Altered serum metabolome as an indicator of paraneoplasia or concomitant cancer in patients with rheumatic disease.

OBJECTIVES: A timely diagnosis is imperative for curing cancer. However, in patients with rheumatic musculoskeletal diseases (RMDs) or paraneoplastic syndromes, misleading symptoms frequently delay cancer diagnosis. As metabolic remodelling characterises both cancer and RMD, we analysed if a metabolic signature can indicate paraneoplasia (PN) or reveal concomitant cancer in patients with RMD. METHODS: Metabolic alterations in the sera of rheumatoid arthritis (RA) patients with (n=56) or without (n=52) a history of invasive cancer were quantified by nuclear magnetic resonance analysis. Metabolites indicative of cancer were determined by multivariable regression analyses. Two independent RA and spondyloarthritis (SpA) cohorts with or without a history of invasive cancer were used for blinded validation. Samples from patients with active cancer or cancer treatment, pulmonary and lymphoid type cancers, paraneoplastic syndromes, non-invasive (NI) precancerous lesions and non-melanoma skin cancer and systemic lupus erythematosus and samples prior to the development of malignancy were used to test the model performance. RESULTS: Based on the concentrations of acetate, creatine, glycine, formate and the lipid ratio L1/L6, a diagnostic model yielded a high sensitivity and specificity for cancer diagnosis with AUC=0.995 in the model cohort, AUC=0.940 in the blinded RA validation cohort and AUC=0.928 in the mixed RA/SpA cohort. It was equally capable of identifying cancer in patients with PN. The model was insensitive to common demographic or clinical confounders or the presence of NI malignancy like non-melanoma skin cancer. CONCLUSIONS: This new set of metabolic markers reliably predicts the presence of cancer in arthritis or PN patients with high sensitivity and specificity and has the potential to facilitate a rapid and correct diagnosis of malignancy.

Negative impact of Interleukin-9 on synovial regulatory T cells in rheumatoid arthritis.

In Rheumatoid Arthritis (RA), regulatory T cells (Tregs) have been found to be enriched in the synovial fluid. Despite their accumulation, they are unable to suppress synovial inflammation. Recently, we showed the synovial enrichment of interleukin-9 (IL-9) producing helper T cells and its positive correlation with disease activity. Therefore, we investigated the impact of IL-9 on synovial Tregs in RA. Here, we confirmed high synovial Tregs in RA patients, however these cells were functionally impaired in terms of suppressive cytokine production (IL-10 and TGF-ß). Abrogating IL-9/ IL-9 receptor interaction could restore the suppressive cytokine production of synovial Tregs and reduce the synovial inflammatory T cells producing IFN-γ, TNF-α, IL-17. However, blocking these inflammatory cytokines failed to show any effect on IL-9 producing T cells, highlighting IL-9's hierarchy in the inflammatory network. Thus, we propose that blocking IL-9 might dampen synovial inflammation by restoring Tregs function and inhibiting inflammatory T cells.

Sex and anti-inflammatory treatment affect outcome of melanoma and non-small cell lung cancer patients with rheumatic immune-related adverse events.

BACKGROUND: Rheumatic immune-related adverse events (R-irAEs) occur in 5-15% of patients receiving immune checkpoint inhibitors (ICI) and, unlike other irAEs, tend to be chronic. Herein, we investigate the factors influencing cancer and R-irAEs outcomes with particular focus on adverse effects of anti-inflammatory treatment. METHODS: In this prospective, multicenter, long-term, observational study, R-irAEs were comprehensively analyzed in patients with malignant melanoma (MM, n=50) and non-small cell lung cancer (NSCLC, n=41) receiving ICI therapy who were enrolled in the study between August 1, 2018, and December 11, 2022. RESULTS: After a median follow-up of 33 months, progressive disease or death occurred in 66.0% and 30.0% of MM and 63.4% and 39.0% of patients with NSCLC. Male sex (progression-free survival (PFS): p=0.013, and overall survival (OS): p=0.009), flare of a pre-existing condition (vs de novo R-irAE, PFS: p=0.010) and in trend maximum glucocorticoid (GC) doses >10 mg and particularly ≥1 mg/kg prednisolone equivalent (sex-adjusted PFS: p=0.056, OS: p=0.051) were associated with worse cancer outcomes. Patients receiving disease-modifying antirheumatic drugs (DMARDs) showed significantly longer PFS (n=14, p=0.011) and OS (n=20, p=0.018). Effects of these variables on PFS and/or OS persisted in adjusted Cox regression models. Additionally, GC treatment negatively correlated with the time from diagnosis of malignancy and the latency from ICI start until R-irAE onset (all p<0.05). R-irAE features and outcomes were independent of other baseline patient characteristics in both studied cancer entities. CONCLUSION: Male sex, flare of pre-existing rheumatologic conditions and extensive GC treatment appeared to be linked with unfavorable cancer outcomes, while DMARD use had a favorable impact. These findings challenge the current dogma of restrictive DMARD use for R-irAE and thus may pave the way to better strategies and randomized controlled trials for the growing number of patients with R-irAE.

