Gene Conversion-Like Events in the Diversification of Human Rearranged IGHV3-23*01 Gene Sequences

Gene conversion (GCV), a mechanism mediated by activation-induced cytidine deaminase (AID) is well established as a mechanism of immunoglobulin diversification in a few species. However, definitive evidence of GCV-like events in human immunoglobulin genes is scarce. The lack of evidence of GCV in human rearranged immunoglobulin gene sequences is puzzling given the presence of highly similar germline donors and the presence of all the enzymatic machinery required for GCV. In this study, we undertook a computational analysis of rearranged IGHV3-23*01 gene sequences from common variable immunodeficiency (CVID) patients, AID-deficient patients, and healthy individuals to survey “GCV-like” activities. We analyzed rearranged IGHV3-23*01 gene sequences obtained from total PBMC RNA and single-cell polymerase chain reaction of individual B cell lysates. Our search identified strong evidence of GCV-like activity. We observed that GCV-like tracts are flanked by AID hotspot motifs. Structural modeling of IGHV3-23*01 gene sequence revealed that hypermutable bases flanking GCV-like tracts are in the single stranded DNA (ssDNA) of stable stem-loop structures (SLSs). ssDNA is inherently fragile and also an optimal target for AID. We speculate that GCV could have been initiated by the targeting of hypermutable bases in ssDNA state in stable SLSs, plausibly by AID. We have observed that the frequency of GCV-like events is significantly higher in rearranged IGHV3-23-*01 sequences from healthy individuals compared to that of CVID patients. We did not observe GCV-like events in rearranged IGHV3-23-*01 sequences from AID-deficient patients. GCV, unlike somatic hypermutation (SHM), can result in multiple base substitutions that can alter many amino acids. The extensive changes in antibody affinity by GCV-like events would be instrumental in protecting humans against pathogens that diversify their genome by antigenic shift

Cell-Free DNA as a Biomarker in Autoimmune Rheumatic Diseases

Endogenous DNA is primarily found intracellularly in nuclei and mitochondria. However, extracellular, cell-free (cf) DNA, has been observed in several pathological conditions, including autoimmune diseases, prompting the interest of developing cfDNA as a potential biomarker. There is an upsurge in studies considering cfDNA to stratify patients, monitor the treatment response and predict disease progression, thus evaluating the prognostic potential of cfDNA for autoimmune diseases. Since the discovery of elevated cfDNA levels in lupus patients in the 1960s, cfDNA research in autoimmune diseases has mainly focused on the overall quantification of cfDNA and the association with disease activity. However, with recent technological advancements, including genomic and methylomic sequencing, qualitative changes in cfDNA are being explored in autoimmune diseases, similar to the ones used in molecular profiling of cfDNA in cancer patients. Further, the intracellular origin, e.g., if derived from mitochondrial or nuclear source, as well as the complexing with carrier molecules, including LL-37 and HMGB1, has emerged as important factors to consider when analyzing the quality and inflammatory potential of cfDNA. The clinical relevance of cfDNA in autoimmune rheumatic diseases is strengthened by mechanistic insights into the biological processes that result in an enhanced release of DNA into the circulation during autoimmune and inflammatory conditions. Prior work have established an important role of accelerated apoptosis and impaired clearance in leakage of nucleic acids into the extracellular environment. Findings from more recent studies, including our own investigations, have demonstrated that NETosis, a neutrophil cell death process, can result in a selective extrusion of inflammatory mitochondrial DNA; a process which is enhanced in patients with lupus and rheumatoid arthritis. In this review, we will summarize the evolution of cfDNA, both nuclear and mitochondrial DNA, as biomarkers for autoimmune rheumatic diseases and discuss limitations, challenges and implications to establish cfDNA as a biomarker for clinical use. This review will also highlight recent advancements in mechanistic studies demonstrating mitochondrial DNA as a central component of cfDNA in autoimmune rheumatic diseases

T cell memory to evolutionarily conserved and shared hemagglutinin epitopes of H1N1 viruses: a pilot scale study

