Name: Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells (Video) | JoVE Journal
Uploaded: 2025-03-07T13:40:00.000+00:00
Duration: 9 min 4 s
Description: Learn to analyze human T cell activity in allogeneic co-culture with pre-treated tumor cells using flow cytometry. This method reveals how radiotherapy and kinase inhibitors modulate immune responses.

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5 minutes

This research was partly funded by the Interdisciplinary Center for Clinical Research Erlangen (IZKF Erlangen) and the Bayerisches Zentrum f&#252;r Krebsforschung (BZKF).

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</ol>

The authors declare no conflicts of interest.

The protocol presented here offers a fast and easy method to analyze the proliferative capacity of T cells along with their activation status in a co-culture setting with pre-treated tumor cells. Thereby, the effects of different treatment modalities, such as RT, CT, or TT, on T cell activity and proliferation can easily be screened, building the foundation for subsequent deeper immunological analyses of promising approaches. The representative results shown in this manuscript prove that this allogenic T cell co-culture assay is performing well. Significant differences in T cell proliferation, as well as in T cell activity, were observed for the co-culture with differentially pre-treated human HSC4 (HNSCC) cells (Figure 4 and Figure 5). It was found that the treatment of the HSC4 with either RT alone or in combination with TT resulted in an increased proliferation of the T cells, in particular in regard to the highly proliferative fraction of the T cells (Figure 4). In line with the quantification of the proliferation rate, the activation state of the T cells was also differentially affected by the different treatment regimes. In summary, the application of RT resulted in a strong downregulation of the expression of CD25 on T cells, whereas the expression of HLA-DR was upregulated. RT induces DNA damage in the irradiated tumor cells, which in turn leads to cellular stress responses that include the release of stress- and damage-associated molecules and cytokines, as well as the expression of immune-modulating cell surface ligands19. 
The combination of RT with TT in the form of DNA-repair inhibitors is further enhancing and maintaining those effects, as the tumor cells cannot efficiently repair the RT-induced DNA damage. This might further promote and maintain the secretion of immunogenic factors and the expression of immunogenic cell surface ligands20. In accordance with this, we have demonstrated previously that the treatment of tumor cells (HNSCC) with RT and DNA damage inhibitors alters the immune phenotype on the tumor cell surface. The modulation of the immune phenotype included the regulation of the immune-stimulatory molecule ICOS-L as well as the immune-suppressive molecule PD-L111. In response to this modulation of the tumor cell immune phenotype, T cells might regulate the expression of activation markers on their cell surface as well. Further, RT is well-known to stimulate the release of pro-inflammatory cytokines, such as IFN&#947; or IL-6, which in turn impact T cell proliferation and activity21. The expression of activation markers on T cells is a highly dynamic process. Data from Zimmerman et al.22 showed that CD25 is highly expressed 24 h after stimulation but is down-regulated again after 96 h post-stimulation. This is in line with our findings. In contrast, HLA-DR is a late-phase activation marker and is generally preceded by an increase of CD25 and CD6923. Noticeably, the combination of RT + ATRi leads to a significant upregulation of HLA-DR on T cells compared to RT + ATMi. This finding is in line with work of Dillion et al. who demonstrated synergistic effects of ATRi + RT in inducing an inflammatory TME24. A less pronounced modulation of the T cell activity and proliferation was achieved by the tumor cell treatment with the kinase inhibitors alone (Figure 5). This might be due to the fact that the DNA repair inhibitors ATMi and ATRi are functioning as an enhancer of the RT-induced DNA damage and are consequently not very immunogenic when applied as a monotherapy. We already demonstrated the minimal toxicity of ATMi or ATRi alone11. In summary, the results confirm the immunogenic potential of RT (and TT with DNA damage inhibitors) and further indicate that this experimental system is suitable for screening the immunostimulatory capacities of distinct treatment modalities in human head and neck cancer cells.
Even though the method presented here is easy and robust, there are some critical steps in the protocol that have to be considered beforehand. The presence of high EDTA concentrations during the isolation process, as well as during the flow cytometric analysis of the T cells, is necessary. Thus, it is recommended to collect the donor blood into EDTA-coated collection tubes and to use a MACS buffer supplemented with 10 mM EDTA to ensure efficient T cell isolation and the generation of a homogeneous single-cell suspension for the flow cytometric evaluation. In addition, the composition of the T cell medium is of major importance for the proliferative capacity of T cells. After testing different culture media compositions, a medium with low concentrations of L-Arginine and L-Lysine results in the best T cell proliferation signal, as L-Arginine is critical for T cell metabolism and survival and should be supplied in a suitable concentration25,26. Nonetheless, one should keep in mind that L-Arginine and L-lysine solutions are not stable long-term and thus need to be used within 3 months. For optimal stimulation, the T cell medium supplemented with the amino acids needs to be prepared freshly for every T cell cultivation experiment. Moreover, the cell numbers of the T cells, as well as those of the tumor cells, are critical factors that need to be considered as they impact the readout of the experiment. 
