Janny Liautard

The myeloma cell antigen syndecan-1 is lost by apoptotic myeloma cells

Syndecan‐1 is a cell membrane proteoglycan that binds extracellular matrix components and various growth factors. It is expressed only on malignant plasma cells in bone marrow samples from patients with multiple myeloma (MM). Several reports have suggested that syndecan‐1 was present only on a part of the myeloma cells. By using either IL‐6‐dependent myeloma cell lines or primary myeloma cells stained by annexin V, we report here that syndecan‐1 was rapidly lost by myeloma cells undergoing apoptosis. In the same experimental conditions, expression of other cell membrane antigens such as CD38, HLA class‐I or CD49d on apoptotic myeloma cells was not affected. In addition, we show that syndecan‐1 loss was independent of activation of the gp130 IL‐6 transducer. Dexamethasone induced a strong apoptosis of myeloma cells associated with the loss of syndecan‐1. Finally, by using freshly‐explanted tumoural samples, we show that syndecan‐1 rapidly disappeared from myeloma cells in association with induction of apoptosis. In conclusion we showed that syndecan‐1 is a marker for viable myeloma cells which is rapidly lost by apoptotic cells. These results emphasize the usefulness of anti‐syndecan‐1 antibodies to purge tumoural cells from haemopoietic grafts or to purify these cells for further manipulations for immuno or gene therapies.

Production of TNF-α by Human Vγ9Vδ2 T Cells Via Engagement of FcγRIIIA, the Low Affinity Type 3 Receptor for the Fc Portion of IgG, Expressed upon TCR Activation by Nonpeptidic Antigen

Human lymphocytes expressing the γδ TCR represent a minor T cell subpopulation found in blood. The majority of these cells express Vγ9Vδ2 determinants and respond to nonpeptidic phosphoantigens. Several studies have shown that, in vivo, the percentage of Vγ9Vδ2 T cells dramatically increases during pathological infection, leading to the hypothesis that they play an important role in the defense against pathogens. However, the specific mechanisms involved in this response remain poorly understood. It has been established that Vγ9Vδ2 T cells display potent cytotoxic activity against virus-infected and tumor cells, thereby resembling NK cells. In this study, we show that, upon stimulation by nonpeptidic Ags, Vγ9Vδ2 T cells express FcγRIIIA (CD16), a receptor that is constitutively expressed on NK cells. CD16 appears to be an activation Ag for Vγ9Vδ2 T cells. Indeed, ligation of CD16 on Vγ9Vδ2 T cells leads to TNF-α production. This TNF-α production, which is dependent (like that induced via the TCR-CD3 complex) on the activation of the p38 and extracellular signal-regulated kinase-2 mitogen-activated protein kinases, can be modulated by CD94 NK receptors. Therefore, it appears that Vγ9Vδ2 T cells can be physiologically activated by two sequential steps via two different cell surface Ags: the TCR-CD3 complex and the FcγRIIIA receptor, which are specific cell surface Ags for T lymphocytes and NK cells, respectively. This strongly suggests that, in the general scheme of the immune response, Vγ9Vδ2 T cells represent an important subpopulation of cells that play a key role in the defense against invading pathogens.

Release of LL-37 by Activated Human Vγ9Vδ2 T Cells: A Microbicidal Weapon against Brucella suis

Human Vγ9Vδ2 T cells play a crucial role in early immune response to intracellular pathogens. Moreover, in brucellosis, these cells are drastically increased in the peripheral blood of patients during the acute phase of infection. In vitro, Vγ9Vδ2 T cells are capable of inhibiting Brucella growth and development through a combination of mechanisms: 1) cytotoxicity, 2) macrophage activation and bactericidal activity through cytokine and chemokine secretion, and 3) antibacterial effects. We previously described that antibacterial factors were found in supernatants from activated Vγ9Vδ2 T cells. In this study, we show that Vγ9Vδ2 T cells express the human cathelicidin hCAP18 and its mature form, known as LL-37, is released upon activation of Vγ9Vδ2 T cells. We also show that LL-37 has an antibacterial effect on Brucella suis. Overall, our results demonstrate that LL-37 is a soluble factor responsible for a part of the bactericidal activity of Vγ9Vδ2 T cells.

Vγ9Vδ2 T cells use a combination of mechanisms to limit the spread of the pathogenic bacteria Brucella

Human Vγ9Vδ2 T cells pay a crucial role in early immune response to intracellular pathogens. In brucellosis infection, this population of cells is drastically increased in the peripheral blood of patients during the acute phase of infection. In vitro, Vγ9Vδ2 T cells exhibit strong cytolytic activity against Brucella‐infected cells and are able to impair intracellular growth of Brucella suis in autologous macrophages. In this study, we have investigated the relative importance of contact‐dependent mechanisms versus soluble factors in the intracellular growth and viability of B. suis. We show that Vγ9Vδ2 T cells use contact‐dependent mechanisms, such as the release of lytic granules and Fas‐mediated signals, to decrease intracellular B. suis through lysis of infected macrophages, but these mechanisms have little impact on Brucella survival. Moreover, we demonstrate that soluble factors secreted by Vγ9Vδ2 T cells can directly affect B. suis survival through their potent bactericidal effects. From these results, we conclude that Vγ9Vδ2 T cells are able to use a combination of mechanisms that reduce the total numbers of B. suis and thus, may benefit the host by limiting the spread of this intracellular pathogen.

