Diabetes, bDepartment of Immunology and Microbiology, and dProgram in Structural Biology and Biophysics, University of Colorado School of Medicine, Aurora, CO 80045; and cDepartment of Biomedical Research, Howard Hughes Medical Institute, National Jewish Health, Denver, CO 80206

Contributed by John W. Kappler, February 13, 2015 (sent for review October 10, 2014; reviewed by Mark Peakman and Jay Skyler) Certain class II MHC (MHCII) alleles in mice and humans confer risk in the IAg7 β-chain at position 57 changing the conserved Asp(D) for or protection from type 1 diabetes (T1D). Insulin is a major au- to Ser(S) favors the binding of peptides that place an acidic toantigen in T1D, but how its peptides are presented to CD4 T cells amino acid at the p9 position in the peptide binding groove. In by MHCII risk alleles has been controversial. In the mouse model Reg3, the B22 Arg(R) of B:9–23 is a very poor match for this of T1D, CD4 T cells respond to insulin B-chain peptide (B:9–23) pocket, but mutating the R to Glu(E) creates an insulin mimo- mimotopes engineered to bind the mouse MHCII molecule, IAg7,in tope peptide (B22E) that binds to IAg7 almost exclusively in an unfavorable position or register. Because of the similarities Reg3. The B22E mimotope stimulates B:9–23-specific CD4 between IAg7 and human HLA-DQ T1D risk alleles, we examined T cells about 100-fold better than the WT peptide, and fluorescent control and T1D subjects with these risk alleles for CD4 T-cell IAg7 tetramers made with the altered peptide detect CD4 T cells responses to the same natural B:9–23 peptide and mimotopes. A high in the pancreas and pancreatic lymph nodes of prediabetic NOD proportion of new-onset T1D subjects mounted an inflamma- mice (12). In addition, this mimotope but not the WT B:9–23 tory IFN-γ response much more frequently to one of the mimotope peptide is capable of inducing tolerance at low doses and com- peptides than to the natural peptide. Surprisingly, the control sub- pletely preventing diabetes onset in the NOD mouse (13). jects bearing an HLA-DQ risk allele also did. However, these control In humans, particular HLA alleles are also associated with T1D, subjects, especially those with only one HLA-DQ risk allele, very fre- explaining more than 50% of the genetic risk for disease de- quently made an IL-10 response, a cytokine associated with regu- velopment (14). For example, the HLA-DQ8 (DQB*03:02)and latory T cells. T1D subjects with established disease also responded DQ2 (DQB*02:01 and DQB*02:02) alleles greatly increase the – to the mimotope rather than the natural B:9 23 peptide in pro- risk of disease development, with ∼90% of all T1D individuals liferation assays and the proliferating cells were highly enriched in having one or both alleles (14–16). Strikingly, the polymorphic certain T-cell receptor sequences. Our results suggest that the risk of HLA-DQ6 (DQB*06:02)alleleprovidesdominantprotection T1D may be related to how an HLA-DQ genotype determines the from diabetes development (14). DQ is the homolog of mouse IA, balance of T-cell inflammatory vs. regulatory responses to insulin, and the β-chains of DQ2 and DQ8 share with IAg7 the unique having important implications for the use and monitoring of insulin- non-D amino acid polymorphism at position 57 that favors an specific therapies to prevent diabetes onset. acidic amino acid at the p9 position of their binding grooves (17). We hypothesized that diabetogenic insulin-reactive CD4 T cells in diabetes | CD4 T cells | autoimmunity | self-tolerance | insulin Significance ype 1 diabetes (T1D), the autoimmune form of diabetes, Tresults from T cell-mediated destruction of insulin-producing β-cells within pancreatic islets (1). The disease is dramatically Certain class II major histocompatibility alleles confer disease increasing in incidence, doubling in the last two decades (2, 3), risk for type 1 diabetes (T1D). Insulin-specific and other auto- and now predictable with the measurement of antibodies di- antibodies often precede T1D development, but major efforts at rected against insulin and other proteins found in β-cells (4). disease