Abstract
Although clonal expansion is a hallmark of adaptive immunity, the location(s) where antigen-responding T cells enter cell cycle and complete it have been poorly explored. This lack of knowledge stems partially from the limited experimental approaches available. By using Ki67 plus DNA staining and a novel data analysis technique, we distinguished antigen-specific CD8 T cells in G0, in G1, and in S-G2-M phases after intramuscular vaccination of BALB/c mice with antigen-expressing viral vectors. We discovered an entire population of cycling cells that are usually missed. This “extra” population was present early after vaccination in lymph nodes, spleen and, surprisingly, also in the blood, which is not expected to be a site for mitosis of normal non-leukemic cells. These results have implications for previous and future immunological studies in animal models, and potentially in humans. They might also inspire hematologists to seek for other missed populations of dividing cells in blood.
Introduction
Cell-to-cell interactions within tissue niches in solid organs and hematopoietic bone marrow (BM) regulate proliferation of stem cells and differentiated progenitors (1, 2), along with structural, physical, paracrine and neural cues provided by the microenvironment (3). Similarly, clonal expansion of T cells during adaptive immune responses is driven by antigen presenting cells within specialized niches in lymphoid organs, where local chemokines and cytokines guide T cell responses (4).
We nevertheless still lack essential spatial information on clonal expansion, particularly as to the location of T cells during each phase of the cell cycle. To date, T cell expansion in animal models has been mostly measured by dyes that label cells proliferating over time (e.g. CFSE; BrdU) (5, 6), without the ability to assess whether the labeled cells found in a particular location proliferated locally or rather migrated into that organ after dividing elsewhere. Another common method is staining for the intranuclear protein Ki67, after cell fixation and permeabilization (7–10). Though Ki67 is generally considered to label dividing cells, it actually labels all cells not in G0, not distinguishing actively cycling cells committed to mitosis (those in S-G2-M) from those in G1, which may quickly proceed into S, or stay in a prolonged G1, or even revert to G0 without dividing (11).
Here we used Ki67 plus DNA staining to track rare naïve antigen-specific CD8 T cells responding to vaccination in wild-type mice (12, 13). The naïve CD8 T cells clonally expanded, and we analyzed the resulting polyclonal population.
We found a significant number of antigen-responding CD8 T cells cycling in lymph nodes (LNs), spleen and (surprisingly) in the blood, a finding that opens new directions for the analysis of immune responses.
Results
BALB/c mice were vaccinated intramuscularly (i.m.) against the model antigen, HIV-1 gag, using a recombinant chimpanzee-derived adenoviral vector (ChAd3-gag) and a Modified Virus Ankara (MVA-gag) for priming and boosting, respectively (12). The cell cycle stages of gag-specific CD8 T cells were analyzed using Hoechst 33342, a DNA dye, and anti-Ki67 mAb (14, 15).
Fig 1A-B shows the steps for analysing gag-specific CD8 T cells by flow cytometry, fig. 1B an example of spleen and LN cell analysis at day (d) 3 post-boost. Steps 1-2 identify single cells by DNA analysis, and live cells by dead cell marker exclusion. Step 3 uses Forward Scatter-A (FSC-A) and Side Scatter-A (SSC-A) profiles to identify certain leukocyte populations. Lymphocytes tend to have low SSC-A and medium-low FSC-A, whereas granulocytes have high SSC-A, and are normally excluded from the canonical ‘narrow’ gate used for lymphocyte studies (16–19) (Fig. 1B, Step 3, ‘narrow’). However, we noticed an unusual population of cells with high SSC-A that appeared only in vaccinated spleens and contained a significant number of antigen-specific lymphocytes (Fig. 1B, Step 3, arrow). When we enlarged our FSC-A/ SSC-A gate (Step 3 ‘relaxed’), before labeling CD8 T cells (Step 4) and antigen-specific T cells (Step 5), we found a 2-6 fold greater proportion of gag-specific CD8 T cells in the ‘relaxed’ gate population than in the ‘narrow’ gate population: not only in the spleen but also in the LNs (Fig. 1B-D). Although this gating strategy is novel for standard ex vivo studies of lymphocytes (Fig. 1-S1), cells with high FSC-A and high SSC-A are often included when examining in vitro activated T cells (20).
