Follicular Lymphoma Lymph Research Articles

Patient-derived follicular lymphoma spheroids recapitulate lymph node signaling and immune profile uncovering galectin-9 as a novel immunotherapeutic target

Follicular lymphoma (FL), the most common indolent non-Hodgkin lymphoma, constitutes a paradigm of immune tumor microenvironment (TME) contribution to disease onset, progression, and heterogenous clinical outcome. Here we present the first FL-Patient Derived Lymphoma Spheroid (FL-PDLS), including fundamental immune actors and features of TME in FL lymph nodes (LNs). FL-PDLS is organized in disc-shaped 3D structures composed of proliferating B and T cells, together with macrophages with an intermediate M1/M2 phenotype. FL-PDLS recapitulates the most relevant B-cell transcriptional pathways present in FL-LN (proliferation, epigenetic regulation, mTOR, adaptive immune system, among others). The T cell compartment in the FL-PDLS preserves CD4 subsets (follicular helper, regulatory, and follicular regulatory), also encompassing the spectrum of activation/exhaustion phenotypes in CD4 and CD8 populations. Moreover, this system is suitable for chemo and immunotherapy testing, recapitulating results obtained in the clinic. FL-PDLS allowed uncovering that soluble galectin-9 limits rituximab, rituximab, plus nivolumab/TIM-3 antitumoral activities. Blocking galectin-9 improves rituximab efficacy, highlighting galectin-9 as a novel immunotherapeutic target in FL. In conclusion, FL-PDLS maintains the crosstalk between malignant B cells and the immune LN-TME and constitutes a robust and multiplexed pre-clinical tool to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.

Single-Cell Longitudinal Characterization of FL Heterogeneity and Residual Disease in the Bone Marrow from Low-Tumor Burden FL Enrolled in the Flirt Clinical Trial

Follicular Lymphoma (FL) remains a clinically challenging disease as most patients undergo a succession of clinical response/relapse cycles suggesting that current therapies do not eradicate residual tumor cells. Genetic studies suggest that FL relapses seed from a reservoir of rare Cancer Precursor Cells (CPCs), which likely remain resistant to treatment due to their homing to propitious niches such as the bone marrow (BM) and/or to the acquisition of a specific functional B-cell state. Yet, the rarity of such CPCs and the use of bulk sequencing technologies have so far hampered their direct characterization. Profiling CPC remains thus a major challenge in the FL field. Here, we identify and characterize FL cells before/after rituximab monotherapy within the BM of low tumor burden FL, looking for CPC-like cells supporting minimal residual disease. We harnessed a unique collection of longitudinal BM aspirates from 15 newly diagnosed low-tumor burden FL patients enrolled in the phase III clinical trial FLIRT (NCT02303119) evaluating Rituximab monotherapy and sampled at diagnosis (M0) and at one-year post-treatment start (M12). We generated a multi-modal single-cell dataset combining gene expression, B-cell receptor (BCR) and T-cell receptor (TCR) repertoire (Fig1A). We used BCR sequence analysis as a pivotal method to assign the clonal malignant identity within variably infiltrated BM at diagnosis and track the clonally related residual disease FL cells at M12. We compared FL B cell transcriptional heterogeneity after rituximab therapy. We also integrated BM T cell transcriptomes from both timepoints to study compositional changes of the BM microenvironment linked to a B-cell depletion treatment. We profiled 9,222 BM infiltrating FL cells. Single cell clustering showed a unique transcriptional signature for malignant cells of each FL patient, indicating a major inter-patient heterogeneity. By contrast, the non-malignant B cell clusters were mixed across patients and organized into transcriptional signatures encompassing normal B-cell differentiation steps (Fig1B, left). We then used unsupervised metaclustering of malignant B cells to uncover shared transcriptional states across patients, yielding 6 metaclusters (Mcl) expressing distinct gene expression programs. Mcl gene expression signatures observed in FL BM shared some similarities with the transcriptional programs observed in FL lymph nodes such as non-cycling Germinal Center-like (Mcl.1) or Mem-like (Mcl.3 and Mcl.4) states. This suggests that FL transcriptional heterogeneity is conserved between distinct niches. BM FL cells also upregulated several pathways, including glycolytic metabolism ( GAPDH), NF-κB signaling, negative regulators of cell cycle, or entry into quiescence programs. By analyzing BM samples obtained at M12, we were able to track and profile very rare clonally-related FL cells in 80% of FL patients with a range of 2 to 65 residual FL cells per patient. Those residual FL cells present a heterogeneous cellular phenotype partly redundant with the transcriptional heterogeneity found at diagnosis indicating that residual cells cover a plurality of cell states whose links with clinical outcome is underway. Differential expression analysis in one case also revealed a transcriptional shift in residual FL cells (including overexpression of BCL2) compared to BM tumor cells at diagnosis (Fig1B, right), providing novel insights into putative CPC identity with possible implications for personalized therapies. Finally, exploration of the FL T-cell microenvironment revealed abundant TFH-like cells at diagnosis that were barely detectable in healthy donors. Interestingly, high frequency of the TFH-like subset was maintained at remission suggesting a potential role as a niche for therapy-resistant B cells and later relapses. In summary, our single cell RNAseq analysis in the frame of a clinical trial provide the first longitudinal map of FL heterogeneity in the BM and characterize putative CPCs resistant to Rituximab-therapy. Ongoing efforts are now focusing on the microenvironment drivers of this intra-patient BM diversity and on specific molecular features of BM residual cells that could represent therapeutic vulnerabilities in this particular low-tumor burden FL population.

Follicular Lymphoma Microenvironment Traits Associated with Event-Free Survival.

The majority of patients with Follicular Lymphoma (FL) experience subsequent phases of remission and relapse, making the disease "virtually" incurable. To predict the outcome of FL patients at diagnosis, various clinical-based prognostic scores have been proposed; nonetheless, they continue to fail for a subset of patients. Gene expression profiling has highlighted the pivotal role of the tumor microenvironment (TME) in the FL prognosis; nevertheless, there is still a need to standardize the assessment of immune-infiltrating cells for the prognostic classification of patients with early or late progressing disease. We studied a retrospective cohort of 49 FL lymph node biopsies at the time of the initial diagnosis using pathologist-guided analysis on whole slide images, and we characterized the immune repertoire for both quantity and distribution (intrafollicular, IF and extrafollicular, EF) of cell subsets in relation to clinical outcome. We looked for the natural killer (CD56), T lymphocyte (CD8, CD4, PD1) and macrophage (CD68, CD163, MA4A4A)-associated markers. High CD163/CD8 EF ratios and high CD56/MS4A4A EF ratios, according to Kaplan-Meier estimates were linked with shorter EFS (event-free survival), with the former being the only one associated with POD24. In contrast to IF CD68+ cells, which represent a more homogeneous population, higher in non-progressing patients, EF CD68+ macrophages did not stratify according to survival. We also identify distinctive MS4A4A+CD163-macrophage populations with different prognostic weights. Enlarging the macrophage characterization and combining it with a lymphoid marker in the rituximab era, in our opinion, may enable prognostic stratification for low-/high-grade FL patients beyond POD24. These findings warrant validation across larger FL cohorts.

