Edoardo Migliori

CXCL13-producing TFH cells link immune suppression and adaptive memory in human breast cancer

T follicular helper cells (TFH cells) are important regulators of antigen-specific B cell responses. The B cell chemoattractant CXCL13 has recently been linked with TFH cell infiltration and improved survival in human cancer. Although human TFH cells can produce CXCL13, their immune functions are currently unknown. This study presents data from human breast cancer, advocating a role for tumor-infiltrating CXCL13-producing (CXCR5-) TFH cells, here named TFHX13 cells, in promoting local memory B cell differentiation. TFHX13 cells potentially trigger tertiary lymphoid structure formation and thereby generate germinal center B cell responses at the tumor site. Follicular DCs are not potent CXCL13 producers in breast tumor tissues. We used the TFH cell markers PD-1 and ICOS to identify distinct effector and regulatory CD4+ T cell subpopulations in breast tumors. TFHX13 cells are an important component of the PD-1hiICOSint effector subpopulation and coexpanded with PD-1intICOShiFOXP3hi Tregs. IL2 deprivation induces CXCL13 expression in vitro with a synergistic effect from TGFβ1, providing insight into TFHX13 cell differentiation in response to Treg accumulation, similar to conventional TFH cell responses. Our data suggest that human TFHX13 cell differentiation may be a key factor in converting Treg-mediated immune suppression to de novo activation of adaptive antitumor humoral responses in the chronic inflammatory breast cancer microenvironment.

Immune Checkpoint Molecules on Tumor-Infiltrating Lymphocytes and Their Association with Tertiary Lymphoid Structures in Human Breast Cancer.

There is an exponentially growing interest in targeting immune checkpoint molecules in breast cancer (BC), particularly in the triple-negative subtype where unmet treatment needs remain. This study was designed to analyze the expression, localization, and prognostic role of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, and TIM3 in primary BC. Gene expression analysis using the METABRIC microarray dataset found that all six immune checkpoint molecules are highly expressed in basal-like and HER2-enriched compared to the other BC molecular subtypes. Flow cytometric analysis of fresh tissue homogenates from untreated primary tumors show that PD-1 is principally expressed on CD4+ or CD8+ T cells and CTLA-4 is expressed on CD4+ T cells. The global proportion of PD-L1+, PD-L2+, LAG3+, and TIM3+ tumor-infiltrating lymphocytes (TIL) was low and detectable in only a small number of tumors. Immunohistochemically staining fixed tissues from the same tumors was employed to score TIL and tertiary lymphoid structures (TLS). PD-L1+, PD-L2+, LAG3+, and TIM3+ cells were detected in some TLS in a pattern that resembles secondary lymphoid organs. This observation suggests that TLS are important sites of immune activation and regulation, particularly in tumors with extensive baseline immune infiltration. Significantly improved overall survival was correlated with PD-1 expression in the HER2-enriched and PD-L1 or CTLA-4 expression in basal-like BC. PD-1 and CTLA-4 proteins were most frequently detected on TIL, which supports the correlations observed between their gene expression and improved long-term outcome in basal-like and HER2-enriched BC. PD-L1 expression by tumor or immune cells is uncommon in BC. Overall, the data presented here distinguish PD-1 as a marker of T cell activity in both the T and B cell areas of BC associated TLS. We found that immune checkpoint molecule expression parallels the extent of TIL and TLS, although there is a noteworthy amount of heterogeneity between tumors even within the same molecular subtype. These data indicate that assessing the levels of immune checkpoint molecule expression in an individual patient has important implications for the success of therapeutically targeting them in BC.

