On the other hand, attempting immunomodulation in individuals who exhibit clinical disease is better justifiable as the autoimmunity is not speculative (unlike in prevention approaches) but a fact. This then leads to the question of what is considered the point of “too late” at which immunomodulation is ineffective and only adverse events will plague the patient without any possibility of real benefit.

In the absence of bona fide biomarkers of immune efficacy, physiologic measurements become of paramount importance. Examples, for T1DM, include maintenance or increase of pretreatment C-peptide levels, improvement of mixed meal-stimulated insulin production and consequent reduction of glucose levels in blood, improvements in glycated HbA1c and, in certain instances, reduction of exogenously administered insulin or complete cessation thereof for a period of time. As will be discussed later, the values between pretreatment and posttreatment physiological measurements in a number of recent immunomodulation clinical trials, in particular if promoted and implemented close to the clinical onset of the disease, have required stringent statistical analysis to identify differences, and in many respects, the pronouncements of success for some immunomodulation treatments require considerable faith in the outcome of statistical treatments of the data.

In 1985, Eisenbarth and colleagues reported that steroid-supplemented antithymocyte globulin administration into new-onset T1DM patients reduced insulin requirements. However, this approach was abandoned due to the thrombocytopenia that the procedure was generating [105]. Since then, antithymocyte globulin has been reconsidered and, in new-onset patients, carefully adjusted dosing slowed C-peptide decline [106]. Antithymocyte globulin binds to the CD3 complex on T cells as one of its many targets and causes significantly greater T-cell depletion. Thus, its justification is hampered by significantly greater adverse events and immunosuppression compared to anti-CD3 antibodies.

The initial excitement generated over insulin administration was tempered by the outcome of a major clinical study which showed few, if any, beneficial outcomes [87]. More recent clinical studies using monoclonal antibodies targeting CD3 and CD20 have also been disappointing despite the convincing and robust successes in preclinical studies [65, 89] (http://www.rttnews.com/Content/BreakingNews.aspx?Id=1451366&SM=1&SimRec=1 and http://www.bizjournals.com/washington/quick_news/2010/10/macrogenics-lilly-abandon-diabetes-drug.html).

Long before confirmation in genetic models, insulin and proinsulin were strong contenders as the T1DM-initiating autoantigens [107–114]. As autoantigens, many proposed that the exposure of the T1DM immune system to exogenous insulin in a manner that could modulate immunity towards insulin-specific tolerance could prevent disease in prediabetic states and perhaps delay the progression to overt clinical hyperglycemia in more advanced, but subclinical, states [115–118]. Persistent oral insulin treatment of NOD mice delayed T1DM onset and reduced disease incidence in NOD mice [119]. The addition of adjuvants provided a practical benefit in reducing the amount of exogenous insulin required to achieve the beneficial effects [120]. Largely based on the data supporting the view that mucosal delivery of soluble peptides promotes tolerance to them, a number of efforts targeted, in addition to oral, intranasal insulin or insulin-derived peptide, aerosolisation into prediabetic NOD mice. In these studies, diabetes was significantly delayed [71, 117, 121, 122]. These very promising studies with what essentially was a simple intervention led to the DPT-1 multicenter trial which determined the efficacy of oral insulin in first- and second-degree relatives of T1DM patients deemed to fall into high-risk status based on metabolic, immune, and genetic evidence [73, 123]. Other than a possible benefit in individuals with the highest autoantibody titers, the DPT-1 study failed to delay or prevent T1DM. A similarly disappointing outcome resulted in another prevention trial where insulin was administered intranasally Näntö-Salonen et al, [124]. However, a very small subgroup of autoantibody-positive patients was identified in which some effect was shown. The general view of nonpharmacologic insulin therapy is that, if beneficial, it probably will be restricted to very well-defined and characterised subpopulations of patients. It is unclear what can distinguish these patients from a general at-risk population.

