Persis P. Wadia

B cells promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies

Chronic inflammation characterized by T cell and macrophage infiltration of visceral adipose tissue (VAT) is a hallmark of obesity-associated insulin resistance and glucose intolerance. Here we show a fundamental pathogenic role for B cells in the development of these metabolic abnormalities. B cells accumulate in VAT in diet-induced obese (DIO) mice, and DIO mice lacking B cells are protected from disease despite weight gain. B cell effects on glucose metabolism are mechanistically linked to the activation of proinflammatory macrophages and T cells and to the production of pathogenic IgG antibodies. Treatment with a B cell–depleting CD20 antibody attenuates disease, whereas transfer of IgG from DIO mice rapidly induces insulin resistance and glucose intolerance. Moreover, insulin resistance in obese humans is associated with a unique profile of IgG autoantibodies. These results establish the importance of B cells and adaptive immunity in insulin resistance and suggest new diagnostic and therapeutic modalities for managing the disease.

Identifying compartment-specific non-HLA targets after renal transplantation by integrating transcriptome and “antibodyome” measures

We have conducted an integrative genomics analysis of serological responses to non-HLA targets after renal transplantation, with the aim of identifying the tissue specificity and types of immunogenic non-HLA antigenic targets after transplantation. Posttransplant antibody responses were measured by paired comparative analysis of pretransplant and posttransplant serum samples from 18 pediatric renal transplant recipients, measured against 5,056 unique protein targets on the ProtoArray platform. The specificity of antibody responses were measured against gene expression levels specific to the kidney, and 2 other randomly selected organs (heart and pancreas), by integrated genomics, employing the mapping of transcription and ProtoArray platform measures, using AILUN. The likelihood of posttransplant non-HLA targets being recognized preferentially in any of 7 microdissected kidney compartments was also examined. In addition to HLA targets, non-HLA immune responses, including anti-MICA antibodies, were detected against kidney compartment-specific antigens, with highest posttransplant recognition for renal pelvis and cortex specific antigens. The compartment specificity of selected antibodies was confirmed by IHC. In conclusion, this study provides an immunogenic and anatomic roadmap of the most likely non-HLA antigens that can generate serological responses after renal transplantation. Correlation of the most significant non-HLA antibody responses with transplant health and dysfunction are currently underway.

H-Y antibody development associates with acute rejection in female patients with male kidney transplants.

Background. Human minor histocompatibility antigens (mHA) and clinically relevant immune responses to them have not been well defined in organ transplantation. We hypothesized that women with male kidney transplants would develop antibodies against H-Y, the mHA encoded on the Y-chromosome, in association with graft rejection. Methods. We tested sera from 118 consecutive transplant recipients with kidney biopsies. Antibodies that specifically recognized the recombinant H-Y antigens RPS4Y1 or DDX3Y were detected by IgG enzyme-linked immunosorbent assay and western blotting. Immunogenic epitopes were further identified using overlapping H-Y antigen peptides for both the H-Y proteins. Results. In the 26 female recipients of male kidneys, H-Y antibody development posttransplant (1) was more frequent (46%) than in other gender combinations (P<0.001), (2) showed strong correlation with acute rejection (P=0.00048), (3) correlated with plasma cell infiltrates in biopsied kidneys (P=0.04), and (4) did not correlate with C4d deposition or donor-specific anti-human leukocyte antigen (HLA) antibodies. Of the two H-Y antigens, RPS4Y1 was more frequently recognized (P=0.005). Conclusion. This first demonstration of a strong association between H-Y antibody development and acute rejection in kidney transplant recipients shows that in solid organ allografts, humoral immune responses against well defined mHA have clear clinical correlates, can be easily monitored, and warrant study for possible effects on long-term graft function.

Protein microarrays identify antibodies to protein kinase Cζ that are associated with a greater risk of allograft loss in pediatric renal transplant recipients

Antibodies to human leukocyte antigens (HLAs) are a risk factor for acute renal allograft rejection and loss. The role of non-HLAs and their significance to allograft rejection have gained recent attention. Here, we applied protein microarray technology, with the capacity to simultaneously identify 5056 potential antigen targets, to assess non-HLA antibody formation in 15 pediatric renal transplant recipients during allograft rejection. Comparison of the pre- and post-transplant serum identified de novo antibodies to 229 non-HLA targets, 36 of which were present in multiple patients at allograft rejection. On the basis of its reactivity, protein kinase Czeta (PKCzeta) was selected for confirmatory testing and clinical study. Immunohistochemical analysis found PKCzeta both within the renal tissue and infiltrating lymphocytes at rejection. Patients who had an elevated anti-PKCzeta titer developed rejection, which was significantly more likely to result in graft loss. The absence of C4d deposition in patients with high anti-PKCzeta titers suggests that it is a marker of severe allograft injury rather than itself being pathogenic. Presumably, critical renal injury and inflammation associated with this rejection subtype lead to the immunological exposure of PKCzeta with resultant antibody formation. Prospective assessment of serum anti-PKCzeta levels at allograft rejection will be needed to confirm these results.

