With the existing therapeutic efforts, the long term survival for lung cancer patients remains low, thus novel therapeutic strategies are needed. Cancer immunotherapy offers an attractive therapeutic option. The concept of immune approaches against lung cancer remains attractive because although surgery, chemotherapy, and radiotherapy alone or in combination produce response rates, relapse is unavoidable. Thus, strategies that harness the immune system against tumors have the potential for long term cancer free survival. Immune therapy for lung cancer has potential; however, there have been limited Phase III trial-documented improvement in survival 
. Tumor-induced immune suppression may contribute to the limited efficacy of the approaches. Many tumors, including lung cancer, have the capacity to promote immune tolerance and escape host immune surveillance. Tumors utilize numerous pathways to inhibit immune responses, including the elaboration of immune inhibitory cytokines as well as inducing host cells to release immune inhibitors 
. In addition to these mechanisms, immune suppression through MDSC has a crucial role in promoting tumor progression. We hypothesized that activating immune cells through therapeutic vaccination with simultaneous disruption of MDSC mediated regulatory mechanisms that limit immune responses will improve the antitumor activity in lung cancer. To test our hypothesis, we evaluated the efficacy of a combined immune based approach consisting of a therapeutic vaccine and MDSC depletion on the impact of immune activation and antitumor activity in an established murine tumor model.
To initiate antigen specific responses in vivo
, we utilized a cellular vaccine, consisting of BMA cells pulsed with the model OVA antigen expressed by the OVA modified 3LL tumor cells. The BMA cells were pulsed with the OVA antigen to allow for antigen processing and presentation and then injected s.c.
on the contralateral flank of the established tumor to initiate antigen specific anti tumor immune responses. The BMA cells expressed cell surface phenotype for APC activity and efficiently processed and presented antigens to antigen specific CD8 T cells in vitro
as well as induced antitumor responses in vivo
. In contrast, although lung cancer cells express tumor antigens, the limited expression of MHC antigens, defective transporter associated with antigen processing (TAP) and lack of co-stimulatory molecules make them ineffective APCs 
To circumvent MDSC mediated immune suppression in the tumor microenvironment, we utilized monoclonal antibodies (anti-Gr1 or anti-Ly6G) to deplete MDSC. We have found that as tumors progress, the frequency and activity of MDSC are enhanced in the tumor microenvironment and systemically. Our results show about 40% of tumor infiltrates are MDSC that have the capacity to turn off the functional antitumor activities of APC, NK and T cell effectors. The tumor cell inoculations in this study did not include matrigel mixed with tumor cells but rather tumor cells in saline. Hence the increase in MDSC in the tumor microenvironment and systemically is due to progressive tumor growth. However injection of matrigel formulations that release MDSC chemotactic factors also have the potential to recruit MDSC. The inadequate function of the host immune system through the down regulation of APC, NK and T cell effectors as well as the elaboration of effector molecules is one of the major mechanisms of tumor immune escape. Similar to increases of MDSC in cancer patients, our data demonstrate that 3LL tumor bearing mice had increased frequency of MDSC in the tumor, blood, spleen and BM as a function of tumor growth. These MDSC functionally suppressed T cell proliferation and APC activities in vitro.
The central importance of functional APC in the immune response against cancer has been well defined 
. The study revealed that even highly immunogenic tumors require host APCs for antigen presentation. Thus, host APC, rather than tumor cells, present tumor antigens. This is consistent with a study indicating that CD8+ T-cell responses can be induced in vivo
by professional APC that present exogenous antigens in a MHC I-restricted manner 
. This has been referred to as cross-priming or representation and may be critical for effective antitumor responses 
. However, in tumor-bearing hosts, there is a state of T-cell unresponsiveness 
. T cell non responsiveness to specific antigens has been shown to be an early event in tumor progression in animal models of cancer and in cancer patients 
. The cellular mechanisms that lead to the induction of the tolerogenic state are not well understood. The dominant mechanism underlying the development of antigen-specific T-cell unresponsiveness is thought to be through tumor-antigen processing and presentation by APC 
. Although T-cell tolerance in cancer has been shown to be mediated by host APC, 
the nature of these APC have not been clear. Recent studies provide evidence that MDSC may represent a population of APC responsible for induction of antigen-specific CD8 T-cell tolerance in cancer 
. The tumor microenvironment has immune suppressive mediators such as PGE2, TGF-β, IL-10, VEGF, GM-CSF, IL-6, S100A8/A9 and SCF that recruit and/or activate MDSC 
. Solid tumors contain a significant proportion of MDSC that maintain an immune suppressive network in the tumor microenvironment.