Lymph node location and retention properties of DC‐SIGN engagement with the Immunoglobulin Oligomannoses of Follicular Lymphoma

Introduction: The oligomannose-type glycans occupying the surface immunoglobulin antigen-binding site (sIg-Mann) are a tumor-founding post-translational requirement of classic follicular lymphoma (FL). These are universally acquired and a clonal necessity during the entire natural history of FL from the early stages throughout transformation into EZB-DLBCL (Chiodin G, Blood 2021, Odabashian M, Blood 2020). The low-affinity interaction of sIg-Mann with its specific ligand DC-SIGN distinguishes from conventional high-affinity antigen:Ig protein interactions for promoting prolonged low-level growth and prosurvival signals via PI3K/AKT and not endocytosis (Linley A, Blood 2015). However, the location and function of DC-SIGN remains to be further elucidated. Here, we investigated the hypothesis that the specific DC-SIGN:Ig-Mann interaction functions to promote retention of FL cells in tissue niches maintaining selective survival advantage. Methods: Immunofluorescence imaging was used to determine DC-SIGN histological location in primary FL lymph nodes. In WSU-FSCCL cells, DC-SIGN effects on sIg redistribution were measured by dSTORM; adhesion to VCAM-1 in presence/absence of inhibitors was measured by flow cytometry. Results: Immunofluorescence of lymph nodes from FL patients revealed that DC-SIGN was expressed on interfollicular CD163+ macrophages and, remarkably, on CD23+ follicular dendritic cells (FDC), claiming an influence on FL cell retention and survival. dSTORM revealed that DC-SIGN induced less dense and more diffuse surface Ig clusters compared to anti-Ig, reflecting an immature sIg redistribution unable to promote endocytosis and death. However, DC-SIGN efficiently induced adhesion of sIg-Mann+ve lymphoma cells to VCAM-1, known to be expressed on both lymph node macrophages and FDC, and inhibited migration towards SDF-1 in vitro. Blocking of DC-SIGN carbohydrate-recognition domain completely abrogated DC-SIGN-induced adhesion. Although intracellular signals were significantly lower, DC-SIGN induced levels of adhesion similar to those induced by anti-Ig. Adhesion to VCAM-1 was observed at concentrations from 20 µg/ml down to 20 ng/ml, even when AKT and ERK phosphorylation was not detectable. Either proximal inhibition of the PI3K/AKT pathway, or distal inhibition of ARP2/3, formin, and Cdc42 for lamellipodium formation at the surface membrane, suppressed adhesion. Conclusions: These results reveal an important mechanism of the DC-SIGN:sIg-Mann interaction that fine-tunes signals for membrane adaptation towards tumor cell adhesion, without promotion of cell death. This possibly facilitates FL cell retention in the lymph node protected tumor niche where selective growth and survival signals are maintained. Interrupting this interaction will be a novel way towards tumour-specific targeted therapy in FL patients. The research was funded by: This research was funded by Blood Cancer UK (grant 18009), Cancer Research UK (ECRIN-M3 accelerator award C42023/A29370, program C2750/A23669, and BTERP project C36811/A29101). D.T. was funded by the Eyles Cancer Immunology PhD scholarship, G.C. was funded by the Eyles Cancer Immunology Fellowship and the Southampton Cancer Immunology Centre Pump-priming award 2021). Keywords: Indolent non-Hodgkin lymphoma, Microenvironment, Targeting the Tumor Microenvironment Conflicts of interests pertinent to the abstract. F. Forconi Employment or leadership position: University of Siena Consultant or advisory role: Beigene Honoraria: Abbvie, Janssen-cilag, Beigene, Astra-Zeneca Research funding: Cancer Research UK Educational grants: Beigene, Abbvie Other remuneration: BC Platform

Deep Peripheral T Cell Immune-Profiling in Relapsed/Refractory Non-Hodgkin Lymphoma: Evaluation of Baseline Samples from the Epcoritamab Epcore NHL-1 Trial

Background: Lymphomas account for approximately 4% of cancer diagnoses worldwide, of which 90% are non-Hodgkin lymphomas (NHL). NHLs encompass a group of heterogeneous hematological malignancies with over 60 different subtypes; the most frequently diagnosed subtypes are diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Treatment regimens incorporating α-CD20 monoclonal antibodies have become the standard-of-care for DLBCL and FL; however, intrinsic or acquired resistance will occur in the majority of patients and therefore newer therapeutic interventions are needed. Epcoritamab is a CD3 redirecting, CD20 targeting bispecific antibody that redirects patient tumor and peripheral T cells to lymphoma cells expressing CD20. Therefore understanding both the baseline T cell immune status and T cell fitness in patients with relapsed/refractory (R/R) disease is essential. Here we utilized a peripheral immune profiling multi-omics approach to enable deep characterization of the T cell immune landscape at baseline in R/R DLBCL and FL patient populations from EPCORE NHL-1, a phase 1/2, open-label, multi-center clinical trial of epcoritamab (NCT03625037; EudraCT 2017-001748-36). Methods:Samples and immune-phenotyping: Peripheral blood mononuclear cells (PBMCs) were prospectively collected from the EPCORE NHL-1 trial from patient whole blood and frozen for subsequent immune profiling. PBMCs were also collected from normal healthy volunteer (NHV) donors as age-matched controls. Multiplexed flow cytometry-based immune-phenotyping panels were developed and validated for clinical exploratory use to profile the major T cell subsets and their activation states, including CD4/CD8 naïve T cells (Tnaive), memory T cells (Tmem) and T-regulatory cells (Tregs). Eighty-seven DLBCL, 78 FL patient samples and 24 NHV controls were included in the study. Transcriptomics and TCRseq: PBMCs were also lysed for HTG EdgeSeq transcriptomic profiling and TCR repertoire sequencing (TCRseq). Normal healthy tissue gene expression was established by profiling NHV tonsils and lymph nodes as controls. FFPE DLBCL resections and core needle biopsies (n=99), FL (n=19) and normal lymph nodes (n=8) were sourced commercially. FFPE sections were processed into lysates and hybridized with HTG's Human Transcriptome Panel (HTP) probes for mRNA quantification. Results: Immune profiling of R/R DLBCL and FL patients uncovered distinct patient clusters and inter-disease specific differences in the Tnaive and Tmem cell subsets. Overall R/R FL patients showed elevated levels of CD19+ B cells at baseline compared to R/R DLBCL. DLBCL patients had increased levels of CD8+CD107a+ Tnaive /Tmem cells and PD-1+ Tregs compared to FL patients and NHV donors. In the CD4 T cell compartment, FL patients showed elevated levels of activated CD4+ T cells compared to DLBCL. Intriguingly, overall peripheral Tregs were higher in FL patients when compared to NHV donors and DLBCL patients. In good agreement with the EPCORE NHL-1 patient immune-phenotyping data in the periphery, transcriptomic profiling of commercially sourced NHL tumors showed an elevated Treg mRNA signature in FL when compared to DLBCL and NHV tonsil. Finally, the immune repertoire profiling by TCRseq in the periphery demonstrated that NHL patients generally exhibited lower clonal diversity than age-matched NHV donors. Conclusions: Comprehensive immune profiling of prospectively collected baseline PBMC samples from the EPCORE NHL-1 clinical trial using a multi-omics approach uncovered distinct inter- and intra-disease immune states in NHL patients. DLBCL patients had elevated expression of the degranulation and cytotoxic-specific marker CD107a in the periphery, suggesting an active immune state in these R/R patients. Notably, FL patients showed increased Tregs in the periphery and these data correlated with non-matched commercially sourced tumor when compared to DLBCL patients, while DLBCL patient Tregs had elevated T cell activation markers including PD-1. Finally, both DLBCL and FL patients had lower clonal diversity than NHV donors which may be a function of prior treatment. Ongoing studies include measuring the patient immune landscape over time and correlating immune pharmacodynamic/biomarkers with clinical outcomes.

