Recurrence of type 1 diabetes after beta-cell replacement

C H A P T E R

62 Recurrence of type 1 diabetes after beta-cell replacement Paolo Monti Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milan, Italy

O U T L I N E Introduction

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The persistence of autoreactive memory T cells and B cells after the onset of T1D

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The presence of autoreactive memory T cells and autoantibodies before islet transplant

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Introduction Memory is a hallmark of adaptive immunity. When the immune system recognizes and responds to an antigenic challenge in the primary response, it acquires also the ability to specifically react to a secondary antigenic challenge with a quick and powerful reaction. This property is acquired by a specific subset of leukocytes named memory T cells and B cells. In the context of autoimmunity, a memory autoreactive T-cell and B-cell response is acquired in patients who developed type 1 diabetes (T1D) and remains for decades after the onset of the disease. If patients are transplanted with a novel source of beta cells, this represents a reexposure of a pre-sensitized host to beta-cell antigens and can cause reactivation of memory T cells and B cells to generate a recurrence of T1D. Seminal were identical twin transplants performed by David Sutherland in which transplant of pancreas segment from an unaffected twin to the twin with longterm T1D in the absence of immune suppression resulted in the loss of graft beta-cell function and insulitis reminiscent of what is seen at diabetes onset.1, 2 Observations

Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 1 https://doi.org/10.1016/B978-0-12-814833-4.00062-9

Autoimmunity recurrence after islet or pancreas transplantation Pancreas transplantation Islet transplantation

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References

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of relapse of autoimmunity as assessed by autoantibodies and occasionally T cells have also been reported following allogeneic pancreas transplant under immune suppression.3 These cases appear to be infrequent, and although suggestive, it is not fully proven whether this has clinical relevance. In the clinical practice, beta-cell replacement is performed with transplantation of allogeneic islets or pancreas. When islets or pancreas are transplanted in patients with T1D, it represents an immunological challenge where both allogeneic rejection and beta-cell-specific autoimmunity coexist, but with the potential for reactivation of autoreactive memory T cells and B cells, posing an additional set of therapeutic obstacles.4 Activation and expansion of T cell under immunosuppression can be driven by pathways that are different from those that drive conventional immune responses. Therefore, autoimmunity recurrence is difficult to control with standard immunosuppression and novel approaches are needed in order to improve the outcome of beta-cell replacement therapies, including replacement therapies with beta cell generated from autologous or allogenic precursors like stem cells.

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© 2020 Elsevier Inc. All rights reserved.

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The persistence of autoreactive memory T cells and B cells after the onset of T1D Proteins such as glutamic acid decarboxylase 65 (GAD65), insulin, islets-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), IA2, and ZnT8 are utilized within islets for normal physiological function, but during the progression of T1D, they become highly antigenic in humans both for T cells and B cells.5 To date, immune markers for beta-cell autoimmunity have primarily centered on the measurement of autoantibodies to beta-cell antigens, and measurement of autoantibodies has been shown to be useful markers to predict and diagnose T1D.6 In contrast, measurements of T-cell responses to beta-cell antigens have been inconsistent in their diabetes specificity. It is now commonly accepted that T-cells reactive to beta-cell antigens can be detected in both healthy patients with T1D but also in subjects with no signs of autoimmunity.7 A crucial difference, however, is that autoreactive T cells in patients with T1D have characteristics indicating prior antigen experience, whereas those in healthy individuals never encountered beta-cell antigens in an immunogenic context and therefore retain a naïve phenotype.8 Early studies showed that T cells isolated from patients with T1D can react with a proliferative response when stimulated with beta-cell antigens in the absence of co-stimulation by CD28 and B7–1.9 The presence of co-stimulatory signals is required for activation of naïve T cells, whereas memory cells can be stimulated to proliferate in the absence of co-stimulation. These data suggested that the autoreactive T-cell repertoire include cells with a memorylike phenotype and T-cell proliferation test in the a­ bsence of co-stimulation by CD28 and B7–1 is one of the ways patients with T1D can be distinguished from healthy controls. Further studies showed that at least a proportion of T cell that proliferates to beta-cell antigens in patients with T1D expressed the memory marker CD45RO.10 The expression of the CD45RA marker identifies naïve T cells. When CD45RO+ memory T cells were isolated from patients with T1D and healthy controls, a positive proliferative response was detected in patients, but not in healthy controls. In contrast in the CD45RO negative fraction, the proliferative response was present both in patients and healthy controls. These data demonstrated that only patients with T1D developed memory T cells reactive to beta-cell antigens, even though all subjects have naïve T cells that can potentially recognize beta-cell antigens. The development of memory T cells responsive to beta-cell antigens in already present before the onset of the disease in subjects that are autoantibody positive and therefore at risk to develop the disease. The development of a memory phenotype involves the activation of a naïve precursor that developed a memory phenotype after extensive proliferation. It is

