OT1-GFP cells infiltrate and reject OVA-expressing tumors
To analyze the role of antigen recognition in tumor infiltration and in CTL motility within tumors, we inoculated individual C57BL/6 mice s.c. with two tumors: the EL4 thymoma in one flank and the EG7 (OVA-expressing EL4 cells) in the other flank. Naive GFP-expressing, OVA-specific, TCR-transgenic OT1 cells (OT1-GFP) were adoptively transferred to the mice when the EL4 and EG7 tumors were 500–1,000 mm3 (around day 10). OT1-GFP cells induced the complete rejection of EG7, but not of EL4 tumors, within 6–8 d (). After the adoptive transfer of OT1-GFP cells, the tumors continued to grow for 3 d. After 3–4 d, the tumor stops growing before the size actually starts decreasing at days 5–6. At days 7–8, the tumors continued to shrink and disappeared completely. We defined two periods for the analysis of the active rejection process: the “early” rejection phase corresponds to days 3–4 after adoptive transfer when the tumor stops growing; the “late” rejection phase corresponds to days 5–6 when the size of the tumors starts to decrease, but before complete rejection (which occurs at days 7–8).
Figure 1. Activation of specific T cells in antigen-expressing tumors. C57Bl6 mice were injected s.c. with five 105 EG7 and EL4 tumor cells in either flank. 8–10 d later, the mice were adoptively transferred with 107 purified naive CD8+ cells from (more ...)
We first sought to analyze the activation of the transferred OT1 cells in the draining lymph node or nondraining lymph node EG7 tumors. As shown in Fig. S1 (available at http://www.jem.org/cgi/content/full/jem.20061890/DC1
), OT1-GFP cells are primed in EG7 draining lymph nodes but not in the nondraining ones. The transferred cells transiently up-regulate CD69, maintain high levels of CD44, and display decreased CD62L expression (Fig. S1, A and B and reference 30
). OT1 activation resulted in the expansion of the OT1-GFP cells up to fivefold at day 3 (0.37% ± 0.02 versus 1.8% ± 0.3 of total cells; P = 0.005) (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20061890/DC1
). The slight increase of OT1-GFP cell numbers in the spleen (0.20% ± 0.11 to 0.55% ± 0.1; P = 0.006) is probably caused by the recirculation of activated cells rather than a local antigen-induced expansion (30
). Therefore, transferred OT1 cells are selectively activated in the antigen-expressing tumor draining lymph nodes.
The infiltration of the tumors by the OT1 cells was analyzed next by measuring the density of OT1-GFP cells in frozen sections of either tumor at days 3–4 after adoptive transfer. Both EG7 and EL4 tumors were infiltrated with OT1-GFP cells (), consistent with the observation that activated primary CD8+
T cells migrate to nonlymphoid tissue regardless of their site of activation (31
). The number of OT1-GFP cells infiltrating EG7 was, as expected, higher than the number infiltrating EL4, especially at day 6 after adoptive transfer (327 ± 50 cells/mm2
versus 27 ± 2 cells/mm2
) (). The OT1-GFP cells infiltrating both EL4 and EG7 tumors expressed low levels of CD62L, attesting that both cell populations had been previously activated (). In contrast, only the OT1-GFP cells infiltrating OVA-expressing EG7 tumors, but not those infiltrating the EL4 tumors, reexpressed high levels of the early activation marker CD69 () and produced IFN-γ (). The EG7-infiltrating OT1-GFP cells, but not OT1-GFP cells infiltrating EL4, also down modulated their TCR, as attested by the decreased expression of Vβ5 (Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20061890/DC1
). We conclude that the parallel inoculation of antigen-expressing and antigen-nonexpressing tumors to the same mouse induced the infiltration of both tumors, although the T cells accumulated preferentially in the antigen-expressing tumors. T cell activation and effector function (CD69 expression, IFN-γ production, and killing), however, were only detectable in CTLs infiltrating the antigen-bearing tumors.
