Loss of intra-islet CD20 expression may complicate efficacy of B-cell-directed type 1 diabetes therapies

OBJECTIVE

Consistent with studies in NOD mice, early clinical trials addressing whether depletion of B cells by the Rituximab CD20-specific antibody provides an effective means for type 1 diabetes reversal have produced promising results. However, to improve therapeutic efficacy, additional B-cell–depleting agents, as well as attempts seeking diabetes prevention, are being considered.

RESEARCH DESIGN AND METHODS

Autoantibodies, including those against insulin (IAAs), are used to identify at-risk subjects for inclusion in diabetes prevention trials. Therefore, we tested the ability of anti-CD20 to prevent diabetes in NOD mice when administered either before or after IAA onset.

RESULTS

The murine CD20-specific 18B12 antibody that like Rituximab, depletes the follicular (FO) but not marginal zone subset of B cells, efficiently inhibited diabetes development in NOD mice in a likely regulatory T-cell–dependent manner only when treatment was initiated before IAA detection. One implication of these results is that the FO subset of B cells preferentially contributes to early diabetes initiation events. However, most important, the inefficient ability of anti-CD20 treatment to exert late-stage diabetes prevention was found to be attributable to downregulation of CD20 expression upon B cell entry into pancreatic islets.

CONCLUSIONS

These findings provide important guidance for designing strategies targeting B cells as a potential means of diabetes intervention.While the autoimmune destruction of pancreatic β-cells that results in type 1 diabetes is ultimately mediated by both CD4 and CD8 T cells, in the NOD mouse model and potentially in humans, disease pathogenesis also requires contributions from B cells (reviewed in Silveira et al. [1]). Studies in NOD mice indicate B cells likely contribute to diabetes by serving as a subset of antigen presenting cells (APCs) that most efficiently support the expansion of pathogenic CD4 T-cell responses (2–4). This is because unlike other APC subsets, B cells express plasma membrane-bound Ig molecules, allowing for their specific and efficient capture of pancreatic β-cell proteins (5,6). Indeed, some diabetes susceptibility genes in NOD mice mechanistically contribute to disease pathogenesis by impairing immunological tolerance induction mechanisms normally deleting or inactivating B cells expressing autoreactive Ig specificities (7–9). Secreted autoreactive Ig molecules may also contribute to diabetes pathogenesis in NOD mice (10,11). In addition, B cells may contribute to diabetes in NOD mice by supporting development in the vicinity of pancreatic islets of tertiary lymphoid structures where pathogenic T cells might be activated (12).Eliminating B cells from birth by either genetic or antibody-mediated approaches inhibits diabetes development in NOD mice (13,14). Partly on the basis of these findings, early phase clinical trials were initiated to determine whether depletion of B cells using the human CD20-specific Rituximab antibody provided beneficial effects, including preservation of C-peptide production, for recent-onset diabetes patients (15,16). Hope for these trials was bolstered by several reports suggesting that in addition to a capacity to block progression to overt diabetes when initiated at an early prodromal stage of disease development, anti-CD20–mediated B-cell depletion (and in one case, using anti-CD22) can also reverse recently established hyperglycemia in at least a subset of NOD mice (17–19). However, it is unclear if CD20- and CD22-specific antibodies with a reported ability to reverse recent-onset diabetes in NOD mice exert the same pattern of B-cell subset deletion as Rituximab. In this regard, it should be noted that Rituximab efficiently depletes the follicular (FO) but not the marginal zone (MZ) subset of mature B cells (20). Such a characteristic is of potential importance given reports that MZ subset B cells can exert potent APC activity and may preferentially contribute to diabetes development in NOD mice (21,22). Furthermore, the capacity of anti-CD20 treatment to eliminate B cells that become activated within pancreatic insulitic infiltrates during diabetes development is also unknown.Another factor to consider is the short time frame after onset of overt hyperglycemia in which anti-CD20–mediated B-cell depletion can reportedly exert a disease reversal effect in NOD mice (18). It is unclear how frequently anti-CD20 treatment could be undertaken in an analogous time frame after diabetes onset in humans. Furthermore, the first reports from human diabetes intervention trials indicate Rituximab treatment retards the rate but does not eliminate the further erosion of residual pancreatic β-cell mass in recent disease onset patients (23). With this result, while promising, it has been questioned whether anti-CD20 treatment might prove more effective in preventing the progression to overt diabetes when initiated in individuals at late prodromal stages of disease development. Here, such trials would take advantage of a continual refinement of genetic and immunological susceptibility markers (24,25).One key marker considered predictive for future diabetes development in humans is the appearance of insulin autoantibodies (IAAs) (26). The presence of IAAs also reportedly marks individual NOD mice that will first develop overt diabetes (27). Hence, to model a potential clinical use setting, we determined if when first initiated in already IAA-positive NOD mice, treatment with a murine CD20-specific antibody sharing B-cell deletional characteristics similar to Rituximab retained a capacity to inhibit diabetes development. We also assessed whether during progression of diabetes development, anti-CD20 treatment could eliminate B cells within pancreatic islet leukocytic infiltrates. Diabetes resistance elicited in NOD mice treated with a B-cell activating factor (BAFF)-blocking reagent depleting all mature B cells reportedly results from an enhanced ability of residual remaining myeloid-type APCs to support a regulatory T-cell (Treg) expansion (28). Hence, we also evaluated if diabetes protection resulting from the possible partial pattern of anti-CD20–mediated B-cell depletion was due to a Treg expansion.     