possible to measure the proliferative history of somatic cells by measuring the length of telomeres. Telomeres are regions of repetitive nucleotide sequences at each end of a chromosome, and they have a pivotal role in protecting the end of the chromosome from deterioration or from fusion with other chromosomes. In human germline cells, telomeres are approximately 20-kb long11 and most cells, including leukocytes, undergo the loss of 50–100 bp at each cell division.12 This is a result of the balance between telomeric DNA loss during cell division and the activity of telomerase, a unique reverse transcriptase that has the ability to extend the 3′ end of telomeres. Consistent with a prior history of proliferation, shorter telomeres were detected in memory T cells that respond to GAD65 and insulin in patients with T1D as compared with the naive counterpart in the same subjects, suggesting a previous history of in vivo proliferation.10 With respect to the recurrence of T1D after transplantation of islets or pancreas, an important question is to determine how the autoreactive memory T-cell response can persist for many years after the onset of the disease. Memory is a hallmark of adaptive immunity and it is well established that memory T cells can persist for years or decades after the first antigen encounter, and in the absence of additional antigenic restimulation. However, the memory compartment is composed of a heterogeneous pool of T cells.13 Different memory T-cell subsets can now be identified according to their phenotype, gene expression, and anatomical localization. The differential expression of CD45RA and CD62L is frequently used to identify CD45RA-CD62L+ central memory (Tcm), CD45RA-CD62L-effector memory (Tem), and CD45RA + CD62L+ effector memory (Temra) T cells.14 Based on the assumption that long-term memory is maintained by long-lived memory T cells, studies have been performed in order to determine the in vivo lifespan of different memory T-cell subsets. In vivo labeling with stable isotopes in combination with appropriate mathematical analysis of these data provides a way to obtain T-cell decay and production rates and to follow the fate of recently produced T cells.15 These data clearly showed that while naïve T cells are characterized by a median lifespan of 4–6 years, memory T cells are characterized by a median lifespan of only 5–8 months. Since conventional memory, T-cell subsets do not preserve the memory response for a long time it has been hypothesized that the known memory subsets can be generated from a rarer precursor with some characteristics of stem cells, such as long life span and self-renewal potential. This precursor was named stem memory T cell (Tscm) and subsequently described to exist in mice,16 nonhuman primates,17 and humans.18 All memory subsets differentiate from a naïve T-cell precursor according to a progressive differentiation model.19 In this model, depending on the strength and quality of

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The presence of autoreactive memory T cells and autoantibodies before islet transplant

stimulatory signals, Tn progress along a differentiation pathway in the order of Tn, Tcm, Tem, and Temra and culminates in the generation of terminally differentiated short-lived effector T cells. In this linear differentiation model, Tscm can be placed between naïve and central memory T cells. Earlier studies showed that Tscm express markers of naïve T cells such as CD45RA, CD62L, and CCR7 but also markers of memory T cells such as CD122 (IL-2 receptor beta chain) and CD95. There is now a consensus that the phenotypic requirements for Tscm are the simultaneous expression of CD45RA, CD62L, and CD95.20 With respect to the functions of the Tscm subset, perhaps the most compelling evidence for Tscm stemness comes from experiments in mice showing the ability of these cells to reconstitute the full diversity of the memory T-cell compartment on serial transplantation.18, 21 Generation of Tscm from naïve T cells involves the homeostatic cytokines such as IL-7 and IL-15. In the classical antigen-specific activation pathway, T-cell receptor engagement and IL-2 provides strong signals for T-cell differentiation toward short-lived effector cells. In contrast, the homeostatic cytokine IL-7 sustain T-cell proliferation without the robust differentiating activity of IL-2. In vitro priming of naïve T cells in the presence of IL-7 results in the generation of T cells with phenotypic, functional, and gene expression attributes found in naturally arising Tscm cells.22 This is relevant to T1D as the IL-7/IL-7 receptor axis was associated with the pathogenesis of beta-cell autoimmunity both in mice and humans.23 Exogenous administration of IL-7 accelerates diabetes onset in the nonobese diabetic (NOD) mouse,24 while blockade of the IL-7R can reverse diabetes in the same model.25, 26 In humans, single-nucleotide polymorphisms of IL-7R alpha were associated with an increased risk for developing T1D.27 However, the most compelling evidence of the role of IL-7 in autoreactive T-cell expansion comes from the clinical setting of transplanting islets into patients with T1D. Lymphopenia-induced by the immunosuppressive regimen was associated with an increase of circulating IL-7 and homeostatic expansion of memory T cells, including autoreactive clones.28 In the context of T1D, the existence of CD8+ Tscm precursors of autoreactive T cells specific for GAD65, insulin and IGRP peptides was recently reported.29 Autoreactive Tscm were present in a patient with T1D at the onset, but also in patients with a long duration of disease candidated to undergo to islet transplantation. Similarly to Tscm studied in other clinical settings, autoreactive Tscm specific for beta-cell antigens showed a superior capacity to generate a clonal progeny of autoreactive T cells and preserve telomeric DNA during expansion. In vitro expansion of autoreactive Tscm generated all the other memory subsets but, as self-renewing cells a fraction of cells after proliferation retained the Tscm phenotype. All these studies suggest that a memory T-cell response is