Antigen expression determines the motility of CTLs in tumors
To investigate the role of antigen recognition in the motility of antigen-specific CTLs within tumors, we next performed intravital time-lapse two-photon imaging. 8–10 d after s.c. injection of the tumors, OT1-GFP cells were adoptively transferred into tumor-bearing mice. At days 3–4 (early phase of rejection) or 5–6 (late phase of rejection) after adoptive transfer of OT1-GFP cells, the mice were anesthetized and injected i.v. with fluorescent dextran to visualize blood vessels. As shown in Videos S1 and S2 (available at http://www.jem.org/cgi/content/full/jem.20061890/DC1
), during the early phase of rejection OT1-GFP moved actively in EL4 tumors but had reduced motility in EG7 tumors. During the late phase of rejection, OT1-GFP cells moved actively in both tumors (Videos S3 and S4, available at http://www.jem.org/cgi/content/full/jem.20061890/DC1
). Individual cell trajectories were tracked (representative examples are shown in ), and the mean velocity, arrest coefficient (the proportion of time every individual remains arrested), and confinement ratio (the ratio of the distance between the initial and the final positions of each cell to the total distance covered by that cell) were determined ().
Figure 2. CTLs motility during tumor infiltration and clearance. (A) TPLSM images (260 × 260 μm) of OT1-GFP cells (green) within tumors (EG7 and EL4) of anesthetized mice at early (day 3) and late (day 5) time points after adoptive transfer. Vascular (more ...)
During the early phase of rejection, OT1-GFP cells moved with a mean velocity of 4 ± 2 μm/min in the EG7 tumors compared with 10 ± 4 μm/min in the EL4 tumors. The migration trajectories were more restrained in the EG7 compared with the EL4 tumors (confinement ratios of 0.4 ± 0.2 versus 0.6 ± 0.2). Consistently, the arrest coefficient was higher in EG7 (33 ± 17%) than in EL4 (13 ± 17%) tumors. During the late phase of rejection, the motility of OT1-GFP cells in EG7 increased to 8 ± 3 μm/min−1 to become similar to the mean velocity of OT1-GFP cells in EL4 tumors (10 ± 5 μm/min−1). The migration trajectories also became less confined (0.6 ± 0.2) and identical to the ones observed in EL4 tumors. Finally, the arrest coefficient in EG7 tumors dropped to 14 ± 16%, which is close to that of OT1-GFP cells in EL4 tumors (16 ± 21%). We conclude that CTLs transiently stop moving in antigen-expressing tumors during the early phase and then resume their migration during the late phase when the size of the tumor starts to decrease.
OT1-GFP stop in contact with OVA-expressing tumor cells
To investigate if this transient arrest of migration in the antigen-expressing tumors is related to the killing of tumor cells by the CTLs, EG7 and EL4 tumor cells were transfected with GFP-encoding plasmid. The GFP-expressing tumors behaved like their GFP-negative counterparts in terms of tumor growth and rejection after transfer of OT1 cells. Tumor cell viability in vivo was determined at different time points by the level and the distribution of intracellular GFP using two-photon microscopy. During the early phase of tumor rejection, tumor cells formed a dense network of bright living cells in both EL4-GFP and EG7-GFP tumors, similar to that observed in the absence of OT1 cells (). In the late phases of rejection, in contrast, EG7 tumors, but not EL4 tumors, were mainly composed of residual dead tumor cells— i.e., cells displaying lower GFP levels and a GFP-negative, compact nucleus. In EG7 tumors during the late phase, the enhanced second harmonic generation (SHG) signal reflected an increase in the density of collagen fibers (), suggesting increased fibrosis in this region of the tumor.
Figure 3. Tumor cell clearance during early and late phase. (A) Representative TPLSM images (160 × 160 μm) of GFP-EG7 or GFP-EL4 tumors during early (day 3) and late phase (day 6) of tumor rejection after adoptive transfer of 107 OT1 cells. Vessels (more ...)