Tumor Microenvironment and Microvascular Density in Follicular Lymphoma.

Follicular lymphoma (FL) is a slowly progressive disease and constitutes the second most common non-Hodgkin lymphoma. Biological factors, such as the tumor microenvironment and the host response, are determinants in the outcome of FL but the experimental data about microenvironment and tumor cells in FL are variable and contradictory. In this morphometric study, we analyzed by immunohistochemistry the cellular components of the tumor microenvironment and correlated these data with the microvascular vascular density in three different grades of FL lymph node biopsies, comparing the results to healthy lymph node controls. The results indicated a significant increase in the number of CD68+ and CD163+ macrophages in all three analyzed FL grades. Tryptase+ mast cells resulted in an increase only in grade 1. PDL-1+ cells, CD4- and CD8-lymphocytes number results were reduced in FL samples. The higher number of CD34+ microvessels in the FL grades 1 and 2 of samples positively correlated with CD68+ and CD163+ cells, underlining the important angiogenic potential of this subset of macrophages.

Follicular Lymphoma Regulatory T Cell Function and Origin

Follicular Lymphoma (FL) is the most frequent indolent lymphoma and results from the malignant transformation of germinal center (GC) B cells. Among FL recurrent genetic alterations, some are not only oncogenic per se but favor the interactions of FL B cells with their microenvironment including in particular surrounding follicular helper T cells (Tfh). During normal GC reaction Tfh support early GC B-cell selection and differentiation whereas follicular regulatory T cells (Tfr) expand later and inhibit both Tfh activity and B-cell antibody secretion. Whereas the role of Tfh in human diseases is an intense matter of interest, very few data are available concerning the origin and function of human Tfr. We previously demonstrated that Tfh and Tfr proportions are increased within FL lymph nodes and FL-Tfh contribute, through a specific polarization profile, to FL B cell survival and drug resistance. Interestingly FL-Tfr expansion is correlated to the one of FL-Tfh but not FL-Treg. The aim of this work was to characterize human Tfr in non-malignant (chronically inflamed tonsils) and malignant (FL) settings through transcriptomic, DNA methylation, TCR repertoire, and functional studies. We demonstrated for the first time that human Tfr purified from FL samples and tonsils directly inhibit autologous Tfh proliferation and trigger a downregulation of the co-stimulatory markers CD86 and HLA-DR on autologous normal and malignant GC B cells. In agreement FL-Tfh proliferated less than tonsil-Tfh in situ and after in vitro restimulation. Moreover, we found that Tfr clustered with Treg at both transcriptomic and methylome levels including for the high demethylation level of the FOXP3 TSDR, suggesting a stable regulatory phenotype. However, TCR sequencing demonstrated that tonsil-Tfr shared a common repertoire only with tonsil-Tfh unlike tonsil-Treg, arguing for their peripheral unlike thymic origin. Conversely in FL, even if the majority of common clones were found between Tfr and Tfh, a small proportion of TCR repertoire was shared between Tfr and Treg. Altogether, these results are the first detailed characterization of human Tfr and challenge our current understanding of Tfr commitment pathway. FL-Tfr retain their capacity to inhibit Tfh thus questioning their role in FL and their potential as therapeutic target in this fatal malignancy. DisclosuresTarte:Roche: Consultancy; Novimmune: Research Funding; Celgene: Consultancy, Research Funding. Lamy:Roche: Consultancy, Honoraria.

The architecture of neoplastic follicles in follicular lymphoma; analysis of the relationship between the tumor and follicular helper T cells.

CD4+ T-follicular helper cells are essential for the survival, proliferation, and differentiation of germinal center B cells and have been implicated in the pathogenesis of follicular lymphoma (FL). To further define the role of these cells in FL, we used multiparameter confocal microscopy to compare the architecture of normal and neoplastic follicles and next generation sequencing to analyze the T-cell receptor repertoire in FL lymph nodes (LN). Multiparameter analysis of LN showed that the proportion of T-follic-ular helper cells (TFH) in normal and neoplastic follicles is the same and that the previously reported increase in TFH numbers in FL is thus due to an increase in the number and not content of follicles. As in normal germinal centers, TFH were shown to have a close spatial correlation with proliferating B cells in neoplastic follicles, where features of immunological synapse formation were observed. The number of TFH in FL correlate with the rate of B-cell proliferation and TFH co-localized to activation induced cytidine deaminase expressing proliferating B cells. T-cell receptor repertoire analysis of FL LN revealed that follicular areas are significantly more clonal when compared to the rest of the LN. These novel findings show that neoplastic follicles and germinal centers share important structural features and provide further evidence that TFH may play a role in driving B-cell proliferation and genomic evolution in TFH. Our results also suggest that targeting this interaction would be an attractive therapeutic option.

