Pemphigus vulgaris (PV) is a potentially fatal blistering disease caused by autoantibodies against desmoglein 3 (Dsg3). Here, we clone anti-Dsg3 antibodies from four PV patients and identify pathogenic VH1-46 autoantibodies from all four patients. Unexpectedly, VH1-46 autoantibodies had relatively few replacement mutations. We reverted antibody somatic mutations to their germline sequences to determine the requirement of mutations for autoreactivity. Three of five VH1-46 germline-reverted antibodies maintain Dsg3 binding, compared to zero of five non-VH1-46 germline-reverted antibodies. Site-directed mutagenesis of VH1-46 antibodies demonstrate that acidic amino acid residues introduced by somatic mutation or heavy chain VDJ recombination are necessary and sufficient for Dsg3 binding. Our data suggest that VH1-46 autoantibody gene usage is commonly found in PV because VH1-46 antibodies require few to no mutations to acquire Dsg3 autoreactivity, which may favor their early selection. Common VH gene usage indicates common humoral immune responses, even among unrelated patients.

Human tryptase-beta (HTbeta) is a serine protease that is isolated as a tetramer of four identical, catalytically active subunits (HTbeta-AT). Tetramer activity is not affected by protein-based physiological inhibitors but instead may be regulated by an autoinactivation process we have called spontaneous inactivation. Unless stabilized by heparin or high salt, the active tetramer converts to an inactive state consisting of an inactive-destabilized tetramer that reversibly dissociates to inactive monomers upon dilution. We refer to this mixture of inactive species as siHTbeta and show in this study that previous reports of monomeric catalytic forms are derived from this mixture. siHTbeta itself did not hydrolyze model substrates but unlike the tetramer did react slowly with the serpin alpha2-antiplasmin (alpha2-AP), suggesting a highly limited catalytic potential. In the presence of heparin (or other highly charged polysaccharides), we demonstrate that siHTbeta formed a well-defined complex with the heparin (siHTbeta-HC) that reacted 70-fold faster with alpha2-AP than siHTbeta and also hydrolyzed model substrates and fibrinogen. Formation of siHTbeta-HC was limited to dilute subunit solutions since high subunit concentrations resulted in the reformation of the active tetramer. By compensating for changes in the strength of heparin binding, siHTbeta-HC could be formed over the pH range of 6.0-8.5. The activity dependence on pH was bell-shaped with highest activity between pH 6.8 and pH 7.5. In contrast, HTbeta-AT activity showed no dependence upon heparin, increased over the pH range of 6.0-8.5, and was much higher than that of siHTbeta-HC especially above pH 6.8. HTbeta-AT incubated with excess heparin of different size (3-15 kDa) was functionally stable at 25 degrees C but lost activity regardless of heparin size at 37 degrees C above pH 6.8. The change in stability, which is likely due to weakened heparin binding, did not result in the formation of a stable catalytic monomer. These results confirm that siHTbeta is for the most part an inactive species and that any active monomer is a consequence of heparin binding to siHTbeta under dilute conditions where unfavorable thermodynamics and/or kinetics restrict formation of active tetramer. Heparin binding under these conditions drives a limited reorganization of the active site to a conformation that is catalytic but not the equivalent of a subunit within the active tetramer.

One of the challenges in caring for patients with mycosis fungoides, the most common cutaneous T-cell lymphoma (CTCL), is early identification of patients who will develop progressive disease. While TNM staging is prognostic, identification of high-risk patients earlier in their disease remains an unmet clinical need. We hypothesized that the balance between anti-tumor CD8 + T cell and anti-inflammatory FOXP3 + T regulatory cell infiltration in the initial CTCL skin biopsy could be predictive of disease progression. To examine this, a cohort of 33 CTCL patients (9 I, 4 II) with a median follow-up of 78 months were divided into two groups depending on whether they developed progressive disease (n¼13). We studied their skin biopsy specimens by multiplexed tyramide signal amplification based staining for CD4, CD8, FoxP3, PD-1 and DAPI expression, followed by image deconvolution and automated cell analysis. Patients with greater CD8 + T cell infiltration had significantly improved progression-free survival in univariate (HR¼0.6, CI 0.4-0.97, p¼0.04) and multivariate analysis (HR 0.4, CI 0.2-0.7, p¼0.01). When we restricted the analysis to early stage patients, this finding remained significant (HR¼0.6, CI 0.4-0.98, p¼0.04) and did not correlate with malignant clone frequency or age. The number of FoxP3 + regulatory T cells, PD-1 + T cells and total CD4 + T cells were not associated with improved survival. Ratios between these cell types were also examined for potential associations with prognosis. We found that an increased ratio of CD4 + T cells:FoxP3 + T cells was associated with worse PFS (HR 1.02, CI 1.0-1.03, p¼.01) but this result did not remain significant in multivariate analysis (p¼0.7). We are in the process of confirming this finding in a larger cohort. In summary, CD8 + T cell density in the lesional skin of CTCL patients is predictive of disease progression and may be a useful adjunct in risk-stratifying early stage patients.