To determine if the cells expressing low levels of GFP observed in the late phase were dead tumor cells or macrophages having engulfed dead tumor cells, we injected the tumor-bearing mice with propidium iodide (PI). As shown in , in EG7 tumors during the early phase, when most tumor cells are still alive, few cells are stained with PI. During the late phase, in contrast, all cells displaying low levels of GFP with dark GFP-negative nuclei are labeled by PI (). We conclude that during tumor rejection, dead tumor cells accumulate in the periphery of the tumors, most likely before being cleared by macrophages.
The change in the dynamics of OT1 cell motility in the EG7 tumors between the early and late phases could therefore correspond to the arrests of OT1 cells during tumor cell killing. To investigate this possibility, we analyzed the dynamics of OT1 cells expressing cyan fluorescent protein (OT1-CFP) in tumors expressing GFP during the early or late rejection phases (Videos S5 and S6, available at http://www.jem.org/cgi/content/full/jem.20061890/DC1
). As shown in and Video S5, during the early phase the majority of the arrested OT1-CFP cells are in contact with living EG7-GFP tumor cells. During the late phase, OT1-CFP cells seem to resume motility in regions where the tumor cells are dead ( and Video S6). We therefore compared the dynamic of the OT1 cells in regions where the tumor cells are alive or dead. As shown in , the OT1-CFP cells displayed lower mean velocity (1.1 ± 0.4 μm/min) and confinement ratios (0.15 ± 0.05) and higher arrest coefficients (82 ± 11%) in regions where the tumor cells are alive during early phase than in regions where the tumor cells are dead during late phase (mean velocity, 6.4 ± 2.5 μm/min; confinement ratio, 0.7 ± 0.2; and arrest coefficient, 17 ± 18%). During the late phase, we eventually found regions of the tumors where at least parts of the EG7-GFP cells are still alive. OT1-CFP cells in these regions only partially resumed motility compared with regions where most of the tumor cells were dead ().
Figure 4. Dynamics of CTL interactions with tumor cells. TPLSM images of OT1-CFP cells (A, blue; B, magenta) within EG7-GFP tumors (green) during early phase (day 4) and late phase (day 5) of tumor rejection. Collagen fibers (blue) are imaged by SHG. Examples of (more ...)
The interactions between OT1-CFP and tumor cells were analyzed in more details by defining four types of trajectories. shows representative examples of cell trajectories falling into each of these four categories. The first trajectory corresponds to cells arrested in close contact with GFP-positive tumor cells (, Stable). The second one corresponds to cells that are not completely arrested, but move around a GFP-positive tumor cell, or interact with several neighboring tumor cells (, Confined). The third one includes OT1 cells that interact transiently with several distant tumor cells (, Serial), and the fourth one corresponds to OT1 cells that do not establish any measurable interaction with a tumor cell (, Fleet). After sorting the cells into these four categories, we analyzed their mean velocity, confinement ratio, and arrest coefficient (Fig. S4). As expected, the two former parameters increased from categories 1 to 4, whereas the arrest coefficient decreased.
The relative proportions of each type of OT1 categories were quantified during the early phase (in regions of the tumors where tumor cells are alive) and during the late phase (in regions where most tumor cells are dead). As shown in , the proportion of OT1 cells in the first two categories (, Stable and Confined) represents 95% in regions of the tumor where the tumor cells are alive. In contrast, in regions where most tumor cells are dead, categories 3 and 4 (, Serial and Fleet) represent ~90% of the OT1 cells. Importantly, during the late phase, in regions of the tumor where part of the tumor cells were alive, OT1-CFP cells adopted an intermediate behavior, indicating again that resumed motility OT1-CFP cells was dictated by the viability of the tumor cells rather than the day after adoptive transfer.
