Lipid Motif of a Bacterial Antigen Mediates Immune Responses via TLR2 Signaling

The cross-talk between the innate and the adaptive immune system is facilitated by the initial interaction of antigen with dendritic cells. As DCs express a large array of TLRs, evidence has accumulated that engagement of these molecules contributes to the activation of adaptive immunity. We have evaluated the immunostimulatory role of the highly-conserved outer membrane lipoprotein P6 from non-typeable Haemophilus influenzae (NTHI) to determine whether the presence of the lipid motif plays a critical role on its immunogenicity. We undertook a systematic analysis of the role that the lipid motif plays in the activation of DCs and the subsequent stimulation of antigen-specific T and B cells. To facilitate our studies, recombinant P6 protein that lacked the lipid motif was generated. Mice immunized with non-lipidated rP6 were unable to elicit high titers of anti-P6 Ig. Expression of the lipid motif on P6 was also required for proliferation and cytokine secretion by antigen-specific T cells. Upregulation of T cell costimulatory molecules was abrogated in DCs exposed to non-lipidated rP6 and in TLR2−/− DCs exposed to native P6, thereby resulting in diminished adaptive immune responses. Absence of either the lipid motif on the antigen or TLR2 expression resulted in diminished cytokine production from stimulated DCs. Collectively; our data suggest that the lipid motif of the lipoprotein antigen is essential for triggering TLR2 signaling and effective stimulation of APCs. Our studies establish the pivotal role of a bacterial lipid motif on activating both innate and adaptive immune responses to an otherwise poorly immunogenic protein antigen

Loss of CXCR3 expression on memory B cells in individuals with long-standing type 1 diabetes

Aims/hypothesis Islet-specific autoantibodies can predict the development of type 1 diabetes. However, it remains unclear if B cells, per se, contribute to the causal pancreatic immunopathology. We aimed to identify phenotypic signatures of disease progression among naive and memory B cell subsets in the peripheral blood of individuals with type 1 diabetes. Methods A total of 69 participants were recruited across two separate cohorts, one for discovery purposes and the other for validation purposes. Each cohort comprised two groups of individuals with type 1 diabetes (one with newly diagnosed type 1 diabetes and the other with long-standing type 1 diabetes) and one group of age- and sex-matched healthy donors. The phenotypic characteristics of circulating naive and memory B cells were investigated using polychromatic flow cytometry, and serum concentrations of various chemokines and cytokines were measured using immunoassays. Results A disease-linked phenotype was detected in individuals with long-standing type 1 diabetes, characterised by reduced C-X-C motif chemokine receptor 3 (CXCR3) expression on switched (CD27+IgD−) and unswitched (CD27intermediateIgD+) memory B cells. These changes were associated with raised serum concentrations of B cell activating factor and of the CXCR3 ligands, chemokine (C-X-C motif) ligand (CXCL)10 and CXCL11. A concomitant reduction in CXCR3 expression was also identified on T cells. Conclusions/interpretation Our data reveal a statistically robust set of abnormalities that indicate an association between type 1 diabetes and long-term dysregulation of a chemokine ligand/receptor system that controls B cell migration

Human Antibody Response to Outer Membrane Protein G1a, a Lipoprotein of Moraxella catarrhalis

Moraxella catarrhalis is an important cause of respiratory infections in adults with chronic obstructive pulmonary disease (COPD) and of otitis media in children. Outer membrane protein (OMP) G1a is an ∼29-kDa surface lipoprotein and is a potential vaccine candidate. The gene that encodes OMP G1a was expressed and purified using a novel plasmid vector. [(3)H]palmitic acid labeling demonstrated that both native and recombinant OMP G1a contain covalently bound palmitic acid. To assess the expression of OMP G1a during human infection, paired sera and sputum supernatants from adults with COPD followed prospectively were studied by enzyme-linked immunosorbent assays with recombinant lipidated OMP G1a to detect antibodies made specifically during carriage of M. catarrhalis. Overall, 23% of patients developed either a serum immunoglobulin G (IgG) response (9%) or sputum IgA response (21%) to OMP G1a, following 100 episodes of acquisition and clearance of M. catarrhalis. Patients developed antibody responses at similar rates following episodes of clinical exacerbation compared to asymptomatic colonization. Serum IgG antibodies following natural infection were directed predominantly at OMP G1a epitopes that are not exposed on the bacterial surface. These data show that OMP G1a is expressed during infection of the human respiratory tract and is a target of systemic and mucosal antibodies. These observations indicate that OMP G1a, a highly conserved surface protein, should be evaluated further as a vaccine candidate

