METHOD FOR PREDICTING THE SURVIVAL TIME OF A PATIENT SUFFERING FROM A CANCER
20230086718 · 2023-03-23
Inventors
- Ghita CHABAB (Cedex 5 Montpellier, FR)
- Virginie LAFONT (Cedex 5 Montpellier, FR)
- Nathalie BONNEFOY (Cedex 5 Montpellier, FR)
Cpc classification
G01N33/57484
PHYSICS
G01N2800/52
PHYSICS
International classification
Abstract
The present invention relates to the prediction of the survival time of a patient suffering from a cancer. The inventors identified in blood from healthy donors a subpopulation of WIT cells that expresses CD73 and displays immunosuppressive phenotype and functions (i.e., production of immunosuppressive molecules and inhibition of αßT cell proliferation). Furthermore, they detected the presence of CD73+ γδ T cells in immune infiltrates of freshly resected breast cancer specimens. Altogether, these data suggest that part of γδ T cells infiltrated in the breast cancer microenvironment presents a regulatory phenotype characterized by CD73 expression, and are likely to display pro-tumor functions through the mechanisms they described in vitro. Thus, the invention relates to a method for predicting the survival time of a patient suffering from a cancer comprising i) determining in a sample obtained from the patient the level of Gamma/Delta T cells expressing CD73 ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the level of Gamma/Delta T cells expressing CD73 determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the level of Gamma/Delta T cells expressing CD73 determined at step i) is higher than its predetermined reference value.
Claims
1. A method for predicting the survival time of and treating a patient suffering from a cancer comprising i) measuring the level of Gamma/Delta T cells expressing CD73 in a sample obtained from the patient; ii) determining that the level of Gamma/Delta T cells expressing CD73 is higher than a reference value and iii) treating the subject determined to have a higher level of Gamma/Delta T cells expressing CD73.
2. The method according to claim 1 wherein the Gamma/Delta 1 T cells expressing CD73 are Gamma/Delta 1 T cells expressing CD73 or Gamma/Delta 2 T cells expressing CD73.
3. The method according to claim 1, wherein the Gamma/Delta T cells expressing CD73 also express CD39 and/or an immune checkpoint selected from the group consisting of PD-L1, CTLA-4, PD1 and TIGIT.
4. The method according to claim 3 wherein the Gamma/Delta T cells expressing CD73 also express CD39 and an immune checkpoint selected from the group consisting of PD-L1, CTLA4, PD1 and TIGIT.
5. The method according to claim 4 wherein the Gamma/Delta T cells expressing CD73 are Gamma/Delta 1 T cells and also express CD39 and PD-L1.
6. The method according to claim 4 wherein the Gamma/Delta T cells expressing CD73 are Gamma/Delta 2 T cells and also express CD39 and PD-L1.
7. The method according to claim 1, wherein the sample is blood, peripheral-blood, a cancer biopsy or surgical pieces.
8. The method according to claim 1, wherein the step of measuring the Gamma/Delta T cells is done by flow cytometry.
9. (canceled)
Description
FIGURES
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EXAMPLES
Example 1
[0101] Material & Methods
[0102] Antigens, Antibodies and Reagents
[0103] Phytohemagglutinin (PHA) was purchased from Thermofisher Scientific. The anti-human CD3 (UCHT1), and the anti-human TCR γδ and TCR Vδ1 antibodies were purchased from Beckman Coulter (Brea, Calif., USA). Anti-human CD3, CD73, CD39 and their isotypically matched control mouse antibodies were from BD Biosciences (San Jose, Calif., USA). The anti-human TCR γδ antibody used for IHC was from Santa Cruz Biotechnology (USA). Recombinant human IL-2 (rhIL-2) was from Novartis Pharma (Rueil-Malmaison, France), and recombinant IL-21 (rhIL-21) was from Miltenyi Biotec (Paris, France). ELISA kits for detecting human IL-10 and IL-8 were from BD Biosciences (San Jose, Calif., USA). ELISA kit for the detection of human TGFβ was from RD system.
