IntroductionFollicular lymphoma (FL) is a slowly progressive and largely incurable human B-cell malignancy. Transformation to a more aggressive lymphoma, such as diffuse large B-cell lymphoma, is common and strongly associated with an increase in morbidity and mortality. A chromosomal translocation t(14:18) is the hallmark of this disease, and it is found in 85%-90% of cases. It results in the juxtaposition of the BCL2 proto-oncogene with the immunoglobulin (Ig) heavy chain gene, IGH, leading to deregulated overexpression of Bcl-2 protein, a major inhibitor of apoptosis. However, the t(14:18) translocation is insufficient to cause malignancy as it is detectable in rare B cells from healthy persons. 1-3 Thus, FL pathogenesis requires additional signals beyond that imparted by the deregulation of BCL2. The observation that FL cells isolated from patients fail to survive in vitro and undergo spontaneous apoptosis supports the hypothesis that extrinsic microenvironmental factors are required for maintenance and expansion of FL. 4 Phenotypically, FL tumor cells resemble antigen-experienced germinal center B cells. Their Ig genes, which are rearranged to produce a functional B-cell receptor (BCR), have numerous point mutations compared with their germline counterparts, and this process of somatic hypermutation (SHM) is ongoing as the malignant clone expands and diversifies. Thus, individual tumor cells can each have slightly different Ig variable region sequences. 5 Random mutations should eventually result in stop codons and loss of BCR protein expression. However, FL tumors maintain a surface BCR, indicating a selective force favoring retention of a functional BCR. Furthermore, therapy with anti-idiotype antibodies directed against the BCR did not select for the outgrowth of BCR-negative variants. Rather, this therapy selected for the outgrowth of cells that had amino acid substitutions in the targeted V region sequence, making them unrecognizable by the anti-idiotype antibody. 6 Other in vitro studies with malignant B-cell lines have shown that experimental knockdowns of the BCR and members of its signaling pathway result in growth arrest, implicating their importance in tumor cell survival. 7 The BCR can transmit a tonic survival signal, but this is greatly augmented on its binding to a cognate antigen. 8 There is indirect evidence to suggest that antigen recognition plays a role in the pathogenesis of FL. SHM can introduce silent or replacement mutations, the latter leading to an amino acid substitution. In a normal immune response, B cells with mutations resulting in higher binding affinity for the inciting antigen preferentially survive. This selective pressure leads to enrichment of replacement mutations in the complementarity determining regions (CDRs) of the BCR, and an under representation of replacement mutations in the framework regions (FWRs). 9 This same distribution of replacement and silent mutations has been reported for the BCRs of FL cells, 5 and the intraclonal diversity resulting from ongoing SHM...

Summary The U1 small nuclear ribonucleoprotein particle (snRNP) is a target of autoreactive B cells and T cells in several rheumatic diseases including systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). We propose that inherent structural properties of this autoantigen complex, including common RNA-binding motifs, B and T-cell epitopes, and a unique stimulatory RNA molecule, underlie its susceptibility as a target of the autoimmune response. Immune mechanisms that may contribute to overall U1-snRNP immunogenicity include epitope spreading through B and T-cell interactions, apoptosis-induced modifications, and Toll-like receptor (TLR) activation through stimulation by U1-snRNA. We conclude that understanding the interactions between U1-snRNP and the immune system will provide insights into why certain patients develop anti-U1-snRNP autoimmunity, and more importantly how to effectively target therapies against this autoimmune response.

BackgroundExisting methods to measure influenza vaccine immunogenicity prohibit detailed analysis of epitope determinants recognized by immunoglobulins. The development of highly multiplex proteomics platforms capable of capturing a high level of antibody binding information will enable researchers and clinicians to generate rapid and meaningful readouts of influenza-specific antibody reactivity.MethodsWe developed influenza hemagglutinin (HA) whole-protein and peptide microarrays and validated that the arrays allow detection of specific antibody reactivity across a broad dynamic range using commercially available antibodies targeted to linear and conformational HA epitopes. We derived serum from blood draws taken from 76 young and elderly subjects immediately before and 28±7 days post-vaccination with the 2008/2009 trivalent influenza vaccine and determined the antibody reactivity of these sera to influenza array antigens.ResultsUsing linear regression and correcting for multiple hypothesis testing by the Benjamini and Hochberg method of permutations over 1000 resamplings, we identified antibody reactivity to influenza whole-protein and peptide array features that correlated significantly with age, H1N1, and B-strain post-vaccine titer as assessed through a standard microneutralization assay (p<0.05, q <0.2). Notably, we identified several peptide epitopes that were inversely correlated with regard to age and seasonal H1N1 and B-strain neutralization titer (p<0.05, q <0.2), implicating reactivity to these epitopes in age-related defects in response to H1N1 influenza. We also employed multivariate linear regression with cross-validation to build models based on age and pre-vaccine peptide reactivity that predicted vaccine-induced neutralization of seasonal H1N1 and H3N2 influenza strains with a high level of accuracy (84.7% and 74.0%, respectively).ConclusionOur methods provide powerful tools for rapid and accurate measurement of broad antibody-based immune responses to influenza, and may be useful in measuring response to other vaccines and infectious agents.