Abstract Background The 2009 pandemic influenza was milder than expected. Based on the apparent lack of pre-existing cross-protective antibodies to the A (H1N1)pdm09 strain, it was hypothesized that pre-existing CD4+ T cellular immunity provided the crucial immunity that led to an attenuation of disease severity. We carried out a pilot scale study by conducting in silico and in vitro T cellular assays in healthy population, to evaluate the pre-existing immunity to A (H1N1)pdm09 strain. Methods Large-scale epitope prediction analysis was done by examining the NCBI available (H1N1) HA proteins. NetMHCIIpan, an eptiope prediction tool was used to identify the putative and shared CD4+ T cell epitopes between seasonal H1N1 and A (H1N1)pdm09 strains. To identify the immunogenicity of these putative epitopes, human IFN-γ-ELISPOT assays were conducted using the peripheral blood mononuclear cells from fourteen healthy human donors. All donors were screened for the HLA-DRB1 alleles. Results Epitope-specific CD4+ T cellular memory responses (IFN-γ) were generated to highly conserved HA epitopes from majority of the donors (93%). Higher magnitude of the CD4+ T cell responses was observed in the older adults. The study identified two HA2 immunodominant CD4+ T cell epitopes, of which one was found to be novel. Conclusions The current study provides a compelling evidence of HA epitope specific CD4+ T cellular memory towards A (H1N1)pdm09 strain. These well-characterized epitopes could recruit alternative immunological pathways to overcome the challenge of annual seasonal flu vaccine escape

Preexisting CD4+ T-Cell Immunity in Human Population to Avian Influenza H7N9 Virus: Whole Proteome-Wide Immunoinformatics Analyses

<div><p>In 2013, a novel avian influenza H7N9 virus was identified in human in China. The antigenically distinct H7N9 surface glycoproteins raised concerns about lack of cross-protective neutralizing antibodies. Epitope-specific preexisting T-cell immunity was one of the protective mechanisms in pandemic 2009 H1N1 even in the absence of cross-protective antibodies. Hence, the assessment of preexisting CD4+ T-cell immunity to conserved epitopes shared between H7N9 and human influenza A viruses (IAV) is critical. A comparative whole proteome-wide immunoinformatics analysis was performed to predict the CD4+ T-cell epitopes that are commonly conserved within the proteome of H7N9 in reference to IAV subtypes (H1N1, H2N2, and H3N2). The CD4+ T-cell epitopes that are commonly conserved (∼556) were further screened against the Immune Epitope Database (IEDB) to validate their immunogenic potential. This analysis revealed that 45.5% (253 of 556) epitopes are experimentally proven to induce CD4+ T-cell memory responses. In addition, we also found that 23.3% of CD4+ T-cell epitopes have ≥90% of sequence homology with experimentally defined CD8+ T-cell epitopes. We also conducted the population coverage analysis across different ethnicities using commonly conserved CD4+ T-cell epitopes and corresponding HLA-DRB1 alleles. Interestingly, the indigenous populations from Canada, United States, Mexico and Australia exhibited low coverage (28.65% to 45.62%) when compared with other ethnicities (57.77% to 94.84%). In summary, the present analysis demonstrate an evidence on the likely presence of preexisting T-cell immunity in human population and also shed light to understand the potential risk of H7N9 virus among indigenous populations, given their high susceptibility during previous pandemic influenza events. This information is crucial for public health policy, in targeting priority groups for immunization programs.</p></div

