Peripheral blood mononuclear cells reactivity in recent-onset type I diabetes patients is directed against the leader peptide of preproinsulin, GAD65271-285 and GAD65431-450

Introduction T cell reactivity against pancreatic autoantigens is considered one of the main contributors to the destruction of insulin-producing cells in type 1 diabetes (T1D). Over the years, peptide epitopes derived from these autoantigens have been described in NOD mice and in both HLA class II transgenic mice and humans. However, which ones are involved in the early onset or in the progressive phases of the disease is still unclear. Methods In this work we have investigated, in early-onset T1D pediatric patients and HLA-matched controls from Sardinia, the potential of preproinsulin (PPI) and glutamate decarboxylase 65 (GAD65)-derived peptides to induce spontaneous T cell proliferation responses of peripheral blood mononuclear cells (PBMCs). Results Significant T cell responses against PPI1-18, PPI7-19 and PPI31-49, the first two belonging to the leader sequence of PPI, and GAD65271-285 and GAD65431-450, were found in HLA-DR4, -DQ8 and -DR3, -DQ2 T1D children. Conclusions These data show that cryptic epitopes from the leader sequence of the PPI and GAD65271-285 and GAD65431-450 peptides might be among the critical antigenic epitopes eliciting the primary autoreactive responses in the early phases of the disease. These results may have implications in the design of immunogenic PPI and GAD65 peptides for peptide-based immunotherapy.


Introduction
Type 1 diabetes mellitus (T1D) is the result of a slow progressive multistep autoimmune destruction of pancreatic insulin-producing cells (1)(2)(3). Early studies in European and derived populations have shown that susceptibility to T1D is strongly associated with HLA-DR3, -DQ2 and HLA-DR4, -DQ8 haplotypes, while protection is associated with HLA-DR2, -DQ6 haplotypes (4,5). Subsequent studies have shown that within these haplotypes certain HLA-DQB1, and -DRB1 alleles and residues confer susceptibility, while others provide resistance to the disease (6)(7)(8)(9)(10)(11). For instance, several studies have shown that differences of a few amino acid residues in HLA-DRB1*04 alleles are per se sufficient to modify the risk of developing T1D conferred by the high-risk HLA-DQB1*03:02 allele (12-15). Indeed, the spectrum of HLA-DQB1 and -DRB1 association, which constitutes the major component of T1D risk, is more complex than initially outlined and can be primarily grouped into very high risk, intermediate and very low risk haplotypes which, in turn, are the result of the structure of the peptide-binding pockets (15,16).
In recent years, the intricacy and the molecular nature of immunogenic T cell epitopes of pancreatic autoantigens have been further elucidated (17). It is generally believed that T cell responses against autoantigens may become less evident with the progressive destruction of the islets of Langerhans (18,19). Moreover, the spreading of T cell reactivity to other not primarily involved regions, of preproinsulin (PPI) or glutamate decarboxylase 65 (GAD65) autoantigens or to other pancreatic proteins/neoantigens generated from post-translational modifications may be an important confounding factor complicating the identification of pathogenically relevant T cells (19)(20)(21)(22). Secondly, autoreactive immune responses are not completely disease-specific, since T cell reactivity against autoantigenic proteins is detected also in control subjects who carry disease-associated HLA-DR and -DQ molecules (18, 23,24).
Thus, we tested in children with recent-onset T1D and HLAmatched healthy controls from Sardinia a set of PPI and GAD65 peptides derived from previous studies in both HLA-DR4 and -DQ8 transgenic mice and humans (26-28, 33, 35).
In all T1D patients the diagnosis was performed 3 days before enrollment. Fourteen HLA-DR4, -DQ8 positive and four HLA-DR4, -DQ8 negative healthy blood donors without history of autoimmune diseases served as controls. The 18 healthy individuals were HLA typed in connection with other studies (36). All T1D patients with recent-onset disease were recruited from our clinic in the Microcitemico Hospital A. Cao, ASL8, Cagliari. The mean age was 7.4 ± 3.3 years, in line with the mean age of T1D onset in Sardinia (37). The mean age of healthy controls was 43.6 ± 5.4. Table 1 shows the HLA-DR, -DQ typing of T1D patients and controls. An ethics committee approval was obtained for this study (authorization no. PG/2016/7815).