For the tumor cells, on the one hand, cell confluency of 70% to 80% is desired at the end of the co-culture experiment. An overgrowth of the tumor cells might result in the secretion of factors that inhibit cell growth and would subsequently also impact the T cell proliferation rate. As the tumor cell growth is highly dependent on the individual cell line, we recommend thoroughly testing the growth behavior of the respective tumor cell line in different well plates and for the different treatment regimes. Further, the tumor cells and the T cells should be co-cultured in a ratio of 1:1. Therefore, it is obligatory to seed an extra well of tumor cells for each treatment condition, which can be used for the determination of the cell count on the day of the onset of the co-culture. Thereby, one can ensure that a suitable number of T cells will be seeded for the different treatment conditions. Concerning the cell number of the T cells, it is necessary to consider that the amount of T cells that can be isolated from a healthy donor is very individual. The T cells account for around 45% to 70% of the PBMCs of a healthy donor27. Thus, the required amount of T cells should already be broadly estimated at the time of the blood withdrawal. Moreover, the T cell density is important for the efficiency of the T cell activation and their survival. Consequently, the T cells should be seeded in a concentration of at least 1.5 million cells per well of a 6-well plate in 3 mL of T cell medium for the initial stimulation before the co-culture onset. A higher T cell density is possible, but a lower density should be avoided. The activation of the T cells can also be evaluated morphologically under the microscope, as activated T cells tend to form cell clusters.
As this method is a simplified experimental system for studying the immunostimulatory capacities of different tumor cell lines and treatment modalities, it has some limitations that need to be taken into consideration. First, this assay is based on an allogeneic system, meaning that the donor T cells are not HLA-matched with the respective tumor cell line. Thus, this HLA-mismatch might already induce T cell stimulation and, subsequently, T cell proliferation without further stimulation from the tumor cell treatment28. To quantify this unwanted effect, isolated T cells were co-cultured with the HSC4 cell line without prior stimulation with CD28 and CD3 antibodies. It was found that almost no proliferation was induced in this setting, which indicates that the HLA-mismatch only has minor effects on the T cell activation in this specific experimental setting. Besides, significant differences in the T cell proliferation and activation based on the tumor cell treatment despite the allogenic setting were found. Nevertheless, for the establishment of this assay, the potential allogeneic-induced proliferation of T cells should be tested and quantified once at the beginning of the experiments. The recommended "only T cell" and "only tumor cell" controls are needed for sufficient gating and are mandatory in every replication of the experiments. Nonetheless, one needs to keep in mind that an allogeneic co-culture system lacks tumor-specific antigen recognition. Thus, this system may not accurately reflect the specific anti-tumor response as it would occur in vivo in patients29,30. For further improvement, a more sophisticated setting would be an autologous co-culture approach. In this setting, patient-derived tumor biopsies need to be cultivated and brought into culture with T cells isolated from the same patient's peripheral blood28. This experimental approach, however, might be challenging not only in terms of culturing primary tumor cells but also in terms of the availability of patient-derived biomaterial. A further limitation that needs to be considered is the ratio of the tumor cells and the T cells in the co-culture. As the recommended ratio of the cells is 1:1, the ratio does not reflect the physiological situation of the TME in patients31. However, this limitation has to be accepted, as with lower T cell counts, the changes in the proliferation rate and activation status are not quantifiable. 
In the field of tumor immunology, this assay offers the opportunity to screen for the immunogenicity of different treatment modalities in an easy and fast experimental in vitro setting. Therefore, not only can time be saved by pre-screening the most promising approaches in terms of immunogenicity, but also in vivo experiments. Animal models can be reduced, as only promising treatment schemes might be further pursued in animal models to reflect the immune system as a whole in an organism. Further, as this assay is based on human primary cells and human cancer cell lines, the results might be more translatable into the clinic than assays that are based on other model systems and species. 
In the future, this assay might be modulated and adapted to answer more specific research questions. For instance, one could include a treatment of the T cells to reflect a treatment scenario that is closer to the situation in the patients. Since tumor-reactive T cells are mostly found in the TME, they are equally affected by local therapies such as RT and are likely also affected by systemic therapies such as CT or TT32. A further enhancement of the assay is the co-cultivation of T cells with tumor spheroids from tumor cell lines or even the co-cultivation with patient-derived tumor organoids. These three-dimensional cultures are more comparable to the structure of a tumor in a patient28. Finally, also the readout of the experiment via flow cytometry can be adapted easily in order to investigate further molecules on the surface of T cells or by analyzing the expression of immune checkpoint molecules on the tumor cells. In addition to the determination of the immune phenotype of the T cells, one could use the cell culture supernatants from the co-culture experiments for the quantification of secreted cytokines or chemokines to gain more insights into the T cell activity. In summary, this protocol delivers a comprehensive, robust, and easy T cell and tumor cell co-culture assay that allows screening for the immunogenicity of different cancer treatment modalities. As this assay is adaptable to specific research questions, it is well-suited for application in the broad field of tumor immunology.