PS1304 FOLLICULAR HELPER T‐CELLS FORM MUTUALLY SUPPORTIVE INTERACTIONS WITH FOLLICULAR LYMPHOMA B‐CELLS THAT MAY SUPPORT TUMOUR GROWTH

Background:Follicular lymphoma (FL) is a germinal centre (GC) B‐cell neoplasm that is dependent on interactions with its non‐malignant tumour microenvironment (TME). Follicular helper T‐cells (Tfh) play a pivotal role in supporting healthy GC B‐cell development, proliferation, and maturation. In FL, Tfh are present in similar proportions to reactive lymphoid tissue (Townsend, ASH 2014 abs 144) and can support survival of FL cells in vitro (Ame Thomas, Blood 2015). It remains unclear to what extent Tfh are able to drive FL growth and transformation.Aims:To explore whether mutual FL‐TFH interactions influence the survival and phenotype of both FL cells and infiltrating TFH.Methods:Disaggregated FL lymph node (LN) tissue and fine needle aspirates were obtained from untreated or relapsed FL patients (n = 19). Total FL LN cells (n = 10) were cultured unmodified, or with depletion of either TFH or B‐cells with immunomagnetic anti‐CD3 or anti‐CD19 beads (n = 10), respectively. To study in situ spatial interactions, 4‐colour confocal immunofluorescence microscopy was performed on archival FL tissue (n = 20).Results:In FL LN, TFH (PD‐1hiICOS+CXCR5+) comprise 28.6% (95% CI: 21.4–35.7) of CD4+ T‐cells by flow cytometry. Only a minority (5.0%; 95% CI: 0.6–9.4) had a FoxP3+ regulatory phenotype. After CD19 (FL cell) depletion, TFH partially lost their phenotype, with reduced CXCR5 and PD‐1 expression, as well as increased TFH cell death. There was no change in TFH ICOS expression after CD19 depletion, whilst ICOS‐L was upregulated in FL cells after CD3 (TFH) depletion. This partly mirrors findings with healthy TFH and B‐cells), demonstrating mutually beneficial and dynamic TFH‐FL interactions.These findings were confirmed in cultures with flow‐sorted TFH and FL B‐cells (n = 6). FL cells had higher expression of B‐cell activation markers (CD86 and HLA‐DR) when cultured in the presence of flow‐sorted TFH compared to those without. Additionally, we observed a reciprocal increase in TFH activation, with higher CD69 expression (but not CD25 or Tim3) in those cultured with FL cells. These findings were specific to B‐TFH interactions and were either not seen or diminished when FL cells were cultured with non‐TFH CD4 T‐cells.In FL tissue, TFH within closely interacted and correlated in number with proliferating FL cells: 58.5% Ki67+ (95% CI: 50.8–66.2) cells were in contact with TFH. This mirrored findings in reactive lymph node tissue (n = 5) and suggests the number of TFH cells may determine FL proliferation rate. We also found a close spatial relationship between TFH and FL‐cell expression of AID and MYC, both of which have been implicated in FL progression and transformation. Further characterisation of these complex cell populations within the FL TME requires highly multiplexed imaging techniques. We have designed an imaging mass cytometry panel to enable simultaneous assessment of 23 antigens in the same FL tissue section. We show that IMC is a feasible method for visualising TFH in FFPE tissue, allowing multiparameter analysis with surrounding FL cells and other TME cells.Summary/Conclusion:Here we provide evidence that TFH form mutually supportive, reciprocal interactions with FL B‐cells, implying a key role in promoting FL growth. TFH have a close relationship with FL proliferation and markers suggesting genomic dysregulation, which may drive FL progression. In an immunotherapy era, better understanding of these interactions will suggest future targets and pathways for therapeutic intervention in FL.

Multiple Polypoid Lesions in the Ileum After Treatment for Primary Ileal Follicular Lymphoma

Question: A 79-year-old man with a history of primary follicular lymphoma (FL) of the ileum was referred to our department for a follow-up endoscopy. He was diagnosed with stage II primary ileal FL and inguinal lymph node dissemination 13 years before. Ileocolonoscopy performed at the time of the FL diagnosis revealed multiple lymphomatous polypoid lesions in the terminal ileum (Figure A). A histologic diagnosis of FL was made by endoscopic biopsy revealing neoplastic cells positive for CD20, CD10 (Figure B), and BCL2 (Figure C), which disrupted the architecture of the lymphoid follicles and infiltrated the mucosa and submucosa.

T Cells Expressing Checkpoint Receptor TIGIT Are Enriched in Follicular Lymphoma Tumors and Characterized by Reversible Suppression of T-cell Receptor Signaling.

Purpose: T cells infiltrating follicular lymphoma (FL) tumors are considered dysfunctional, yet the optimal target for immune checkpoint blockade is unknown. Characterizing coinhibitory receptor expression patterns and signaling responses in FL T-cell subsets might reveal new therapeutic targets.Experimental Design: Surface expression of 9 coinhibitory receptors governing T-cell function was characterized in T-cell subsets from FL lymph node tumors and from healthy donor tonsils and peripheral blood samples, using high-dimensional flow cytometry. The results were integrated with T-cell receptor (TCR)-induced signaling and cytokine production. Expression of T-cell immunoglobulin and ITIM domain (TIGIT) ligands was detected by immunohistochemistry.Results: TIGIT was a frequently expressed coinhibitory receptor in FL, expressed by the majority of CD8 T effector memory cells, which commonly coexpressed exhaustion markers such as PD-1 and CD244. CD8 FL T cells demonstrated highly reduced TCR-induced phosphorylation (p) of ERK and reduced production of IFNγ, while TCR proximal signaling (p-CD3ζ, p-SLP76) was not affected. The TIGIT ligands CD112 and CD155 were expressed by follicular dendritic cells in the tumor microenvironment. Dysfunctional TCR signaling correlated with TIGIT expression in FL CD8 T cells and could be fully restored upon in vitro culture. The costimulatory receptor CD226 was downregulated in TIGIT+ compared with TIGIT- CD8 FL T cells, further skewing the balance toward immunosuppression.Conclusions: TIGIT blockade is a relevant strategy for improved immunotherapy in FL. A deeper understanding of the interplay between coinhibitory receptors and key T-cell signaling events can further assist in engineering immunotherapeutic regimens to improve clinical outcomes of cancer patients. Clin Cancer Res; 24(4); 870-81. ©2017 AACR.

