Key implications of this work

4.2.1 VH1-46 utilization is observed in the Dsg3-reactive Ab repertoires across all patients analyzed.

The study described in Chapter 2, while not the first to describe the autoAb repertoire in PV patients281, 283_{, is the most comprehensive in terms of encompassing all B cell} subsets in the peripheral blood. In addition, the utilization of multiple methods of Ab repertoire cloning underscores the validity of the VH1-46 phenotype we observe in the anti-Dsg3 Ab repertoires of our four patient cohort. Lastly, we provide strong evidence of these VH1-46 Abs in contributing to the pathogenesis of PV via ex vivo human skin injection.

4.2.2 A subset of VH1-46 mAbs are able to bind Dsg3 upon reversion of somatic mutations.

Somatic mutation analyses revealed a relative dearth of somatic mutations in the VH1- 46 Abs compared to non-VH1-46 Abs, leading us to the hypothesis that perhaps these VH1-46 Abs do not largely depend on somatic mutation for Dsg3 reactivity. Germline (GL) reversion experiments confirmed that three of the five VH1-46 Abs maintained the ability to bind Dsg3 upon reversion, compared to zero of five non-VH1-46 Abs. This GL reactive phenotype is also in direct contrast to four non-VH1-46 Abs isolated from two

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other PV patients281_{, wherein all four also required somatic mutations in the heavy chain} for Dsg3 reactivity.

4.2.3 Those VH1-46 mAbs that did require somatic mutations to bind depend on one or two somatic mutations that led to acidic amino residues.

Given that two VH1-46 Abs lost the ability to bind Dsg3 upon reversion, we wanted to better understand why this was the case. We observed an enrichment of replacement somatic mutations leading to acidic amino acid residues, and upon reintroduction of these residues into the GL Ab, we observed a partial rescue of these two non-GL- reactive Abs in terms of Dsg3 binding. Furthermore, removal of acidic residues in two GL-reactive Abs led to a complete loss of binding, suggesting that acidic residues are both necessary and partially sufficient to confer Dsg3 reactivity in the VH1-46 Abs we have tested, and can be generate by either V(D)J recombination, or somatic

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4.2.4 Despite shared VH1-46 gene usage in the antibody responses to both Dsg3 and VP6, cross-reactivity in the IgG compartment is rare due to disparate amino acid characteristics.

The striking VH1-46 phenotype we observed in the autoAb response to Dsg3 led us to determine whether other VH1-46-biased Ab responses may play a role in the etiology of PV. The only other reported VH1-46-biased Ab response to an antigen is towards the rotavirus protein VP6. Both of the VH1-46 responses demonstrated lower levels of somatic mutations, as well as reactivity towards Dsg3 and VP6 in the absence of these somatic mutations, respectively. We hypothesized that PV could be initiated by a cross-reactive VH1-46 B cell clone. Focused testing of our Dsg3-

reactive VH1-46 Abs largely did not reveal strong levels of cross-reactivity, with one of seven IgG clones being cross-reactive. In addition, screening four IgG repertoires by antibody phage display did not identify cross-reactive clones.

Further analyses of the amino acid characteristics in the Dsg3 and VP6 VH1-46 responses pointed to distinct patterns of acidic and basic residues245_{, respectively.} Indeed, a majority of the mutagenesis experiments conducted on the heavy chain CDR2 to introduce rotavirus-specific somatic mutations did not confer cross-reactivity to three VH1-46 clones isolated from PV patients. However, in one case, there was a moderate increase in cross-reactivity to both Dsg3 and VP6, suggesting that while rare, it is possible to heighten cross-reactivity of VH1-46 clones via somatic hypermutation.

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4.2.5 A majority of VH1-46 heavy chains in the IgM compartment are cross-reactive in a PV patient in remission, but not in two patients with active disease or a

healthy individual.

In order to maximize the probability of isolating a cross-reactive clone, we screened the IgM repertoires of four individuals; three PV patients, and one healthy individual. We chose a diverse cohort of individuals because the hypothesis of a cross-reactive VH1-46 clone alludes to a secondary assumption that perhaps everyone exhibits cross-reactive VH1-46 heavy chains, especially in their naïve IgM compartment. While we did isolate polyreactive clones from three IgM libraries, the only library wherein we isolated heavy chains reactive specifically to Dsg3 and VP6 was in a PV patient in remission after four rounds of rituximab, which may influence the B cell repertoire. Interestingly, all six cross- reactive heavy chains were VH1-46.

4.2.6 A subset of cross-reactive VH1-46 IgM heavy chains can both inhibit rotavirus replication and keratinocyte adhesion in vitro.

Upon characterizing these cross-reactive VH1-46 heavy chains more closely, we observed that all stained the surface of keratinocytes by immunofluorescence, confirming their Dsg3 specificity. Secondly, two heavy chains were able to inhibit

rotavirus replication in vitro. Interestingly, these same two VH1-46 heavy chains inhibited keratinocyte adhesion in vitro. This highlights that a VH1-46 heavy chain can possess the ability to both inhibit rotavirus and Dsg3 function in the skin, pointing to the possibility

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of these cross-reactive heavy chains playing pivotal roles in both rotavirus infection and in PV.