HLA class II typing and haplotype analysis
HLA-DRB1, -DQA1 and -DQB1 genotypes were determined by polymerase chain reaction with sequence-specific primers (SSP-PCR) using Olerup SSP typing kits (Olerup SSP AB, Stockholm, Sweden). The HLA class II haplotypes were predicted on the basis of the known linkage disequilibrium in Sardinians (12, 13).

Proliferation assays
Blood was drawn from recent-onset T1D patients within two to three days after diagnosis. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient. Blood was diluted 1:1 with complete medium (RPMI 1640, Life Technologies Italia, Monza, Italy) containing 2% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml of streptomycin, 50 mM 2-mercaptoethanol and HEPES. Diluted blood was then layered 1:1 on a Lymphoprep ™ gradient (Stem Cell Technologies, Monza, Italy) and centrifuged at 400 g for 30 min at room temperature.
Cells were harvested and washed in complete medium containing 10-15% autologous human serum, and 2 x 10 5 cells/ 200 µl per well were incubated in the presence or absence of 20 µg/ ml of PPI or GAD65 peptides. Proliferative responses against antigens were determined in 6 replicate cultures in roundbottomed 96 well plates by [ 3 H]-thymidine incorporation after 6 days of culture, following a 6-hour pulse with 0.5 µCi [ 3 H]thymidine. T cell reactivity was attributed using a relative ratio (RR) of counts per minute (cpm) of radioactivity incorporated by PBMCs plus peptide, compared to PBMCs alone. A RR of 3 or higher was considered positive and used in the statistical calculation. T cell reactivity has been controlled by stimulation with concanavalin A (data not shown).

Statistical analysis
A Chi-square test was used to compare PPI and GAD65 proliferative responses between T1D and controls. In the case of samples size lower than 5, a two-tailed Fisher's exact test was used. A p value lower than 0.05 was considered statistically significant.

T cell reactivity against PPI peptides
PBMCs from patients and HLA-matched healthy individuals were cultured with or without peptides and their proliferations measured. The eleven peptides tested cover the PPI protein sequence almost completely. Data were analyzed as a whole and after grouping patients and control individuals according to HLA-DR4 haplotype. Tables S1, S2 summarize the proliferative responses and RR obtained in T1D patients and controls respectively.
We have found that 75% of T1D patients and 33.3% of controls did respond to PPI peptides (p= 0.0204). Response to more than one PPI peptide was found in 62.5% of T1D patients and in 11.1% of controls (p= 0.0033).
Next, we evaluated which epitopes accounted for this reactivity by comparing the frequency of responses against specific peptides of PPI in T1D patients and controls. Figure 1A illustrates the frequency of responses against PPI peptides, while Figure 1B provides an example of responses against PPI peptides in a T1D patient (n. 11, Table S1). Tables S1, S2 summarize the proliferative responses and the RR obtained in T1D patients and controls respectively. Significant differences between patients and controls were found for PPI 1-18 (p=0.0348; Figure 1A), PPI 7-19 peptides (p=0.0348; Figure 1A) and PPI 31-49 (p=0.0392; Figure 1A). The statistical significance for PPI peptides was lost after stratification for DR4 haplotypes.
In terms of global reactivity, our results indicate a higher response against peptides of the PPI protein and less reactivity against GAD65 peptides. In T1D patients, we observed 46 out of 176 responses for PPI peptides versus 40 out of 224 responses for GAD65 (p=0.045).

