The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools
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The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.
KeywordsIntracellular cytokine staining Flow cytometry Monocytes Immunomonitoring T cells
A large body of data generated by mechanistic studies in animal models showed that both tumor-specific CD4+ T-helper type (Th) 1 and cytotoxic CD8+ T cells (CTL) play a major role in controlling tumor growth [1, 2, 3]. Cohort studies indicating an increased incidence of cancer in immune-suppressed patients or showing that the presence of memory CD4+ Th1 and CTL in tumors is predictive for a beneficial clinical outcome as well as clinical trials in which patients display clinical benefit after adoptive transfer of tumor-specific T cells or after therapeutic vaccination sustain these conclusions . Recent data from immunotherapy studies suggest that the expansion of antigen-specific infused T cells [5, 6] or the magnitude, type and breadth of the vaccine-induced T-cell reaction [7, 8, 9, 10, 11] may correlate with success or failure to respond to treatment and reinforces the notion that the ability to type and enumerate T-cell reactivity within clinical samples is an important asset in the development of new treatments for cancer. Evidently, the number and type of assays that can be used are determined by logistics. In most cases, a number of only relatively small (50 mL) blood samples are taken because patients have no problems consenting to this and because it is easy to isolate peripheral blood mononuclear cells (PBMC) and to store them in liquid nitrogen for later studies. Different techniques have been developed to measure and enumerate the T-cell response to as many as possible epitopes within one sample directly ex vivo. One such method to gain information about T-cell reactivity at feasible extent constitutes the stimulation of PBMC with overlapping peptide pools of defined antigens. This allows for simultaneous testing of functional reactivity of both CD4+ and CD8+ T cells after vaccination or during viral infection [12, 13], both by IFN-γ ELISPOT assay and by the flow cytometry-based intracellular cytokine staining (ICS), with the latter being less equipped to measure low-level responses . In addition, pools containing peptides that are 5–6 amino acids longer than the exact HLA class I-restricted T-cell epitopes resulted in the detection <80 % of the frequencies found than when T cells were stimulated with the exact HLA-fitting peptide epitope, although they were appropriate for the stimulation of CD4+ T cells . We have used single or pooled 30-mer peptides for the detection of CD4+ and CD8+ T-cell responses against influenza matrix protein 1 (M1), following one round of enrichment and expansion , under the premise that this would also allow us to screen T-cell reactivity against a high number of pooled antigens if patient samples are limiting. While this allowed us to stimulate both CD4 and CD8 T cells during 10-day cultures as well as to measure CD4+ T-cell reactivity, we needed to return to large pools of 10-mer peptides to appropriately measure CD8+ T-cell reactivity against HPV antigens [10, 11], as it was difficult to separate populations of responding CD8+ T cells from the background.
In order to optimize the simultaneous detection of both IFN-γ-producing CD8+ and CD4+ T cells using pools of long peptides directly ex vivo, we exploited the T-cell response to influenza M1, as these responses are present in the majority of humans at similar frequencies that are expected to arise after vaccination with cancer vaccines. Based on our previous IFN-γ-ELISPOT analyses, influenza M1-specific CD4+ T-cell responses and the HLA-A*0201-restricted influenza M1 CD8+ T-cell epitope are present in frequencies between 1/10,000 and 1/1,000 [17, 18, 19, 20], which is about the frequency of tumor-specific T cells after vaccination [8, 11].
Materials and methods
The authors acknowledge the concept of the Minimal Information About T cell Assays (MIATA) framework, which was recently published [21, 22]. Therefore, detailed information is provided as structured in the proposed 5 modules by MIATA: the sample, the assay, the data acquisition, the data analysis and the laboratory environment in which the human T-cell assays were performed.