Identification of Tigit on Intra-Tumor T Cells As a New Target for Immune Checkpoint Blockade in Follicular Lymphoma

Background: Follicular lymphoma (FL) is the most common subtype of indolent non Hodgkin’s lymphoma (NHL). Median survival is long (>10 years), but current chemo-immunotherapy regimens used for FL are usually not curative. While T cells in the FL tumor microenvironment are considered dysfunctional and associated with disease progression, a better understanding of T-cell signaling may reveal how to productively engage tumor-infiltrating T cells to kill lymphoma B cells. Our previous study showed that expression of the immune checkpoint receptor PD-1 was directly correlated with reduced cytokine signaling in FL T cells (Myklebust et al., Blood 2013). Antibody immunotherapy targeting the PD-1/PD-L1 pathway has shown significant activity in solid tumors, but these benefits have not been as profound in NHLs, including FL. Co-blockade of checkpoint inhibitors may therefore be necessary to generate optimal anti-tumor responses in FL. The hypothesis underlying this study was that characterizing signaling responses in FL tumor-infiltrating T cells will identify new targets for combination of checkpoint blockade.Methods: Surface expression of 9 checkpoint receptors governing T cell function was measured in subsets of CD4 and CD8 T cells from FL lymph node tumors (n = 14) and from healthy donor tonsils (n= 11) and peripheral blood samples (n = 7) using fluorescence flow cytometry. Patterns of checkpoint receptor expression were compared with 1) intracellular phospho-protein signaling response and 2) cytokine production for subsets of T cells infiltrating FL tumors and the corresponding T-cell populations in healthy tonsils. Phospho-specific flow cytometry measured phosphorylation of STATs and T cell receptor (TCR) signaling effectors within minutes following stimulation by IL-4, IL-7, IL-21, or α-CD3+α-CD28 (TCR stimulation) antibodies.Results: CD4 and CD8 T cells infiltrating FL tumors were gated into subsets defined by PD-1 and ICOS protein expression, and compared to cognate T cell subsets in healthy tonsils. FL and tonsil T cells closely matched in their signaling responses to IL-4, IL-7, and IL-21 stimulation, with PD-1 expressing cells (CD4+PD-1hiICOS+ (TFH) and CD8+PD-1int T cells) exhibiting modest phospho-protein signaling responses compared to T cells not expressing PD-1. Furthermore, TCR membrane proximal signaling events (p-CD3ζ, p-SLP76) following TCR stimulation were comparable in FL and tonsil T cells. This contrasted reduced phospho-ERK signaling in all CD4 and CD8 T cell subsets infiltrating FL tumors which distinguished them from tonsillar T cells. IFN-γ production also differed between FL and tonsils, as CD8 T cells infiltrating FL tumors produced less IFN-γ. Reduced IFN-γ production was independent of PD-1 expression, suggesting suppressed function in these T cells which could be due to inhibitory receptors other than PD-1. Of the 9 checkpoint receptors measured, PD-1 and T cell Ig and ITIM domain (TIGIT) were expressed at the highest frequency. In FL, TIGIT was expressed in 58% and 80% of CD8 effector and effector memory cells, respectively, as compared to 43% and 68% of the cognate healthy tonsillar subsets. TIGIT was also frequently expressed in CD4 FL T cells, as 52% and 79% of effector and effector memory cells expressed TIGIT, respectively, as compared to 16% and 59% of the corresponding subsets from healthy tonsils. viSNE analysis demonstrated that TIGIT and PD-1 were frequently co-expressed in FL T cells, and a large fraction of PD-1int T cells had high expression of TIGIT (Figure 1). These results provide a rationale for co-blockade of PD-1 and TIGIT in FL and for investigation of how co-blockade impacts T cell functions.Conclusions: These results reveal specific suppression of cytokine signaling in CD8 effector T cells infiltrating FL tumors and identify TIGIT and PD-1 as strong candidates for co-checkpoint blockade in FL. A deeper understanding of the interplay between checkpoint receptors and key T cell cytokine signaling events in FL will further assist in engineering immuno-therapeutic regiments that improve FL patient clinical outcomes. [Display omitted] DisclosuresKolstad:Nordic Nanovector: Other: Membership of Scientific Advisory Board. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Irish:Incyte: Research Funding; Janssen: Research Funding; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

ADCC Induced By Monoclonal Anti-CD20 Antibodies in a 3D Follicular Lymphoma model : Signaling and Spatial Localization

Follicular lymphoma (FL) is the second most common type of B non-Hodgkin’s lymphomas (NHL) and makes up to 40% of all adult lymphomas. Like most carcinomas, NHL grow as spherical tumors. Based on carcinoma 3D models (often described as spheroids) it is acknowledged that spatial organization may profoundly affect tumor cell behavior since important functions are dictated by the collective properties of a cell population rather than those of a single cell. These include growth, metastasis, cell-to-matrix and cell-to-cell interaction, as well as intracellular signaling and resistance to anti-tumor agents or even immune escape. Spatial organization can exert these effects in cancer cells by affecting gene expression profiles or influencing major signaling pathways such as those driven by MAPK, Akt and oncogenic products such as HER-2. More importantly, spatial organization is known to influence the response to antibodies such as Trastuzumab or Pertuzumab.Conventional 2D NHL cell culture models do not reflect the true effects of antibodies as they occur in vivo and may be of limited use. Thus, we recently created a NHL 3D culture system, the so-called Multicellular Aggregates of Lymphoma Cells (MALC) model, using a modification of the “hanging drop” method. This model, which displays similar transcriptomic profiles compared to biopsies from FL patients, is not only useful for studying FL biology, but also for evaluating tumor sensitivity to antibodies since diffusion/distribution is different within a solid 3D tumor.Thus, we recently showed that 3D organization influences direct response to Rituximab (RTX) and GA101 (Obinutuzumab), two monoclonal anti-CD20 antibodies, as we observed a more potent efficiency in MALC model compared to conventional 2D cell culture in term of cell death induction (apoptosis, senescence, lysosomal cell death) and intracellular signaling targeting.Here, we evaluate antibody-dependent cell cytotoxicity in MALC in presence of immune effectors cells such as NK cells and Tgd lymphocytes. We showed that RTX and GA101 induce immune cells activation as attested by CD69 and IFNg increase and immune cells cytotoxic activity as attested by CD107 increase as well as granzyme B and perforine decrease, reflecting granules release. GA101 appears to be more efficient than RTX in effectors cells activation. We correlate this phenomenon with a more potent effect on target cells as attested by MALC volume decrease and cell death induction observed by flow cytometry and confocal microscopy. By investigating intracellular signaling pathways induced during ADCC on both effector (NK) and target cells (MALC), we observed on NK cells the activation of CD16 downstream signaling molecules such as Syk, PLCg2 and Akt, with a more potent effect in presence of GA101 compared to RTX for proximal proteins activation. On the other hand, we showed that RTX and GA101 induced inhibition of classical overactivated signaling pathways such as Syk, Akt, ERK and p38 on target cells. Finally, we asked the question of the presence of immune cells in lymph node isolated from FL patients. By flow cytometry, we were able to determine the percent of NK and gdT cells and present evidences that immune cells number is higher in FL lymph node compared to normal tonsil.Altogether, this study completes our previous works on the identification of MAbs mechanisms of action in a relevant 3D FL model.A better understanding of these mechanisms is necessary to improve therapy by identifying mechanisms of resistance and new therapeutic targets. Moreover, we believe that MALC is a powerful and relevant model for further investigation on FL biology, and also useful for the development/screening of new genotoxic drugs and therapeutic monoclonal antibodies in lymphoma. DisclosuresBezombes:Roche: Research Funding. Savina:roche: Employment. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

