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Keywords:

  • bacillus Calmette-Guérin;
  • bladder cancer;
  • dendritic cells;
  • γδT cells;
  • natural killer cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References

Background:  Immunotherapy with bacillus Calmette-Guérin (BCG) for bladder cancer is successful, although the precise mechanism is unclear. Natural killer (NK) cells are a candidate for BCG-activated killer cells, but the roles of other T lymphocytes, such as NKT cells and γδT cells, are not fully understood. Mycobacterium tuberculosis is a potent activator of both NKT cells and γδT cells. However, it is known that the patient's prognosis is good if there are increased numbers of dendritic cells (DCs) in the urine after BCG therapy. Therefore, we investigated whether DCs are matured by BCG and whether BCG-pulsed DCs stimulate NKT cells and γδT cells.

Methods:  Naïve Pan T cells were isolated form peripheral blood mononuclear cells (PBMCs) and DCs were obtained by culturing CD14+ monocytes with granulocyte–macrophage colony-stimulating factor and interleukin-4. The DCs were pulsed with BCG and their maturation was measured by fluorescence-activated cell sorter analysis using the CD86 antibody. Naïve T lymphocytes were stimulated by coculture with BCG-pulsed DCs in vitro, followed by FACS analysis to estimate the ratio and activation of NKT cells and the ratio of γδT cells. The 51Cr (chromium) release assay was used to estimate the cytotoxic activity of the stimulated T cells. Cytolytic proteins in the patient's PBMCs were measured during BCG therapy using semiquantitative reverse transcriptase-polymerase chain reaction.

Results:  The DCs were matured by BCG stimulation and the number of NKT cells and γδT cells increased after culturing with BCG-pulsed DCs. The BCG-pulsed DCs also activated the NKT cells and γδT cells. Also, the lymphocytes that were cocultured with the BCG-pulsed DCs showed unspecific cytotoxic activity against a bladder cancer cell line.

Conclusion:  Sensitization of NKT cells and γδT cells by BCG-pulsed DCs might be one of the mechanisms of BCG immunotherapy.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References

Intravesical immunotherapy with bacillus Calmette-Guérin (BCG) was first used by Morales in 1976.1 Since then, several investigators have obtained favorable results in preventing and treating superficial tumors of the urinary bladder. Although the immunotherapeutic approach is clinically effective, its precise mechanism has not been adequately clarified,and various theoretical and practical questions remain unsolved. It has been reported that BCG-induced cytokines affect lymphocyte-mediated cytotoxicity in bladder cancers2–5 and that killer T lymphocytes or natural killer (NK) cells are activated and eventually release cytokines into the patient's urine.6–8 Such evidence strongly suggests that cell-mediated immunity plays a key role in the mechanism of BCG's beneficial effect. Likewise, during repeated BCG instillations, the levels of cytolytic proteins (e.g. perforin, glanzyme B) are elevated in the peripheral blood of patients and perforin has been shown to be the key molecule in lymphocyte-mediated cytotoxicity.9 Perforin is expressed by activated T cells, NK cells, and NKT lymphocytes when activated.10 In cancer immunology, cytotoxic activity mediated by NK cells, NKT cells or γδT cells is very important and it shares not only innate immunity, but also facilitates acquired immunity. However, a favorable prognosis in cases of bladder cancer with a relatively large number of dendritic cells (DCs) in the urine of patients treated with BCG has been reported.11 The DCs play a central role in the immune system; they not only prime naïve T cells to generate antigen-specific cytotoxic T lymphocytes (CTLs), but also increase the cytotoxic activity of T cells, including NKT cells and γδT cells. As bacteria induce the maturation of DCs, DC-mediated immune responses might be affected by BCG instillation to the bladder; however, such killing activity induced by BCG-pulsed DCs has not been clarified. Therefore, in this study we investigated whether DCs are matured by BCG and whether BCG-pulsed DCs stimulate NKT cells and γδT cells for sustained innate immune responses.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References

Lymphocyte activation with bacillus Calmette-Guérin or bacillus Calmette-Guérin-pulsed dendritic cells

Preparation of naïve T cells

Peripheral blood mononuclear cells (PBMCs) from heparinized blood samples of healthy human donors were obtained by the Ficoll-Paque method (Pharmacia Biotech, Tokyo, Japan) and concentrated to 4.0 × 106/mL in RPMI-1640 medium (Gibco-BRL, Tokyo, Japan) containing 10% autologous human serum, penicillin (100 U/mL), and streptomycin (100 μg/mL). Naïve T cells were isolated as follows: the naïve T cells were isolated from the PBMCs by collecting a negative selection of CD45RO+ cells using CD45RO MicroBeads (Miltenyi Biotec, Auburn, CA, USA). Subsequently, the naïve T cells were isolated from these CD45RO cells by the Pan T Cell Isolation Kit II (Miltenyi Biotec, Auburn, CA, USA) and the T cells were cultured with autologous BCG-pulsed DCs for 7 days and used for fluorescence-activated cell sorter (FACS) analysis of the NK and γδT cells.

