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Keywords:

  • Mycobacterium indicus pranii;
  • myeloma;
  • thymoma;
  • T cells;
  • IFNγ

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Mycobacterium indicus pranii (MIP) is approved for use as an adjuvant (Immuvac/Cadi-05) in the treatment of leprosy. In addition, its efficacy is being investigated in clinical trials on patients with tuberculosis and different tumors. To evaluate and delineate the mechanisms by which autoclaved MIP enhances anti-tumor responses, the growth of solid tumors consisting of Sp2/0 (myeloma) and EL4 (thymoma) cells was studied in BALB/c and C57BL/6 mice, respectively. Treatment of mice with a single intra-dermal (i.d.) injection of MIP 3 days after Sp2/0 implantation greatly suppresses tumor growth. MIP treatment of tumor bearing mice lowers Interleukin (IL)6 but increases IL12p70 and IFNγ amounts in sera. Also, increase in CD8+ T cell mediated lysis of specific tumor targets and production of high amounts of IL2 and IFNγ by CD4+ T cells upon stimulation with specific tumor antigens in MIP treated mice is observed. Furthermore, MIP is also effective in reducing the growth of EL4 tumors; however, this efficacy is reduced in Ifnγ−/− mice. In fact, several MIP mediated anti-tumor responses are greatly abrogated in Ifnγ−/− mice: increase in serum Interleukin (IL)12p70 amounts, induction of IL2 and lysis of EL4 targets by splenocytes upon stimulation with specific tumor antigens. Interestingly, tumor-induced increase in serum IL12p70 and IFNγ and reduction in growth of Sp2/0 and EL4 tumors by MIP are not observed in nonobese diabetic severe combined immunodeficiency mice. Overall, our study clearly demonstrates the importance of a functional immune network, in particular endogenous CD4+ and CD8+ T cells and IFNγ, in mediating the anti-tumor responses by MIP.

The exciting area of cancer immunotherapy has emerged as a powerful method to treat tumors and numerous agents that enhance the potential of the host immune system are being evaluated in clinical trials: Toll-like receptor (TLR) agonists, recombinant cytokines, etc. In addition, the use of bacteria to potentiate the host anti-tumor response is well known. This approach was first championed by William Coley who used extracts of pyogenic bacteria to treat sarcomas. Bacteria belonging to the genus Mycobacterium exhibit powerful adjuvant activity to enhance cancer therapy,1 and the best known example is live bacillus Calmette-Guérin (BCG) to treat superficial bladder cancer.2 Also, BCG vaccination reduces the risk of developing childhood leukemia and melanoma.1 In addition, heat-killed suspension of M. vaccae (SRL172) is efficacious against other diseases and induces potent anti-tumor responses.1, 3 Immunotherapy with SRL172 in patients with cancer has significantly improved the quality of life and enhanced overall survival time.4–7

A saprophytic bacterium Mycobacterium indicus pranii (MIP), previously known as Mycobacterium w, stimulates cell mediated responses in patients suffering from leprosy.8 Further biochemical, molecular and phylogenic analysis has shown MIP to be distinct but closely related to M. avium intracellulare.9 MIP treatment, together with standard multidrug treatment, increases clearance of bacilli and reduces the recovery time of leprosy patients.10, 11 MIP retains its immunologic potential even after it is killed, unlike BCG, and has been found to share antigens with Mycobacterium leprae and Mycobacterium tuberculosis. In an experimental model of tuberculosis, MIP immunizations have been shown to be protective.12, 13 Importantly, MIP immunization in humans has shown prophylactic efficacy against pulmonary tuberculosis.14 Also, MIP is a general immunomodulator and enhances immunity to other diseases, e.g., HIV,15 psoriasis.16

