Abstract
Background: The aim of this study was to analyse changes in levels of memory T-lymphocytes during growth of SL2 tumours in DBA/2 mice and to evaluate whether these lymphocytes may have an inhibitory effect on tumour growth. Materials and Methods: Percentages of naïve (CD8+CD44lowCD62L+), central memory (CD8+CD44high CD62L+) and effector memory (CD8+CD44highCD62L−) lymphocytes in the CD8+ subset in peripheral blood, spleen and lymph nodes of tumour-bearing and control mice were analysed by flow cytometry. Results: The percentage of effector memory lymphocytes in the CD8+ subset increased during growth of tumours, whereas that of naïve CD8+ lymphocytes decreased. No correlation between the levels of effector memory lymphocytes in peripheral blood and the mass of tumours was found. Conclusion: SL2 tumours induce expansion of effector memory lymphocytes in DBA/2 mice. However, expanded effector memory lymphocytes do not inhibit the growth of tumours.
Recent evidence indicates that expansion of some T-lymphocyte subsets, including both CD4+ and CD8+ cells, may take place during tumour growth. Regarding CD4+ lymphocytes, an increase in numbers of regulatory T-cells (CD4+CD25+FOXP3+) in cancer patients is well-established. These cells contribute to the evasion of antitumour immune response (1, 2). To improve immunotherapy, it is thought that the number of regulatory T-cells and their functions should be inhibited in patients with advanced cancer (3).
Regarding CD8+ lymphocytes, increased numbers of CD8+CD57+ (CD8+CD28−) lymphocytes were found both in the tumour microenvironment and in the peripheral blood of patients with solid tumours and haemato-oncological diseases (4). Interestingly, these lymphocytes seem to play both positive and negative roles in various types of cancers (5). Similarly to CD8+CD57+ in humans, CD8+ lymphocyte expansion in tumour-bearing mice can be demonstrated by an increase in numbers of CD8+CD44high lymphocytes. It has been demonstrated that long-term tumour immunity after adoptive transfer of tumour antigen-reactive T-cell subpopulations is accompanied by an elevation in the number of CD8+CD44high lymphocytes in spleen and lungs of mice (6).
We have reported that CD8+CD44high lymphocyte levels in peripheral blood increase during growth of SL2 tumours in DBA/2 mice (7). However, these expanded CD8+CD44high lymphocytes do not seem to have an antitumour effect, as tumours grow, and all tumour-bearing mice die during the second or third week after tumour implantation.
The CD8+CD44high lymphocyte subset can be further analysed according to the expression of the cell-trafficking molecule CD62L (L-selectin). At least two memory T-cell subsets can be identified based on the CD62L expression: central memory T-cells (TCM) are of CD8+CD44high CD62L+ phenotype, whereas effector memory T-cells (TEM) are of CD8+CD44highCD62L− phenotype (8, 9). TCM lymphocytes have little or no effector function. These lymphocytes home to T-cell areas of secondary lymphoid organs and readily proliferate and differentiate to TEM cells in response to antigenic stimulation. TEM lymphocytes migrate to inflamed peripheral tissues and display immediate effector function (8, 10). Naïve CD8+ (TN) cells express the CD8+CD44low CD62L+ phenotype (11).
In this study, we analysed changes in levels of TN, TCM and TEM lymphocytes during growth of SL2 tumours in DBA/2 mice. We were also interested to investigate whether any of these lymphocyte subsets may have an inhibitory effect on the growth of tumours.
Analysis of CD8, CD44 and CD62L antigens in peripheral blood lymphocytes in DBA/2 mice. A: Side scatter (SSC)/CD8 dot plot was used to gate CD8+ (R1) lymphocytes. B: Analysis of CD44 and CD62L antigens on gated CD8+ T-lymphocytes. CD8+CD44lowCD62L+ (R2) lymphocytes were identified as naïve (TN) cells. CD8+CD44highCD62L+ (R3) lymphocytes were identified as central memory (TCM) cells. CD8+CD44highCD62L− (R4) cells were identified as effector memory (TEM) lymphocytes.
Materials and Methods
Mice and tumours. Male DBA/2 mice at the age of 8-12 weeks, weighing 20-26 g, were obtained from the local breeding facility at the State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania.
