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

Adoptive T cell transfer for therapy of highly malignant lymphomas in the c-myc transgenic mouse model

Zusammenfassung Überwiegend wird mit der Überexpression des Apoptoseinhibitors Bcl-2 oder dem Verlust des zentralen Tumorsuppressorproteins p53 bei Tumoren eine negative Prognose assoziiert, da sie eine erhöhte Aggressivität aufweisen und Resistenzen gegenüber Chemotherapie und Strahlentherapie besitzen. Nach allogener Stammzelltransplantation, deren Graft-versus-Tumor-Effekt auf der zytotoxischen Aktivität von T-Zellen beruht, ließ sich bei Chemotherapie- resistenten Lymphomen eine erhöhte Wahrscheinlichkeit für die Progression der Krankheit beobachten. Daraus ergab sich die Fragestellung, ob Chemotherapie- resistente Lymphome und durch Chemotherapie induzierte seneszente Lymphomzellen für T-Zelltherapie schlechter zugänglich sind. Mit Hilfe der λ -MYC-Maus wurden hochmaligne c-myc-getriebene Lymphomzelllinien mit bcl-2-Überexpression und p53-Defizienz hergestellt und mit dem Modellantigen Ovalbumin (OVA) versehen. Es konnte gezeigt werden, dass diese Zelllinien Chemotherapie-resistent sind und in vitro Seneszenz als zweite DNA- Schadensstrategie ausbilden. Sowohl Chemotherapie-resistente als auch seneszente Lymphomzellen wurden von OVA-spezifischen OT-1-T-Zellen lysiert. Mit der λ-OVA-Maus, die OVA als B-zellspezifisches Antigen exprimiert, wurde eine OVA-tolerante Maus generiert, die das Antigen in normalen B-Zellen, aber auch nach Transplantation von OVA-positiven Lymphomzellen in der Neoplasie selbst trägt. Nach Chemotherapie und adoptivem T-Zelltransfer zeigten Mäuse mit transplantierten bcl-2-überexprimierenden Lymphomen statt des erwarteten verkürzten progressionsfreien Überlebens eine deutlich verlängerte Remissionsdauer. In weniger ausgeprägter Form konnte diese Verbesserung auch bei ersten Versuchen mit p53-defizienten Tumoren beobachtet werden. Da Rezidive OVA-negativ vorlagen, war davon auszugehen, dass bcl-2-Überexpression die Wahrscheinlichkeit für die Selektion von Antigenverlustvarianten nach Chemotherapie verringerte. Im zweiten Teil der Arbeit wurden autochthone Lymphome von Mäusen der Kreuzung λ-OVA- x λ-MYC-Maus mit Chemotherapie und adoptivem OT-1-T-Zelltransfer behandelt. Es zeigte sich hier, dass der adoptive T-Zelltransfer, im Vergleich zur Behandlung mit Chemotherapie allein, insgesamt keine Verbesserung des Überlebens bewirkte. Allerdings war bei einer Gruppe von Mäusen mit verlängerter Latenz des Primärlymphoms ein verlängertes progressionsfreies Überleben zu beobachten. Rezidive waren in diesem Fall OVA- Antigen-positiv, weshalb die Induktion von T-Zelltoleranz vermutet wurde. Diese Arbeit zeigt, dass bcl-2-Überexpression und eventuell auch p53-Defizienz in Lymphomen mögliche positive prognostische Faktoren für den adoptiven T-Zelltransfer nach Chemotherapie, zumindest aber kein Hindernis darstellen. Die Ergebnisse aus dem autochthonen Modell weisen darauf hin, dass im jungen Alter auftretende Lymphome eine höhere Aggressivität aufweisen können und dass in der klinischen Situation des adoptiven T-Zelltransfers mit B-zellspezifischem Zielantigen die Induktion von T-Zelltoleranz eher ein Problem darstellt als die Selektion von Antigenverlustvarianten.Over-expression of the apoptosis inhibitor Bcl-2 or loss of the central tumor suppressor p53 in tumors is generally associated with negative prognosis, as these tumors show increased aggressiveness and resistance to chemotherapy and radiotherapy. It has been observed that after allogeneic stem cell transplantation (whose graft-versus-tumor effect is based on cytotoxic activity of T-cells), the probability of progression of the disease was elevated for chemotherapy resistant lymphomas. Consequently the question has been raised whether chemotherapy-resistant lymphomas and therapeutically induced senescent lymphoma cells are less accessible to T-cell therapy. With the help of the λ-MYC mouse model, highly malignant c-myc-driven lymphoma cell lines were generated and supplied with the model antigen ovalbumin (OVA). It could be shown that these cell lines are chemotherapy-resistant and develop senescence as a second DNA damage strategy. Both chemotherapy-resistant and senescent cells were lysed by OVA-specific OT-1 T-cells. With the λ-OVA mouse (which expresses OVA as B-cell specific antigen) an OVA tolerant mouse was generated, which carries the antigen in normal B-cells as well as in the neoplasia itself — the transplanted OVA-positive lymphoma cells. After chemotherapy and adoptive T-cell transfer, mice with transplanted bcl-2 over- expressing lymphomas clearly showed a prolonged remission instead of the expected decline in progression-free survival. This improvement was also observed (albeit less obviously) in p53-deficient tumors. As relapsed tumors were OVA-negative, it could be concluded that bcl-2-over-expression reduces the probability for the selection of antigen-loss variants after chemotherapy. In the second part of this work autochthonous lymphomas of mice generated by intercrossing λ-OVA with λ-MYC mice were treated with chemotherapy and OT-1 T-cell transfer. Here it could be shown that adoptive T-cell transfer compared with chemotherapy treatment alone did not result in improved survival. However, a group of mice with prolonged latency of the primary tumor showed prolonged progression-free survival. In this case relapsed tumors were OVA- positive. Therefore induction of T-cell tolerance was presumed. This work shows that bcl-2-over-expression and optionally p53-deficiency in lymphomas may represent positive prognostic factors for adoptive T-cell transfer after chemotherapy and at least do not seem to impede remission. The results from the autochthonous model point towards higher aggressiveness in tumors which develop at a young age, and lead to the conclusion that the induction of T-cell tolerance in the case of adoptive T-cell transfer with B-cell specific target antigen poses more of a problem than selection of antigen loss variants

