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ABSTRACT: Myeloid-derived Suppressor Cells (MDSC) have been identified as tumor-induced immature myeloid cells (IMC) with potent immune suppressive activity in cancer. Whereas strict phenotypic classification of MDSC has been challenging due to the highly heterogeneous nature of cell surface marker expression, use of functional markers such as Arginase and inducible nitric oxide synthase (iNOS) may represent a better categorization strategy. In this study we investigated whether iNOS could be utilized as a specific marker for the identification of a more informative homogenous MDSC subset.
Single-cell suspensions from tumors and other organs were prepared essentially by enzymatic digestion. Flow cytometric analysis was performed on a four-color flow cytometer. Morphology, intracellular structure and localization of iNOS+ ring cells in the tumor were determined by cytospin analysis, immunofluorescence microscopy and immunohistochemistry, respectively. For functional analysis, iNOS+ ring subset were sorted and tested in vitro cell culture experiments. Pharmacologic inhibition of iNOS was performed both in vivo and in vitro.
The results showed that intracellular iNOS staining distinguished a granular iNOS+ SSChi CD11b+ Gr-1dim F4/80+ subset with ring-shaped nuclei (ring cells) among the CD11b+ Gr-1+ cell populations found in tumors. The intensity of the ring cell infiltrate correlated with tumor size and these cells constituted the second major tumor-infiltrating leukocyte subset found in established tumors. Although phenotypic analysis demonstrated that ring cells shared characteristics with tumor-associated macrophages (TAM), morphological analysis revealed a neutrophil-like appearance as detected by cytospin and immunofluorescence microscopy analysis. The presence of distinct iNOS filled granule-like structures located next to the cell membrane suggested that iNOS was stored in pre-formed vesicles and available for rapid release upon activation. Tumor biopsies showed large areas with infiltrating ring cells primarily surrounding necrotic areas. Importantly, these cells significantly impaired CD8+ T-cell proliferation and induced apoptotic death. The intratumoral accumulation and suppressive activity of ring cells could be blocked through pharmacologic inhibition of iNOS, demonstrating the critical role of this enzyme in mediating both the differentiation and the activity of these cells.
In this study, iNOS expression was linked to a homogeneous subset; ring cells with a particular phenotype and immune suppressive function, in a common and well-established murine tumor model; 4T-1. Since the absence of a Gr-1 homolog in humans has made the identification of MDSC much more challenging, use of iNOS as a functional marker of MDSC may also have clinical importance.
Journal of Translational Medicine 07/2012; 10:152. · 3.41 Impact Factor
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ABSTRACT: Sustained intratumoral delivery of IL-12 and GM-CSF can overcome tumor immune suppression and promote T cell-dependent eradication of established disease in murine tumor models. However, the antitumor effector response is transient and rapidly followed by a T suppressor cell rebound. The mechanisms that control the switch from an effector to a regulatory response in this model have not been defined. Because dendritic cells (DC) can mediate both effector and suppressor T cell priming, DC activity was monitored in the tumors and the tumor-draining lymph nodes (TDLN) of IL-12/GM-CSF-treated mice. The studies demonstrated that therapy promoted the recruitment of immunogenic DC (iDC) to tumors with subsequent migration to the TDLN within 24-48 h of treatment. Longer-term monitoring revealed that iDC converted to an IDO-positive tolerogenic phenotype in the TDLN between days 2 and 7. Specifically, day 7 DC lost the ability to prime CD8(+) T cells but preferentially induced CD4(+)Foxp3(+) T cells. The functional switch was reversible, as inhibition of IDO with 1-methyl tryptophan restored immunogenic function to tolerogenic DC. All posttherapy immunological activity was strictly associated with conventional myeloid DC, and no functional changes were observed in the plasmacytoid DC subset throughout treatment. Importantly, the initial recruitment and activation of iDC as well as the subsequent switch to tolerogenic activity were both driven by IFN-γ, revealing the dichotomous role of this cytokine in regulating IL-12-mediated antitumor T cell immunity.
