Elsevier

Leukemia Research

Volume 26, Issue 6, June 2002, Pages 577-590
Leukemia Research

Interleukin-12-induced cytotoxicity against syngeneic B cell lymphomas of SJL/J mice

https://doi.org/10.1016/S0145-2126(01)00179-5Get rights and content

Abstract

The B cell lymphomas (RCS) that develop spontaneously in 90% of aging SJL/J mice stimulate syngeneic CD4+ Vβ16+ Th2 cells to produce cytokines, such as IL-4 and IL-5, which promote lymphoma growth. Although RCS cells express a unique superantigen (vSAg) encoded by an endogenous MMTV (Mtv-29) provirus that also elicits IFN-γ production from naı̈ve syngeneic lymphoid cells, there is no development of RCS-specific cytotoxicity. However, addition of IL-12 to co-cultures of SJL spleen and irradiated (γ-)RCS cells resulted in the appearance of effector cells that killed RCS and NK-susceptible target cells. Antibody depletion studies revealed at least two types of RCS/IL-12-induced cytotoxic cells: (1) NK cells (Asialo GM1+) and (2) CD8+ CTL. Despite high titers of IFN-γ in the SN of co-culture of SJL spleen and γ-RCS cells, cytotoxicity only developed if IL-12 was also included in the co-cultures. The results of RNAse protection assays and multi-parameter FACS analysis demonstrated an upregulation of IFN-γ and decrease in IL-4 by activated Th cells in co-cultures with IL-12. These results indicate that inclusion of IL-12 in primary co-cultures of SJL spleen and γ-RCS cells influences the qualitative nature of the response to favor use of RCS-responsive Th1 rather than Th2 cells to facilitate the production of cytotoxic effector cells. Results of in vivo experiments support this hypothesis, as judged by tumor growth assays and FACS analysis of the tumor cell content of lymphoid tissues. Inhibition of lymphoma growth was observed in mice given γ-RCS/IL-12-induced effector cells prior to injection of viable RCS cells. These results demonstrate that IL-12 can be used to alter the host immune response leading to induction of cytotoxic effector cells that inhibit the development and/or progressive growth of otherwise resistant B cell lymphomas in SJL/J mice.

Introduction

SJL/J mice exhibit a 90% incidence of B cell lymphomas (originally called reticulum cell sarcomas or RCS) at approximately 13 months of age. Since the first report by Murphy in 1963 [1], the results of studies by us and others (reviewed in [2]) have not only identified the phenotypic characteristics of these B cell lymphomas, but have also revealed a novel lymphomagenic mechanism, termed reverse immune surveillance [3], [4], [5]. This phenomenon refers to the inability of RCS cells to grow progressively in immunocompromised SJL recipients, and their dependence on tumor-activated Th2 cells to provide tumor growth-promoting cytokines, such as IL-4 and IL-5 [6], [7].

The discovery by Tsiagbe et al. [8], [9] that a unique superantigen (vSAg) encoded by an endogenous MMTV (Mtv-29) provirus in RCS tumor cells stimulated TCR Vβ16+ Th cells, offered a new perspective of the SJL/RCS lymphoma model. The results of these studies unequivocally demonstrated that the Mtv-29 provirus transcribes a vSAg, using the META promoter/enhancer [10], which is different from, and additional to, those produced by resting or LPS-activated, non-malignant SJL B cells. Moreover, by stimulating Vβ16+ T cells to produce the requisite tumor growth-promoting cytokines [9], this vSAg-29-induced host response represents a major contributing factor to the phenomenon of reverse immune surveillance.

In complementary studies, Lasky and co-workers [6], [11] showed that the cytokines that facilitated RCS tumor cell growth in vitro were those produced by Th2 cells (chiefly, IL-5). However, in addition to the Th2 cytokines that clearly supported RCS cell growth, numerous other cytokines, such as IL-2 and IFN-γ, were also present in the SN of cultures containing SJL lymphoid cells and syngeneic γ-RCS stimulator cells [12].

SJL mice exhibit a constitutively low natural killer (NK) phenotype [13], [14]. However, moderate to high levels of NK activity were seen in effector cells obtained from primary co-cultures of SJL lymphoid cells and γ-RCS stimulator cells [3], [15], as well as from lymphoid tissues of SJL mice injected with γ-RCS cells [15], and from tumor bearing SJL mice [15], [16]. Although these RCS-activated NK cells killed a variety of NK-susceptible tumor target cells (such as YAC-1 and RBL-3) no cytotoxicity was obtained when LAK-sensitive target cells (such as L1210) or syngeneic RCS lymphoma target cells were tested [15]. Therefore, the ability to stimulate NK activity in SJL mice appeared to be a unique property of RCS tumor cells, since similar cytotoxic effector function was not obtained following stimulation of SJL lymphoid cells with potent NK inducers, such as poly I:C and IFN-γ [14], [17].

