Impact of viral hepatitis therapy in multiple myeloma and other monoclonal gammopathies linked to hepatitis B or C viruses.

Subsets of multiple myeloma (MM) and monoclonal gammopathies of undetermined significance (MGUS) present with a monoclonal immunoglobulin specific for hepatitis C virus (HCV), thus are presumably HCV-driven, and antiviral treatment can lead to the disappearance of antigen stimulation and improved control of clonal plasma cells. Here we studied the role of hepatitis B virus (HBV) in the pathogenesis of MGUS and MM in 45 HBV-infected patients with monoclonal gammopathy. We analyzed the specificity of recognition of the monoclonal immunoglobulin of these patients and validated the efficacy of antiviral treatment (AVT). For 18 of 45 (40%) HBV-infected patients, the target of the monoclonal immunoglobulin was identified: the most frequent target was HBV (n=11), followed by other infectious pathogens (n=6) and glucosylsphingosine (n=1). Two patients whose monoclonal immunoglobulin targeted HBV (HBx and HBcAg), implying that their gammopathy was HBV-driven, received AVT and the gammopathy did not progress. AVT efficacy was then investigated in a large cohort of HBV-infected MM patients (n=1367) who received or did not receive anti-HBV treatments and compared to a cohort of HCV-infected MM patients (n=1220). AVT significantly improved patient probability of overall survival (P=0.016 for the HBV-positive cohort, P=0.005 for the HCV-positive cohort). Altogether, MGUS and MM disease can be HBV- or HCV-driven in infected patients, and the study demonstrates the importance of AVT in such patients.

Comparison of Monoclonal Gammopathies Linked to Poliovirus or Coxsackievirus vs. Other Infectious Pathogens.

Chronic stimulation by infectious pathogens or self-antigen glucosylsphingosine (GlcSph) can lead to monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). Novel assays such as the multiplex infectious antigen microarray (MIAA) and GlcSph assays, permit identification of targets for >60% purified monoclonal immunoglobulins (Igs). Searching for additional targets, we selected 28 purified monoclonal Igs whose antigen was not represented on the MIAA and GlcSph assays; their specificity of recognition was then analyzed using microarrays consisting of 3760 B-cell epitopes from 196 pathogens. The peptide sequences PALTAVETG and PALTAAETG of the VP1 coat proteins of human poliovirus 1/3 and coxsackievirus B1/B3, respectively, were specifically recognized by 6/28 monoclonal Igs. Re-analysis of patient cohorts showed that purified monoclonal Igs from 10/155 MGUS/SM (6.5%) and 3/147 MM (2.0%) bound to the PALTAVETG or PALTAAETG epitopes. Altogether, PALTAV/AETG-initiated MGUS are not rare and few seem to evolve toward myeloma.

Efficacy of Antiviral Treatment in Hepatitis C Virus (HCV)-Driven Monoclonal Gammopathies Including Myeloma.

Multiple myeloma (MM) remains an incurable plasma cell malignancy. While its origin is enigmatic, an association with infectious pathogens including hepatitis C virus (HCV) has been suggested. Here we report nine patients with monoclonal gammopathy of undetermined significance (MGUS) or MM with previous HCV infection, six of whom received antiviral treatment. We studied the evolution of the gammopathy disease, according to anti-HCV treatment and antigen specificity of purified monoclonal immunoglobulin, determined using the INNO-LIA™ HCV Score assay, dot-blot assays, and a multiplex infectious antigen microarray. The monoclonal immunoglobulin from 6/9 patients reacted against HCV. Four of these patients received antiviral treatment and had a better evolution than untreated patients. Following antiviral treatment, one patient with MM in third relapse achieved complete remission with minimal residual disease negativity. For two patients who did not receive antiviral treatment, disease progressed. For the two patients whose monoclonal immunoglobulin did not react against HCV, antiviral treatment was not effective for MGUS or MM disease. Our results suggest a causal relationship between HCV infection and MGUS and MM progression. When HCV was eliminated, chronic antigen-stimulation disappeared, allowing control of clonal plasma cells. This opens new possibilities of treatment for MGUS and myeloma.