Reduction of Proinflammatory Effector Functions Through Remodeling of Fatty Acid Metabolism in CD8+ T Cells From Rheumatoid Arthritis Patients.

OBJECTIVE: Rheumatoid arthritis (RA) CD8+ T cells maintain their effector proinflammatory phenotype by changing their metabolism toward aerobic glycolysis. However, their massive energy and biosynthesis needs may require additional substrates other than glucose. Since systemic alterations in lipid metabolism have been reported in RA patients, we explored the role of fatty acid (FA) metabolism in CD8+ T cells to identify potential targets to curb their proinflammatory potential. METHODS: The expression of FA metabolism-related genes was analyzed for total CD8+ T cells and CD8+ T cell subsets in the data of RA patients and healthy controls retrieved from the GEO database. Functional assays were performed using peripheral blood CD8+ T cells isolated from RA (n = 31), psoriatic arthritis (n = 26), and spondyloarthritis (n = 21) patients receiving different therapies (disease-modifying antirheumatic drugs, biologics, and JAK inhibitors) and from healthy controls (n = 14). We quantified the expression of FA transporters, lipid uptake, intracellular FA content, cytokine production, activation, proliferation, and capacity to inhibit tumor cell growth, either with or without FA metabolism inhibitors. RESULTS: The CD8+ T cell gene expression profile of FA metabolism-related genes was significantly different between untreated RA patients and healthy controls. RA patients who had a good clinical response after 6 months of methotrexate therapy had significantly increased expression of FA metabolism-related genes. Cell surface expression of the FA transporters FA binding protein 4 (FABP4) and G protein-coupled receptor 84 (GPR84) and FA uptake were higher in effector and memory CD8+ T cells from RA patients compared to those from healthy controls. In vitro blockade of FA metabolism significantly impaired CD8+ T cell effector functions. CONCLUSION: RA CD8+ T cells present an altered FA metabolism, which could provide potential therapeutic targets to control their proinflammatory profile, particularly therapies directed against the transport and oxidation of free FA.

Effect of Increased Lactate Dehydrogenase A Activity and Aerobic Glycolysis on the Proinflammatory Profile of Autoimmune CD8+ T Cells in Rheumatoid Arthritis.

OBJECTIVE: CD8+ T cells contribute to rheumatoid arthritis (RA) by releasing proinflammatory and cytolytic mediators, even in a challenging hypoxic and nutrient-poor microenvironment such as the synovial membrane. This study was undertaken to explore the mechanisms through which CD8+ T cells meet their metabolic demands in the blood and synovial membrane of patients with RA. METHODS: Purified blood CD8+ T cells from patients with RA, patients with psoriatic arthritis (PsA), and patients with spondyloarthritis (SpA), as well as healthy control subjects, and CD8+ T cells from RA synovial membrane were stimulated in medium containing 13 C-labeled metabolic substrates in the presence or absence of metabolic inhibitors, under conditions of normoxia or hypoxia. The production of metabolic intermediates was quantified by 1 H-nuclear magnetic resonance. The expression of metabolic enzymes, transcription factors, and immune effector molecules was assessed at both the messenger RNA (mRNA) and protein levels. CD8+ T cell functional studies were performed. RESULTS: RA blood CD8+ T cells met their metabolic demands through aerobic glycolysis, production of uniformly 13 C-enriched lactate in the RA blood (2.6 to 3.7-fold higher than in patients with SpA, patients with PsA, and healthy controls; P < 0.01), and induction of glutaminolysis. Overexpression of Warburg effect-linked enzymes in all RA CD8+ T cell subsets maintained this metabolic profile, conferring to the cells the capacity to proliferate under hypoxia and low-glucose conditions. In all RA CD8+ T cell subsets, lactate dehydrogenase A (LDHA) was overexpressed at the mRNA level (P < 0.03 versus controls; n = 6 per group) and protein level (P < 0.05 versus controls; n = 17 RA patients, n = 9 controls). In RA blood, inhibition of LDHA with FX11 led to reductions in lipogenesis, migration and proliferation of CD8+ T cells, and CD8+ T cell effector functions, while production of reactive oxygen species was increased by 1.5-fold (P < 0.03 versus controls). Following inhibition of LDHA with FX11, RA CD8+ T cells lost their capacity to induce healthy B cells to develop a proinflammatory phenotype. Similar metabolic alterations were observed in RA CD8+ T cells from the synovial membrane. CONCLUSION: Remodeling glucose and glutamine metabolism in RA CD8+ T cells by targeting LDHA activity can reduce the deleterious inflammatory and cytolytic contributions of these cells to the development of autoimmunity.