[0104] Cell Culture
[0105] Peripheral Blood Mononuclear Cells (PBMCs) were obtained by density centrifugation on Ficoll-Paque (Eurobio, Les Ulis, France) of blood samples from healthy donors and breast cancer patients. Healthy donor samples were provided by the Etablissement Français du Sang (Convention EFS-OCPM no 21PLER2018-0069) and the blood of patients was provided by the ICM (BCB-EC-1-FR-ICM-ENR-269-004-CRB). To analyze comparable cohorts between cancer patients and healthy donors, we selected EFS blood samples from healthy women with an age ranging from 18 to 70 years. Vδ1 T cells were isolated from PBMCs by a positive immunoselection using the anti-human Vδ1 antibody (Beckman Coulter) and anti-IgG1 magnetic beads (Miltenyi Biotec). Briefly, 300.106 cells were incubated with 10 μg of anti-human Vδ1 antibody in 5 ml of PBS supplemented with 2% SVF and EDTA (2 mM) for 1 hour at 4° C., then washed and incubated with 200 μl of anti-IgG1 magnetic beads for 1 hour at 4° C., then washed and collected on column according to the manufacturer's instructions (Miltenyi Biotec). Reproducible high purity of Vδ1 T cells (>90%) was obtained with this protocol. Purified Vδ1 T cells (2.106 cells/ml) were stimulated with 2 μg/mL of PHA in the presence of syngeneic macrophages isolated using their adherence properties. Briefly, PBMC (2.106/ml) were incubated in RPMI 10% FCS for 1 hour at 37° C. in 96 well-plates to allow to monocytes to adhere and differentiate in macrophages. Non-adherent cells were removed by 2 washes with RPMI medium. Purified Vδ1 T cells were added to macrophages, activated by PHA and expanded in the complete medium containing RPMI 1640/Glutamax medium supplemented with 5% human AB serum and 5% fetal calf serum (FCS) in the presence of rhIL-2 (control) or rhIL-2+rhIL-21 (experimental condition) at 37° C. in humidified atmosphere with 5% CO2. Every 2 days, fresh medium is added and after 1 week Vδ1 T cells were separated from adherent macrophages and amplified in the complete medium with cytokines for 2 more weeks before phenotyping and analysis.
[0106] Flow Cytometry Analysis
[0107] Cells were first incubated at 4° C. for 30 min with Fc-block solution to minimize non-specific binding of antibodies to Fc receptors, then incubated with a dye cell viability and the panel of antibodies in the staining buffer (PBS-2% FCS) at 4° C. for 30 min, cells were then washed and fixed in 1% paraformaldehyde. Data were acquired with a Cytoflex cytometer (Beckman-Coulter) and analyzed with the FlowJo software.
[0108] ELISA
[0109] Expanded Vδ1 T cells (2.106 cells/mL) were incubated in fresh medium without cytokines in wells coated or not with the anti-CD3 antibody (UCHT1) for 6 h, and then supernatants were collected. IL-8 and IL-10 protein levels were assessed using the relevant BD Biosciences Opteia Kits. The mean values of duplicate samples from the same experiment are shown for each data point with their standard error of the mean (SEM).
[0110] Adenosine Measurement by MALDI TOF Spectrometry
[0111] Amplified Vδ1 T cells (2.106 cells/mL) were washed in cold PBS and resuspended in PBS supplemented with 50 mM AMP (Sigma) at 4° C. for 30 min. After centrifugation, adenosine levels in supernatants were analyzed by MALDI-TOF mass spectrometry, as previously described by Bastid et al. [44].
[0112] T Cell Proliferation Assay
[0113] Peripheral blood from healthy donors was obtained from the EFS and mononuclear cells were isolated on a Ficoll gradient. PBMC were stained with 2.5 μM CFSE for 11 min at 37° C. 4×104 CFSE stained PBMC were distributed in 96 well flat-bottom plates coated with the anti-CD3 antibody (UCHT1 10 μg/ml). Sorted CD73- and CD73+ Vδ1 T cells were added at ratio 1:1. Proliferation of αβ T cells was analyzed by flow cytometry at day 5.
[0114] Tumor Dissociation
[0115] Freshly resected tumors from patients were cut into small pieces (around 1 mm3). Tissues were resuspended in digestion solution (10 mg/ml collagenase IV from Sigma and 10 mg/ml DNase I from Roche) in Hanks modified balanced salt solution and alternate between enzymatic digestion (15 min at 37° C.) and mechanical dissociation using the gentle MACS dissociator (Miltenyi Biotec) for 3 rounds. The obtained single cell suspensions were washed in PBS/2% FCS, and resuspended in PBS/2% FCS with FcBlock (Miltenyi Biotec) at 4° C. in the dark for 30 min. Then, cells were washed and incubated with a panel of conjugated antibodies and results analyzed as described in the previous sections. The cession of fresh samples was approved by the Montpellier Cancer Institute Review Board (ICM-CORT-2018-34).
