- Email: [email protected]
Granzyme B-expressing treg cells are enriched in colorectal cancer and present the potential to eliminate autologous T conventional cells
Journal Pre-proof Granzyme B-expressing Treg cells are enriched in colorectal cancer and present the potential to eliminate autologous T conventional cells Bing Sun, Mingtao Liu, Meng Cui, Tao Li
PII:
S0165-2478(19)30297-4
DOI:
https://doi.org/10.1016/j.imlet.2019.10.007
Reference:
IMLET 6380
To appear in:
Immunology Letters
Received Date:
5 June 2019
Revised Date:
11 September 2019
Accepted Date:
5 October 2019
Please cite this article as: Sun B, Liu M, Cui M, Li T, Granzyme B-expressing Treg cells are enriched in colorectal cancer and present the potential to eliminate autologous T conventional cells, Immunology Letters (2019), doi: https://doi.org/10.1016/j.imlet.2019.10.007
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Granzyme B-expressing Treg cells are enriched in colorectal cancer and present the potential to eliminate autologous T conventional cells
Short title: granzyme B-expressing Treg cells in colorectal cancer
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Bing Sun1, Mingtao Liu2, Meng Cui1, Tao Li1
Department of Colorectal Surgery, Shandong Provincial Qianfoshan Hospital, the First
Department of Colorectal Surgery, People’s Hospital of Xiajin, Shandong, China.
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2
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Hospital Affiliated with Shandong First Medical University, Jinan, Shandong, China.
Corresponding author
Department of Colorectal Surgery,
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Tao Li
First Medical University,
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Shandong Provincial Qianfoshan Hospital, the First Hospital Affiliated with Shandong
16766 Jingshi Rd, Jinan, Shandong, China.
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Email: [email protected]
Highlight
granzyme B+ Tregs were present in resected CRC tumor. Expression of granzyme B in Treg cells required stimulation with bacterial products. Granzyme B expression was enriched in TIM-3+ Treg cells. TIM-3+ Treg presented higher cytolytic capacity toward autologous Tconv cells. This effect was depended on granzyme B but not TIM-3.
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Abstract In addition to expressing inhibitory cytokines and suppressive molecules, Treg cells could downplay inflammation by releasing cytotoxic molecules and eliminating proinflammatory immune cells. Colorectal cancer (CRC) is a common malignancy that
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has led to many cancer-related deaths. In this study, we investigated the cytotoxic aspect of Treg cells in CRC patients. Data showed that tumor-infiltrating FOXP3+ Treg cells
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expressed granzyme B immediately following resection, indicating that granzyme B-expressing Treg cells were present directly ex vivo. In the tumor-associated lymph
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nodes (LNs) and circulating lymphocytes, however, granzyme B-expressing Treg cells were only scarcely found. We then attempted to stimulate granzyme B expression in
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circulating Treg cells. Granzyme B upregulation in Treg cells could not be activated by standard T cell receptor (TCR) activation through anti-CD3/CD28 and IL-2 but required
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stimulation with bacterial products, such as with heat-killed Staphylococcus aureus. Interestingly, granzyme B expression was highly concentrated in TIM-3+ Treg cells, a Treg subset previously shown to be enriched in the tumor microenvironment and
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presented increased suppressive capacity. These TIM-3+ Treg cells presented higher cytolytic capacity toward autologous T conventional cells than the TIM-3- Treg cells, in a manner that was dependent on granzyme B but not TIM-3. Overall, we found that granzyme B-expressing Treg cells were enriched in the tumors from CRC patients and
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had the potential to eliminate autologous T conventional cells.
Keywords
Granzyme B; Treg cells; TIM-3; colorectal cancer.
Introduction Treg cells are double-edged swords in the immune system. They are critical in maintaining tolerance and preventing autoimmunity. Yet during infections, Treg cells may hinder the clearance of pathogens, and in malignancies, Treg cells can suppress antitumor promote
the
differentiation
of
suppressor
cells,
and
mediate
of
inflammation, angiogenesis[1].
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Multiple mechanisms are employed by Treg cells to mediate immune suppression[2,3]. Treg cells constitutively express the IL-2 receptor alpha chain CD25,
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which can reduce the availability of IL-2 to T effector (Teff) cells. By expressing CTLA-4, which downregulates the expression of costimulatory molecules CD80 and
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CD86 on the surface of antigen-presenting cells (APCs)[4], Treg cells may prevent the optimal T cell activation by APCs. Also, CTLA-4-induced signaling can increase the
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expression of indoleamine 2,3-dioxygenase (IDO), which degrades tryptophan and leads to Teff starvation and cell cycle arrest[5,6]. LAG-3, a ligand of MHC class II expressed by Treg cells and T regulatory type 1 (Tr1) cells, is shown to inhibit dendritic cell (DC)
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activation, though the precise mechanism remains unclear[7,8]. In addition, Treg cells express various regulatory cytokines, such as IL-10, IL-35, and TGF-β, and mediate immune suppression through their signal transduction pathways[3,9]. Granzyme B is a serine protease expressed by activated cytotoxic CD8+ T cells and
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natural killer (NK) cells, among other immune cells. When delivered inside the cytoplasm, granzyme B activates the caspase cascade, cleaves BID and Mcl-1 to facilitate cytochrome C release from the mitochondria, and promotes the generation of reactive oxygen species[10]. As all of these mechanisms will lead to fast and efficient cell death, granzyme B is thought to be indispensable for the immunity-mediated elimination of tumor cells[11]. Interestingly, multiple studies have demonstrated that granzyme B can be
expressed by Treg cells to mediate elimination of Teff cells and downplay antiviral and antitumor immunity. In acute respiratory syncytial virus (RSV)-infection, lung Treg cells expressed granzyme B, and loss of granzyme B in Treg cells resulted in more extensive cellular infiltration into the lung[12]. In a murine tumor model, Cao et al. demonstrated that the granzyme B-deficient mice could eliminate syngeneic and allogeneic tumors more efficiently than wild-type mice, and adoptive transfer of wild-type, but not
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granzyme B-deficient, Treg cells could confer susceptibility to tumor[13]. In addition, graft survival was significantly shorter in mice with granzyme B-deficient Treg cells than
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in mice with wild-type Treg cells[14]. Mechanistically, granzyme B-expressing Treg cells could mediate the apoptosis of Teff cells[15].
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Colorectal cancer (CRC) remains one of the most common cancers and a significant contributor to cancer-related deaths[16]. Although numerous studies were conducted to
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investigate the role of Treg cells in CRC[17], the granzyme B-expressing aspect of Treg cells has not been examined. In this study, we examined granzyme B-expressing Treg
Materials and methods
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Sample collection
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cells in CRC patients.
Resected tumors and lymph node (LN) tissues were collected from 10 stage II CRC patients and 10 stage III CRC patients. The Ethics Board of the First Hospital Affiliated with Shandong First Medical University approved this study. For tumor-infiltrating
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lymphocyte (TIL) collection, non-tumor surrounding tissues were removed immediately after resection, and the tumors were sterilized in ice cold sterile PBS supplemented with 10 × penicillin-streptomycin (Invitrogen). The tumors were then placed in a biosafety cabinet, rinsed with 1 × penicillin-streptomycin in PBS, and dissected into small pieces. Digestion was performed in 1 × Collagenase/Hyaluronidase (Stemcell) in EpiCult-C Medium (Human) (Stemcell) under 37˚C overnight, and the supernatant was poured over
a 70-µm strainer (Corning) to remove undigested tissue and collect lymphocytes. LNs were disrupted using sterile forceps and the lymphocytes were rinsed through a 70-µm strainer. Peripheral blood samples were collected from the CRC patients who donated tumor and LN samples, and from 10 healthy individuals who were matched with the CRC patients in age and sex. Peripheral blood mononuclear cells (PBMCs) were collected
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using standard Ficoll (Sigma) density gradient centrifugation.