Antibodies specifically target AML antigen NuSAP1 after allogeneic bone marrow transplantation

Identifying the targets of immune response after allogeneic hematopoietic cell transplantation (HCT) promises to provide relevant immune therapy candidate proteins. We used protein microarrays to serologically identify nucleolar and spindle-associated protein 1 (NuSAP1) and chromatin assembly factor 1, subunit B (p60; CHAF1b) as targets of new antibody responses that developed after allogeneic HCT. Western blots and enzyme-linked immunosorbent assays (ELISA) validated their post-HCT recognition and enabled ELISA testing of 120 other patients with various malignancies who underwent allo-HCT. CHAF1b-specific antibodies were predominantly detected in patients with acute myeloid leukemia (AML), whereas NuSAP1-specific antibodies were exclusively detected in patients with AML 1 year after transplantation (P < .001). Complete genomic exon sequencing failed to identify a nonsynonymous single nucleotide polymorphism (SNP) for NuSAP1 and CHAF1b between the donor and recipient cells. Expression profiles and reverse transcriptase-polymerase chain reaction (RT-PCR) showed NuSAP1 was predominately expressed in the bone marrow CD34(+)CD90(+) hematopoietic stem cells, leukemic cell lines, and B lymphoblasts compared with other tissues or cells. Thus, NuSAP1 is recognized as an immunogenic antigen in 65% of patients with AML following allogeneic HCT and suggests a tumor antigen role.

Protein microarrays discover angiotensinogen and PRKRIP1 as novel targets for autoantibodies in chronic renal disease

Biomarkers for early detection of chronic kidney disease are needed, as millions of patients suffer from chronic diseases predisposing them to kidney failure. Protein microarrays may also hold utility in the discovery of auto-antibodies in other conditions not commonly considered auto-immune diseases. We hypothesized that proteins are released as a consequence of damage at a cellular level during end-organ damage from renal injury, not otherwise recognized as self-antigens, and an adaptive humoral immune response to these proteins might be detected in the blood, as a noninvasive tracker of this injury. The resultant antibodies (Ab) detected in the blood would serve as effective biomarkers for occult renal injury, enabling earlier clinical detection of chronic kidney disease than currently possible, because of the redundancy of the serum creatinine as a biomarker for early kidney injury. To screen for novel autoantibodies in chronic kidney disease, 24 protein microarrays were used to compare serum Ab from patients with chronic kidney disease against matched controls. From a panel of 38 antigens with increased Ab binding, four were validated in 71 individuals, with (n = 50) and without (n = 21) renal insufficiency. Significant elevations in the titer of novel auto-Ab were noted against angiotensinogen and PRKRIP1 in renal insufficiency. Current validation is underway to evaluate if these auto-Ab can provide means to follow the evolution of chronic kidney disease in patients with early stages of renal insufficiency, and if these rising titers of these auto-Ab correlate with the rate of progression of chronic kidney disease.

Recombinant Antigen Microarrays for Serum/Plasma Antibody Detection

Recombinant antigen arrays represent a new frontier in parallel analysis of multiple immune response profiles requiring only minute blood samples. In this article, we review the benefits and pitfalls of recombinant antigen microarrays developed for multiplexed antibody quantification. In particular, we describe the development of antigen arrays presenting a set of Y chromosome-encoded antigens, called H-Y antigens. These H-Y antigens are immunologically recognized as minor histocompatibility antigens (mHA) following allogeneic blood and organ transplantation. Clinically relevant B-cell responses against H-Y antigens have been demonstrated in male patients receiving female hematopoietic stem cell grafts and are associated with chronic graft versus host development. This chapter discusses our recombinant antigen microarray methods to measure these clinically relevant allo-antibodies.

Humoral Immunity to Cytomegalovirus and Chronic Graft-Versus-Host Disease

Human cytomegalovirus (HCMV) is an important cause of morbidity and mortality in patients with chronic graft-versus-host disease (cGVHD), but the underlying mechanisms are not understood. The aim of this investigation was to determine whether humoral immune responses to the HCMV antigens were quantitatively different in hematopoietic cell transplant (HCT) recipients who developed cGVHD from those who did not. Antibodies to HCMV and its proteins UL94 and UL70 were quantitated in 79 cGVHD and 30 non-cGVHD patients by enzyme-linked immunosorbent assays (ELISAs). Mean levels of antibodies to the whole HCMV and to its protein UL94 were not significantly different between the cGVHD and the non-cGVHD subjects. However, the levels of antibodies to HCMV UL70 were significantly higher in non-cGVHD subjects than in those with cGVHD (20.91±15.63 versus 15.00±10.35 ng/mL; p=0.03). This suggests that anti-UL70 antibodies might play a protective role in the development of cGVHD.