Anti-Glucosylsphingosine Autoimmunity, JAK2V617F-Dependent Interleukin-1ß and JAK2V617F-Independent Cytokines in Myeloproliferative Neoplasms.

Inflammatory cytokines play a major role in myeloproliferative neoplasms (MPNs) as regulators of the MPN clone and as mediators of clinical symptoms and complications. Firstly, we investigated the effect of JAK2V617F on 42 molecules linked to inflammation. For JAK2V617F-mutated patients, the JAK2V617F allele burden (%JAK2V617F) correlated with the levels of IL-1ß, IL-1Rα, IP-10 and leptin in polycythemia vera (PV), and with IL-33 in ET; for all other molecules, no correlation was found. Cytokine production was also studied in the human megakaryocytic cell line UT-7. Wild-type UT-7 cells secreted 27/42 cytokines measured. UT-7 clones expressing 50% or 75% JAK2V617F were generated, in which the production of IL-1ß, IP-10 and RANTES was increased; other cytokines were not affected. Secondly, we searched for causes of chronic inflammation in MPNs other than driver mutations. Since antigen-driven selection is increasingly implicated in the pathogenesis of blood malignancies, we investigated whether proinflammatory glucosylsphingosine (GlcSph) may play a role in MPNs. We report that 20% (15/75) of MPN patients presented with anti-GlcSph IgGs, distinguished by elevated levels of 11 cytokines. In summary, only IL-1ß and IP-10 were linked to JAK2V617F both in patients and in UT-7 cells; other inflammation-linked cytokines in excess in MPNs were not. For subsets of MPN patients, a possible cause of inflammation may be auto-immunity against glucolipids.

Analysis of the Targets and Glycosylation of Monoclonal IgAs From MGUS and Myeloma Patients.

Previous studies showed that monoclonal immunoglobulins G (IgGs) of "monoclonal gammopathy of undetermined significance" (MGUS) and myeloma were hyposialylated, thus presumably pro-inflammatory, and for about half of patients, the target of the monoclonal IgG was either a virus-Epstein-Barr virus (EBV), other herpes viruses, hepatitis C virus (HCV)-or a glucolipid, lysoglucosylceramide (LGL1), suggesting antigen-driven disease in these patients. In the present study, we show that monoclonal IgAs share these characteristics. We collected 35 sera of patients with a monoclonal IgA (6 MGUS, 29 myeloma), and we were able to purify 25 of the 35 monoclonal IgAs (6 MGUS, 19 myeloma). Monoclonal IgAs from MGUS and myeloma patients were significantly less sialylated than IgAs from healthy volunteers. When purified monoclonal IgAs were tested against infectious pathogens and LGL1, five myeloma patients had a monoclonal IgA that specifically recognized viral proteins: the core protein of HCV in one case, EBV nuclear antigen 1 (EBNA-1) in four cases (21.1% of IgA myeloma). Monoclonal IgAs from three myeloma patients reacted against LGL1. In summary, monoclonal IgAs are hyposialylated and as described for IgG myeloma, significant subsets (8/19, or 42%) of patients with IgA myeloma may have viral or self (LGL1) antigen-driven disease.

Characteristics of MGUS and Multiple Myeloma According to the Target of Monoclonal Immunoglobulins, Glucosylsphingosine, or Epstein-Barr Virus EBNA-1.

: Chronic stimulation by infectious or self-antigens initiates subsets of monoclonal gammopathies of undetermined significance (MGUS), smoldering multiple myeloma (SMM), or multiple myeloma (MM). Recently, glucosylsphingosine (GlcSph) was reported to be the target of one third of monoclonal immunoglobulins (Igs). In this study of 233 patients (137 MGUS, 6 SMM, 90 MM), we analyzed the GlcSph-reactivity of monoclonal Igs and non-clonal Igs. The presence of GlcSph-reactive Igs in serum was unexpectedly frequent, detected for 103/233 (44.2%) patients. However, GlcSph was targeted by the patient's monoclonal Ig for only 37 patients (15.9%); for other patients (44 MGUS, 22 MM), the GlcSph-reactive Igs were non-clonal. Then, the characteristics of patients were examined: compared to MM with an Epstein-Barr virus EBNA-1-reactive monoclonal Ig, MM patients with a GlcSph-reactive monoclonal Ig had a mild presentation. The inflammation profiles of patients were similar except for moderately elevated levels of 4 cytokines for patients with GlcSph-reactive Igs. In summary, our study highlights the importance of analyzing clonal Igs separately from non-clonal Igs and shows that, if autoimmune responses to GlcSph are frequent in MGUS/SMM and MM, GlcSph presumably represents the initial pathogenic event for ~16% cases. Importantly, GlcSph-initiated MM appears to be a mild form of MM disease.