Interestingly, MIP decreases tumor size in lung cancer and improves lung function17 and is also effective against bladder cancer.18 Despite these observations, the mechanisms by which MIP enhances anti-tumor responses is not known. Our study was initiated to evaluate and understand the mechanisms involved in the anti-tumor effects of MIP, using two different tumor models: Sp2/0 myeloma and EL4 thymoma. Notably, multiple myeloma afflicts 1–5 per 100,000 individuals and is characterized by the uncontrolled proliferation of malignant plasma cells, bone destruction and production of monoclonal immunoglobulins.19 On the other hand, the overall incidence of thymoma is relatively rare with 0.15 cases per 100,000 individuals.20 Drugs such as thalidomide, Velcade, etc. are used for treatment of myeloma but are also associated with a high level of toxicity. Nevertheless, complete cure is rarely achieved due to persistence of residual tumor cells and emphasizes the need of newer strategies.21

Autoclaved MIP is approved for treatment of leprosy patients undergoing chemotherapy.11 Currently, trials to study the efficacy of MIP are on going with respect to tuberculosis14 and several cancers. Therefore, it is important to study the mechanisms by which MIP functions as an immunomodulator. Here, the ability of MIP to enhance anti-tumor T cell responses that are dependent on a functional immune network, in particular T cells and endogenous Interferon-gamma (IFNγ), is shown.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Mice

BALB/c, C57BL/6 [wild-type (WT)] and Ifnγ−/− mice (BL6 background), weighing ∼18–25 g and ∼7 weeks old, were obtained from the Central Animal Facility, IISc. NOD.CB17-Prkdcscid/NCrCrl [nonobese diabetic severe combined immunodeficiency (NOD-SCID]22 male mice were obtained from ACTREC (Navi Mumbai, India) and were maintained under pathogen-free conditions. All animal procedures were performed in accordance with institutional animal care and use guidelines (http://www.cpcsea.in).

Reagents

Autoclaved MIP (Immuvac/Cadi-05) was obtained from Cadila Pharmaceuticals, Ahmedabad, India. Antibodies used for neutralizing or fluorescent activated cell sorter (FACS) were purchased from eBioscience. Murine recombinant IFNγ and TNFα were purchased from Peprotech.

Cell culture

Sp2/0, a myeloma cell line, was maintained in IMDM (Sigma) with 10% (v/v) heat-inactivated fetal bovine serum (fetal bovine serum (FBS); Invitrogen) and other supplements. EL4, a thymoma cell line, was maintained in RPMI supplemented with 10% (v/v) FBS. H6, a hepatoma cell line23 and mouse splenocytes were cultured in RPMI supplemented with 5% (v/v) FBS. Cells were cultured in a humidified atmosphere of 5% CO2 at 37°C in an incubator.

In vivo anti-tumor studies

Cohorts of BALB/c mice received s.c. injection of ∼107 Sp2/0 cells in the loose area around the neck and shoulder region.24 EL4 cells (5 × 105) were injected s.c. into cohorts of C57BL/6 WT and Ifnγ−/− mice. Mice were administered with Phosphate Buffered Saline (PBS) or MIP (∼5 × 108) usually 3 days after tumor inoculation via i.d. route in the ventral abdominal wall adjacent to inguinal lymph nodes. Tumor size was measured bidimensionally with calipers every 2 days, and tumor volume was calculated using the formula: (a × b2)/2, where a is the largest diameter and b is its perpendicular. In general, mice were euthanized after 14–17 days after tumor size increased (>∼5,000 mm3) and were surgically excised.

Cytokine assays

Sera samples were collected after termination of experiments. Blood was collected by cardiac puncture, allowed to clot at room temperature and centrifuged (∼10,000g for 10 min) to separate serum. Cytokine amounts in sera (diluted 1:5) were determined using Enzyme Linked Immunosorbent Assay (ELISA) (eBioscience). The linear range of detection for Interleukin (IL)12p70, IFNγ, IL6 and IL4 is 62.5–2,000 pg/mL, 62.5–2,000 pg/mL, 4–500 pg/mL and 15–500 pg/mL, respectively.