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generated in patients who develop T1D and the immunity to beta-cell antigens can be maintained probably lifelong by memory subsets with stem cell-like properties. B lymphocytes belong to the humoral arm of the adaptive immune system. Similarly to T cells, they exist as naïve and memory B cells, the latest able to preserve immunity to antigens for several years after the first encounter. B-cell memory is mediated through long-lived memory B cells and sustained antibody titers produced from long-lived plasma cells.30 These cells are predominantly produced in the germinal centers of secondary lymphoid organs, but the precise mechanisms that drive differentiation from naive B cells to memory B cells or long-lived plasma cells are not entirely known.31 In humans, memory B cells were traditionally identified as class-switched immunoglobulin D negative cells and more recently by the expression of CD27.32 However, subsets of B cells that display memory characteristics but do not express CD27 have also been described.32 Long-lived plasma cells are a fundamental component of humoral immunological memory and produce a large amount of antibodies. Long-lived plasma cells can persist for several years and express high levels of the chemokine receptor CXCR4, which regulate their homing to specialized niches in the bone marrow that express at high levels the CXCR4 ligand, CXCL12.33 In the context of T1D, B-cell autoreactivity can be monitored by measuring of autoantibody in the serum of patients. Autoantibodies against (pro)insulin, GAD65, IA-2, and ZnT8, appear before the onset of type I diabetes, and they have a clear and important prognostic and diagnostic value. Autoantibody measurements have undergone an intensive effort for standardization among different laboratories making humoral immune monitoring highly reliable also among different patient cohorts.34 Circulating autoantibodies can persist for a long time after the onset of the disease. An important study measured the persistence of autoantibodies in patients with long duration of T1D (>50 years) and found a high frequency of autoantibody-positive subjects for GAD65 (48.4%), IA2 (5.8%), and ZnT8 (24.6%).35 B-cell immunity and autoantibodies have been considered nonpathogenic in the process of beta-cell destruction that leads to T1D. However, recent studies showed that B-cell depletion can be a therapeutic option to control beta-cell autoimmunity.36

The presence of autoreactive memory T cells and autoantibodies before islet transplant One important aspect of autoimmunity recurrence is the immunological status of patients before they are transplanted with islets or pancreas. The large majority of these studies have been performed in the setting of

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allogeneic islet transplantation in patients with T1D. The aim of these studies was to identify immunological correlates for islet or pancreas transplant survival, including markers of allo-and autoimmunity. A possible implication of these studies is the possibility to select patients with higher or lower chances to develop allo or autoimmunity before transplantation. Alternatively, for patients with higher chances to develop an unwanted immune response to islet or pancreas grafts, there would be also the possibility for immunological pretreatment to reduce allo or autoreactivity posttransplant. Traditionally, immune monitoring of islet or pancreas recipient has been performed by measuring changes in autoantibodies before and after transplant.37 The appearance of circulating autoantibodies may occur very early in life and precedes the clinical onset of the disease by years. Thereafter, autoantibody levels tend to decline and disappear within a few years in some patients, while in others, they persist at detectable levels indefinitely.38 Early studies tried to address whether autoantibodies titers pretransplant can predict the outcome of islet or pancreas transplantation. An important study evaluated the humoral and cellular immunity against auto- and alloantigens before and during 1 year after transplantation in 21 patients undergoing to islet allotransplantation.39 Humoral reactivity was measured by auto- and alloantibodies. The autoreactive T-cell response was evaluated by proliferation based lymphocyte stimulation tests against autoantigens GAD65 and IA-2. The alloreactive T-cell response was measured by cytotoxic T lymphocyte precursor assay. A multivariate analysis was performed in order to identify immunological determinants of clinical outcome. The study showed that the presence of cellular autoimmunity before and after transplantation is associated with delayed insulin-independence and lower circulating C-peptide levels during the first year after transplantation. On the other hand, seven out of eight patients without preexistent T-cell autoreactivity became insulin independent, vs none of the four patients reactive to both islet autoantigens GAD and IA-2 before transplantation. Autoantibody levels and cellular alloreactivity had no significant association with the outcome of islet allotransplantation. A subsequent study from the same research group focused their research on the correlation between baseline autoreactivity and the outcome of islet allotransplantation in a cohort of 30 patients.39 Baseline patient characteristics were compared with outcome parameters, including circulating C-peptide, the variability of glycemia, and achievement and duration of insulin independence, during the first 6 months posttransplant. They found a significant association between a high T-cell autoreactivity pretransplant and a lower graft function. Interestingly, the study also found an association between higher total and B-cell counts with a poorer transplantation outcome. The authors also had