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Identification of granzyme B-expressing Treg cells
Tumor and LN lymphocytes and PBMCs were incubated under 37˚C for 5 hours in 5
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µg/mL GolgiStop and 5 µg/mL GolgiPlug (BD), washed, and incubated under 4˚C for 30 minutes with LIVE/DEAD Violet stain (Invitrogen) and fluorescent anti-human CD3 and
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CD4 antibodies (eBioscience). Cells were washed, fixed, permeabilized, and stained with fluorescent anti-human granzyme B and FOXP3 antibodies (eBioscience) using human
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FOXP3 / TF Staining Buffer set (eBioscience), following the manufacturer’s instructions. Flow cytometry was performed in an LSR instrument (BD).
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Stimulation of granzyme B expression in circulating Treg cells PBMCs were incubated in medium containing 1 µg/mL anti-CD3 antibody OKT3 (BioLegend), 1 µg/mL anti-CD28 antibody CD28.2 (BioLegend), and 20 U/mL recombinant human IL-2 (R&D Systems), or in medium containing heat-killed S. aureus
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(100 bacteria per Treg cell; EMD Millipore), for 12 hours under 37˚C. 5 µg/mL GolgiStop and 5 µg/mL GolgiPlug (BD) were added during the final 5 hours. Flow cytometry was performed as described above.
CD4 T cell fractionation and mRNA quantification Total CD4 T cells were separated from PBMCs using Human CD4 T Cell
Enrichment Kit (Stemcell), through the manufacturer’s protocol. The purity was verified using flow cytometry staining and was consistently over 97%. The CD4 T cells were then incubated under 4˚C for 30 min with fluorescent anti-human CD25 and TIM-3 antibodies (eBioscience) and sorted into CD25-, CD25+TIM-3+, and CD25+TIM-3- cells in a FACSAria instrument (BD). For mRNA analysis, sorted cells were stimulated with heat-killed S. aureus (100 bacteria per Treg cell) in the presence of irradiated feeder cells
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for 12 hours. Feeder cells were made through the following procedure: CD4 T cells were removed from PBMCs using Human CD4 Positive Selection Kit (Stemcell), and the
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remaining cells were given 40 Gy of gamma radiation and used as feeder cells. Messenger RNA was extracted using TRIZOL reagent (Invitrogen) and transcribed into
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cDNA using SuperScript III Reverse Transcriptase Kit (Invitrogen). The quantification of FOXP3 and GZMB was then performed using TaqMan assays Hs01085834_m1 and
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Hs00188051_m1, respectively.
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Cr release assay
1 × 106 CD4+CD25- Tconv cells were incubated for 1 hour under 37˚C with 50 µCi sodium chromate (Perkin Elmer), washed, and plated at 1 × 104 cells per well in a 96-well
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plate. CD25-, CD25+TIM-3+, and CD25+TIM-3- CD4 T cells were sorted and stimulated with Heat-killed Staphylococcus aureus as described above, and added to the Tconv cells at 1 to 10 Treg / Tconv ratios. After 6 hours at 37˚C, the plates were centrifuged to collect the supernatant.
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Cr from the supernatant was counted in a gamma counter.
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Z-AAD-CMK (EMD Millipore) or TIM-3 blocking antibody 2E2 (BioLegend) were added at the beginning of the 6-hour incubation in select experiments.
Statistical analysis The statistics were performed in Prism software (GraphPad), using tests specified in the figure legend. P < 0.05 (two-tailed) was considered significant.
Results Expression of granzyme B by tumor and lymph node Treg cells To identify Treg cells, we first performed intracellular staining of FOXP3 in CD3+CD4+
tumor-infiltrating
lymphocytes
(Figure
1A).
Although
Treg
cells
constitutively express FOXP3, activated effector T cells may also express FOXP3
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transiently, especially in the microenvironment of CRC tumors where T cell stimuli were likely enriched. We examined the expression of other Treg-associated markers, including
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CD25, CD39, and CD127. Compared to FOXP3- T cells, FOXP3+ T cells were high in CD25 and CD39 but low in CD127 (Figure 1A), which was consistent with canonical
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Treg phenotype[18,19]. To investigate the Treg-mediated expression of granzyme B, tumor samples and nearby lymph nodes (LNs) were obtained from 10 stage II patients
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and 10 stage III patients. The tumor-infiltrating lymphocytes and the LN lymphocytes were then stained with fluorophore-conjugated antibodies directly ex vivo and examined
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using flow cytometry. Pre-gated CD4 T cells were distinguished into Treg cells and T conventional (Tconv) cells by FOXP3+ and FOXP3- expression, respectively, and the frequencies of granzyme B+ cells were examined and compared (Figure 1B). In the
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tumor-infiltrating CD4 T cells from stage II CRC patients (Figure 1C), the frequencies of granzyme B+ cells was significantly higher in FOXP3+ Treg cells than in FOXP3- Tconv cells. In the LN from stage II CRC patients, however, the frequencies of granzyme B+ cells were significantly lower in FOXP3+ Treg cells than in FOXP3- Tconv cells. The
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tumor and LN FOXP3- Tconv cells presented similar levels of granzyme B+ cells, while the tumor FOXP3+ Treg cells presented significantly higher granzyme B expression than the LN FOXP3+ Treg cells. In tumor CD4 T cells from stage III CRC patients (Figure 1D), the frequencies of granzyme B+ cells were comparable between FOXP3+ Treg cells and FOXP3- Tconv cells, while in LN CD4 T cells from stage III patients, the frequencies of granzyme B+ cells were significantly lower in FOXP3+ Treg cells than in FOXP3-
Tconv cells. Both the FOXP3+ Treg cells and the FOXP3- Tconv cells in the tumor presented significantly higher levels of granzyme B+ cells than their counterparts in the LN.
Granzyme B and perforin expression in circulating Treg cells. Ten healthy volunteers, with matching age and sex with stage II and stage III CRC
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patients, were recruited as controls. Peripheral blood was obtained from healthy controls and stage II and stage III CRC patients who donated tumor and LN samples. We
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examined the granzyme B expression in circulating FOXP3+ Treg cells and FOXP3Tconv cells (Figure 2A). Unlike tumor-infiltrating Treg cells and Tconv cells, the
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circulating Treg cells and Tconv cells presented very few granzyme B+ cells directly ex vivo (Figure 2B), with frequencies of lower than 0.3% in all subjects. No significant
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differences between healthy controls, stage II CRC patients, and stage III CRC patients were observed.
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Subsequently, we attempted to upregulate granzyme B expression in Treg cells by stimulation with anti-CD3/CD28 antibodies and supplementing with IL-2. After 12-hour stimulation, granzyme B was only observed in a small proportion of FOXP3- CD4 T cells
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and a smaller proportion of FOXP3+ CD4 T cells (Figure 2A and 2C). No significant differences between healthy controls, stage II patients, and stage III patients were observed.
The colorectal tissues are constantly exposed to the gut microbiota, and microbial
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products have been found inside resected colorectal tumors[20]. In this part, we stimulated CD4 T cells using heat-killed Staphylococcus aureus (S. aureus), a member of the Firmicutes[21]. Interestingly, in both FOXP3- and FOXP3+ CD4 T cells, heat-killed S. aureus was more effective at promoting granzyme B expression than anti-CD3/CD28 and IL-2 (Figure 2A and 2D). No significant differences between healthy controls, stage II CRC patients, and stage III CRC patients were observed.
In addition, the expression of FOXP3 in CD4 T cells in unstimulated and stimulated conditions was examined. Overall, the frequency of FOXP3-expressing CD4 T cells remained stable throughout the 12-hour incubation period in unstimulated media, in anti-CD3/CD28 and IL-2 stimulation, and in S. aureus stimulation (Figure 2E). This stable trend was observed in healthy controls as well as in stage II and stage III CRC patients.
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We also investigated the expression of perforin in the circulating FOXP3+ Treg cells and FOXP3- Tconv cells (Figure 3A). Directly ex vivo, few perforin-expressing cells
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were found with no significant differences between between healthy controls, stage II CRC patients, and stage III CRC patients (Figure 3B). After 12-hour anti-CD3/CD28 and
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IL-2, perforin was increased in both FOXP3- CD4 T cells and FOXP3+ CD4 T cells (Figure 3C). Overall, FOXP3- CD4 T cells presented significantly higher perforin than
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FOXP3+ CD4 T cells. In FOXP3- CD4 T cells, the perforin expression was significantly higher in healthy controls than in stage III patients.