Spleen cell isolation and depletion of CD4+ and CD8+ T cells

Single-cell suspensions of splenocytes from different groups of mice were subjected to Histopaque 1083 (Sigma) density centrifugation. Cells at the interface were washed and different T cell subsets were depleted using monoclonal antibodies GK1.5 and Mar18.5 for CD4+ T cells and 3.155 for CD8+ T cells for 60–90 min followed by treatment with rabbit complement (1:40 dilution) for 30 min.25 The efficacy of depletion (>95%) of desired T cell population was tested by staining with noncross reactive anti-CD4 or anti-CD8 and confirmed by flow cytometry.

Cell proliferation assay

To measure cell mediated cytotoxicity, DNA synthesis was measured as an indication of cell proliferation, using bromodeoxyuridine (BrdU) ELISA kit (Calbiochem). Briefly, effector splenocytes (∼5 × 105) from control and treated mice were stimulated with tumor lysate for 5 days. Tumor cell targets (Sp2/0, EL4 and H6) were cultured with BrdU for 18 hr, washed and ∼5 × 103 were added to effectors followed by incubation for 12 hr. The plates were centrifuged and supernatants were discarded. Cells were incubated with 100 μl of fixative/denaturing solution for 30 min followed by incubation with a mouse monoclonal anti-BrdU for 1 hr. After washing the unbound antibody, horseradish peroxidase-conjugated goat anti-mouse was added and incubated for 30 min. After washing, the chromogenic substrate [3,3′,5,5′-tetramethylbenzidine (TMB)] was added and the intensity of the colored reaction product, which is proportional to the amount of BrdU incorporated into the cells, was read with a spectrophotometer at 450 nm. The percentage of BrdU incorporation was established by applying the formula, % proliferation = (experimental OD − background OD)/control OD × 100. Control OD refers to OD obtained for tumor cells after subtracting background (OD for unlabeled tumor cells) and was considered as 100% BrdU incorporation.

Tumor specific cytokine production

Sp2/0, EL4 or H6 cells were grown and lysed by five freeze/thaw cycles in liquid nitrogen and then in 37°C water-bath, centrifuged at ∼10,000g for 15 min, supernatant (lysate) was filtered and protein concentration was determined by Bradford assay. Mice were sacrificed and splenocytes (∼0.5 × 106) were restimulated in vitro with 50 μg/ml tumor lysate in 96-well flat-bottomed tissue culture plates for 60 hr. Cell-free supernatants were harvested at 60 hr and IL2, IL4 and IFNγ amounts were analyzed by ELISA (eBioscience).

Cell viability assays

Cell free supernatants obtained from cultures mentioned above were tested for the ability to specifically mediate target cell lysis by Trypan blue exclusion assay. Briefly, supernatants (5 or 25 μl) were added to ∼5 × 103 tumor cells and viable cells were counted after 48 hr of incubation. To determine which cytokines are involved, 10 μg/ml of neutralizing antibodies were added 1 hr before addition of supernatants.

Statistical analysis

Data were analyzed using commercial software (GraphPad Prism 5 Software). Unpaired Student's t-test was used to determine statistically significant differences between two groups.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

MIP treatment reduces in vivo solid tumor growth

To evaluate the role of MIP in tumor progression, BALB/c mice were injected i.d with MIP 1 day before or 3 or 6 days after Sp2/0 cell implantation (Fig. 1a). A significant reduction in subcutaneous tumor growth was noted in groups treated with MIP (−1 and +3 day) compared to PBS treated mice. However, injection of MIP 6 days after tumor inoculation did not reduce tumor growth (Figs. 1b and 1c). Significant abrogation of tumor growth was observed in mice treated with higher doses (5–50 × 107) of MIP (Supporting Information Fig. 1). Overall, these data demonstrates that MIP efficiently reduced tumor growth and the therapeutic treatment (+3 days) was most effective.

