Jessica Stolp

General strategy for the generation of human antibody variable domains with increased aggregation resistance

The availability of stable human antibody reagents would be of considerable advantage for research, diagnostic, and therapeutic applications. Unfortunately, antibody variable heavy and light domains (VH and VL) that mediate the interaction with antigen have the propensity to aggregate. Increasing their aggregation resistance in a general manner has proven to be a difficult and persistent problem, due to the high level of sequence diversity observed in human variable domains and the requirement to maintain antigen binding. Here we outline such an approach. By using phage display we identified specific positions that clustered in the antigen binding site (28, 30–33, 35 in VH and 24, 49–53, 56 in VL). Introduction of aspartate or glutamate at these positions endowed superior biophysical properties (non-aggregating, well-expressed, and heat-refoldable) onto domains derived from common human germline families (VH3 and Vκ1). The effects of the mutations were highly positional and independent of sequence diversity at other positions. Moreover, crystal structures of mutant VH and VL domains revealed a surprising degree of structural conservation, indicating compatibility with VH/VL pairing and antigen binding. This allowed the retrofitting of existing binders, as highlighted by the development of robust high affinity antibody fragments derived from the breast cancer therapeutic Herceptin. Our results provide a general strategy for the generation of human antibody variable domains with increased aggregation resistance.

B cell-directed therapies in type 1 diabetes

B cells play a pathogenic role as antigen-presenting cells and autoantibody secretors in the lead up to T cell-mediated autoimmune destruction of insulin-producing β cells in type 1 diabetes (T1D). This has led to significant interest in the use of B cell depletion therapies as a treatment for T1D. In this review, we compare results from five recent studies that used distinct B cell-depleting agents and protocols to successfully prevent and even reverse T1D in the non-obese diabetic (NOD) mouse model. We discuss how information gained from animal studies could be used to improve on the positive outcomes of a completed phase II clinical trial of the B cell-depleting drug rituximab in humans with recent-onset T1D.

Hypoxia-inducible factor-1α (HIF-1α) potentiates β-cell survival after islet transplantation of human and mouse islets.

A high proportion of β-cells die within days of islet transplantation. Reports suggest that induction of hypoxia-inducible factor-1α (HIF-1α) predicts adverse transplant outcomes. We hypothesized that this was a compensatory response and that HIF-1α protects β-cells during transplantation. Transplants were performed using human islets or murine β-cell-specific HIF-1α-null (β-HIF-1α-null) islets with or without treatment with deferoxamine (DFO) to increase HIF-1α. β-HIF-1α-null transplants had poor outcomes, demonstrating that lack of HIF-1α impaired transplant efficiency. Increasing HIF-1α improved outcomes for mouse and human islets. No effect was seen in β-HIF-1α-null islets. The mechanism was decreased apoptosis, resulting in increased β-cell mass posttransplantation. These findings show that HIF-1α is a protective factor and is required for successful islet transplant outcomes. Iron chelation with DFO markedly improved transplant success in a HIF-1α-dependent manner, thus demonstrating the mechanism of action. DFO, approved for human use, may have a therapeutic role in the setting of human islet transplantation.

Genes within the Idd5 and Idd9/11 Diabetes Susceptibility Loci Affect the Pathogenic Activity of B Cells in Nonobese Diabetic Mice

Autoreactive T cells clearly mediate the pancreatic β cell destruction causing type 1 diabetes (T1D). However, studies in NOD mice indicate that B cells also contribute to pathogenesis because their ablation by introduction of an Igμnull mutation elicits T1D resistance. T1D susceptibility is restored in NOD.Igμnull mice that are irradiated and reconstituted with syngeneic bone marrow plus NOD B cells, but not syngeneic bone marrow alone. Thus, we hypothesized some non-MHC T1D susceptibility (Idd) genes contribute to disease by allowing development of pathogenic B cells. Supporting this hypothesis was the finding that unlike those from NOD donors, engraftment with B cells from H2g7 MHC-matched, but T1D-resistant, nonobese-resistant (NOR) mice failed to restore full disease susceptibility in NOD.Igμnull recipients. T1D resistance in NOR mice is mainly encoded within the Idd13, Idd5.2, and Idd9/11 loci. B cells from NOD congenic stocks containing Idd9/11 or Idd5.1/5.2-resistance loci, respectively, derived from the NOR or C57BL/10 strains were characterized by suppressed diabetogenic activity. Immature autoreactive B cells in NOD mice have an impaired ability to be rendered anergic upon Ag engagement. Interestingly, both Idd5.1/5.2 and Idd9/11-resistance loci were found to normalize this B cell tolerogenic process, which may represent a mechanism contributing to the inhibition of T1D.