The impact of MDSC depletion on the APC, T and NK effector activities was evaluated to determine if antitumor innate and adaptive T cell activities could be restored in lung cancer. Broad depletion of MDSC by antibodies that targeted the Gr1 or Ly6G led to decreased MDSC population in the tumor and systemically in the blood, spleen and BM. The APC activity in the tumor was reduced as the tumors progressed, however, MDSC depletion led to increased APC activity in the tumor. Consistent with an increase in APC activity with MDSC depletion, there were increased frequency and activity of NK and T cell effectors in the tumor in comparison to controls. The increased activity of NK cell effectors is significant since these innate immune cells are the first line of defense against tumors and inhibit tumor growth in a non-MHC restricted manner and without prior sensitization to an antigen 
. CTL and NK cells possess similar cytolytic mechanisms including secretion of perforin and granzyme. Our data indicates that the activated NK and T cell effectors had increased expression of the cytolytic markers granzyme and perforin as well as an increase in IFNγ but reduced IL-10. The cytokine signature of increased IFNγ and reduced IL-10 in the tumor further facilitates antitumor activity. While IFNγ induces anti-angiogenesis through the induction of CXCL9 and CXCL10 
, reduction in IL-10 can improve APC activity and promote Type I responses 
. This data indicates that while 3LL tumor growth increased MDSC and reduced APC, NK, and T cell activities, MDSC depletion released the brakes on the functional activities of APC, NK and T cell effectors. In accord with the increased NK and T cell activities, there was an increase in the frequency of apoptotic tumor cells and a concomitant inhibition in tumor burden and migration of tumor cells from the primary tumor site to the lung. The reduction in tumor growth and abrogation in tumor cell migration may be explained by an increase in the frequency of activated T and/or NK effector mediated tumor apoptosis and/or T or NK IFNγ mediated anti-angiogenesis. Based on our observations on increased T and NK cell IFNγ production in the tumor, we evaluated the angiogenic profiles in the tumor. The anti-angiogenic (CXCL9, CXCL10) and pro-angiogenic markers (VEGFa, CXCL2, CXCL5, Angiopoietin 1 and Angiopoietin 2) were quantified in the tumor by RTQPCR. Following MDSC depletion, the anti-angiogenic chemokines CXCL9 and CXCL10 were increased but the pro-angiogenic cytokines VEGFa, Angiopoietin1, Angiopoietin2, CXCL2 and CXCL5 were markedly reduced. Accompanying this profile was a reduction in the endothelial markers MECA 32 in the tumor but an increase in CXCR3 expression. This data indicates that following MDSC depletion there is an influx of IFNγ producing activated T and NK cells that promote angiostasis in the tumor microenvironment by altering the balance of pro and anti angiogenic chemokines. This further suggests that MDSC depletion not only improves APC, NK and T cell immune activities but promotes anti angiogenesis in the tumor that is more effective at controlling tumor growth.
Based on increases in the APC, NK and T cell activities following MDSC depletion, therapeutic vaccination responses were evaluated in vivo. In comparison to controls, therapeutic vaccination with BMA-OVA led to decreased tumor burden without complete eradication of the tumors. However following the depletion of MDSC with anti-Gr1 in combination with BMA-OVA vaccine, treated mice had the most substantial reduction in tumor burden with 50% of the mice completely eradicating the tumors and the remainder of mice with a 20 fold reduction in tumor burden in comparison to control. The data indicates that therapeutic vaccination is more effective when combined with MDSC depletion. Long term immunological memory was induced in these mice that were efficient in rejecting a secondary tumor challenge. Flow cytometric evaluation of spleens of mice that had rejected a secondary tumor rechallenge showed an enhancement in the CD4+CD44+ and CD8+CD44+ T cell memory markers as well as an enhanced secretion of IFNγ by memory T cells in response to OVA stimulation in vitro. Our findings demonstrate that to engage a coordinated and effective attack against tumors, multiple components of the immune system need to evolve in parallel that require mechanisms leading to immune cell activation with the coordinated disruption of the regulatory mechanisms that limit antitumor immune responses.
Taken together, our data indicate that MDSC depletion reprograms the tumor niche by altering the inflammatory infiltrates in the tumor microenvironment making it permissive for immune destruction of tumors. The benefit of MDSC depletion is further augmented when combined with therapeutic vaccination leading to tumor eradication and immunological memory. Targeting MDSC can improve antitumor immune responses suggesting a broad applicability of combined immune based approaches against a wide range of solid malignancies. This multifaceted approach may prove useful for cancer therapeutics against solid tumors where MDSC play a major role in tumor immune evasion. These results are encouraging and warrant further evaluation of combined MDSC targeting with vaccination approaches for the full therapeutic potential of this strategy in lung cancer and other malignancies.