Flow cytometry analysis

For FACS, 1 × 106 PBMC/100 μL were stained with Cy5-labeled CD56 (Immunotech, Marseille, France), phycoerythrin (PE)-labeled CD56 (Becton Dickinson, San Jose, CA, USA), Cy5-labeled CD3 (Becton Dickinson, San Jose, CA, USA), Cy5-labeled perforin (Becton Dickinson, San Jose, CA, USA), PE-labeled perforin (Becton Dickinson, San Jose, CA, USA), fluorescein-labeled interferon (IFN)-γ (Becton Dickinson, San Jose, CA, USA), and PE-labeled T cell receptor (TCR)ãδ (E-Bioscience, San Diego, CA, USA) using the FIX and PERM Cell Permeabilization Kit (Caltag Laboratories, San Francisco, CA, USA). The FIX and PERM reagent allows intracellular antigens to be analysed as easily as surface antigens. Tri-color FACS was performed using a FACS Calibur (Becton Dickinson, San Jose, CA, USA) and the cells were analysed with Cell Quest (BD Bioscience, San Jose, CA, USA). For all staining, isotope antibodies (BD Bioscience, San Jose, CA, USA) were used as the controls. For each antigenic determinant analysed, the percentage of positive cells and the intensity of expression (evaluated as the mean fluorescence intensity [MFI] expressed in arbitrary relative linear units on a scale from 0–10 000) were recorded.

In vitro cytotoxicity assay

Dendritic cell generation from peripheral blood

The PBMCs were isolated from heparinized blood samples by centrifugation over Ficoll-Hypaque gradients and CD14+ cells were isolated using CD14 MicroBeads (Miltenyi Biotec, Auburn, CA, USA). The CD14+ cells were stimulated with granulocyte–macrophage colony-stimulating factor (20 μg/mL; R & D Systems, Minneapolis, MN, USA) and interleukin-4 (10 μg/mL; E-Bioscience, San Diego, CA, USA) for 7 days for DCs that would be used as antigen-presenting cells. Some DCs were cultured with 1 μg/μL of BCG for 3 days and used for CTL generation.12

T effector cell preparation

Naïve T cells were used for CTL generation. Pan T cells were isolated from PBMCs using the Pan T Cell Isolation Kit II (Miltenyi Biotec, Auburn, CA, USA) and, subsequently, the naïve T cells were isolated by collecting a negative selection of CD45RO+ cells using CD45RO MicroBeads (Miltenyi Biotec, Auburn, CA, USA). The cells were cultured with autologous BCG-pulsed DCs for 7 days and used as T effector cells at a stimulator/effector ratio of 1:10.

Chromium release assay

Two types of target cells (T24 bladder cancer cell lines infected or uninfected by BCG; Fig. 1) were prepared. The target cells were labeled with 51Cr (chromium) in saline solution by incubating 2 × 106 of the cells in 2 mL RPMI 1640 with 18.5 Bq/mL of 51Cr (Amersham Bioscience, Pittsburgh, PA, USA) for 3 h at 37°C in 5% carbon dioxide (CO2). After three washes, 1 × 104/100 μL 51Cr-labeled targets and serial dilutions of effector cells at various effector : target ratios were incubated in 200 μL of RPMI 1640 (100 μL was RPMI containing effector and DC cells) in 96-well plates (NUNC, Rochester, NY, USA). These plates were incubated for 24 h at 37°C in 5% CO2, after which 100 μL of the supernatant was harvested and the released 51Cr was measured using a scintillation counter (Cobra B5005; Packard Instruments Company, Meriden, CT, USA).

image

Figure 1. After a 24-h culture with bacillus Calmette-Guérin (BCG), a cytospin of the T24 bladder cancer cell line was prepared for Ziehl-Neelsen staining. (a) Bacillus Calmette-Guérin-uninfected T24 bladder cancer cell line. (b) Bacillus Calmette-Guérin-infected T24 bladder cancer cell line. The arrows indicate infected BCG.

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Confirmation of bacillus Calmette-Guérin-infected bladder cancer cells

T24 bladder cancer cells were cultured with 0.1 mg/mL of BCG in RPMI 1640 for 24 h. After washing with phosphate-buffered saline (PBS), cytospins of the bladder cancer cells were prepared and then Ziehl-Neelsen staining for BCG-infected T24 cells was performed (Fig. 1).