Media and reagents
IMDM (Lonza, Verviers, Belgium), supplemented with 100 U/mL penicillin/100 μL/mL streptomycin (Invitrogen, Grand Island, NY, USA), 2 mM l-glutamine (Cambrex, East Rutherford, NJ, USA) and human AB serum (Greiner, Alphen aan den Rijn, the Netherlands), assigned as complete IMDM, or X-Vivo 15 medium (Lonza) were used as indicated. The following peptides were used in this study: CMVpp65 495-503 (CMV Short Peptide or CMV SP), CMVpp65 483-512 (CMV Single Long Peptide or CMV SLP), Influenza M1 58-66 (SP) and peptides spanning the whole M1 protein derived from influenza, consisting of 16 peptides with a length of 30 amino acids and an overlap of 15 amino acids (C-terminal peptide with an overlap of 18 amino acids; SLP, Long Peptide Pool 1, 2, 3, 4 or LPP1, 2, 3, 4, each pool consist of 4 long peptides), were synthesized with >95 % purity  and dissolved in DMSO at the concentration of 50 mg/mL and then further diluted to a concentration of 1 mg/mL in phosphate buffered saline (PBS) and stored at −20 °C. The cytokines used in this study were GM-CSF (800 IU/mL; Immunotools, Friesoythe, Germany) and interferon alpha (Roferon-A, which is IFN-α2a). Memory Response Mix (MRM; stock 4×), consisting of tetanus toxoid (0.06 LF/mL; National Institute of Public Health and the Environment, Bilthoven, The Netherlands), mycobacterium tuberculosis sonicate (0.4 μg/mL; Royal Tropical Institute, Amsterdam, The Netherlands) and Candida Albicans (0.0012 %; HAL Allergenen Lab, Haarlem, The Netherlands).
PBMC used in this study were derived from anonymous HLA-A*0201 healthy blood bank donors (Sanquin, The Netherlands) and from 3 patients vaccinated with a p53 vaccine in the LUMC, after informed consent. PBMC were isolated within 24 h after blood drawl by Ficoll density gradient centrifugation and cryopreserved in 90 % Fetal Calf Serum (FCS; PAA laboratories, Pasching, Austria) and 10 % DMSO (Sigma, St Louis, MO, USA). Cells were stored in the vapor phase of the liquid nitrogen vessel until further use. The handling and storage of the blood samples were performed according to the standard operating procedure (SOP) of the department of Clinical Oncology, section Experimental Cancer Immunology and Therapy at the Leiden University Medical Center by well-trained personnel.
The cryopreserved PBMC were thawed and subjected to the T-helper ELISPOT assay, both according to SOPs and as described previously [8, 10, 11, 19, 20]. Briefly, PBMC were seeded at a density of 2 × 106 cells per well in a 24-wells plate (Costar) in 1 mL of complete IMDM in the presence or absence of 5 μg/mL of indicated influenza M1-derived 30-mers peptides combined in pools (LPP1, 2, 3 and 4). As a positive control, PBMC were cultured in the presence of MRM. After 4 days of stimulation in the incubator (37 °C, 5 % CO2, 92 % RH), PBMC were harvested, washed, resuspended in complete IMDM (however, the 10 % human AB serum was exchanged for 10 % FCS) and seeded in four replicate wells at a density of 105 cells per well in a Multiscreen 96-well plate (MAHAS45, Millipore, Billarica, MA, USA) coated with the IFN-γ-catching antibody (Mab-1-D1K, Mabtech, Nacka Strand, Sweden). Further antibody incubations and development of the ELISPOT was done according to the manufacturer’s instructions (Mabtech). Spots were counted with a fully automated computer-assisted video-imaging analysis system (BioSys 5000). Specific spots were calculated by subtracting the mean number of spots + 2 × SD of the medium only control from the mean number of spots in experimental wells. Antigen-specific T-cell frequencies were considered to be positive when specific T-cell frequencies were ≥1 of 10,000 .