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45(+) cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45(+) cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

Quantifying Tertiary Lymphoid Structure-Associated Genes in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissues

Tertiary lymphoid structures (TLS) have been detected in several types of human solid tumors. These structures are thought to regulate local adaptive immune responses that can promote or antagonize tumor progression. Despite positive prognostic values associated with a TLS presence in several studies, discrepancies still exist. TLS are structurally organized entities composed of varying numbers of multiple cell types making their assessment in tumor tissues, particularly biopsies, challenging. Immunohistochemical staining of TLS-related cell populations is the most frequently used method for identifying and scoring them; however, TLS-related gene expression has also been explored. The protocols described are detailed to allow the user to quantify TLS-related gene expression on formalin-fixed paraffin-embedded human breast tumor tissues.

Abstract A62: Investigating the role of follicular helper T cells, B cells and CXCL13 in breast cancer-associated tertiary lymphoid structures

Patient outcomes have been linked to the presence of tumor infiltrating lymphocytes (TIL) in solid tumors. In human breast cancer (BC), higher TIL infiltration is associated with a better prognosis and also predicts relevant responses to pre-operative chemotherapy. TIL are primarily composed of T cells, albeit around 20% of BC patients show significant B cell infiltration, and can organize in tertiary lymphoid structures (TLS) located in the peritumoral stroma 1, which are associated with survival in HER2+ and triple negative BC patients. Further, these studies revealed that CD4+ follicular helper T (Tfh) cells producing CXCL13 were specifically associated with peritumoral TLS. CXCL13 is an important B cell chemoattractant whose function is to recruit B cells to the germinal center (GC) in secondary lymphoid organs and TLS, where they can mature and differentiate into memory or antibody-producing B cells. Our recent efforts have focused on exploring the role of Tfh, B cells and CXCL13 play in the development and/or maintenance of GC-like structures in BC-associated TLS. We first derived a GC-B cell gene signature for integration in our published Tfh cell gene signature 1. The combined gene signature was tested for its ability to sensitively detect BC-associated TLS using a qRT-PCR-based assay and a retrospective series (n=54) of formalin-fixed paraffin-embedded (FFPE) BC tissues. These data revealed a correlation between gene signature expression and the extent of TIL and TLS scored by trained pathologists on dual-immunohistochemistry stained (CD3+CD20 for T and B cells, respectively) FFPE tissue sections. The detection of TLS using our combined GC-B cell/Tfh cell gene signature was subsequently confirmed using tissues from our prospective BC cohort (n=83). In addition, CXCL13 gene expression was well correlated with genes associated with GC-B cells and Tfh, indicating these parameters are closely related, as confirmed by immunofluorescence staining on FFPE tissues. Further understanding the factors that promote TLS formation in vivo could provide important insight for treatment decisions in BC. CXCL13 expression was originally identified as an important signal associated with TLS that was predictive for patient outcomes 1. Factors capable of inducing CXCL13 expression in CD4+ T cells isolated from peripheral blood were investigated using flow cytometry. TGFβ1 alone or together with several cytokines (IL4, IL12, IL21, IL23 and in particular IL2 blockade) increased CXCL13 expression in activated CD4+ T cells. Similar to our characterization of Tfh TIL in fresh tumor tissues, these CXCL13-producing CD4+ T cells were CXCR5 negative and expressed the Tfh marker ICOS; however, they only had low levels of PD-1 expression compared to PD-1hi Tfh cells. CD8+ T cells were also found to produce CXCL13 albeit at low levels. The currently ongoing identification of critical genes involved in regulating CXCL13 production in treated CD4+T cells will help to elucidate the mechanism(s) underlying chemokine induction. The increased accuracy in TIL and TLS detection in BC together with a better understanding of the role Tfh and CXCL13 play in these structures (and GC) development should help to identify the critical immune components involved in BC TLS formation. 1Gu-Trantien et al. J Clin Invest. 2013 Citation Format: Edoardo Migliori, Chunyan Gu-Trantien, Soizic Garaud, Gert Van den Eynden, Alexandre De Wind, Pushpamali De Silva, Cinzia Solinas, Anais Boisson, Celine Naveaux, Denis Larsimont, Martine Piccart-Gebhart, Karen Willard-Gallo. Investigating the role of follicular helper T cells, B cells and CXCL13 in breast cancer-associated tertiary lymphoid structures. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A62.