In FOXP3+ CD4 T cells, no
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significant differences between healthy controls, stage II patients, and stage III patients were observed. Heat-killed S. aureus could also elevate perforin expression in FOXP3CD4 T cells and FOXP3+ CD4 T cells (Figure 3D). No significant differences between
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healthy controls, stage II CRC patients, and stage III CRC patients were observed.
Granzyme B-expressing Treg cells were enriched in CD25+TIM-3+ CD4 T cells TIM-3 is mainly known as a negative regulator of proinflammatory immune
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responses[22]. TIM-3 expression is found on terminally differentiated IFN-γ-producing Th1 cells, Th17 cells, activated/exhausted CD8 T cells, and intratumoral Treg cells[23]. Multiple sources indicate that TIM-3+ Treg cells present higher expression of granzymes and perforin than TIM-3- Treg cells[24–26]. Using the findings from previous reports[23], we obtained unstimulated CD4 T cells from PBMCs using magnetic negative sorting (Figure 3A). Purified CD4 T cells were then sorted into CD25- Tconv cells,
CD25+TIM-3+ cells, and CD25+TIM-3- cells using fluorescence-activated cell sorting (FACS) (Figure 4A). Each fraction was then stimulated using heat-killed S. aureus for 12 hours. The expression of FOXP3 was examined to verify the identity of Treg cells (Figure 4B). As predicted, CD25- Tconv cells presented significantly lower FOXP3 than CD25+TIM-3- and CD25+TIM-3+ CD4 T cells. On average, CD25+TIM-3+ CD4 T cells presented higher FOXP3 expression than CD25+TIM-3- CD4 T cells, but the difference
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was not statistically significant. No significant differences between healthy subjects, stage II CRC patients, and stage III CRC patients were observed (Figure 4B). The
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granzyme B (GZMB) transcript expression was then quantified in each cell type (Figure 4C). CD25+TIM-3+ CD4 T cells presented significantly higher GZMB expression than
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both CD25- Tconv cells and CD25+TIM-3- CD4 T cells. On average, CD25- Tconv cells presented higher GZMB than CD25+TIM-3- CD4 T cells, but the difference was not
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statistically significant. No significant differences between healthy subjects, stage II CRC
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patients, and stage III CRC patients were observed.
CD25+TIM-3+ CD4 T cells mediated stronger lysis of Tconv cells than CD25+TIM-3CD4 T cells
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Subsequently, we investigated whether Treg cells could directly mediate lysis of Tconv cells. CD4+CD25- Tconv cells were labeled with
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Cr. Heat-killed S.
aureus-stimulated CD25+TIM-3- CD4 Treg cells and CD25+TIM-3+ CD4 Treg cells were then added at various Treg / Tconv ratios for 6 hours, after which the specific lysis
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mediated by Treg cells were calculated. Both CD25+TIM-3- CD4 Treg cells and CD25+TIM-3+ CD4 Treg cells were capable of mediating Tconv lysis in a manner that was positively associated with the Treg / Tconv ratio (Figure 5A to 5C). At Treg / Tconv ratios of equal to or greater than 3, the lysis efficiency of CD25+TIM-3+ CD4 Treg cells was significantly higher than CD25+TIM-3- CD4 Treg cells in healthy controls, stage II patients, and stage III patients.
Inhibition of granzyme B, but not TIM-3, was capable of suppressing CD25+TIM-3+ CD4 Treg-mediated lysis of Tconv cells To demonstrate the involvement of granzyme B in Treg-mediated lysis, we used a small molecule inhibitor Z-AAD-CMK to inhibit the activity of granzyme B. In both CD25+TIM-3- CD4 Treg cells and CD25+TIM-3+ CD4 Treg cells, Z-AAD-CMK reduced
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the specific lytic capacity in a dose-dependent manner (Figure 6A). In addition, at a concentration of 1 µM, Z-AAD-CMK eliminated the differences between CD25+TIM-3-
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CD4 Treg cells and CD25+TIM-3+ CD4 Treg cells.
CD25+TIM-3- CD4 Treg cells and CD25+TIM-3+ CD4 Treg cells not only differed in
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granzyme B production but also differed in TIM-3 expression. To investigate whether TIM-3 had a role in Treg-mediated lysis, we inhibited TIM-3 using the blocking antibody
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2E2. In neither CD25+TIM-3- CD4 Treg cells nor CD25+TIM-3+ CD4 Treg cells, the addition of 2E2 presented a significant effect on Treg-mediated lysis of Tconv cells
Discussion
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(Figure 6B).
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Granzyme B is one of the most abundant granzymes and is mainly expressed by activated cytotoxic T cells and NK cells. Due to its role in directly promoting cell death, granzyme B is critical to the elimination of precancerous cells and malignant cells during immunosurveillance and cancer[11]. However, it is also employed by Treg cells to
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eliminate proinflammatory immune cells and downplay inflammation[2,3]. In this study, we investigated Treg-mediated granzyme B expression in healthy individuals and in patients with CRC. Overall, we presented following findings. First, we found that granzyme B-expressing FOXP3+ Treg cells were present in resected tumor, and to a much lesser extent in tumor-associated LN and in circulating Treg cells. Second, the expression of granzyme B in Treg cells could not be activated by standard T cell receptor (TCR)
activation through anti-CD3/CD28 and IL-2, but required stimulation with heat-killed S. aureus. Third, granzyme B expression was enriched in TIM-3+ Treg cells. And fourth, the TIM-3+ Treg cells presented higher cytolytic capacity toward autologous Tconv cells than the TIM-3- Treg cells, in a manner that depended on granzyme B but not TIM-3. Given the enrichment of granzyme B-expressing Treg cells in tumor, and the observation that granzyme B-expressing circulating Treg cells mediated lysis of
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autologous Tconv cells, future studies should investigate whether tumor-infiltrating Treg cells actively eliminate proinflammatory T cells in vivo, and what impacts they might
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have on the progression of cancer. Many other tumors, especially those associated with the digestive system or are in contact with mucosal surfaces, such as pancreatic, gastric,
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lung, breast, ovarian, and prostate carcinomas, are found to harbor bacteria in the tumor microenvironment, and in some cases, the presence of bacteria may confer stronger
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resistance to chemotherapy[20,27]. In this study, S. aureus may weaken proinflammatory immune responses by activating granzyme B expression in Treg cells. However, S. aureus
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is rarely found in the intestinal tract. Whether it is a coincidence or S. aureus essentially affects the immunity of CRC requires further study. Of note, the inhibition of TIM-3 did not significantly alter the cytotoxic capacity of
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TIM-3+ Treg cells. This may be problematic for immunotherapeutic strategies that target TIM-3. It is increasingly recognized that TIM-3 inhibits antitumor immunity and the blockade of TIM-3, together with other inhibitory molecules such as PD-1/PD-L1 and CTLA-4, may be required for optimal results[28,29]. However, as TIM-3 inhibition was
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unable to reduce Treg-mediated cytotoxicity, the potency of TIM-3 inhibition may be limited. Further studies into the regulatory mechanisms of Treg-mediated cytotoxicity are required.
Another observation is that the granzyme B expression by LN FOXP3+ Treg cells was severely impaired compared to the granzyme B expression by LN FOXP3- CD4 T cells. Whether LN FOXP3+ Treg cells express additional inhibitory molecules that
prevent granzyme B expression remains unclear and should be studied further. A major limitation of this study is the use of S. aureus as a model bacterium in Treg stimulation. Although this bacterium is common as a commensal and can become a pathogen in the blood, the respiratory tract, and the urinary tract, it is rarely found in the intestinal tract. Bacterial species that were previously found in the resected colorectal tumor tissues and/or shown to have carcinogenic activity in colorectal cancer, such as
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Fusobacterium and Campylobacter, should be used in Treg stimulation[30,31]. In addition, in both FOXP3- CD4 T cells and FOXP3+ Treg cells, about 1% to 2% cells
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expressed granzyme B following S. aureus stimulation. This high frequency, combined with the fact that S. aureus has surface proteins that could act as T cell mitogens[32],
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suggested that the observed stimulatory effects were not antigen-specific. The underlying mechanism for granzyme B expression mediated by S. aureus and other bacterial species
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should be further studied. In addition, this study focused on granzyme B expression. However, the pore-forming protein perforin is also critical to cytolysis. Further studies
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should also investigate the expression of perforin, along with degranulation marker CD107a, in Treg cells from the tumor and LN tissues and the blood of CRC patients.