The role of cellular immunity in Influenza H1N1 population dynamics

Abstract Background Pre-existing cellular immunity has been recognized as one of the key factors in determining the outcome of influenza infection by reducing the likelihood of clinical disease and mitigates illness. Whether, and to what extent, the effect of this self-protective mechanism can be captured in the population dynamics of an influenza epidemic has not been addressed. Methods We applied previous findings regarding T-cell cross-reactivity between the 2009 pandemic H1N1 strain and seasonal H1N1 strains to investigate the possible changes in the magnitude and peak time of the epidemic. Continuous Monte-Carlo Markov Chain (MCMC) model was employed to simulate the role of pre-existing immunity on the dynamical behavior of epidemic peak. Results From the MCMC model simulations, we observed that, as the size of subpopulation with partially effective pre-existing immunity increases, the mean magnitude of the epidemic peak decreases, while the mean time to reach the peak increases. However, the corresponding ranges of these variations are relatively small. Conclusions Our study concludes that the effective role of pre-existing immunity in alleviating disease outcomes (e.g., hospitalization) of novel influenza virus remains largely undetectable in population dynamics of an epidemic. The model outcome suggests that rapid clinical investigations on T-cell assays remain crucial for determining the protection level conferred by pre-existing cellular responses in the face of an emerging influenza virus

Comparison of amino acid sequence identity of 11 protein segments of newly emerged avian influenza (H7N9) viruses in China with H1N1, H2N2, and H3N2 virus subtypes.

<p>PB2: RNA polymerase subunit B2; PB1: RNA polymerase subunit B1; PA: RNA polymerase subunit A; HA: hemagglutinin; NP: nucleoprotein; NA: neuraminidase; M1 and M2: matrix proteins; NS1 and NS2: nonstructural protein 1 and 2. ^#: partial genes available.</p

Population coverage analysis of identified commonly conserved CD4+ T-cell epitopes - 3A) all ethnicities groups, and 3B) only indigenous groups.

<p>The identified commonly conserved CD4+ T-cell epitopes provide broad population coverage. Based on the binding data for each HLA class II-restricted DRB1 alleles, theoretical population coverage was calculated. The number of possible epitope-HLA allele combinations as a function of the fraction of each ethnic population (%) is shown.</p

Information CD4+ T-cell epitopes that are commonly conserved between H7N9 viruses and human influenza A virus (IAV) subtypes (H1N1, H2N2, H3N2).

<p>Italicized numbers refer to conserved regions between H7N9 and each of 2009 H1N1 and H3N2.</p

HLA-DRB1 alleles restriction of predicted commonly conserved CD4+ T-cell epitopes.

<p>Shown in each panel from A to I are commonly conserved CD4+ T-cell epitopes of nine influenza A virus proteins. Shown on the X-axis are fourteen HLA-DRB1 alleles. On Y-axis are the numbers of predicted CD4+ T-cell epitopes. Each solid circle denotes the HLA-DRB1 allele restriction and promiscuity of identified epitopes.</p

N-Formyl Methionine Peptide-Mediated Neutrophil Activation in Systemic Sclerosis

Exaggerated neutrophil activation and formation of neutrophil extracellular traps (NETs) are reported in systemic sclerosis (SSc) but its involvement in SSc pathogenesis is not clear. In the present study we assessed markers of neutrophil activation and NET formation in SSc patients in relation to markers of inflammation and disease phenotype. Factors promoting neutrophil activation in SSc remain largely unknown. Among the neutrophil activating factors, mitochondrial-derived N-formyl methionine (fMet) has been reported in several autoinflammatory conditions. The aim of the current study is to assess whether SSc patients have elevated levels of fMet and the role of fMet in neutrophil-mediated inflammation on SSc pathogenesis. Markers of neutrophil activation (calprotectin, NETs) and levels of fMet were analyzed in plasma from two SSc cohorts (n=80 and n=20, respectively) using ELISA. Neutrophil activation assays were performed in presence or absence of formyl peptide receptor 1 (FPR1) inhibitor cyclosporin H. Elevated levels of calprotectin and NETs were observed in SSc patients as compared to healthy controls (p<0.0001) associating with SSc clinical disease characteristics. Further, SSc patients had elevated levels of circulating fMet as compared to healthy controls (p<0.0001). Consistent with a role for fMet-mediated neutrophil activation, fMet levels correlated with levels of calprotectin and NETs (r=0.34, p=0.002; r=0.29, p<0.01 respectively). Additionally, plasma samples from SSc patients with high levels of fMet induced de novo neutrophil activation through FPR1-dependent mechanisms. Our data for the first time implicates an important role for the mitochondrial component fMet in promoting neutrophil-mediated inflammation in SSc