Human anti-mouse antibody response to the injection of murine monoclonal antibodies against IL-6

We analysed human anti‐mouse antibodies (HAMA) in 12 patients (six with multiple myeloma (MM) and six with metastatic renal cell carcinoma (MRCC)) who were treated with B‐E8, an IgG1 MoAb against IL‐6. Efficiency of the treatment was evidenced by the drop in the serum levels of C‐reactive protein (CRP), the in vivo production of which is under the control of IL‐6. Three patients with MM and the six patients with MRCC became immunized m the injected MoAb. HAMA appeared between days 7 and 15 after the beginning of the treatment. The nine patients made IgG antibodies: four also made IgM. All immunized patients made anti‐biotype antibodies specific to B‐E8. Two of them also developed HAMA directed to murine IgG1 isotype: in these two patients B‐E8 MoAb cleared rapidly from the circulation with loss of treatment efficiency. In the patients who developed only anti‐biotype antibodies, serum levels of B‐E8 remained unchanged and CRP production remained inhibited, indicating that treatment remained efficient in the presence of HAMA, Circulating B‐E8 MoAbs were still able to bind to iL‐6 and to inhibit IL‐6‐dependent proliferation despite the presence of anti‐idiotypic HAMA, Therefore, in contrast to HAMA produced against MoAb directed against cellular targets, HAMA against anti‐IL‐6 MoAb idiotopes led neither to clearance nor to functional inactivation of the injected MoAb. This was further shown by resuming the B‐E8 treatment with success in a patient who still had anti‐idiotypic HAMA.

Immunomodulating IL-6 activity by murine monoclonal antibodies

The human anti-mouse immunoglobulin antibody (HAMA) response, which occurs frequently after injection of murine monoclonal antibodies (MAb) directed against cellular targets, has been reported extensively in several studies. We analysed here HAMA in 12 patients (six with multiple myeloma, MM, and six with metastatic renal cell carcinoma, MRCC) who were treated with B-E8, an IgG1 MAb against interleukin-6 (IL-6). Efficiency of the treatment was evidenced by the drop in the serum levels of C reactive protein (CRP), of which the in vivo production is under the control of IL-6. Three patients with MM and the six patients with MRCC became immunized to the injected MAb. HAMA appeared between days 7 and 15 after the beginning of the treatment. The nine patients made IgG antibodies; four also made IgM. All of immunized patients made anti-idiotype antibodies specific to B-E8. Two of them also developed HAMA directed to murine IgG1 isotype; in these two patients B-E8 MAb cleared rapidly from the circulation with loss of treatment efficiency. In the patients who developed only anti-idiotype antibodies, serum levels of B-E8 remained unchanged and CRP production remained inhibited, indicating that treatment efficiency was not affected by the presence of HAMA. Circulating B-E8 MAb were still able to bind to IL-6 and to inhibit IL-6-independent proliferation despite the presence of anti-idiotypic HAMA. Therefore, in contrast to HAMA against MAb directed against cellular targets, HAMA against anti-IL-6 MAb idiotopes led neither to clearance nor to functional inactivation of the injected MAb.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific Signaling Pathways Triggered by IL-2 in Human Vγ9Vδ2 T Cells: An Amalgamation of NK and αβ T Cell Signaling

The global immune response can be simplified into two components: the innate and the acquired systems. The innate immune response comprises primarily macrophages and NK cells, while B and T cells orchestrate the acquired response. Human Vγ9Vδ2 T cells represent a minor T cell subpopulation in blood (1–5%) that is activated via the TCR by small nonpeptidic molecules. Their percentage dramatically increases during the early phase of infection by intracellular pathogens, and they display many characteristics of NK cells, which places them at a unique position within the immune system. Our aim was to explore the behavior of these cells when they are activated by a receptor that is common to NK and αβ T cells, and to determine signaling pathways and biological responses induced in these cells through this receptor. Thus, we investigated whether Vγ9Vδ2 T cells behave as NK cells or as αβ T cells. We demonstrated that IL-2 activates not only STAT3, STAT5, the phosphatidylinositol 3-kinase pathway, and extracellular signal-regulated kinase-2 pathway, but also STAT4 as in NK cells, and the p38 mitogen-activated protein kinase pathway as in αβ T cells. Moreover, IL-2 induces the production of IFN-γ in Vγ9Vδ2 T cells as observed in NK cells. Due to their double profiles, Vγ9Vδ2 T cells are at the interface of the innate and the acquired immune response and may therefore not only modulate the activity of innate cells, but also influence Th1/Th2 differentiation.