the chance to study a liver biopsy from a patient who received two intraportal islet infusions. They identified in the portal tract two insulin-positive islets that were surrounded by CD3+ T cells and a dominant population of CD20+ B cells a high frequency of circulating B cells. This patient did not achieve insulin independence and the circulating c-peptide was low (0.62 ng/mL). A second liver biopsy was obtained from a patient with a low number of circulating B cells, showing islets in the liver surrounded by a cell infiltrate with much less CD20+ B cells. This patient had a mean circulating c-peptide of 2.2 ng/mL. This observation about a possible correlation between B cells and the clinical outcome of islet transplantation shed new light about a possible role of other leukocyte population in a phenomenon that was thought to be dominated by T cells. That T1D can occur also in subjects with severe hereditary B-cell deficiency indicate that the autoimmune process of insulin-producing ­beta-cell destruction can occur without participation of B cells,40 but does not exclude a possible role for B cells in other patients. In animal models of diabetes, B cells appear implicated in the destruction of insulin-producing beta cells.41, 42 There are two main pathways to which B cells can contribute to autoimmunity. On one hand, they produce auto and alloantibodies. On the other hand, they can act as antigen presenting cells for T-cell activation. With respect to the clinical setting of allogenic islet transplantation, it has to be determined whether B cells play a role through production of antibodies and/or through antigen presentation. In this study, the authors did not find correlations between antibodies and islet graft function. Moreover, autoantibodies can be produced in niches distant from the target tissue, whereas in this case a B-cell infiltrate was found in the tissue surrounding islet grafts into the liver. These findings and particularly the anatomical location of B cells are suggestive of a role of B cells as antigen presenting cells more that antibody production, even though none of these mechanisms can be concluded from the present data. The role of baseline autoreactive T-cells pretransplant was initially addressed by measuring the proliferative response to beta-cell antigens in pretransplant setting and see how it changes posttransplant and correlating the results with the clinical outcome. These tests provide information about the immune reactivity to a given antigen but they are poorly informative with respect to the baseline precursor frequency and changes posttransplant. Specifically, posttransplant and during immune-suppression tests based on proliferation can be biased by the effect of immune suppressive molecules as the precursor frequency can be unchanged but their capacity to proliferate in an in vitro test can be impaired by immune-suppressive drugs. Technologies for direct detection of antigen-specific T cells have become available in the last decade and were used to determine the

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The presence of autoreactive memory T cells and autoantibodies before islet transplant

frequency of both CD4+ and CD8+ T cells in peripheral blood samples. MHC-peptide multimers are MHC molecules that are bound to a peptide from a beta-cell antigen and a fluorescent molecule.43 They bind to T-cell receptors that specifically recognize the MHC-peptide complex allowing a direct enumeration of T cells specific for beta-cell antigens. A more sophisticated methods based on combinatorial approach of a pool of MHC multimers with a multicolor flow cytometry technology was used to measure simultaneously the frequency of 10 antigen-specific T cells, including beta-cell antigens such as GAD65, insulin B, PPI, IA-2, IGRP, and viral antigens including CMV, EBV, and Measles.44 Using combinatorial MHC Multimers technology, Velthius et  al. measured changes of autoreactive T-cell frequencies against multiple islet cell-derived epitopes that were associated with disease activity and correlated with clinical outcome.44 An important information generated from this study was that, despite disease durations up to several decades, beta-cell-specific autoreactive CD8+ T cells were still detectable in the majority of patients with T1D at the time of islet transplantation. Even though the test

β-cell mass

Changes in β-cell mass

Primary autoimmunity

was conducted only 7 islet recipients, no particular islet epitope or pattern of reactivity emerged from the study. Using a similar approach with MHC multimers, another study determined the phenotype in terms of naïve and memory T-cell subsets of CD8+ T cells specific for GAD65, insulin B, and IGRP in 8 patients with T1D with a mean diabetes duration of 27 years, before islet transplantation.29 Autoreactive CD8+ T cells were found in all patients and, even though at a lower frequency, in all nondiabetic controls. In patients with T1D, the majority of autoreactive T cells had a memory phenotype, including the central memory and effector memory phenotype, whereas in nondiabetic controls the majority of T cells showed a naïve, nonantigen experienced phenotype. Of note, in patients with T1D, but not in nondiabetic controls, a small but significant fraction of autoreactive T cells had a stem cell memory T-cell phenotype that can be implicated in the long-term persistence of autoreactive T-cells decades after the onset of the disease. Overall, these studies suggest that autoimmunity is still present at the time of transplantation both for the humoral and the cellular component (Fig. 1). However, it is

