Mechanisms of Rejection of High Grade B cell Lymphoma in Mice

The incidence of high grade B cell lymphoma in western countries has increased over the last decades. Improvement of conventional chemotherapy regimens has significantly contributed to prolonged 5-year survival rates which currently reach around 60%. However, relapse after conventional chemotherapy is an important challenge, especially in high grade B cell lymphomas. The potential benefit of immunological approaches for the elimination of such lymphomas still remains unclear. In this study, we attempted to address whether the forced expression of foreign antigens in a tumor of B cell origin leads to immune recognition and elimination of the tumor and to assess the potential role of IFN-gamma (IFN-g) in tumor rejection. To this end, we used a transgenic mouse lymphoma model, where the human proto-oncogene c-myc (a foreign antigen for the mouse host) is under the control of regulatory elements of the immunoglobulin lambda locus, thereby recapitulating the important features of a t(8;22) translocation as found in human Burkitt’s lymphoma. From these spontaneously developing tumors, lymphoma cell lines were established that either express (line 291) or are deficient (line 9) in Stat1- a key signaling molecule in the response to interferons. We found that the expression of foreign antigens such as chicken ovalbumin (OVA) and green fluorescent protein (GFP) in Stat1-competent 291 cells led to immune responses that delayed tumor progression and improved survival of wild-type animals. Consistent with this, loss of foreign antigen inevitably led to accelerated tumor progression upon transfer into immunocompetent wild-type mice. Transfer of immunogenic 291-OVA-GFP lymphoma cells led to increased tumor progression without loss of foreign antigen upon transfer into IFN-γ-/- and Stat1-/- mice indicating that no selection of antigen loss-variants occurred in these mice. The rejection of 291-OVA-GFP cells in wild-type mice was at least in part mediated by CD8+ T cells as measured by enrichment of the OVA antigen-derived MHC class I-restricted SIINFEKL epitope-specific cells in wild-type recipients.. Interestingly, Stat1-deficient lymphoma cells (9-GFP and 9-OVA-GFP) were rejected by immunocompetent UBQ-GFP transgenic wild-type C57BL/6 mice irrespectively of the presence of a foreign antigen, indicating the existence of immunosurveillance against these Stat1-deficient lymphomas. To evaluate the key players behind lymphoma rejection, we transferred 9-GFP cells into IFN-γ-/- and Stat1-/- recipients. This led to enhanced tumor growth indicating that endogenous IFN-γ production and Stat1 signaling are critical for tumor rejection. To gain an insight into the mechanistic aspects of innate immunosurveillance against the Stat1-competent and Stat1-deficient lymphomas, NK cell functionality was evaluated. We found that NK cells could efficiently lyse both Stat1-competent and Stat1-deficient lymphoma cell lines in vitro. Treatment with IFN-γ increased the susceptibility of Stat1-deficient lymphoma cells to NK cell killing, but decreased that of Stat1-competent cells, presumably by upregulating MHC class I expression. The results of this work show that host IFN-γ and Stat1 signaling are important for tumor clearance, and that paradoxically, the absence of Stat1 within the lymphoma is required for rejection

Mechanisms of Rejection of High Grade B cell Lymphoma in Mice

The incidence of high grade B cell lymphoma in western countries has increased over the last decades. Improvement of conventional chemotherapy regimens has significantly contributed to prolonged 5-year survival rates which currently reach around 60%. However, relapse after conventional chemotherapy is an important challenge, especially in high grade B cell lymphomas. The potential benefit of immunological approaches for the elimination of such lymphomas still remains unclear. In this study, we attempted to address whether the forced expression of foreign antigens in a tumor of B cell origin leads to immune recognition and elimination of the tumor and to assess the potential role of IFN-gamma (IFN-g) in tumor rejection. To this end, we used a transgenic mouse lymphoma model, where the human proto-oncogene c-myc (a foreign antigen for the mouse host) is under the control of regulatory elements of the immunoglobulin lambda locus, thereby recapitulating the important features of a t(8;22) translocation as found in human Burkitt’s lymphoma. From these spontaneously developing tumors, lymphoma cell lines were established that either express (line 291) or are deficient (line 9) in Stat1- a key signaling molecule in the response to interferons. We found that the expression of foreign antigens such as chicken ovalbumin (OVA) and green fluorescent protein (GFP) in Stat1-competent 291 cells led to immune responses that delayed tumor progression and improved survival of wild-type animals. Consistent with this, loss of foreign antigen inevitably led to accelerated tumor progression upon transfer into immunocompetent wild-type mice. Transfer of immunogenic 291-OVA-GFP lymphoma cells led to increased tumor progression without loss of foreign antigen upon transfer into IFN-γ-/- and Stat1-/- mice indicating that no selection of antigen loss-variants occurred in these mice. The rejection of 291-OVA-GFP cells in wild-type mice was at least in part mediated by CD8+ T cells as measured by enrichment of the OVA antigen-derived MHC class I-restricted SIINFEKL epitope-specific cells in wild-type recipients.. Interestingly, Stat1-deficient lymphoma cells (9-GFP and 9-OVA-GFP) were rejected by immunocompetent UBQ-GFP transgenic wild-type C57BL/6 mice irrespectively of the presence of a foreign antigen, indicating the existence of immunosurveillance against these Stat1-deficient lymphomas. To evaluate the key players behind lymphoma rejection, we transferred 9-GFP cells into IFN-γ-/- and Stat1-/- recipients. This led to enhanced tumor growth indicating that endogenous IFN-γ production and Stat1 signaling are critical for tumor rejection. To gain an insight into the mechanistic aspects of innate immunosurveillance against the Stat1-competent and Stat1-deficient lymphomas, NK cell functionality was evaluated. We found that NK cells could efficiently lyse both Stat1-competent and Stat1-deficient lymphoma cell lines in vitro. Treatment with IFN-γ increased the susceptibility of Stat1-deficient lymphoma cells to NK cell killing, but decreased that of Stat1-competent cells, presumably by upregulating MHC class I expression. The results of this work show that host IFN-γ and Stat1 signaling are important for tumor clearance, and that paradoxically, the absence of Stat1 within the lymphoma is required for rejection

miR-155 augments CD8(+) T-cell antitumor activity in lymphoreplete hosts by enhancing responsiveness to homeostatic gamma(c) cytokines