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Figure 1. MIP treatment suppresses tumor growth and induces a Th1 cytokine response. (a) General outline of the in vivo experimentation protocol performed. (b) Comparison of the anti-tumor effects of MIP administered at different time points. Cohorts of ten mice were inoculated s.c. with ∼107 Sp2/0 cells. Mice were injected i.d. with a single dose of MIP (∼5 × 108) either one day (−1D) before or 3 (+3D) or 6 (+6D) days after tumor inoculation. Mice injected i.d. with PBS on day 3 were included as controls. The growth of tumors (mean ± SD mm3) at indicated days postimplantation is shown. (c) Representative photographs of solid tumors from different treatment groups dissected on day 14 are shown. (d) Mice (n = 5) treated with PBS or MIP on day 3 were sacrificed 7 or 14 days postinoculation of Sp2/0 cells and serum amounts of cytokines were determined by ELISA. Significance between MIP and PBS treated groups are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

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MIP treatment reveals a Th1 preference

Cytokines are important mediators of the immune response and play significant roles in cancer progression and therapy.26 To evaluate whether MIP treatment modulated cytokine amounts during tumor progression, mice injected with MIP or PBS were sacrificed on days 7 and 14 after implantation of Sp2/0. Basal cytokine amounts in sera of nontumor mice injected with MIP or PBS were comparable to that of control uninjected mice (Fig. 1d). With tumor progression, the serum IL6 amounts increased from day 7 to 14 but MIP treatment led to significant reduction. Although IL4 amounts were lowered in some mice, there was no consistent and significant difference observed with MIP treatment (Fig. 1d, lower panel). Interestingly, the amounts of two T helper 1 (Th1)-type cytokines, IL12p70 and IFNγ, were significantly increased in the sera from mice injected with MIP compared with PBS treated mice (Fig. 1d, upper panel).

To determine whether MIP treatment of tumor bearing mice influences overall T cell activation, spleen cells were incubated with different concentrations of soluble anti-CD3. However, no difference in terms of 3H-Thymidine incorporation was observed in spleen cells from PBS and MIP mice (Supporting Information Fig. 2). Our results indicate that overall T cell activation was not altered by MIP.

MIP treatment increases CD8+ T cell mediated killing

Induction of tumor cell cytotoxicity during therapeutic treatment of cancers is known to play an important role.27 To investigate whether MIP treatment increases anti-tumor cytotoxic T lymphocytes (CTL), splenocytes from treated mice were used as effectors after stimulation with tumor lysates. The extent of drop in Sp2/0 cell proliferation was more with splenocytes from MIP-treated mice (Figs. 2a and 2b). Importantly, the proliferation of H6 targets was unaffected upon culture with splenocytes, thereby demonstrating tumor specificity (Fig. 2a and Supporting Information Fig. 3). Splenic T cell populations were depleted in vitro (Supporting Information Fig. 4) and depletion of CD8+ or CD4 plus CD8 T cells of MIP treated mice led to almost complete rescue in Sp2/0 cell proliferation (Fig. 2b). Together, these results indicate that CD8+ T cells are primarily involved in MIP mediated killing of tumor targets.

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Figure 2. Decrease in tumor targets in presence of CD8+ T cells is observed upon MIP treatment. (a) Approximately 0.5 × 106 splenocytes (effectors) from PBS and MIP treated mice (n = 3) as well as control nontumor injected mice were stimulated for 5 days with 50 μg/ml of tumor lysates followed by incubation with ∼5 × 103 BrdU-labeled tumor targets, either H6 (nonspecific) or Sp2/0 (specific). (b) Splenocytes were depleted of different T cell subsets and stimulated with indicated tumor lysates for 5 days and cell proliferation was determined, using labeled targets. Statistical analysis was performed using paired Student's t-test: *p < 0.05; **p < 0.01; ***p < 0.001 was considered to be significant when compared to tumor cells alone.