Nowadays, it is becoming ever more evident that the outgrowth and progress of tumors are strongly dependent on the effective manipulation and suppression of the host&#39;s immune system. Transformed cells emerge every day, even in a healthy organism. However, the formation of macroscopic tumors is a rather rare event, as emerging transformed cells are removed from the organism with a high efficiency. For the removal of malignant cells, different immune cell types such as cytotoxic T cells, natural killer (NK)T cells, NK cells, or macrophages come into action1,2,3. Nonetheless, sometimes transformed cell clones may emerge that survive in an equilibrium state with the host&#39;s immune system, which is characterized by different immunosuppressive strategies of the tumor cell clone4. Eventually, some transformed cells acquire further functions that enable the tumor cells to actively suppress the immune response, which consequently leads to the outgrowth of the tumor. This immunosuppression is mediated by numerous mechanisms, which include the expression of immunosuppressive ligands on the tumor cells or the active recruitment or priming of immunoregulatory or immunosuppressive immune cell populations. This so-called immune editing concept demonstrates the key role of immune modulation during tumor formation and outgrowth5.
Thus, it is not surprising that the immune system is nowadays a major focus not only in cancer therapy but also as a predictive and prognostic factor in many tumor entities and therapeutic settings. During the last years, immune checkpoint inhibitors (ICIs) came up as promising therapeutic options in different solid cancer entities, such as head-and-neck squamous cell carcinoma (HNSCC) or non-small cell lung cancer (NSCLC) with the goal of modulating the tumor microenvironment (TME) towards a more efficient anti-tumor immune response and a diminished immunosuppression of the tumor cells6. The immune checkpoint inhibitors aim to boost the T cell-mediated tumor cell killing by targeting immune checkpoint molecules such as those of the programmed death-protein 1 (PD-1) and its ligand 1 (PD1/PD-L1) axis. This fact stresses the key role of T cells in anti-tumor immunity. In HNSCC, for example, ICIs have successfully been approved as first-line therapy in recurrent and metastasized HNSCC7. In line with that, the presence of cytotoxic T cells in the TME, as well as the expression of PD1 and PD-L1 on the tumor cells and T cells accordingly, can serve as predictive biomarkers in HNSCC8,9,10.
Even though T cells have a crucial role in tumor immunology and tumor therapy, many open questions about their interactions with the tumor still need to be addressed. Nowadays, it is well-known that the tumor immune response is a dynamic process and that the immunogenicity of tumors can change throughout the course of the disease and of the treatments. Different treatment modalities, such as chemotherapy (CT), radiation therapy (RT), or targeted therapy (TT), are especially widely known to modulate the immunological phenotype of tumor cells. RT can drive the upregulation of immune checkpoint molecules in the tumor tissue and alter the frequency of tumor-infiltrating cells11,12. TT, on the other hand, can also endorse favorable changes within the tumor and the TME by direct modulation of the adaptive immune response13,14,15,16. However, these modulations are challenging to study in patients, as this would require the repetitive examination of the tumor tissue during the course of treatment. Thus, conclusive experimental model systems are needed to study the dynamic immunological phenotype of tumor cells and T cells and, more importantly, their interactions.
Hence, for the purpose of analyzing T cell activity and the interactions of tumor cells and T cells, a comprehensive ex vivo co-culture assay is needed that is easy to implement in any laboratory based on straightforward cell culture work and commonly used flow cytometry analyses. Based on the existing and available literature, no easy-to-use and commonly used protocol regarding the co-culture of T cells and tumor cells has been published so far. Even though several co-culture assays for T cells and tumor organoids have been published lately, the 3D cell culture technique is nowadays still not implemented as a standard technique in every laboratory. Thus, we provide a protocol for the use in 2D cell culture, which might as well be established for 3D cell culture in the future. Other 2D co-culture protocols are often more complex, as they require, for example, the transduction of tumor cells with luciferase17 or are only suitable for hematological malignancies (co-culture of unmatched T cells with leukemia cells)18. In the assay described here, T cells from normal healthy donors are isolated from peripheral blood and stimulated with anti-CD3 and anti-CD28 antibodies. Subsequently, the T cells are co-cultured with (pre-)treated tumor cells in order to analyze the proliferative capacity of the T cells as well as their activity via flow cytometry. Thereby, the effects of different treatment modalities on the tumor cell immune phenotype, such as RT, CT, or TT, which in turn influences T cell activity and proliferation, can easily be screened and used as a basis for deeper mechanistic analyses and consecutive selected pre-clinical in vivo analyses. The assay described here offers an easy-to-use setup, as no unconventional devices, techniques, or materials are necessary. In addition, the assay can be easily adapted to different tumor cell lines or specific T cell subsets (e.g., CD4+ T cells). Using this technique, high standardization and reproducibility are achieved.