Quiescence

Autoreactive T cells

Autoimmunity recurrence β-cell replacement

T1D onset

Time (years) Activation

Autoantibodies

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Activation

Memory T cells

Effector T cells IL-7 Immunesuppression

Seroconversion Multiple autoAbs Drop of autoAbs titers

Multiple autoAbs

Naive T cells Effector T cells IL-2

FIG. 1  Immunological mechanism of primary autoimmunity and recurrence of autoimmunity. Primary autoimmunity starts when autoreactive T cells that are present in the immune system in a naïve state are activated by islet antigens and start to proliferate in an IL-2-dependent manner to generate effector T cells with the capacity to target and destroy β cells. The time of activation of T cells also corresponds to the seroconversion with the appearance of one or more autoantibodies. Primary autoimmunity leads to a substantial loss of the initial β-cell mass until the onset of the disease when 70%–80% of the β-cell mass is lost. When the β-cell mass is completely destroyed, the immune system enters in a phase of quiescent in which memory T cells and B cells survive long-term in the immune system in the absence of immunological stimuli. Islet or pancreas transplantation (β-cell replacement) initiate the process of autoimmunity recurrence. Despite immunosuppression, quiescent memory T cells are reactivated by antigeneic exposure in the presence of high levels of circulating IL-7. Autoantibody titers rise concomitantly and the transplanted β-cell mass is progressively destroyed.

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not clear yet whether signs of autoimmunity persistence before islet transplantation are predictive of the outcome of islet or pancreas transplantation. Considering the four main components of the immune response studied (autoantibodies, alloantibodies, autoreactive T cells, and alloreactive T cells) as baseline parameter before transplantation there is no consensus of whether one or more components are predictive of subsequent graft function. Most studies are limited by the relatively low number of patients studied as islet allotransplantation is performed in small cohort of ­ patients in each ­ transplantation ­center. This is even more problematic when studies are performed with MHC-peptide multimers, in which reagents are usually available for specific HLAs, such as the HLA-A*0201 which is expressed in 38% of subjects. Further studies are needed to clearly determine whether baseline immunological measurements can be used as predictive factors of beta-cell graft function and therefore can be used for preselection of patients that may have an advantage from T-cell or B-cell depletion or suppression.

Autoimmunity recurrence after islet or pancreas transplantation Islet or pancreas transplantation outcome is limited by several factors. In addition to transplant-associated morbidity and concerns about the toxicity of immunosuppressive regimens, recent studies have reported a number of transplant patients who experience autoimmunity recurrence that leads to immunological failure of the graft also in the absence of allogeneic rejection. Although this appears to occur in a minority (<10%) of patients, autoimmunity recurrence raise a fundamental concern regarding immunological mechanisms, with implications for immunotherapies to specifically target autoimmunity.

Pancreas transplantation The recurrence of autoimmunity in clinical pancreas transplantation patients was first reported by Dr. David Sutherland in 1984 in living related HLA identical siblings.1 In this study, a pancreas segment from a twin with no sign of autoimmunity and diabetes was transplanted to the twin with long-term T1D. Given the HLA identity that excludes an allogeneic rejection, the procedure was performed with no or minimal immunosuppression. The result of the transplantation was the loss of graft beta-cell function and insulitis reminiscent of what is seen at diabetes onset. This observation also provided a fundamental evidence of the autoimmune origin of T1D. Subsequent studies reported failures of pancreas grafts obtained from deceased donors and p ­ erformed under immunosuppression. Despite it was clear the