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Tumor antigen-specific cytokine secretion by CD4+ T cells

To investigate the ability of MIP to induce tumor specific recall responses, splenocytes were stimulated in vitro with tumor lysates and supernatants were collected for cytokine analysis. Splenocytes from MIP-treated mice produced higher amounts of IL2 and IFNγ, but no IL4, upon incubation with Sp2/0 lysate as compared with mice treated with PBS or control nontumor injected mice (Fig. 3a). Interestingly, IL2 and IFNγ amounts from MIP-treated mice with tumors were considerably downregulated with depletion of CD4+ T cells or both CD4+ and CD8+ T cells. On the other hand, these cytokines were enhanced upon depletion of CD8+ T cells, presumably due to increased numbers of CD4+ T cells (Fig. 3a, upper and middle panel). The induction of IL2 and IFNγ by CD4+ T cells was tumor-specific as shown with studies with H6 lysate. Together, our results demonstrate that CD4+ T cells from MIP treated tumor bearing mice produce high amounts of IFNγ and IL2.

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Figure 3. CD4+ T cells from MIP treated mice produce high amounts of IL-2 and IFNγ in a tumor-specific manner. (a) Two weeks after transplantation of Sp2/0, splenocytes were isolated and depleted of specific T cell populations from PBS or MIP injected mice (n = 8–10) as well control nontumor injected mice and stimulated in vitro with H6 (nonspecific) or Sp2/0 (specific) tumor lysates (50 μg/ml). Culture supernatants were collected after 60 hr and indicated cytokines were measured by ELISA. “Ctrl” refers to basal amounts of the respective cytokine in nontumor injected mice. Data are representative of three independent experiments. Significance between MIP and PBS groups is indicated as follows: *p < 0.05; ***p < 0.001. (b) CD4+ T cells play a major role in growth suppression of Sp2/0 cells. Undepleted splenocytes or depleted of CD4+ or/and CD8+ T cells, from PBS or MIP injected mice (n = 5), were stimulated with indicated tumor lysates (50 μg/ml) for 60 hr. Cell free supernatants were collected and incubated with ∼5 × 103 Sp2/0 cells and cultured for another 48 hr. Viability was determined by Trypan blue exclusion assay. (c) Effect of different neutralizing antibodies to cytokines on growth suppression. Sp2/0 cells were 1 hr before incubation with cell free supernatants treated with 10 μg/ml of isotype control or neutralizing antibodies to IFNγ or TNFα and viability was determined 48 hr later. Statistical analysis was performed using paired Student's t-test: **p < 0.01; ***p < 0.001. (d) Effect of recombinant IFNγ and TNFα on growth suppression of Sp2/0 cells. 5 × 103 Sp2/0 cells were treated with varying concentrations of recombinant IFNγ or TNFα and cultured in vitro for 48 hr. Significance between untreated and IFNγ or TNFα treated groups are indicated as follows: **p < 0.01; ***p < 0.001.

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MIP-induced IFNγ reduces Sp2/0 growth in vitro

Next, cell-free supernatants from stimulated splenocytes were harvested at 60 hr and incubated with Sp2/0. Supernatants from splenocytes from mice treated with MIP induced a considerable decrease in cell viability at 48 hr in comparison to PBS treated mice (Fig. 3b). Furthermore, depletion of CD4+ T cells reduced the growth suppressive effect observed with MIP. In contrast, depletion of CD8+ T cells, i.e., resulting in higher number of CD4+ T cells, led to further decrease in cell viability (Fig. 3b).

To identify the cytokine(s) involved in growth suppression, the effect of neutralizing antibodies to cytokines was studied. No effect was observed with an isotype control (anti-IgG2a) but significant reduction in cell growth suppression was observed with a neutralizing Ab against IFNγ but not against TNFα (Fig. 3c). Also, the growth of Sp2/0 cells was clearly inhibited with recombinant IFNγ in a dose-dependent manner compared to TNFα (Fig. 3d). These results revealed that IFNγ plays a major role in the suppression of Sp2/0 growth in vitro.