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Conflict of interests
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The authors declare no conflict of interest.
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Human regulatory T cells undergo self-inflicted damage via granzyme pathways upon activation. JCI Insight. 2017; doi:10.1172/jci.insight.91599 Sakuishi K, Ngiow SF, Sullivan JM, Teng MWL, Kuchroo VK, Smyth MJ, et al.
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TIM3 + FOXP3 + regulatory T cells are tissue-specific promoters of T-cell dysfunction in cancer. Oncoimmunology. 2013;2: e23849. doi:10.4161/onci.23849 27.
Geller LT, Barzily-Rokni M, Danino T, Jonas OH, Shental N, Nejman D, et al.
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Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine. Science (80- ). 2017; doi:10.1126/science.aah5043
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Shayan G, Ferris RL. PD-1 blockade upregulate TIM-3 expression as a compensatory regulation of immune check point receptors in HNSCC TIL. J Immunother Cancer. BioMed Central; 2015;3: P196.
doi:10.1186/2051-1426-3-S2-P196 29.
Romero D. Immunotherapy: PD-1 says goodbye, TIM-3 says hello. Nat Rev Clin Oncol. Nature Research; 2016;13: 202–203. doi:10.1038/nrclinonc.2016.40
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Castellarin M, Warren RL, Freeman JD, Dreolini L, Krzywinski M, Strauss J, et al. Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. Genome Res. 2012; doi:10.1101/gr.126516.111 He Z, Gharaibeh RZ, Newsome RC, Pope JL, Dougherty MW, Tomkovich S, et al.
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Campylobacter jejuni promotes colorectal tumorigenesis through the action of
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cytolethal distending toxin. Gut. 2019; doi:10.1136/gutjnl-2018-317200
Sakane T, Green I. Protein A from Staphylococcus aureus-a mitogen for human T
-p
lymphocytes and B lymphocytes but not L lymphocytes. J Immunol. American Association of Immunologists; 1978;120: 302–11. Available:
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http://www.ncbi.nlm.nih.gov/pubmed/304871
Figure Legend Figure 1. Granzyme B expression by tumor and LN Treg cells in CRC patients. (A) Expression of Treg markers by Foxp3+ and Foxp3- T cells, pre-gated in live CD3+CD4+ tumor-infiltrating lymphocytes. (B) In pre-gated CD4 T cells, granzyme B+ and granzyme B- cells were gated in FOXP3- and FOXP3+ cells, as shown in the representative figures from one stage II CRC patient and one stage III CRC patient.
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Staining was performed in unstimulated tumor and LN samples directly ex vivo and without stimulation. (C) The frequencies of granzyme B+ cells in CD4+FOXP3- (filled)
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and CD4+FOXP3+ (open) T cells from stage II tumor and LN samples. (D) The frequencies of granzyme B+ cells in CD4+FOXP3- (filled) and CD4+FOXP3+ (open) T
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cells from stage III tumor and LN samples. (B) and (C) Two-way ANOVA and Tukey’s
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multiple comparisons. ns, not significant. *P < 0.05. ***P < 0.001.
of ro -p re lP ur na Jo Figure 2. Granzyme B expression by circulating Treg cells in CRC patients and healthy
controls. (A) The expression of granzyme B by FOXP3- and FOXP3+ CD4 T cells directly ex vivo, after 12-hour stimulation with anti-CD3/CD28 antibodies (1 µg/mL each) and IL-2 (20 U/mL), or after 12-hour stimulation with heat-killed S. aureus (100 bacteria per Treg cell). Representative results from one stage II CRC patient is shown. (B) The frequencies of granzyme B+ cells in FOXP3- and FOXP3+ circulating CD4 T cells from healthy (circle),
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stage II CRC (square), and stage III CRC (triangle) subjects, examined directly ex vivo. (C) The frequencies of granzyme B+ cells in FOXP3- and FOXP3+ circulating CD4 T
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cells from healthy (circle), stage II CRC (square), and stage III CRC (triangle) subjects, examined after 12-hour stimulation with anti-CD3/CD28 and IL-2. (D) The frequencies
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of granzyme B+ cells in FOXP3- and FOXP3+ circulating CD4 T cells from healthy (circle), stage II CRC (square), and stage III CRC (triangle) subjects, examined after
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12-hour stimulation with heat-killed S. aureus. (E) The frequencies of Foxp3+ CD4 T cells in healthy subjects (open circle), stage II CRC subjects (square), and stage III CRC
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subjects (cross). N = 10 for each group. (B) to (E) Two-way ANOVA and Tukey’s
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multiple comparisons. ns, not significant. ***P < 0.001.
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Figure 3. Perforin expression by circulating Treg cells in CRC patients and healthy controls.
(A) The expression of perforin by FOXP3- and FOXP3+ CD4 T cells directly ex vivo,
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after 12-hour stimulation with anti-CD3/CD28 antibodies (1 µg/mL each) and IL-2 (20 U/mL), or after 12-hour stimulation with heat-killed S. aureus (100 bacteria per Treg cell). Representative results from one stage II CRC patient is shown. (B) The frequencies of perforin+ cells in FOXP3- and FOXP3+ circulating CD4 T cells from healthy (circle), stage II CRC (square), and stage III CRC (triangle) subjects, examined directly ex vivo. (C) The frequencies of perforin+ cells in FOXP3- and FOXP3+ circulating CD4 T cells
from healthy (circle), stage II CRC (square), and stage III CRC (triangle) subjects, examined after 12-hour stimulation with anti-CD3/CD28 and IL-2. (D) The frequencies of perforin+ cells in FOXP3- and FOXP3+ circulating CD4 T cells from healthy (circle), stage II CRC (square), and stage III CRC (triangle) subjects, examined after 12-hour stimulation with heat-killed S. aureus. (B) to (D) Two-way ANOVA and Tukey’s multiple
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comparisons. ns, not significant. *P < 0.05. **P < 0.01. ***P < 0.001.
Figure 4. FOXP3 and GZMB expression by CD25-, CD25+TIM-3-, and CD25+TIM-3+
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CD4 T cells.
Total CD4 T cells from healthy (circle), stage II CRC (square), and stage III CRC (triangle) subjects were first isolated via magnetic negative selection and then separated into CD25-, CD25+TIM-3-, and CD25+TIM-3+ subsets using FACS. The CD4 T cell subsets were then stimulated for 12 hours with heat-killed S. aureus (100 bacteria per Treg cell). (A) The efficiency of CD4 T cell isolation and CD25 vs. TIM-3 gating
strategy in one representative sample. (B) The FOXP3 mRNA expression by each subset. (C) The GZMB mRNA expression by each subset. Two-way ANOVA and Tukey’s
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multiple comparisons. ns, not significant. **P < 0.01. ***P < 0.001.
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Figure 5. Specific lysis by CD25+TIM-3- and CD25+TIM-3+ Treg cells. 51
Cr-labeled CD4+CD25- Tconv cells were plated at 1 × 104 cells per well in a 96-well
plate. CD25+TIM-3- CD4 Treg cells (filled) and CD25+TIM-3+ CD4 Treg cells (open)
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were stimulated with heat-killed S. aureus (100 bacteria per Treg cell) for 12 hours, and were then added at various Treg / Tconv ratios. Spontaneous release had no Treg cells, and maximum release had 100% water in place of culture medium. After 6 hours, the supernatant was collected and the 51Cr level was measured. Specific lysis was calculated
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by the formula (experiment - spontaneous) / (maximum - spontaneous). (A) The Treg-mediated specific lysis in healthy subjects (N = 10). (B) The Treg-mediated specific lysis in stage II CRC patients (N = 10). (C) The Treg-mediated specific lysis in stage III CRC patients (N = 10). Two-way ANOVA and Tukey’s multiple comparisons. ns, not significant. **P < 0.01. ***P < 0.001.
