T cell immunity to islets of Langerhans : relevance for
immunotherapy and transplantation to cure type 1 diabetes
Huurman, V.A.L.
Citation
Huurman, V. A. L. (2009, March 4). T cell immunity to islets of Langerhans : relevance for immunotherapy and transplantation to cure type 1 diabetes.
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General discussion
NOVEL THERAPEUTIC APPROACHES FOR TYPE 1 DIABETES
T cells play a central role in the development of type 1 diabetes (T1D) and therefore should be considered the foremost target for disease intervention1,2. Their additional role in al- lograft rejection emphasizes the detrimental influence they may exert in β-cell replacement therapy3. This thesis underscores the importance of T cells and provides leads that may im- prove guidance and outcome of immunotherapeutic and transplantation strategies aiming to cure T1D.
All known genes associating with development of T1D encode for different and indepen- dent aspects of the T cell-driven immunological process that eventually causes autoimmune β-cell destruction or the regulation thereof. Many non-specific types of immunosuppression have been tested to cure human T1D, but they have not shown long-term benefit4. More recently, blockade of T cell activation pathways such as CD3 or CD255-7 has gained inter- est, as well as enhancement of inhibitory costimulatory pathways as ICOS and CTLA4 (also described in Chapter 2 of this thesis)8-11.
These immunotherapies are aimed at inhibiting a pro-inflammatory, antigen-specific memory T cell response or prevention of T-cell priming, respectively. Other strategies, such as peptide immunization, target islet-specific autoreactivity by utilizing the regulatory potential of auto-antigen-specific T cells rather than inhibiting their destructive potential (Chapter 3). The rationale for this is that exposure of T1D-specific peptide to the immune system will lead to activation of a T cell subset with homeostatic or regulatory properties12,13, analogous to the blood transfusion effect in transplantation14.
However, immunotherapy is currently only possible after diagnosis, when most β-cells are already destroyed. Establishment of biomarkers identifying autoimmune destruction of β-cells in an earlier stage may therefore improve therapeutic potential. Replacement of β-cells, by either pancreas or islet cell transplantation, is less hampered by this limitation.
However, shortage of organ donors is the most important among multiple factors limiting widespread application of allotransplantation.
Pancreas transplantation can be considered the most successful β-cell replacement therapy today15. Transplantation procedures and immunosuppressive regimens have greatly im- proved over time, leading to a well controlled and largely successful treatment for a subgroup of T1D patients. Its clinical outcome is presently superior to that of islet cell transplantation, a more novel and experimental procedure in which a smaller graft is transplanted in a less in- vasive manner. Although such conditions seem preferable, the factors influencing islet graft survival are not yet clearly understood. These conceivably relate to either the transplanted islets, the recipients’ immune system or the immunosuppressive regimen16. Previously, it was shown that the amount of transplanted β-cells (rather than the number of islet equivalents) influences outcome17: size matters indeed. The standardized preparation of β-cell grafts in Brussels enabled study of additional variables such as immune factors, which may affect
204 Chapter 8
transplantation outcome. These include recurrent cytotoxic and pro-inflammatory T cell autoimmunity (Pinkse et al.18, Chapter 4) and anti-HLA antibodies19. Alloimmune cytotoxic- ity can be important if immunosuppression is inadequate while alloimmune T helper cell reactivity is related to outcome as well (Chapter 5 and 6). Adaptation of these responses may lead to increased graft survival. Moreover, β-cell survival will also need improvement if the donor shortage issue can be solved by using β-cells from novel sources (e.g. differentiated from pancreatic duct or embryonic stem cells, or xenogeneic porcine β-cells).
PREVENTION VERSUS INTERVENTION
The data presented in this thesis suggest that intervention in a pre-existing, memory T cell response is different from and more difficult than the prevention of a naïve immune response. CTLA4Ig, which can effectively prevent naïve responses, was less effective for intervention in pre-existent autoimmunity in vitro (Chapter 2). This may be due to secondary activation signals being less dependent on costimulatory molecules, or because costimula- tion is redundant in pre-committed T cell responses. Such redundancy has been described in the literature20-22, but has been overshadowed by the general dogma that costimulation is a necessity for T cell activation.
