Roles for TbDSS-1 in RNA surveillance and decay of maturation by-products from the 12S rRNA locus

The Trypanosoma brucei exoribonuclease, TbDSS-1, has been implicated in multiple aspects of mitochondrial RNA metabolism. Here, we investigate the role of TbDSS-1 in RNA processing and surveillance by analyzing 12S rRNA processing intermediates in TbDSS-1 RNAi cells. RNA fragments corresponding to leader sequence upstream of 12S rRNA accumulate upon TbDSS-1 depletion. The 5′ extremity of 12S rRNA is generated by endonucleolytic cleavage, and TbDSS-1 degrades resulting upstream maturation by-products. RNAs with 5′ ends at position −141 and 3′ ends adjacent to the mature 5′ end of 12S rRNA are common and invariably possess oligo(U) tails. 12S rRNAs with mature 3′ ends and unprocessed 5′ ends also accumulate in TbDSS-1 depleted cells, suggesting that these RNAs represent dead-end products normally destined for decay by TbDSS-1 in an RNA surveillance pathway. Together, these data indicate dual roles for TbDSS-1 in degradation of 12S rRNA maturation by-products and as part of a mitochondrial RNA surveillance pathway that eliminates stalled 12S processing intermediates. We further provide evidence that TbDSS-1 degrades RNAs originating upstream of the first gene on the minor strand of the mitochondrial maxicircle suggesting that TbDSS-1 also removes non-functional RNAs generated from other regions of the mitochondrial genome

9G4 Autoreactivity Is Increased in HIV-Infected Patients and Correlates with HIV Broadly Neutralizing Serum Activity

The induction of a broadly neutralizing antibody (BNAb) response against HIV-1 would be a desirable feature of a protective vaccine. Vaccine strategies thus far have failed to elicit broadly neutralizing antibody responses; however a minority of HIV-infected patients do develop circulating BNAbs, from which several potent broadly neutralizing monoclonal antibodies (mAbs) have been isolated. The findings that several BNmAbs exhibit autoreactivity and that autoreactive serum antibodies are observed in some HIV patients have advanced the possibility that enforcement of self-tolerance may contribute to the rarity of BNAbs. To examine the possible breakdown of tolerance in HIV patients, we utilized the 9G4 anti-idiotype antibody system, enabling resolution of both autoreactive VH4-34 gene-expressing B cells and serum antibodies. Compared with healthy controls, HIV patients had significantly elevated 9G4+ serum IgG antibody concentrations and frequencies of 9G4+ B cells, a finding characteristic of systemic lupus erythematosus (SLE) patients, both of which positively correlated with HIV viral load. Compared to the global 9G4−IgD− memory B cell population, the 9G4+IgD− memory fraction in HIV patients was dominated by isotype switched IgG+ B cells, but had a more prominent bias toward “IgM only" memory. HIV envelope reactivity was observed both in the 9G4+ serum antibody and 9G4+ B cell population. 9G4+ IgG serum antibody levels positively correlated (r = 0.403, p = 0.0019) with the serum HIV BNAbs. Interestingly, other serum autoantibodies commonly found in SLE (anti-dsDNA, ANA, anti-CL) did not correlate with serum HIV BNAbs. 9G4-associated autoreactivity is preferentially expanded in chronic HIV infection as compared to other SLE autoreactivities. Therefore, the 9G4 system provides an effective tool to examine autoreactivity in HIV patients. Our results suggest that the development of HIV BNAbs is not merely a consequence of a general breakdown in tolerance, but rather a more intricate expansion of selective autoreactive B cells and antibodies

9G4+ antibodies isolated from HIV-infected patients neutralize HIV-1 and have distinct autoreactivity profiles.

Potent HIV-1 specific broadly neutralizing antibodies (BNA) are uncommon in HIV infected individuals, and have proven hard to elicit by vaccination. Several, isolated monoclonal BNA are polyreactive and also recognize self-antigens, suggesting a breach of immune tolerance in persons living with HIV (PLWH). Persons with systemic lupus erythematosus (SLE) often have elevated levels of autoreactive antibodies encoded by the VH4-34 heavy chain immunoglobulin gene whose protein product can be detected by the 9G4 rat monoclonal antibody. We have recently found that levels of these "9G4+" antibodies are also elevated in PLWH. However, the putative autoreactive nature of these antibodies and the relationship of such reactivities with HIV neutralization have not been investigated. We therefore examined the autoreactivity and HIV neutralization potential of 9G4+ antibodies from PLWH. Results show that 9G4+ antibodies from PLWH bound to recombinant HIV-1 envelope (Env) and neutralized viral infectivity in vitro, whereas 9G4+ antibodies from persons with SLE did not bind to Env and failed to neutralize viral infectivity. In addition, while 9G4+ antibodies from PLWH retained the canonical anti-i reactivity that mediates B cell binding, they did not display other autoreactivities common to SLE 9G4+ antibodies, such as binding to cardiolipin and DNA and had much lower reactivity with apoptotic cells. Taken together, these data indicate that the autoreactivity of 9G4+ antibodies from PLWH is distinct from that of SLE patients, and therefore, their expansion is not due to a general breakdown of B cell tolerance but is instead determined in a more disease-specific manner by self-antigens that become immunogenic in the context of, and possibly due to HIV infection. Further studies of 9G4+ B cells may shed light on the regulation of B cell tolerance and interface between the generation of specific autoreactivities and the induction of antiviral immunity in persons living with HIV

9G4+ antibodies purified from HIV- infected patients bind to HIV gp140 and neutralize HIV-1.