High Level of CD71 Expression On Neoplastic B Cells At Diagnosis Is Predictive of Overall Survival After Rituximab and Anthracyclines Based Regimen in Follicular Lymphoma

AbstractAbstract 1601 Background:The prognosis of follicular lymphoma (FL) remains a challenge despite stratification on the FLIPI scoring, histological grading, and microenvironement cell type infiltration. Thus, new biomarkers are needed to identify patients (pts) who require a more aggressive therapeutic strategy or targeted therapies. Transferrin receptor (TfR) is highly expressed in proliferating cells including non Hodgkin lymphoma cells. High TfR level has been associated with disease progression in chronic lymphocytic leukaemia and is observed at relapse in mantle cell lymphoma. Murine anti human TfR (CD71) antibody has been validated for histological and flow cytometric evaluation of TfR expression. The aim of this work was to assess expression and prognosis value of TfR expression on FL cells. Material and Methods:We retrospectively analyzed the expression level of CD71 in neoplastic B cells of FL lymph node biopsies at diagnosis of 45 pts who received first-line treatment with Rituximab and anthracyclines based chemotherapy at the hematology department of Necker Hospital, Paris, France, and assessed its prognosis value (second treatment free survival (TFS) and overall survival (OS)). High CD71 and Ki67 expression in FL lymph nodes biopsies were defined as upper than 25% of FL cells. CD71 expression was compared with FLIPI at diagnosis and the expression and prognosis value of Ki67 expression. Results:Characteristics of the cohort are shown in table 1. FLIPI risk was low in 7 pts (16%), intermediate in 19 (43%) and high in 18 (41%). FLIPI stratification did not show correlation with both OS and TFS in this small cohort of pts. Among 44 evaluable pts, CD71 expression was heterogeneous ranging from 0% to 90% of FL cells. T cell labeling was used as a quality control. CD71 low expression (225% of tumor cells) was observed in 31 samples (69%) and KI67 low expression in 27 (60%). After a median follow-up of 40 months (range 1 – 94) for the all cohort, TFS was 68.9 % (95 % CI 0.55–0.82) and OS was 91.1% (95% CI 0.83 – 0.99). The mean TFS after R-chemotherapy was 86 months in the low CD71 level group vs. 18 months in the high CD71 level group (p=0,075). The mean estimated OS after first R-chemotherapy was 89 months in the low CD71 group vs. 54 months in the high CD71 group (p=0,006) (figure 1). By univariate analysis, high Ki67 expression was associated with significant lower OS (p=0.035) but not TFS (p=0,162). In multivariate analysis including CD71 level, FLIPI risk, FLIPI parameter groups, CD71 was the only factor independently associated with OS (p=0,019) and TFS (p=0,037). Furthermore, Cox regression analysis did not reveal any significant correlation between Ki67 and TFS or survival. Conclusion:CD71 expression is an important independent histological prognostic factor in this monocentric retrospective study predicting OS in FL patients after first-line treatment with R anthracyclin based regimen. Ki 67 was also associated with OS in univariate analysis, but CD71 was the only independent factor associated with OS and TFS in multivariate analysis. These promising results warrant further studies in a larger prospective cohort and provide a rationale to target Tfr in relapsing refractory FL. [Display omitted] Table 1Characteristics of the pts at baseline and evolution after first line R-CHOP like regimen according to the CD71 risk groupsCharacteristicsCD71 lowCD71 highp.n (%)31 (69%)14 (31%)At DiagnosisAge< 60 ans1930,0453 60 ans1210SexMale1671Female157Clinical stageI-II411III-IV2713BM involvementYes2270,173No97No. of involved nodes area (FLIPI)241540,335>4169LDHN22120,313> N92Hemoglobin3 12 g/dl120,204< 12 g/dlFLIPI risk groupLow610,251Intermediate154High108Ki67 expressionLow (225%)2380,007High (>25%)410After treatmentMedian FU (months)47.8 [10.5-92.5]30 [1.1-75.1]Need second line of treatment: No. (%)10 (32)7 (50)nsDeceased: No. (%)1 (3)4 (29)0.03 Disclosures:No relevant conflicts of interest to declare.

The Transcriptional Profile of Follicular Lymphoma Associated Regulatory T-Cells (Tregs) Is More Consistent with Tregs Having a Germinal Center Localization and the Ability to Suppress Follicular Helper T-Cells