Subclonal Variation of RAG-Mediated Diversification in B Cell Acute Lymphoblastic Leukemia

Background: Recombination-activating gene (RAG) mediates recombination of the immunoglobulin heavy chain variable gene (IGHV) in immature B lymphocytes. Aberrant targeting of RAG to non-IGH sites in B cell acute lymphoblastic leukemia (B-ALL) contributes to the development of driver mutations and clonal evolution (Papaemmanuil et al. Nat. Genet. 2014). This finding suggests that patients whose ALL involves increased RAG-mediated clonal diversification would exhibit more aggressive disease. As an initial step toward addressing this hypothesis, we asked whether the extent of RAG-mediated diversification in a patient's leukemia is consistent across all cells or variable between subclones. To assess variation in RAG-mediated subclone diversification, we interrogated rearranged IGH sequences from diagnostic B-ALL specimens to identify early subclones (SC) and to quantify RAG-derived sub-subclones (SSC) from each. We hypothesized that if RAG activity is a consistent feature of the leukemia, all SC within a single patient will have a comparable extent of SSC evolution. Methods: Amplicon-based IGHV sequencing identified the number of clonal IGH SC and their RAG-derived SSC in 22 pre-treatment adult and pediatric patients with newly-diagnosed B-ALL. Analysis was performed on peripheral blood (PB) for all patients in addition to diagnostic bone marrow (BM) for 16 of the 22 patients studied. Ultra-deep sequencing of IGH utilized 500 ng genomic DNA (representing approximately 80,000 cells/specimen) for the MiSeqDx platform, generating ~300 bp reads surrounding the VDJ junctional region. The NCBI IgBlast sequence analysis tool assigned IGH VDJ identities according to germline reference. Methods for determining subclones (SC) of shared clonal lineage involved classifying reads with a common Jh identity and 6 shared upstream Dh-Jh junctional nucleotides (termed "6N_Jx") according to defined methods (Gawad et al. Blood 2012). Further evolved sub-subclones (SCC) - as evidence of ongoing RAG activity - were defined by unique junctional sequences upstream of the common Dh-Jh junction (termed the "NDN" region). SSC were quantified according to their relative proportions within each SC family. Results: VDJ-rearranged SC families were detected for 20 of the 22 patients studied (median 2/patient; range 1-7); further analysis to assess variation in RAG-mediated diversification was limited to the 18 cases with ≥ 2 SC. In 9 of these 18, numerous evolved SSCs were identified from at least one SC in the specimen. In 4/9, there were starkly distinct levels of RAG-mediated diversity observed between intrapatient SC families, with some clonal precursors giving rise to numerous SSCs (up to 2,200 SSC per SC) while others showed minimal-to-no RAG-derived evolution (Table 1). Fifteen of 16 patients with matched BM and PB diagnostic specimens had detectable SCs. Of these, 73% (11/15) shared the dominant SC, while in 27% (4/15) the dominant SC differed between sites. However, regardless of which SC predominated, the extent of SSC diversification within each SC was preserved between the BM and the PB, with similar evolution patterns observed regardless of disease site. There was no relationship between SC read frequency and number of SSCs. Conclusions: Using deep sequencing of a single IGHV locus, wide variation in the extent of subclone diversification was observed in 4 of 20 patients with B-ALL. These findings indicate that the degree of RAG-mediated heterogeneity in B-ALL can range from minimal to extensive among distinct subclones in a single patient. The data underscore the relevance of single cell investigation of tumor characteristics to improving our understanding of the mechanisms of clonal evolution in lymphoid malignancies. Disclosures No relevant conflicts of interest to declare. </jats:sec

Haemophilus influenzae Outer Membrane Protein P6 Molecular Characterization May Not Differentiate All Strains of H. Influenzae from H. haemolyticus▿

Distinguishing nontypeable Haemophilus influenzae and Haemophilus haemolyticus isolates by outer membrane protein (OMP) P6 gene sequencing is complicated by sequence variants in isolates. Further testing using RapID NH and multilocus sequence analysis may not help identify some isolates. Translated OMP P6 gene sequences are not conserved among all isolates presumed to be H. influenzae

Regional Differences in Lymphomagenesis Identified through Quantification of AICDA-Mediated Subclonal Evolution in Follicular Lymphoma: Do Pesticides Play a Role?

