Sergei Kusmartsev

Antigen-Specific Inhibition of CD8+ T Cell Response by Immature Myeloid Cells in Cancer Is Mediated by Reactive Oxygen Species

Tumor growth is associated with the accumulation of immature myeloid cells (ImC), which in mice are characterized by the expression of Gr-1 and CD11b markers. These cells suppress Ag-specific CD8+ T cells via direct cell-cell contact. However, the mechanism of immunosuppressive activity of tumor-derived ImC remains unclear. In this study we analyzed the function of ImC isolated from tumor-free control and tumor-bearing mice. Only ImC isolated from tumor-bearing mice, not those from their control counterparts, were able to inhibit the Ag-specific response of CD8+ T cells. ImC obtained from tumor-bearing mice had significantly higher levels of reactive oxygen species (ROS) than ImC isolated from tumor-free animals. Accumulation of H2O2, but not superoxide or NO, was a major contributor to this increased pool of ROS. It appears that arginase activity played an important role in H2O2 accumulation in these cells. Inhibition of ROS in ImC completely abrogated the inhibitory effect of these cells on T cells, indicating that ImC generated in tumor-bearing hosts suppress the CD8+ T cell response via production of ROS. Interaction of ImC with Ag-specific T cells in the presence of specific Ags resulted in a significant increase in ROS production compared with control Ags. That increase was independent of IFN-γ production by T cells, but was mediated by integrins CD11b, CD18, and CD29. Blocking of these integrins with specific Abs abrogated ROS production and ImC-mediated suppression of CD8+ T cell responses. This study demonstrates a new mechanism of Ag-specific T cell inhibition mediated by ROS produced by ImCs in cancer.

STAT1 Signaling Regulates Tumor-Associated Macrophage-Mediated T Cell Deletion

It is well established that tumor progression is associated with the accumulation of myeloid suppressive cells, which in mice include Gr-1+ immature myeloid cells and F4/80+ macrophages. The paradox is that with the exception of terminal stages of the disease or chemotherapy treatment, tumor-bearing mice or cancer patients do not display a profound systemic immune suppression. We therefore raised the question as to whether myeloid cell-mediated T cell suppression is controlled at a local level at the site of the tumor. We have demonstrated that after adoptive transfer to tumor-bearing recipients, Gr-1+ (immature myeloid cells) freshly isolated from spleens of tumor-bearing mice become F4/80+ tumor-associated macrophages (TAM). These TAM, but not F4/80+ macrophages or Gr-1+ cells freshly isolated from spleens of tumor-bearing or naive mice were able to inhibit T cell-mediated immune response in vitro via induction of T cell apoptosis. Arginase and NO were both responsible for the apoptotic mechanism, and were seen only in TAM, but not in freshly isolated Gr1+ cells. Using the analysis of STAT activity in combination with STAT knockout mice, we have determined that STAT1, but not STAT3 or STAT6, was responsible for TAM-suppressive activity.

Hyperactivation of STAT3 Is Involved in Abnormal Differentiation of Dendritic Cells in Cancer

Abnormal differentiation of myeloid cells is one of the hallmarks of cancer. However, the molecular mechanisms of this process remain elusive. In this study, we investigated the effect of tumor-derived factors on Janus kinase (Jak)/STAT signaling in myeloid cells during their differentiation into dendritic cells. Tumor cell conditioned medium induced activation of Jak2 and STAT3, which was associated with an accumulation of immature myeloid cells. Jak2/STAT3 activity was localized primarily in these myeloid cells, which prevented the differentiation of immature myeloid cells into mature dendritic cells. This differentiation was restored after removal of tumor-derived factors. Inhibition of STAT3 abrogated the negative effects of these factors on myeloid cell differentiation, and overexpression of STAT3 reproduced the effects of tumor-derived factors. Thus, this is a first demonstration that tumor-derived factors may affect myeloid cell differentiation in cancer via constitutive activation of Jak2/STAT3.