i­mmunological basis of pancreas graft failures, these cases were typically ascribed to chronic rejection. Later reports provided initial but incomplete evidence that autoimmunity recurrence occurred in unrelated SPK transplant recipients despite the HLA mismatch and the immunosuppressive regimen.45, 46 Autoimmunity recurrence in pancreas transplantation was considered for long time a rare complication so that most centers do not routinely monitor islet autoimmunity. It was also traditionally assumed that immunosuppressive regimens that are clinically efficient to control allograft rejection should be also sufficient to control autoimmunity recurrence.47 In the last 15 years, however, an increasing body of evidence suggests that autoimmunity recurrence is an underestimated cause of graft loss in patients who presented years after transplantation with hyperglycemia and loss of circulating C-peptide, but with preserved pancreas exocrine function typically such as stable urine amylase levels. These patients that are usually transplanted simultaneously with kidney (simultaneous ­pancreas-kidney transplantation, SPK) also showed stable kidney transplant function, as an additional indicator of the lack of an allogenic rejection.47 Clear cases of autoimmunity recurrence in a SPK patient were reported and documented by Vendrame in 2010. Of these, one patient achieved stable euglycemia and insulin independence for 5 years after SPK transplantation and subsequently presented with severe hyperglycemia requiring insulin therapy.48 This patient was monitored for exocrine pancreas function (stable levels of urine amylase) and kidney function (normal/stable creatinine), both suggesting the absence of ongoing allograft rejection. A biopsy of both kidney and pancreas transplants was performed. The analysis of the kidney biopsy did not reveal abnormalities that could be ascribed to allograft rejection. However, biopsy of the pancreas transplant revealed a reduced insulin staining of the beta cells, and an insulitis with infiltrates of CD20+ B cells and CD3+ T cells both of the CD4+ and CD8+ subsets. Measures of autoantibodies showed a conversion with autoantibodies to GAD65 and IA-2 and staining of peripheral blood T cells with MHC-peptide multimers showed the presence of circulating GAD65-specific, autoreactive CD4+ T cells. The patient was treated with thymoglobulin and daclizumab (a monoclonal antibody to CD25) that lead to the disappearance of autoreactive CD4 T cells for over 1  year. Circulating GAD65-specific autoreactive CD4 T cells later reappeared with a concomitant decline of the pancreas transplant endocrine function and loss of c-peptide. Other similar cases were described, in which some still presenting a residual beta-cell function and some in which beta-cell function was completely lost. The same authors completed a retrospective analysis of 223 SPK recipients who were transplanted between 1990 and 2012.49 Of these, approximately 80% were

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Autoimmunity recurrence after islet or pancreas transplantation

­ ormoglycemic whereas approximately 20% had develn oped hyperglycemia (n = 47). Of these hyperglycemic recipients, 7.6% (n = 17) were classified as having developed autoimmunity recurrence, while 4.5% (n = 10) were classified as having developed allogenic rejection of pancreas transplants. The study showed that autoimmunity can be more frequent than pancreas chronic rejection as the cause of pancreas graft failure. Moreover, while in most cases autoimmunity recurrence was detected in the absence of allogenic rejection, in some patients, both autoimmunity and rejection coexisted. These s­tudies also provided important insights with respect to the efficiency of immunosuppression therapies to prevent autoimmunity recurrence and/allogenic rejection in SPK patients. Approximately half of these patients were transplanted under an immunosuppression regimen that included an induction therapy with thymoglobulin and anti-CD25, and a maintenance therapy based on steroids, tacrolimus, and either rapamycin or mycophenolate mofetil. This represents a classical modern immunosuppressive regimen for SPK. As compared to older immunosuppressive schemes (anti-CD25 or no induction and maintenance therapy with steroids, tacrolimus, and mycophenolate mofetil), the new regimen did not substantially changed the prevalence of autoimmunity recurrence, suggesting that other mechanisms and therapeutic targets are needed to keep autoimmunity recurrence under control after SPK.

Islet transplantation Islet allotransplantation can be performed after kidney transplantation [islet after kidney (IAK)] or as islet transplantation alone (ITA). As IAK takes advantage of the immunosuppressive regimen needed to preserve kidney graft and is similar to that described before in the SPK setting, we will focus of ITA, in which the immunosuppressive regimen is different and modern protocols do not include steroids. There is good evidence to indicate that transplantation of isolated allogeneic islets can be associated with a relapse of autoimmunity. As for pancreas transplantation, autoimmunity recurrence occurs in a small but significant proportion of patients. Occasionally, it was reported that islet-transplanted patients had dramatic rises in islet autoantibodies within a few weeks after transplant.50 Weaker immune suppression regimens such as 1,25 dihydroxy vitamin D3 in association with mycophenolate mofetil were more frequently associated with autoantibody rises. In some cases, this was associated with evidence of an allogenic rejection.50 Apparently, islet allograft recipients who had an antibody rise after transplant showed similar initial performance of islet grafts in term of achievements of euglycemia and insulin independence. However, the long-term islet graft function was significantly lower in

