Jean-Jacques Fournié

Bromohydrin pyrophosphate enhances antibody-dependent cell-mediated cytotoxicity induced by therapeutic antibodies

In human blood, 1% to 5% of lymphocytes are gammadelta T cells; they mostly express the gammadelta T-cell receptor (TCR)Vgamma9, recognize nonpeptide phosphoantigens (PAgs) produced by microbes and tumor cells, and mediate different modes of lytic activities directed against tumor target cells. Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by cytolytic lymphoid cells is essential for the clinical activity of anticancer monoclonal antibodies (mAbs), but whether PAgs affect ADCC by gammadelta T cells is unknown. Here we report that, in association with the CD20(+)-specific mAb rituximab (RTX), the synthetic PAg bromohydrin pyrophosphate (BrHPP) increased TCRVgamma9(+) cell binding to CD20(+) lymphoma cells in vitro. This combination activated phospho-ZAP70 and phospho-ERK1/2 signaling in TCRVgamma9(+) cells and strongly enhanced their ADCC activity. We obtained similar results with BrHPP in the context of the mAbs alemtuzumab and trastuzumab. Furthermore, BrHPP enhanced RTX-mediated depletion of CD20(+) cells in vitro from peripheral blood mononuclear cells of healthy subjects and enhanced ADCC by gammadelta T cells from patients with chronic lymphocytic leukemia. In cynomolgus macaques, a regimen combining RTX, BrHPP, and IL2 activated TCRVgamma9(+) lymphocytes and enhanced B-cell depletion from blood and lymph nodes. Thus, the combination with BrHPP PAg is able to improve the efficacy of cancer immunotherapy by therapeutic mAbs.

Rituximab inhibits B-cell receptor signaling.

Rituximab (RTX), a monoclonal antibody directed against the CD20 protein, is a drug commonly used in the treatment of B-cell-derived lymphoid neoplasias and of antibody-mediated autoimmune diseases. In addition to cell- and complement-mediated B-cell depletion, RTX is thought to inhibit B-cell survival and proliferation through negative regulation of canonical signaling pathways involving Akt, ERK, and mammalian target of rapamycin. However, surprisingly, although B-cell receptor (BCR) signaling has been considered critical for normal and more recently, for neoplastic B cells, the hypothesis that RTX could target BCR has never been investigated. Using follicular lymphoma cell lines as models, as well as normal B cells, we show here, for the first time, that pretreatment with RTX results in a time-dependent inhibition of the BCR-signaling cascade involving Lyn, Syk, PLC gamma 2, Akt, and ERK, and calcium mobilization. The inhibitory effect of RTX correlates with decrease of raft-associated cholesterol, complete inhibition of BCR relocalization into lipid raft microdomains, and down-regulation of BCR immunoglobulin expression. Thus, RTX-mediated alteration of BCR expression, dynamics, and signaling might contribute to the immunosuppressive activity of the drug.

Genomic and phenotypic characterization of nurse-like cells that promote drug resistance in chronic lymphocytic leukemia

Here, we examined both genetic and phenotypic determinants of nurse-like cells (NLCs) to better predict their putative functions in chronic lymphocytic leukemia (CLL). We showed that NLCs belong to the wound-healing macrophage subset (so-called ‘M2 subset’), but most of all, unsupervised clustering analysis positions NLCs as CLL-specific tumor associated macrophages (TAMs). Chemokinome assays confirmed that NLCs secrete prototypical M2 cytokines and chemokines. Extending previous reports of fludarabine resistance, NLCs are able to promote multidrug resistance. Altogether, we propose that NLCs are not just nursing, but actively participate in the setting of an environment-mediated drug resistance (EM-DR) and immune escape for CLL cells.

Pitfalls on the roadmap to γδ T cell-based cancer immunotherapies

Human TCRVgamma9Vdelta2+ gammadelta cells are unconventional T lymphocytes which had not been specifically mobilized in any cancer vaccine or immunotherapy so far, although they present the major advantage of mediating an HLA-unrestricted highly cytotoxic activity directed against most human tumor cell types. The rapid development of highly selective small molecule agonists targeting these lymphocytes has recently paved the way to clinical trials assessing their activity against cancer. Despite the serious hopes borne by these new weapons however, many pathways usually enable tumor cells to escape conventional immune responses. Here this minireview discusses how cancer immunoevasion pathways might be relevant to counteract the therapeutic bioactivity of TCRVgamma9Vdelta2+ gammadelta cells.

Hammada scoparia flavonoids and rutin kill adherent and chemoresistant leukemic cells

In search for compounds able to reduce cell adhesion-mediated drug resistance (CAM-DR), we studied effects of Hammada scoparia extracts on leukemic cells adherent or in suspension. We show that H. scoparia flavonoidic fraction and its compound rutin induce apoptosis specifically in adherent leukemic cells and abolish CAM-DR. Importantly, rutin inhibited survival of adherent leukemic progenitors (CD34(+)38(-)123(+)) but spared normal progenitors (CD34(+)38(-)). The pro-apoptotic effects of rutin were correlated with a decrease of active GSK3β and inhibitors of GSK3β reproduced rutin-induced cytotoxicity. This study uncovers the potential of H. scoparia flavonoids and rutin to overcome CAM-DR in acute myeloid leukemia.

