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 PBMCs. Our data support that this is a robust and reproducible system that reliably expands the frequency of precursor T cells present in the peripheral blood proportionally.
Many other T cell culture methods have been described in the literature. Notable examples include a similar protocol using autologous PBMCs as the APC, as reported by Jackson et al [23
]. An important difference from our protocol is the fact that the peptide-pulsed autologous PBMCs were not irradiated. Instead, peptide was added to the entire PBMC population without subsequent irradiation. The authors did note that irradiation of peptide-pulsed PBMCs facilitated T cell expansion but only if <50% of the entire cell population was irradiated, which would be consistent with our observation. The mechanism by which irradiation of the peptide-pulsed APCs leads to increased T cell expansion is unclear but is presumably related to augmentation of antigen-presentation and/or the generation of a mitogenic signal.
Other T cell culture protocols include the use of cell lines to act as APCs, such as the K562/A*0201 cell line, as previously reported by our group [24
]. While the use of a cell line has the theoretical benefit of offering a “standardized” APC with known frequency and characteristics, disadvantages include the fact that only T cells from individuals with the same HLA type as the HLA molecule expressed on the cell line can be assayed. Maintaining multiple cell lines for routine immune monitoring may be challenging even for research laboratories. Patient PBMCs may also mount responses to the multiple allogenic epitopes present on the foreign cell line, which may mask or overwhelm the expansion of the low-frequency antigen-specific T cell population of interest.
Finally, other T cell culture protocols have utilized autologous dendritic cells. While dendritic cells serve as professional APCs that can present antigen more efficiently than non-professional APCs, the need to first generate and expand them in vitro from precursors in the peripheral blood prior to use in an assay is both cumbersome and time-consuming but may also be impractical in many situations where only a limited number of patient PBMCs are available for manipulation and analysis.
In our series of experiments, we demonstrated that culture with IL-2 and IL-15 led to greater T cell expansion than with IL-2 and IL-7 and was not further enhanced by the addition of IL-7. These results are in broad agreement with the findings of other investigators, who have demonstrated that several cytokines other than or in addition to IL-2 alone (IL-7, IL-12 and IL-15) can enhance T cell proliferation in various culture systems [25
More importantly, we also verified that the proportion of effector/ memory CD4+ and CD8+ T cells – as defined by the markers CCR7 and CD45RA – was not affected by the different cytokine culture conditions. Similarly, the proportion of effector/memory populations in tetramer-reactive CD8+ T cells was not influenced by the cytokine milieu. Finally, we further subdivided the CD8+CCR7-CD45RA- with the markers CD27 and CD28 and determined that the proportion of these subpopulations was similarly unaffected by cytokine conditions.
Our experiments also demonstrated that we were able to generate NY-ESO-1 specific polyfunctional CD4+ and CD8+ responses in a melanoma patient who was NY-ESO-1 seropositive. These T cells were capable of producing multiple cytokines, consisting of various combinations of IFN-γ, TNF-α, MIP-1β, IL-2 and CD107a. We did note that the staining pattern of these combinations differ from that of other T cell culture systems, where IL-2 and TNF-α production are often more abundant. One possible explanation is that the polyfunctional cytokine profile of a spontaneous response against a tumor-specific antigen such as NY-ESO-1 may differ from the response produced by viral antigens or by other strategies, e.g. prime-boost vaccination.
IL-2, TNF-α, MIP-1β and IFN-γ represent a relatively simple set of cytokines and chemokines that can be used to define a vaccine-elicited response. In addition, T cells mediate cytolytic activity through the release of perforin or granzymes. CD107a is an indirect measure of degranulation and provides further insight into important effector pathways[28
]. While immunization with vaccinia virus has been shown to induce polyfunctional CD8 T cell responses, a recent study by our group showed that tumor antigen-specific T cells are also polyfunctional[8
]. There are increasing data that such polyfunctional T cell responses, which correlate with the “quality” of the response, are associated with improved disease control in such chronic infections as HIV, tuberculosis and Hepatitis C [17
]. Murine experiments involving various vaccine formulations against Leishmania major
have correlated the development of a polyfunctional response with protection against subsequent challenge while, in humans, vaccinia vaccination, which is protective against smallpox, has been shown to generate a polyfunctional virus-specific CD8+ response.
Based on these observations, it is thought that vaccination strategies that can elicit polyfunctional responses are desirable and may ultimately be associated with increased protection/efficacy. While this concept has yet to be rigorously proven in human cancers, there are nevertheless clear implications for monitoring in any immune-based strategy in cancer trials. The ability to generate polyfunctional T cell responses will allow for correlation with clinical outcome to determine if a particular subset of T cells with a specific combination of effector functions is associated with clinical benefit. Such polyfunctional responses have the potential to become a surrogate biomarker by which the effectiveness of cancer immunotherapy can be judged.
Utilizing the T cell culture methodology described in this paper, our group has subsequently provided monitoring for 15 patients with metastatic melanoma receiving therapy with ipilimumab, a monoclonal antibody against cytotoxic T-lymphocyte antigen-4, a negative regulator of immune function. Our data indicate that patients who derive clinical benefit from ipilimumab experience an increase in NY-ESO-1 specific polyfunctional cytokine responses [29
In summary, we have presented a protocol for human T cell culture using peptide-pulsed irradiated autologous PBMCs as APCs. We believe that the use of autologous PBMCs offers theoretical and practical advantages over other systems that employ cell lines or expanded dendritic cells. To our knowledge, we are also the first to demonstrate that our T cell culture system is able to efficiently expand low-frequency precursors without biasing their baseline proportions and that our system is capable of monitoring antigen-specific polyfunctional T cell responses, which may play an important role in future immune monitoring.