SL2 lymphoma cells were maintained by weekly intraperitoneal passage in syngeneic DBA/2 mice. SL2 cells were collected from the peritoneal cavity, then washed and diluted in RPMI-1640 medium. Solid tumours were induced by subcutaneous injection of 107 SL2 cells in phosphate-buffered saline (PBS). The phenomenon of concomitant tumour immunity is observed in the DBA/2–SL2 model (12). This means that the immune reaction towards primary and secondary tumours may be different. Therefore, each mouse received two injections of SL2 tumour cells, one on the left (primary tumour) and one on the right side (secondary tumour) of the chest with an interval of two days.
Mice were sacrificed by cervical dislocation on day 9 of the experiment, tumours were removed and weighed (Denver Instrument GmbH, Göttingen, Germany). Experimental research on animals has been conducted according to recommendations of the Lithuanian Ethics Committee for the Laboratory Animal Use.
Preparation of lymphocytes for flow cytometric analysis. Peripheral blood was collected from the tail vein of individual mice on day 0 (prior to tumour implantation), on day 5 and on day 9 after implantation of the primary tumour. Heparin was used as an anti-coagulant. After sacrifice, spleens and lymph nodes were removed from tumour-bearing and naïve DBA/2 mice and lymphocyte suspensions were obtained by pressing spleens and lymph nodes through a nylon mesh. Contaminating red blood cells from spleen cell suspensions were removed by buffered ammonium chloride (5 min at room temperature). After centrifugation, the supernatant was decanted and the pellet resuspended in PBS.
Flow cytometry. Twenty μl of whole blood or 6 μl of cell suspension from spleens and lymph nodes were incubated for 30 min on ice in the dark with 0.2 μl of anti-CD8-peridinin chlorophyll protein (PerCP), 0.2 μl of anti-CD44-fluorescein isothiocyanate (FITC) (PharMingen, San Diego, USA) and 0.2 μl of anti-CD62L-allophycocyanin (APC) (Miltenyi Biotec GmbH, Teterow, Germany) monoclonal antibodies. All stainings were performed in the presence of purified anti-CD16/32 (PharMingen), at saturation to block nonspecific staining via FcRII/III. After incubation of blood samples, red blood cells were lysed by FACS Lysing Solution (Becton Dickinson, San Jose, CA, USA). The samples were analysed on a FACScalibur flow cytometer (Becton Dickinson) with a laser tuned at 488 nm. Data were acquired and analysed with the CellQuest software (Becton Dickinson). List mode files were collected for 10,000 cells from each sample. TN TCM and TEM lymphocytes were identified as shown in Figure 1.
Statistics. Comparisons between the percentages of TN, TCM and TEM lymphocytes in the CD8+ subset, at different time points of the experiment, were made using the non-parametric sign test for dependent samples. Comparisons between the variables of tumour-bearing and control mice were made using the non-parametric Mann–Whitney U-test. Correlations between the percentage of TEM lymphocytes in the CD8+ subset in peripheral blood and tumour mass were evaluated by Spearman's rank correlation test. The level of significance was set at p=0.05.
Results
Changes in peripheral blood levels of TN, TCM and TEM lymphocytes during tumour growth. Changes in the percentages of TN, TCM and TEM lymphocytes in the CD8 subset during growth of SL2 tumours in DBA/2 mice are shown in Figure 2. The percentage of TN lymphocytes in the CD8+ subset decreased, whereas that of TEM lymphocytes was higher on day 9 compared to day 0 and day 5. The percentage of TCM lymphocytes decreased from day 0 to day 5, then slightly increased at day 9, but did not reach the level of day 0.
Changes in naïve (TN), central memory (TCM) and effector memory (TEM) lymphocyte levels in peripheral blood of DBA/2 mice during growth of SL2 tumours. Peripheral blood was collected from the tail vein of 9 mice on days 0, 5 and 9. Lymphocytes were analysed by flow cytometry. The increase in the percentage of TEM lymphocytes in the CD8+ subset on day 9 is statistically significant compared to day 0 and day 5 (p=0.004). The decrease in the percentage of TN lymphocytes in the CD8+ subset on day 9 is statistically significant compared to day 0 and day 5 (p=0.004). The percentage of TCM lymphocytes in the CD8+ subset decreases from day 0 to day 5, then slightly increases at day 9 (p=0.008), but does not reach the level of day 0 (p=0.046). Values are given as means±SD.