We conclude that during the early phase of tumor rejection, OT1 cells are in contact with living tumor cells, either completely arrested or following very constrained trajectories, suggesting that they interact with several neighboring tumor cells. During the late phase of tumor rejection, in the regions where most tumor cells are dead, OT1 cells either make very short arrests or migrate rapidly within the tumor with no apparent stops. Therefore, resumed T cell motility in antigen-expressing tumors occurs preferentially in regions where the tumor cells are dead.
Modes of CTL migration in tumors
We next sought to characterize this resumed CTL migration in more detail. Resumed CTL motility in regions of the tumor where the tumor cells are dead followed an amoeboid pattern (Videos S6 and S7, available at http://www.jem.org/cgi/content/full/jem.20061890/DC1
). We observed important changes in the cell shape, suggesting a strong adaptation of the CTLs to the extracellular matrix and their ability to squeeze through narrow spaces as observed in artificial three-dimensional collagen matrix (32
) or around stromal and tumor cells. CTLs occasionally followed collagen fibers, which were visualized by their SHG signals (Video S6). We also observed OT1 cells dividing inside the EG7 tumors (0.8% of the tracked OT1 cells, n
= 1,249; Video S8). Because our videos last 30 min, the proportion of divisions over 24 h could be relatively high (up to 30–40% of the OT1 cells, assuming the rate of divisions remains the same over this period of time). Divisions might have gone unnoticed in the EL4 tumors because of the near 10-fold lower T cell population. Accumulation of tumor cells around blood vessels was observed occasionally (Video S9), suggesting either regions of preferential CTL extravasation or chemotactism of CTLs for the blood vasculature. Three-dimensional reconstitution shows that CTLs are in close contact to blood vessels (Video S10). Strikingly, CTLs moved frequently along blood vessels (Video S7), adopting a more elongated morphology and undergoing less important changes in their shape.
To characterize and quantify the cells migrating along blood vessels in more detail, we took advantage of certain regions of the tumor periphery that displayed a highly organized blood vessel pattern orientated along one major axis (). In these areas, numerous CTLs maintained close contact with vessels during migration (Video S11, available at http://www.jem.org/cgi/content/full/jem.20061890/DC1
) for relatively long distances (114 ± 63 μm, n
= 17). The CTLs adopted an anisotropic migration pattern along the axis of the blood vessels as indicated by a higher correlation coefficient (no-contact, r = 0.31 and contact, r = 0.73; P < 0.001) (, bottom). The ability of several cells to migrate from a blood vessel to another confirmed that the CTLs are probably not in the lumen of the vessels, but either in the tissue or within the “sheath” around the vessels. Migration along blood vessels proceeds with a mean velocity similar to that of the CTLs migrating at distance of visible blood vessels (8.2 ± 2.4 μm/min and 8.6 ± 2.8 μm/min, respectively; ).
Figure 5. Distinct migratory patterns of T cells within tumors. (A) TPLSM images of OT1-GFP cells (green) within an EG7 tumor during the late phase of tumor rejection (day 5). Vessels (red) are labeled by 70 kD rhodamine-dextran. Several representative migration (more ...)
The shape of CTLs during migration along blood vessels seemed to differ from their morphology in other areas of the tumor (examples are shown in , top, and Video S12, available at http://www.jem.org/cgi/content/full/jem.20061890/DC1
). , top, shows the evolution of the elongation index (ratio between the length and the breadth of a cell) of two representative cells migrating either in contact or at distance from a blood vessel. Cells in contact with blood vessels were more elongated, displaying a higher elongation index (1.9 ± 0.2) than cells crawling at distance from visible blood vessels (1.6 ± 0.1) (, bottom). To investigate if the shape of individual cells changes when they migrate in contact or at distance of a blood vessel, we followed the elongation index of individual cells migrating in the same videos in contact or at distance of a vessel (, bottom). As long as the cell is at distance from any visible vessel, its elongation index is <2 for most of the time, and occasionally rises over this value for short periods of time (<1 min). When the cell starts migrating in close vicinity to the blood vessel, its elongation index rises to values >2 for several min. The elongation index then falls to ~2, whereas the cell is still migrating along the same blood vessel. For this particular cell, the difference in the elongation index before and after the initiation of the contact with the blood vessel is statistically significant (P = 0.0015). A significant increase in the elongation index before and after the initiation of the contact with the blood vessel, however, was only observed in three out of five cells analyzed.