Antigen-specific CD4 + T cells are implicated in the autoimmune disease systemic lupus erythematosus (SLE), but little is known about the peptide antigens that they recognize and their precise function in disease. We generated a series of MHC class II tetramers of I-E k -containing peptides from the spliceosomal protein U1-70 that specifically stain distinct CD4 + T-cell populations in MRL/lpr mice. The T-cell populations recognize an epitope differing only by the presence or absence of a single phosphate residue at position serine 140 . The frequency of CD4 + T cells specific for U1-70(131-150):I-E k (without phosphorylation) correlates with disease severity and anti-U1-70 autoantibody production. These T cells also express RORγt and produce IL-17A. Furthermore, the U1-70-specific CD4 + T cells that produce IL-17A are detected in a subset of patients with SLE and are significantly increased in patients with mixed connective tissue disease. These studies provide tools for studying antigen-specific CD4 + T cells in lupus, and demonstrate an antigen-specific source of IL-17A in autoimmune disease.tetramer | autoimmunity | lupus | SLE | IL-17

4124 There is accumulating evidence that FL is a non-autonomous disease. Firstly, the t(14:18) translocation, which places the bcl-2 proto-oncogene under control of the IgH promotor, is a cytogenetic hallmark of FL, yet it can be detected in B cells from healthy individuals, indicating that this translocation is not sufficient for disease. Phenotypically, follicular lymphoma tumor cells resemble antigen experienced germinal center B cells in that they have highly mutated B cell receptors (BCRs) and are continually undergoing somatic hypermutation (SHM). Despite the high risk of SHM leading to the introduction of nonsense mutations, follicular lymphoma cell continue to express functional BCRs on their surface. These features suggest a selective pressure for the maintenance of BCR expression and indicate the possibility that follicular lymphoma cells may be receiving survival signals through the BCR via antigen recognition. We hypothesize that follicular lymphoma BCRs can recognize self-antigens. To determine the frequency of tumors with self-reactive BCRs, recombinant tumor immunoglobulins were utilized in an indirect immunofluorescence assay with the HEp-2 human cell line. With this assay self-reactivity was detected in 43/99 (43%) of tested tumor immunoglobulins. Within the self-reactive tumor immunoglobulins there was a great diversity of staining patterns, indicating the absence of a unifying antigen that is recognized by all follicular lymphoma BCRs. Autoantigen protein microarrays were utilized to determine the frequency of tumor immunoglobulins reactive for known autoantibody targets associated with autoimmune diseases. Irrespective of HEp-2 reactivity status, none of the tumor immunoglobulins tested (0/50) were reactive against known auto-antigens. These observations indicate that the self-antigens recognized by tumor immunoglobulins might be categorically different from those recognized by the autoantibodies present in patients with autoimmune disease. In an effort to identify specific self-antigens being recognized we utilized the recombinant follicular lymphoma immunoglobulins to immunoprecipitate targets from HEp-2 cell lysates. For one patient's tumor immunoglobulin we identified myoferlin as a recognized self-antigen. The presence of ongoing SHM in follicular lymphoma tumors leads to the existence of multiple tumor clones. To assess how antigen recognition changed as this patient's tumor evolved through SHM we performed a rescue fusion, which immortalizes the tumor cells, halts SHM, and allows for the secretion of the tumor immunoglobulins. Recovered clones differed by a number of silent and replacement mutations, yet all remained HEp-2 reactive. Additionally, all clones maintained their ability to bind myoferlin, though with variable avidity. The observation that ongoing SHM did not break the self-reactivity of the patient's tumor supports the possibility that there is a selective pressure to preserve antigen recognition. Disclosures: No relevant conflicts of interest to declare.