Abstract Background: Environmental risk factors for lymphoma are ill-defined and controversial. Epidemiologic studies have demonstrated an association between pesticide exposure and an increase in non-Hodgkin lymphomas (NHL), particularly those with a 14;18 translocation. However, in the absence of a mechanistic explanation for the effects of pesticide on lymphomagenesis, the data remain correlative and subject to alternative explanations. Mutagenesis due to activation-induced cytidine deaminase (AICDA) is key to lymphomagenesis, particularly in follicular lymphoma (FL). AICDA exerts physiologic effects by creating mutations in the immunoglobulin heavy locus (IGH) for somatic hypermutation in order to produce highly specific antibodies. AICDA's off-target genomic damage creates "driver" and "passenger" mutations, which can be used to quantify the subclonal evolution of each tumor specimen. Using deep sequencing (10000-fold) with appropriate error-thresholding, our lab previously demonstrated that the number of AICDA-mediated mutations in the 5' UTR of BCL2 is linearly related to the sum of AICDA-mediated mutations at other off-target sites, implying that mutations within the 5'UTR of BCL2 can function as a surrogate marker for the genome-wide aberrant targeting (Spence, J Immunology, 2014). We have also demonstrated that the number of mutations within IGHV is unrelated to the sum of genome-wide aberrant mutations. The sequencing data can thus be used to determine the minimum number of subclonal populations within a specimen as defined by each region (BCL2 and IGHV). We hypothesized that the number of AICDA-generated subclones would differ in patients with FL living in high and low pesticide use regions, elucidating whether pesticide-associated lymphomagenesis acts through AICDA or an alternative mechanism. We sought to assess the feasibility of demonstrating a difference in extent of AICDA-mediated subclonal evolution in patients with FL residing in regions with high and low pesticide usage by quantifying AICDA-mediated subclones in the 5' UTR of BCL2 and within IGHV. Methods: Using a state-wide database of pesticide application organized by ZIP code, we identified 35 patients with FL seen at our institution in Western New York in locales with high (N=19) and low (N=16) pesticide usage and quantified AICDA-mediated subclones in the 5' UTR of BCL2 and within IGHV for these patients' FL specimens. This sample size provided 75-92% power to detect a difference in the number of subclones by pesticide usage, using a 2-sided 0.05 level Wilcoxon test, assuming an effect size of 75-80% probability of more subclones in one pesticide usage group. Results: We identified 19 patients from high pesticide use (1000+ gigaliters (GL)) and 16 from low pesticide use ZIP codes (&amp;lt;1000 GL). Pesticide volume use by ZIP code in the low group ranged from 10-664 GL with a median of 263 GL, and in the high group ranged from 1636 to 67,140 GL with a median of 4787 GL. Patients living in regions of high and low pesticide use were similar in terms of race and ethnicity. The proportion of women was also similar: 43% among high versus 50% among low. Mean age at diagnosis was 62 in the high versus 60 in the low group. Three patients were diagnosed at age &amp;lt; 45 in the high group while no one in the low group was diagnosed at age &amp;lt; 45. BCL2 subclone count was lower (p = 0.046) among those in high pesticide ZIP codes (median 3.0), compared with those in low pesticide ZIP codes (median 6.5). Although the median IGH subclone count was 278 among those in the high group versus 91.5 in the low group, there was insufficient evidence of an association between IGH subclone count and the level of pesticide usage (p = 0.36). Conclusions: The extent of subclonal evolution was significantly different between lymphoma specimens of patients residing in ZIP codes with high versus low pesticide usage. Our data suggest that AICDA-mediated subclonal evolution is negatively correlated with residence in a ZIP code with high pesticide use, raising the question of whether pesticides could induce lymphoma through an alternate mechanism. This observation calls for both corroboration and examination of alternative hypotheses. We demonstrate a feasible approach to assessing these questions in a larger dataset. Disclosures Peterson: Abbott: Consultancy. Casulo: Verastem: Research Funding; BMS: Research Funding; Genentech: Research Funding; Gilead: Research Funding. </jats:sec