<table><tbody><tr><td>15 mL Cellstar tubes</td><td>Greiner Bio-One GmbH</td><td>188271</td><td></td></tr><tr><td>50 mL Cellstar tubes</td><td>Greiner Bio-One GmbH</td><td>227261</td><td></td></tr><tr><td>6 well cell culture plate sterile, with lid</td><td>Greiner Bio-One GmbH</td><td>657160</td><td></td></tr><tr><td>96 well cell culture plate sterile, F-bottom, with lid</td><td>Greiner Bio-One GmbH</td><td>655180</td><td></td></tr><tr><td>AZD0156</td><td>Selleck Chemicals GmbH</td><td>S8375</td><td></td></tr><tr><td>Berzosertib (VE-822)</td><td>Selleck Chemicals GmbH</td><td>S7102</td><td></td></tr><tr><td>CASYcups</td><td>OMNI Life Science GmbH &amp;amp; Co KG&amp;nbsp;</td><td>5651794</td><td></td></tr><tr><td>CASYton</td><td>OMNI Life Science GmbH &amp;amp; Co KG&amp;nbsp;</td><td>5651808</td><td></td></tr><tr><td>CD25a, PE-Dazzle594, Mouse IgG1</td><td>Biolegend</td><td>356126</td><td></td></tr><tr><td>CD28-UNLB</td><td>Beckmann Coulter, Inc.</td><td>IM1376</td><td></td></tr><tr><td>CD3a,Krome Orange, Mouse IgG1</td><td>Beckmann Coulter, Inc.</td><td>B00068</td><td></td></tr><tr><td>CD3e Monoclonal Antibody</td><td>Thermo-Fisher Scientific, Inc.</td><td>MA1-10176</td><td></td></tr><tr><td>CD4, APC, Mouse Anti-Human Mouse IgG1</td><td>BD Pharmingen</td><td>555349</td><td></td></tr><tr><td>CD8 MicroBeads, human</td><td>Miltenyi Biotec, Inc.</td><td>130-045-201</td><td></td></tr><tr><td>CD8a, PerCP-Cy5.5, Mouse IgG1</td><td>Biolegend</td><td>300924</td><td></td></tr><tr><td>CellTracker Deep Red Dye</td><td>Thermo Fisher Scientific, Inc.</td><td>C34565</td><td></td></tr><tr><td>CFSE</td><td>&amp;nbsp;Merck KGaA (Sigma-Aldrich)</td><td>21888</td><td></td></tr><tr><td>DMEM (Dulbecco&amp;acute;s Modified Eagle&amp;acute;s Medium)</td><td>PAN-Biotech GmbH</td><td>P04-02500</td><td></td></tr><tr><td>DxFlex Flow Cytometer (with Auto Loader)</td><td>Beckmann Coulter, Inc.</td><td>C44326, C02846</td><td></td></tr><tr><td>EDTA disodium salt dihydrate</td><td>Carl Roth GmbH + Co. KG</td><td>8043.2</td><td></td></tr><tr><td>FBS superior&amp;nbsp;</td><td>&amp;nbsp;Merck KGaA (Sigma-Aldrich)</td><td>S0615-500ML</td><td></td></tr><tr><td>FBS superior&amp;nbsp;</td><td>&amp;nbsp;Merck KGaA (Sigma-Aldrich)&amp;nbsp;</td><td>S0615-100ML</td><td>For production of heat-inactivated FBS. Heat up for 30 min at 56 &amp;deg;C with mixing to inactivate complement proteins.</td></tr><tr><td>Graph Pad Prism (version number 9)</td><td>GraphPad Software</td><td>-</td><td></td></tr><tr><td>HLA-DR, DP, DQ Antibody, anti-human, APC-Vio770</td><td>Miltenyi Biotec, Inc.</td><td>130-123-550</td><td></td></tr><tr><td>Kaluza (version number 2.1)</td><td>Beckmann Coulter, Inc.</td><td>-</td><td></td></tr><tr><td>L-Arginin</td><td>Merck KGaA (Sigma-Aldrich)</td><td>A8094-25G</td><td></td></tr><tr><td>L-Lysin-monohydrochloride</td><td>Merck KGaA (Sigma-Aldrich)</td><td>L5626-100G</td><td></td></tr><tr><td>MACS BSA Stock Solution</td><td>Miltenyi Biotec, Inc.</td><td>130-091-376</td><td></td></tr><tr><td>MS Columns</td><td>Miltenyi Biotec, Inc.</td><td>130-042-201</td><td></td></tr><tr><td>Neubauer-improved counting chamber</td><td>Paul Marienfeld GmbH &amp;amp; Co. KG&amp;nbsp;</td><td>640010</td><td></td></tr><tr><td>PBS</td><td>Merck KGaA (Sigma-Aldrich)</td><td>D8537-500mL</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo-Fisher Scientific, Inc.</td><td>1514-122</td><td></td></tr><tr><td>ROTISep 1077</td><td>Carl Roth GmbH + Co. KG</td><td>0642.2</td><td></td></tr><tr><td>RPMI-1640 Medium</td><td>Merck KGaA (Sigma-Aldrich)</td><td>R1790</td><td></td></tr><tr><td>SepMate 50mL tubes</td><td>Stemcell Technologies</td><td>85450</td><td></td></tr><tr><td>Trypan blue</td><td>Merck KGaA (Sigma-Aldrich)</td><td>T6146-25G</td><td></td></tr><tr><td>Trypsin</td><td>Thermo-Fisher Scientific, Inc.</td><td>15400054</td><td></td></tr></tbody></table>