Lymphodepleting regimens are used before adoptive immunotherapy to augment the antitumor efficacy of transferred T cells by removing endogenous homeostatic "cytokine sinks." These conditioning modalities, however, are often associated with severe toxicities. We found that microRNA-155 (miR-155) enabled tumor-specific CD8(+) T cells to mediate profound antitumor responses in lymphoreplete hosts that were not potentiated by immune-ablation. miR-155 enhanced T-cell responsiveness to limited amounts of homeostatic gamma c cytokines, resulting in delayed cellular contraction and sustained cytokine production. miR-155 restrained the expression of the inositol 5-phosphatase Ship1, an inhibitor of the serine-threonine protein kinase Akt, and multiple negative regulators of signal transducer and activator of transcription 5 (Stat5), including suppressor of cytokine signaling 1 (Socs1) and the protein tyrosine phosphatase Ptpn2. Expression of constitutively active Stat5a recapitulated the survival advantages conferred by miR-155, whereas constitutive Akt activation promoted sustained effector functions. Our results indicate that overexpression of miR-155 in tumor-specific T cells can be used to increase the effectiveness of adoptive immunotherapies in a cell-intrinsic manner without the need for life-threatening, lymphodepleting maneuvers.112922Ysciescopu

BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers.

T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs

Stromal Interferon-γ Signaling and Cross-Presentation Are Required to Eliminate Antigen-Loss Variants of B Cell Lymphomas in Mice

To study mechanisms of T cell-mediated rejection of B cell lymphomas, we developed a murine lymphoma model wherein three potential rejection antigens, human c-MYC, chicken ovalbumin (OVA), and GFP are expressed. After transfer into wild-type mice 60–70% of systemically growing lymphomas expressing all three antigens were rejected; lymphomas expressing only human c-MYC protein were not rejected. OVA expressing lymphomas were infiltrated by T cells, showed MHC class I and II upregulation, and lost antigen expression, indicating immune escape. In contrast to wild-type recipients, 80–100% of STAT1-, IFN-γ-, or IFN-γ receptor-deficient recipients died of lymphoma, indicating that host IFN-γ signaling is critical for rejection. Lymphomas arising in IFN-γ- and IFN-γ-receptor-deficient mice had invariably lost antigen expression, suggesting that poor overall survival of these recipients was due to inefficient elimination of antigen-negative lymphoma variants. Antigen-dependent eradication of lymphoma cells in wild-type animals was dependent on cross-presentation of antigen by cells of the tumor stroma. These findings provide first evidence for an important role of the tumor stroma in T cell-mediated control of hematologic neoplasias and highlight the importance of incorporating stroma-targeting strategies into future immunotherapeutic approaches

Retinoic acid controls the homeostasis of pre-cDC–derived splenic and intestinal dendritic cells

Dendritic cells (DCs) comprise distinct populations with specialized immune-regulatory functions. However, the environmental factors that determine the differentiation of these subsets remain poorly defined. Here, we report that retinoic acid (RA), a vitamin A derivative, controls the homeostasis of pre-DC (precursor of DC)–derived splenic CD11b(+)CD8α(−)Esam(high) DCs and the developmentally related CD11b(+)CD103(+) subset within the gut. Whereas mice deprived of RA signaling significantly lost both of these populations, neither pre-DC–derived CD11b(−)CD8α(+) and CD11b(−)CD103(+) nor monocyte-derived CD11b(+)CD8α(−)Esam(low) or CD11b(+)CD103(−) DC populations were deficient. In fate-tracking experiments, transfer of pre-DCs into RA-supplemented hosts resulted in near complete conversion of these cells into the CD11b(+)CD8α(−) subset, whereas transfer into vitamin A–deficient (VAD) hosts caused diversion to the CD11b(−)CD8α(+) lineage. As vitamin A is an essential nutrient, we evaluated retinoid levels in mice and humans after radiation-induced mucosal injury and found this conditioning led to an acute VAD state. Consequently, radiation led to a selective loss of both RA-dependent DC subsets and impaired class II–restricted auto and antitumor immunity that could be rescued by supplemental RA. These findings establish a critical role for RA in regulating the homeostasis of pre-DC–derived DC subsets and have implications for the management of patients with immune deficiencies resulting from malnutrition and irradiation

Host IFN-γ and host IFN-γ signaling are required for rejection of 291OVA cells.

<p>1×10⁵ 291 parental cells (291PC) and retrovirally transduced 291OVA cells were injected s.c. into IFN-γ-deficient (A) and into IFN-γ-receptor- and STAT1-deficient recipient mice (B). Survival (left panels) and corresponding cumulative tumor growth (right panels) were monitored over 100 days. The data are compiled from two independent experiments. (C) Lymphomas developing in IFN-γ-receptor- and STAT1-deficient mice after inoculation of 1×10⁵ 291 parental or 291OVA cells were analyzed by immunohistochemistry for infiltration of CD3-positive cells (peroxidase brown staining) and perforin expressing cells (alkaline phosphatase staining) (left panel) in the same fashion as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034552#pone-0034552-g002" target="_blank">Figure 2C</a>. 10 animals per group were analyzed and Mann Whitney test was used for comparison.</p