Reactive Oxygen Species Deficiency Due to Ncf1-Mutation Leads to Development of Adenocarcinoma and Metabolomic and Lipidomic Remodeling in a New Mouse Model of Dextran Sulfate Sodium-Induced Colitis.

Inflammatory bowel disease is characterized by chronic relapsing idiopathic inflammation of the gastrointestinal tract and persistent inflammation. Studies focusing on the immune-regulatory function of reactive oxygen species (ROS) are still largely missing. In this study, we analyzed an ROS-deficient mouse model leading to colon adenocarcinoma. Colitis was induced with dextran sulfate sodium (DSS) supplied via the drinking water in wild-type (WT) and Ncf1-mutant (Ncf1) B10.Q mice using two different protocols, one mimicking recovery after acute colitis and another simulating chronic colitis. Disease progression was monitored by evaluation of clinical parameters, histopathological analysis, and the blood serum metabolome using 1H nuclear magnetic resonance spectroscopy. At each experimental time point, colons and spleens from some mice were removed for histopathological analysis and internal clinical parameters. Clinical scores for weight variation, stool consistency, colorectal bleeding, colon length, and spleen weight were significantly worse for Ncf1 than for WT mice. Ncf1 mice with only a 7-day exposure to DSS followed by a 14-day resting period developed colonic distal high-grade dysplasia in contrast to the low-grade dysplasia found in the colon of WT mice. After a 21-day resting period, there was still ß-catenin-rich inflammatory infiltration in the Ncf1 mice together with high-grade dysplasia and invasive well-differentiated adenocarcinoma, while in the WT mice, high-grade dysplasia was prominent without malignant invasion and only low inflammation. Although exposure to DSS generated less severe histopathological changes in the WT group, the blood serum metabolome revealed an increased fatty acid content with moderate-to-strong correlations to inflammation score, weight variation, colon length, and spleen weight. Ncf1 mice also displayed a similar pattern but with lower coefficients and showed consistently lower glucose and/or higher lactate levels which correlated with inflammation score, weight variation, and spleen weight. In our novel, DSS-induced colitis animal model, the lack of an oxidative burst ROS was sufficient to develop adenocarcinoma, and display altered blood plasma metabolic and lipid profiles. Thus, oxidative burst seems to be necessary to prevent evolution toward cancer and may confer a protective role in a ROS-mediated self-control mechanism.

CD38, CD81 and BAFFR combined expression by transitional B cells distinguishes active from inactive systemic lupus erythematosus.

In view of its heterogeneous presentation and unpredictable course, clinical management of systemic lupus erythematosus (SLE) is difficult. There is a need for biomarkers and diagnostic aids to monitor SLE disease activity and severity prior to, during and after treatment. We undertook this study to search for unique phenotypic patterns in each peripheral blood (PB) B cell subset, capable of distinguishing SLE patients with inactive disease versus SLE patients with active disease versus controls by using an automated population separator (APS) visualization strategy. PB was collected from 41 SLE patients and 28 age- and gender-matched controls. We analyzed the cell surface markers (in a tube CD20/CD27/CD19/CD45/CD38/CD81/BAFFR combination) expression on PB B cell subsets using principal component analysis, implemented in the APS software tool. Overall, our analysis indicates that active SLE can be distinguished from inactive SLE on the basis of a single tube analysis, focused on the decreased expression of CD38, CD81 and BAFFR in transitional B cells. The cluster analysis of immunophenotypic profiles of B cell subsets highlighted disease-specific abnormalities on transitional B cells that emerge as promising surrogate markers for disease activity. Further validation is needed with larger samples and prospective follow-up of patients.