A number of other trials were initiated since then without any significant benefits. The IMDIAB trial showed that oral insulin provided no benefit over placebo after a one-year followup in terms of mean C-peptide secretion and insulin requirements. The ORALE trial, comparing low- with high-dose oral insulin (2.5mg/day versus 7.5mg/day), after one year could not discern any benefit in decelerating the loss of physiologic beta cell function [125]. It was recently shown that combined intranasal insulin with CD3 antibody was able to reverse new-onset T1DM in NOD mice, although the effect was quite likely due to the CD3 antibody and the characteristics of the NOD mouse cohorts and not due to the insulin [126].

The most recent attempts at using insulin to improve functional beta cell mass involve intramuscular injection of human insulin B chain in incomplete Freund's adjuvant as well as subcutaneous injection of a DNA plasmid vector encoding proinsulin see [127] and http://www.bayhilltherapeutics.com/. The outcomes of these proposals rest on the publication of phase II studies which are currently at various stages of implementation.

As strong an argument for GAD can be made in the etiopathogenesis of T1DM as for insulin. The 65KDa isoform of GAD has been demonstrated to be a target of early-insulitic T cells in vitro and in vivo [128, 129]. Administration of GAD into very young NOD mice suppresses anti-GAD T-cell reactivity as well as disease onset [129, 130]. GAD is equally effective when administered into older NOD mice [130]. The relevance of GAD to human T1DM relies on the presence of GAD autoantibodies in prediabetic humans and is one of three reliable markers of susceptibility [131].

Two clinical trials have used human GAD65 with alum as adjuvant to determine improvement of physiologic beta cell function. One was conducted in LADA patients [132] and determined that this formulation increased fasting and stimulated C-peptide at 24 weeks compared to baseline, a benefit that was associated with an increase of CD4+ CD25+ Tregs. The other study considered the effects of GAD65 in alum in recent-onset T1DM individuals (10–18 years of age) on the rate of decline of stimulated C-peptide [133]. The study revealed a slower rate of decline of stimulated C-peptide in GAD-treated diabetics compared to the placebo group.

Early studies showed that intrathymic administration of Hsp60-derived peptides was prophylactic in NOD mice even though Hsp60 was not considered a bona fide autoantigen involved in T1DM initiation [134]. More experiments in NOD mice revealed that the p277 peptide of Hsp60 was quite effective in suppressing disease progression [135]. These data compelled the development of a human equivalent of p277 (DiaPep277; a 24 amino acid synthetic peptide derived from the C terminus of human hsp60).

DiaPep277 has been evaluated in phase II studies [136], and data demonstrate some degree of preservation of stimulated C-peptide secretion.

Since then, three other studies have been conducted in new-onset T1DM patients [137–139]. In two of these studies, adult patients were the study population, and these recipients exhibited better, yet limited, C-peptide production than placebo-treated individuals. In contrast, DiaPep277 did not offer any objective benefits in young new-onset patients [137].

T-cell depletion targeting the CD3 complex with the OKT3 monoclonal antibody was successfully employed about two decades ago [91]. Administration of the antibody into diabetic NOD mice resulted in complete remission of disease [91, 92]. One caveat of this particular antibody was that, under some situations, it could activate T cells and therefore it was not considered suitable for human use. Soon thereafter, FcR nonbinding embodiments were manufactured which exhibited either IgG1 Fc chain or which eliminated glycosylation sites of the original OKT3 clone. These variants were found to be less activating than OKT3 [140, 141]. Herold and colleagues initially demonstrated that a short-term administration of the nonglycosylated variant into new-onset T1DM patients suppressed the loss of beta cell function as measured indirectly by surrogate physiologic markers [90, 142]. Some patients maintained improved physiologic markers of beta cell function for as much as two years following the treatment in the absence of systemic immunosuppression. A second round of anti-CD3 antibody injections was postulated as necessary to maintain the beneficial outcome. However, additional injections of the antibody were shown to confer more serious, previously undocumented clinical complications.