prevention using insulin preparations (subcutaneous, Major efforts at disease prevention have been undertaken using oral, and intranasal) to induce tolerance have not been effective. preparations of insulin (s.c., oral, and intranasal) to induce tol- Measuring insulin-specific T-cell responses from the peripheral erance and delay the onset of clinical symptoms (5–7). Measur- blood has been a challenging feat but would allow for assess- ing insulin-specific T-cell responses from the peripheral blood ment of therapeutic response in these trials. In our study, we has been challenging but would allow for assessment of thera- report CD4 T-cell responses to a mutated insulin B-chain peptide in new-onset and established T1D as well as control subjects de- peutic response, which has been a major obstacle in these trials. pendent on HLA-DQ genotype. Our results have important im- In our study, we sought to detect peripheral T-cell responses to plications for the application and monitoring of insulin-specific insulin in T1D patients and nondiabetic controls using a modified therapies to prevent diabetes onset. insulin B-chain peptide. Insulin is a major self-antigen for both T and B cells in murine Author contributions: M.N., P.G., J.W.K., and A.M. designed research; K.M., L.F.-M., C.K., INFLAMMATION

– – IMMUNOLOGY AND and human T1D, with insulin B-chain amino acids 9 23 (B:9 23) and L.Y. performed research; M.N., J.K.S.-B., J.W.K., and A.M. analyzed data; and M.N., being a key epitope presented by major histocompatibility complex H.W.D., M.R., P.G., J.W.K., and A.M. wrote the paper. class II (MHCII) molecules to CD4 T cells targeting pancreatic Reviewers: M.P., King’s College London; and J.S., University of Miami. β-cells (8–10). There is strong evidence from the nonobese diabetic The authors declare no conflict of interest. (NOD) mouse model of spontaneous autoimmune diabetes that Freely available online through the PNAS open access option. g7 the NOD MHCII molecule, IA , is required for development of 1To whom correspondence may be addressed. Email: [email protected] or T1D and that pathogenic CD4 T cells recognize insulin B:9–23 [email protected]. presented in an unfavorable position or register (Reg3) in the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. g7 IA peptide binding groove (9, 11, 12). A unique polymorphism 1073/pnas.1502967112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1502967112 PNAS | April 7, 2015 | vol. 112 | no. 14 | 4429–4434 Downloaded by guest on October 1, 2021 T1D patients may also recognize B:9–23 bound to DQ2 or DQ8 in the unfavorable Reg3. If so, the B:9–23 (B22E) mimotope might detect these T cells much better than the WT peptide. Therefore, we examined T cells in the peripheral blood of new-onset T1D individuals and then, those with established disease for their responses to the mimotope vs. WT B:9–23 peptide. We observed vigorous responses to the mimotope but not the WT insulin peptide in numerous T1D patients. Analysis of the T-cell receptors (TCRs) in CD4 T cells proliferating in vitro in response to the mimotope peptide revealed the presence of a highly enriched oligoclonal population. Surprisingly, we also observed responses to the mimotope peptide in control subjects, especially those in which β57D DQ alleles were present, opening the possibility that this peptide may stimulate both inflammatory and regulatory CD4 T cells. Results Subjects. The subjects in this study are listed in Fig. S1A.The subjects with new-onset T1D (n = 28) and established T1D (n = 7) were recruited from the Barbara Davis Center for Diabetes Clinics. Nondiabetic controls (n = 27) were healthy adult volun- Fig. 1. Strong IFN-γ responses to the insulin B22E mimotope in new-onset T1D teers negative for all islet autoantibodies. The study protocol was and control subjects. PBMC IFN-γ ELISPOT results from (A)T1Dand(B) control. approved by the Institutional Review Board, and written informed DQβ57D−/− (red), DQβ57D+/− (blue), and DQβ57D+/+ (green) subjects are shown consent was obtained from all study participants. A detailed de- either without stimulation or with stimulation with (Left)WTB:9–23, (Center) scription of the new-onset and control subjects is given in Table the B22E mimotope, or (Right) the B21G,22E mimotope. In each case, freshly S1. The new-onset T1D subjects had a very short duration of isolated PBMCs were cultured in the presence or absence of the peptide for 48 h, washed, and then transferred to an IFN-γ mAb-coated plate for overnight diabetes, with the mean time from diagnosis being only 15 d; 26 of culture followed by development and enumeration of ELISPOTs. A control 28 (93%) T1D individuals had diabetes less than 3 wk before response to the Pentacel vaccine was also performed (Tables S3 and S4). The cytokine enzyme-linked immunosorbent spot (ELISPOT) assays number of subjects in each DQβ57D category is shown. The geometric averages were performed. All of the new-onset and control subjects were and SEs of the IFN-γ responses of the T1D (red) and control (blue) subjects, − − + − HLA-genotyped. The nondiabetic control subjects were slightly which were either (C)DQβ57D / or (D)DQβ57D / , are shown. Cntrl, control; older than the T1D patients and included more individuals having no Ag, no antigen. risk DQ alleles (i.e., those lacking β57D) than expected in the general population to allow for comparison with T1D subjects, β (7 of 28) and the B21G,22E mimotope (7 of 28) was much most of which had at least one DQ allele lacking 57D. The 6 seven subjects with established T1D are described in Table S2. lower, and only one response was above 100 spots per 10 cells P < They had diabetes ranging from 1.5 to 29 y and already known ( 0.01 comparing B22E responders with WT or B21G,22E) A HLA-DQ genotypes before performing T-cell proliferation assays (Fig. 1 ). Unexpectedly, the control subjects also mounted an γ B–D on all subjects and TCR sequencing for three subjects. IFN- response to the peptides (Fig. 1 and Table S4). Again, responses to the B22E mimotope (17 of 27) were much more Insulin Peptides. The native amino acid sequence of insulin B:9–23 frequent than those to the WT B:9–23 (2 of 27) or the B21G,22E is listed in Fig. S1B along with those of two altered B:9–23 mimotope (6 of 27; P < 0.01 comparing control B22E responders mimotopes. These were designed based on T1D studies in the NOD with WT or B21G,22E) (Fig. 1B). Only three T1D subjects and mouse (9, 11, 12) and the structural features shared between IAg7 two controls had a high responder frequency with >100 spots per 6 and human DQ8 and DQ2 (18–20). Both had a Glu substituted 10 PMBCs, indicating a frequency of ∼1 spot per 10,000 PBMCs. for Arg at B22 to enhance binding in Reg3 by replacing the dis- In general, the average magnitude of the response to the insulin favored p9 anchor amino Arg with a highly favorable Glu. The first peptides and Pentacel, a childhood vaccine, was similar in the T1D (B22E) had no other changes, but the second (B21G,22E) also had and control groups, with the exception that T1D subjects lacking the Glu at B21 changed to Gly. In the mouse, B:9–23-specific CD4 DQ β57D on both alleles responded significantly better to the T cells fall into two categories, A and B. Both react to the peptide B22E mimotope than the corresponding subjects in the con- bound to IAg7 in Reg3; however, type A T cells prefer the B22E trol group (Fig. 1C)(P = 0.02). mimotope, and type B prefer the B21G,22E mimotope (12). Using the strongly stimulating insulin B22E mimotope, we were able to follow the persistence of the IFN-γ response at 6 and 16 wk Detection of Robust IFN-γ Responses to an Insulin Mimotope. We after initial diagnosis in a new-onset T1D subject (18 in Table S3). determined the cytokine ELISPOT IFN-γ responses of unfrac- The B22E mimotope response was consistently seen at both later tionated peripheral blood mononuclear cells (PBMCs) measured time points (Fig. S2). During this time, the subject remained after short-term in vitro stimulation with no antigen vs. the WT unresponsive to WT insulin B:9–23 and the B21G,22E mimotope. insulin B:9–23 peptide or the two insulin mimotopes (Materials A positive control response to Pentacel was observed for each and Methods). The raw data for