IFN-γ responsiveness of lymphoma cells is not required for antigen-dependent lymphoma rejection.

<p>IFN-γ-receptor-deficient λ-huMYC lymphoma cells, either retrovirally transduced (50OVA) (solid line) or untransduced (50PC) (dashed line), were injected s.c. into wild-type mice. STAT1-deficient mice inoculated with retrovirally transduced 50OVA cells served as positive control (dotted line). Mice were monitored for survival (left panel) and cumulative tumor growth (right panel). The data are combined from 2 independent experiments.</p

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

OVA and GFP serve as foreign antigens and mediate rejection of the λ-huMYC lymphoma cell line 291 in wild-type mice.

<p>(A) 291PC cells were transduced with retroviruses expressing IRES-GFP or OVA-IRES-GFP. Fluorescence activated cell sorting for GFP resulted in a purity of 96 to 98% GFP-positive cells, respectively (left panel of A). Expression of OVA was confirmed by Western blot analysis (A, right panel). (B and C) 1×10⁵ λ-huMYC 291PC (parental cells) lymphoma cells (dashed line), retrovirally transduced with IRES-GFP (291GFP) (dotted line), or OVA-IRES-GFP (291OVA) (solid line) were injected s.c. into either wild-type (B) or into GFP-transgenic UBI-GFPtg mice (C). Overall survival (left panels) and tumor growth (right panels) were monitored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034552#s4" target="_blank">Material & Methods</a> (data are compiled from 3 independent experiments).</p

Inoculation of OVA-expressing lymphoma cells into wild-type mice elicits an OVA-specific CD8+ T cell response.

<p>(A) 1×10⁵ CD90-selected splenic T cells of mice challenged with 291OVA cells were restimulated with peritoneal macrophages (APC, antigen presenting cells) in the presence or absence of SIINFEKL peptide (p = 0.020) or with 291 parental (291PC) or OVA-expressing lymphoma cells (291OVA, p = 0.021, Mann-Whitney test, n = 5 animals/group). (B) T cell responses against OVA were monitored at different time points by flow cytometry using SIINFEKL-specific pentamers in the peripheral blood of wild-type mice inoculated with 291OVA cells. The percentage of SIINFEKL pentamer-positive CD8+ T cells increased over time as shown for one representative mouse (B, left panel). Mean values (+/− standard deviation) of percentage of SIINFEKL pentamer-positive CD8+ T cells increased over time as compiled from 5 mice (B, right panel). (C) After inoculation of 1×10⁵ 291 parental or 291OVA cells, the developing lymphomas were analyzed by immunohistochemistry for infiltration of CD3-positive (peroxidase brown staining) and perforin expressing (alkaline phosphatase red staining) cells (C, left panel). CD3− and perforin-positive cells were quantified in blinded fashion by counting 10 high power fields (HPF, 400×) per section. Values are given as a mean ± standard error of the mean (C, right panel). Only CD3-positive and CD3/perforin-double positive cells (CD3/HPF) were regarded as T cells, whereas single perforin-positive cells were considered to be NK cells and excluded from the analysis. For each group 8–15 sections were analyzed. Mann Whitney-U test was used for comparison.</p

Transfer of OVA-expressing tumor cells into wild-type mice results in loss of antigen and induction of MHC on lymphoma cells.

