Immunoglobulin (Ig)M antibodies to proteinase 3 in granulomatosis with polyangiitis and microscopic polyangiitis.

Anti-neutrophil cytoplasmic antibodies (ANCA) appear to play an important role in the pathogenesis of ANCA-associated vasculitis (AAV). However, ANCA alone are not sufficient to generate disease, and some evidence suggests that infectious triggers may serve as inciting events for AAV disease activity. Antibodies of the immunoglobulin (Ig)M isotype often serve as markers of recent infection, and IgM ANCA have been identified previously in patients with AAV, although the frequency and clinical relevance of IgM ANCA is not well established. We sought to characterize IgM ANCA more clearly by creating a novel enzyme-linked immunosorbent assay (ELISA) for IgM antibodies to proteinase 3 [IgM proteinase 3 (PR3)-ANCA], which we applied to two large, clinically well-characterized trial cohorts of patients with granulomatosis with polyangiitis and microscopic polyangiitis. In the first cohort, IgM PR3-ANCA occurred with a frequency of 15·0%, and were associated with a higher degree of disease severity and a trend towards a higher rate of alveolar haemorrhage (29·6 versus 15·7%, P = 0·10). Analysis of follow-up samples in this cohort showed that the presence of IgM PR3-ANCA was transient, but could recur. In the second cohort, IgM PR3-ANCA occurred with a frequency of 41·1%, and were also associated with a higher degree of disease severity. A higher rate of alveolar haemorrhage was observed among those with IgM PR3-ANCA (45·3 versus 15·8%; P < 0·001). The association of transient IgM PR3-ANCA with an acute respiratory manifestation of AAV suggests a possible link between an infectious trigger and AAV disease activity.

Comparability of patients with ANCA-associated vasculitis enrolled in clinical trials or in observational cohorts.

OBJECTIVES: To analyse the differences between patients with granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA) entered into randomised clinical trials (RCTs) and those followed in large observational cohorts. METHODS: The main characteristics and outcomes of patients with generalised and/or severe GPA or MPA with a five-factor score ≥ 1 enrolled in the French Vasculitis Study Group (FVSG) or the US-Canadian-based Vasculitis Clinical Research Consortium cohorts were compared to those enrolled in one of 2 FVSG clinical RCTs (WEG91, WEGENT) or 3 European Vasculitis Society clinical trials (CYCLOPS, CYCAZAREM, IMPROVE). RESULTS: 657 patients (65.3% with GPA) in RCTs were compared to 437 in cohorts (90.6% with GPA). RCT patients were older at diagnosis than the cohort patients (56.6 ± 13.9 vs. 46.8 ± 17.3 years), had higher Birmingham vasculitis activity score (19.5 ± 9.1 vs. 16.9 ± 7.4), and more frequent kidney disease (84.0% vs. 54.9%) but fewer ear, nose, and throat symptoms (56.8% vs. 72.2%). At 56 months post-diagnosis, mortality and relapse rates, adjusted for age and renal function, were higher for patients with GPA in RCTs vs. cohorts (10.7% vs. 2.5% [p=0.001] and 22.5% vs. 15.6% [p=0.03], respectively) but similar for patients with MPA (6.2% vs. 6.6% [p=0.92] and 16.6% vs. 10.1% [p=0.39], respectively). CONCLUSIONS: Patients with GPA or MPA in RCTs and those in observational cohorts show important differences that should be remembered when interpreting results based on these study populations.