To determine the frequency of antigen-specific CD8+ T cells, a CTL ELISPOT was conducted as according to our SOP, which is also provided on the CIMT website (http://www.cimt.eu/dl/sop_elispot.pdf). Briefly, after thawing, the PBMC were rested overnight in complete IMDM in the incubator (37 °C, 5 % CO2, 92 % RH). The following day, the cells were washed, resuspended in complete IMDM (however, the 10 % human AB serum was exchanged for 10 % FCS) and seeded at a density of 5 × 105 cells per well in the with IFN-γ-catching antibody-coated ELISPOT plate (Millipore), in the presence or absence of 1 μg/mL of indicated influenza M1 or CMV short peptide (SP). As a positive control, PBMC were stimulated on the plate with PHA. The staining and the analysis were similar as described above for the T-helper ELISPOT.
Antigen-specific T-cell stimulation and ICS
The T-cell stimulation and staining as it was finally performed are described in the SOP shown as online resource 1. A positive response was defined as a frequency of antigen-specific T cells in the test sample, which was at least twice that of the non-stimulated PBMC (negative control). Although the definition of clearly clustered population is subjective, this was taken along in the interpretation of whether a response was found to be positive or not.
In all experiments, the mean of the triplets of IFN-γ+ T cells (CD8+ or CD4+) for each donor and antigen and the coefficient of variation (CV) was calculated. To compare the influence of IFN-α on the percentage of IFN-γ+ CD8+ cells against SLP, a paired students t test was used.
The laboratory of the Clinical Oncology, section Experimental Cancer Immunology and Therapy at the Leiden University Medical Center, is a research laboratory where the assays are performed according to SOPs, including the predefined criteria for positive responses, by well-trained personnel.
High-, intermediate- and low-frequency IFN-γ-producing CD8 T cells are detectable by intracellular cytokine staining and flow cytometry analysis when exact CTL-epitope peptides are used
The use of IFN-α increases the detection of CD8+ T-cell reactivity against LPP in cultures of stimulated PBMC
The detection of CD8+ T-cell responses ex vivo requires APC pulsed with much higher concentrations of single long peptides than short peptides and may be improved by the use of poly(I:C)
Responses observed with the adapted protocol for SLP are comparable to that detected with SP tested according to the original protocol
Both CD8+ and CD4+ T-cell responses are directly ex vivo detected in one single ICS assay
In order to test whether the number of peptides used in the LPP would influence the percentage of cells recognizing a particular epitope, we tested the PBMC from one donor against the SP, SLP and different LPPs comprising 4, 8, 12 or 16 of the Influenza long peptides. In another case, the influenza SLP was mixed with 4, 8, 12 or 15 different p53 SLP  to form LPPs of different sizes. In both cases, the response to the SLP was not altered by the addition of extra “irrelevant” peptides (Fig. 5c).
All together, our results show that the adapted ICS protocol allows the ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T cells to both known and unknown epitopes. Importantly, the reaction is not altered by the addition of extra peptides, which indicates that the antigen-specific reactions in PBMC samples can be screened by using pools of peptides and can, therefore, be very useful in the immunomonitoring of vaccination trials.
The adapted ICS protocol is superior in detecting low-frequency p53-specific CD4+ T-cell responses in PBMC from patients participating in a p53 vaccine trial
In this study, we have improved an ICS protocol to directly ex vivo detect low to high frequencies of antigen-specific CD4+ and CD8+ T-cell responses, by using pools of long peptides. Previously, we had shown that SLP and LPP could perfectly stimulate antigen-specific CD4+ and CD8+ T cells as well as could be used to detect T-cell reactivity in activated T-cell cultures by ICS or other assays that lasted a couple of days [10, 11, 16], but the existing ICS protocol failed to detect T-cell reactivity directly ex vivo in cryopreserved samples. The ability to directly ex vivo measure low-frequency CD8+ and CD4+ T-cell responses using SLP or LPP depended on the concentration of the long peptides used, which needed to be at least tenfold higher, and was enhanced by compounds that increased processing and presentation of these peptides by the APC, in our hands IFN-α and poly(I:C). Furthermore, our data indicated that the presence of other peptides (up to 16 different long peptides)—which theoretically may congest the uptake and processing machinery—did not alter the processing and presentation of SLP. All together, this suggests that especially the capacity to ingest and process enough amounts of peptide within the time frame used here is the rate-limiting factor for sufficient presentation of peptide epitopes in HLA class I and II. This is not a factor when exact HLA-fitting peptide epitopes are used as they can bind to the HLA molecules at the cell surface without a requirement for uptake and processing. Also for the mouse OVA model, it has been shown that a higher protein concentration resulted in a more efficient cross-presentation to antigen-specific CD8+ T cells [32, 33].