[0116] Breast Tumor Tissue Microarray
[0117] A TMA that included breast tumors from 50 patients was constructed using two malignant tissue cores (1 mm diameter) per tumor. Tissue samples were from patients who underwent surgery at our institution between 2001 and 2011 and received no neoadjuvant treatment. They were informed and gave their consent for using their tissue samples for biological research. Tumor samples were collected following the French laws under the supervision of an investigator and declared to the French Ministry of Higher Education and Research (declaration number DC-2008-695). The study was approved by the Montpellier Cancer Institute Review Board (ICM-CORT-2015-32).
[0118] Immunohistochemistry
[0119] After epitope retrieval in EDTA buffer (pH 9) and neutralization of endogenous peroxidase, 3 μm TMA sections were incubated with the mouse monoclonal anti-TCR γδ antibody (Clone H-41, Santa Cruz) at room temperature for 30 min. This was followed by an amplification step with a mouse linker and the standard detection system (Flex, Dako), consisting of a dextran backbone to which a large number of peroxidase molecules and secondary anti-mouse and anti-rabbit antibodies were coupled. 3,30-Diaminobenzidine was used as substrate. The NanoZoomer slide scanner system (Hamamatsu Photonics) was used to digitalize the stained TMA sections with a ×20 objective. Immunoreactive cells were manually identified and counted on the digitalized slides with the NDP.view software. When both samples from the same tumor were assessable (39 out of 50), the mean value was calculated and data expressed as number of TCR γδ-positive cells per spot. Salgado's method was used to determine the immune infiltrate in TMA spots [45].
[0120] Statistical Analysis
[0121] Results were compared using the paired Wilcoxon test or a chi2 test depending on the experiment. A P value <0.05 was considered as statistically significant. Analyses were performed using the GraphPad Prism software, version 6.
[0122] Results
[0123] A Subset of Vδ1 T Cells Expresses the CD39 and CD73 Ectonucleotidases
[0124] Extracellular ATP and adenosine act as positive and negative regulators of the immune response, respectively. We previously reported that ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1 or CD39), which catalyzes the phosphohydrolysis of extracellular ATP into ADP and of ADP into AMP, is expressed by Vγ9Vδ2 T cells after TCR activation [43]. Conversely, the ecto-5′-nucleotidase CD73, which completes AMP conversion into adenosine, is only expressed by a subset of activated Vγ9Vδ2 T cells, a phenotype that can be amplified in the presence of IL-21 [43]. Here, we investigated CD39 and CD73 expression in the Vδ1 T cell subpopulation. To this aim, we isolated Vδ1 T cells from the blood of healthy donors, and activated and amplified them in vitro in the presence of rhIL-2 (control) or rhIL-2+rhIL21 (experimental condition). We observed that most Vδ1 T cells (>85%) expressed CD39. Moreover, exposure to rhIL-21 did not modify the percentage of CD39-positive cells, suggesting that IL-21 does not influence its expression (data not shown). Conversely, rhIL-21 significantly increased the fraction of CD73-positive Vδ1 T cells to 50% (vs 20% in the control condition), although results were heterogeneous among donors (data not shown). The fraction of CD39/CD73-positive Vδ1 T cells also was significantly higher in cells exposed to rhIL-21 compared with control cells (40% vs 18%) (data not shown).
[0125] Adenosine Production by Vδ1 T Cells
[0126] Around 60-70% of CD73-positive Vδ1 T cells expressed also CD39 (data not shown), suggesting that these cells could produce adenosine in an autonomous manner in an ATP-rich environment. As the percentage of CD39-positive Vδ1 T cells was identical in the presence/absence of rhIL-21, we hypothesized that CD73 expression was the only parameter influencing adenosine production. Therefore, we quantified adenosine levels in the supernatants of Vδ1 T cells by MALDI-TOF mass spectrometry, as previously described [44]. IL-21-amplified Vδ1 T cells generated higher amounts of adenosine in the presence of AMP compared with control cells (only rhIL-2) (˜1.4 μM vs ˜0.4 μM) (data not shown). The presence of adenosine 5′-(α,β-methylene)-diphosphate sodium salt (APCP), a pharmacological inhibitor of CD73, strongly impaired adenosine production by control and IL-21-amplified Vδ1 T cells (data not shown), confirming that adenosine synthesis by these cells depends on CD73 activity. Taken together, these results suggest that CD73-positive Vδ1 T cells display an immunosuppressive function through adenosine production. Moreover, the presence of some CD73-positive control cells suggests that rhIL-21 promotes the development of a preexisting immunosuppressive population, rather than just inducing CD73 expression.