Clinical outcomes of remission induction therapy for severe antineutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVE: To evaluate the reasons that complete remission is not achieved or maintained with original treatment in some patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) treated with rituximab (RTX) or with cyclophosphamide/azathioprine (CYC/AZA). METHODS: The Rituximab in AAV trial was a randomized, double-blind, placebo-controlled trial comparing the rate of remission induction among patients treated with RTX (n = 99) and patients treated with CYC followed by AZA (n = 98). Glucocorticoids were tapered over a period of 5 months. The primary outcome measure was lack of disease activity without glucocorticoid treatment at 6 months. To determine the most important reason for failure to achieve the primary outcome, 7 hierarchical categories of reasons were defined retrospectively (uncontrolled disease, adverse event leading to therapy discontinuation, severe flare, limited flare, Birmingham Vasculitis Activity Score for Wegener's Granulomatosis >0, prednisone treatment at any dosage, and other). RESULTS: Although remission (lack of disease activity) was achieved in 170 of the 197 patients (86%) in the first 6 months, the primary outcome measure was not achieved in 42%. There were 3 deaths. Twenty-four percent of the patients failed to achieve the primary end point due to active disease: 10 (5%) experienced uncontrolled disease in the first month and 37 (19%) experienced flares after initial improvement. In the majority of such patients, treatment with blinded crossover or according to best medical judgment led to disease control. Ninety-one percent of patients who had uncontrolled disease or experienced a severe flare had proteinase 3 (PR3)-ANCA. When patients with uncontrolled disease were excluded from analysis, those who were PR3-ANCA positive were found to experience fewer flares when treated with RTX compared to CYC/AZA (8 of 59 [14%] versus 20 of 62 [32%]; P = 0.02). Neither ANCA titers nor B cell counts predicted disease flare. CONCLUSION: Current treatment regimens are largely successful in controlling AAV, but in approximately one-fourth of patients, active disease persists or recurs in the first 6 months despite treatment. PR3-ANCA positivity is a risk factor for recurrence or persistence of severe disease. ANCA titers and B cell detectability are poor predictors of both disease relapse and disease quiescence in the first 6 months.

Point mutation in essential genes with loss or mutation of the second allele: relevance to the retention of tumor-specific antigens.

Antigens that are tumor specific yet retained by tumor cells despite tumor progression offer stable and specific targets for immunologic and possibly other therapeutic interventions. Therefore, we have studied two CD4(+) T cell-recognized tumor-specific antigens that were retained during evolution of two ultraviolet-light-induced murine cancers to more aggressive growth. The antigens are ribosomal proteins altered by somatic tumor-specific point mutations, and the progressor (PRO) variants lack the corresponding normal alleles. In the first tumor, 6132A-PRO, the antigen is encoded by a point-mutated L9 ribosomal protein gene. The tumor lacks the normal L9 allele because of an interstitial deletion from chromosome 5. In the second tumor, 6139B-PRO, both alleles of the L26 gene have point mutations, and each encodes a different tumor-specific CD4(+) T cell-recognized antigen. Thus, for both L9 and L26 genes, we observe "two hit" kinetics commonly observed in genes suppressing tumor growth. Indeed, reintroduction of the lost wild-type L9 allele into the 6132A-PRO variant suppressed the growth of the tumor cells in vivo. Since both L9 and L26 encode proteins essential for ribosomal biogenesis, complete loss of the tumor-specific target antigens in the absence of a normal allele would abrogate tumor growth.

Outcomes of nonsevere relapses in antineutrophil cytoplasmic antibody-associated vasculitis treated with glucocorticoids.

OBJECTIVE: Nonsevere relapses are more common than severe relapses in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), but their clinical course and treatment outcomes remain largely unexamined. We undertook this study to analyze the outcomes of patients with nonsevere relapses in the Rituximab in ANCA-Associated Vasculitis (RAVE) trial who were treated with prednisone according to a prespecified protocol. METHODS: RAVE was a randomized, double-blind, placebo-controlled trial comparing rituximab (RTX) to cyclophosphamide (CYC) followed by azathioprine (AZA) for induction of remission. Patients who experienced nonsevere relapses between months 1 and 18 were treated with a prednisone increase without a concomitant change in their nonglucocorticoid immunosuppressants, followed by a taper. RESULTS: Forty-four patients with a first nonsevere relapse were analyzed. In comparison to the 71 patients who maintained relapse-free remission over 18 months, these patients were more likely to have proteinase 3-ANCAs, diagnoses of granulomatosis with polyangiitis (Wegener's), and a history of relapsing disease at baseline. A prednisone increase led to remission in 35 patients (80%). However, only 13 patients (30%) were able to maintain second remissions through the followup period (mean 12.5 months); 31 patients (70%) had a second disease relapse, 14 of them with severe disease. The mean time to second relapse was 9.4 months (4.7 months in the group treated with RTX versus 13.7 months in the group treated with CYC/AZA; P < 0.01). Patients who experienced nonsevere relapses received more glucocorticoids than those who maintained remission (6.7 grams versus 3.8 grams; P < 0.01). CONCLUSION: Treatment of nonsevere relapses in AAV with an increase in glucocorticoids is effective in restoring temporary remission in the majority of patients, but recurrent relapses within a relatively short interval remain common. Alternative treatment approaches are needed for this important subset of patients.