Abstract A124: Tertiary lymphoid structures: Predictors of effective antitumor immunity in human breast cancer?

The clinical relevance of tumor infiltrating lymphocytes (TIL) in breast cancer (BC) is now broadly accepted; however, the relationship between protective immunity, observed in some patients, and critical features of lymphoid subset composition and organization are largely unknown. Our recent work revealed that tertiary lymphoid structures (TLS), principally composed of T and B cells, present in the peri-tumoral regions of BC are associated with increased TIL infiltration in the tumor bed and good clinical outcomes in both the neo-adjuvant and adjuvant settings. To gain insight into the immune components linked with a significant TIL and TLS presence, we initiated a prospective study of T and B cell TIL. Breast tissues from tumors (current n=483) and matched non-adjacent non-tumor tissues (NANT) as well as normal tissue from mammary reductions (n=49) were systematically immunophenotyped by flow cytometry on the day of surgery. Primary tumor supernatants derived from the tissue homogenates (non-enzymatic) and plasma from all patients were stored for cytokine/chemokine and immunoglobulin analysis. TIL organization and spatial distribution was analyzed on paraffin sections using immunohistochemistry (IHC) and immunofluorescence (IF). The fresh tissue analyses revealed that TIL distribution in BC forms a continuum. The infiltration levels detected in normal tissue and NANT were used to define cutoffs that discriminate between TIL positive (TILpos) and negative (TILneg) tumors. TILneg tumors are defined as those with a TIL density 99th percentile of lymphocyte density in NANT and TIL-intermediate (TILint). Applying these thresholds to the TIL infiltration levels identified approximately 25% of tumors as TILneg with infiltrating lymphocytes similar to normal breast tissue. The TILpos tumors (75%) are subdivided into 36% TILint and 39% TILhi tumors. TILpos tumors are characterized by an increase in CD4+ T cells and CD19+/CD20+ B cells. The median CD4/CD8 ratio was >1 in TILpos compared to Citation Format: Soizic Garaud, Laurence Buisseret, Chunyan Gu-Trantien, Gert Van den Eynden, Alexandre de Wind, Edoardo Migliori, Roberto Salgado, Denis Larsimont, Martine Piccart, Karen Willard-Gallo. Tertiary lymphoid structures: Predictors of effective antitumor immunity in human breast cancer? [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A124.

Abstract 4139: TfhX13 cells link breast cancer immune suppression and adaptive memory