T1DM is unquestionably a T-cell-mediated disease; however, other cell populations have been implicated in the onset, and early progression, of the disease, including B-lymphocytes. Accumulated findings in NOD mice demonstrated that B-cell depletion could be beneficial [143–145], even some studies suggested the contrary [146] where adoptive transfer of T1DM into NOD-SCID mice was possible in the absence of B cells and antibodies. B cells can also participate in disease exacerbation by promoting epitope spreading as shown by Tian and colleagues [147]. Indeed, capture of autoantigens such as GAD65 by B-cell surface immunoglobulin (Ig) is followed by processing and presentation of T-cell determinants by B cells, a step that is crucial for activation of autoreactive T cells and induction of diabetes [148–150]. B-cell-mediated processing of self-Ags may contribute to the generation of an inflammatory microenvironment in the pancreas, which is critical for overcoming the regulatory barrier(s) to initiation of diabetes in NOD mice [151]. Antigen-specific B cells and their antibodies are essential in catalyzing determinant-spreading reaction via (a) generation of novel previously cryptic epitopes through altered antigen processing or (b) facilitation of T-cell activation through generation of ligands with higher affinity for TCR and delivery of costimulatory signals. Ag processing through surface receptor-mediated internalization, for example, Fc receptors and soluble Ig, is different from that occurring through pinocytosis and phagocytosis [152].

Pescovitz and colleagues demonstrated a small but statistically significant improvement in physiologic markers of beta cell function in new-onset patients [65]. Nevertheless, the improvement was transient as placebo and rituximab-treated patients eventually (by two years) exhibited an identical decline in beta cell function. One possible mechanism could involve the restriction of epitope spreading by B cells after rituximab-mediated deletion, or the repopulation by B-cells that process and present epitopes that compete with diabetes-relevant epitopes.

Autoreactive T cells have been frequently associated with the production of TH1-type proinflammatory cytokines. It is reasonable, therefore, to use cytokine blockade as a unique or part of a combination therapeutic strategy. IL-1beta, TNFalpha, and type 1 interferons have been historically the first to exhibit direct beta cell cytotoxicity [153–155].

Recently, administration of etanercept, a soluble TNFalpha receptor, in an early-phase efficacy trial resulted in a reduction in the loss of C-peptide in T1DM patients [156]. It is important to note, however, that TNFalpha blockade may have unwanted outcomes; TNFalpha can prevent diabetes in older NOD mice even when splenocytes from diabetic NOD mice are adoptively transferred [157]. Furthermore, TNFalpha blockade may require careful consideration of the age of the individuals being treated since blockade in younger NOD mice prevented disease, while in older NOD mice it accelerated T1DM.

Cell therapy, especially with dendritic cells, is a clinical reality that has also recently been accepted by health providers (http://www.provenge.com/). Overwhelming studies indicate that downregulated costimulation capacity in DC can subserve a tolerogenic therapeutic outcome [169–175]. Costimulation-impaired (or downregulated), functionally-immature DC administration achieves long term and stable allograft survival in a variety of mouse and rat models and prevents a number of autoimmune diseases [176–183]. Mechanistically, functionally immature DCs. act by inducing anergy either via direct cell contact and/or cytokines [172, 184, 185] or, as described more recently, by upregulating the number and function of immune cell subsets, especially the regulatory populations which include Foxp3+ CD25+ CD4+ T cells and a class of CD8+ immunosuppressive T cells [186–194].

