Optimization and validation of a robust human T-cell culture method for monitoring phenotypic and polyfunctional antigen-specific CD4 and CD8 T-cell responses
Introduction
The discovery over the last several decades of human cancer antigens (Ag) has ushered in a new era of Ag-specific cancer immunotherapy. These Ag include differentiation Ag expressed only on certain cell types/tumors, for example MART-1/Melan-A in melanoma, and cancer-testis Ag, such as NY-ESO-1, which are expressed on certain tumors but not in normal tissue, with the exception of the placenta and testes [1,2]. Optimal anti-tumor activity is achieved with the induction of both CD4+ and CD8+ tumor-specific T cells [3., 4., 5., 6., 7., 8.].
A critical component of the success of such trials is the close monitoring of Ag-specific CD4+ and CD8+ responses to quantify the full phenotypic and functional characteristics of the T cells. This quantification allows analysis of the effectiveness of the immunization strategy, correlation with any clinical benefit and, ultimately, development of an improved vaccine formulation that can be deployed clinically. Ideally, the most accurate form of T-cell monitoring would involve ex vivo measurement without the need for in vitro manipulation. However, most vaccine- and immune-based strategies produce relatively low frequencies of Ag-specific precursors in the peripheral blood [9,10] that are difficult to quantitate accurately even with current technology. As such, in vitro expansion of these precursors is frequently necessary.
In terms of phenotype, four major subsets of human CD8+ T cells have been delineated with the help of two cell-surface markers, CCR7 and CD45RA [11]. These are naive cells (CD45RA+ CCR7+), central memory cells (TCM, CD45RA− CCR7+), effector memory cells (TEM, CD45RA− CCR7−) and effector cells (TEMRA, CD45RA+ CCR7−) [12., 13., 14.]. Recently, two additional surface markers, CD27 and CD28, have proven useful in defining four additional subsets of CD8+ CD45RA− CCR7− T cells that have different effector and cytolytic functions: EM1 (CD27+ CD28+), EM2 (CD27+ CD28−), EM3 (CD27− CD28−) and EM4 (CD27− CD28+) [15].
Until recently, T-cell responses have largely been determined by measuring the expression of a single effector function, such as interferon (IFN)-γ production. However, there are increasing data that a single parameter does not reflect the full functional potential of a T cell [16]. Recent studies have demonstrated that polyfunctional T cells, which produce multiple cytokines and chemokines in response to Ag stimulation, are in fact associated with improved viral control in pre-clinical models of infectious diseases, in patients infected with human immunodeficiency virus (HIV) or immunized using vaccinia constructs [17,18] and in cancer patients receiving vaccination or immunotherapy [8]. Flow cytometry is an important methodology that can simultaneously characterize multiple functions, enabling a broad assessment of the phenotype and functional capacity of T-cell effector functions described above [19,20]. Advances in the number of parameters that can be evaluated simultaneously now allow even more extensive characterization of T cells at the single-cell level.
In this study, we undertook flow cytometry studies to determine the phenotypic and polyfunctional responses of Ag-specific T cells both before and after in vitro stimulation and expansion. To establish the parameters for optimal in vitro T-cell stimulation, we evaluated the ability of autologous peripheral blood mononuclear cells (PBMC) to function as antigen-presenting cells (APC) for T-cell stimulation and expansion. We examined four phenotype markers (CCR7, CD45RA, CD28 and CD27) and five functional markers [surface CD107a, IFN-γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-1β] in T cells stimulated by several Ag (fluMP, Melan-A and NY-ESO-1).
Section snippets
Blood donation from healthy donors and melanoma patients
Healthy donors provided blood samples for this study, after giving informed consent, as participants in protocols approved by the Memorial Sloan-Kettering Cancer Center (MSKCC, New York, NY, USA) Institutional Review Board (IRB). Blood cells from five patients with completely resected high-risk melanoma, who received either a gp100 or tyrosinase DNA vaccine construct on IRB-approved trials, were used in this study. Four patients were HLA-A*0201 positive and NY-ESO-1 serum antibody (Ab)
Autologous PBMC function as potent stimulators to expand Ag-specific CD8+ T cells during short-term in vitro culture
Thawed PBMC were stimulated during a 10-day culture with irradiated autologous PBMC pulsed with fluMP, Melan-A and NY-ESO-1 peptides at a 1:1 ratio, with the addition of IL-2 and IL-15 cytokines. PBMC from healthy donors were stimulated with fluMP peptides, while PBMC from patients with a history of melanoma were stimulated with Melan-A or NY-ESO-1 peptides. The percentages of fluMP/HLA-A*0201 tetramer, Melan-A/HLA-A*0201 tetramer and NY-ESO-1/HLA-B*3501 tetramers were increased after the in
Discussion
In this paper, we present a protocol for the in vitro culture of human T cells obtained from peripheral blood with stimulation by peptide-pulsed autologous irradiated PBMC. Our data support this as a robust and reproducible system that reliably expands proportionally the frequency of precursor T cells present in the peripheral blood.
Many other T-cell culture methods have been described in the literature. Notable examples include a similar protocol using autologous PBMC as APC, as reported by
Acknowledgments
We thank Dr M. Roederer for providing the SPICE software, Dr Immanuel Luescher from the Tetramer Core, Lausanne Branch, Ludwig Institute of Cancer Research, for providing all the tetramers used in these experiments, Dr Bo Dupont and Alice Yeh from his laboratory for performing HLA analysis on one patient, and Dr. Miguel Perales, who provided editorial advice. This work was supported by Swim Across America, the Experimental Therapeutics Center of MSKCC and Ludwig Foundation. J. D. Wolchok was
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The last two authors contributed equally to this study. Yun Lin, Lloyd J. Old, James P. Allison, Alan N. Houghton, Jedd D. Wolchok and Jianda Yuan designed research. Yun Lin, Humilidad F. Gallardo, Hao Li, Gregor Manukian, Teresa S. Rasalan, Yinyan Xu, Stephanie L. Terzulli and Jianda Yuan performed research. Yun Lin and Jianda Yuan analyzed data. Yun Lin, Geoffrey Y. Ku, Jedd D. Wolchok and Jianda Yuan wrote the paper.