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those patients, as compared to patients without an increase in islet autoantibodies.51 T-cell responses to islet autoantigens are often increased after islet allotransplantation.4 These conclusions were drawn either by analyzing proliferative T-cell responses in  vitro after stimulation with islet-specific antigens or by staining peripheral blood mononuclear cells with HLA multimers bound to peptides for beta-cell-associated antigens. Data obtained from the expansion of autoreactive T-cell clones in the context of islet allotransplantation suggested that ­expansion of autoreactive T cells in this setting substantially differs from the one that occurs in conventional immune responses. The conventional model of T-cell expansion typical of the immune response to acute infections is based on the IL-2/IL-2R axis and may not be applicable to autoimmunity recurrence post islet transplantation under immunosuppression. An example of the conventional way for T-cell activation is the T-cell response to acute viral infections. In this model, T cells specific for viral antigens are activated by high-affinity interactions between the T-cell receptor and the MHC-peptide complex. This results in the up-regulation of the IL-2R α chain (CD25) and autocrine production of IL-2 by activated T cells. The IL-2/IL-2R autocrine pathway is sufficient to sustain the expansion phase of the clonal population.8 Following the expansion phase, a massive apoptotic death of activated T cells is the driver of the contraction phase terminates the immune response after the antigenic treat is completely cleared. Although the molecular determinants of the autoreactive T-cell response to beta-cell antigens are complex and not fully understood, it is known that the autoreactive T-cell response is raised against low to medium affinity antigen epitopes,52 and that autoreactive T cells can persist in chronically activated status, instead of undergoing to contraction. The T-cell receptor affinity can pose a checkpoint for T-cell activation in response to beta-cell antigens. Since antigen-specific T-cell proliferation requires autocrine production of IL-2 that in turn engages the IL-2 receptor on the T-cell surface, the alpha chain of the IL-2 receptor (CD25) is upregulated only in cells that recognized the antigen to limit the proliferation of CD25 negative bystander T cells when IL-2 is secreted. When the T-cell receptor affinity is low, it is possible that the IL-2/IL-2R axis is not properly activated affecting both the expansion and the contraction phase of the immune response. With respect to the persistence of autoreactive T cells it is established that naive T cells rely on survival signals through contact with self-peptide-loaded MHC molecules, and requires signals from with homeostatic cytokines such as IL-7 and IL-15.53, 54 On the other hand, the memory T-cell pool is typically MHC independent, and they survive and undergo periodic homeostatic ­proliferation through IL-7 and IL-15, whose receptors are

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62.  Recurrence of type 1 diabetes after beta-cell replacement

expressed at high levels.55, 56 The slow homeostatic turnover of quiescent T cells can be dramatically increased in conditions of lymphopenia. The immune system can sense T-cell loss and respond with a vigorous cytokinemediated expansion of remaining T cells driven by increasing the circulating concentration of IL-7.57 Unlike steady-state homeostatic proliferation, that does not change the phenotype and the activation state of T cells, “acute” homeostatic proliferation induce T cells to acquire characteristics of antigen-activated T cells.58 In the context of islet transplantation, immune suppression is design to preferentially target the IL-2/IL-2R axis. Monoclonal antibodies to CD25 binds to and block the IL-2 receptor, calcineurin inhibitors affect the production of IL-2 and rapamycin affect the signaling of the IL-2R. Despite exposure to cognate antigens, under immunosuppression autoreactive T cells cannot use the IL-2/IL-2R axis in order to expand. A possible mechanism for autoreactive T-cell expansion under immunosuppression involves the homeostatic cytokine IL-7. The increased circulating concentration of IL-7 has been measured in patients with reduced T-cell counts in several pathologic (e.g., viral infections) and iatrogenic (e.g., chemotherapy, radiotherapy, and immunosuppression) conditions.59 High circulating concentrations were measured also in patients with T1D undergoing islet allotransplantation.28 In this setting, a mild lymphopenia consequent to the immunosuppressive regimen is associated with a rise in the IL-7 levels and homeostatic T-cell proliferation, which is detectable in the peripheral blood by staining with the proliferation marker Ki-67. T cells that proliferate in response to IL-7 are mostly memory T cells, including autoreactive T cells clones. T cells specific for beta-cell antigens are typically cell with a low to medium affinity T-cell receptor, with lower chances to expand in steadystate conditions where competition with high-affinity T cells, such as those responding to viral antigens, severely limits their capacity to compete for nutrients and cytokine signals. On the other hand, in the context of high IL-7 post islet transplantation, autoreactive T cells have a selective advantage for proliferation also because islet grafts provide at the same time an antigenic stimulus for expansion. Different from IL-2-dependent proliferation, IL-7dependent homeostatic proliferation is largely ignored by immunosuppression. Calcineurin inhibitors and rapamycin do not affect homeostatic proliferation.28 Anti-CD25 monoclonal antibodies were shown to increase the sensitivity of T cells to IL-7. As the alpha chains of the IL-2 receptor (CD25) and the IL-7 receptor (CD127) share the common gamma chain (CD132) to form high-affinity receptors, blocking CD25 with a monoclonal antibody, render more common gamma chain available to interact with CD127.60, 61 This was associated with an increased sensitivity of T cells to IL-7. Of the immunosuppressive compound tested, mycophenolate mofetil