analysis of human t cell activity in an allogeneic co-culture setting of pre-treated tumor cells

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

HSC4 tumor cells derived from an HNSCC were seeded and incubated overnight. After 24 h, the cells were treated with a kinase inhibitor. After 3 h, the first two doses of 5 Gy per fraction were applied. After 24 h, the second dose was applied, and cells were again incubated overnight. In parallel, 48 h prior to the start of the co-culture, T cells were isolated from the blood of a healthy donor. First, PBMCs were isolated using density gradient centrifugation tubes and a sterile separation medium. PBMCs were counted using a cell counting chamber, and CD8+ T cells were isolated using the CD8+ T cell isolation kit. Isolated CD8+ T cells were then stained with CFSE (1 µM) and subsequently counted. T cells were seeded in CD3/CD28 pre-coated well-plates with a density of 1.5 x 106 cells/3 mL. After 48 h of stimulation, the T cells were harvested, counted, and resuspended in a density of 10,000 cells/10 µL. Additionally, representative wells of the seeded tumor cells were harvested and counted, and the medium was exchanged for all remaining wells. T cells were added to the tumor cells in a 1:1 ratio. The co-culture was harvested after 96 h, the supernatant was stored at -20 °C, and the cells were stained and measured by flow cytometry (Figure 1).
T cells were identified by size exclusion and CD3/CD8 positivity. CFSE-signal of CD3+/CD8+ positive cells showed the distribution of non-proliferative T cells (high CFSE-signal) and distinct subpopulations of proliferating T cells (loss of CFSE-signal intensity). The proportion of proliferating T cells was exemplarily measured for the HSC4 HNSCC cell line (Figure 4A). All T cells which showed at least one division were defined as "proliferative". T cells that showed more than 3 divisions were defined as "highly proliferative" (Figure 4B). For the HPV-negative cell line HSC4, a slight increase in the proliferation rate was detected when T cells were co-cultivated with irradiated tumor cells. RT and RT plus inhibition of ATR resulted in a significantly increased consecutive T cell proliferation when compared to co-cultures of RT plus ATM inhibition treated HSC4 cells with T cells. Regarding the "highly proliferative" fraction of T cells, pretreatment of HSC4 cells with RT plus inhibition of ATR was most effective in stimulating T cell proliferation (Figure 4B).
<img alt="HSC4 T cell proliferation bar chart; shows ATM, ATR inhibition effects; statistical significance noted." class="xfigimg" src="/files/ftp_upload/67643/67643fig04v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Proliferation of CFSE-stained T cells for analyses of HSC4 treatment-dependent changes in T cell proliferation. (A) Proportion of proliferating T cells after 96h of co-culture with pre-treated HPV-negative HSC4 tumor cells. T cell proliferation was significantly lower after co-culture with RT+ATMi treated HSC4 tumor cells when compared to RT or RT plus ATRi treatment. (B) RT of HSC4 tumor cells induced a higher fraction of highly proliferating T cells (more than three cell divisions. After co-culture of RT+ATRi pre-treated HSC4 tumor cells with T cells, the percentage of highly proliferative T cells was the highest. The bars show data from four independent experiments with the T cells from four independent, healthy donors (n = 4; Mean ± SD). (*p≤ 0.05, **p≤ 0.01, ***p≤ 0.001, ****p≤ 0.0001; statistical significance tested by comparing all experimental conditions against