Perforin and granzyme B expression in peripheral blood mononuclear cells after bacillus Calmette-Guérin therapy

Patients and protocol for intravesical bacillus Calmette-Guérin instillation

The study group comprised two patients who had undergone transurethral resection of the bladder because of stage T1 transitional cell carcinoma (Case 1: a 51-year-old woman with a tumor of grade G2; Case 2: a 73-year-old man with a tumor of grade G1). Each patient's bladder was catheterized and 80 mg of Tokyo 172 strain BCG (Japan BCG Company, Tokyo, Japan) was instilled each week for eight consecutive weeks.

Blood samples and isolation of peripheral blood mononuclear cells

Blood samples were taken 4 h after each BCG instillation and serologically screened for the HIV and hepatitis B and C viruses. Immediately after collection, the PBMCs were isolated from the heparinized blood samples by centrifugation over Ficoll-Hypaque gradients. The PBMCs were washed three times with PBS and the total RNA was extracted. Informed consent was given prior to drawing the blood samples.

Total RNA extraction and cDNA generation

The PBMCs were suspended with 250 μL of tris-hydrochloride and ethylenediaminetetraacetic acid buffer and the total RNA was extracted with 750 μL of ISOGEN-LS (Wako, Tokyo, Japan), then eluted with RNase-free water. For the cDNA synthesis, 1 μg of total RNA was transcribed using a TaKaRa RNA LA PCR Kit (AMV version 1.1; Takara Bio, Tokyo, Japan) and random 9mer and stored at −21°C until analysis. The cDNA standards were generated using reverse transcriptase and primer-specific amplification of the mRNA of the relevant genes using a technique identical to that used for the previous preparation of the test cDNA. The amplified cDNA was then purified and quantified by spectrophotometry (OD260).

Semiquantitative, real-time polymerase chain reaction

Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) can detect low quantities of mRNA and many investigators are using this powerful method to better understand the molecular mechanisms and assess the different markers of immunological activity. Semiquantitative, real-time PCRs for perforin and granzyme B (GB) were performed using the ABI prism 5700 Sequence Detection System (Applied Biosystems, Tokyo, Japan). Primer and TaqMan probes were designed to span exon–intron junctions to prevent the amplification of genomic DNA and to produce amplicons of <150 base pairs, enhancing the efficacy of PCR amplification. All three TaqMan probes (Applied Biosystems, Tokyo, Japan) (for β-actin, perforin, and GB) were labeled at the 5′-end with the reporter dye molecule FAM (6-carboxyfluorescein; emission: γmax = 518 nm); the probes also were labeled at the 3′-end with the quencher dye molecule TAMRA (6-carboxytetramethylrhodamine; emission: γmax = 582 nm). The PCRs of the cDNA specimens and cDNA standards were conducted in a total volume of 25 μL with 1 × TaqMan Master Mix (Applied Biosystems, Tokyo, Japan) and the primers and probes at optimized concentrations. The thermal cycle parameters consisted of 2 min at 50°C, 10 min at 95°C, and 40 cycles involving denaturing at 95°C for 15 s, with annealing/extension at 60°C for 1 min. Real-time monitoring of the fluorescent emission from the cleavage of the sequence-specific probes by the nuclease activity of Taq polymerase allowed the threshold cycle during the exponential phase of amplification to be defined. Standard curves were generated for each gene and the slopes of the curves confirmed the efficiencies of the PCR amplifications. Linear regression analyses showed the correlation coefficient of all the standard curves to be >0.99. Perforin and GB were normalized to β-actin.

Statistical analysis

The results were expressed as the mean ± SD for the groups. The statistical analyses were performed using the Mann–Whitney's U-test for DC maturation, the Bonferroni test for the difference in number and activated state of NKT cells and γδT cells, and post-hoc comparison (two-factor factorial anova) was used for the cytotoxicity of T cells against the bladder cancer cell line, using SPSS software (SPSS, Tokyo, Japan).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References

Maturation of dendritic cells by bacillus Calmette-Guérin

To test the immunogenicity of BCG, DC maturation induced by BCG stimulation was investigated by FACS analysis using the CD86 antibody, which is a maturation marker of DCs. After 24 h stimulation with 10 μg/mL BCG, the MFI of CD86 was ≈ 7-fold higher (68.9 ± 2.8–468.2 ± 37.3, P = 0.0495) than that achieved before BCG administration (Fig. 2a,b).

image

Figure 2. (a) Representative flow cytometry result of dendritic cell maturation caused by bacillus Calmette-Guérin (BCG) stimulation. (b) Maturation of dendritic cells after culturing with BCG for 24 h.