The gene expression profile of phosphoantigen-specific human γδ T lymphocytes is a blend of αβ T-cell and NK-cell signatures

Global transcriptional technologies have revolutionised the study of lymphoid cell populations, but human γδ T lymphocytes specific for phosphoantigens remain far less deeply characterised by these methods despite the great therapeutic potential of these cells. Here we analyse the transcriptome of circulating TCRVγ+ γδ T cells isolated from healthy individuals, and their relation with those from other lymphoid cell subsets. We report that the gene signature of phosphoantigen‐specific TCRVγ+ γδ T cells is a hybrid of those from αβ T and NK cells, with more ‘NK‐cell’ genes than αβ T cells have and more ‘T‐cell’ genes than NK cells. The expression profile of TCRVγ+ γδ T cells stimulated with phosphoantigen recapitulates their immediate physiological functions: Th1 cytokine, chemokine and cytotoxic activities reflect their high mitotic activity at later time points and do not indicate antigen‐presenting functions. Finally, such hallmarks make the transcriptome of γδ T cells, whether resting or clonally expanding, clearly distinctive from that of NK/T or peripheral T‐cell lymphomas of the γδ subtype.

Several immune escape patterns in non-Hodgkin's lymphomas

Follicular Lymphomas (FL) and diffuse large B cell lymphomas (DLBCL) must evolve some immune escape strategy to develop from lymphoid organs, but their immune evasion pathways remain poorly characterized. We investigated this issue by transcriptome data mining and immunohistochemistry (IHC) of FL and DLBCL lymphoma biopsies. A set of genes involved in cancer immune-evasion pathways (Immune Escape Gene Set, IEGS) was defined and the distribution of the expression levels of these genes was compared in FL, DLBCL and normal B cell transcriptomes downloaded from the GEO database. The whole IEGS was significantly upregulated in all the lymphoma samples but not in B cells or other control tissues, as shown by the overexpression of the PD-1, PD-L1, PD-L2 and LAG3 genes. Tissue microarray immunostainings for PD-1, PD-L1, PD-L2 and LAG3 proteins on additional biopsies from 27 FL and 27 DLBCL patients confirmed the expression of these proteins. The immune infiltrates were more abundant in FL than DLBCL samples, and the microenvironment of FL comprised higher rates of PD-1+ lymphocytes. Further, DLBCL tumor cells comprised a higher proportion of PD-1+, PD-L1+, PD-L2+ and LAG3+ lymphoma cells than the FL tumor cells, confirming that DLBCL mount immune escape strategies distinct from FL. In addition, some cases of DLBCL had tumor cells co-expressing both PD-1, PD-L1 and PD-L2. Among the DLBCLs, the activated B cell (ABC) subtype comprised more PD-L1+ and PD-L2+ lymphoma cells than the GC subtype. Thus, we infer that FL and DLBCL evolved several pathways of immune escape.

Endogenous IL-8 acts as a CD16 co-activator for natural killer-mediated anti-CD20 B cell depletion in chronic lymphocytic leukemia

Rituximab (RTX, anti-CD20 antibody) combined with chemotherapy is currently standard treatment for chronic lymphocytic leukemia (CLL). Serum level of IL-8 is a prognostic factor for CLL that correlates with disease stage. We investigated whether endogenous IL-8 affects RTX or Obinutuzumab (GA101) B-leukemic depletion mediated by natural killers (NK). Using whole peripheral blood lymphocytes from untreated CLL patients, RTX, but most significantly GA101, were effective in B-cell depletion and NK activation. IL-8 inhibition completely inhibited B-cell depletion by RTX and reduced GA101-induced B-cell depletion. Altogether results underline IL-8 as an endogenous NK co-activator and confirm GA101 therapeutic potential for CLL treatment.

Phosphoantigens Overcome Human TCRVγ9+ γδ Cell Immunosuppression by TGF-β: Relevance for Cancer Immunotherapy

Human γδ cells expressing TCRVγ9 are HLA-unrestricted CTLs with high relevance for cancer immunotherapy. Many tumor cell types produce TGF-β, however, a cytokine strongly immunosuppressive for conventional T CD4, CD8, and NK cells. Whether TGF-β also inhibits TCRVγ9+ lymphocytes was unknown. Because phosphoantigens (PAgs), such as bromohydrin pyrophosphate, selectively activate the antitumor functions of TCRVγ9+ T cells, in this study, we investigated whether TGF-β modulates these functions. We report that TGF-β does not block activation of TCRVγ9+ T cells but inhibits their PAg/IL-2–induced proliferation and maturation into effector cells and finally reduces the cytotoxic activity of these γδ T cells when exposed to lymphoma target cells. TGF-β did not bias their differentiation pattern toward γδ Th17 or γδ regulatory T cells. Nevertheless, increasing doses of PAg stimulus countered TGF-β inhibition. So, although TGF-β impairs TCRVγ9+ γδ cells like other cytolytic lymphocytes, PAg alone or combined to therapeutic mAb has the ability to bypass its immunosuppressive activity.

How tumors might withstand γδ T-cell attack.

Several clinical trials are currently assessing the therapeutic activity of human TCRVγ9Vδ2+ lymphocytes in cancer. Growing tumors usually follow a triphasic “Elimination, Equilibrium, Escape” evolution in patients. Thus, at diagnostic, most tumors have already developed some means to escape to immune protection. We review here the conventional immunoescape mechanisms which might also protect against cytolytic TCRVγ9Vδ2+ lymphocytes activated by phosphoantigens. Neutralization of these deleterious processes might prove highly valuable to improve the efficacy of ongoing γδ cell-based cancer immunotherapies.