Table 1 lists the models where immature DC administration has proven successful. In addition to anergy, Foxp3+ CD25+ CD4+ T-cell numbers have been observed to be increased by the exogenous supply of tolerogenic DC [195–199]. Immature or semimature DC can induce the differentiation of naturally-occurring (thymic) T regulatory cells (CD4+ CD25+) as well as IL-10-producing CD4+ T cells in vitro and in vivo [195, 198]. Overexpression of the Jagged-1 gene in DC results in the augmentation of antigen-specific TGFbeta-producing CD4+ CD25+ CD4+ T-cells [200]. Functionally immature DCs convert naive T cells into IL-10-secreting cells in vitro, and antigen-pulsed DCs injected subcutaneously into humans augment the number of IL-10-producing CD8+ T cells and trigger a reduction in IFNgamma+ T cells [201, 202]. Despite the abundance of data in support of immature DCs as functional inducers of regulatory immune cells, other investigators have discovered that the state of maturity may not be relevant for the induction of immunosuppressive endogenous T cells. For example, similar to the studies by Morel and colleagues [2], mature DCs were shown to expand CD4+ CD25+ CD4+ Foxp3+ T cells which were functionally suppressive and capable of preventing diabetes in the NOD mouse [203–205]. Whether this reflects a restimulation of existing CD4+ CD25+ T cells or their expansion is unknown. We have shown that DCs maintained in a functionally immature state following in vitro treatment with NF-kappaB decoys and antisense oligodeoxyribonucleotides (AS-ODNs) to the CD40, CD80, and CD86 costimulatory molecules are diabetes preventive in the NOD mouse [6, 8, 206] and may involve short-range IL-7 signaling, quite likely inside the pancreatic lymph node of NOD mice, to augment the number of CD4+ CD25+ Treg via suppression of apoptosis of a preexisting pool [206]. Multiple injections of AS-ODN-treated DCs in NOD mice maintained the animals as diabetes free without affecting the overall T-cell activity against alloantigens. Furthermore, repeated administrations of co-stimulation-deficient DC reverses hyperglycemia in new-onset diabetes NOD mice. The promising results from this study, together with the low risk of the procedure, helped in gaining approval by the Food and Drug Administration (FDA) office for a phase I clinical trial which has recently ended with very interesting outcomes, the most important being the complete absence of any adverse events. Adult (18 years or older) volunteers with a documented evidence of insulin-requiring T1D of at least 5 years duration were enrolled. Leukocytes of the patients were obtained by apheresis, and DCs were generated in vitro and engineered in GMP facilities with the addition of AS-ODN. These DCs expressed low levels of CD80, CD86, and CD40 and were then injected into the patient by intradermal/subcutaneous administration at an anatomical site proximal to the pancreas. The choice of this anatomic region for DC delivery was based on the location of the lymphatic conduits (microvessels) that drain the injection site and favor migration to the pancreatic and para-aortic lymph nodes. We conjecture, based on previous rodent studies, that once inside the pancreatic lymph nodes, the injected DCs will acquire molecules that have drained from the pancreas. These molecules can be acquired by passive drainage or can be transferred to the exogenously supplied DC by antigen-presenting cells coming into the pancreatic lymph nodes from apoptotic islets. Therefore, either by direct uptake of “naked” molecules or antigen transfer by migratory antigen-presenting cells, the exogenously administered DC will acquire the antigen specificity. Alternatively, our administered DC could be recruited from the lymph nodes draining the injection site to the apoptotic islets where they could indirectly deliver an anergizing signal to the T cells they encounter. They can also induce regulatory immune cells. The recent concept of contrasuppression, or alternatively, aggregational suppression (network of intercommunicating DC:Treg) could be operative in our system [207–211]. The vicious cycle that promotes the T cell-mediated anti-beta-cell-antigen-spreading phenomenon will be interrupted this way, enabling the recovery of the physiologic endocrine function of the gland with time. The abrogation of the autoimmune diabetogenic insult should be sufficient to promote rescue or regeneration of the insulin-producing beta cells in the host endocrine pancreas, even after the onset of the disease. Currently, there are no methods to directly test this mechanism in vivo in humans; however, mouse and nonhuman primate models offer a number of opportunities. Table 1 lists the many methods to generate tolerogenic DCs. Some methods converge upon identical cellular and molecular pathways (augmentation of Treg numbers, NF-kappaB inhibition, costimulation blockade). Others are not so obvious. A cautionary detail needs insertion herein: it is important to understand that transplantation immunobiology may not be identical to autoimmunity and vice versa. Indeed, there are numerous instances where a therapeutic regimen achieving long-term allograft survival has failed to abrogate autoimmunity [212–219]. Therefore, DCs that are able to suppress donor-specific antigen alloreactivity may not evoke the appropriate regulatory mechanisms capable of controlling and reversing autoimmunity. Also, although different DC products may exhibit similar cell surface phenotypes and nuclear proteome signatures, by no means can this be a predictor of mechanism of action. Cell viability has rarely been examined in these DC products, and not all potential mechanisms of action were considered. It is possible that all listed DC embodiments may intersect at immune regulatory cell levels, but this remains to be determined. The site of action of many DC products, exogenously administered, is currently unknown. While it is generally believed that the DC activity will occur inside the pancreatic lymph nodes, there are equally likely possibilities that the activity of immune tolerance may in fact occur extralymphatically and around the islets, or inside many lymph nodes, anatomically distant from the pancreas. The study by Ludewig and colleagues demonstrating a nascent peri-islet lymphoid ultrastructure during diabetes onset is very interesting and instructive and awaits the discovery of a mechanism [220].

