High level of soluble programmed cell death ligand 1 in blood impacts overall survival in aggressive diffuse large B-Cell lymphoma: results from a French multicenter clinical trial.

The dosage of soluble programmed cell death ligand 1 (sPD-L1) protein in the blood of adults with cancer has never been performed in a prospective patient cohort. We evaluated the clinical impact of sPD-L1 level measured at the time of diagnosis for newly diagnosed diffuse large B-cell lymphoma (DLBCL). Soluble PD-L1 was measured in the plasma of 288 patients enrolled in a multicenter, randomized phase III trial that compared R-high-dose chemotherapy with R-CHOP. The median follow-up was 41.4 months. A cutoff of 1.52 ng/ml of PD-L1 level was determined and related to overall survival (OS). Patients with elevated sPD-L1 experienced a poorer prognosis with a 3-year OS of 76% versus 89% (P<0.001). Considering clinical characteristics, the multivariate analysis retained this biomarker besides bone marrow involvement and abnormal lymphocyte-monocyte score as independently related to poor outcome. sPD-L1 was detectable in the plasma and not in the serum, found elevated in patients at diagnosis compared with healthy subjects and its level dropped back to normal value after CR. The intention-to-treat analysis showed that elevated sPD-L1 was associated with a poorer prognosis for patients randomized within the R-CHOP arm (P<0.001). Plasma PD-L1 protein is a potent predicting biomarker in DLBCL and may indicate usefulness of alternative therapeutic strategies using PD-1 axis inhibitors.

Monocytes and T cells cooperate to favor normal and follicular lymphoma B-cell growth: role of IL-15 and CD40L signaling.

Interleukin-15 (IL-15) has been extensively studied for its role in the survival and proliferation of NK and T cells through a unique mechanism of trans-presentation by producer cells. Conversely, whereas activated B cells have been described as IL-15-responding cells, the cellular and molecular context sustaining this effect remains unexplored. In this study, we found that, whereas human B cells could not respond to soluble IL-15, monocytes and lymphoid tissue-derived macrophages but not stromal cells efficiently trans-present IL-15 to normal B cells and cooperate with T-cell-derived CD40L to promote IL-15-dependent B-cell proliferation. Furthermore, CD40L signaling triggers a Src-independent upregulation of STAT5 expression and favors a Src-dependent phosphorylation of STAT5 in response to IL-15. In follicular lymphoma (FL), immunohistochemical studies reported a strong relationship between malignant B cells, infiltrating macrophages and T cells. We show here an overexpression of IL-15 in purified tumor-associated macrophages, and STAT5A in purified tumor B cells. Moreover, FL B cells respond to IL-15 trans-presented by monocytes/macrophages, in particular, in the presence of CD40L-mediated signaling. This cooperation between IL-15 and CD40L reinforces the importance of tumor microenvironment and unravels a mechanism of FL growth that should be considered if using IL-15 as a drug in this disease.

Characterization of intratumoral follicular helper T cells in follicular lymphoma: role in the survival of malignant B cells.

Accumulating evidences indicate that the cellular and molecular microenvironment of follicular lymphoma (FL) has a key role in both lymphomagenesis and patient outcome. Malignant FL B cells are found admixed to specific stromal and immune cell subsets, in particular CD4(pos) T cells displaying phenotypic features of follicular helper T cells (T(FH)). The goal of our study was to functionally characterize intratumoral CD4(pos) T cells. We showed that CXCR5(hi)ICOS(hi)CD4(pos) T cells sorted from FL biopsies comprise at least two separate cell populations with distinct genetic and functional features: (i) CD25(pos) follicular regulatory T cells (T(FR)), and (ii) CD25(neg) T(FH) displaying a FL-B cell supportive activity without regulatory functions. Furthermore, despite their strong similarities with tonsil-derived T(FH), purified FL-derived T(FH) displayed a specific gene expression profile including an overexpression of several genes potentially involved directly or indirectly in lymphomagenesis, in particular TNF, LTA, IL4 or CD40LG. Interestingly, we further demonstrated that these two last signals efficiently rescued malignant B cells from spontaneous and rituximab-induced apoptosis. Altogether, our study demonstrates that tumor-infiltrating CD4(pos) T cells are more heterogeneous than previously presumed, and underlines for the first time the crucial role of T(FH) in the complex set of cellular interactions within FL microenvironment.

Follicular lymphoma cell niche: identification of a preeminent IL-4-dependent T(FH)-B cell axis.

Follicular lymphoma (FL) B cells contract tight connections with their microenvironment, which governs the pathogenesis and progression of the disease. Indeed, specific immune response gene signatures, obtained from whole biopsy samples, have been associated with patient survival. In this study, we performed gene expression profiling of purified B cell and non-B cell compartments obtained from FL and reactive lymph nodes. We identified 677 non-redundant genes defining the FL interface and involving 26 FL-specific functional networks. This approach highlighted an interleukin-4 (IL-4)-centered pathway associated with an activation of signal transducer and activator of transcription 6 (STAT6), which favors overexpression of IL-4-target genes. In addition, FL microenvironment was characterized by a strong enrichment in follicular helper T cells (T(FH)), as demonstrated through transcriptomic and flow cytometry analyses. The majority of phospho-STAT6(pos) B cells were located at the vicinity of cells expressing the programmed death 1 (PD-1) T(FH) marker. Moreover, purified FL-derived T(FH), expressed IL4 at very high levels compared with purified tonsil-derived T(FH) or non-T(FH) microenvironment. Altogether, our study demonstrated that tumor-infiltrating T(FH) specifically express functional IL-4 in FL, creating an IL-4-dependent T(FH)-B cell axis. This cross talk could sustain FL pathogenesis and represent a new potential therapeutic target.