Changes in levels of TN, TCM and TEM lymphocytes in spleen and lymph nodes of tumour-bearing mice. TCM lymphocytes are known to proliferate in secondary lymphoid organs. Therefore, we wanted to determine whether the tumour-bearing state is associated with an increase in TCM lymphocyte levels in spleen and lymph nodes. The percentages of TN, TCM and TEM lymphocytes in spleen and in lymph nodes of tumour-bearing and control mice are shown in Figure 3. Figure 3 shows that the percentage of TCM lymphocytes in spleen and in lymph nodes of tumour-bearing mice was lower than that in control mice, as was the percentage of TN lymphocytes. However, the percentage of TEM lymphocytes in the CD8+ subset was increased in the spleen of tumour-bearing mice, whereas changes of TEM lymphocytes in lymph nodes were not significant.
Relationship between the level of TEM lymphocytes in peripheral blood and tumour mass. To determine whether expansion of TEM lymphocytes may inhibit the growth of tumours, correlation between the percentage of TEM cells in the CD8+ subset and tumour mass in individual mice was analysed. As can be seen in Figure 4, no significant correlation between the level of TEM lymphocytes in peripheral blood and the mass of primary or secondary tumour was found.
Percentages of naïve (TN), central memory (TCM) and effector memory (TEM) lymphocytes in the CD8+ subset in spleen (A) and lymph nodes (B) of tumour-bearing and control mice. Control mice (n=5) were killed on day 0 and tumour-bearing mice (n=10) were killed on day 9 of the experiment. Spleen and lymph nodes were removed and cell suspensions were prepared for flow cytometric analysis. The percentage of TCM lymphocytes in the CD8+ subset decreased in the spleen (p=0.005) and in lymph nodes (p=0.003) of tumour-bearing mice compared to control mice. The percentage of TN cells in the CD8+ subset is significantly lower in the spleen and in lymph nodes of tumour-bearing mice compared to control mice (p=0.002). The percentage of TEM lymphocytes in the CD8+ subset was significantly higher in the spleen of tumour-bearing mice compared to control mice (p=0.002). Values are given as means±SD.
Analysis of correlation between the percentage of peripheral blood TEM lymphocytes in the CD8+ subset and the mass of primary or secondary tumour. Peripheral blood was collected from tumour-bearing mice (n=8) on day 9. The mice were killed and tumours were weighed.
Thus, growth of SL2 tumours induced expansion of TEM lymphocytes in peripheral blood and in the spleen of DBA/2 mice. However, these lymphocytes did not inhibit the growth of tumours.
Discussion
Our results show that the percentage of TEM lymphocytes in the CD8+ subset increases during growth of SL2 tumours in DBA/2 mice, whereas the percentage of TN lymphocytes decreases. This suggests that tumours induce the development of TN lymphocytes into TEM cells. This type of change in CD8+ lymphocytes is observed both in peripheral blood and in secondary lymphoid organs of mice. TEM lymphocytes are known to exert immediate effector functions (13), mediated by the pre-expression of granzyme B and perforin within intracellular cytotoxic granules (8). However, lack or correlation between the levels of TEM lymphocytes in peripheral blood and tumour mass in our experiments indicates that TEM cells do not play a critical role in the inhibition of growth of SL2 tumours. Thus, cytotoxic function in vitro may not be important for the in vivo antitumour activity of T-lymphocytes. This finding is in agreement with the findings of other authors who did not find a correlation between cytotoxic T-cell function in vitro and the effect of cancer immunotherapy in vivo (14, 15).
Several previous studies using the adoptive transfer of T-lymphocytes have demonstrated that TCM cells are more effective at eliminating established tumours than are TEM cells (16), despite the fact that TCM cells lack immediate effector function (13). The major difference between TCM and TEM lymphocytes seems to be the ability of TCM, but not of TEM lymphocytes, to rapidly proliferate and expand after re-encountering antigens (17). These data suggest that the proliferative potential of T-lymphocytes is more important than cytotoxicity for an effective antitumour response. Interestingly, the proliferative activity of intratumoural CD8+ T-lymphocytes was found to be important as a prognostic factor in human renal cell carcinoma (18).
The available data indicate that CD8+ T-lymphocytes follow a progressive pathway of differentiation from TN cells into TCM and TEM cells (13). However, in our study, we did not observe an increase in TCM lymphocyte levels. Thus, it can be hypothesised that progressive growth of SL2 tumours in DBA/2 mice is associated with the failure to generate TCM lymphocytes.
- Received May 30, 2012.
- Revision received June 19, 2012.
- Accepted June 20, 2012.
- Copyright © 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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