Chunyan Gu-Trantien

CD4+ follicular helper T cell infiltration predicts breast cancer survival

CD4⁺ T cells are critical regulators of immune responses, but their functional role in human breast cancer is relatively unknown. The goal of this study was to produce an image of CD4⁺ T cells infiltrating breast tumors using limited ex vivo manipulation to better understand the in vivo differences associated with patient prognosis. We performed comprehensive molecular profiling of infiltrating CD4⁺ T cells isolated from untreated invasive primary tumors and found that the infiltrating T cell subpopulations included follicular helper T (Tfh) cells, which have not previously been found in solid tumors, as well as Th1, Th2, and Th17 effector memory cells and Tregs. T cell signaling pathway alterations included a mixture of activation and suppression characterized by restricted cytokine/chemokine production, which inversely paralleled lymphoid infiltration levels and could be reproduced in activated donor CD4⁺ T cells treated with primary tumor supernatant. A comparison of extensively versus minimally infiltrated tumors showed that CXCL13-producing CD4⁺ Tfh cells distinguish extensive immune infiltrates, principally located in tertiary lymphoid structure germinal centers. An 8-gene Tfh signature, signifying organized antitumor immunity, robustly predicted survival or preoperative response to chemotherapy. Our identification of CD4⁺ Tfh cells in breast cancer suggests that they are an important immune element whose presence in the tumor is a prognostic factor.

Tumor-infiltrating lymphocyte composition, organization and PD-1/ PD-L1 expression are linked in breast cancer

ABSTRACT The clinical relevance of tumor-infiltrating lymphocytes (TIL) in breast cancer (BC) has been clearly established by their demonstrated correlation with long-term positive outcomes. Nevertheless, the relationship between protective immunity, observed in some patients, and critical features of the infiltrate remains unresolved. This study examined TIL density, composition and organization together with PD-1 and PD-L1 expression in freshly collected and paraffin-embedded tissues from 125 patients with invasive primary BC. Tumor and normal breast tissues were analyzed using both flow cytometry and immunohistochemistry. TIL density distribution is a continuum with 25% of tumors identified as TIL-negative at a TIL density equivalent to normal breast tissues. TIL-positive tumors (75%) were equally divided into TIL-intermediate and TIL-high. Tumors had higher mean frequencies of CD4+ T cells and CD19+ B cells and a lower mean frequency of CD8+ T cells compare with normal tissues, increasing the CD4+/CD8+ T-cell ratio. Tertiary lymphoid structures (TLS), principally located in the peri-tumoral stroma, were detected in 60% of tumors and correlated with higher TIL infiltration. PD-1 and PD-L1 expression were also associated with higher TIL densities and TLS. TIL density, TLS and PD-L1 expression were correlated with more aggressive tumor characteristics, including higher proliferation and hormone receptor negativity. Our findings reveal an important relationship between PD-1/PD-L1 expression, increased CD4+ T and B-cell infiltration, TIL density and TLS, suggesting that evaluating not only the extent but also the nature and location of the immune infiltrate should be considered when evaluating antitumor immunity and the potential for benefit from immunotherapies.

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.

Tumor-infiltrating follicular helper T cells: The new kids on the block

By analyzing CD4+ lymphocytes in human breast carcinomas, we have recently uncovered the presence of follicular helper T cells in lesions exhibiting an extensive immune infiltrate. The presence of these specialized CD4+ T cells, which localize to the germinal centers of peritumoral tertiary lymphoid structures found in extensively infiltrated neoplastic lesions, predicts improved disease outcome among breast carcinoma patients.

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.

FOXP1 is a regulator of quiescence in healthy human CD4+ T cells and is constitutively repressed in T cells from patients with lymphoproliferative disorders

The forkhead box P1 (FOXP1) transcription factor has been shown to regulate the generation and maintenance of quiescent naïve murine T cells. In humans, FOXP1 expression has been correlated with overall survival in patients with peripheral T‐cell lymphoma (PTCL), although its regulatory role in T‐cell function is currently unknown. We found that FOXP1 is normally expressed in all human leukocyte subpopulations. Focusing on primary human CD4+ T cells, we show that nuclear expression of FOXP1 predominates in naïve cells with significant downregulation detected in memory cells from blood and tonsils. FOXP1 is repressed following in vitro T‐cell activation of naïve T cells, and later re‐established in memory CD4+ T cells, albeit at lower levels. DNA methylation analysis revealed that epigenetic mechanisms participate in regulating the human FOXP1 gene. ShRNA‐mediated FOXP1 repression induces CD4+ T cells to enter the cell cycle, acquire memory‐like markers and upregulate helper T‐cell differentiation genes. In patients with lymphoproliferative disorders, FOXP1 expression is constitutionally repressed in the clonal T cells in parallel with overexpression of helper T‐cell differentiation genes. Collectively, these data identify FOXP1 as an essential transcriptional regulator for primary human CD4+ T cells and suggest its potential important role in the development of PTCL.

Circulating (CD3−CD19+CD20−IgD−CD27highCD38high) Plasmablasts: A Promising Cellular Biomarker for Immune Activity for Anti-PLA2R1 Related Membranous Nephropathy?