CD8+ T cell profiles in patients with rheumatoid arthritis and their relationship to disease activity.

OBJECTIVE: CD8+ T cells are abundant in rheumatoid arthritis (RA). However, their role in disease pathogenesis is poorly defined. This study was undertaken to investigate the relationship between disease activity and CD8+ T cell phenotypes, production of cytokines, and production of cytotoxic molecules in the peripheral blood (PB) and synovial fluid (SF) of patients with RA. METHODS: CD8+ T cell phenotypes were determined in 96 patients with RA (44 with disease in remission, 34 with active disease, 18 with low disease activity) and in 64 sex- and age-matched healthy controls. Ten paired PB and SF samples from patients with active RA were analyzed. The expression of surface markers, cytokines, and proteolytic enzymes in CD8+ T cells was evaluated using flow cytometry. RESULTS: PB CD8+ T cells from RA patients with active disease exhibited an effector (CD27-CD62L-) phenotype (P = 0.005), with elevated expression of proinflammatory cytokines (tumor necrosis factor α [TNFα], interferon-γ [IFNγ], interleukin-6 [IL-6], IL-17A) when compared to healthy controls. In a state of remission, the same phenotype observed in patients with active disease persisted, including a significant increase in the frequency of CD69 (P < 0.001), but lower cytokine production was observed. SF CD8+ T cells from RA patients expressed more robust effector memory (CD27+CD62L-) and activated (CD69+) profiles compared to the T cell subsets in paired PB samples. Production of cytokines (IL-6, IL-17A, and IFNγ) by CD8+ T cells from RA PB was positively correlated within individual donors. Moreover, production of cytokines (TNFα, IFNγ, and IL-17A) by CD8+ T cells from RA PB positively correlated with the Disease Activity Score in 28 joints. CONCLUSION: The activation status and proinflammatory potential of CD8+ T cell subsets observed in the RA patients in this study strongly suggest that a phenotype of local and systemic cytotoxic effector T cells plays a role in this disease.

Deficient production of reactive oxygen species leads to severe chronic DSS-induced colitis in Ncf1/p47phox-mutant mice.

BACKGROUND: Colitis is a common clinical complication in chronic granulomatous disease (CGD), a primary immunodeficiency caused by impaired oxidative burst. Existing experimental data from NADPH-oxidase knockout mice propose contradictory roles for the involvement of reactive oxygen species in colitis chronicity and severity. Since genetically controlled mice with a point-mutation in the Ncf1 gene are susceptible to chronic inflammation and autoimmunity, we tested whether they presented increased predisposition to develop chronic colitis. METHODS: Colitis was induced in Ncf1-mutant and wild-type mice by a 1st 7-days cycle of dextran sulfate sodium (DSS), intercalated by a 7-days resting period followed by a 2nd 7-days DSS-cycle. Cytokines were quantified locally in the colon inflammatory infiltrates and in the serum. Leukocyte infiltration and morphological alterations of the colon mucosa were assessed by immunohistochemistry. RESULTS: Clinical scores demonstrated a more severe colitis in Ncf1-mutant mice than controls, with no recovery during the resting period and a severe chronic colitis after the 2nd cycle, confirmed by histopathology and presence of infiltrating neutrophils, macrophages, plasmocytes and lymphocytes in the colon. Severe colitis was mediated by increased local expression of cytokines (IL-6, IL-10, TNF-α, IFN-γ and IL-17A) and phosphorylation of Leucine-rich repeat kinase 2 (LRRK2). Serological cytokine titers of those inflammatory cytokines were more elevated in Ncf1-mutant than control mice, and were accompanied by systemic changes in functional subsets of monocytes, CD4+ T and B cells. CONCLUSION: This suggests that an ineffective oxidative burst leads to severe chronic colitis through local accumulation of peroxynitrites, pro-inflammatory cytokines and lymphocytes and systemic immune deregulation similar to CGD.

Reactive oxygen species deficiency induces autoimmunity with type 1 interferon signature.