Patients transplanted with either whole pancreas or isolated β-cells will have to overcome both development of allograft rejection and recurrence of pre-existent T1D-specific autoim- munity. Recurrent autoimmunity is observed rarely after pancreas transplantation23,24 but more often after transplantation of isolated β-cells18,25,26. The potential of CTLA4Ig in this context needs to be assessed more extensively, but our results point to limited suitability in inhibiting memory T-cell reactivity.
In a cohort of standardized islet graft recipients that we analyzed with regard to immune- related factors, both pre-existent and post-transplantation T cell autoimmunity (proliferation in response to diabetes-specific antigens GAD and/or IA-2) correlated significantly and inde- pendently with worse transplantation outcome (Chapter 4). A relation with the significantly higher overall B- and T-cell counts observed in these patients seems apparent. In our cohort transplanted under ATG-tacrolimus-MMF immunosuppression (the ‘Brussels’ protocol), we cannot confirm earlier claims27 that associate production of auto-antibodies with outcome, This underscores the notion that T cells dominate T1D pathogenesis, while auto-antibodies may reflect epiphenomena.
With regard to allograft rejection, the presence of CD8+ alloreactivity was associated with impaired outcome in patients transplanted under sirolimus-based immunosuppression, but surprisingly not under ATG-tacrolimus-MMF (Chapter 5). The earlier established associations of pre-transplant autoreactivity and graft size with outcome were confirmed in a combined
cohort with three different immunosuppressive regimens, implying that immunosuppression did not affect the importance of these factors. Clearly, novel regimens are needed that are able to deal with autoreactive T cell responses in islet cell transplantation more adequately.
The impact of pre-existing autoimmune T cells is comparable that of pre-sensitization with anti-HLA allo-antibodies (or a positive serum crossmatch): both act as absolute contraindica- tions for transplantation19. No doctor will waste scarce grafts in transplantations destined to fail, or subject patients to the risks associated with such a procedure. However, T-cell autoim- munity need not be detrimental, since auto-antigen-specific therapeutic immunization leads to tolerogenic autoimmune T cell responses associated with preservation of remaining β-cell function in recent-onset T1D patients, as demonstrated in Chapter 3.
IMPACT OF IMMUNOSUPPRESSION
Alloreactive CTLs could be successfully neutralized after transplantation under the ‘Brussels’
protocol. However, this was not the case under alternative immunosuppressive regimens using sirolimus. Additionally, the type of T helper alloreactivity was shown to correlate with transplantation outcome (Chapter 5): where allograft specific IL10 production improved clinical prognosis, proliferative responses correlated with worse outcome. Alloimmunity may also become important when successful immunosuppression is tapered. In the Brussels islet transplantation program patients are tapered starting one year after transplantation because immunosuppressive side effects are considered a burden to islet recipients that may exceed the disadvantages of life-long insulin therapy. Especially in insulin independent recipients, a state of tolerance (i.e. homeostasis between pro- and anti-inflammatory immune factors preserving the graft) may be achieved. Such homeostasis has been described in both kidney and liver transplant recipients with stable graft function without a need for immunosup- pression28,29. In a pilot study on the effect of tapering on immunity and islet graft function, some patients showed an increase in autoreactive T cell responses, others in alloreactive responses or in both (Chapter 6). The percentage of highly avid alloreactive CTLs increased significantly as the immunosuppressive load decreased. The changes in auto- and alloim- munity were largely responsible for the generally observed decrease in graft function. Under full immunosuppression autoreactive responses were at least partly inhibited, judging from their increase upon tapering in some cases. No evidence for long-term graft acceptance after islet transplantation was observed, but this pilot study suggested that tapering of immuno- suppression may be guided by follow-up of immune factors. As this concerns pilot studies performed on patients with diverse graft function and immune reactivity, it is not yet clear whether immunological monitoring will enable guided tapering of immunosuppression in the future. Still, our results underscore the value of measuring immunological endpoints as correlates of efficacy of immune suppression and/or preservation of β-cell function.