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Figure 6. Effect of granzyme B and TIM-3 inhibition on the specific lysis by CD25+TIM-3- and CD25+TIM-3+ Treg cells. Cr-labeled CD4+CD25- Tconv cells were plated at 1 × 104 cells per well in a 96-well
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51
plate. CD25+TIM-3- CD4 Treg cells (filled) and CD25+TIM-3+ CD4 Treg cells (open) were stimulated with heat-killed S. aureus (100 bacteria per Treg cell) for 12 hours, and were then added at a Treg / Tconv ratio of 3. Granzyme B inhibitor Z-AAD-CMK or TIM-3 blocking antibody clone 2E2 were added accordingly. (A) The Treg-mediated specific lysis in the presence of various levels of Z-AAD-CMK. (B) The Treg-mediated
specific lysis in the presence of various levels of 2E2. Two-way ANOVA and Tukey’s
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multiple comparisons. ns, not significant. *P < 0.05. **P < 0.01. ***P < 0.001.
PII:
S0165-2478(19)30297-4
DOI:
https://doi.org/10.1016/j.imlet.2019.10.007
Reference:
IMLET 6380
To appear in:
Immunology Letters
Received Date:
5 June 2019
Revised Date:
11 September 2019
Accepted Date:
5 October 2019
Please cite this article as: Sun B, Liu M, Cui M, Li T, Granzyme B-expressing Treg cells are enriched in colorectal cancer and present the potential to eliminate autologous T conventional cells, Immunology Letters (2019), doi: https://doi.org/10.1016/j.imlet.2019.10.007
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Granzyme B-expressing Treg cells are enriched in colorectal cancer and present the potential to eliminate autologous T conventional cells
Short title: granzyme B-expressing Treg cells in colorectal cancer
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Bing Sun1, Mingtao Liu2, Meng Cui1, Tao Li1
Department of Colorectal Surgery, Shandong Provincial Qianfoshan Hospital, the First
Department of Colorectal Surgery, People’s Hospital of Xiajin, Shandong, China.
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2
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Hospital Affiliated with Shandong First Medical University, Jinan, Shandong, China.
Corresponding author
Department of Colorectal Surgery,
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Tao Li
First Medical University,
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Shandong Provincial Qianfoshan Hospital, the First Hospital Affiliated with Shandong
16766 Jingshi Rd, Jinan, Shandong, China.
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Email: [email protected]
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granzyme B+ Tregs were present in resected CRC tumor. Expression of granzyme B in Treg cells required stimulation with bacterial products. Granzyme B expression was enriched in TIM-3+ Treg cells. TIM-3+ Treg presented higher cytolytic capacity toward autologous Tconv cells. This effect was depended on granzyme B but not TIM-3.
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Abstract In addition to expressing inhibitory cytokines and suppressive molecules, Treg cells could downplay inflammation by releasing cytotoxic molecules and eliminating proinflammatory immune cells. Colorectal cancer (CRC) is a common malignancy that
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has led to many cancer-related deaths. In this study, we investigated the cytotoxic aspect of Treg cells in CRC patients. Data showed that tumor-infiltrating FOXP3+ Treg cells
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expressed granzyme B immediately following resection, indicating that granzyme B-expressing Treg cells were present directly ex vivo. In the tumor-associated lymph
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nodes (LNs) and circulating lymphocytes, however, granzyme B-expressing Treg cells were only scarcely found. We then attempted to stimulate granzyme B expression in
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circulating Treg cells. Granzyme B upregulation in Treg cells could not be activated by standard T cell receptor (TCR) activation through anti-CD3/CD28 and IL-2 but required
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stimulation with bacterial products, such as with heat-killed Staphylococcus aureus. Interestingly, granzyme B expression was highly concentrated in TIM-3+ Treg cells, a Treg subset previously shown to be enriched in the tumor microenvironment and
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presented increased suppressive capacity. These TIM-3+ Treg cells presented higher cytolytic capacity toward autologous T conventional cells than the TIM-3- Treg cells, in a manner that was dependent on granzyme B but not TIM-3. Overall, we found that granzyme B-expressing Treg cells were enriched in the tumors from CRC patients and
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had the potential to eliminate autologous T conventional cells.
Keywords
Granzyme B; Treg cells; TIM-3; colorectal cancer.
Introduction Treg cells are double-edged swords in the immune system. They are critical in maintaining tolerance and preventing autoimmunity. Yet during infections, Treg cells may hinder the clearance of pathogens, and in malignancies, Treg cells can suppress antitumor promote
the
differentiation
of
suppressor
cells,
and
mediate
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inflammation, angiogenesis[1].
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Multiple mechanisms are employed by Treg cells to mediate immune suppression[2,3]. Treg cells constitutively express the IL-2 receptor alpha chain CD25,
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which can reduce the availability of IL-2 to T effector (Teff) cells. By expressing CTLA-4, which downregulates the expression of costimulatory molecules CD80 and
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CD86 on the surface of antigen-presenting cells (APCs)[4], Treg cells may prevent the optimal T cell activation by APCs. Also, CTLA-4-induced signaling can increase the
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expression of indoleamine 2,3-dioxygenase (IDO), which degrades tryptophan and leads to Teff starvation and cell cycle arrest[5,6]. LAG-3, a ligand of MHC class II expressed by Treg cells and T regulatory type 1 (Tr1) cells, is shown to inhibit dendritic cell (DC)
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activation, though the precise mechanism remains unclear[7,8]. In addition, Treg cells express various regulatory cytokines, such as IL-10, IL-35, and TGF-β, and mediate immune suppression through their signal transduction pathways[3,9]. Granzyme B is a serine protease expressed by activated cytotoxic CD8+ T cells and
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natural killer (NK) cells, among other immune cells. When delivered inside the cytoplasm, granzyme B activates the caspase cascade, cleaves BID and Mcl-1 to facilitate cytochrome C release from the mitochondria, and promotes the generation of reactive oxygen species[10]. As all of these mechanisms will lead to fast and efficient cell death, granzyme B is thought to be indispensable for the immunity-mediated elimination of tumor cells[11]. Interestingly, multiple studies have demonstrated that granzyme B can be
expressed by Treg cells to mediate elimination of Teff cells and downplay antiviral and antitumor immunity. In acute respiratory syncytial virus (RSV)-infection, lung Treg cells expressed granzyme B, and loss of granzyme B in Treg cells resulted in more extensive cellular infiltration into the lung[12]. In a murine tumor model, Cao et al. demonstrated that the granzyme B-deficient mice could eliminate syngeneic and allogeneic tumors more efficiently than wild-type mice, and adoptive transfer of wild-type, but not
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granzyme B-deficient, Treg cells could confer susceptibility to tumor[13]. In addition, graft survival was significantly shorter in mice with granzyme B-deficient Treg cells than
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in mice with wild-type Treg cells[14]. Mechanistically, granzyme B-expressing Treg cells could mediate the apoptosis of Teff cells[15].
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Colorectal cancer (CRC) remains one of the most common cancers and a significant contributor to cancer-related deaths[16]. Although numerous studies were conducted to
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investigate the role of Treg cells in CRC[17], the granzyme B-expressing aspect of Treg cells has not been examined. In this study, we examined granzyme B-expressing Treg
Materials and methods
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Sample collection
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cells in CRC patients.