In order to discriminate between gag-specific CD8 T cells in G0, G1, and S-G2-M, we examined Ki67 expression plus DNA content, using either the ‘narrow’ or the ‘relaxed’ gate (Fig. 2). We observed a striking difference in the percentages of proliferating cells between the two strategies. The ‘narrow’ gate missed most of the dividing cells in S-G2-M (<2%), whereas the ‘relaxed’ gate revealed that these cells made up to 42% of the gag-specific cells in LNs and 26% in spleen (Fig. 2A, C). Cell cycle entry and progression was accompanied by a graded increase of FSC-A, and more prominently of SSC-A (Fig. 2B-C). Proliferation was also seen after a single priming dose, though the kinetics were slower and there were fewer cells in S-G2-M (Fig. 2-S1).
The ‘narrow’ gate missed up to a third of gag-specific CD8 T cells in the blood (Fig. 3A), which —with the ‘relaxed’ gate— averaged 2% at d3, 36% at d7, and 13% at d44 post-boost (Fig. 3B). As expected (12), gag-specific cells down-modulated CD62L (Fig 3-S1A-B). A well-defined population of mitotic gag-specific CD8 T cells was revealed uniquely using the ‘relaxed’ gate. Cells in S-G2-M were obvious at d3 (up to 13%) and less evident at d7 when Ki67+ peaked (up to 94%), suggesting that Ki67+ cells (non G0) persist in blood after mitotic cells disappear (Fig. 3C-D; 3-S1C-D). By day 44, almost all gag-specific cells were in G0 (Fig. 3C), suggesting that they had mostly switched to a resting memory state. We also saw mitotic antigen-specific CD8 T cells in blood after a single priming shot of vaccine (Fig. 3-S2).
Hypothesizing that the increased DNA content of the expanding CD8 T cells could be exploited as a marker to identify antigen-responding cells in the blood, we focussed on CD62L(−) cells, as CD62L is generally down-regulated upon activation (Fig. 3-S1A-B). We evaluated the frequency of gag-specific cells among the following 4 populations of CD8 T cells (Fig. 4A): 1) total CD8 T (including naïve, memory and recently activated cells), 2) CD62L(−) (non-naïve cells), 3) CD62L(−) Ki67+ (non-Go non-naïve cells), 4) CD62L(−) in S-G2-M (dividing non-naïve cells). At day 3, the average percentage of gag-specific cells among the dividing non-naïve cells was 15-fold higher than among total CD8 T cells (Fig. 4C), sometimes up to 70% (Fig 4B), a much higher proportion than observed among the other 3 populations (Fig 4B-C). By d7, the gag-specific cells comprised 40% of the dividing non-naïve and 84% of the non-G0 non-naïve population. By d44 gag-specific cells were decreased in all the populations, though less evidently in the CD62L(−) population (Fig. 4C). Results were similar, though the kinetics slower, after a single priming dose (Fig 4-S1).
Since CD62L is a cell membrane molecule, and DNA can be visualized using vital dyes, our results suggest that the dividing CD62L(−) CD8 T cells in blood could potentially be a valuable source of live antigen-specific CD8 T cells at early times of response.
Discussion
Long ago, Sprent showed that, within days of an immunogenic stimulus, antigen-specific ‘blast’ T cells circulated in the thoracic duct lymph (21). There was no way of knowing at that time whether these were proliferating or simply activated cells. Here we show that mitotic antigen-specific T cells circulate in the blood stream, challenging the current view that the S-G2-M phases of clonal expansion occur only in lymphoid organs, or sometimes in BM, or in extra-lymphoid follicles in tissues (22, 23). Thus proliferation is not always limited to supportive tissues sites, but cells that have been stimulated in one organ can expand while circulating to other sites.