Background: Previously, we identified PD-1hiCD200hiCD4+ follicular helper T (Tfh) cells infiltrating human breast cancer (BC TIL) as principal producers of the B cell chemoattractant CXCL13 and correlated their presence with improved patient outcome. Our recent efforts have focused on understanding the relationship between Tfh, Th1 and Treg BC TIL, investigating the role(s) that CXCL13+CD4+ TIL play in tumor immunity and determining the condition(s) that favor their differentiation. Methods: Ten-color flow cytometry was used to characterize CD4+ TIL subpopulations in fresh tissues. Confocal microscopy was employed to study the in situ interaction between CXCL13+CD4+ T cells and B cells. CXCL13 was induced in normal PBMC in vitro. Results: Our data reveal that an exacerbated Treg response accompanies high CXCL13 expression within the activated CD4+ TIL compartment in BC. Th1 responses, using IFN-γ expression as a barometer, are limited. CXCL13+CD4+ TIL display both similar and distinct characteristics compared to their tonsillar counterparts. Tumor bed-localized CXCL13+ TIL, external to tertiary lymphoid structure (TLS) germinal centers (GC), are abundant in some tumors with extensive lymphoid infiltrates. Based on our observations, we designate the CXCL13+CD4+ TIL as TfhX13 cells. A combination of PD-1 and ICOS can clearly segregate the CD4+ TIL into three distinct subpopulations: PD-1loICOSlo, PD-1hiICOSint and PD-1intICOShi. The PD-1hiICOSint TIL are enriched in TfhX13 and Th1 cells while the PD-1intICOShi TIL are principally FoxP3hi Treg. A linear correlation is observed between these two TIL subpopulations in 90% of BC while the remaining 10% contain an unbalanced, higher level of PD-1intICOShi Treg. qRT-PCR data confirmed the significant positive prognostic value of CXCL13 gene expression with ICOS expression signaling the adverse effects of Treg cell dominance observed in some BC. TfhX13 TIL abundance appears to parallel both a GC and post-TLS memory B cell presence. Confocal microscopy revealed TfhX13 TIL interact directly with B cells and plasma cells, potentially guiding B cell migration through the tumor and promoting TLS formation with functional GC. Finally, we show that IL-2 deprivation is critical for inducing CXCL13 while TGF-β1 treatment reduces IFN-γ and increases FoxP3 expression in normal CD4+ T cells. Conclusion: IL-2 consumption by Treg cells was shown to be essential for murine Tfh cell development and a subsequent GC response. Supported by this observation, our data indicate that TfhX13 cell differentiation is an important element of the Tfh cell program and suggests their propagation in BC occurs in response to Treg accumulation. CXCL13 expression may activate adaptive memory responses dependent upon in situ B cell maturation and thereby initiate a secondary attempt at protective anti-tumor immunity in the face of Treg-mediated immune suppression. Citation Format: Chunyan Gu-Trantien, Edoardo Migliori, Denis Larsimont, Karen Willard-Gallo. TfhX13 cells link breast cancer immune suppression and adaptive memory. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4139.

Abstract 1669: Characterization of B-cells infiltrating human breast cancer and their presence in peritumoral tertiary lymphoid structures

Recent advances in tumor immunology show an important link exists between tumor infiltrating lymphocytes (TIL) and patient outcome. In human breast cancer (BC) more extensive lymphocyte infiltration is associated with a better prognosis and can also predict responses to pre-operative chemotherapy. Tertiary lymphoid structures (TLS) detected at the tumor periphery have been correlated with a good prognosis in lung and colorectal cancer. Our recent work was the first to demonstrate that TLS are present in BC, located adjacent to the tumor bed in extensively infiltrated tumors. TLS organization is similar to a lymph node, with a CD3+ T-cell zone adjacent to a CD20+ B-cell follicle where germinal centers containing CD23+ follicular dendritic cells, Bcl6+ TFH cells and Ki67+ cells are found. Our laboratory recently discovered that TLS-associated CD4+ Tfh cells are present in extensively infiltrated tumors and showed they signal a good prognosis. This important presence of Tfh-containing TLS adjacent to the tumor bed suggests that B-cells may also play a role in generating effective anti-tumor immune responses. We therefore undertook to fully characterize the B-cells infiltrating human BC. Flow cytometric analysis of fresh breast tumor homogenates detected an increase in CD45+ leukocytes in all BC subtypes compared with normal and non-tumor non-adjacent (NANT) breast tissue irrespective of lymphocyte infiltration levels. The infiltrate was dominated by CD3+ T-cells (65-86% of CD45+) in normal and malignant tissues. In contrast, B-cells were elevated in tumors compared to normal and NANT tissue, particularly in extensively infiltrated high proliferative BC subtypes. This increase was paralleled by a relative decrease in CD8+ T-cells. CD45/CD19/CD38/IgD labeling revealed that approximately 50% of the infiltrating B-cells are memory cells in contrast to normal tissues, which contain less than 15%. Centroblasts and centrocytes, which are follicular B cells, were significantly increased in extensively infiltrated tumors in association with Tfh cells. We assessed immunoglobulin isotypes in primary supernatants from breast tissue homogenates, detecting decreased IgA and increased IgG and IgM per mm3 in the tumor. Patient sera, particularly from high proliferative subtypes, had elevated IgG4 compared with low proliferative subtypes and healthy donors. Immunofluorescent analysis of embedded tissues show that TLS contain a marginal zone region(CD20+CD27+IgD-), a follicular mantle zone (CD20+IgD+) and a germinal center zone (CD20+Ki67+CD35+CD21+PD-1+). Tumor-associated TLS are surrounded by T-cells (CD4+ and CD8+). These data reveal that when B-cells are present in breast tumors they are principally associated with organized TLS and may therefore play an important functional role in comprehensive anti-tumor immune responses, an aspect currently under further investigation. Citation Format: Soizic Garaud, Laurence Buisseret, Chunyan Gu, Edoardo Migliori, Jean-Nicolas Lodewyckx, Hugues Duvillier, Ligia Craciun, Denis Larsimont, Karen Willard-Gallo. Characterization of B-cells infiltrating human breast cancer and their presence in peritumoral tertiary lymphoid structures. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1669. doi:10.1158/1538-7445.AM2014-1669