<p>9G4+ antibodies isolated from SLE patients (n=6) and PLWH (n=8) normalized to equal concentrations, were serially diluted in triplicate and added to ELISA plates coated with oligomeric YU2 gp140 (<b>A</b>) Influenza vaccine (<b>C</b>) or CMV lysate (<b>D</b>) and binding detected by anti-IgG. (<b>B</b>) 9G4+ antibodies were tested for inhibition of pseudotyped HIV-1 SF-162 infection of TZM-bl cells. Infectivity was measured by luciferase assay, and relative inhibitions were calculated after normalizing to internal controls. Each symbol represents the mean value of a patient. Significance determined by Mann-Whitney test.</p

9G4+ antibodies from SLE and HIV-infected patients bind B cells.

<p>Tonsil cells were incubated with either patient plasma, purified 9G4+ antibodies from patients’ plasma or a positive control 9G4+ monoclonal antibody then stained with fluorescently -labeled antibody cocktails. (<b>A</b>) Gating strategy: lymphocytes were gated based on size and granularity followed by an exclusion of doublets. Dead cells were excluded using a viability stain then live cells were then gated by expression of CD3 and CD19. CD3-CD19+ B cells were then further divided by IgD and CD27 markers. The open histogram represents the no antibody source control (PBS alone). The solid histogram is representative of 9G4+ antibody binding B cells. (<b>B</b>) B cell binding of 9G4+ antibodies purified from SLE (n=6) and HIV- infected (n=8) patients was assessed. Significance was determined by Mann-Whitney test.</p

Auto-antigen microarray profiles of 9G4+ IgG isolated from HIV-infected patients.

<p>Plasma, 9G4+ and 9G4- antibodies were screened for IgG binding to 85 auto-antigens. Columns represent HIV samples, rows represent antigens and color maps to log₁₀ signal (gray indicates lower limit of detection, LLOD). Columns are organized by sample type (unfractionated plasma, 9G4+, 9G4-) and subject order within groups is the same for each type. Rows are clustered based on Euclidean distance to facilitate visualization. Antigens that resulted in no signal are listed to the right of the heat map. Antigens in red indicate higher 9G4+ binding relative to 9G4- based on a Wilcoxon signed-rank test at p<0.05. Average signal data where either the signal intensity was greater than 100 or the signal-to-noise ratio was greater than 2 were considered to be above the LLOD; these data were background-subtracted (based on PBS control per antigen) and then log₁₀ transformed.</p

9G4+ antibodies from HIV-1 infected patients bind apoptotic lymphocytes.

<p>Unfractionated serum from normal donors (n=16), SLE (n=21) and HIV- infected (n=57) were used in apoptotic binding assays. (<b>A</b>-<b>C</b>) The Jurkat Human T cell line was treated with camptothecin to induce apoptosis then incubated with patient serum. Serum binding to cells was detected using FITC- labeled 9G4 rat anti-human monoclonal antibody. Apoptotic Cell Gate was drawn around cells stained positive for viability dye, representing the dying cell population. (<b>C</b>) The shaded histogram represents cells incubated with PBS in the absence of patient serum. The thin-line represents the antibody binding from a normal serum donor and the Bold-line is representative of 9G4+ antibody binding from an HIV infected patient. (<b>D</b>) Data from all samples are plotted. The dashed-line represents the normal donor mean plus 2 standard deviations. Significance determined by Mann-Whitney test.</p

9G4+ antibodies isolated from HIV-1 infected patients exhibit less Cardiolipin and ANA autoreactivity than 9G4+ isolated from SLE patients.

<p>Purified 9G4+ antibodies (from SLE [n=8] and HIV-1 infected patients [n=8]) were added to Cardiolipin (<b>A</b>) and HEp2 lysate (<b>B</b>) coated ELISA plates. Anti-IgG was used to detect antibody binding. (<b>B</b>) Cardiolipin IgG (GPLU/ml) and (<b>C</b>) ANA IgG (arbitrary units) were calculated based on manufacturer guidelines, and data were plotted. Each dot represents a sample. Significant differences between HIV and SLE patients were observed in 9G4+ antibody binding to cardiolipin and ANA as determined by Mann-Whitney test. (<b>C</b>) Slides coated with HEp-2 cells were incubated with patient antibodies, detected with FITC conjugated anti-IgG, and visualized with a fluorescence microscope. Dilutions of patient plasma were selected based on manufacturer guidelines while fractionated samples were used at 100 µg/ml. Representative images for unfractionated plasma, 9G4+ and 9G4- antibody fractions from an SLE patient and HIV patient are presented. </p