Pro-inflammatory State in Monoclonal Gammopathy of Undetermined Significance and in Multiple Myeloma Is Characterized by Low Sialylation of Pathogen-Specific and Other Monoclonal Immunoglobulins.

Multiple myeloma (MM) and its pre-cancerous stage monoclonal gammopathy of undetermined significance (MGUS) allow to study immune responses and the chronology of inflammation in the context of blood malignancies. Both diseases are characterized by the production of a monoclonal immunoglobulin (mc Ig) which for subsets of MGUS and MM patients targets pathogens known to cause latent infection, a major cause of inflammation. Inflammation may influence the structure of both polyclonal (pc) Ig and mc Ig produced by malignant plasma cells via the sialylation of Ig Fc fragment. Here, we characterized the sialylation of purified mc and pc IgGs from 148 MGUS and MM patients, in comparison to pc IgGs from 46 healthy volunteers. The inflammatory state of patients was assessed by the quantification in serum of 40 inflammation-linked cytokines, using Luminex technology. While pc IgGs from MGUS and MM patients showed heterogeneity in sialylation level, mc IgGs from both MGUS and MM patients exhibited a very low level of sialylation. Furthermore, mc IgGs from MM patients were less sialylated than mc IgGs from MGUS patients (p < 0.01), and mc IgGs found to target an infectious pathogen showed a lower level of sialylation than mc IgGs of undetermined specificity (p = 0.048). Regarding inflammation, 14 cytokines were similarly elevated with a p value < 0.0001 in MGUS and in MM compared to healthy controls. MM differed from MGUS by higher levels of HGF, IL-11, RANTES and SDF-1-α (p < 0.05). MGUS and MM patients presenting with hyposialylated pc IgGs had significantly higher levels of HGF, IL-6, tumor necrosis factor-α, TGF-ß1, IL-17, and IL-33 compared to patients with hyper-sialylated pc IgGs (p < 0.05). In MGUS and in MM, the degree of sialylation of mc and pc IgGs and the levels of four cytokines important for the anti-microbial response were correlated, either positively (IFN-α2, IL-13) or negatively (IL-17, IL-33). Thus in MGUS as in MM, hyposialylation of mc IgGs is concomitant with increased levels of cytokines that play a major role in inflammation and anti-microbial response, which implies that infection, inflammation, and abnormal immune response contribute to the pathogenesis of MGUS and MM.

Self/non-self discrimination by human gammadelta T cells: simple solutions for a complex issue?

Although gammadelta T cells express clonally distributed T-cell receptors (TCRs), a hallmark of adaptive immunity, they are classically considered as innate-like effectors, owing to the high frequency of preactivated gammadelta T cells, with restricted antigen recognition repertoire in particular tissue locations. Actually, such features are shared only by a fraction of gammadelta T-cell subsets located in the skin and reproductive organ mucosa in rodents or in peripheral blood in humans. By contrast, other gammadelta subsets, e.g. those found in rodent and human spleen, show diverse antigenic reactivity patterns and mixed naive/memory phenotypes. Thus, gammadelta T cells are made of both 'primitive' subsets endowed with innate-like properties and 'evolved' subsets able to mount anamnestic responses like conventional major histocompatibility complex-restricted alphabeta T cells. In this article, we show that human gammadelta T cells, although heterogeneous, do share recurrent innate features that distinguish them from mainstream alphabeta T cells. In particular, most of them are activated on TCR- or natural killer receptor-mediated recognition of a restricted set of conserved yet poorly defined endogenous stress determinants. This rather simple recognition mechanism allows human gammadelta T cells to discriminate healthy cells from altered cells and to exert a variety of immunostimulatory or regulatory functions. The recent availability of synthetic gammadelta T-cell agonists mimicking these natural stress-induced ligands have fostered development of immunotherapeutic strategies, with broad indications against infectious and tumor diseases, which are briefly reviewed here.