AIMS: Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the phagocyte reactive oxygen species (ROS)-producing NOX2 enzyme complex and characterized by recurrent infections associated with hyperinflammatory and autoimmune manifestations. A translational, comparative analysis of CGD patients and the corresponding ROS-deficient Ncf1(m1J) mutated mouse model was performed to reveal the molecular pathways operating in NOX2 complex deficient inflammation. RESULTS: A prominent type I interferon (IFN) response signature that was accompanied by elevated autoantibody levels was identified in both mice and humans lacking functional NOX2 complex. To further underline the systemic lupus erythematosus (SLE)-related autoimmune process, we show that naïve Ncf1(m1J) mutated mice, similar to SLE patients, suffer from inflammatory kidney disease with IgG and C3 deposits in the glomeruli. Expression analysis of germ-free Ncf1(m1J) mutated mice reproduced the type I IFN signature, enabling us to conclude that the upregulated signaling pathway is of endogenous origin. INNOVATION: Our findings link the previously unexplained connection between ROS deficiency and increased susceptibility to autoimmunity by the discovery that activation of IFN signaling is a major pathway downstream of a deficient NOX2 complex in both mice and humans. CONCLUSION: We conclude that the lack of phagocyte-derived oxidative burst is associated with spontaneous autoimmunity and linked with type I IFN signature in both mice and humans.

Alterations in the peripheral blood B cell subpopulations of multidrug-resistant tuberculosis patients.

The function of B cells in the immune response against Mycobacterium tuberculosis (Mtb) is still regarded as secondary, although major findings in mouse models of tuberculosis (TB) support their participation as regulators and antibody producers. However, studies in cohorts of TB or multidrug-resistant TB (MDR-TB) patients have failed to clearly identify changes in the circulating B cell pool. Therefore, in the present study we aimed at identifying alterations in the different B cell subpopulations in peripheral blood samples of HIV-negative pulmonary MDR-TB patients when compared to healthy donors. The data show, for the first time, that MDR-TB patients, similarly to what has been observed in other chronic inflammatory diseases, have a much lower frequency of peripheral blood unswitched IgD(+)CD27(+) memory B cells. Equally novel are the findings that in MDR-TB patients there is a reduction in the circulating plasma cell pool and that in MDR-TB there is an increased frequency of circulating type 1 transitional IgD(+)CD38(++), CD69(+) and TLR9(+) B cells. These results document disease-related shifts in peripheral blood B cell subsets in MDR-TB and suggest that such changes should be taken into account when designing new strategies to boost the cellular and humoral immune response against Mtb.

Potential roles for CD8(+) T cells in rheumatoid arthritis.

CD8(+) T cells have long been suggested to play a role in rheumatoid arthritis (RA). The current paradigm on the pathogenesis and maintenance of the disease would endorse these cells with predominantly protective and minor influences. However, several animal studies suggest that these cells may have a predominantly proinflammatory (cytotoxic) effect in the disease. Other studies claim otherwise, that they have a mainly regulatory role in inflammatory joints. The evidence in human disease is remarkably scarce. Studies in human samples indicate that CD8(+) T cells play an important role in the establishment of germinal centers observed in nearly 50% of RA patients, which may have a decisive role in the initiation and maintenance of the disease process. The conflicting results of experimental studies, the scarcity of data and the complexity of research needed to unravel these complex interactions may explain the relative oblivion of CD8 cells in the field of arthritis over recent decades. Is this a wise decision or may we run the risk of not finding the key to RA because we search for it where there is light as opposed to its probable location? The present review brings together available data on the potential role of CD8(+) T cells in inflammation, with emphasis on rheumatoid arthritis, hoping to foster interest and fresh research in this area.

Monoclonal anti-CD8 therapy induces disease amelioration in the K/BxN mouse model of spontaneous chronic polyarthritis.