each using two-tailed Mann-Whitney-U for not normally-distributed data). <a href="https://www.jove.com/files/ftp_upload/67643/67643fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Proliferation-based CFSE-loss can be used to quantify the T cell proliferation rate. Further, several cell surface markers are described as being associated with T cell activity, such as CD25 and HLADR. Therefore, analysis of CD25 and HLA-DR on the surface of all CD3+ and CD8+ T cells by flow cytometry was performed (Figure 5). The expression can be quantified if distinct CD25high or HLA-DRhigh sub-populations are detectable and can be discriminated by gating these highly positive cells (Figure 3B). If no distinct sub-populations are detectable, the mean fluorescence intensity (MFI) of the whole population can be measured, and condition-based shifts of the MFI can be quantified (Figure 3C).
Exemplarily, the expression identified by the MFI of CD25 and HLA-DR was analyzed after 96 h of co-culture with pre-treated HSC4 tumor cells (Figure 5). After co-culture of T cells with RT-treated HSC4 tumor cells, the expression of CD25 on the T cells was strongly down-regulated. Notably, pretreatment of the tumor cells with only ATMi resulted in a significantly decreased expression of CD25 when compared to ATRi pretreatment. In the combined setting with RT, even though RT resulted in decreased expression of CD25 on T cells, the combination of RT with ATMi resulted in an increased expression of CD25 when compared to RT plus ATRi (Figure 5A). Regarding the expression of HLA-DR on T cells, RT generally led to upregulation of HLA-DR, but again, in combination with either ATMi or ATRi, a different behavior was observed (Figure 5B). T cells co-cultured with HSC4 cells that were pre-treated with a combination of RT+ATRi increased the expression of HLA-DR in comparison to RT alone or RT+ATMi.
<img alt="Box plot comparing HLA-DR and CD25 expression on T cells; MFI analysis in co-cultured tumor cells." class="xfigimg" src="/files/ftp_upload/67643/67643fig05v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Expression of the activation markers CD25 and HLA-DR on the surface of T cells after 96 h of co-culture with pre-treated HSC4 tumor cells. The expression of CD25 and HLA-DR was analyzed based on the shift of the MFI of the whole T cell population. RT of HSC4 resulted in a decreased expression of CD25 on T cells and an increased expression of HLA-DR. The combination of RT with ATMi resulted in significantly different expression patterns when compared to RT plus ATRi. The boxplots show data from four independent experiments with the T cells from four independent, healthy donors (n = 4; Mean ± SD). The data was analyzed by comparing all experimental conditions against each other using a two-tailed Mann-Whitney-U test for not normally distributed data (*p≤ 0.050). <a href="https://www.jove.com/files/ftp_upload/67643/67643fig05largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
These data exemplarily indicate that the treatment of tumor cells, in this case of HSC4 HNSCC cells, impacts the immunogenicity of the tumor cells. This can be monitored by co-cultivation of the tumor cells with pre-stimulated T cells, which leads to a diverse proliferation behavior and expression of activation markers on human T cells based on the applied tumor cell treatment regime.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

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Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

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