Abstract P5-01-02: Characterization of follicular helper CD4 T cells and B cells resident in peritumoral tertiary lymphocyte structures as specific markers for an immunological grade in breast cancer

Molecular approaches such as gene expression profiling have improved the classification of human breast cancer (BC) by identifying molecular subtypes and demonstrating that the immune infiltrate is an important prognostic or predictive factor. Our prospective study of freshly isolated CD4+ T cells infiltrating (TIL) BC discovered that along with Th1, Th2 and Th17 effector memory and Treg subpopulations the newest CD4+ Th subset, T follicular helper (Tfh) cells, was also present in the tumor. Comparison of extensive versus minimally-infiltrated tumors led to our finding that extensive immune infiltrates are distinguished by Tfh cells located in tertiary lymphoid structures (TLS) next to the tumor bed. TLS are highly organized with a CD3+ T cell zone adjacent to a CD20+ B cell follicle with germinal centers containing Bcl6+ Tfh cells, CD23+ follicular dendritic cells and Ki67+ cells. Tfh cells are known to play a critical role in generating antibody producing plasma cells and memory B cells. We demonstrated that Tfh cells are the principal cellular source of the B cell chemoattractant CXCL13 in BC. Thus, a Tfh presence paralleled the TLS incidence and together they were associated with an increased peritumoral B cell presence and a higher frequency of CD8+ T cells in the tumor bed. Our recent work shows that approximately 50% of the infiltrating B cells are memory cells in contrast to normal and non-tumor non-adjacent breast tissue (controls, less than 15%). A significant B cell presence is found in extensively infiltrated high proliferative BC subtypes with germinal center centroblasts and centrocytes consistently associated with a Tfh cell presence. We assessed immunoglobulin isotypes in supernatants of fresh breast tissue homogenates, detecting decreased IgA and increased IgG and IgM in the tumor tissue compared to controls. Patient sera, particularly from patients with high proliferative BC subtypes, had elevated IgG3 and IgG4 compared to low proliferative tumors and healthy donors. Immunofluorescent analysis found the majority of B cells infiltrating tumors have germinal center characteristics (IgD+Ki67+CD35+CD21+PD-1+) and are clustered with Tfh in the B cell follicles surrounded by a T cell zone, which together create the tumor-associated TLS. Based on their specificity for TLS, we derived a scoring system, referred to as immunological grade, to measure the extent of the lymphocytic infiltrate and the level of immune organization. This system is based on a morphological score (the density of the lymphocyte infiltrate determined by CD3/CD20 immunohistochemistry plus the number and size of peritumoral TLS assessed by two pathologists) together with a molecular score [an 8 gene qRT-PCR Tfh signature that predicts long term survival in an untreated patient cohort with >10-year survival (n = 794) or pathological complete response in patients treated with preoperative chemotherapy (n = 996)]. We are testing this immunological grading system on a large retrospective series of patients to determine its value in evaluating a patients anti-tumor immune response in pre-operative biopsies and/or the primary tumor at surgery as a new marker for use in treatment decisions. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-02.