Modulation of HLA-G antigens expression by human cytomegalovirus: specific induction in activated macrophages harboring human cytomegalovirus infection.

After infection, human CMV (HCMV) establishes a latent and persistent infection in immature myeloid progenitors and peripheral blood monocytes. Completion of the HCMV life cycle is possible upon maturation of monocytes to tissue macrophages and under permissive circumstances, e.g., immunosuppression. We investigated the hypothesis that HLA-G molecules could be induced during HCMV reactivation in activated macrophages to favor virus dissemination. In this study, we provide evidence that HLA-G Ags are produced during viral reactivation in macrophages generated after allogeneic stimulation of HCMV latently infected monocytes. While HLA-G surface expression is up-regulated, classical MHC-I molecules are partially down-regulated by HCMV. In vivo, bronchoalveolar macrophages collected from patients suffering from acute HCMV pneumonitis also express HLA-G molecules. The direct correlation between HLA-G Ag induction and HCMV infection was confirmed in U-373 MG astrocytoma cells. Soluble HLA-G expression is stimulated upon HCMV infection, and this modulation depends on the cooperative action of the two immediate-early-1 pp72 and immediate-early-2 pp86 products. Because HLA-G transcription is active in macrophages and U-373 MG astrocytoma cells, it is likely that the modulation of HLA-G protein expression during HCMV replication occurs at a post-transcriptional level. Our data suggest that induction of HLA-G molecules could be an additional mechanism that helps HCMV to subvert host defenses.

Modulation of HLA-G antigens expression in myelomonocytic cells.

As trophoblast cells and macrophages share cellular characteristics, we investigated the expression of HLA-G antigens during the myelomonocytic differentiation. Analyses with the 87G and 16G1 monoclonal antibodies demonstrated that HLA-G was not expressed in peripheral blood monocytes, in in vitro differentiated dendritic cells and macrophages, and in resident mononuclear phagocytes infiltrating healthy tissues. Conversely, activated macrophages and dendritic cells localized in tumoral biopsies of some lung carcinomas expressed HLA-G antigens. Induction of HLA-G expression at the cell surface of the monohistiocytic cell line U 937 with different cytokines strongly suggests that cytokines secreted during inflammation may be involved in this specific upregulation. Bronchoalveolar macrophages collected from patients suffering from acute HCMV pneumonitis also expressed HLA-G molecules. In vitro, we thus demonstrated that HLA-G antigens are produced during viral reactivation in the macrophages generated after allogeneic stimulation of HCMV latently infected monocytes. Our data suggest that inflammatory processes in lung tissues, like tumoral transformation and HCMV acute infection, are likely to induce HLA-G molecules in infiltrating macrophages and dendritic cells. The expression of molecules capable of downregulating both the innate and adoptive immunity could be a mechanism that helps tumoral and HCMV infected cells to escape immune response.

HLA-G, -E, -F preworkshop: tools and protocols for analysis of non-classical class I genes transcription and protein expression.

Non-classical MHC class I HLA-E, -F, and -G molecules differ from classical class I histocompatibility antigens by specific patterns of transcription, protein expression, and immunological functions. Restriction of the expression pattern of these non-classical antigens may play a key role in modulation of immune responses during pregnancy and diseases but remains to be additionally defined. A specific component of the second International Conference on HLA-G and the 13th HLA-G Histocompatibility Workshop will be dedicated to the analysis of transcription and expression of non-classical class I genes in normal and pathological tissues. In a first step, referred to as the preworkshop, we here report the analysis and conclusions of a working group which was constituted to gather and validate optimal reagents and protocols allowing RT-PCR analysis of HLA-E, -F, -G transcript levels and flow cytometry and immunochemistry analysis of HLA-G expression in cells and tissues. As a result of this work, use of specific primers and probes detecting alternative transcripts of HLA-E, -F, and G have been validated in transfected cells expressing differential pattern of HLA class I antigens. Analysis of the specificity and affinity of collected antibodies has allowed definition of reagents to be proposed for immunochemistry and flow cytometry analysis of HLA-G expression in normal and pathological tissues during the workshop. This work has allowed constitution of an extended workshop group which is now initiating analysis of non-classical class I transcription and expression in various cells and tissues, a collective contribution that will additionally refine our view of the expression of these antigens in normal and pathological situations.

HLA-G protein expression is not induced during malignant transformation.

To evaluate the biological relevance of HLA-G expression during tumoral transformation, we analyzed its expression in different malignant cells and immune effector cells infiltrating solid tumors. Our analysis of 33 tumor cell lines and 53 tumoral biopsies demonstrated that: i) six tumor cell lines display HLA-G transcription with differential alternative splicing patterns and only Jeg3 choriocarcinoma and MCF-7 breast adenocarcinoma cell lines express HLA-G translated products; and ii) HLA-G antigens are not expressed in malignantly transformed cells derived from lung (n=18), liver (n=5), colon (n=5), breast (n=10), kidney (n=5), ovary (n=5), and larynx (n=5) tissues ex vivo. The healthy tissues surrounding these tumor tissues do not express HLA-G molecules either. On the other hand, surprisingly, HLA-G products were detected in activated macrophages and dendritic cells localized in tumoral biopsies of 5 out of 18 different lung carcinomas. No HLA-G labelling was observed in resident mononuclear phagocytes of surrounding healthy tissues. Our observations clearly demonstrate that HLA-G is not a marker of malignant cells but appears as a gene expressed in tumor-associated macrophages and dendritic cells, preferentially in those recruited in lung carcinomas. Our findings suggest that specific environmental factors around lung tumors could be involved in the induction of HLA-G protein expression.