was the only effective on homeostatic T-cell proliferation.28 Mycophenolate mofetil act as a purine synthesis inhibitor, interfering with the synthesis of DNA downstream to cytokine signaling. Patients transplanted with islet with rapamycin and FK506 showed high rate of circulating Ki-67 positive T cells. When patients switched from rapamycin to mycophenolate mofetil for adverse side effects of rapamycin, the percentage of Ki67 positive T cells dropped to the pretransplant levels. The limited ­efficacy of standard immunosuppression on homeostatic T-cell proliferation indicates that there is a need to specifically target the IL-7/IL7R axis. Therapeutic targeting of pro-inflammatory cytokines is clinically beneficial in several autoimmune disorders.62, 63 So far, no efforts have been made to target IL-7. Monoclonal antibodies that bind to and block the IL-7 receptor have been developed in the murine system and it has been shown that they can prevent diabetes in the NOD mouse model.25, 26 In the absence of a monoclonal antibody or biomolecule for specific targeting of IL-7 or the IL-7R in humans, other molecules that act as endogenous regulators of the IL-7/IL-7R axis can be considered for therapeutic purpose. One molecule appears to be the soluble form of the IL-7 receptor alpha chain (sCD127). Soluble forms of cytokine receptors have been described and they can have both antagonistic functions or agonistic functions. For example, the tumor necrosis factor (TNF) receptor I and II64 and the IL-6R subunit gp13065 were reported to exert an inhibitory action on their respective cytokine signaling. In contrast, the soluble IL-15 receptor α can increase IL-15-mediated proliferation of CD8+ T cells and natural killer cells up to 50-fold when it is pre-complexed to IL-15.66 The sCD127 was showed to bind to and inhibit the bioavailability of circulating IL-7 and can represent an endogenous regulator of the IL-7 biological activity.67, 68 The affinity of IL-7 for CD127 is relatively low (Kd = 10–8 M), approximately 3 logs lower than the affinity of IL-7 to the surface CD127/CD132 complex.69 However, sCD127 concentration in serum samples (70–80 ng/mL) was reported to be 10,000-fold higher than that of IL-7 (5 pg/mL) measured.70 Even in the presence of 10-fold increases in IL-7 concentration as found in patients undergoing to islet transplantation,28 is expected that sCD127 can significantly affect IL-7 bioactivity. Moreover, the use of modified sCD127 with higher IL-7 binding affinity could have therapeutic application. Overall these data suggest that the expansion of autoreactive T-cells post islet transplantation can be driven by mechanisms that are different from those expanding autoreactive T cells in the natural history of T1D. The use of immunosuppressive drugs to control the host immune response to the graft, also induce lymphopenia and the consequent rise of IL-7. Homeostatic T-cell proliferation in response to IL-7 is not efficiently controlled with immune-suppressive drugs and specific targeting

B. Islet allo-transplantation



References

of the IL-7/IL-7R axis could substantially improve the efficacy of immunosuppressive therapy in the context of transplantation. Another important issue in the context of allogeneic pancreas or islet transplantation is the capacity of T cells generated during the autoimmune process to recognize antigen of the mismatched HLA of the donor and cause damage to the graft. The donor/recipient HLA mismatch issue is important for CD8+ T cells that once activated into cytotoxic lymphocytes can recognize their cellular target by the expression of the same HLA-peptide complex to which they were initially instructed to recognize by an autologous antigen presenting cell. Therefore, although associations with reduced graft function have been occasionally reported, it is not proven that autoimmunity recurrence equals to autoimmune-mediated destruction of transplanted beta cells. The T-cell receptor of memory autoreactive T cells before transplantation specifically recognizes antigens in the form of peptides presented on the recipient MHC class I (for CD8+ T cells) or class II (for CD4+ T cells) context. Studies performed using MHC-peptide multimers in which the target HLA of multimers is the HLA of the recipients, demonstrated that the preexisting T cell is specific to the islet antigen peptides from the graft, but presented to T cells on the recipient antigen presenting cells. This is somewhat similar to the presentation of allogeneic antigens, which occurs through indirect allo-recognition pathways.44 In the setting of whole pancreas transplantation, a persistent memory response expanded from preexisting autoreactive T cells was clearly shown by analyzing the T-cell receptors through the identification of Vbeta sequences obtained from T-cells stained with MHC-peptide multimers.48 These data clearly suggest that autoimmunity recurrence is the consequence of expansion and activation of autoreactive memory T-cell clones already present pretransplant, but does not provide information on recipient CD8+ cytotoxic clones can recognize and destroy donor HLA mismatched beta cells.

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B. Islet allo-transplantation