Mitotic gag-specific CD8 T cells were found in the blood after a single dose of ChAd3-gag, although they were fewer than after a boost with MVA-gag, possibly due to slower kinetics, and/or differences in spatial distribution of antigen-responding CD8 T cells inside the LNs (24) that were reflected in the blood. Further studies will be necessary to elucidate whether naïve and memory cells behave differently upon stimulation in vivo, whether vaccination route matters, and whether the cycling CD8 T cell clones in the blood comprise a special highly dividing subset and/or express high affinity TCRs.
The majority of dividing CD8 T cells in blood, spleen and LNs showed increased FSC-A and unusually high SSC-A, likely due to changes in mitochondria, chromatin condensation, etc. (25, 26). Cells with these characteristics are usually excluded from the analysis of normal lymphocytes ex vivo, for example human blood lymphocytes in conditions apart from cancer. Considering that nearly all immunological studies in humans use blood samples, important informations are likely to be missed, perhaps leading to incorrect conclusions. For example, it was found that up to 70% of virus-specific CD8 T cells were Ki67+ in the blood of patients at early phases of primary infections (8, 9), whereas memory-phenotype CD8 T cells from the blood of donors with no apparent infections comprised about 2-10% of Ki67+ cells (27). Furthermore, an early increase of Ki67+ PD-1+ CD8 T cells was observed in the blood of a subset of lung cancer patients treated with checkpoint inhibitors, and it was proposed that this could be relevant for antitumor effects (28). In all these studies it was suggested that the Ki67+ cells were proliferating in response to a recent immunogenic stimulus (8, 9, 27, 28), however cells with high side scatter were discarded (9, 28), and DNA content was not evaluated (8, 9, 27, 28), thus it cannot be distinguished whether the Ki67+ were actively cycling, or rather they were non-proliferating cells in G1, possibly on their way back to G0. Furthermore, the proliferation —when present— was likely greatly underestimated. A single study in humans did use DNA staining, and found that an average of <0.1% of memory-phenotype CD8 T cells were in S-G2-M in the blood of donors with no systemic diseases (27). The interpretation at that time was that blood-derived memory CD8 T cells are resting (27, 29, 30). We suggest instead that the cells in S-G2-M could have been newly activated cells responding to an environmental antigen.
Our results have several potential translational uses. For example, human blood might be the source of enriched populations of recently activated CD8 T cells, proliferating in response to vaccines, infections, transplantation and cancers, that could be studied, cloned and used therapeutically, even without knowing the antigen to which they are responding, or as a way of searching for that antigen. And, in cases where a patient presents with symptoms of immune activation, but no obvious infection, an analysis of the mitotic cells in the blood could reveal clues as to the cause of the symptoms and/or the target of the response.
Material and Methods
Adenoviral and MVA vectors
Replication defective, ΔE1 ΔE2 ΔE3 ChAd3 vector encoding HIV-1 gag protein under HCMV promoter (ChAd3-gag, 21) (31) and Modified Vaccinia Ankara encoding the HIV-1 gag protein under the control of vaccinia p7.5 promoter (MVA-gag) were used in all experiments.
Vaccination
Six-week-old female BALB/c mice from Envigo (S. Pietro al Natisone, Udine, Italy) were housed at Plaisant animal facility (Castel Romano, Rome, Italy), and divided into experimental groups of at least 40 mice each (untreated and vaccinated). All mice of the vaccinated group were primed with ChAd3-gag, and a subset was analyzed after priming only. The remaining primed mice were boosted once with MVA-gag, at either d60 (range 60-67) or d100 (range 95-109) post-prime. Results of d60 and d100 boosts were similar, thus we combined them. Viral vectors were administered i.m. in the quadriceps at a dose of 107 viral particles (vp) for ChAd3-gag and 106 plaque-forming units (pfu) for MVA-gag, in a volume of 50 μl per side (100 μl total). All experimental procedures were approved by the local animal ethics council and performed in accordance with national and international laws and policies (UE Directive 2010/63/UE; Italian Legislative Decree 26/2014). Vaccination procedures were performed under anesthesia, and all efforts were made to reduce animal numbers and minimize suffering.