Role Of Immature Myeloid Cells in Mechanisms of Immune Evasion In Cancer

Tumor affects myelopoiesis by inhibiting the process of differentiation/maturation of antigen-presenting cells from their myeloid precursors and by stimulating an accumulation of immature myeloid cells in cancer patients and tumor-bearing mice. These immature myeloid cells can contribute greatly to tumor progression and promote tumor evasion from immune attack: i) by inhibiting development of adaptive immune responses against tumor in lymphoid organs; ii) by migrating into tumor site and differentiating there into highly immune suppressive tumor-associated macrophages. Immature myeloid cells and tumor-associated macrophages utilize different JAK/STAT signaling pathways and different mechanisms to control T cell responses, which include increased production of TGF-beta, reactive oxygen species, peroxynitrites, as well as enhanced L-arginine metabolism. Understanding of precise mechanisms, which tumors use to affect differentiation of APC from myeloid cell precursors and inhibit T cell responses, could help to develop new approaches for cancer therapy and substantially improve efficiency of existing cancer vaccination strategies.

Immature myeloid cells and cancer-associated immune suppression.

Abstract. Impaired balance between mature and immature myeloid cells is one of the hallmarks of cancer. In cancer patients as well as in mouse models there is increasing evidence that progressive tumor growth is associated with an accumulation of immature myeloid cells, monocytes/macrophages, and with a decreased number and function of dendritic cells (DC). This review examines recent findings on the contribution of immature myeloid cells (ImC) to cancer-induced immune suppression.

Tumor-Associated CD8+ T Cell Tolerance Induced by Bone Marrow-Derived Immature Myeloid Cells

T cell tolerance is a critical element of tumor escape. However, the mechanism of tumor-associated T cell tolerance remains unresolved. Using an experimental system utilizing the adoptive transfer of transgenic T cells into naive recipients, we found that the population of Gr-1+ immature myeloid cells (ImC) from tumor-bearing mice was able to induce CD8+ T cell tolerance. These ImC accumulate in large numbers in spleens, lymph nodes, and tumor tissues of tumor-bearing mice and are comprised of precursors of myeloid cells. Neither ImC from control mice nor progeny of tumor-derived ImC, including tumor-derived CD11c+ dendritic cells, were able to render T cells nonresponsive. ImC are able to take up soluble protein in vivo, process it, and present antigenic epitopes on their surface and induce Ag-specific T cell anergy. Thus, this is a first demonstration that in tumor-bearing mice CD8+ T cell tolerance is induced primarily by ImC that may have direct implications for cancer immunotherapy.

Inhibition of myeloid cell differentiation in cancer: the role of reactive oxygen species

It is well established that tumor growth is associated with accumulation of immature myeloid cells (ImC). They play an important role in tumor‐associated immune suppression. ImC accumulate not only in tumor‐bearing hosts but also in immunized, tumor‐free hosts or hosts infected with bacterial pathogens. The kinetics of ImC in these mice is different. If in tumor‐bearing mice, the number of ImC continues to increase with tumor progression in tumor‐free mice after an initial spike, it decreases to the control level. Here, we have investigated the mechanisms of ImC accumulation in tumor‐bearing hosts by comparing differentiation of ImC obtained from tumor‐free and tumor‐bearing mice. In the presence of appropriate growth factors, ImC isolated from tumor‐free mice quickly differentiated in vitro into mature dendritic cells (DC), macrophages, and granulocytes. In contrast, differentiation of ImC from tumor‐bearing mice was significantly delayed. Similar results were obtained in vivo after adoptive transfer of ImC into naïve, congeneic mice. ImC transferred into tumor‐bearing recipients failed to differentiate into DC or macrophages. ImC from tumor‐bearing mice had significantly higher levels of reactive oxygen species (ROS) than ImC obtained from tumor‐free mice. Hydrogen peroxide (H2O2) but not superoxide radical anions was found to be the major part of this increased ROS production. In vitro experiments demonstrated that scavenging of H2O2 with catalase induced differentiation of ImC from tumor‐bearing mice into macrophages. Thus, this is a first demonstration that tumors may prevent differentiation of antigen‐presenting cells by increasing the level of endogenous H2O2 in immature myeloid cells.

Effect of tumor-derived cytokines and growth factors on differentiation and immune suppressive features of myeloid cells in cancer

It is well established that cancers affect differentiation of dendritic cells and promote systemic expansion of immune suppressive immature myeloid cells. This phenomenon may represent a mechanism of tumor escape from immune attack and could have significant impact on tumor progression. In this review we discuss the role of different tumor-derived factors, which were implicated in abnormal myeloid cell differentiation. The role of reactive oxygen species as well as JAK/STAT signaling in mechanisms of the effects of tumor-derived factors on myeloid cells is also discussed.