Table 1 highlights the studies where animals were treated with tolerogenic DCs pulsed with putative autoantigens. Although the ongoing insulitis drives migration of exogenously administered DCs into the islets, where they acquire the antigen specificity to provide beta cell antigens (from apoptotic/necrotic beta cells) to pancreatic lymph-node-resident regulatory immune cells like Foxp3+ CD25+ CD4+ T cells, it is currently unknown if ex vivo pulsing with putative autoantigen(s) could stabilize class I MHC to yield more specific Treg in vivo. Our data, however, indicate that the supply of putative autoantigens may not be necessary given the endogenous supply acquired by migratory exogenous DC inside the insulitic lesion. Fathman and colleagues have shown that exogenous DC administration results in preferential accumulation of the DC inside the pancreatic lymph nodes and the omentum [3, 221, 222]. The critical factor in any tissue-targeted approach will be to maintain the DCs in a functionally immature state during and after their accumulation inside that tissue [6, 8, 178, 180, 206].

That our CD40-, CD80-, and CD86-impaired DCs exhibited exemplary safety motivates us to proceed with phase II efficacy trials in new-onset T1DM patients with the objective of decelerating the rate of decline of residual beta cell mass, improving glucose profiles, and perhaps reducting insulin requirements. We anticipate beginning such a trial in the next 12–18 months.

T regulatory cells (Tregs) are another potential cell population that can be used for suppressing the autoimmunity of T1DM. A variety of methods have been employed to expand natural Tregs and to differentiate induced Tregs from peripheral blood progenitors. These cells have been shown to control ongoing autoimmunity and promote diabetes reversal in NOD mice [5, 223–225]. It is unclear whether antigen specificity is relevant in this approach because both antigen-specific and nonspecific expanded Tregs are equally efficacious in suppressing T1DM. Furthermore, Tregs recognising one antigen can have a broad effect on suppression effectors reactive to other antigens [5, 205]. Clinical protocols for T-cell administration into humans exist and patient-specific polyclonal Tregs can, under current conditions, be employed in at least phase I studies.

A particularly interesting discovery very recently indicating that aryl-hydrocarbon receptor-activated CD4+ T cells preferentially differentiate into stably suppressive Foxp3 Tregs, offers therapeutic opportunities that can be exploited by oral route of drug administration [226, 227]. If safe, orally stable aryl hydrocarbon receptor agonists are developed, one could envisage a situation where mucosal CD4+ T-cells could be induced to differentiate into stably suppressive Foxp3+ Tregs. This could be achieved with chemicals or small molecules alone or formulated into nanodelivery particles.

Reports from recently completed phase III trials using one embodiment of the humanised CD3 antibody were not encouraging. The underlying reasons for why the primary end-points were not achieved, even previous studies were demonstrated to be successful, yet limited in time, are not at all clear at present but could involve one or more of the following: (i) variation among the T1DM patients in actual residual beta cell mass, in that the outcome reflects the enrollment of individuals with a more aggressive autoimmunity and hence poorer residual beta cell mass and (ii) the possibility that the time of administration varied between this phase III and previous trials; it is well known that administration of immunomodulatory agents during different times of the day as well as during different points of biologic cycles can profoundly affect the immune response [234–237].