OBJECTIVE: CD8+ T cells are part of the T cell pool infiltrating the synovium in rheumatoid arthritis (RA). However, their role in the pathogenesis of RA has not been fully delineated. Using the K/BxN mouse model of spontaneous chronic arthritis, which shares many similarities with RA, we studied the potential of CD8+ T cell depletion with monoclonal antibodies (mAb) to stop and reverse the progression of experimental arthritis. METHODS: CD8+ T cells from the blood and articular infiltrate of K/BxN mice were characterized for cell surface phenotypic markers and for cytokine production. Additionally, mice were treated with specific anti-CD8 mAb (YTS105 and YTS169.4), with and without thymectomy. RESULTS: CD8+ T cells from the peripheral blood and joints of K/BxN mice were mainly CD69+ and CD62L-CD27+ T cells expressing proinflammatory cytokines (interferon-γ [IFNγ], tumor necrosis factor α [TNFα], interleukin-17a [IL-17A], and IL-4), and granzyme B. In mice receiving anti-CD8 mAb, the arthritis score improved 5 days after treatment. Recovery of the CD8+ T cells was associated with a new increase in the arthritis score after 20 days. In thymectomized and anti-CD8 mAb-treated mice, the arthritis score improved permanently. Histologic analysis showed an absence of inflammatory infiltrate in the anti-CD8 mAb-treated mice. In anti-CD8 mAb-treated mice, the serologic levels of TNFα, IFNγ, IL-6, and IL-5 normalized. The levels of the disease-related anti-glucose-6-phosphate isomerase antibodies did not change. CONCLUSION: These results indicate that synovial activated effector CD8+ T cells locally synthesize proinflammatory cytokines (IFNγ, TNFα, IL-17, IL-6) and granzyme B in the arthritic joint, thus playing a pivotal role in maintaining chronic synovitis in the K/BxN mouse model of arthritis.

Alterations in peripheral blood memory B cells in patients with active rheumatoid arthritis are dependent on the action of tumour necrosis factor.

INTRODUCTION: Disturbances in peripheral blood memory B cell subpopulations have been observed in various autoimmune diseases, but have not been fully delineated in rheumatoid arthritis (RA). Additionally, the possible role of tumour necrosis factor (TNF) in regulating changes in specific peripheral blood memory B cell subsets in RA is still unclear. METHODS: The frequency and distribution of B cell subsets in the peripheral blood and synovial membrane of active RA patients with long-standing disease have been analysed. Additionally, the possible role of TNF in causing disturbances in memory B cell subsets in RA patients was assessed in a clinical trial with the specific TNF-neutralising antibody, infliximab. RESULTS: RA patients, independent of disease duration, have a significantly lower frequency of peripheral blood pre-switch IgD+CD27+ memory B cells than healthy individuals, whereas post-switch IgD-CD27+ accumulate with increased disease duration. Notably, both pre-switch IgD+CD27+ and post-switch IgD-CD27+ memory B cells accumulate in the synovial membrane of RA patients. Finally, anti-TNF therapy increased the frequency of pre-switch IgD+CD27 memory B cells in the peripheral blood. CONCLUSIONS: The data suggest that decreases in peripheral blood IgD+CD27+ pre-switch memory B cells in RA reflect their accumulation in the synovial tissue. Moreover, the significant increase in the peripheral blood pre-switch memory B cells in patients who underwent specific TNF-blockade with infliximab indicates that trafficking of memory B cells into inflamed tissue in RA patients is regulated by TNF and can be corrected by neutralising TNF.

The NF-kappaB canonical pathway is involved in the control of the exonucleolytic processing of coding ends during V(D)J recombination.

V(D)J recombination is essential to produce an Ig repertoire with a large range of Ag specificities. Although NF-kappaB-binding sites are present in the human and mouse IgH, Igkappa, and Iglambda enhancer modules and RAG expression is controlled by NF-kappaB, it is not known whether NF-kappaB regulates V(D)J recombination mechanisms after RAG-mediated dsDNA breaks. To clarify the involvement of NF-kappaB in human V(D)J recombination, we amplified Ig gene rearrangements from individual peripheral B cells of patients with X-linked anhidrotic ectodermal dysplasia with hyper-IgM syndrome (HED-ID) who have deficient expression of the NF-kappaB essential modulator (NEMO/Ikkgamma). The amplification of nonproductive Ig gene rearrangements from HED-ID B cells reflects the influence of the Ikkgamma-mediated canonical NF-kappaB pathway on specific molecular mechanisms involved in V(D)J recombination. We found that the CDR3(H) from HED-ID B cells were abnormally long, as a result of a marked reduction in the exonuclease activity on the V, D, and J germline coding ends, whereas random N-nucleotide addition and palindromic overhangs (P nucleotides) were comparable to controls. This suggests that an intact canonical NF-kappaB pathway is essential for normal exonucleolytic activity during human V(D)J recombination, whereas terminal deoxynucleotide transferase, Artemis, and DNA-dependent protein kinase catalytic subunit activity are not affected. The generation of memory B cells and somatic hypermutation were markedly deficient confirming a role for NF-kappaB in these events of B cell maturation. However, selection of the primary B cell repertoire appeared to be intact and was partially able to correct the defects generated by abnormal V(D)J recombination.