Involvement of IFNγ in MIP induced anti-tumor responses in vivo

The effect of MIP on an aggressive tumor, EL4 thymoma, which is relatively less sensitive to IFNγ in vitro was studied (Supporting Information Fig. 5). In addition, the role of IFNγ in the MIP induced host responses in vivo was evaluated using Ifnγ−/− mice. MIP treatment in WT mice decreased tumors compared to PBS treated mice whereas this difference was not observed in Ifnγ−/− mice (Fig. 4a). Interestingly, the significant increase in the serum IL12p70 and IFNγ amounts observed in MIP-treated WT were considerably reduced in Ifnγ−/− mice (Fig. 4b). On the other hand, IL6 amounts in PBS or MIP treated Ifnγ−/− mice were enhanced compared to WT mice and the extent of decline observed upon MIP treatment was impaired. These observations suggest a relevant role of IFNγ as a mediator of the immune response triggered by MIP.

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Figure 4. IFNγ plays an important role in MIP mediated reduction of tumors. (a) WT C57BL/6 and Ifnγ−/− mice (n = 10) were injected s.c. with ∼ 5 × 105 EL4 cells. Three days later, mice were injected i.d. with MIP (∼5 × 108) or PBS. The mean tumor volume in each group was estimated on indicated days. (b) Determination of serum cytokine amounts in EL4 tumor bearing WT and Ifnγ−/− mice (n = 5) after treatment with MIP. Cytokine amounts in sera collected 15 days postinoculation of EL4 cells were determined by ELISA. Control nontumor injected mice (n = 2) were included to depict the basal cytokine amounts in the sera. Significance between MIP and PBS groups is indicated as follows: *p < 0.05; ***p < 0.001.

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MIP-induced anti-tumor T cell responses are lower in Ifnγ−/− mice

Further studies on the anti-tumor T cell responses induced by MIP in Ifnγ−/− mice were performed. Splenocytes from MIP treated mice activated with EL4 lysate (specific) showed reduced proliferation of EL4 targets in MIP treated WT but not Ifnγ−/− mice (Fig. 5a). In addition, splenocytes from MIP-treated WT mice upon treatment with specific tumor lysate produced higher amounts of IL2 and IFNγ but this was not observed in Ifnγ−/− mice (Fig. 5b). These results demonstrate that MIP induced anti-tumor T cell responses were lower in mice lacking IFNγ.

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Figure 5. MIP induced IFNγ enhances anti-tumor T cell responses. (a) Splenocytes isolated from PBS or MIP injected and control nontumor injected mice (n = 3) were restimulated in vitro with 50 μg/ml H6 (nonspecific) or EL4 (specific) tumor lysate for 5 days followed by incubation with BrdU-labeled tumor cells for 12 hr. (b) Splenocytes were restimulated in vitro with indicated tumor lysates. Culture supernatants were collected 60 hr after activation and cytokine amounts were measured. Significance between MIP and PBS groups is indicated as follows: **p < 0.01; ***p < 0.001.

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Anti-tumor activities of MIP are reduced in NOD-SCID mice

To address the in vivo roles of a functional immune network in the anti-tumor responses mediated by MIP in both tumor models, experiments were performed in NOD-SCID mice. These mice possess greatly reduced numbers of T and B lymphocytes and lack functional NK cells.22 MIP treatment significantly reduced tumor growth in both tumors in WT mice. However, NOD-SCID mice injected with either PBS or MIP exhibited almost the same degree of tumor growth (Figs. 6a and 6b). Additionally, the serum IL12p70 and IFNγ amounts that were enhanced upon MIP treatment in WT mice bearing tumors were significantly reduced in NOD-SCID mice. Although increased IL6 amounts in sera were detected in NOD-SCID mice compared to BALB/c or C57BL/6 mice, there was no difference between PBS and MIP treated groups (Figs. 6c and 6d). These results show the crucial involvement of a functional immune cellular network in anti-tumor responses mediated by MIP.