Differential Responsiveness of Innate-like IL-17– and IFN-γ–Producing γδ T Cells to Homeostatic Cytokines

γδ T cells respond to molecules upregulated following infection or cellular stress using both TCR and non-TCR molecules. The importance of innate signals versus TCR ligation varies greatly. Both innate-like IL-17–producing γδ T (γδT-17) and IFN-γ–producing γδ T (γδT-IFNγ) subsets tune the sensitivity of their TCR following thymic development, allowing robust responses to inflammatory cytokines in the periphery. The remaining conventional γδ T cells retain high TCR responsiveness. We determined homeostatic mechanisms that govern these various subsets in the peripheral lymphoid tissues. We found that, although innate-like γδT-17 and γδT-IFNγ cells share elements of thymic development, they diverge when it comes to homeostasis. Both exhibit acute sensitivity to cytokines compared with conventional γδ T cells, but they do not monopolize the same cytokine. γδT-17 cells rely exclusively on IL-7 for turnover and survival, aligning them with NKT17 cells; IL-7 ligation triggers proliferation, as well as promotes survival, upregulating Bcl-2 and Bcl-xL. γδT-IFNγ cells instead depend heavily on IL-15. They display traits analogous to memory CD8+ T cells and upregulate Bcl-xL and Mcl-1 upon cytokine stimulation. The conventional γδ T cells display low sensitivity to cytokine-alone stimulation and favor IL-7 for their turnover, characteristics reminiscent of naive αβ T cells, suggesting that they may also require tonic TCR signaling for population maintenance. These survival constraints suggest that γδ T cell subsets do not directly compete with each other for cytokines, but instead fall into resource niches with other functionally similar lymphocytes.

Intrinsic Molecular Factors Cause Aberrant Expansion of the Splenic Marginal Zone B Cell Population in Nonobese Diabetic Mice

Marginal zone (MZ) B cells are an innate-like population that oscillates between MZ and follicular areas of the splenic white pulp. Differentiation of B cells into the MZ subset is governed by BCR signal strength and specificity, NF-κB activation through the B cell–activating factor belonging to the TNF family (BAFF) receptor, Notch2 signaling, and migration signals mediated by chemokine, integrin, and sphingosine-1-phosphate receptors. An imbalance in splenic B cell development resulting in expansion of the MZ subset has been associated with autoimmune pathogenesis in various murine models. One example is the NOD inbred mouse strain, in which MZ B cell expansion has been linked to development of type 1 diabetes and Sjögren’s syndrome. However, the cause of MZ B cell expansion in this strain remains poorly understood. We have determined that increased MZ B cell development in NOD mice is independent of T cell autoimmunity, BCR specificity, BCR signal strength, and increased exposure to BAFF. Rather, mixed bone marrow chimeras showed that the factor(s) responsible for expansion of the NOD MZ subset is B cell intrinsic. Analysis of microarray expression data indicated that NOD MZ and precursor transitional 2-MZ subsets were particularly dysregulated for genes controlling cellular trafficking, including Apoe, Ccbp2, Cxcr7, Lgals1, Pla2g7, Rgs13, S1pr3, Spn, Bid, Cd55, Prf1, and Tlr3. Furthermore, these B cell subsets exhibited an increased steady state dwell time within splenic MZ areas. Our data therefore reveal that precursors of mature B cells in NOD mice exhibit an altered migration set point, allowing increased occupation of the MZ, a niche favoring MZ B cell differentiation.

Enhanced responsiveness to T‐cell help causes loss of B‐lymphocyte tolerance to a β‐cell neo‐self‐antigen in type 1 diabetes prone NOD mice

Self‐reactive B lymphocytes contribute to type 1 diabetes pathogenesis as APC and auto‐Ab producers in NOD mice and humans. To shed light on the mechanisms responsible for the breakdown in B‐lymphocyte self‐tolerance to β‐cell Ag, we utilised a model whereby hen‐egg lysozyme (HEL)‐specific Ig Tg (IgHEL‐Tg)‐Tg B lymphocytes were allowed to develop in or were transferred into mice expressing the HEL Tg under an insulin promoter (insHEL‐Tg). IgHEL‐Tg B lymphocytes enhanced type 1 diabetes susceptibility of insHEL‐Tg NOD mice. A comparison of the tolerogenic activity of IgHEL‐Tg B lymphocytes with NOD and non‐autoimmune‐prone C57BL/6 genetic backgrounds showed that both were rendered anergic in the presence of insHEL when competing with polyclonal B lymphocytes. Nevertheless, NOD IgHEL‐Tg B lymphocytes transferred into insHEL‐Tg mice were more readily susceptible to rescue from anergy than their C57BL/6 counterparts, following provision of in vivo T‐cell help. The different tolerogenic outcomes were an intrinsic property of B lymphocytes rather than being related to the quality of T‐cell help, with the defective response being at least partially controlled by genes mapping to insulin‐dependent diabetes (Idd) susceptibility loci on Chromosome 1 (Idd5) and 4 (Idd9/11).