Despite the stimulation of Th cells and NK cells, and the accompanying production of cytokines following exposure of naı̈ve SJL lymphoid cells to RCS tumor cells, development of tumor specific cytotoxic effector cells was not observed [18]. However, early transplantation studies [19], [20], using primary RCS lymphomas, suggested that tumor specific immunity could be generated against these lymphoma cells, although the basis for this immunity was not investigated. Moreover, following immunization of SJL mice with irradiated, long-term transplantable RCS cells, moderate levels of CTL-mediated, tumor-specific cytotoxic activity were obtained when RCS-primed lymphoid cells were restimulated in secondary co-cultures with γ-RCS cells [18].

In view of the involvement of Th subsets and distinct cytokines in humoral (Th2; IL-4, IL-5) and cell-mediated (Th1; IFN-γ, IL-12) immune responses [21], [22], we reasoned that the failure to develop tumor-specific CTL activity against RCS lymphomas following primary exposure might be due to an imbalance in the differentiation of Th cells, which favored Th2 over Th1 cells. Therefore, we reassessed the question of CTL development in the SJL/RCS lymphoma model, using IL-12 to modulate the response of SJL lymphoid cells to γ-RCS stimulator cells. These recent cytotoxicity experiments were facilitated by the development (in the years following the earlier CTL studies) of in vitro RCS cell lines for use as target cells in cytotoxicity assays [11], [23].

The results demonstrate that the presence of IL-12 during stimulation of naı̈ve SJL lymphoid cells with γ-RCS cells not only caused higher levels of NK activity, but the development of tumor-specific CTL also occurred. Moreover, while IFN-γ was necessary for the development of the observed CTL activity, without IL-12 it was insufficient, by itself, to trigger the generation of tumor-specific cytotoxic effector cells. Inclusion of IL-12 did not alter other quantitative parameters (e.g. proliferation or total number of “blast” cells) of the γ-RCS-induced response. Therefore, the qualitative changes that occurred in the presence of IL-12 most likely involved a switch from a predominantly Th2 to a Th1 response, resulting in production of tumor-specific cytotoxic effector cells. This conclusion was supported by the results of complementary in vitro experiments that showed an increase in the number of cells with intracytoplasmic IFN-γ in cultures containing IL-12 and a decrease in cells containing IL-4, and in vivo experiments that demonstrated reduced lymphoma cell growth in mice given γ-RCS/IL-12-induced effector cells prior to challenge with viable RCS cells. Overall, the results of our study indicate that IL-12 can modulate the RCS-induced response to favor the development of effector mechanisms that lead to tumor cell destruction.

Section snippets

Mice

SJL/J, BALB.B, C57BL/6J, C57L/J and CBA mice were purchased from the Jackson Laboratories (Bar Harbor, ME), and were housed in the AAALAC-accredited research animal facility (RAF) at the University of Medicine and Dentistry of New Jersey (Newark, NJ).

Antibodies and cytokines

Monoclonal antibodies (mAbs) were prepared as supernatants (SN) or as mouse ascites from hybridomas obtained from the American Type Culture Collection (ATCC, Rockville, MD) or colleagues, as indicated: 3.155 (recognizes all CD8 alleles), 83-12.5

Inclusion of IL-12 stimulates development of cytotoxic effector cells

Table 1 presents the results of a representative experiment that demonstrates the vigorous proliferative response of SJL lymphoid cells to syngeneic RCS lymphoma cells. This characteristic response has been well characterized by us and others [2], [3], and is primarily due to recognition of vSAg29 determinants on the RCS cells by TCR Vβ16+ cells within the responder population [8], [9]. As shown by the data in Table 1, the magnitude of this response is significantly higher than allostimulation

Discussion and conclusion

It is well documented that vSAg29 expressed by RCS cells stimulates primarily CD4+ Vβ16+ cells to produce tumor growth-promoting cytokines, such as IL-4 and IL-5 [6], [7]. It is thought that this form of host response, termed reverse immune surveillance [3], [4], [5], not only promotes the growth of transplantable tumor cell lines after adoptive transfer to young SJL recipients, but is also a central component in the etiology of these B cell lymphomas in aging SJL mice [2]. The observation of

Acknowledgements

The authors wish to thank Dr. Vincent Tsiagbe (New York University Medical Center, New York, NY) for help in performing the RNAse protection assay for cytokine gene expression and Dr. Roberto Chiarle (New York University Medical Center, New York, NY) for describing the histopathology of tissues obtained from tumor injected mice. We also wish thank Meg O’Donnell and the External Research Department of the Genetics Institute (Cambridge, MA) for providing the recombinant IL-12 for our studies, Dr.

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