Rapid destruction of encapsulated islet xenografts by NOD mice is CD4-dependent and facilitated by B-cells: innate immunity and autoimmunity do not play significant roles

BACKGROUND: Spontaneously diabetic NOD mice rapidly reject microencapsulated islet xenografts via an intense pericapsular inflammatory response. METHODS: Tilapia (fish) islets were encapsulated in 1.5% alginate gel microspheres. Recipients in series 1 were spontaneously diabetic NOD mice and streptozotocin-diabetic nude, euthymic Balb/c, prediabetic NOD, and NOR (a recombinant congenic strain not prone to autoimmune diabetes) mice. Recipients in Series 2 were STZ-diabetic NOD, NOD-scid, NOD CD4 T-cell KO, NOD CD8 T-cell KO, and NOD B-cell KO mice. RESULTS: In Series 1, encapsulated fish islet grafts uniformly survived long-term in nude mice but were rejected in Balb/c and, at a markedly accelerated rate, in spontaneously diabetic NOD, streptozotocin-diabetic NOD and NOR recipients. Histologically, intense inflammation (macrophages and eosinophils) surrounding the microcapsules was seen only in NOD and NOR recipients. In Series 2, encapsulated fish islets uniformly survived long-term in NOD-scid and NOD CD4 KO mice; graft survival was markedly prolonged in B-cell KO (P<0.001) but not CD8 KO mice. CONCLUSIONS: The rapid rejection of alginate encapsulated islet xenografts by NOD mice is not solely a consequence of beta-cell directed autoimmunity nor is it merely a vigorous innate immune response. Graft rejection requires CD4 T-cells, is facilitated by B-cells, and does not require CD8 T-cells.     

Unusual resistance of ALR/Lt mouse beta cells to autoimmune destruction: role for beta cell-expressed resistance determinants

Genetic analysis of autoimmune insulin-dependent diabetes mellitus (IDDM) has focused on genes controlling immune functions, with little investigation of innate susceptibility determinants expressed at the level of target beta cells. The Alloxan (AL) Resistant (R) Leiter (Lt) mouse strain, closely related to the IDDM-prone nonobese diabetic (NOD)/Lt strain, demonstrates the importance of such determinants. ALR mice are unusual in their high constitutive expression of molecules associated with dissipation of free-radical stress systemically and at the beta-cell level. ALR islets were found to be remarkably resistant to two different combinations of beta-cytotoxic cytokines (IL-1beta, tumor necrosis factor alpha, and IFN-gamma) that destroyed islets from the related NOD and alloxan-susceptible strains. The close MHC relatedness between the NOD and ALR strains (H2-Kd and H2-Ag7 identical) allowed us to examine whether ALR islet cells could survive autoimmune destruction by NOD-derived Kd-restricted diabetogenic cytotoxic T lymphocyte clones (AI4 and the insulin-reactive G9C8 clones). Both clones killed islet cells from all Kd-expressing strains except ALR. ALR resistance to diabetogenic immune systems was determined in vivo by means of adoptive transfer of the G9C8 clone or by chimerizing lethally irradiated ALR or reciprocal (ALR x NOD)F1 recipients with NOD bone marrow. In all in vivo systems, ALR and F1 female recipients of NOD marrow remained IDDM free; in contrast, all of the NOD recipients became diabetic. In conclusion, the ALR mouse presents a unique opportunity to identify dominant IDDM resistance determinants expressed at the beta cell level.     