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Figure 6. MIP mediated anti-tumor effects are abrogated in NOD-SCID mice. (a) BALB/c and NOD-SCID mice were injected s.c. with ∼107 Sp2/0 cells and (b) C57BL/6 and NOD-SCID mice were injected s.c. with ∼ 5 × 105 EL4 cells on day 0. MIP and PBS were injected on day 3 and tumor growth was monitored. Data are represented as mean ± SD of four mice per group. Indicated cytokine amounts in the sera of different treated groups of Sp2/0 (c) and EL4 (d) tumor-bearing mice (n = 3–4) were measured by ELISA. Significance between MIP and PBS groups is indicated as follows: **, p < 0.01; ***, p < 0.001. (e) A model for the mechanism of MIP mediated anti-tumor response. Treatment of tumor bearing mice with MIP results in high amounts of IL12 and IFNγ and increased anti-tumor immune responses are mediated by CD4+ and CD8+ T cells.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Several studies have shown the induction of Th1 responses during anti-tumor responses by mycobacterial strains.4, 28, 29 The present study is the first detailed analysis of anti-tumor T cell responses using multiple immunological assays upon i.d. injection of dead bacteria. Importantly, the efficacy was shown in two different tumors (Sp2/0 and EL4) and using two mutant mice (Ifnγ−/− and NOD-SCID). The major highlights are: First, MIP enhanced both anti-tumor CD4+ T helper and CD8+ CTL responses. Second, MIP modulated serum cytokine amounts of IL12, IFNγ and IL6, but not IL4 and TNFα, in tumor bearing mice (Supporting Information Fig. 6). Third, the use of NOD-SCID and Ifnγ−/− mice underscores the critical roles of a functional immune network and IFNγ in mediating the in vivo anti-tumor actions of MIP. Based on our results, a model has been proposed for the mechanisms of MIP mediated anti-tumor responses (Fig. 6e).

Adjuvant effects mediated by mycobacteria and their constituents are known.1 However, there may be differences in the mechanism of action by different mycobacterial species. Direct contact between “live” BCG and tumor cells is important for the anti-tumor efficacy of BCG.2 On the other hand, “dead” MIP was injected at a site distant from tumor inoculation. The epidermis contains numerous professional antigen presenting cells, which migrate into lymph nodes and activate T cells.30 Upon i.d. injection with BCG, there is massive infiltration of neutrophils which transport BCG to the draining lymph nodes.31 The site of administration of adjuvant is an important determining factor during anti-tumor responses.32 Anti-tumor studies with M. vaccae use the i.d. route for anti-tumor studies in patients with cancer.6, 7 Also, both mouse and patient trials studying the adjuvant efficacy of MIP have used the i.d. route of injection. MIP appears to boost overall anti-tumor responses and this aspect is important as tumors may metastasize to different organs. We have, therefore, used the i.d. route for MIP immunizations.

The anti-tumor efficacy of MIP was dependent on the time of injection and the best anti-tumor effect of MIP in both tumor models was observed 3 days after tumor injection (Fig. 1). Macrophages are known to migrate to tumor sites in response to chemoattractants secreted by the tumor, which is followed by migration of activated T cells.33 Possibly, MIP treatment 3 days after tumor inoculation may be the optimal time for activation of innate cells to process and present tumor antigens leading to the activation of tumor-specific T cells. It has been well documented that CD8+ CTLs eradicate the growth and metastasis of malignant tumor cells.34 CD8+ CTLs are generated upon MIP treatment and are cytotoxic to tumor targets (Fig. 2b). It is known that CD4+ and CD8+ T cells participate in the MIP-enhanced immune protection against M. tuberculosis.35, 36 Interestingly, significant amounts of IL2 and IFNγ were released by CD4+ T cells from mice treated with MIP upon stimulation with tumor lysates (Fig. 3a). We conclude that cooperation of both T cell populations is required for MIP-induced antigen-specific cytokine production as well as tumor cytotoxicity.