Resected tumors and lymph node (LN) tissues were collected from 10 stage II CRC patients and 10 stage III CRC patients. The Ethics Board of the First Hospital Affiliated with Shandong First Medical University approved this study. For tumor-infiltrating
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lymphocyte (TIL) collection, non-tumor surrounding tissues were removed immediately after resection, and the tumors were sterilized in ice cold sterile PBS supplemented with 10 × penicillin-streptomycin (Invitrogen). The tumors were then placed in a biosafety cabinet, rinsed with 1 × penicillin-streptomycin in PBS, and dissected into small pieces. Digestion was performed in 1 × Collagenase/Hyaluronidase (Stemcell) in EpiCult-C Medium (Human) (Stemcell) under 37˚C overnight, and the supernatant was poured over
a 70-µm strainer (Corning) to remove undigested tissue and collect lymphocytes. LNs were disrupted using sterile forceps and the lymphocytes were rinsed through a 70-µm strainer. Peripheral blood samples were collected from the CRC patients who donated tumor and LN samples, and from 10 healthy individuals who were matched with the CRC patients in age and sex. Peripheral blood mononuclear cells (PBMCs) were collected
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using standard Ficoll (Sigma) density gradient centrifugation.
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Identification of granzyme B-expressing Treg cells
Tumor and LN lymphocytes and PBMCs were incubated under 37˚C for 5 hours in 5
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µg/mL GolgiStop and 5 µg/mL GolgiPlug (BD), washed, and incubated under 4˚C for 30 minutes with LIVE/DEAD Violet stain (Invitrogen) and fluorescent anti-human CD3 and
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CD4 antibodies (eBioscience). Cells were washed, fixed, permeabilized, and stained with fluorescent anti-human granzyme B and FOXP3 antibodies (eBioscience) using human
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FOXP3 / TF Staining Buffer set (eBioscience), following the manufacturer’s instructions. Flow cytometry was performed in an LSR instrument (BD).
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Stimulation of granzyme B expression in circulating Treg cells PBMCs were incubated in medium containing 1 µg/mL anti-CD3 antibody OKT3 (BioLegend), 1 µg/mL anti-CD28 antibody CD28.2 (BioLegend), and 20 U/mL recombinant human IL-2 (R&D Systems), or in medium containing heat-killed S. aureus
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(100 bacteria per Treg cell; EMD Millipore), for 12 hours under 37˚C. 5 µg/mL GolgiStop and 5 µg/mL GolgiPlug (BD) were added during the final 5 hours. Flow cytometry was performed as described above.
CD4 T cell fractionation and mRNA quantification Total CD4 T cells were separated from PBMCs using Human CD4 T Cell
Enrichment Kit (Stemcell), through the manufacturer’s protocol. The purity was verified using flow cytometry staining and was consistently over 97%. The CD4 T cells were then incubated under 4˚C for 30 min with fluorescent anti-human CD25 and TIM-3 antibodies (eBioscience) and sorted into CD25-, CD25+TIM-3+, and CD25+TIM-3- cells in a FACSAria instrument (BD). For mRNA analysis, sorted cells were stimulated with heat-killed S. aureus (100 bacteria per Treg cell) in the presence of irradiated feeder cells
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for 12 hours. Feeder cells were made through the following procedure: CD4 T cells were removed from PBMCs using Human CD4 Positive Selection Kit (Stemcell), and the
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remaining cells were given 40 Gy of gamma radiation and used as feeder cells. Messenger RNA was extracted using TRIZOL reagent (Invitrogen) and transcribed into
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cDNA using SuperScript III Reverse Transcriptase Kit (Invitrogen). The quantification of FOXP3 and GZMB was then performed using TaqMan assays Hs01085834_m1 and
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Hs00188051_m1, respectively.
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Cr release assay
1 × 106 CD4+CD25- Tconv cells were incubated for 1 hour under 37˚C with 50 µCi sodium chromate (Perkin Elmer), washed, and plated at 1 × 104 cells per well in a 96-well
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plate. CD25-, CD25+TIM-3+, and CD25+TIM-3- CD4 T cells were sorted and stimulated with Heat-killed Staphylococcus aureus as described above, and added to the Tconv cells at 1 to 10 Treg / Tconv ratios. After 6 hours at 37˚C, the plates were centrifuged to collect the supernatant.
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Cr from the supernatant was counted in a gamma counter.
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Z-AAD-CMK (EMD Millipore) or TIM-3 blocking antibody 2E2 (BioLegend) were added at the beginning of the 6-hour incubation in select experiments.
Statistical analysis The statistics were performed in Prism software (GraphPad), using tests specified in the figure legend. P < 0.05 (two-tailed) was considered significant.
Results Expression of granzyme B by tumor and lymph node Treg cells To identify Treg cells, we first performed intracellular staining of FOXP3 in CD3+CD4+
tumor-infiltrating
lymphocytes
(Figure
1A).
Although
Treg
cells
constitutively express FOXP3, activated effector T cells may also express FOXP3
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transiently, especially in the microenvironment of CRC tumors where T cell stimuli were likely enriched. We examined the expression of other Treg-associated markers, including
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CD25, CD39, and CD127. Compared to FOXP3- T cells, FOXP3+ T cells were high in CD25 and CD39 but low in CD127 (Figure 1A), which was consistent with canonical
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Treg phenotype[18,19]. To investigate the Treg-mediated expression of granzyme B, tumor samples and nearby lymph nodes (LNs) were obtained from 10 stage II patients
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and 10 stage III patients. The tumor-infiltrating lymphocytes and the LN lymphocytes were then stained with fluorophore-conjugated antibodies directly ex vivo and examined
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using flow cytometry. Pre-gated CD4 T cells were distinguished into Treg cells and T conventional (Tconv) cells by FOXP3+ and FOXP3- expression, respectively, and the frequencies of granzyme B+ cells were examined and compared (Figure 1B). In the
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tumor-infiltrating CD4 T cells from stage II CRC patients (Figure 1C), the frequencies of granzyme B+ cells was significantly higher in FOXP3+ Treg cells than in FOXP3- Tconv cells. In the LN from stage II CRC patients, however, the frequencies of granzyme B+ cells were significantly lower in FOXP3+ Treg cells than in FOXP3- Tconv cells. The
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tumor and LN FOXP3- Tconv cells presented similar levels of granzyme B+ cells, while the tumor FOXP3+ Treg cells presented significantly higher granzyme B expression than the LN FOXP3+ Treg cells. In tumor CD4 T cells from stage III CRC patients (Figure 1D), the frequencies of granzyme B+ cells were comparable between FOXP3+ Treg cells and FOXP3- Tconv cells, while in LN CD4 T cells from stage III patients, the frequencies of granzyme B+ cells were significantly lower in FOXP3+ Treg cells than in FOXP3-
Tconv cells. Both the FOXP3+ Treg cells and the FOXP3- Tconv cells in the tumor presented significantly higher levels of granzyme B+ cells than their counterparts in the LN.
Granzyme B and perforin expression in circulating Treg cells. Ten healthy volunteers, with matching age and sex with stage II and stage III CRC
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patients, were recruited as controls. Peripheral blood was obtained from healthy controls and stage II and stage III CRC patients who donated tumor and LN samples. We
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examined the granzyme B expression in circulating FOXP3+ Treg cells and FOXP3Tconv cells (Figure 2A). Unlike tumor-infiltrating Treg cells and Tconv cells, the
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circulating Treg cells and Tconv cells presented very few granzyme B+ cells directly ex vivo (Figure 2B), with frequencies of lower than 0.3% in all subjects. No significant
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differences between healthy controls, stage II CRC patients, and stage III CRC patients were observed.
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Subsequently, we attempted to upregulate granzyme B expression in Treg cells by stimulation with anti-CD3/CD28 antibodies and supplementing with IL-2. After 12-hour stimulation, granzyme B was only observed in a small proportion of FOXP3- CD4 T cells
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and a smaller proportion of FOXP3+ CD4 T cells (Figure 2A and 2C). No significant differences between healthy controls, stage II patients, and stage III patients were observed.
The colorectal tissues are constantly exposed to the gut microbiota, and microbial
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products have been found inside resected colorectal tumors[20]. In this part, we stimulated CD4 T cells using heat-killed Staphylococcus aureus (S. aureus), a member of the Firmicutes[21]. Interestingly, in both FOXP3- and FOXP3+ CD4 T cells, heat-killed S. aureus was more effective at promoting granzyme B expression than anti-CD3/CD28 and IL-2 (Figure 2A and 2D). No significant differences between healthy controls, stage II CRC patients, and stage III CRC patients were observed.