Abnormal prothrombin in the plasma of rats carrying hepatic tumors

Vitamin K is required for the posttranslational formation of gamma- carboxyglutamyl residues in a number of plasma clotting factors. Interference with vitamin K action results in the appearance of abnormal (des-gamma-carboxy) forms of prothrombin in human plasma. Vitamin K-sufficient patients with primary hepatocellular carcinoma also secrete significant quantities of abnormal prothrombin; this response has now been studied in a rat model. Normal Buffalo strain rats had 9 micrograms/mL of circulating plasma abnormal prothrombin, whereas Buffalo strain rats carrying the transplantable Morris hepatoma tumor no. 7777 had 33 micrograms/mL at 3 weeks after transplant. Vitamin K-dependent carboxylase activity was normal in the liver of these rats, but very low in the tumor tissue. Rats carrying Morris hepatoma tumors no. 9618A and 5123D did not secrete significant amounts of abnormal prothrombin. Carboxylase activity in these tumors was 15 times that of the 7777 tumor. The data suggest that the secretion of abnormal prothrombin by hepatocellular tumors is the result of normal expression of the prothrombin gene by those tumors and a failure of the tumor to express the carboxylase gene.     

Diagnosis and treatment of a child with the syndrome of apparent mineralocorticoid excess type 1

We report the case of a 16-month-old boy who presented with chronic vomiting, failure to thrive, arterial hypertension and medullary nephrocalcinosis. Laboratory results revealed hypokulaeniin. metabolic alkalosis, increased urinary potassium excretion and ii hyporeninaeniic hypo~ildostei-onisiii. Chromatographic determination of urinary steroid metabolites showed a n abnormal elevation of tetrahydi-ocortisol and do-tetrahydrocortisol compared to tetrahydrocortisone; this pattern of urin- ary steroid excretion is essential for the diagnosis of the syndrome of apparcnt mineralocorticoid excess type I and believed to be a result of the underlying metabolic defect, a decreased activity of the II & hydroxysteroid dehydrogenase. A second variant, called syndrome of apparent mineralocorticoid excess type 2, has similiar clinical features but lacks the typical urinary steroid profile. Therapy with spironolaetone resulted in growth, weight gain and blood pressure control.     

Cytokines in haemolytic uraemic syndrome associated with verocytotoxin-producing Escherichia coli infection

The proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) interleukin (IL)-1 beta, IL-6, and IL-8 were measured in plasma and urine samples from 19 children with verocytotoxin-producing Escherichia coli (VTEC) induced haemolytic uraemic syndrome (HUS) and 30 controls. TNF-alpha was detected in the plasma of two cases and one control; IL-6 in the plasma of one, and the urine of two cases, and in the plasma of one control. IL-1 beta and IL-8 were each identified in eight of the 19 cases and in one and two controls respectively. Urinary IL-8 was found in seven cases, four of whom had plasma concentrations below the limit of detection suggesting renal secretion of this cytokine. Cytokine concentrations did not correlate with peripheral blood neutrophil count at onset of disease. These data confirm the systemic release of cytokines responsible for the coordination of acute inflammatory processes in some children with VTEC induced HUS.     

Cholera toxin subunit B peptide fusion proteins reveal impaired oral tolerance induction in diabetes‐prone but not in diabetes‐resistant mice

The cholera toxin B subunit (CTB) has been used as adjuvant to improve oral vaccine delivery in type 1 diabetes. The effect of CTB/peptide formulations on Ag‐specific CD4⁺ T cells has remained largely unexplored. Here, using tetramer analysis, we investigated how oral delivery of CTB fused to two CD4⁺ T‐cell epitopes, the BDC‐2.5 T‐cell 2.5mi mimotope and glutamic acid decarboxylase (GAD) 286–300, affected diabetogenic CD4⁺ T cells in nonobese diabetic (NOD) mice. When administered i.p., CTB‐2.5mi activated 2.5mi⁺ T cells and following intragastric delivery generated Ag‐specific Foxp3⁺ Treg and Th2 cells. While 2.5mi⁺ and GAD‐specific T cells were tolerized in diabetes‐resistant NODxB6.Foxp3^EGFP F1 and nonobese resistant (NOR) mice, this did not occur in NOD mice. This indicated that NOD mice had a recessive genetic resistance to induce oral tolerance to both CTB‐fused epitopes. In contrast to NODxB6.Foxp3^EGFP F1 mice, oral treatment in NOD mice lead to strong 2.5mi⁺ T‐cell activation and the sequestration of these cells to the effector‐memory pool. Oral treatment of NOD mice with CTB‐2.5mi failed to prevent diabetes. These findings underline the importance of investigating the effect of oral vaccine formulations on diabetogenic T cells as in selected cases they may have counterproductive consequences in human patients.