The Journal of Immunology 07/2011; 187(1):126-32. · 5.79 Impact Factor
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ABSTRACT: Interleukin-12 (IL-12) can promote tumor regression via activation of multiple lymphocytic and myelocytic effectors. Whereas the cytotoxic mechanisms employed by T/NK/NKT cells in IL-12-mediated tumor kill are well defined, the antitumor role of macrophage-produced cytotoxic metabolites has been more controversial. To this end, we investigated the specific role of nitric oxide (NO), a major macrophage effector molecule, in post-IL-12 tumor regression. Analysis of tumors following a single intratumoral injection of slow-release IL-12 microspheres showed an IFNγ-dependent sevenfold increase in inducible nitric oxide synthase (iNOS) expression within 48 h. Flow cytometric analysis of tumor-resident leukocytes and in vivo depletion studies identified CD11b(+) F4/80(+) Gr1(lo) macrophages as the primary source of iNOS. Blocking of post-therapy iNOS activity with N-nitro-L: -arginine methyl ester (L-NAME) dramatically enhanced tumor suppression revealing the inhibitory effect of NO on IL-12-driven antitumor immunity. Superior tumor regression in mice receiving combination treatment was associated with enhanced survival and proliferation of activated tumor-resident CD8+ T-effector/memory cells (Tem). These findings demonstrate that macrophage-produced NO negatively regulates the antitumor activity of IL-12 via its detrimental effects on CD8+ T cells and identify L-NAME as a potent adjuvant in IL-12 therapy of cancer.
Cancer Immunology and Immunotherapy 03/2011; 60(6):839-45. · 3.70 Impact Factor
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ABSTRACT: Tumor-resident CD8(+) T cells display a quiescent effector/memory phenotype that is maintained in part by infiltrating CD4(+) CD25(+) Foxp3(+) T-suppressor cells. Intratumoral delivery of IL-12, in contrast, can restore cytotoxic function to tumor-associated CD8(+) T cells and induce the apoptotic death of T-suppressor cells. Depletion of CD8(+) T cells from tumors before IL-12 treatment resulted in the abrogation of treatment-mediated T-suppressor cell apoptosis revealing a link between CD8(+) T cell activation and T-suppressor elimination. Furthermore, IL-12 failed to induce T-suppressor cell loss in IFN-gamma- or FasL-deficient mice demonstrating a requirement for IFN-gamma and FasL in this process. Adoptive transfer of wild-type CD8(+) T cells to FasL-knockout mice restored posttherapy T-suppressor cell elimination from tumors establishing that expression of FasL on CD8(+) T cells was sufficient to promote T-suppressor cell death. IL-12 failed to induce FasL on T-effectors in IFN-gamma-knockout mice demonstrating a requirement for IFN-gamma in FasL up-regulation. Adoptive transfer of wild-type CD8(+) T cells induced T-suppressor cell death in IFN-gamma-knockout mice confirming that autocrine IFN-gamma was sufficient for CD8(+) T cell FasL expression. These findings reveal a mechanism by which cytotoxic T cells can abrogate regulatory cell activity.
The Journal of Immunology 11/2009; 183(12):7656-60. · 5.79 Impact Factor
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ABSTRACT: Sustained delivery of IL-12 and GM-CSF to tumors induces the activation of tumor-resident CD8(+) T effector/memory cells (Tem) followed by cytotoxic CD8(+) T effector cell expansion. To determine whether the secondary effectors expanded from tumor-associated Tem or were primed de novo, activation kinetics of tumor-draining lymph node (TDLN) CD8(+) T cells were analyzed. Treatment promoted a 4-fold increase in the numbers of TDLN CD8(+) T cells displaying a CD69(+)CCR5(+)CD62L(-) periphery-homing effector phenotype by day 4 posttherapy. Pulse labeling of tumor and TDLN T cells with BrdU confirmed that proliferation occurred exclusively within the draining lymph nodes between days 1 and 4 with subsequent migration of primed CD8(+) T effectors to tumors on day 7. Day 4 CD8(+) T effector cells preferentially homed to and lysed experimental, but not control, tumors, establishing tumor specificity. To determine whether the secondary CD8(+) T effector cell response was dependent on activation of tumor-resident CD8(+) Tem, mice that were selectively depleted of tumor-infiltrating CD8(+) T cells were treated and monitored for T effector priming. In the absence of tumor-resident CD8(+) Tem, T effector cell expansion was completely abrogated in the TDLN, revealing that restoration of CD8(+) Tem function was critical to the induction of secondary T effectors. T cell priming failed to occur in IFN-gamma or perforin knockout mice, demonstrating that the requirement for Tem activation was associated with induction of Tem cytotoxicity. These data confirm that intratumoral IL-12 plus GM-CSF induces de novo priming of tumor-specific CD8(+) T effector cells in the TDLN and establish the critical role of preexisting intratumoral CD8(+) Tem in driving this process.
The Journal of Immunology 05/2009; 182(7):4217-25. · 5.79 Impact Factor