T cell reactivity against GAD65 peptides
Sufficient PBMCs for measurement of proliferative responses against GAD65 peptides were available from 14 T1D patients and 18 control individuals. Tables S3, S4 summarize the proliferative responses and the RR obtained in T1D patients and controls respectively. Fifty-six percent of T1D patients and 27.7% of controls did respond against GAD65 peptides (p: NS). The responses to more than one GAD65 peptide did not differ significantly between T1D patients (37.5%) and controls (16.6%). Only for GAD65 271-285 and GAD65 431-450 peptides a significant difference between patients and controls was observed (p=0.0278; Figure 2). The statistical significance for these two GAD65 peptides was lost after stratification for DR4 haplotypes.
In terms of global reactivity, our results indicate a higher response against peptides of the PPI protein and less reactivity against GAD65 peptides.
Since we found significant responses for both peptides, we hypothesize that the sequence responsible for reactivity in T1D patients is included in the PPI 7-19 shared sequence LLPLLALLALWG. Prevalent reactivity of T1D PBMCs against the leader sequence may indicate that precursors of insulin, normally confined to the ER of the b-cells, are involved in targeting T cell autoreactivity to the islets. Interestingly, the same region of PPI is recognized by CD8+ T cells from recent-onset T1D patients (38,39), which may suggest that the leader sequence of PPI is targeted by both CD4+ and CD8+ autoreactive T cells. In addition to HLA-DQ8, the leader sequence of PPI (LALLALWGPDPAAAFV) may be presented also by HLA-DRB1*04:01 as previously shown in HLA-DRB1*04:01 transgenic mice (27). Therefore, in analogy to other autoantigens such as GAD65 (26, 28), the leader sequence of PPI may show overlapping peptide sequences that are presented by both HLA-DR and -DQ molecules.
Our study has several limitations that we tried to mitigate. The large number of potential T cell epitopes of pancreatic autoantigenic proteins that need to be tested challenges the relatively small amount of blood that can be drawn from children. To circumvent this limitation, we restricted the number of epitopes by prior analysis of the T cell responses in transgenic mice carrying human HLA haplotypes. Another limitation is the T cell spreading in long standing T1D patients (19)(20)(21), which is why we focused on early onset T1D pediatric patients recruited within three to four days after diagnosis. Percentage of T1D patients (solid bars) and controls (hatched bars) responding to GAD65 peptides. Significant p values (<0.05) are indicated above the corresponding bars. Statistical analysis was performed using the Fisher's exact test.  Table S1).

Discrepancies in the interpretation of T cell responses from
PBMCs, the usage of diverse experimental methodologies among laboratories and the variability in the frequencies of HLA-DR4 subtypes in different T1D populations, may lead to different results. In this work we tried to reduce these variables taking advantage of the genetically homogenous Sardinian population, less prone to both genetic and clinical confounds that are present in more cosmopolitan collections (13,51). Finally, a further possible limitation of this work was the significant age difference between T1D patients and controls. However, the 18 controls were healthy blood donors who were selected for a negative history of autoimmune diseases, thus making spontaneous not specific responses less likely than what could have been derived from age matched random pediatric controls attending the outpatient clinic for different disorders.
In summary, we identified for both PPI and GAD65 specific proliferative responses addressed to the PPI leader sequence and to GAD65 271-285 , GAD65 431-450 in T1D patients. We suggest that these could be primary epitopes involved in the early phase of the clinical onset of T1D. Finally, these data hint towards the possibility that the leader sequence of PPI is targeted by both CD8+ and CD4+ T cells, with HLA class I and class II presenting slightly different overlapping peptide epitopes and thus inducing a stronger pathogenic immune response involved in the bcell destruction.
These results may have implications in the design of immunogenic PPI and GAD65 peptides for peptide-based immunotherapy. Finally, this work could pave the way to test these epitopes in early stage T1D patients (stage 1 or 2) who have islet autoantibodies but no overt clinical diabetes requiring treatment with exogenous insulin.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Ethics statement
The studies involving human participants were reviewed and approved by Comitato Etico Indipendente AOUCA. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

Author contributions
RJ and MC conceptualized, designed and led the research. DB, ES, SM contributed to data interpretation and drafting the manuscript. RR provided HLA typing. MMan, MMar and CC collected and processed PBMC. CR and MR provided T1D patients. FC critically revised the manuscript. MC is the guarantor of this study. All authors contributed to the article and approved the submitted version.

Funding
The research was funded by: Regione Autonoma della Sardegna (CRP-59539, MC, SM and DB); non-profit association Associazione Diabete Zero ODV supported also by Fondazione Sardegna (DB) and Thyssen Foundation, Germany (RJ).