AbstractAbstract 1536Recent studies have suggested that regulatory T-cells (Tregs) play a critical role in the pathogenesis of follicular lymphoma (FL). We have previously shown that the Tregs infiltrating FL lymph nodes (FLN Tregs) are quantitatively and qualitatively different than those infiltrating normal and reactive nodes (NLN, RLN, respectively). To gain insight into how such Treg populations differ, and how FLN Tregs may impact FL progression and response to therapy, we performed RNA sequence analyses on flow sorted Tregs (CD3+CD4+CD25brightCD127−) from NLN (n=10), FLN (n=12) and RLN (n=7).Using the RefSeq annotated set of mRNA we first identified 11686 annotated genes that were expressed in each of the Treg groups. Using pairwise unequal variance t-tests at p<0.01, we identified 798 genes that differed between NLN and FLN, 589 that differed between NLN and RLN, and 475 genes that differed between FLN and RLN. The transcriptome profile of the FLN Tregs differed most from that of NLN Tregs, with 281 genes increased in FLN relative to NLN, and 517 genes decreased in FLN Tregs relative to NLN Tregs. In contrast, 404 genes were increased and 70 genes decreased in RLN Tregs relative to FLN Tregs suggesting that differences in the transcriptome profile between NLN and FLN Tregs are due, in part, to inflammatory signals shared between FLN and RLN. Tregs, which express FOXP3, are a heterogeneous population which share a “core suppressive module” of CD25 and CTLA-4, but which adapt to microenvironmental signals by co-opting/or up-regulating the canonical transcription factor (TF) of the T-cell population the Treg needs to suppress. This results in modulation of the Treg chemokine receptors in such a fashion that allows these Tregs to traffic to and suppress the specific T-cell population defined by their TF expression. Relative to NLN, FLN Tregs have similar expression of FOXP3 and CD25 but greater expression of CTLA-4 (confirmed at the protein level), which may account, in part, for the greater suppressive activity of FLN Tregs compared to NLN Tregs. Expression of other genes that differ between NLN and FLN which encode for proteins that modulate Treg suppression include IL-10 (up in FLN relative to NLN), S1PR1 and ENTPD1 (both down in FLN relative to NLN), which all favor greater suppression by FLN Tregs. In contrast LGALS1 and NT5E expression (both down in FLN relative to NLN) would favor less suppression by FLN Tregs. Relative to NLN, FLN Tregs have greater BCL-6 expression, consistent with a population that suppress follicular helper T-cells (Tfh), which support normal germinal center (GC) and FL-B cell survival. Consistent with their increased expression of BCL-6, FLN Tregs have greater expression of CXCR5, CXCR4 and CXCL13 and decreased expression of CCR7 compared to that of NLN Tregs, a repertoire associated with GC trafficking. The significant lower expression of both S1PR1 and SELL (CD62L; p<0.000002, and fold changes of −9.0 and −2.8, respectively) that is found in FLN Tregs compared to NLN Tregs is consistent with the greater nodal retention. The lower expression of RORc found in the FLN Tregs would predict for decreased IL-17 secretion and their elevated levels of STAT3 which would support the ability of the FLN Tregs to inhibit Th17 cells.In conclusion, the comparison of the transcriptome profiles of FLN and NLN Tregs; a) suggest that the observed differences are shaped, in part, by inflammatory signals also present in RLN; b) lay the foundation of a hypothesis to explain the molecular basis for their greater immunosuppressive capacity; and c) suggest that FL Tregs are ‘poised' to localize in the GC, be strongly retained in the lymph node and have enhanced potential to suppress Tfh and Th17 cells. Such findings offer new insights into how FL Tregs may contribute to the biology of these B cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

Mesenchymal Stromal Cells Support Follicular Lymphoma-Associated Follicular Helper T-Cells, in Part, Through an IL-6 and IL-21-Dependent Mechanism.

Abstract 2707The biology of follicular lymphoma (FL) is largely dictated by the immune-effector and stromal cells that comprise its tumor microenvironment. Recent studies have demonstrated that various infiltrating T-cell populations can be predictive of clinical outcome, with two such populations, follicular helper T-cells (TFH) and regulatory T-cells (Treg) likely playing critical roles in the biology of this disease. TFH are normally responsible for the activation and maintenance of germinal center B-cells while Tregs suppress effector T-cell priming and function as well as suppress normal B-cell proliferation and differentiation. There is also data to support the notion that both of these cells have direct and/or indirect effects on FL B-cells. These cells and other immune-effector cells within the FL microenvironment have dynamic interactions with mesenchymal stromal cells (MSC), multipotent cells residing in most adult tissues, which can be recruited by FL B-cells into the tumor microenvironment where they inhibit anti-lymphoma T-cell responses and FL B-cell apoptosis. We hypothesized that such MSC play a role in modulating TFH viability, similar to what has been previously reported for Treg, and herein we show such a novel role for MSC, that being the selective support of FL-derived TFH populations.To determine the effect of MSC on the viability of discrete T-cell populations infiltrating FL lymph nodes (FLN), un-separated single cell suspensions (SCS) from FLN (n=13) were cultured for 48 hours alone or with normal tonsillar-derived MSC. The fold change in the proportion of CD4 T-cells that are TFH (CXCR5+PD-1+Bcl-6+), Tregs (CD25+FoxP3+), TH1 (T-bet+), TH2 (GATA-3+) or TH17 (ROR-gamma-t+) cells in SCS incubated with MSC was compared to that of SCS incubated without MSC. A 2.3-fold increase in the percentage of TFH (p=0.006) cells and a 2-fold increase in the percentage of Tregs (p=0.007) were observed in the SCS cultured with MSC compared to those cultured without MSC. The effect of MSC in maintaining TFH and Tregs was selective, as in contrast, the proportion of T-cells expressing the canonical TH1, TH2 and TH17 transcription factors was not increased after culture with MSC. B-cells provide survival signals to TFH and stromal cells provide survival signals to B-cells, therefore we next determined whether the stromal support of TFH was mediated via B-cells. Both purified FL T-cells and B-cell-depleted FL SCS cultured on MSC showed a similar increase in TFH (and Treg) populations as seen with un-separated FL-B SCS, indicating that MSC support of TFH and Tregs was independent of B-cells. MSC only partially supported TFH and Treg populations in transwell experiments suggesting a role for both cell-cell contact and soluble factors. In this regard, IL-6 and IL-21 are known to be secreted by MSC and to signal through gp130 receptors on TFH to induce the transcription of Bcl-6, which is required for TFH differentiation. Blocking IL-6 and IL-21 decreased MSC support of TFH by 42% (p=0.021), while reducing MSC support of Tregs by only 17% (p=0.018) suggesting that IL-6 and IL-21 mediate, in part, the protective effect that MSC have on TFH but that other cytokines are likely to play a role in supporting Tregs. Finally, we have shown that FLN-derived MSC support both FL T-cells and normal lymph node T-cells to a similar extent as the tonsillar MSC used in these experiments. This provides support for the use of tonsillar MSC in these experiments as we were able to generate and expand these more consistently than MSC from FLN, which allowed us to use a consistent MSC product for all experiments.These findings therefore demonstrate a new role for MSC in FL, that being to support FL TFH in addition to Treg populations in the tumor microenvironment. MSCs have been shown to support FL B-cell viability and suppress anti-lymphoma T-cell responses. This finding that MSCs support TFH cells, a population that may provide survival signals to FL B-cells, further supports the potential of targeting MSC as a novel therapeutic strategy for patients with FL. Disclosures:No relevant conflicts of interest to declare.