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Induction of natural killer T cells by bacillus Calmette-Guérin-pulsed dendritic cells

To test whether matured DCs (mDCs, i.e. BCG-pulsed DCs) augmented the number of NKT cells in the T cells, naïve T cells were cultured with BCG-pulsed DCs for 7 days. The immature DCs did not increase the number of NKT cells, but the BCG-pulsed DCs increased the number of NKT cells ≈ 3-fold (0.52 ± 0.1–1.7 ± 0.49%, P = 0.0025) (Fig. 3a).

image

Figure 3. (a) Increased number of natural killer T cells (CD56+/CD3+cells) after culturing with bacillus Calmette-Guérin (BCG)-pulsed dendritic cells for 7 days. (b) Increased number of activated (interferon-γ-positive) natural killer T cells (CD56+/CD3+cells) after culturing with BCG-pulsed dendritic cells for 7 days.

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Induction of activated natural killer T cells by bacillus Calmette-Guérin-pulsed dendritic cells

To test the sensitizing effect of BCG-pulsed DCs for NKT cells, naïve T cells were cultured with BCG-pulsed DCs for 7 days, after which the number of activated (IFN-γ-producing) NKT cells increased (5.78 ± 0.90–20.06 ± 2.30%, P < 0.0001) (Fig. 3b).

Induction of γδT cells by bacillus Calmette-Guérin-pulsed dendritic cells

To test whether BCG-pulsed DCs augmented the number of γδT cells, naïve T cells were cultured with BCG-pulsed DCs for 7 days and compared with the effect of immature DCs. The BCG-pulsed DCs increased the number of γδT cells in the Pan T cells (6.71 ± 0.41–16.38 ± 3.05%, P = 0.0006) (Fig. 4a).

image

Figure 4. (a) Increased number of γδT cells after culturing with bacillus Calmette-Guérin (BCG)-pulsed dendritic cells. (b) Increased number of activated (interferon-γ-positive) γδT cells after culturing with BCG-pulsed dendritic cells for 7 days.

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Induction of activated γδT cells by bacillus Calmette-Guérin-pulsed dendritic cells

To test the sensitizing effect of BCG-pulsed DCs for γδT cells, naïve T cells were cultured with BCG-pulsed DCs for 7 days, after which the number of activated (IFN-γ-producing) γδT cells increased (2.55 ± 0.24–32.14 ± 2.37%, P < 0.0001) (Fig. 4b).

Cytotoxic activity of activated T cells against bladder cancer cells

The cytotoxic activity of T lymphocytes stimulated with BCG-pulsed DCs was tested using a standard 51Cr release assay. As shown in Figure 5, the T cells cultured with BCG-pulsed DCs showed cytotoxic activity against both T24 bladder cancer cell lines (BCG-infected and uninfected; Fig. 1). The cytotoxic activity against non-BCG-infected cancer cells represents unspecific cytotoxic activity of the T cells. Furthermore, the trend that BCG-infected cancer cells were killed more efficiently than uninfected cells was observed (P < 0.0001), which might suggest that BCG infection increases the susceptibility of bladder cancer cells to T cell activity.

image

Figure 5. T cells cultured with bacillus Calmette-Guérin (BCG)-pulsed dendritic cells show cytotoxic activity against both BCG-infected and BCG-uninfected T24 bladder cancer cell lines.

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Cytolytic proteins in patients' peripheral blood after bacillus Calmette-Guérin instillation

The expression of both perforin and GB mRNA on the PBMCs was elevated throughout the BCG therapy, according to the results of the semiquantitative RT-PCR. In Case 1, the fifth BCG instillation elevated the expression of perforin 13.3-fold compared to that achieved before the instillation. Although this peak level of expression did not continue after the sixth instillation, it remained >2.5-fold higher than before the instillation. The same phenomenon was observed for the expression of GB mRNA; that is, the level of GB expression after the fifth instillation was 130.5-fold higher than before the instillation. Similarly, that peak level did not last after the sixth instillation, but continued to be >11.3-fold higher than after the fifth instillation. In Case 2, the highest expression of perforin mRNA occurred after the second instillation (33.9-fold higher than before the instillation) and the highest expression of GB also occurred at that time (Fig. 6).

image

Figure 6. Expression of cytolytic proteins (perforin and granzyme B) on peripheral blood mononuclear cells after bacillus Calmette-Guérin (BCG) instillation was measured throughout the series of BCG instillations (once weekly × 8 weeks) in two patients.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References