new-onset T1D patients (n = 28) ELISPOT assay. and control subjects (n = 27) are listed in Tables S3 and S4,respectively, and shown graphically in Fig. 1. Among 55 subjects, the Relation Between HLA-DQ Genotype and IFN-γ/IL-10 Responses in background responses without peptide stimulation were low (five or Control Subjects. In addition to the IFN-γ response, we were fewer spots per 106 input cells), except in 3 of the T1D subjects. able to track IL-10 responses of the control subjects and a few Among T1D subjects, the strongest IFN-γ responses (Fig. 1 A, C, of the new-onset T1D patients (Fig. 2 and Table S3) in response and D) were seen to the B22E mimotope, with 20 of 28 responding to the insulin peptides. These data might offer an explanation for with more than five ELISPOTS per 106 cells. The magnitude of the unexpectedly strong IFN-γ response seen in the control the responses varied from barely above background to well over subjects, despite the absence of T1D. As in the IFN-γ responses, 100 spots per 106 cells, with a geometric average of about 11. The the background IL-10 ELISPOT response in the absence of an- frequency of individuals responding to the WT B:9–23 peptide tigen stimulation was mostly five or fewer. As seen in Fig. 2B and

4430 | www.pnas.org/cgi/doi/10.1073/pnas.1502967112 Nakayama et al. Downloaded by guest on October 1, 2021 Enrichment of TCR Sequences Among the T Cells Stimulated by the B22E Mimotope. To examine the diversity of CD4 T cells proliferating in response to the B22E mimotope, we sequenced TCRs in the CD4 T cells from several of the established T1D subjects (1,2,and4)describedinTable S2. We sorted unstimulated + CFSE T cells and the corresponding CFSElo-proliferating CD4 T cells for TCR-α (subjects 1, 2, and 4) and TCR-β (subjects 2 and 4) sequencing. The statistics for the productive V(D)J sequences in each sample are shown in Table S5. There was little overlap in sequences among the subjects and between the unstimulated vs. CFSElo T cells for any subject (Fig. S3). In Fig. 4, we show the cumulative contribution of repeated sequences to the total unique sequences from those that repeat a particular number of times. In addition, the predicted contributions based on a cumulative Poisson distribution calculated from the average number of repeats for that sample are shown. All of the sequence sets from the unstimulated T cells (Fig. 4, Lower) matched the predicted distributions reasonably well, Fig. 2. The IL-10 ELISPOT responses of the (A) T1D and (B) control subjects indicating a fairly random set of sequences in PBMCs with are presented. (C) A comparison of the IFN-γ with IL-10 ELISPOT responses of only a few sequences dramatically overrepresented, which was individual control subjects is shown colored as in A and B. The (D) IL-10 and − − not case for stimulated T-cell samples (Fig. 4, Upper). These (E) IFN-γ responses of the control DQβ57D / subjects compared with those + − + + that were either β57D / or β57D / . Colors are the same as in Fig. 1. No Ag, sequence sets, particularly for subjects 1 and 4, showed a biphasic no antigen. distribution deviating dramatically from that predicted for a

Table S4, many of the control subjects had a robust IL-10 response. As in the case of the IFN-γ response, the B22E mimotope yielded the highest frequency of responses (24 of 26) compared with the WT B:9–23 (11 of 26) or B21G,22E (16 of 26) peptide, with more than five ELISPOTS per 106 cells (P < 0.01 comparing B22E with WT and P = 0.02 comparing B22E with B21G,22E). In the response to the B22E mimotope, there was a correlation between the magnitudes of IFN-γ vs. IL-10 (Fig. 2C), and strikingly, the subjects with at least one nonrisk DQ allele having β57D mounted a stronger response to both IL-10 and IFN-γ than those subjects lacking β57D on both DQ alleles (Fig. 2 D and E). We were only able to obtain IL-10 data for eight of the T1D patients. There was only one IL-10 response to the WT peptide or B21G, B22E mimotope, but six of eight patients had a detectable IL-10 response to the B22E mimotope. Notably, these six were among the poorest in the IFN-γ response (Table S3).