In addition, the expression of FOXP3 in CD4 T cells in unstimulated and stimulated conditions was examined. Overall, the frequency of FOXP3-expressing CD4 T cells remained stable throughout the 12-hour incubation period in unstimulated media, in anti-CD3/CD28 and IL-2 stimulation, and in S. aureus stimulation (Figure 2E). This stable trend was observed in healthy controls as well as in stage II and stage III CRC patients.
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We also investigated the expression of perforin in the circulating FOXP3+ Treg cells and FOXP3- Tconv cells (Figure 3A). Directly ex vivo, few perforin-expressing cells
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were found with no significant differences between between healthy controls, stage II CRC patients, and stage III CRC patients (Figure 3B). After 12-hour anti-CD3/CD28 and
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IL-2, perforin was increased in both FOXP3- CD4 T cells and FOXP3+ CD4 T cells (Figure 3C). Overall, FOXP3- CD4 T cells presented significantly higher perforin than
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FOXP3+ CD4 T cells. In FOXP3- CD4 T cells, the perforin expression was significantly higher in healthy controls than in stage III patients.
In FOXP3+ CD4 T cells, no
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significant differences between healthy controls, stage II patients, and stage III patients were observed. Heat-killed S. aureus could also elevate perforin expression in FOXP3CD4 T cells and FOXP3+ CD4 T cells (Figure 3D). No significant differences between
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healthy controls, stage II CRC patients, and stage III CRC patients were observed.
Granzyme B-expressing Treg cells were enriched in CD25+TIM-3+ CD4 T cells TIM-3 is mainly known as a negative regulator of proinflammatory immune
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responses[22]. TIM-3 expression is found on terminally differentiated IFN-γ-producing Th1 cells, Th17 cells, activated/exhausted CD8 T cells, and intratumoral Treg cells[23]. Multiple sources indicate that TIM-3+ Treg cells present higher expression of granzymes and perforin than TIM-3- Treg cells[24–26]. Using the findings from previous reports[23], we obtained unstimulated CD4 T cells from PBMCs using magnetic negative sorting (Figure 3A). Purified CD4 T cells were then sorted into CD25- Tconv cells,
CD25+TIM-3+ cells, and CD25+TIM-3- cells using fluorescence-activated cell sorting (FACS) (Figure 4A). Each fraction was then stimulated using heat-killed S. aureus for 12 hours. The expression of FOXP3 was examined to verify the identity of Treg cells (Figure 4B). As predicted, CD25- Tconv cells presented significantly lower FOXP3 than CD25+TIM-3- and CD25+TIM-3+ CD4 T cells. On average, CD25+TIM-3+ CD4 T cells presented higher FOXP3 expression than CD25+TIM-3- CD4 T cells, but the difference
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was not statistically significant. No significant differences between healthy subjects, stage II CRC patients, and stage III CRC patients were observed (Figure 4B). The
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granzyme B (GZMB) transcript expression was then quantified in each cell type (Figure 4C). CD25+TIM-3+ CD4 T cells presented significantly higher GZMB expression than
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both CD25- Tconv cells and CD25+TIM-3- CD4 T cells. On average, CD25- Tconv cells presented higher GZMB than CD25+TIM-3- CD4 T cells, but the difference was not
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statistically significant. No significant differences between healthy subjects, stage II CRC
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patients, and stage III CRC patients were observed.
CD25+TIM-3+ CD4 T cells mediated stronger lysis of Tconv cells than CD25+TIM-3CD4 T cells
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Subsequently, we investigated whether Treg cells could directly mediate lysis of Tconv cells. CD4+CD25- Tconv cells were labeled with
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Cr. Heat-killed S.
aureus-stimulated CD25+TIM-3- CD4 Treg cells and CD25+TIM-3+ CD4 Treg cells were then added at various Treg / Tconv ratios for 6 hours, after which the specific lysis
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mediated by Treg cells were calculated. Both CD25+TIM-3- CD4 Treg cells and CD25+TIM-3+ CD4 Treg cells were capable of mediating Tconv lysis in a manner that was positively associated with the Treg / Tconv ratio (Figure 5A to 5C). At Treg / Tconv ratios of equal to or greater than 3, the lysis efficiency of CD25+TIM-3+ CD4 Treg cells was significantly higher than CD25+TIM-3- CD4 Treg cells in healthy controls, stage II patients, and stage III patients.
Inhibition of granzyme B, but not TIM-3, was capable of suppressing CD25+TIM-3+ CD4 Treg-mediated lysis of Tconv cells To demonstrate the involvement of granzyme B in Treg-mediated lysis, we used a small molecule inhibitor Z-AAD-CMK to inhibit the activity of granzyme B. In both CD25+TIM-3- CD4 Treg cells and CD25+TIM-3+ CD4 Treg cells, Z-AAD-CMK reduced
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the specific lytic capacity in a dose-dependent manner (Figure 6A). In addition, at a concentration of 1 µM, Z-AAD-CMK eliminated the differences between CD25+TIM-3-
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CD4 Treg cells and CD25+TIM-3+ CD4 Treg cells.
CD25+TIM-3- CD4 Treg cells and CD25+TIM-3+ CD4 Treg cells not only differed in
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granzyme B production but also differed in TIM-3 expression. To investigate whether TIM-3 had a role in Treg-mediated lysis, we inhibited TIM-3 using the blocking antibody
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2E2. In neither CD25+TIM-3- CD4 Treg cells nor CD25+TIM-3+ CD4 Treg cells, the addition of 2E2 presented a significant effect on Treg-mediated lysis of Tconv cells
Discussion
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(Figure 6B).
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Granzyme B is one of the most abundant granzymes and is mainly expressed by activated cytotoxic T cells and NK cells. Due to its role in directly promoting cell death, granzyme B is critical to the elimination of precancerous cells and malignant cells during immunosurveillance and cancer[11]. However, it is also employed by Treg cells to
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eliminate proinflammatory immune cells and downplay inflammation[2,3]. In this study, we investigated Treg-mediated granzyme B expression in healthy individuals and in patients with CRC. Overall, we presented following findings. First, we found that granzyme B-expressing FOXP3+ Treg cells were present in resected tumor, and to a much lesser extent in tumor-associated LN and in circulating Treg cells. Second, the expression of granzyme B in Treg cells could not be activated by standard T cell receptor (TCR)
activation through anti-CD3/CD28 and IL-2, but required stimulation with heat-killed S. aureus. Third, granzyme B expression was enriched in TIM-3+ Treg cells. And fourth, the TIM-3+ Treg cells presented higher cytolytic capacity toward autologous Tconv cells than the TIM-3- Treg cells, in a manner that depended on granzyme B but not TIM-3. Given the enrichment of granzyme B-expressing Treg cells in tumor, and the observation that granzyme B-expressing circulating Treg cells mediated lysis of
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autologous Tconv cells, future studies should investigate whether tumor-infiltrating Treg cells actively eliminate proinflammatory T cells in vivo, and what impacts they might
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have on the progression of cancer. Many other tumors, especially those associated with the digestive system or are in contact with mucosal surfaces, such as pancreatic, gastric,
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lung, breast, ovarian, and prostate carcinomas, are found to harbor bacteria in the tumor microenvironment, and in some cases, the presence of bacteria may confer stronger
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resistance to chemotherapy[20,27]. In this study, S. aureus may weaken proinflammatory immune responses by activating granzyme B expression in Treg cells. However, S. aureus
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is rarely found in the intestinal tract. Whether it is a coincidence or S. aureus essentially affects the immunity of CRC requires further study. Of note, the inhibition of TIM-3 did not significantly alter the cytotoxic capacity of
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TIM-3+ Treg cells. This may be problematic for immunotherapeutic strategies that target TIM-3. It is increasingly recognized that TIM-3 inhibits antitumor immunity and the blockade of TIM-3, together with other inhibitory molecules such as PD-1/PD-L1 and CTLA-4, may be required for optimal results[28,29]. However, as TIM-3 inhibition was
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unable to reduce Treg-mediated cytotoxicity, the potency of TIM-3 inhibition may be limited. Further studies into the regulatory mechanisms of Treg-mediated cytotoxicity are required.