Potentiation of antigen-stimulated V gamma 9V delta 2 T cell cytokine production by immature dendritic cells (DC) and reciprocal effect on DC maturation.

Vgamma9Vdelta2 T cells, a major gammadelta PBL subset in human adults, have been previously implicated in dendritic cell (DC) licensing, owing to their high frequency in peripheral tissues and their ability to produce inflammatory cytokines upon recognition of a broad array of conserved Ags. Although these observations implied efficient recognition of Ag-expressing immature DC (iDC) by Vgamma9Vdelta2 T cells, the role played by DC subsets in activation of these lymphocytes has not been carefully studied so far. We show that iDC, and to a lesser extent mature DC, potentiated Th1 and Th2 cytokine, but not cytolytic or proliferative responses, of established Vgamma9Vdelta2 T cell clones and ex vivo memory Vgamma9Vdelta2 PBL stimulated by synthetic agonists. The ability of iDC to potentiate Vgamma9Vdelta2 production of inflammatory cytokines required for their own maturation suggested that Vgamma9Vdelta2 T cells, despite their strong lytic activity, could promote efficient iDC licensing without killing at suboptimal Ag doses. Accordingly Vgamma9Vdelta2 cells induced accelerated maturation of Ag-expressing iDC but not "bystander" DC, even within mixed cell populations comprising both Ag-expressing and nonexpressing iDC. Furthermore Vgamma9Vdelta2 cells induced full differentiation into IL-12-producing cells of iDC infected by Vgamma9Vdelta2-stimulating mycobacteria that were otherwise unable to induce complete DC maturation. In conclusion the ability of iDC to selectively potentiate cytokine response of memory Vgamma9Vdelta2 T cells could underlie the adjuvant effect of these lymphocytes, and possibly other natural memory T cells, on conventional T cell responses.

Tumor recognition following Vgamma9Vdelta2 T cell receptor interactions with a surface F1-ATPase-related structure and apolipoprotein A-I.

Vgamma9Vdelta2 T lymphocytes, a major gammadelta T lymphocyte subset in humans, display cytolytic activity against various tumor cells upon recognition of yet uncharacterized structures. Here, we show that an entity related to the mitochondrial F1-ATPase is expressed on tumor cell surface and promotes tumor recognition by Vgamma9Vdelta2 T cells. When immobilized, purified F1-ATPase induces selective activation of this lymphocyte subset. The Vgamma9Vdelta2 T cell receptors (TCR) and the F1-ATPase also bind a delipidated form of apolipoprotein A-I (apo A-I), as demonstrated by surface plasmon resonance. Moreover, the presence of apo A-I in the culture medium is required for optimal activation of Vgamma9Vdelta2 T cells by tumors expressing F1-ATPase. This study thus describes an unanticipated tumor recognition mechanism by Vgamma9Vdelta2 lymphocytes and a possible link between gammadelta T cell immunity and lipid metabolism.

Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells.

A group of T cells recognizes glycolipids presented by molecules of the CD1 family. The CD1d-restricted natural killer T cells (NKT cells) are primarily considered to be self-reactive. By employing CD1d-binding and T cell assays, the following structural parameters for presentation by CD1d were defined for a number of mycobacterial and mammalian lipids: two acyl chains facilitated binding, and a polar head group was essential for T cell recognition. Of the mycobacterial lipids tested, only a phosphatidylinositol mannoside (PIM) fulfilled the requirements for CD1d binding and NKT cell stimulation. This PIM activated human and murine NKT cells via CD1d, thereby triggering antigen-specific IFN-gamma production and cell-mediated cytotoxicity, and PIM-loaded CD1d tetramers identified a subpopulation of murine and human NKT cells. This phospholipid, therefore, represents a mycobacterial antigen recognized by T cells in the context of CD1d.