Modulation of cytokines is known to occur with tumor growth.26 It is important to point out that the modulation of serum cytokines upon MIP treatment occurred in mice with tumors (Fig. 1d). IL6 is important for the proliferation and survival of myelomas37 and MIP treatment reduced serum amounts of IL6 (Figs. 1d, 4b, 6c and 6d). The high amounts of serum IL12 and IFNγ induced by MIP during anti-tumor responses suggests that it is a pro-Th1 inducer (Fig. 1d). This aspect is important as IFNγ and IL12 are two principal cytokines that play crucial roles in anti-tumor immunity.38, 39 In addition, the amounts of IL12p70 were reduced to a considerable extent in Ifnγ−/− mice (Fig. 4b). Interestingly, both IL12 and IFNγ were not induced in NOD-SCID mice harboring tumors (Figs. 6c and 6d). These observations are consistent with reports that IL12 and IFNγ regulate each other during anti-tumor responses.38, 39

The anti-tumor activity of IFNγ has been shown against a number of experimental tumors.38–42 Although clear differences were observed with Sp2/0 (sensitive) and EL4 (more resistant) to IFNγ in vitro (Fig. 3d and Supporting Information Fig. 5), MIP treatment reduced solid tumor formation in vivo in both models. Studies with EL4 cells demonstrated that MIP lowered solid tumor formation was dependent on IFNγ (Fig. 4a). Accordingly, MIP-induced anti-tumor T cell responses, e.g., CD8+ T cell mediated cytotoxicity and antigen-specific cytokine production, were reduced in Ifnγ−/− mice (Figs. 5a and 5b). There may be several mechanisms by which IFNγ lowers anti-tumor responses in vivo: T cell mediated macrophage activation,34, 41 migration of tumor-sensitized T cells to tumor site,40 etc. Further studies are required to understand the detailed mechanisms by which MIP-induced IFNγ lowers tumor growth.

There are several notable features of the use of MIP for tumor therapy. First, the efficacy of MIP in reducing tumors in two distinct tumor models is important as it suggests that MIP may be effective against a wide range of tumors. This aspect is important as M. vaccae, together with chemotherapy, was shown to be ineffective against squamous carcinoma of the lung although efficacy was observed with adenocarcinoma.7 Second, the use of “dead” MIP in patients reduces the risk of bacterial proliferation and this aspect is relevant in case of immunocompromised patients as observed with “live” BCG vaccination.43 Third, the observation that the immunotherapeutic efficacy of MIP is optimum early after tumor inoculation suggests that it may be useful in patients detected early with cancer (Fig. 1). Fourth, the quality of life appears to improve in leprosy patients immunized with MIP.11 Also, treatment with M. vaccae has been shown to improve quality of life even in cases where no survival benefit was observed.5 It is possible that MIP treatment may improve the quality of life of patients with tumors. Fifth, the cost of production of this biological agent is low and this aspect is important in times when health care costs are under increasing scrutiny. Our findings are important as the clinical efficacy of MIP in treatment of different tumors is in progress. Further studies on the anti-tumor actions of MIP will shed mechanistic light on its action that may result in better efficacy, either alone or in combination with other drugs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We are grateful for the encouragement by Prof. G.P. Talwar in whose laboratory MIP was discovered. We appreciate the help and support by Profs. R. Dighe, A. Karande and R. Manjunath and thank Dr. B. Khamar for the generous gift of autoclaved MIP and discussions. In addition, we thank Dr. Ramachandra and Ms. Rosa, Central Animal Facility-IISc and Dr. Ingle, ACTREC (NOD-SCID) for providing mice. The support by the FACS facility-IISc and members of the DpN laboratory deserve a special mention.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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