Proliferation of CD4 T Cells to the Insulin Peptides. To complement our ELISPOT experiments from new-onset T1D subjects, we also examined T1D patients with established disease that had − known HLA-DQ genotypes that included two β57D DQ alleles (Table S2). Fig. 3 shows the proliferation results after bulk unfractionated PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) and cultured for 7 days in the presence of no antigen, Pentacel, WT B:9–23 peptide, or the B22E mimotope peptides with no additional stimulus. Similar to the IFN-γ ELISPOT assays, the B22E mimotope resulted in proliferation of CD4 T cells much more than the WT peptide as Fig. 3. Proliferation of unfractionated PBMCs with insulin peptides from − judged by the appearance of cells that had a loss of CFSE subjects with established T1D bearing two β57D DQ alleles. Unfraction- fluorescence (Fig. 3A), which was true for all of the tested T1D ated PBMCs were isolated from the patients listed in Table S2 and labeled patients (Fig. 3 B and C). To determine whether these pro- with CFSE. They were cultured with no antigen, Pentacel, WT B:9–23, or the B22E mimotope without any other stimulus; then, after 7 d, they were an- liferative responses were DQ-restricted, an anti-DQ blocking INFLAMMATION alyzed by flow cytometry for CD4 T-cell CFSE fluorescence dilution as an IMMUNOLOGY AND antibody was added to the culture during proliferation of CD4 indication of proliferation. (A) Representative data from two of the T1D T cells. The antibody was able to block the proliferative response subjects (1 and 2 from Table S2). (B) For all subjects, the percentage of CFSElo of CD4 T cells from subject 5 to the B22E mimotope in a dose- CD4 T cells after culture without antigen is shown vs. after culture with the dependent fashion (Fig. 3D), and all of the tested T1D subjects B22E mimotope. (C)SameasB but comparing stimulation with the response + – bearing at least one DQ8 allele had fewer CD4 CFSElo cells to the WT B:9 23 peptide with that to the B22E mimotope. (D) The percentage of CFSElo CD4 T cells remaining for subject 5 in Table S2 (DQ8/8 homozygote) after proliferation to the insulin B22E mimotope in the pres- after blocking activation by the B22E mimotope by adding various concen- ence of the DQ antibody, indicating that the T-cell response to trations of an anti-DQ antibody throughout the culture period. (E) Inhibition the peptide is at least partially DQ-restricted (Fig. 3E). of the response to the B22E mimotope by six DQ8 subjects in Table S2.

Nakayama et al. PNAS | April 7, 2015 | vol. 112 | no. 14 | 4431 Downloaded by guest on October 1, 2021 − the shared β57D polymorphism among mouse IAg7 and human T1D DQ risk alleles predicts that the B22 Arg to Glu mutation should dramatically improve presentation of the peptide to these T cells. To test this idea, we examined PBMCs from 28 new-onset T1D subjects for an IFN-γ response to the WT vs. mutant B:9–23 peptide using an ELISPOT assay. Nearly all of the patients had − at least one β57D DQ allele. In agreement with our prediction, most of these subjects had a positive response to the mutant insulin peptide but responded much less well to the WT peptide, − which was true whether they had one or two β57D DQ alleles. A few of these subjects responded to the mutant peptide as strongly as they did to the Pentacel vaccine control antigen, but on average, their response was a more modest 10% of the response to Pentacel. We also tested an insulin peptide that had a second mutation of B21 Glu to Gly, because in the NOD mouse, the side chain of this p8 Glu interferes with recognition by some T cells. This double-mutant peptide was much less well-recognized in these human patients.

lo We also examined patients with more established T1D and Fig. 4. Enrichment of particular TCRs in CFSE CD4 T cells responding to found that they too had stronger proliferative DQ-restricted the insulin B22E mimotope. As described in Materials and Methods and Table S5, the TCR repertoires of the CFSElo CD4 T cells proliferating in re- responses in vitro to the mutant than the WT peptide. In several of sponse to the B22E mimotope in Fig. 3 were compared with those of the these experiments, we were able to isolate the proliferating CD4 CFSE+ CD4 T cells from the same subject cultured without antigen. We an- T cells and show that their TCR-α and -β repertoires were very alyzed TCR-α and -β data from two subjects (2 and 4) and TCR-α data only much skewed compared with the starting population, indicating from a third subject (1). The number of unique sequences occurring a particular number of times in the dataset vs. the cumulative contribution (%) of those sequences to the total unique sequences obtained is shown. Upper shows data for the proliferating CFSElo CD4 T cells (red), and Lower shows data for the total CFSE+ unstimulated cells (blue). Also shown (dotted lines) are the cumulative Poisson distribution curves predicted based on the average number of occurrences of unique sequences in the dataset.