<p>(A) Systemically growing lymphomas isolated from the spleen after s.c. inoculation of 291GFP or 291OVA cells were analyzed for GFP expression as a marker for antigen expression in comparison to the inoculated cell lines. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034552#s1" target="_blank">Introduction</a> of either GFP alone (291GFP) or OVA-IRES-GFP (291OVA) into lymphoma cells resulted in loss of or strong decrease in GFP expression in outgrowing lymphomas after transfer into wild-type mice (left panel, n = 6). Outgrowing lymphomas in GFP-transgenic recipients (tolerant for GFP) likewise lost GFP expression after inoculation of 291OVA cells, but retained the antigen when 291GFP cells had been inoculated (right panel). Flow cytometric histogram inlets show representative examples. Grey lines represent GFP expression at the time of injection, black lines after harvest of lymphomas from spleen. (B) Left: western Blot and RT-PCR analysis (arbitrary units, A.U.) of lymphomas harvested after s.c. inoculation of 291 OVA cells from either OVA tolerant (actOVA) or wild type (WT) recipients. In contrast to WT recipients OVA tolerant actOVA animals did not select for antigen (OVA) loss variants. Middle: Explanted lymphomas were cocultured with unprimed OT-I cells (1∶1 ratio). OVA positive lymphomas from actOVA recipients activated OT-I T-cells to secrete large amounts of IFN-γ (ELISA of supernatant, n = 4, Mann Whitney test) whereas OVA negatively selected lymphomas form wild type recipients did not stimulate OT-I cells. Right: Coculture of OT-I T-cells with lymphoma cells dervived from actOVA or wild type recipients resulted in induction of the T-cell activation marker CD69 expansion of CD8+ (OT-I) cells and reduction of the number of GFP expressing cells CD19+ lymphoma cells. Representative analysis from lymphoma cells harvested from individual mice. (C) Inoculation of 291OVA cells, but not of 291PC cells, into wild-type (n = 5) and GFP-transgenic mice (n = 5) significantly induced MHC class I (left panel) and class II (right panel) in outgrowing lymphoma cells. The strong increase in MHC class I and II expression in outgrowing lymphomas depended on the presence of OVA in the lymphoma inoculum (Mann Whitney test). Fold induction was calculated by comparing MHC expression on freshly explanted lymphoma cells with the corresponding cell line on the same day.</p

Defective cross-presentation in cells of the recipient impairs T cell response and enhances lymphoma growth.

<p>Recipient animals were T cell depleted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034552#s4" target="_blank">Materials and Methods</a> and T cell depletion was continued for 28 days. Wild-type (WT) or bm1 mutant recipient mice received 1×10⁵ 291OVA lymphoma cells s.c. together with 1×10⁶<i>in vivo</i> primed OT-I cells. (A) Development of an OVA-specific T cell response is impaired in bm1 recipients. Peripheral blood of animals inoculated with 1×10⁵ 291OVA and 1×10⁶ OT-I cells were analyzed by flow cytometry for the presence of CD90.1-positive cells (OT-I) on the days indicated. Numbers of OT-1 T cells are expressed as percentage of lymphocytes in the peripheral blood. In wild-type mice (solid line, n = 10) adoptively transferred OT-I cells expand more readily (Mann Whitney test) than in bm1 recipients (dashed line, n = 9, p = 0.001 for day 8–27). (B) Disease-free survival and cumulative tumor growth after lymphoma transfer: bm1 recipient mice (dashed line) developed tumors significantly faster and cumulative lymphoma growth was enhanced (right panel). (C) Mean fluorescence of GFP in lymphomas arising in wild-type mice or bm1 mice in the presence and absence of OT-I cells. T cell depletion in the absence of OT-I cells (WT no OT-I, n = 9) resulted in preservation of antigen expression, while adoptive transfer of OT-I cells (WT+OT-I, n = 5) led to selection of antigen-negative lymphoma cells.</p