[0127] CD73+ Vδ1 T Cells Produce IL-10 and IL-8
[0128] Conventional regulatory T cells inhibit the immune response by producing adenosine and also by secreting immunoregulatory cytokines, such as IL-10 and TGF-β [46]. Therefore, we determined whether Vδ1 T cells produce regulatory cytokines. In absence of activation we detected basal level of IL-10 in the supernatants of IL-21-amplified Vδ1 T cells whether they expressed or not CD73, but not of control cells. TCR/CD3 activation strongly increased IL-10 production by IL-21-amplified CD73+ but not in the CD73− Vδ1 T cells. (data not shown). Conversely, we did not detect TGF-β secretion in any of the tested conditions (data not shown).
[0129] As many studies showed that immunosuppressive T cells produce CXCL8 or IL-8, a chemokine implicated in angiogenesis and the recruitment of neutrophils and MDSCs in the tumor microenvironment, we investigated IL-8 production by Vδ1 T cells [40, 47, 48]. As observed for IL-10, IL-8 was highly present only in activated IL-21-amplified CD73+ Vδ1 T cells (data not shown). Thus, CD73+ Vδ1 T cell subset amplified in the presence of IL-21 were the main producer of IL-10 and IL-8.
[0130] These results suggest that through the secretion of IL-10 and IL-8, Vδ1 T cells could contribute to create a tumor microenvironment rich in suppressive factors that favor tumor development and growth.
[0131] CD73+ Vδ1 T Cells Inhibit αβ T Cell Proliferation
[0132] To determine whether CD73+ Vδ1 T cells display regulatory functions, we analyzed their capacity to inhibit γδ T cells proliferation. We sorted Vδ1 T cells in function to their CD73 expression and then evaluated proliferation of CFSE-labeled T cells grown in the presence or not of immobilized anti-CD3 monoclonal antibodies and co-cultured with CD73− or CD73+ Vδ1 T cells. Around 90% of αβ T cell proliferated when co-cultured with CD73− Vδ1 T cells compared to only 60% in the presence of CD73+ subset (data not shown). Overall, these results suggested that CD73+ Vδ1 T cells display regulatory functions that can affect the proliferative capacity of other immune cells, such as αβ T cells.
[0133] Identification of γδ T cells in human breast cancer samples
[0134] To evaluate the in vivo relevance of our findings in human solid cancers, we first assessed by IHC γδ TCR expression in a TMA that included 50 human breast samples at different SBR grades and phenotypes based on HER2 and hormone receptor expression status. All tumor samples used to build TMA were collected at surgery from patients who did not receive any neoadjuvant treatment. Among the 50 tumors analyzed after IHC, 41 showed at least one γδT lymphocyte per mm2. Although γδ T lymphocyte density was highly heterogeneous among samples (from 1 to >500 γδ T cells per mm2) their density progressively increased from grade I to grade III (data not shown). This suggests, as previously reported by others [49], that in breast cancer, the presence of γδ T cells is associated with late tumor grade and/or poor prognosis. Nevertheless, in parallel the analysis of immune infiltrate by Salgado's method showed that lower γδ TCR expression are associated with lower immune infiltrates and inversely. Moreover, although the difference did not reach significance, we observed a higher frequency of γδ T cells in triple-negative breast cancer (TNBC) compared to HER2- or hormone receptor breast cancer (data not shown). TNBCs comprise a very heterogeneous group of cancers. The general prognosis is rather similar with other breast cancer of same stage, except that more aggressive treatment is required due to the inefficacy of hormone- or HER2-targeting therapies. Some types of triple-negative breast cancer are known to be more aggressive, with poor prognosis, while other types have very similar or better prognosis than hormone receptor positive breast cancers. This suggests that other parameters must intervene in the prognostic and evolution of breast cancer.
