Induction of antigen‐specific effector‐phase tolerance following vaccination against a previously ignored B‐cell lymphoma

Abstract

The mechanisms of immune evasion during haematological malignancies are poorly understood. As lymphomas grow in lymphoid organs, it would be expected that if these lymphomas express neo-antigens they should be readily detected by the immune system. To test this assumption, we generated a new non-Hodgkin B-cell lymphoma model expressing the model tumour neo-antigen Ovalbumin (OVA), and analysed the endogenous antigen-specific CD8 + T-cell response that it elicited in recipient mice. The OVA+ lymphoma cells were eliminated by cytotoxic T lymphocytes (CTL) in mice that had been previously vaccinated against OVA. In contrast, the immune system of naïve mice ignored the malignant cells even though these continuously expressed and presented OVA on their MHC class I molecules. This state of ignorance could be overcome by therapeutic vaccination, which led to the expansion of endogenous anti-OVA-specific CD8 + T cells. However, the cytotoxic and interferon-c secretion capacity of these T cells were impaired. The tumour model that we describe thus reproduces several key aspects of human lymphoma; tumor ignorance can be broken by vaccination but the ensuing immune response remains ineffective. This model can be exploited to further understand the mechanisms of lymphoma immunoevasion and devise effective immunotherapy. In numerous human cancers, the presence of tumour-specific CD8 + cytotoxic T lymphocytes (CTL) correlates with better prognosis. 1 These CTL are directed against neo-antigens that are generated during tumour cell transformation. 2 Although sometimes the presence of CTL can lead to spontaneous tumour clearance, 3 in most cases antitumour immunity is either suppressed or the malignant cells are ignored by the host immune system.Most studies investigating mechanisms of tumour immune evasion in pre-clinical animal models have primarily been undertaken using solid tumours inoculated in peripheral tissues. Reports addressing immunoescape mechanisms in the context of blood-derived malignancies, such as lymphoma and/or leukaemia, are rather scarce. This is important because the anatomic localization might dictate the immunoescape strategy exploited by the tumour. For instance, solid tumour cells and/or tumour antigens can be 'walled off' and thus ignored by the immune system. 4-7 Furthermore, tumour-specific CTL may be incapable of infiltrating solid tumours, thwarting their anti-tumour potential. 8 These evasion mechanisms are less likely to occur in the case of blood-derived malignancies because such tumours grow within the secondary lymphoid organs and hence should be readily accessible to T cells. Several studies have addressed the induction of tumour antigen-specific CD4 + T-cell responses against blood-derived tumour models. [9][10][11][12][13] Evidence indicates that tumour antigen-specific transgenic CD4 + T cells interact with antigen presenting cells that display tumour neo-antigens, however, this interaction results in tolerance. In contrast to anti-tumour CD4 + T cells, little is known about the capacity...

Target Density, Not Affinity or Avidity of Antigen Recognition, Determines Adoptive T Cell Therapy Outcomes in a Mouse Lymphoma Model

“…To investigate this in a preclinical setting, we established two distinct mouse lymphoma models varying solely with regard to their levels of expression of the model TAA OVA. Em-myc-GFP-OVA high was similar to our previously described Em-myc-GFP-OVA tumor (11,12), whereas Em-myc-GFP-OVA low expressed significantly lower levels of TAA such that it elicited poor proliferation of naive OT-I T cells in vitro. Indeed, half of these tumor cells escaped killing by highaffinity OT-I CTL, presumably because their level of OVA expression was below the threshold of recognition.…”

“…Em-myc lymphomas expressing GFP and GFP-OVA were described previously (11). Tumors with different TAA expression levels were generated from the original Em-myc-GFP-OVA lymphoma by sorting tumor cells by flow cytometry based on their GFP expression into GFP high, intermediate, and low.…”

Rapid Deletion and Inactivation of CTLs upon Recognition of a Number of Target Cells over a Critical Threshold

“…They express GFP alone (Em-myc-GFP control tumor) or with OVA as a model neoantigen (Em-myc-OVA tumor) (27). The Em-myc-OVA tumor recapitulates several features described for human lymphomas: it is susceptible to anti-OVA CTL killing but is ignored by the immune system of tumor-bearing mice (i.e., it does not elicit anti-OVA CD8 + T cell priming spontaneously), and although it is possible to prime endogenous anti-OVA CD8 + T cells in Em-myc-OVA tumor-bearing mice with an anti-OVA vaccine, the T cells do not become effective CTLs (27). In this scenario, adoptive cell therapy with high-avidity anti-OVA CTLs (OT-I) might be a suitable approach to overcome immunosuppression of the endogenous repertoire.…”

2B4-SAP signaling is required for the priming of naive CD8+ T cells by antigen-expressing B cells and B lymphoma cells

“…To test whether SAP is important for CD8 C T cell responses directed against B lymphoma cells, CFSE-labeled WT and Sh2d1a ¡/¡ OT-I CD8 C T cells were stimulated with previously described Ag-negative (B-GFP) or Ag-expressing EmMyc B lymphoma cells (B-OVA). 34,35 After 4 d culture, WT CD8…”

“…B lymphoma cells expressing GFP (B-GFP) or OVA (B-OVA) were derived from terminally ill Em-myc transgenic mice transfected with expression cassettes containing GFP alone or a membrane-bound ovalbumin, passaged through mice and cryogenically preserved. 34,35 For in vitro assays, B lymphoma cells were cultured from lymph nodes of transplanted mice and expanded as cell lines in DMEM containing 10% FCS, non-essential amino acids, 100 U/mL of penicillin/streptomycin and 55 mM b-mercaptoethanol (all Thermo Fisher Scientific). Antitumor proliferation assays were performed by stimulating 10 4 CFSE-labeled WT, Sh2d1a ¡/¡ and 2B4 ¡/¡ OT-I CD8 C T cells with 10 5 B lymphoma cells, 10 4 B16-OVA or 10 4 NOP12 cells.…”