CD4+ and B lymphocytes in transplantation immunity. II. Augmented rejection of tumor allografts by mice lacking B cells.

To test the importance of B lymphocytes in immunity to major histocompatibility complex class I alloantigens, B cell-deficient mice were generated by reconstituting severe combined immunodeficiency mice, which lack functional B and T lymphocytes, with T cells or with both T and B cells. The reconstituted mice were challenged with a cancer that expresses an MHC class I alloantigen at a low level and is susceptible to killing by CD8+ cytotoxic T lymphocytes. Tumors grew more slowly in and were rejected more frequently by the mice lacking B cells. Understanding the mechanism by which B cells suppress tumor allograft rejection may lead to new approaches for suppressing immune attack on transplanted tissues.

Rituximab for the treatment of relapses in antineutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVE: Disease relapses are frequent in antineutrophil cytoplasmic antibody-associated vasculitis (AAV). This study was undertaken to evaluate outcomes in patients with AAV who are re-treated with rituximab (RTX) and prednisone for severe disease relapses. METHODS: The Rituximab in AAV trial was a randomized, double-blind, placebo-controlled trial comparing the rates of remission induction among patients treated with RTX (n = 99) and patients treated with cyclophosphamide (CYC) followed by azathioprine (AZA) (n = 98). Prednisone was tapered to discontinuation after 5.5 months. After remission was achieved, patients who experienced a severe disease relapse between months 6 and 18 were eligible to receive RTX and prednisone on an open-label basis according to a prespecified protocol. Investigators remained blinded with regard to the original treatment assignment. RESULTS: Twenty-six patients received RTX for disease relapse after remission had initially been achieved with their originally assigned treatment. Fifteen of these patients were initially randomized to receive RTX and 11 to receive CYC/AZA. Thirteen (87%) of the patients originally assigned to receive RTX and 10 (91%) originally assigned to receive CYC/AZA achieved remission again with open-label RTX (an overall percentage of 88%). In half of the patients treated with open-label RTX, prednisone could be discontinued entirely. Patients in this cohort experienced fewer adverse events compared to the overall study population (4.7 adverse events per patient-year versus 11.8 adverse events per patient-year). CONCLUSION: Re-treatment of AAV relapses with RTX and glucocorticoids appears to be a safe and effective strategy, regardless of previous treatment.

A unique tumor antigen produced by a single amino acid substitution.

Mice immunized against a cancer recognize antigens unique to that cancer, but the molecular structures of such antigens are unknown. We isolated CD4+ T cell clones recognizing an antigen uniquely expressed on the UV-induced tumor 6132A; some clones inhibited the growth of tumors bearing the specific antigen. A T cell hybridoma was used to purify this antigen from nuclear extracts by RP-HPLC and SDS-PAGE using T cell immunoblot assays. A partial amino acid sequence was nearly identical to a sequence in ribosomal protein L9. The cDNA sequence of L9 from 6132A PRO cells differed from the normal sequence at one nucleotide; this mutation encoded histidine instead of leucine at position 47. A synthetic peptide containing this mutation was over 1000-fold more stimulatory of T cells than was the wild-type peptide. These results indicate that this unique tumor antigen is derived from a single amino acid substitution in a cellular protein.

Immunodominance deters the response to other tumor antigens thereby favoring escape: prevention by vaccination with tumor variants selected with cloned cytolytic T cells in vitro.