Distribution, function, and prognostic value of cytotoxic T lymphocytes in follicular lymphoma: a 3-D tissue-imaging study

AbstractCD8+ CTLs are thought to play a role in the control of follicular lymphoma (FL). Yet, the link between CTL tissue distribution, activation status, ability to kill FL cells in vivo, and disease progression is still elusive. Pretreatment lymph nodes from FL patients were analyzed by IHC (n = 80) or by 3-color confocal microscopy (n = 10). IHC revealed a rich infiltrate of CD8+ granzyme B+ (GrzB) cells in FL interfollicular spaces. Accordingly, confocal microscopy showed an increased number of CD3+CD8+GrzB+ CTLs and a brighter GrzB staining in individual CTL in FL samples compared with reactive lymph nodes. CTLs did not penetrate tumor nodules. In 3-dimensional (3-D) image reconstructions, CTLs were detected at the FL follicle border where they formed lytic synapse-like structures with FL B cells and with apoptotic cells, suggesting an in situ cytotoxic function. Finally, although GrzB expression in CTLs did not correlate with risk factors, high GrzB content correlated with prolonged progression free-survival (PFS) after rituximab-combined chemotherapy. Our results show the recruitment of armed CTLs with a tumor-controlling potential into FL lymph nodes and suggest that CTL-associated GrzB expression could influence PFS in FL patients having received rituximab-combined chemotherapy.

Follicular lymphoma cell niche: identification of a preeminent IL-4-dependent TFH–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.

In Follicular Lymphoma (FL), γδt-Lymphocytes (γδT) Are Present and Expandable from Peripheral Blood and Rare in Tumour Lymph Nodes, Mostly in Peri-Follicular Areas.

Background: The tumour microenvironment plays an important role in the biology of FL. Different cell populations have been explored, including T-regulatory lymphocytes, macrophages, and T-cell subpopulation. The involvement of γδT in lymph nodes from FL patients or from inflammatory diseases has been rarely documented. So far, their histological pattern and prognosis significance are unknown and must be defined in order to develop new therapeutic programs including in vivo or ex vivo expansion of γδT, as developed particularly in B-cell malignancies by us and different groups. In this study, we analyzed from FL patients 1/the number of circulating γδT and their ex vivo expansion, 2/ the presence and distribution of γδT in tumour lymph nodes, and different chemokines, in comparison to inflammatory lymph nodes (ILN), by immunochemistry.Patients and Methods: Circulating γδT cells were counted in peripheral blood from patients having FL by FACS analysis. Blood samples from 34 patients were collected and expanded in vitro by using γδT ligands, referred to as “phosphoantigens”, including IPH1101 (used in clinical trials) and interleukin 2. Tumour samples from 51 patients (35 at diagnosis and 16 at relapse) having FL were collected from a single institution. Immunochemistry was used to study numbers and distribution of CD8, γδT cells, and the expression of CCL19, 21 and SDF1 chemokines. CCR7/γδT cells were analyzed by double immunofluorescence. Results were compared to 28 samples from patients having ILN.Results: The mean of circulating γδT was 0.36% (0.03–2.5) representing a mean of 2.2% of the CD3 cells. The mean percentage of γδT cells obtained after in vitro culture was 85% (2.1–95) with a mean 220-fold expansion (2-1050). The median number of γδT cells (cells/mm2) in FL lymph node was 18 versus 47.5 in the ILN (mean: 30 versus 82,5), P<.00001. The median of CD8 cells was 1235 in FL as compared to 1503 in ILN (mean: 1290 versus 1524). CD8+ cells had different localization (i.e. intra-and /or extra-follicular localization), but γδT were strictly peri-follicular in both clinical situations. Immunohistochemistry of the high endothelial venules (HEVs) and lymphatic vessels (LV) of 14 FL and 14 ILN were performed. We observed a significant difference (P= 2.10−7) in the expression of only the CCL19 chemokine between FL and ILN, with a poor staining for CCL19 in FL lymph nodes. CCL21 and CXCL12 do not present a difference in their expression levels. The stroma was reactive for all these chemokines, while the SDF1/CXCL12 chemokine shows a topographic difference in the distribution of stromal cells around HEV.Conclusions: These observations suggest that γδT cells are present and expandable in PB from patients having FL including patients with advanced disease. In addition, γδT are not abundant in lymph nodes of patients with FL compared to ILN, but γδT conserve their CCR7+ phenotype. This deficiency could be explained by migratory problems provoked by a lack of CCL19 chemokine expression. As γδT have been demonstrated to kill tumour cells, including B-malignant cells, they could be considered as essential targets for immune therapy in different cancers including B-cell malignancies, but their activation and trafficking has to be considered.

Combination Immunotherapy with a CpG Oligonucleotide and Rituximab (R) Augments the Immunological Response and Favorably Alters the Malignant Microenvironment of Follicular Lymphoma (FL).

The malignant microenvironment of FL, including T cells, macrophages and follicular dendritic cells, contributes to disease pathogenesis and represents a rational therapeutic target. Gene expression profiling and immunohistochemistry studies have confirmed that degree and type of immune response in the malignant FL lymph node impacts clinical outcome. CpG oligonucleotides have pleotropic effects on both the innate and adaptive immune systems, including increased antigen expression, enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and shifting the immune response in a TH-1 direction. These agents therefore have significant potential to manipulate the immune response to FL, and alter the malignant microenvironment in a favorable way. We combined a CpG oligonucleotide (1018 ISS, 0.2 mg/kg sc weekly x 4 beginning day 8) with standard R in patients (pts) with relapsed/refractory, histologically confirmed FL, and comprehensively evaluated immunological changes following the combination. 22 pts (10 female; median age 55) with FL sensitive or naive to R were enrolled. FcγRIIIA polymorphism status was FF n=9, VF n=12, VV n=1. Treatment was well-tolerated with no significant adverse events attributable to therapy. Baseline and post therapy evaluations were performed on blood and the CpG injection sites in all pts. Compared to baseline, 7 pts demonstrated significant increases in ADCC after therapy using a conventional chromium-release assay. Circulating CD3-positive cells increased in 11 pts. Increased IFN-γrelease by Elispot occurred in 5 pts. 10 pts had significant increases in neutrophils, T cells, and macrophages in skin biopsies of CpG injection sites performed 24 hours post therapy compared with baseline. Pre- and post-biopsies of tumor tissue (including lymph nodes and bone marrow) were performed in 5 pts, and in all cases a significant infiltration of T cells (CD8 > CD4) and macrophages were observed following treatment. Clinical responses per Cheson criteria were observed in 45% of pts; median PFS was 18 months in responding pts, and 9 months for entire group; 12 pts have not yet required further therapy for FL. In conclusion, this group of pts with relapsed FL had favorable clinical outcome despite adverse prognostic factors. The addition of CpG to R had measurable and reproducible local and systemic immune effects in a substantial subset of pts. Unlike other cytokine/antibody combinations, this study is the first to histologically confirm perturbation of the local immune malignant microenvironment following systemic biological therapy of FL.