Subcongenic Analyses Reveal Complex Interactions between Distal Chromosome 4 Genes Controlling Diabetogenic B Cells and CD4 T Cells in Nonobese Diabetic Mice

Autoimmune type 1 diabetes (T1D) in humans and NOD mice results from interactions between multiple susceptibility genes (termed Idd) located within and outside the MHC. Despite sharing ∼88% of their genome with NOD mice, including the H2g7 MHC haplotype and other important Idd genes, the closely related nonobese resistant (NOR) strain fails to develop T1D because of resistance alleles in residual genomic regions derived from C57BLKS mice mapping to chromosomes (Chr.) 1, 2, and 4. We previously produced a NOD background strain with a greatly decreased incidence of T1D as the result of a NOR-derived 44.31-Mb congenic region on distal Chr. 4 containing disease-resistance alleles that decrease the pathogenic activity of autoreactive B and CD4 T cells. In this study, a series of subcongenic strains for the NOR-derived Chr. 4 region was used to significantly refine genetic loci regulating diabetogenic B and CD4 T cell activity. Analyses of these subcongenic strains revealed the presence of at least two NOR-origin T1D resistance genes within this region. A 6.22-Mb region between rs13477999 and D4Mit32, not previously known to contain a locus affecting T1D susceptibility and now designated Idd25, was found to contain the main NOR gene(s) dampening diabetogenic B cell activity, with Ephb2 and/or Padi2 being strong candidates as the causal variants. Penetrance of this Idd25 effect was influenced by genes in surrounding regions controlling B cell responsiveness and anergy induction. Conversely, the gene(s) controlling pathogenic CD4 T cell activity was mapped to a more proximal 24.26-Mb region between the rs3674285 and D4Mit203 markers.

The majority of murine γδ T cells at the maternal-fetal interface in pregnancy produce IL-17.

Compared with lymphoid tissues, the immune cell compartment at mucosal sites is enriched with T cells bearing the γδ T‐cell receptor (TCR). The female reproductive tract, along with the placenta and uterine decidua during pregnancy, are populated by γδ T cells predominantly expressing the invariant Vγ6+Vδ1+ receptor. Surprisingly little is understood about the function of these cells. We found that the majority of γδ T cells in the non‐pregnant uterus, pregnant uterus, decidua and placenta of mice express the transcription factor RORγt and produce interleukin‐17 (IL‐17). In contrast, IFNγ‐producing γδ T cells were markedly reduced in gestational tissues compared with uterine‐draining lymph nodes and spleen. Both uterine‐resident invariant Vγ6+ and Vγ4+ γδ T cells which are more typically found in lymphoid tissues and circulating blood, were found to express IL‐17. Vγ4+ γδ T cells were particularly enriched in the placenta, suggesting a pregnancy‐specific recruitment or expansion of these cells. A small increase in IL‐17‐producing γδ T cells was observed in allogeneic compared with syngeneic pregnancy, suggesting a contribution to regulating the maternal response to paternally‐derived alloantigens. However, their high proportions also in non‐pregnant uteri and gestational tissues of syngeneic pregnancy imply a role in the prevention of intrauterine infection or quality control of fetal development. These data suggest the need for a more rigorous evaluation of the role of IL‐17 in sustaining normal pregnancy, particularly as emerging data points to a pathogenic role for IL‐17 in pre‐eclampsia, pre‐term birth, miscarriage and maternal immune activation‐induced behavioral abnormalities in offspring.

The CD19 signalling molecule is elevated in NOD mice and controls type 1 diabetes development

Aims/hypothesisType 1 diabetes is characterised by early peri-islet insulitis and insulin autoantibodies, followed by invasive insulitis and beta cell destruction. The immunological events that precipitate invasive insulitis are not well understood. We tested the hypothesis that B cells in diabetes-prone NOD mice drive invasive insulitis through elevated expression of CD19 and consequent enhanced uptake and presentation of beta cell membrane-bound antigens to islet invasive T cells.MethodsCD19 expression and signalling pathways in B cells from NOD and control mice were compared. Expansion of CD8+ T cells specific for insulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) were compared in CD19-deficient and wild-type NOD mice and this was correlated with insulitis severity. The therapeutic potential of anti-CD19 treatment during the period of T cell activation was assessed for its ability to block invasive insulitis.ResultsCD19 expression and signalling in B cells was increased in NOD mice. CD19 deficiency significantly diminished the expansion of CD8+ T cells with specificity for the membrane-bound beta cell antigen, IGRP. Conversely the reduction in CD8+ T cells with specificity for the soluble beta cell antigen, insulin, was relatively small and not significant.Conclusions/interpretationElevated CD19 on NOD B cells promotes presentation of the membrane-bound antigen, IGRP, mediating the expansion of autoreactive T cells specific for antigens integral to beta cells, which are critical for invasive insulitis and diabetes. Downregulating the CD19 signalling pathway in insulin autoantibody-positive individuals before the development of type 1 diabetes may prevent expansion of islet-invasive T cells and preserve beta cell mass.