random set of sequences. These results indicate that some proliferating T cells expanded dramatically, whereas others did not, leading to a heterogeneous pattern of T-cell enrichment. We hypothesized that the highly expanded T cells were most likely to contain those with higher affinity for the B22E mimotope, whereas the less-expanded T cells were more likely to contain low-affinity T cells or were bystander T cells proliferating nonspecifically (for example to cytokines). Therefore, we examined the five most frequent α- and β-sequences present in the expanded T cells, each found hundreds of times (Fig. 5). They accounted for 4% to over 20% of the thousands of sequences present in five enriched samples (Table S5). None of these sequences were shared among the patients. The majority of the sequences (22 of 25) were absent in the sequences obtained from unstimulated T cells in the same subject, indicating that these sequences were expanded from an infrequent precursor. The CDR3 regions of these T cells were encoded by a dominant (≥95%) DNA sequence, despite the presence of several N region- encoded amino acids that could have used alternate codons, consistent with each T cell having arisen from a single or limited set of T-cell clones. Most strikingly, 6 of the 15 most frequently occurring α-chain sequences contained one of two highly related TRAV-38 family members, whereas the use of this family among three unstimulated T-cell samples was between 6.0% and 8.4%. The CDR3 sequences were unrelated among the six. These results are reminiscent of the T cells in NOD mice specific for the insulin B:9–23 Fig. 5. Analysis of frequent TCRs in B:9–23(B22E)-stimulated CD4 T cells. peptide, in which there is disproportionate use of the mouse From five sets of TCR Vα and Vβ sequences from the B22E-stimulated CFSElo TRAV-5 family with various CDR3 sequences (21, 22). We will T cells described in Fig. 4 and Table S5, the five most frequent Vα and Vβ pursue the specificities of these highly expanded T cells in future sequences are listed with their V and J genes identified and the protein sequence of the CDR3 loops shown. Also shown is the number of times that experiments. a the sequence was found and its overall frequency (%) in the dataset. The b Discussion immunogenetics nomenclature is used for V and J genes. The N(D)N portion of the CDR3 protein sequence was defined as any amino acid The rationale for our experiments here was that, if the B:9–23 encoded by a codon containing at least one non-V/J germ-line gene. cPortion peptide contained CD4 T-cell epitopes important in human T1D, (%) of the entire dataset accounted for by five sequences.

4432 | www.pnas.org/cgi/doi/10.1073/pnas.1502967112 Nakayama et al. Downloaded by guest on October 1, 2021 an enrichment of antigen-reactive T cells. Although thousands of or inhibiting binding in other irrelevant registers. In the case of unique sequences were identified in the expanded T cells, five MHCI, the mutant peptide could favor the processing of a CD8 sequences accounted for a large percentage (4–20%) of the total T-cell epitope. It is known that, within the insulin B:9–23 peptide sequences obtained. Furthermore, although each patient showed sequence, amino acids 10–18 activate CD8 T cells from T1D auniqueα-andβ-CDR3 signature among the most expanded patients (24). It is also worth noting that our experiments involved T cells, there was an overenrichment of T cells bearing members short-term immediate ex vivo stimulation of the T cells, and of the TRAV-38 Vα family, reminiscent of the skewing in the use therefore, it is likely that these responses, potentially driven by non- − of the TRAV-5 Vα family among NOD CD4 B:9–23-reactive DQ β57D MHC molecules, most likely arose from memory α T cells involved in T1D, despite heterogeneity in their V CDR3 T cells with previous antigen experience. β sequences and V use (21, 22). Most models for autoimmunity suggest that, in the non- Because we used bulk populations of T cells, we have not for- autoimmune steady state, there is a balance between antigen- mally shown in our experiments that the weak responses to the driven inflammatory and regulatory responses in the target organ WT peptide and the strong responses to the mutant peptide in- that needs to be disrupted for autoimmunity to blossom out. The volved the same T cells. However, in the course of these studies, strong correlation between the IFN-γ and IL-10 responses to the several of our group collaborated with Kwok and coworkers (23) mutant insulin peptide in the control subjects might reflect this at the Benaroya Institute in Seattle. Kwok and coworkers used balance that prevents T1D development. Other investigators have a different approach, in which T cells from established T1D also shown the presence of autoreactive T cells in the peripheral patients with one or two DQ8 alleles were stimulated with WT or blood of healthy donors that can be suppressed by their own B22E mutant insulin peptides for a short time in vitro and then, regulatory T cells (25). Peakman and coworkers (26) reported single cell-sorted with DQ8 tetramers loaded with either peptide to establish a set of CD4 T-cell clones. Regardless of their source a regulatory IL-10 response to the self-antigens insulinoma- for these clones, they responded much more strongly to the mu- associated antigen 2 (IA-2) and proinsulin, with a panel of IA-2 tant than to the WT peptide. Furthermore, using the same tech- peptides able to distinguish diabetic from control subjects inde- niques