Another observation is that the granzyme B expression by LN FOXP3+ Treg cells was severely impaired compared to the granzyme B expression by LN FOXP3- CD4 T cells. Whether LN FOXP3+ Treg cells express additional inhibitory molecules that
prevent granzyme B expression remains unclear and should be studied further. A major limitation of this study is the use of S. aureus as a model bacterium in Treg stimulation. Although this bacterium is common as a commensal and can become a pathogen in the blood, the respiratory tract, and the urinary tract, it is rarely found in the intestinal tract. Bacterial species that were previously found in the resected colorectal tumor tissues and/or shown to have carcinogenic activity in colorectal cancer, such as
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Fusobacterium and Campylobacter, should be used in Treg stimulation[30,31]. In addition, in both FOXP3- CD4 T cells and FOXP3+ Treg cells, about 1% to 2% cells
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expressed granzyme B following S. aureus stimulation. This high frequency, combined with the fact that S. aureus has surface proteins that could act as T cell mitogens[32],
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suggested that the observed stimulatory effects were not antigen-specific. The underlying mechanism for granzyme B expression mediated by S. aureus and other bacterial species
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should be further studied. In addition, this study focused on granzyme B expression. However, the pore-forming protein perforin is also critical to cytolysis. Further studies
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should also investigate the expression of perforin, along with degranulation marker CD107a, in Treg cells from the tumor and LN tissues and the blood of CRC patients.
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Conflict of interests
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The authors declare no conflict of interest.
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Figure Legend Figure 1. Granzyme B expression by tumor and LN Treg cells in CRC patients. (A) Expression of Treg markers by Foxp3+ and Foxp3- T cells, pre-gated in live CD3+CD4+ tumor-infiltrating lymphocytes. (B) In pre-gated CD4 T cells, granzyme B+ and granzyme B- cells were gated in FOXP3- and FOXP3+ cells, as shown in the representative figures from one stage II CRC patient and one stage III CRC patient.
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Staining was performed in unstimulated tumor and LN samples directly ex vivo and without stimulation. (C) The frequencies of granzyme B+ cells in CD4+FOXP3- (filled)
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and CD4+FOXP3+ (open) T cells from stage II tumor and LN samples. (D) The frequencies of granzyme B+ cells in CD4+FOXP3- (filled) and CD4+FOXP3+ (open) T
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cells from stage III tumor and LN samples. (B) and (C) Two-way ANOVA and Tukey’s
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multiple comparisons. ns, not significant. *P < 0.05. ***P < 0.001.
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controls. (A) The expression of granzyme B by FOXP3- and FOXP3+ CD4 T cells directly ex vivo, after 12-hour stimulation with anti-CD3/CD28 antibodies (1 µg/mL each) and IL-2 (20 U/mL), or after 12-hour stimulation with heat-killed S. aureus (100 bacteria per Treg cell). Representative results from one stage II CRC patient is shown. (B) The frequencies of granzyme B+ cells in FOXP3- and FOXP3+ circulating CD4 T cells from healthy (circle),
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stage II CRC (square), and stage III CRC (triangle) subjects, examined directly ex vivo. (C) The frequencies of granzyme B+ cells in FOXP3- and FOXP3+ circulating CD4 T
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cells from healthy (circle), stage II CRC (square), and stage III CRC (triangle) subjects, examined after 12-hour stimulation with anti-CD3/CD28 and IL-2. (D) The frequencies
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of granzyme B+ cells in FOXP3- and FOXP3+ circulating CD4 T cells from healthy (circle), stage II CRC (square), and stage III CRC (triangle) subjects, examined after
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12-hour stimulation with heat-killed S. aureus. (E) The frequencies of Foxp3+ CD4 T cells in healthy subjects (open circle), stage II CRC subjects (square), and stage III CRC
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subjects (cross). N = 10 for each group. (B) to (E) Two-way ANOVA and Tukey’s
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multiple comparisons. ns, not significant. ***P < 0.001.
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Figure 3. Perforin expression by circulating Treg cells in CRC patients and healthy controls.
(A) The expression of perforin by FOXP3- and FOXP3+ CD4 T cells directly ex vivo,
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after 12-hour stimulation with anti-CD3/CD28 antibodies (1 µg/mL each) and IL-2 (20 U/mL), or after 12-hour stimulation with heat-killed S. aureus (100 bacteria per Treg cell). Representative results from one stage II CRC patient is shown. (B) The frequencies of perforin+ cells in FOXP3- and FOXP3+ circulating CD4 T cells from healthy (circle), stage II CRC (square), and stage III CRC (triangle) subjects, examined directly ex vivo. (C) The frequencies of perforin+ cells in FOXP3- and FOXP3+ circulating CD4 T cells
from healthy (circle), stage II CRC (square), and stage III CRC (triangle) subjects, examined after 12-hour stimulation with anti-CD3/CD28 and IL-2. (D) The frequencies of perforin+ cells in FOXP3- and FOXP3+ circulating CD4 T cells from healthy (circle), stage II CRC (square), and stage III CRC (triangle) subjects, examined after 12-hour stimulation with heat-killed S. aureus. (B) to (D) Two-way ANOVA and Tukey’s multiple
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comparisons. ns, not significant. *P < 0.05. **P < 0.01. ***P < 0.001.
Figure 4. FOXP3 and GZMB expression by CD25-, CD25+TIM-3-, and CD25+TIM-3+
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CD4 T cells.
Total CD4 T cells from healthy (circle), stage II CRC (square), and stage III CRC (triangle) subjects were first isolated via magnetic negative selection and then separated into CD25-, CD25+TIM-3-, and CD25+TIM-3+ subsets using FACS. The CD4 T cell subsets were then stimulated for 12 hours with heat-killed S. aureus (100 bacteria per Treg cell). (A) The efficiency of CD4 T cell isolation and CD25 vs. TIM-3 gating
strategy in one representative sample. (B) The FOXP3 mRNA expression by each subset. (C) The GZMB mRNA expression by each subset. Two-way ANOVA and Tukey’s
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multiple comparisons. ns, not significant. **P < 0.01. ***P < 0.001.
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Figure 5. Specific lysis by CD25+TIM-3- and CD25+TIM-3+ Treg cells. 51
Cr-labeled CD4+CD25- Tconv cells were plated at 1 × 104 cells per well in a 96-well
plate. CD25+TIM-3- CD4 Treg cells (filled) and CD25+TIM-3+ CD4 Treg cells (open)
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were stimulated with heat-killed S. aureus (100 bacteria per Treg cell) for 12 hours, and were then added at various Treg / Tconv ratios. Spontaneous release had no Treg cells, and maximum release had 100% water in place of culture medium. After 6 hours, the supernatant was collected and the 51Cr level was measured. Specific lysis was calculated
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by the formula (experiment - spontaneous) / (maximum - spontaneous). (A) The Treg-mediated specific lysis in healthy subjects (N = 10). (B) The Treg-mediated specific lysis in stage II CRC patients (N = 10). (C) The Treg-mediated specific lysis in stage III CRC patients (N = 10). Two-way ANOVA and Tukey’s multiple comparisons. ns, not significant. **P < 0.01. ***P < 0.001.
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Figure 6. Effect of granzyme B and TIM-3 inhibition on the specific lysis by CD25+TIM-3- and CD25+TIM-3+ Treg cells. Cr-labeled CD4+CD25- Tconv cells were plated at 1 × 104 cells per well in a 96-well
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51
plate. CD25+TIM-3- CD4 Treg cells (filled) and CD25+TIM-3+ CD4 Treg cells (open) were stimulated with heat-killed S. aureus (100 bacteria per Treg cell) for 12 hours, and were then added at a Treg / Tconv ratio of 3. Granzyme B inhibitor Z-AAD-CMK or TIM-3 blocking antibody clone 2E2 were added accordingly. (A) The Treg-mediated specific lysis in the presence of various levels of Z-AAD-CMK. (B) The Treg-mediated
specific lysis in the presence of various levels of 2E2. Two-way ANOVA and Tukey’s
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multiple comparisons. ns, not significant. *P < 0.05. **P < 0.01. ***P < 0.001.