206 Chapter 8
Negative effects of non-specific immunosuppression may include impaired engraftment, infectious complications and impaired development of allo-specific regulatory T cells. The extent to which this happens conceivably depends on the type of immunosuppression and the type of immune response. ATG, a well established, broad immunosuppressive agent targeting all T cells, led to earlier and more severe cytomegalovirus (CMV) infections after pancreas transplantation than anti-CD25, which selectively targets activated T cells including the autoreactive (Chapter 7). These infections correlated with significantly lower amounts of circulating CMV-specific memory T cells. A subsequent study showed that other memory T cells (such as anti-influenza) were also impaired by ATG30. Although no direct effects on clinical outcome or autoreactivity were noted, these studies imply careful and possibly selec- tive application of immunosuppression to prevent infectious complications. The fact that immunosuppression may also impair regulatory T cell function in auto- and alloimmunity complicates its use as immunotherapy31-33.
BIOMARKERS FOR THERAPEUTIC SUCCESS OR FAILURE
Biomarkers are essential to evaluate treatment effect of novel T1D therapies that influence immune factors. Many markers for auto- or alloimmune tolerance have been suggested, but until now their suitability for the clinic has remained limited, a major challenge and critical gap to be bridged34. In this thesis, many of such markers were analyzed, enabling us for the first time to define immune correlates of efficacy and safety of immune intervention therapy upon diagnosis of T1D, as well as in the context of islet and pancreas transplantation in type 1 diabetic recipients.
In islet cell transplantation, pre-existent CD4+ T cell autoimmunity proved to be a marker predicting graft failure (Chapter 4). Previously, this was observed for CD8+ autoimmune CTLs after islet transplantation as well, concurrent with chronic loss in islet graft function presumably due to recurrent islet autoimmunity18. The importance of T cells for outcome was further proven in an extended patient cohort that identified lymphocyte count before transplantation as a simple measure discerning successful from non-successful transplanta- tions. Regarding alloimmunity, the impact of CD8+ CTLs remained limited to situations where immunosuppression included sirolimus. The avidity of CTLs (increasing during tapering of immunosuppression, Chapter 6) was a possible indicator of their influence, as shown earlier in kidney transplantation35.
Under adequate immunosuppression, the role of regulatory CD4+ T cells appeared more important. Graft-specific cytokine profiles were skewed towards Th2 or T regulatory pheno- type in patients becoming independent from exogenous insulin, whereas recipients still re- quiring insulin injection elicited either unchanged or Th1-dominated alloimmune responses (Chapter 5). Production of the inhibitory cytokine IL10 in particular was related to low graft-
specific T cell proliferation and mainly observed in patients reaching insulin independence.
Importantly, while earlier studies only identified markers of graft failure, IL10 proved to be a marker for graft-specific tolerance and preserved allograft function and survival. Interest- ingly, IL10 production by alloreactive CD4+ T cells was also increased after in vitro addition of tacrolimus (Chapter 6). IL10-producing cells have been extensively studied in vitro and have been attributed immunoregulatory properties31. However, more specific study of these cells in vivo addressing their relation with autoimmunity or graft function is warranted.
While IL10 proved to be informative after islet transplantation, it predicted good metabolic outcome beforehand in new-onset T1D patients in the DiaPep277 therapy immunization trial, confirming its potential role as a favourable immunological endpoint. Yet, induction of IL10 production upon DiaPep277 therapy was not associated with β-cell preservation, since all treated patients developed IL10 production regardless of clinical efficacy.
Conversely, where proliferative autoimmunity proved most important before islet trans- plantation, and to a lesser extent afterwards, proliferative autoimmunity to p277 treatment proved informative after therapy, but not before. This apparently reciprocal position of IL10 vs. CD4+ autoimmunity (Table 1) indicates that informative immunological endpoints may differ depending on the type of intervention (e.g. peptide immunotherapy versus islet cell transplantation). Furthermore, immune correlates associated with preserved β-cell function may differ between the natural history of T1D and the situation during immunotherapy.