[0135] Presence of CD73+ γδ T Cells in Human Breast Cancer
[0136] Then, to characterize the phenotype of infiltrated γδ T cells in human breast tumors, we dissociated fresh breast cancer biopsies and analyzed the phenotype of tumor-infiltrating γδ T cells by flow cytometry with the gating protocol. Tumor samples (n=16) were from patients with grade I, II and III breast tumor who did not receive any neoadjuvant treatment. Although the fraction of CD45-positive cells in breast cancers was very heterogeneous (0.75% to 25%), the proportion of CD3-positive cells among the CD45-positive cells was homogenous (around ˜80%), and γδ T cells were always present and represented 3% to 13% of CD3+ cells. (
Example 2
[0137] Material & Methods
[0138] Cell Culture
[0139] Peripheral Blood Mononuclear Cells (PBMCs) were obtained by density centrifugation on Ficoll-Paque (Eurobio, Les Ulis, France) of blood samples from healthy donors and breast cancer patients. Healthy donor samples were provided by the Etablissement Français du Sang and the blood of patients was provided by the Institut regional du Cancer de Montpellier (ICM). To analyze comparable cohorts between cancer patients and healthy donors, we selected EFS blood samples from healthy women with an age ranging from 18 to 70 years. Vδ1 T cells were isolated from PBMCs by a positive immunoselection using the anti-human Vδ1 antibody (Beckman Coulter) and anti-IgG1 magnetic beads (Miltenyi Biotec). Briefly, 300.106 cells were incubated with 10 μg of anti-human Vδ1 antibody in 5 ml of PBS supplemented with 2% SVF and EDTA (2 mM) for 1 hour at 4° C., then washed and incubated with 200 μl of anti-IgG1 magnetic beads for 1 hour at 4° C., then washed and collected on column according to the manufacturer's instructions (Miltenyi Biotec). Reproducible high purity of Vδ1 T cells (>90%) was obtained with this protocol. Purified Vδ1 T cells (2.106 cells/ml) were stimulated with 2 μg/mL of PHA in the presence of syngeneic macrophages isolated using their adherence properties. Briefly, PBMC (2.106/ml) were incubated in RPMI 10% FCS for 1 hour at 37° C. in 96 well-plates to allow to monocytes to adhere and differentiate in macrophages. Non-adherent cells were removed by 2 washes with RPMI medium. Purified Vδ1 T cells were added to macrophages, activated by PHA and expanded in the complete medium containing RPMI 1640/Glutamax medium supplemented with 5% human AB serum and 5% fetal calf serum (FCS) in the presence of rhIL-2 (control) or rhIL-2+rhIL-21 (experimental condition) at 37° C. in humidified atmosphere with 5% CO2. Every 2 days, fresh medium is added and after 1 week Vδ1 T cells were separated from adherent macrophages and amplified in the complete medium with cytokines for 2 more weeks before phenotyping and analysis.
[0140] Flow Cytometry Analysis
[0141] Cells were first incubated at 4° C. for 30 min with Fc-block solution to minimize non-specific binding of antibodies to Fc receptors, then incubated with a dye cell viability and the panel of antibodies in the staining buffer (PBS-2% FCS) at 4° C. for 30 min, cells were then washed and fixed in 1% paraformaldehyde. Data were acquired with a Cytoflex cytometer (Beckman-Coulter) and analyzed with the FlowJo software.
[0142] Tumor Dissociation
[0143] Freshly resected tumors from patients were cut into small pieces (around 1 mm3). Tissues were resuspended in digestion solution (10 mg/ml collagenase IV from Sigma and 10 mg/ml DNase I from Roche) in Hanks modified balanced salt solution and alternate between enzymatic digestion (15 min at 37° C.) and mechanical dissociation using the gentle MACS dissociator (Miltenyi Biotec) for 3 rounds. The obtained single cell suspensions were washed in PBS/2% FCS, and resuspended in PBS/2% FCS with FcBlock (Miltenyi Biotec) at 4° C. in the dark for 30 min. Then, cells were washed and incubated with a panel of conjugated antibodies and results analyzed as described in the previous sections. The cession of fresh samples was approved by the Montpellier Cancer Institute Review Board (ICM-CORT-2018-34).