It is considered that BCG instillation shares innate immunity and its antitumor activity has been proved in the treatment of superficial bladder cancer. However, after instillation to the bladder, BCG can selectively migrate into cancer cells through the fibronectin expressed on the cancer cells, which might induce an antitumor response involving humoral and cellular immune mechanisms. Clinical studies show a marked increase in the number of lymphocytes in the urine after BCG therapy13 and elevated levels of T helper cell 1-dependent cytokines in the urine after repeated BCG therapy.14 Additionally, urologists usually encounter fever in the patients after repeated BCG therapy, all of which indicates that cellular immunity is involved in BCG therapy. In the present study, we demonstrated elevated levels of cytolytic proteins in the patients' peripheral blood after BCG instillation (Fig. 6), which implies systemic immunological responses to BCG. An important question is: What are BCG-activated killer cells? Brandau concluded that they are NK cells with the CD3/CD8+/CD56+/CD16 very dim/perforin+ phenotype.15 However, the cytotoxic activity might be caused not only by NK cells, but also by a subpopulation of T lymphocytes, such as NKT cells and γδT cells. The NKT cells and γδT cells are crucial for anticancer immunity and they express cytolytic proteins when activated.10 The down-regulation of the major histocompatibility complex (MHC) class I would allow cancer cells to evade anticancer immune surveillance of the DCs and conventional α/β T cells. The down-regulated MHC class I is reported in many types of cancers, including bladder cancer.16 Thus, we emphasize the importance of NKT cells and γδT cells, which kill MHC class Ilow cancer cells.

Natural killer T cells are a recently described subpopulation of the TCRα/β+ T cells, which have distinctive phenotypic and functional properties17,18 and recognize foreign and self-glycolipids presented by the non-classical MHC class I molecule, CD1d.19 It has been suggested that NKT cells are primarily involved in the detection of bacterial and parasitic pathogens.20 Glycosylceramides from the Gram-negative bacterium, Ehrlichia muris, and certain Sphingomonas spp., as well as phosphatidylinositol mannoside from mycobacterial membranes, also are recognized by human and mouse NKT cells.21–23 Recent studies suggest a major role for murine NKT cells in the rejection of malignant tumors24–28 and in regulating autoimmunity.29 Therefore, NKT cells are currently believed to have promise as an anticancer immune therapy.30

However, γδT cells, which express γδTCRs instead of the αβTCR found on classical T cells, recognize microbial and tumor antigens.31 Similar to the NK and NKT cells, γδT cells can recognize microbial products and self-molecules on infected or tumor cells, including non-classical MHC class 1 molecules. Also, it is known that Mycobacteria facilitate the recognition of tumors by invariant γδT cells.32

The ‘sentinels’ of the immune system are the DCs, which differ from macrophages and monocyte antigen-presenting cells because they are potent initiators of primary naïve T-cell responses.11 Pathogen recognition by DCs leads to their maturation and the initiation of adaptive immunity. In the present study, we showed that after BCG stimulation, the DCs became activated and acquired a more mature phenotype (i.e. CD86+), displaying a potent allostimulatory capacity to foster NKT cells and γδT cells. We also showed that mature DCs (i.e. BCG-DCs) also augmented the number of NKT cells and γδT cells, as well as triggering the IFN-γ production of NKT cells and γδT cells. Recent studies have shown that innate lymphocytes (i.e. NK, NKT, and γδT cells) also trigger DC maturation and this interaction, in turn, expands and activates innate lymphocytes and initiates adaptive T-cell immunity.20 In this system, BCG might work as a stimulant for both DC- and T-cell immunity. As infected DCs appear to undergo maturation and, therefore, might have acquired migratory properties, they might be able to move into the host's draining lymph nodes to initiate protective immunity. Likewise, BCG instillation to the bladder might cause a DC-mediated systemic immune response. Our results suggest the potential of BCG-pulsed DC immunotherapy for bladder cancer.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References

In summary, BCG is a potent stimulator of DCs and BCG-stimulated DCs are a potent stimulator of NKT cells and γδT cells. Our study findings suggest that the anticancer activity and unspecific immunostimulatory activity of BCG therapy might be the result of the direct activation of DCs by BCG and, thus, activation of T-cell immunity (including NKT and γδT cells.) Further investigation of the mechanisms of BCG's beneficial effect against bladder cancer is needed.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References

This work was supported by Showa University Medical Foundation. We thank Ms Yoshiko Tsuda (Department of Pathology, Showa University, Tokyo) for technical assistance with the Ziehl-Neelsen staining.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgment
  9. References