In addition to the anti-CD3 antibody, there are other monoclonal antibodies targeting cytokines as well as fusion proteins interfering with costimulation and serving the role of cytokine decoys. There again, the question is, do these agents represent a promise of a huge leap, an incremental advance, or more of the same? The question is indeed speculative as trials involving some of these agents are ongoing or have not yet started. However, the antibody-based trials are instructive. First, these biologic agents confer variable adverse events. Despite the arguments that these agents can represent antigen-specific approaches, they do not. At best, they promote the homeostatic expansion of new immune cell repertoires that, at various stages of autoimmunity, can act beneficially or can aggravate disease. Recent data indicate that antithymocyte globulin depletion of T cells promotes the homeostatic expansion of T cells with a temporally favorable expansion of naturally occurring (thymic) Foxp3+ Tregs [238]. This observation can guide a better approach to promoting homeostatic expansion of Tregs without considerable T-cell depletion and may be instructive for therapies like the anti-CD3 antibody. This phenomenon also appears possible following B-cell depletion with rituximab (i.e., homeostatic expansion of naive B-cells with favorable expansion of B cells with possible regulatory activity [239]). Thus, if consideration is given to understand the timing of repopulation of the periphery with regulatory cells following T-cell or B-cell depletion, more of the same could yield huge leaps in terms of therapeutic success in new onset T1DM.

Another consideration that has been discussed but seldom implemented experimentally is the route of administration of autoantigens and derivative peptides with or without adjuvants or immunomodulators/anti-inflammatory agents. Accumulating evidence suggests that the pancreatic lymph nodes are the major, if not the most important, site of autoimmunity triggering and progression [240–243]. Indeed, regulatory immune cells have been identified to expand and to differentiate from precursors inside draining lymph nodes of tissues that are targets of autoimmunity [207, 244–247]. These observations should be instructive in how to approach ongoing autoimmunity with the aim of inducing antigen-specific suppression. As most, if not all, of pancreatic beta cell antigens are concentrated inside the pancreatic lymph nodes by fluid dynamic flow or are brought inside the pancreatic lymph nodes by antigen-presenting cells, they would drive the acquisition of antigen specificity by immune cells modulated inside the same lymph nodes even by an antigen-nonspecific approach. For example, one could administer costimulation-blocking agents in a manner where they accumulate preferentially inside the pancreatic lymph nodes and allow the bulk flow, or the cross-presentation of beta-cell-derived antigens by antigen-presenting cells that have traveled along islet-draining lymphatics, to direct the antigen specificity in conferring antigen-specific immune hyporesponsiveness that is known to occur in response to costimulation blockade.

With the disappointing outcomes of the DPT-1 and related insulin-based trials as well as the failure of industry-sponsored phase III trials with one embodiment of the anti-CD3 antibody, one could reasonably question the value in pursuing further immunodepletion interventions as well as autoantigen-alone trials. There is merit in the argument that the lack of biomarkers to more precisely measure residual beta cell mass that is functional imperils these studies. There is also merit in the arguments that the time of administration of the agents in the phase of the disease, even in new-onset-cases, is critical and needs a comprehensive evaluation. We argue that more of the same cannot go on without such biomarkers and a better understanding of the balance of immunoregulatory cells between the time of clinical onset and 6–12 months from this time in T1DM patients. The variability in immunoregulatory cell numbers and their function has to be securely identified in large populations. In the meantime, many of the resources of research support have been skewed heavily in these approaches with trepidation of supporting cell therapies that are otherwise safe. We propose that cell therapies be seriously considered for at least phase I studies. Furthermore, a new avenue of studies has emerged which attempts to promote accumulation of immunoregulatory cells inside the pancreatic lymph nodes. A better understanding of the pancreatic lymphatics, we believe, will be important to develop these approaches.