Membranous nephropathy (MN) is a kidney specific autoimmune disease mainly mediated by anti-phospholipase A2 receptor 1 autoantibody (PLA2R1 Ab). The adequate assessment of chimeric anti-CD20 monoclonal antibody, rituximab (RTX), efficacy is still needed to improve clinical outcome of patient with MN. We evaluated the modification of plasmablasts (CD3−CD19+CD20−IgD−CD27highCD38high), a useful biomarker of RTX response in other autoimmune diseases, and memory (CD3−CD19+CD20+IgD−CD27+CD38−) and naive (CD3−CD19+CD20+IgD+CD27−CD38low) B cells by fluorescence-activated cell sorter analysis in PLA2R1 related MN in one patient during the 4 years of follow-up after RTX. RTX induced complete disappearance of CD19+ B cells, plasmablasts, and memory B cells as soon as day 15. Despite severe CD19+ lymphopenia, plasmablasts and memory B cells reemerged early before naive B cells (days 45, 90, and 120, resp.). During the follow-up, plasmablasts decreased more rapidly than memory B cells but still remained elevated as compared to day 0 of RTX. Concomitantly, anti-PLA2R1 Ab increased progressively. Our single case report suggests that, besides monitoring of serum anti-PLA2R1 Ab level, enumeration of circulating plasmablasts and memory B cells represents an attractive and complementary tool to assess immunological activity and efficacy of RTX induced B cells depletion in anti-PLA2R1 Ab related MN.

PD-1hiCXCR5−CD4+ TFH Cells Play Defense in Cancer and Offense in Arthritis

T follicular helper (TFH) cells are characteristically defined by their CXCR5 positivity and homing to B cell follicles in secondary lymphoid organs (SLO). An expanded subpopulation of functionally comparable and phenotypically similar PD-1hiCXCR5-CD4+ T cells were recently identified in breast cancer (BC) and rheumatoid arthritis (RA) to have beneficial or detrimental roles, respectively, but are they inflammatory tissue effector TFH cells?

Abstract PD1-3: The significance of tumor infiltrating lymphocyte density, subset composition and organization in breast cancer

The clinical relevance of tumor infiltrating lymphocytes (TIL) in breast cancer (BC) has been clearly established by recent large clinical trials (presented at SABCS 2013). The relationship between protective immunity, observed in some patients, and critical features of lymphoid subset composition and organization remain unknown. Our recent work revealed that tertiary lymphoid structures (TLS), principally composed of T cells, B cells and dendritic cells, are present in the peri-tumoral regions of breast tumors and associated with a CD4+ follicular helper T (Tfh) cell presence. Through retrospective analyses, we determined that TLS were associated with 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 flow cytometric study. We systematically immunophenotyped T and B cell TIL in breast tissues from tumors (n=125), non-adjacent non-tumor tissues (NANT, n=115) and normal tissue from mammary reductions (n=26) on the day of surgery. TIL organization and spatial distribution was subsequently analyzed by immunohistochemistry (IHC) and immunofluorescence (IF) on paraffin sections for a subset of patients (n=78). The fresh tissue analyses revealed that TIL density was a continuum across the 125 patients analyzed. A cutoff for TIL-positive and -negative tumors was set at 58 CD45+ TIL per mm3 of tissue based on the number of CD45+ cells present in the remarkably similar normal and NANT tissues. Applying this threshold to BC, 65% were TIL-positive with approximately one-third considered extensively infiltrated. TIL-positive tumors are characterized by an increase in CD4+ T cells and CD19+/CD20+ B cells. The median CD4/CD8 ratio was >1 in TIL-positive compared to 95% of CD45+ cells) IHC and scored by trained pathologists. The best correlation was observed between CD3/CD20 flow cytometry and CD3/CD19 IHC, the latter also associated with lower inter-observer variability. TIL density was positively correlated with proliferation (Ki67 & histological grade) and hormone receptor negativity while TLS were more frequent in younger women. These data suggest that organized immune responses in TLS adjacent to the tumor bed provide an effective location for generating anti-tumor memory T and B cell responses. Citation Format: Karen Willard-Gallo, Laurence Buisseret, Soizic Garaud, Chunyan Gu-Trantien, Alexandre de Wind, Sebastien Duquenne, Denis Larsimont, Christos Sotiriou, Martine Piccart. The significance of tumor infiltrating lymphocyte density, subset composition and organization in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD1-3.

The transcription factors Runx3 and ThPOK cross-regulate acquisition of cytotoxic function by human Th1 lymphocytes

Cytotoxic CD4 (CD4CTX) T cells are emerging as an important component of antiviral and antitumor immunity, but the molecular basis of their development remains poorly understood. In the context of human cytomegalovirus infection, a significant proportion of CD4 T cells displays cytotoxic functions. We observed that the transcriptional program of these cells was enriched in CD8 T cell lineage genes despite the absence of ThPOK downregulation. We further show that establishment of CD4CTX-specific transcriptional and epigenetic programs occurred in a stepwise fashion along the Th1-differentiation pathway. In vitro, prolonged activation of naive CD4 T cells in presence of Th1 polarizing cytokines led to the acquisition of perforin-dependent cytotoxic activity. This process was dependent on the Th1 transcription factor Runx3 and was limited by the sustained expression of ThPOK. This work elucidates the molecular program of human CD4CTX T cells and identifies potential targets for immunotherapy against viral infections and cancer.