Variant cancer cells which arise from the parent tumor during tumor progression can escape immunity but retain antigens. We have mixed highly immunogenic (A+B+) murine parental cancer cells with less immunogenic (A-B+) variant cancer cells to construct a model of a cancer containing escape variants. When such mixtures of cancer cells were injected into normal mice, the variant cells grew out because immune responsiveness to the B antigen on the variant was hindered by dominance of the A antigen on the surrounding parental tumor cells. However, A-B+ variant cells inoculated alone at a separate site induced B specific cytolytic T cells and were rejected. Moreover, mice immunized with A-B+ cells rejected a challenge which contained a mixture of variant and parental cancer cells, while immunization with A+B+ cells was ineffective. Thus, variant tumor cells selected from parental tumor cells by cytolytic T cells in vitro can be used to induce protective immunity against variants expected to escape tumor immunity in vivo. The immunodominance of the A antigen may be related to its ability to induce a much more rapid CTL response than the B antigen, since we show in another model that the pre-existence of a CTL response to one antigen prevented the subsequent induction of CTL to another antigen injected at the same site, even if both antigens were equally efficient at inducing CTL. These results indicate that immunodominance can affect strong as well as weak antigens. Vaccination with individual antigens at separate sites rather than with multiple antigens at one site may, therefore, be needed to prevent tumor escape and tumor recurrence or to counteract infectious diseases.

Activation of oxidative stress genes by mutations at the soxQ/cfxB/marA locus of Escherichia coli.

Exposure of Escherichia coli to superoxide-generating drugs, such as menadione or paraquat, uniquely induces approximately 40 proteins, nine of which are under the positive control of the soxR locus (at min 92). We report here that certain mutations at a separate locus that we have named soxQ (at min 34) confer some of the phenotypes seen in soxR-constitutive strains, including resistance to menadione. A previously known mutation called cfxB, identified through antibiotic resistance, is likely an allele of soxQ. The soxQ1 and cfxB mutations cause transcriptional activation of the genes that encode Mn-containing superoxide dismutase, glucose 6-phosphate dehydrogenase, and the soi-17/19::lac and soi-28::lac fusions. These genes are also activated by soxR, but the soxQ1 and cfxB mutations increase the synthesis of seven other proteins not influenced by soxR. Moreover, the soxQ1- and cfxB-dependent phenotypes do not depend on the soxR gene, and gene induction by soxR in response to redox stress does not depend on the soxQ locus. As well as increasing cellular resistance to some oxidants, the soxQ1 and cfxB mutations confer elevated resistance to various antibiotics, probably via diminished expression of outer membrane protein OmpF. The marA1 multiple-antibiotic resistance mutation (also at min 34) behaves like a weak allele of soxQ but probably resides in a nearby gene that, with soxQ, is part of a regulatory complex. We propose that soxQ helps control some oxidative stress proteins as part of another regulon that responds to an unknown environmental signal.

CD4(+) T cells eliminate MHC class II-negative cancer cells in vivo by indirect effects of IFN-gamma.

CD4(+) T cells can eliminate tumor cells in vivo in the absence of CD8(+) T cells. We have CD4(+) T cells specific for a MHC class II-restricted, tumor-specific peptide derived from a mutant ribosomal protein expressed by the UV light-induced tumor 6132A-PRO. By using neutralizing mAb specific for murine IFN-gamma and adoptive transfer of CD4(+) T cells into severe combined immunodeficient mice, we show that anti-IFN-gamma treatment abolishes the CD4(+) T cell-mediated rejection of the tumor cells in vivo. The tumor cells were MHC class II negative, and IFN-gamma did not induce MHC class II expression in vitro. Therefore, the tumor-specific antigenic peptide must be presented by host cells and not the tumor cells. Tumor cells transduced to secrete IFN-gamma had a markedly reduced growth rate in severe combined immunodeficient mice, but IFN-gamma did not inhibit the growth of the tumor cells in vitro. Furthermore, tumor cells stably expressing a dominant-negative truncated form of the murine IFN-gamma receptor alpha chain, and therefore insensitive to IFN-gamma, nevertheless were rejected by the adoptively transferred CD4(+) T cells. Thus, host cells, and not tumor cells, seem to be the target of IFN-gamma. Together, these results show that CD4(+) T cells can eliminate IFN-gamma-insensitive, MHC class II-negative cancer cells by an indirect mechanism that depends on IFN-gamma.