Organs
Spleen, LNs and blood were obtained at different times after either prime or boost, i.e. d7, d10 and d14 post-prime; d3, d7 and d44 post-boost. At each time, the organs were collected from 3 vaccinated and 3 untreated mice, and cells from the 3 mice of each group were pooled. At d7 (and in one experiment at d3) blood was obtained by submandibular vein puncture in conscious mice. At all the other time points organ harvesting was scheduled, thus blood was obtained by cardiac puncture upon carbon dioxide euthanasia. Blood was immediately put into Heparin or EDTA blood collection tubes and further processed for analysis. Single-cell suspensions were prepared from spleen and LNs (iliac and inguinal) by mechanical disruption and passage through cell strainers (32).
Membrane Staining
Spleen and LN cells were incubated with Fixable Viability Dye conjugated with eFluor780 fluorochrome (Affimetrix, eBioscience, Santa Clara, CA) and background staining was blocked with anti-FcγR mAb (clone 2.4G2). Cells were then incubated for 15 minutes at 4°C with H-2k(d) AMQMLKETI APC-labeled Tetramer (Tetr-gag, NIH Tetramer Core Facility, Atlanta, GA) and PE-labeled Pentamer (Pent-gag, Proimmune, Oxford, UK) to stain for gag197-205(gag)-specific CD8 T cells. Cells were incubated for further 15 minutes at 4°C after addition of the following mAbs: anti-CD3 peridinin chlorophyll protein (PerCP)-Cy5.5 (clone 145-2C11, BD Biosciences), anti-CD8α BUV805 (clone 53-6.7, BD Biosciences), anti-CD62L phycoerythrin (PE)-Cy7 (clone MEL-14, Biolegend, San Diego, CA, USA). Blood samples were incubated for 30 minutes at RT with the above antibodies/reagents that were placed all together. After washing, blood cells were fixed with Cell Fix solution (BD Biosciences). Red cells were lysed with Pharm Lyse solution (BD Biosciences).
Intracellular Staining
Intracellular staining for Ki67 and DNA was performed as previously described, with some modifications (14, 15). Cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer (Affimetrix, eBioscience). Intracellular staining was performed with anti-Ki67 mAb conjugated with Fluorescein isothiocyanate (FITC) or Alexafluor 700 (clone SolA-15; eBioscience). DNA was stained by incubation with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA).
Flow cytometry analysis
Samples were analyzed by LSRFortessa flow cytometer (BD Biosciences), gating out CD3(−) cells when acquiring spleen samples. Data were analysed using FlowJo software, v.10 (FlowJo, Ashland, OR, USA).
Statistical analysis
At each time point, the vaccinated group was compared with its corresponding untreated group by performing a two-tailed unpaired Student t test with Welch’s correction. A two-tailed paired Student t test was used for comparison of N and R gates. Friedman test with Dunn’s multiple comparison was used for comparison of multiple cell subsets within vaccinated mice samples. Differences were considered significant when *p ≤ 0.05; ** p ≤ 0.01; *** p ≤0.001. Statistical analysis was performed using Prism v.6.0f, GraphPad Software (La Jolla, CA, USA).
Author contributions
F.D. designed experiments, interpreted the results and wrote the paper with help by S.S., A. Natalini and A.S.; A.F., S. C. and A. Nicosia prepared the viral vectors and performed mouse treatments, S.S. and A.N. performed/analyzed flow cytometry experiments.
Funding
Work supported by Reithera, by CTN01_00177_962865 (Medintech) grant from Ministero dell’Università e delle Ricerca (MIUR) and by 5 × 1000 grant from Associazione Italiana Ricerca sul Cancro (AIRC).
Conflict of interest disclosure
A.F., S. C. and A. Nicosia are employees of Reithera. Alfredo Nicosia is named inventor on patent application WO 2005071093 (A3) “Chimpanzee adenovirus vaccine carriers”. Authors do not disclose any other conflict of interest.
Acknowledgements
We thank A. Hayday, M. Munoz-Ruiz and F. Granucci for discussion. A special thank to P. Matzinger for providing precious advices and for reading the manuscript. The following tetramer was obtained through the NIH Tetramer Facility: APC-conjugated H-2K(d) HIV gag 197–205 AMQMLKETI