In addition to an enhanced cross-priming , IFN-α also upregulates the co-stimulatory molecule CD80 on monocytes, a molecule essential for T-cell activation [34, 35]. Poly(I:C) is also known to activate and enhance the expression of co-stimulatory molecules on APC [36, 37]. Improved co-stimulation may explain part of the increased reactivity to SLP-/LPP-pulsed monocytes. Recent studies in the field of HIV have shown the use of antibodies to CD28 and CD49d to co-stimulate T cells during in vitro stimulation to enhance their reactivity [38, 39]. Possibly, the use of such antibodies in our protocol may enhance responsiveness also, but this was not tried as also an increase in background reactivity was observed in the aforementioned studies , and this is known to affect the capacity to detect low-frequency responses [18, 24, 40]. Finally, IFN-α is known to enhance the survival of activated T cells , and one can envisage that keeping the activated cells alive during the test increases the detection efficiency of low-frequency T cells.
While comparing the responses against the exact CD8+ peptide epitope (SP) with SLP or LPP of either influenza M1 or CMV, we observed that CD8+ T-cell responses had a reactivity toward a SLP ranging between 50 and 90 % of the response detected by applying the SP as antigen. In situations where the T-cell epitopes are known and patients are included based on the restricting HLA types, it is, therefore, preferred to use the exact HLA-fitting peptides for stimulation of the PBMC. In other cases, for instance when vaccines are used that encode for whole proteins or comprise whole proteins and the patient population constitute a multitude of different HLA types, LPPs may be used to detect a number of CD4+ and CD8+ T-cell responses within one or more patients. This can be important as we have recently shown that both the breadth and the magnitude of a T-cell response was associated with the clinical response to vaccination [8, 10], however, with the trade-off that some responses can be missed.
Applying our previous and adapted protocol to PBMC samples of patients vaccinated in an ongoing p53-vaccination trial revealed that the new protocol also displayed better performance with respect to the detection of CD4+ and CD8+ T-cell responses. The responses measured in cryopreserved PBMC by the old protocol were clearly inferior to those measured when freshly isolated. The adapted protocol, however, restored the measured activity to the levels found with freshly isolated PBMC.
In conclusion, here we present a successfully improved and robust ICS protocol to detect antigen-specific CD4+ and CD8+ T cells directly ex vivo in cryopreserved PBMC samples using one single vial of PBMC. The assay, thus, is highly economical with respect to the usage of the restricted amounts of patient material generally available for immunomonitoring of cancer trials. Whereas other sensitive and quantitative assays such as the ELISPOT assay also allow for ex vivo measurements, it is important to obtain as much information about the reacting T cell as possible since trial samples are unique. The current assay allows for more detailed analysis of the T-cell phenotype as it utilizes flow cytometry. The costs for the assay are higher, but assay costs are less important when one considers the expenditure of a trial. With this protocol, we have consistently measured antigen-specific responses with intra- and inter-assay variation below 30 % as well as were able to detect low-frequency responses. This makes the improved assay suitable for the monitoring of samples from anticancer vaccination trials.
This study was financially supported by a grant from the Wallace Coulter Foundation to SHvdB.
Conflict of interest
The authors declare that they have no conflict of interest.
This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
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