Research paper
A robust human T-cell culture method suitable for monitoring CD8+ and CD4+ T-cell responses from cancer clinical trial samples

https://doi.org/10.1016/j.jim.2004.04.017Get rights and content

Abstract

Many tumor antigenic determinants have been identified and included in cancer clinical trials. Due to low T-cell frequencies even after vaccination, few T-cell responses can be revealed ex vivo without in vitro stimulation. Various expansion protocols have been employed for this purpose and the outcomes tend to be quite variable, partly due to the high complexity involved in the protocols. Here we systematically studied various common culture conditions including sera, cytokines and feeders and describe a reliable “bulk” culture method that is robust, simpler and more economical. We demonstrated that fetal calf serum (FCS) supported T-cell proliferation better than multiple commercially available pooled human AB sera. IL-2 is critical in our cultures, but IL-7, IL-15 and anti-CTLA-4 in combination with IL-2 did not further enhance T-cell expansion. We typically achieve more than a 40-fold expansion within a 10-day culture period for antigen-specific T cells measured by HLA–peptide tetramer before and after culture. This method was not only validated by multiple operators as a standard operating procedure for monitoring T-cell responses but was also successfully used for discovering novel CD8+ and CD4+ T cells specific to previously unknown epitopes from the NY-ESO-1 tumor antigen.

Introduction

Due to the discovery of many tumor antigens and the characterisation of their derived antigenic epitopes for CD8+ and CD4+ T cells (for a review, see Renkvist et al., 2001), therapeutical cancer clinical trials incorporating these T-cell epitopes have entered a new era Jager et al., 2002, Davis et al., 2003. However, few trials have achieved significant clinical outcomes and most are still judged using immunological end points. Among all the immune parameters, cellular responses assessed after a short period of in vitro culture are most often used (Coulie and Bruggen, 2003). Thus, a successful trial outcome is vitally dependent on the T-cell culture methodology.

Traditionally, T-cell culture methods were empirically developed predominantly for the discovery of new antigens. A variety of sera, cytokines and feeder cells has been explored individually or in combination. Over a period of three decades, very few published studies have systematically evaluated the above variables and addressed issues such as reproducibility. It was reported that human T cells cultured in fetal calf serum (FCS)-containing medium could develop some FCS-specific, class I-restricted (Misko et al., 1984) and more often class II-restricted T cells Torsteinsdottir et al., 1986, Misko et al., 1991 due to the xenogeneic nature of the sera. Consequently, the vast majority of human T-cell cultures was performed in the presence of pooled human AB sera Skornick et al., 1990, Yee et al., 1999, Valmori et al., 2000; some used fetal calf sera (FCS; Bonini et al., 2001, Fu et al., 2001) and a few used either autologous sera or serum-free conditions Lamers et al., 1992, Jonuleit et al., 2000. Various cytokines were also explored. In the early days of human T-cell culture, cytokines (mainly IL-2) were added to the culture with a delay to minimize the potential nonspecific T cells and the lymphokine activated killer (LAK) cell expansion, which are still practised by some researchers today Yee et al., 1999, Fu et al., 2001. Various irradiated feeder cells were also utilized, including irradiated autologous B lymphocyte cell line (BLCL), mixed BLCL, autologous peripheral blood mononuclear cells (PBMC) and allogeneic PBMC Van de Griend et al., 1984, Valmori et al., 2000. When autologous PBMC were pulsed with antigenic peptides and used as antigen-presenting cells (APC), they were often irradiated for more sustained stimulation in the absence of feeder cell division (Valmori et al., 1998). As the functions of various cytokines are elucidated in mouse studies, many culture protocols now not only include the general T-cell growth factor IL-2 but also add other growth factors. These include growth factors shown to be essential for naı̈ve T-cell survival and homeostatic proliferation (IL-7; Lalvani et al., 1997, Tan et al., 2001) and memory T cell in vivo renewal (IL-15; Tan et al., 2002), although their in vitro functions have not been well characterized. In other cases, a Th1 polarising cytokine, such as IL-12 (Bertagnolli et al., 1992), was also included in T-cell cultures aiming to further enhance CD8+ T-cell expansion. Anti-CTLA-4 has also been applied to block the potential negative feedback for the proliferating T cells (Hsu and Komarovskaya, 2002). Taken together, human T-cell culture methods have been largely laboratory-specific and empirically practised. As the search for a cure for cancer intensifies, more anticancer clinical trials are either conducted or planned. Simple yet effective T-cell monitoring in a timely and reproducible fashion becomes increasingly important.

We have conducted T-cell monitoring for several cancer clinical trials. Some trials involved multiple peptide vaccines and repeated sampling prior to and after each vaccine injection. We sought to establish a relatively uncomplicated and reproducible method to accomplish the daunting T-cell monitoring task by systematically studying the above-mentioned human T-cell culture conditions. Here we describe a new “bulk” culture method which has been proven in our laboratory to be efficient, robust and reliable. Compared to other reported methods, it is simpler; it uses FCS instead of pooled human AB serum and does not involve feeder cells and irradiation. More importantly, it does not require any cell enrichment and purification, which means multiple cultures can be set up at the same time with ease. It is efficient in using relatively low cell numbers and allows both CD4+ and CD8+ antigen-specific T cells to be assayed simultaneously. The method has been used as a standard operating procedure for monitoring cancer clinical trials and also as means for discovering novel T-cell epitopes from known tumor antigens.

Section snippets

Patients

One subject (patient 7) was a patient with resected melanoma who participated in the NY-ESO-1 ISCOMATRIX™ adjuvant cancer clinical trial conducted at the Ludwig Institute for Cancer Research, Melbourne branch (trial LUD99-008; Davis et al., in press). The other subject (patient 9) was from a peptide clinical trial (trial LUD97-012; unpublished data). Both trials were approved by the Institutional Review boards of the Austin Hospital and Ludwig Institute for Cancer Research. Blood samples,

Various APC/responder ratios affect T-cell expansion

We sought to establish a simpler human T-cell culture method for monitoring the cellular immune responses from either peptide or full-length tumor antigen-vaccinated patients. As a first step, we wanted to adapt a method from murine CD8+ T-cell culture, which normally used a fraction of syngeneic spleen cells as APC to stimulate other spleen-derived antigen-specific CD8+ T cells (Chen et al., 1999). To test the culture conditions properly and sensitively, samples from patient 7 were chosen

Discussion

The major goal for most cancer clinical trials has been CD8+ T-cell induction. Ideally, the most accurate T-cell monitoring should be directly assessed ex vivo without any in vitro culture. In practice, most vaccinated or boosted T-cell responses are normally too low to be directly assessed even when using the best contemporary methodologies. Consequently, in vitro expansion is required and an efficient and reproducible T-cell expansion method thus becomes an essential part of a successful

Acknowledgements

The authors thank the following people without whom this work could not have been done: Lena Miloradovic, Christine Millar and Kelly-Anne Masterman. We also thank Dr. Immanuel Luescher and others at the LICR Tetramer Facility in Lausanne, Switzerland, who provided tetramers for our studies. Dr. Weisan Chen is a Senior International Fellow supported by the Wellcome Trust (066646/Z/01/Z).

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