[0144] Results
[0145] A Subset of Vol T Cells Expresses the CD39, CD73 and PD-L1
[0146] We investigated CD39, CD73 and PD-L1 expression in the Vδ1 T cell subpopulation. To this aim, we isolated Vδ1 T cells from the blood of healthy donors, and activated and amplified them in vitro in the presence of rhIL-2 (control) or rhIL-2+rhIL21 (experimental condition). We observed that most Vδ1 T cells (>85%) expressed CD39. Moreover, exposure to rhIL-21 did not modify the percentage of CD39-positive cells, suggesting that IL-21 does not influence its expression (
[0147] Also, we observed that about 50% of Vol T cells (in the control condition) expressed PD-L1 (
[0148] Presence of Various CD73+ γδ T Cell Subsets in Human Breast Cancer
[0149] Then, to characterize the phenotype of infiltrated γδ T cells in human breast tumors, we dissociated fresh breast cancer biopsies and analyzed the phenotype of tumor-infiltrating γδ T cells by flow cytometry with the gating protocol. Tumor samples (n=16) were from patients with grade I, II and III breast tumor who did not receive any neoadjuvant treatment. Although the fraction of CD45-positive cells in breast cancers was very heterogeneous (0.75% to 25%), the proportion of CD3-positive cells among the CD45-positive cells was homogenous (around ˜80%), and γδ T cells were always present and represented 3% to 13% of CD3+ cells. (
[0150] To conclude, we demonstrated in human breast tumors the presence of various CD73+ γδ T cell subsets expressing immunosuppressive markers such as CD39 and CD73 ectonucleotidases and inhibitory immunocheckpoint PD-L1 (data not shown). These various CD73+ γδ T cell subsets could be used as a prognostic marker of the tumor evolution (data not shown).
Example 3
[0151] Material & Methods
[0152] Tumor Dissociation
[0153] Freshly resected tumors from patients were cut into small pieces (around 1 mm3). Tissues were resuspended in digestion solution (10 mg/ml collagenase IV from Sigma and 10 mg/ml DNase I from Roche) in Hanks modified balanced salt solution and alternate between enzymatic digestion (15 min at 37° C.) and mechanical dissociation using the gentle MACS dissociator (Miltenyi Biotec) for 3 rounds. The obtained single cell suspensions were washed in PBS/2% FCS, resuspended at 10.Math.10.sup.6 cells/ml in RPM/10% FCS and incubated in the presence of Golgi STOP (Bd Biosciences) at 37° C. for 4 hours.
[0154] The cession of fresh samples was approved by the Montpellier Cancer Institute Review Board (ICM-CORT-2018-34).
[0155] Flow Cytometry Analysis
[0156] Cells were harvested and incubated at 4° C. for 30 min with Fc-block solution to minimize non-specific binding of antibodies to Fc receptors. After washing cells were incubated with a dye cell viability and the panel of antibodies (anti-CD45, -CD3, -γδTCR, -CD73) for extracellular staining in the staining buffer (PBS-2% FCS) at 4° C. for 30 min, before fixation and permeabilization with the BD FixPerm Kit and intracellular staining for IL-10 and IL-8. Data were acquired on a Cytoflex cytometer (Beckman Coulter) and results analyzed using the FlowJo software.
[0157] Results
[0158] Expression of the immunosuppressive cytokines IL-10 and IL-8 in CD73- versus CD73+ tumor-infiltrating γδ T lymphocytes (γδ TILs).
[0159] To analyze the functional suppressive activity of CD73+ γδ TILs, we investigated, ex vivo by flow cytometry, the expression of IL-10 and IL-8 in infiltrated γδ T cells in human breast tumors obtained from patients who did not receive any neoadjuvant treatment (n=8 for IL-8 analyses and n=7 for IL-10 analyses). We assessed the expression of IL-10 and IL-8 in CD73− and CD73+ γδ subsets. Around 20% and 30% of CD73− γδ T cells expressed IL-8 and IL-10 respectively (
Example 4
[0160] Material and Methods.
[0161] Sample Collection
[0162] Tissue samples were selected from the biological resource center of Montpellier Cancer Institute (ICM). Clinical data were obtained by reviewing the medical files. Samples were collected following the French laws under the supervision of an investigator and their collection was declared to the French Ministry of Higher Education and Research. The study was approved by the ICM Institutional Review Board (ICM-CORT-2020-32).