that were used to establish the register 3 nature of the pendent of HLA type. More recently, posttranslational modifi- – NOD insulin epitopes, this study confirmed that the mutant cation of a proinsulin peptide (insulin B:30 C:13) with tissue peptide was recognized bound in register 3 to DQ8. transglutaminase was able to bind HLA-DQ8 and identified in- γ One difference between the experiments we report here and flammatory IFN- responses from T1D subjects and regulatory those with Kwok and coworkers is that we used the full-length IL-10 responses from some healthy controls (27). Other studies B:9–23 peptide, whereas Kwok and coworkers (23) used a pep- have documented responses to other insulin epitopes in human tide truncated toward the register 3 core, B:11–23. However, the T1D. For example, the work by Kent et al. (28) identified CD4 B22E mutation strongly promotes binding in register 3 to IAg7 T cells responding to DR4-presented epitopes from the insulin and DQ8, leaving B:9 and B:10 well outside the MHC binding A-chain. Another recent study has described a series of CD4 groove and far away for the usual TCR footprint. Therefore, T-cell clones isolated from the pancreas of a T1D patient that is we conclude that, taken together, these two studies point out reactive to C-peptide epitopes presented by DQ8 or DQ8/DQ2 the presence of a previously underappreciated response to insulin heterodimers (29). Unfortunately, unlike in the case of NOD in T1D patients and provide useful tools to pursue the study of this mouse, the tools are presently lacking to determine the relative response in the future. They also further reinforce the idea that importance of these different antiinsulin responses in the onset the strong association of T1D in mice, rats, and humans with a and progression of the disease in humans. similar MHCII polymorphism may be because of a similar usual In conclusion, the data presented here show a modified insulin mechanism of MHCII allele-specific autoantigen presentation (23). B-chain peptide capable of detecting robust autoreactive T-cell The most unexpected results in our experiments were those responses from the peripheral blood and suggest that T1D risk obtained with control non-T1D subjects, again mostly having one may be related to how an HLA-DQ genotype determines the β − or two 57D DQ alleles. In this case, we had enough of each balance of the T-cell inflammatory vs. regulatory responses to γ sample to perform both IFN- and IL-10 ELISPOT assays (for insulin. With the increasing incidence of T1D over the last two example, cytokines associated with inflammatory vs. regulatory decades, especially in children less than 5 y of age (3), and the T cells, respectively). Surprisingly, for both cytokines, many of ability to predict diabetes development in individuals with two or these individuals also made strong responses to the mutant but more islet autoantibodies (4), diabetes prevention trials are un- mostly poor responses to the WT insulin peptide. Compared with derway (30). Antigen-specific therapy holds the potential for the T1D patients, the main difference in the pattern of response being able to safely and specifically induce tolerance to an ad- was the DQ genotype of the strong responders. Whereas among ministered self-antigen. The insulin B:9–23 (B22E) mimotope the T1D patients, there was no significant difference in the IFN-γ − has been used as antigen-specific therapy in the NOD mouse, in response of patients with one or two β57D DQ alleles, in the which small amounts of the mimotope are able to convert naïve control subjects, both the IFN-γ and IL-10 responses were sig- + − nificantly lower in those with two β57D DQ alleles than those T cells into Foxp3 regulatory T cells and prevent diabetes onset + with one or even two β57D DQ alleles. (13). Therefore, because of its superagonist properties, the po- Our data do not offer any direct explanation for this unexpected tential exists to use the insulin B22E mimotope as a tolerogenic but potentially important observation. At this point, we can only vaccine in those at risk for T1D. In other studies, antibodies – g7 speculate on possibilities that can be tested in future experiments. specific for the register 3 B:9 23/IA complex inhibit T-cell β − responses in vitro and delay T1D onset in vivo in NOD mice (31,

Perhaps the simplest explanation is that the DQ 57D response INFLAMMATION

to the register 3-presented insulin peptide is suppressed in these 32), opening another potential avenue for antigen-specific ther- IMMUNOLOGY AND control subjects, explaining the lack of T1D and the poor response apy. Our results also raise the possibility that individuals with a − + in the patients containing only DQ β57D alleles. The IFN-γ β57D -containing DQ allele may have the ability to produce and IL-10 responses in the other control subjects may require the a regulatory response to the insulin epitope. With the genetics of − MHC alleles that lack the DQ β57D polymorphism. Because T1D shifting toward more new-onset patients with at least one of these responses are strongly enhanced by the B22 Arg to Glu these alleles (33), insulin-specific immune therapies are an ap- mutation, this mutation should be involved in peptide processing pealing approach for diabetes prevention, because there is now or the binding and/or register selection by these other MHC a robust means to assess proinflammatory and regulatory T-cell molecules, perhaps by improving binding in the relevant register responses from the peripheral blood.