Nonetheless, for the first time immune responses are identified that may act as surrogate markers of immunological and clinical efficacy and safety. The robust and informative im- mune biomarkers our studies delivered were previously unmet needs in clinical T1D, im- munotherapy and transplantation. This is an important and clinically valuable development that may facilitate assessment of therapeutic benefit, allowing more rapid translation to clinical practice.
TABLE 1 Importance of IL10 and CD4+ autoimmunity as surrogate markers for clinical efficacy in different treatments for T1D described in this thesis.
Primary immune endpoint of disease activity
Before treatment After treatment
P277 immunotherapy IL10 Cellular autoimmunity
Islet cell transplantation Cellular autoimmunity IL10
FUTURE DIRECTIONS
Application of T1D immunotherapy is presently limited to a stage of the disease where most β-cells are already destroyed. Earlier detection of autoimmunity may lead to earlier applica- tion and increased preservation of β-cells. The results from Chapters 2 and 4 indicate that the potential of any immunotherapy to inhibit committed autoreactivity needs extensive
208 Chapter 8
consideration, especially since prevention and intervention may require distinct strategies.
Tolerance-promoting strategies are of increasing interest as alternatives of the currently employed or evaluated less specific, inhibitory agents. Peptide immunization, as well as DNA vaccination or induction of T regulatory cells by modified, tolerogenic dendritic cells (DC) may improve success and reduce hazard.
The promising results using selective anti-CD3 treatment in recent-onset T1D patients pro- vide a basis for its implementation as induction therapy to counter recurrent autoimmunity in islet cell transplantation. In fact, this intervention strategy is the first to interfere in the pathogenesis of T1D. More rigorous or earlier T cell depleting induction therapy (ATG) may improve conditions for patients with proven pre-transplant lymphocytosis. Outcome would further benefit from a less immunogenic environment for β-cells, probably by injection into an alternative site or the co-injection of cells or agents with immunoregulatory potential.
IL10 is increasingly recognized as a useful biomarker for regulation of both auto- and alloimmune responses. IL10-producing cells need to be identified and their regulatory potential assessed both for specific- and non-specific responses (‘linked suppression’). Other markers that were shown to be important, such as the presence or absence of cellular auto- and alloimmunity in vitro, may be used to expand and confirm earlier analyses and to guide selection of islet and pancreas recipients. Rational use of these immunological tools could eventually facilitate a situation where each individual patient can receive tailored pre- intervention conditioning, maximizing the chance for success for both immunotherapeutic and transplantation-based T1D therapy.
CONCLUDING REMARKS
In the Introduction I proposed that ‘any possible new treatment for T1D will have to fulfil a number of criteria: it must be safe, (cost-) effective, sufficiently available and ultimately less of a burden than intensive insulin treatment’. Immunotherapies as described in Chapter 2 and 3 are readily available, require short-term application only and seem safe. However, their efficacy in the context of established autoimmunity remains questionable. This thesis points to some explanations for this and offers opportunities to improve efficacy that require further assessment in the future. Transplantation-based therapies as studied in Chapters 4 to 7 seem effective, but carry safety risks and suffer from limited availability of donor organs.
Additionally, although most islet transplant recipients benefit tremendously from decreas- ing serum glucose variation and subsequent decrease in hypo- and hyperglycemic episodes, long-term outcome of islet transplantation still needs major improvement. This thesis dem- onstrates unambiguously that the detrimental effect of pre-existent T cell autoimmunity must be dealt with, even if β-cells from novel sources may come of use as insulin-producing grafts in the future.
The results from this thesis therefore endorse a revised view on T1D therapy, which recog- nizes T cell autoimmunity as a separate entity distinct from other types of immune reactivity.
Novel therapeutic agents need to be implemented that are able to specifically intervene in this pre-existent immune state, with acceptable side effects. Eventually, tailored therapy for individual patients guided by close immune monitoring may become possible. This thesis provided suitable tools and compelling evidence for the feasibility of immune monitoring to define causes of failure and correlates of success, helping to revise and improve immune sup- pressive therapy at diagnosis of T1D or after β-cell replacement. Such major improvements are indispensable, because only when autoimmune T cells can be satisfactorily countered, a definitive cure for T1D comes in sight.
210 Chapter 8
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