Elsevier

European Journal of Cancer

Volume 37, Issue 13, September 2001, Pages 1709-1718
European Journal of Cancer

T Lymphocytes infiltrating various tumour types express the MHC class II ligand lymphocyte activation gene-3 (LAG-3): role of LAG-3/MHC class II interactions in cell–cell contacts

https://doi.org/10.1016/S0959-8049(01)00184-8Get rights and content

Abstract

The product of the Lymphocyte Activation Gene-3 (LAG-3, CD223) is a high affinity MHC class II ligand expressed by activated CD4+ and CD8+ T cells, which can associate with the T cell receptor (TCR) and downregulate TCR signalling in vitro. We have also reported that a soluble mLAG-3Ig fusion protein works as a vaccine adjuvant in vivo in mice, enhancing Th1 and CD8 T cell responses. Here, we report that LAG-3 expression was found, using fluorescent activated cell sorting (FACS) analysis, on 11–48% of human tumour-infiltrating lymphocytes (TILs) isolated from eight freshly dissociated renal cell carcinomas (RCCs), and was restricted mostly to CD8+ cells. Immunohistochemical analysis confirmed LAG-3 expression by TILs in 9/11 RCCs, as well as in tumours of different origins, such as melanomas (3/5) and lymphomas (7/7). Since not only antigen presenting cells (APCs), but also TILs themselves strongly express major histocompatibility complex (MHC) class II, we firstly investigated whether LAG-3/MHC class II T–T cell contacts might influence tumour cell recognition. However, cytotoxicity inhibition was not observed in two RCC-specific CD8+ T cell clones in the presence of the LAG-3-specific MAb, and there was also no observed difference in the recognition of LAG-3-transfected or wild-type RCC by these cytotoxic T lymphocytes (CTLs). In contrast, MHC class II engagement by LAG-3Ig was found to enhance the capacity of immature dendritic cells to stimulate naive T cell proliferation and IL-12-dependent IFN-γ production by T cells in vitro. These results therefore provide support for a role for TIL-expressed LAG-3 in the engagement of class II molecules on APCs, thereby contributing to APC activation and Th1/Tc1 commitment, without downregulating cytotoxicity.

Introduction

Recent progress made in the understanding of antitumoral immunity has revealed that tumours of sufficient immunogenicity trigger an immune response in the host resulting in the recruitment of tumour antigen-specific T lymphocytes, which infiltrate the tumour tissue. Among these tumour-infiltrating lymphocytes (TILs), a variable proportion of CD8 T cells exerting cytotoxic functions against tumour cells has been observed 1, 2, 3. Thus, tumour progression in cancer patients may not simply result from an absence of immune response, but rather from the inability of effector cells to control or destroy the tumour cells. Several mechanisms have been proposed to explain tumour resistance to destruction, including the production of immunosuppressive cytokines by the tumour cells, resistance of target cells to tumour necrosis factor alpha (TNFα) or Fas-mediated cytotoxicity [4], and the engagement of Killer Inhibitory Receptors (KIRs) on T cells by tumour MHC class I molecules [5]. In addition to T cells, dendritic cells (DCs), the most potent antigen presenting cells (APCs), have also been detected in tumour tissues 6, 7. In an attempt to develop efficient new strategies to cure cancer, characterisation of these players of antitumour cell immunity, and of how they interact and how their functions are impaired or may be boosted, is of critical importance.

The Lymphocyte Activation Gene-3 (LAG-3), which is embedded in the CD4 locus 8, 9, encodes a protein, also termed CD223 (HLDA7 workshop), that is expressed in activated CD4+ and CD8+ T cells and associated with the CD3/T cell receptor (TCR) complex at the cell surface of these cells 10, 11. LAG-3 has structural similarities with CD4 [8] and like CD4 12, 13, binds major histocompatibility complex (MHC) class II molecules 14, 15. It may oligomerise at the cell surface to interact more efficiently with class II molecules [16]. Several findings indicate that LAG-3 expression participates in the type 1 (Th1/Tc1) T cell response. LAG-3 expression is associated with intracellular interferon-gamma (IFN-γ) production in both CD4+ and CD8+subsets 17, 18 and both IFN-γ and LAG-3 are upregulated by interleukin-12 (IL-12) [18]. In addition, both ectopically-expressed on murine tumour cells, and as a soluble fusion protein (mLAG-3Ig), LAG-3 has been shown to contribute in vivo to the commitment of murine T cells toward the Th1/Tc1 type, by cross-linking MHC class II molecules expressed on APCs 19, 20. In vitro, hLAG-3Ig-induced cross-linking of MHC class II molecules expressed by immature human DCs results in IL-12 secretion, a known Th1 inducer, without the need of a cell determinant 40 ligand (CD40L) signal [21].

However, we reported that LAG-3 participates in the cell–cell interactions between phorbol ester (PHA)-activated CD4 T cells which result in downregulation of their proliferation and cytokine production [15]. Moreover, cross-linking LAG-3 expressed by activated T cells with MAb prior to restimulation led to TCR signalling downmodulation and T cell unresponsiveness [10], indicating that LAG-3 expression by activated T cells renders them susceptible to as yet unclear MHC class II-dependent downregulation of their effector functions. These experiments also suggest that LAG-3 may indeed contribute to the induction of TIL unresponsiveness.

We examined here the possibility that TILs might express the LAG-3 antigen at their surface and that this could contribute to modulate their antitumour effector functions.

Section snippets

Tumour samples, TILs and TIL-derived T cell clones

Tumour samples were obtained from patients undergoing surgical tumour ablation in our institution, and were used to prepare either frozen sections or single-cell suspensions and TIL cell lines as previously described in Ref. [23]. Generation of renal cell carcinoma (RCC) tumour-specific T cell clone 3B8 (TCR α/β+ CD4 CD8+, HLA-B7-restricted and specific for a nonameric intestinal carboxyl esterase epitope) from patient no. 8's TILs and clone 11C2 (TCR α/β+ CD4 CD8+, HLA-A2-restricted and

FACS analysis of RCC-derived TILs

We have previously described the expression of LAG-3 on TILs from 1 RCC patient [23]. We then became interested in assessing whether this observation was an exception or a common feature of most RCC TILs. 8 patients' tumours were therefore enzymatically dissociated and their LAG-3 expression analysed by FACS. As shown in Fig. 1 and summarised in Table 1, all RCC patients had LAG-3-expressing TILs, although the expression varied from 11 to 48%. LAG-3 expression is often quite low, and therefore

Discussion

In this present study, we strengthen and also expand on our previous observation that a subset of TILs express the LAG-3 antigen (CD223), a high affinity MHC class II ligand 14, 29. We had previously reported that LAG-3 was expressed on a subset of TILs derived from enzymatically-dissociated RCCs in 1 patient [23]. Suspecting that LAG-3 was not detected in all analysed cases due to technical reasons, such as tumour dissociation and the staining procedure used, we further examined LAG-3

Acknowledgements

This work was supported by grants from the Ligue contre le Cancer (Comité des Yvelines), Association pour la Recherche contre le Cancer, the Fonds de la Recherche en Santé du Québec, and Serono International S.A.

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