Development of a novel, highly sensitive assay for quantification of minimal residual B cells in autoimmune disease and comparison to traditional methods across B-cell-depleting agents.

Targeted B-cell depletion is a useful therapy for many diseases, including autoimmune disorders and certain cancers. We developed a sensitive blood B-cell depletion assay, MRB 1.1, compared its performance with the T-cell/B-cell/NK-cell (TBNK) assay, and assessed B-cell depletion with different therapies. The empirically defined lower limit of quantification (LLOQ) for CD19+ cells in the TBNK assay was 10 cells/µL, and 0.441 cells/µL for the MRB 1.1 assay. The TBNK LLOQ was used to compare differences between B-cell depletion in similar lupus nephritis patient populations who received rituximab (LUNAR), ocrelizumab (BELONG), or obinutuzumab (NOBILITY). After 4 weeks, 10% of patients treated with rituximab retained detectable B cells vs 1.8% with ocrelizumab and 1.7% for obinutuzumab; at 24 weeks 93% of patients who received obinutuzumab remained below LLOQ vs 63% for rituximab. More-sensitive measurements of B cells may reveal differences in potency among anti-CD20 agents, which may associate with clinical outcomes.

Discovery and Qualification of Candidate Urinary Biomarkers of Disease Activity in Lupus Nephritis.

Lupus nephritis (LN) is a severe clinical manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity and mortality. Assessment of severity and activity of renal involvement in SLE requires a kidney biopsy, an invasive procedure with limited prognostic value. Noninvasive biomarkers are needed to inform treatment decisions and to monitor disease activity. Proteinuria is associated with disease progression in LN; however, the composition of the LN urinary proteome remains incompletely characterized. To address this, we profiled LN urine samples using complementary mass spectrometry-based methods:  protein gel fractionation, chemical labeling using tandem mass tags, and data-independent acquisition. Combining results from these approaches yielded quantitative information on 2573 unique proteins in urine from LN patients. A multiple-reaction monitoring (MRM) method was established to confirm eight proteins in an independent cohort of LN patients, and seven proteins (transferrin, α-2-macroglobulin, haptoglobin, afamin, α-1-antitrypsin, vimentin, and ceruloplasmin) were confirmed to be elevated in LN urine compared to healthy controls. In this study, we demonstrate that deep mass spectrometry profiling of a small number of patient samples can identify high-quality biomarkers that replicate in an independent LN disease cohort. These biomarkers are being used to inform clinical biomarker strategies to support longitudinal and interventional studies focused on evaluating disease progression and treatment efficacy of novel LN therapeutics.

Inhibition of c-Kit is not required for reversal of hyperglycemia by imatinib in NOD mice.

AIM/HYPOTHESIS: Recent studies indicate that tyrosine kinase inhibitors, including imatinib, can reverse hyperglycemia in non-obese diabetic (NOD) mice, a model of type 1 diabetes (T1D). Imatinib inhibits c-Abl, c-Kit, and PDGFRs. Next-generation tyrosine kinase inhibitors for T1D treatment should maintain activities required for efficacy while sparing inhibition of targets that might otherwise lead to adverse events. In this study, we investigated the contribution of c-Kit inhibition by imatinib in reversal of hyperglycemia in NOD mice. METHODS: The T670I mutation in c-Kit, which confers imatinib resistance, was engineered into the mouse genome and bred onto the NOD background. Hematopoietic stem cells (HSCs) from NOD.c-Kit(T670I) mice and NOD.c-Kit(wt) littermates were expanded in the presence or absence of imatinib to verify imatinib resistance of the c-Kit(T670I) allele. Diabetic mice were treated with imatinib at the onset of hyperglycemia for three weeks, and blood glucose was monitored. RESULTS: In vitro expansion of HSCs from NOD.c-Kit(wt) mice was sensitive to imatinib, while expansion of HSCs from NOD.c-Kit(T670I) mice was insensitive to imatinib. However, in vivo treatment with imatinib lowered blood glucose levels in both strains of mice. CONCLUSIONS/INTERPRETATION: The HSC experiment confirmed that, in NOD.c-Kit(T670I) mice, c-Kit is resistant to imatinib. As both NOD.c-Kit(T670I) and NOD.c-Kit(wt) mice responded comparably to imatinib, c-Kit inhibition does not substantially contribute to the efficacy of imatinib in T1D. Thus, we conclude that inhibition of c-Kit is not required in next-generation tyrosine kinase inhibitors for T1D treatment, and may be selected against to improve the safety profile.

Cathepsin L is essential for onset of autoimmune diabetes in NOD mice.

Lysosomal proteases generate peptides presented by class II MHC molecules to CD4+ T cells. To determine whether specific lysosomal proteases might influence the outcome of a CD4+ T cell-dependent autoimmune response, we generated mice that lack cathepsin L (Cat L) on the autoimmune diabetes-prone NOD inbred background. The absence of Cat L affords strong protection from disease at the stage of pancreatic infiltration. The numbers of I-A(g7)-restricted CD4+ T cells are diminished in Cat L-deficient mice, although a potentially diabetogenic T cell repertoire persists. Within the CD4+ T cell compartments of Cat L-deficient mice, there is an increased proportion of regulatory T cells compared with that in Cat L-sufficient littermates. We suggest that it is this displaced balance of regulatory versus aggressive CD4+ T cells that protects Cat L-deficient mice from autoimmune disease. Our results identify Cat L as an enzyme whose activity is essential for the development of type I diabetes in the NOD mouse.

CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion.

CD4+CD25+ T regulatory cells (Tregs) prevent autoimmune disease, yet little is known about precisely where they exert their influence naturally in a spontaneous autoimmune disorder. Here, we report that Tregs and T effector cells (Teffs) coexist within the pancreatic lesion before type 1 diabetes onset. We find that BDC2.5 T cell receptor transgenic animals contain a small subset of FoxP3 positive CD4+CD25+CD69- cells in the pancreas, actively turning over, expressing the clonotypic receptor, and containing functional regulatory activity. Gene expression profiling confirms that the CD4+CD25+CD69- cells in pancreatic tissue express transcripts diagnostic of regulatory cells, but with significantly higher levels of interleukin 10 and inducible costimulator (ICOS) than their lymph node counterparts. Blockade of ICOS rapidly converts early insulitis to diabetes, which disrupts the balance of Teffs and Tregs and promotes a very broad shift in the expression of the T regulatory-specific profile. Thus, CD4+CD25+69- Tregs operate directly in the autoimmune lesion and are dependent on ICOS to keep it in a nondestructive state.