[0163] Ovarian Cancer Tissue Microarray
[0164] Two TMA with a total of 91 ovarian cancer samples were constructed for retrospective studies allowing the comparison of long-term vs short-term ovarian cancer survivors. For each tumor sample, two cores (1 mm in diameter) were sampled from different malignant areas.
[0165] Breast Cancer Tissue Microarray
[0166] A tissue microarray (TMA) with breast tumor samples from 50 chemotherapy-naive patients was constructed using two malignant tissue cores (1 mm diameter) per tumor.
[0167] Immunofluorescence
[0168] After de-paraffinization, TMA sections were subjected to antigen retrieval using 1× Target Retrieval Solution (Dako, S2367), then incubated in 1× Superblock Blocking Buffer (Thermofisher, 37515) for 45 min followed by 1 h incubation in a FcBlock solution (Miltenyi Biotech—130-059-901). After washing, TMA sections were incubated with primary antibodies against TCR γδ (H-41, Santa Cruz, 1/25) and CD73 (D7AF9A, CST, 1/100) overnight at 4° C. After washing, sections were incubated with secondary antibodies: goat anti-Rabbit Alexa Fluor Plus 555 (Thermofisher, A32732) and goat anti-Mouse Alexa Fluor 647 (Thermofisher, A21236). Finally, sections were counterstained with DAPI and were imaged with AxioScan (Zeiss) to obtain high-power field images. TMA sections were analyzed by observer blinded to the clinicopathological characteristics and patient outcomes at the time of scoring.
[0169] Statistical Analysis Data are presented as scatter plots showing the mean values with the standard error of the mean (SEM). Results were compared using Mann-Whitney t test. A P value <0.05 was considered statistically significant. Analyses were performed using GraphPad Prism, version 6. *** p<0.001; ****p<0.0001
[0170] For survival analysis, categorical variables were presented as frequency distributions and continuous variables as medians and ranges. Categorical variables were compared with the Pearson's chi-square test. OS was defined as the time between the date of surgery and the date of death (whatever the cause). Patients lost to follow-up were censored at the last documented visit. The Kaplan-Meier method was used to estimate the OS. Differences in survival rates were compared using the log-rank test. Multivariate analyses were performed using the Cox proportional hazard model. Hazard ratios (HR) are given with their 95% confidence interval (95% CI). Statistical analyses were performed with STATA 16.0 (StatCorp, College Station, Tex., USA).
[0171] Results
[0172] Total γδ and CD73+ γδ Tumor Infiltrating Lymphocyte (TIL) Density are Increased in Short-Term Survival Ovarian Cancer Patients
[0173] We quantified by immunofluorescence both total γδ TILs and CD73+ γδ TILs in tumor samples from 91 ovarian cancer patients. Patients were classified in two groups: long-term (LT) and short-term (ST) survivors. We observed significantly higher densities of total γδ TILs and CD73+ γδ TILs in ST patients than in LT patients suggesting that γδ and CD73+ γδ T cell presence at the tumor site are associated with poor-prognosis in ovarian cancer patients (
[0174] CD73+ γδ TILs Predict Worse Clinical Outcome in Ovarian Cancer
[0175] Because tumor-infiltrating CD73+ γδ T cells were predominant in ST surviving patients, we next investigated the association between these cells and clinical outcome of the patients, focusing on the overall survival (OS). The median follow-up was 13.44 years (95% CI [11.67-15.47]) and the median survival was 5.14 years (95% CI [3.75-6.08]). We analyzed total γ6 TILs, CD73+ γδ TILs and clinical data from 91 patients. Patients with a low density of γδ T cells at the tumor site had significantly longer OS compared with those with a high density of γδ T cells (p=0.028) (
[0176] Tumor-Infiltrating CD73+ γδ T Predict Worse Clinical Outcome in Breast Cancer
[0177] We investigate the association between the presence of CD73+ γδ TILs and the clinical outcome of breast cancer patients. We analyze CD73+ γδ TILs and clinical data from 50 patients at different SBR grades and phenotypes based on HER2 and hormone receptor expression status. Patients with high density of CD73+ γδ T cells have a shorter OS compared with those with a low density of CD73+ γδ T cells. These results on breast cancer patients are corroborated by the study realized by Hu et al. [65] where they also showed that the presence of tumor infiltrating CD73+γδ T cells is a bad prognosis for breast cancer patients. These results reinforce the idea that the CD73+ γδ T cell presence in breast tumor is a strong prognosis factor.
REFERENCES
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