In this study, we have shown clear indications of an antigen-specific T regulatory cell response arising following treatment of new onset type 1 diabetes with a vaccine comprised of insulin B-chain in IFA. This is a unique response not previously observed in type 1 diabetes using antigen-specific agents. Incomplete Freund's adjuvant has been long overlooked in human diabetes mellitus, even though it has been successfully applied in other indications [39
], with a good long-term safety profile [40
]. In recent years IFA-enhanced antigen T cell receptor peptide vaccination trials have been successfully conducted in rheumatoid arthritis [41
] and in multiple sclerosis [42
]. Here, we employed a newer formulation of IFA that has shown excellent tolerability in patients with HIV [43
The entry criteria for this study did not require a minimum C-peptide level at baseline for inclusion in this study. This resulted in an imbalance of baseline C-peptide levels between the two groups. The mean total C-peptide level at study entry was higher in the control group, an imbalance that was sustained throughout the follow-up period, but one that at no time was statistically significant. As this was a pilot study, the sample size was small (n=12 study participants); the study was not originally powered to detect significant differences between the two groups for changes in mean C-peptide production over time. However, there was a better trend in the stimulated C-peptide decline in the insulin B-chain vaccinated group after three months of the vaccination.
Although it is not possible to draw conclusions concerning the vaccine’s clinical efficacy, the vaccine did elicit a clear insulin-specific humoral response. IAA levels, which peaked at 12 weeks then gradually declined, were significantly higher compared to placebo at weeks 8, 12, and 24. Although some elevation of IAA values was noted in the placebo-vaccinated patients, these values stayed within the range typical of subjects receiving exogenous insulin to control diabetes [38
]. The increased IAA levels cannot be attributed to differences in insulin dose, as both arms reported similar doses throughout the study, thus increased IAA showed no adverse effect on insulin usage.
We also observed that immune cells from vaccinated subjects responded to insulin B-chain in an antigen-specific manner. All six study participants in the insulin B-chain vaccinated group and none in the control group showed a high level of T-cell proliferation in responses to insulin B-chain stimulation. This antigen-specific response reached a zenith at 6 months, then slowly declined, but remained positive for up to 2 years. Interestingly, the antigen-specific response showed a modest positive correlation with the level of decline in stimulated C-peptide response (delta C-peptide and T cell SI at 1 year; r=0.75). Only limited responses were observed to the B-chain fragments (OP2 [B5-20,] OP3 [B9-23]) and to pro-insulin, and this response was also restricted to the insulin B-chain vaccinated group. Comparing these limited responses to the robust response elicited by the full length B-chain suggests that a combination of the putative epitopes on the B-chain and/or its specific conformational structure is needed to elicit full scale stimulation. The dynamic and the duration of the humoral and cellular responses evoked are quite different. The humoral response peaked at 3 months and reverted to the level of the controls by one year, meanwhile the cellular response reached zenith at 6 months and remained positive throughout the study.
Regulatory T cells are believed to be critical in the maintenance of immune tolerance [44
] and there are indications that their suppressive function is defective in T1DM [45
]. They exert their effect on other immune cells by cell-to-cell contact as well by a set of powerful cytokines like TGF-β and IL-10. They do not represent a homogeneous population and currently there is no single marker regarded as sine qua non in humans. Most carry surface markers alone or in combination, like CD4+CD25 high, CD127 negative/low, and express transcription factor Foxp3.
Peripheral blood T cells of patients who received insulin B-chain vaccine produced a significant amount of TGF-β upon stimulation with insulin B chain. We further characterized and tested single T cell clones isolated from two patients’ peripheral blood following insulin B-chain stimulation. These human insulin B-chain T cell clones showed phenotypic characteristics of regulatory T cells: CD127 negative/low, Foxp3+ and TGFβ and/or IL-10 positive. As expected, they varied in their capacity to further expand upon insulin B-chain restimulation, some with strong and some with no or little expansion. In case of two clones, we were able to document their inhibitory capacity. The level of inhibition was similar to that observed previously in T1DM subjects. [45
]. These cells were not produced as a result of the cloning process, as CD4+CD25- cells taken from healthy controls and T1DM patients that were subject to the same cloning procedures showed no such characteristics. Therefore, these data suggest that human insulin B-chain vaccination in patients with T1DM evokes insulin B-chain specific regulatory T cells, which have the capacity to exert their regulatory functions. This response is polyclonal as indicated by the results of the TCR typing.
It is unknown whether these regulatory T cells are results of the expansion of the existing regulatory repertoire or represent de novo generation of a peripherally induced new regulatory compartment (from insulin B-chain specific effector cells). It is known that effector T cells can be turned into Foxp3+ regulatory T cells [46
]. In future trials, our intentions are to further investigate the induced T cell population at the single cell level to understand its ontogenesis and expand on characterization of their function [47
The antigen-specificity of these induced regulatory cells is important as they have the capacity to accumulate at the relevant site of autoimmunity [48
]. The regulatory T cells are “multitalented masters of immune regulation” [49
] and can function in many ways. They can control by bystander suppression where one antigen-specific regulatory T cell controls effector T cells with different antigen specificity. They can also exert their control by infectious tolerance whereby they change the micro-milieu to foster generation of regulatory T cells with different antigen specificity. Moreover, regulatory T cells can confer suppressor activity to effector T cells [50
]. The notion of epitope and antigen spreading in T1DM autoimmunity is well accepted [30
], implying that several effector cell populations with different epitope and antigen specificities are present. Thus, one may speculate about the possibility that regulatory T cells generated by this vaccine could control not only insulin B-chain-specific effector T cells, but those specific for the entire panel of islet beta cell antigens involved in the autoimmune process.
Taken together, these data indicate that human insulin B-chain in IFA is a safe intervention and evokes robust, antigen-specific immune response. This vaccine generates type 1 diabetes autoantigen-specific regulatory T cells that appear to retain their functional capacity and thus have the potential to arrest T1DM autoimmunity. As the first report of the generation of an autoantigen-specific regulatory T cell response in human T1DM, it thus warrants continued clinical investigation to further characterize its mechanism of action and to establish the optimal dose, dose regime, optimal formulation, time and age for intervention.