Highlights of Mediterranean clinical immunology.

The immune system continues to fascinate by the complexity of its intricacies. At the First Mediterranean Workshop on Clinical Immunology held in Evora (Portugal), recently identified mechanisms of immune defense and immunoregulation were put under a magnifying glass by an international cast of immunologists. Studies of Bacillus anthracis revealed that this anaerobic bacterium can inhibit type-II A phospholipase synthesis and secretion by alveolar macrophages, thereby subverting the pulmonary host immune response. Investigation of the mode of action of regulatory T cells indicated that FOXP3 binds the heterodimeric transcription factor AML1 and suppresses AML1-enhanced IL-2 production. In an experimental autoimmune model of prostatitis, a non-hypercalcemic vitamin D receptor agonist was able to interfere with key pathogenic events in already established disease. Other studies in the rat suggest that treating arthritis with oxidants, like phytol, may correct the deficient redox level and prevent T cell autoreactivity. With a number of other observations, the sparkling discussions opened new doors for medical immunology around the Mediterranean, but also elsewhere.

Developmental changes in the human heavy chain CDR3.

The CDR3 of the Ig H chain (CDR3(H)) is significantly different in fetal and adult repertoires. To understand the mechanisms involved in the developmental changes in the CDR3(H) of Ig H chains, sets of nonproductive V(H)DJ(H) rearrangements obtained from fetal, full-term neonates and adult single B cells were analyzed and compared with the corresponding productive repertoires. Analysis of the nonproductive repertoires was particularly informative in assessing developmental changes in the molecular mechanisms of V(H)DJ(H) recombination because these rearrangements did not encode a protein and therefore their distribution was not affected by selection. Although a number of differences were noted, the major reasons that fetal B cells expressed Ig H chains with shorter CDR3(H) were both diminished TdT activity in the DJ(H) junction and the preferential use of the short J(H) proximal D segment D7-27. The enhanced usage of D7-27 by fetal B cells appeared to relate to its position in the locus rather than its short length. The CDR3(H) progressively acquired a more adult phenotype during ontogeny. In fetal B cells, there was decreased recurrent DJ(H) rearrangements before V(H)-DJ(H) rearrangement and increased usage of junctional microhomologies both of which also converted to the adult pattern during ontogeny. Overall, these results indicate that the decreased length and complexity of the CDR3(H) of fetal B cells primarily reflect limited enzymatic modifications of the joins as well as a tendency to use proximal D and J(H) segments during DJ(H) rearrangements.

Characterization of the human Ig heavy chain antigen binding complementarity determining region 3 using a newly developed software algorithm, JOINSOLVER.

We analyzed 77 nonproductive and 574 productive human V(H)DJ(H) rearrangements with a newly developed program, JOINSOLVER. In the productive repertoire, the H chain complementarity determining region 3 (CDR3(H)) was significantly shorter (46.7 +/- 0.5 nucleotides) than in the nonproductive repertoire (53.8 +/- 1.9 nucleotides) because of the tendency to select rearrangements with less TdT activity and shorter D segments. Using criteria established by Monte Carlo simulations, D segments could be identified in 71.4% of nonproductive and 64.4% of productive rearrangements, with a mean of 17.6 +/- 0.7 and 14.6 +/- 0.2 retained germline nucleotides, respectively. Eight of 27 D segments were used more frequently than expected in the nonproductive repertoire, whereas 3 D segments were positively selected and 3 were negatively selected, indicating that both molecular mechanisms and selection biased the D segment usage. There was no bias for D segment reading frame (RF) use in the nonproductive repertoire, whereas negative selection of the RFs encoding stop codons and positive selection of RF2 that frequently encodes hydrophilic amino acids were noted in the productive repertoire. Except for serine, there was no consistent selection or expression of hydrophilic amino acids. A bias toward the pairing of 5' D segments with 3' J(H) segments was observed in the nonproductive but not the productive repertoire, whereas V(H) usage was random. Rearrangements using inverted D segments, DIR family segments, chromosome 15 D segments and multiple D segments were found infrequently. Analysis of the human CDR3(H) with JOINSOLVER has provided comprehensive information on the influences that shape this important Ag binding region of V(H) chains.