Nakayama et al. PNAS | April 7, 2015 | vol. 112 | no. 14 | 4433 Downloaded by guest on October 1, 2021 Materials and Methods 7days,thelossofCFSEfromdividingCD4Tcellswasdeterminedby Subjects and Samples. Subjects were recruited from the Barbara Davis Center flow cytometry. for Diabetes Clinics, and written informed consent was obtained after the α nature and possible consequences of the study were explained to individuals. TCR Sequencing. We determined the TCR- sequences of sorted CFSE-labeled The clinical investigation in this study was conducted in accordance with CD4 T cells from three of DQ2/DQ8 subjects (1, 2, and 4) listed in Table S2. TCR-β sequences were also determined for two of these subjects (2 and 4). For the Declaration of Helsinki principles, with study approval provided by the + + Colorado Multiple Institutional Review Board. Peripheral blood was obtained each subject, two sets of T cells were sorted: total unstimulated CD4 CSFE + lo for T-cell assays, islet autoantibodies, and HLA genotyping. Islet autoanti- cells and CD4 CFSE cells after 1 week of in vitro stimulation with the B22E bodies to insulin, GAD65, IA-2, and ZnT8 were measured from the serum by insulin mimotope. Total RNA was directly extracted from sorted cells using radioimmunoassay as previously described (34). HLA-DRB, DQA, and DQB the Rneasy Mini Kit (Qiagen) for cells before proliferation and the PicoPure genotyping was performed using linear arrays of immobilized sequence- RNA Isolation Kit (Life Technologies) for those after proliferation. cDNA was α β specific oligonucleotides similar to previously described methodology (35). prepared from each of the samples, and TCR V and V sequences obtained and analyzed as described in SI Materials and Methods. Antigens. The insulin B:9–23 and mimotope peptides listed in Fig. S1B were obtained from Genemed Synthesis Inc. at >95% purity. For a control antigen, Statistical Analysis. Total spot numbers from ELISPOT assays were analyzed with a nonparametric Mann–Whitney test (rank sum test). ELISPOT response rates to we used Pentacel, a childhood vaccine containing five different immunogens, a given antigen were compared with a two-sided Fisher’s exact text. For CFSE obtained from Sanofi Pasteur. proliferation assays, a Wilcoxon signed rank test compared samples from the same subject. For all statistical tests, a two-tailed P value of <0.05 is considered Human Indirect ELISPOT. ELISPOT analyses were conducted as previously de- significant. Analyses were performed using GraphPad Prism 4.0 software. scribed using the human IFN-γ and IL-10 ELISPOT Kits (UCyTech Biosciences) (29). Details are discussed in SI Materials and Methods. ACKNOWLEDGMENTS. We thank George Eisenbarth for helpful scientific dis- cussions, inspiration, and encouragement. This work was supported by National CFSE Proliferation Assay. As described in SI Materials and Methods, PBMCs Institute of Diabetes and Digestive Kidney Diseases Grants R01DK032083, were isolated from whole blood, labeled with CFSE, and cultured with K08DK095995, R00DK080885, and R01DK099317; the Juvenile Diabetes Research no antigen, Pentacel, the WT B:9–23 peptide, or the B22E mimotope. After Foundation; the Children’s Diabetes Foundation; and the Brehm Coalition.

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