We conclude that single CTLs can migrate along blood vessels using high or low elongation indexes. Elongations indexes higher than 2 lasting for several min were only observed in cells migrating along blood vessels and not in cells migrating at distance of blood vessels. In the latter, the elongation index was very variable and eventually increased over 2 for short periods of time, reflecting a higher deformability of the cells, most likely a result of constraints imposed by the extracellular matrix encountered during the crawling.
Deep infiltration in antigen-expressing tumors
Because two-photon imaging is technically restricted to peripheral area of the tumors, we next sought to analyze the topography of CTL infiltration deeper into the tumor and to analyze the role of antigen recognition in this process. To do so, we had to quantify infiltration by CTLs in regions of the tumor beyond the penetration limit of the two-photon microscope. We therefore made ordered sequential frozen sections of either EG7 or EL4 tumors at different time points after adoptive transfer of GFP-OT1 cells (Fig. S5). The number of GFP-OT1 cells was quantified according to the distance from the surface of the tumor (Fig. S5). Representative examples of OT1-GFP cells in tumor sections are shown in . During the early phase of rejection, most GFP-OT1 cells were found within the first 200-μm peripheral sections of both EL4 and EG7 tumors (, top left). An accumulation of lymphocytes in the periphery of solid tumors has been reported previously in other tumor models (33
), but the actual mechanisms for this preferential infiltration through the periphery are still unclear. To address possible macroscopic heterogeneities in tumor vessel distribution that might account for the peripheral infiltration pattern, we stained endothelial cells in tumor sections using anti-CD31 antibody. We did not detect any significant difference in the density of blood vessels (Fig. S6). This result, of course, does not exclude functional differences in the “permeability” of blood vessels to lymphocytes in different regions of the tumors.
Figure 6. T cell infiltration through the periphery of tumors. OVA-specific OT1-GFP infiltration in EG7 and EL4 tumors (Thymoma) or MCA tumors (Fibrosarcoma), expressing or not the OVA epitope, during the early (day 3) and the late (day 6) phase of tumor rejection. (more ...)
In the late phase of rejection, similar to the early phase, most OT1-GFP cells infiltrating EL4 tumors remain in the periphery (, top right). In contrast, in the OVA-expressing EG7 tumors, an important redistribution of the CTLs is observed during the late phase. The density of OT1-GFP cells in more central regions of the tumor increases, resulting in a homogeneous distribution from the periphery to the tumor center. We conclude that CTLs infiltrate the thymomas through the periphery and that deep tumor infiltration during rejection requires antigen expression by the tumor cells (since the OVA-negative EL4 tumors were not infiltrated deeply).
Antigen expression within the tumor causes the local activation (increased CD69 expression), IFN-γ production, and cytotoxicity (tumor rejection) of OT1 cells (). To investigate if deep tumor infiltration requires antigen recognition by individual T cells or is the consequence of overall modifications in the tumor environment caused by antigen recognition by the CTLs, we analyzed the infiltration of EG7 tumors by polyclonal, GFP-expressing T cells. The polyclonal T cells were activated in vitro and then adoptively transferred to EG7-bearing mice in the presence or absence of OT1 cells. At days 5–6 after transfer, tumor rejection started only in mice injected with polyclonal T cells and OT1 cells (unpublished data). Strikingly, these polyclonal T cells accumulated and infiltrated the tumors deeply only in the presence of OT1 cells (Fig. S7). We conclude that OT1 cells induce a change in the antigen-expressing tumors, which makes them permissive to infiltration by other activated T cells.
If tumor infiltration occurs through the periphery of the tumors, tumor cell killing by CTLs should also start in the periphery. To address this possibility, we performed ordered frozen sections as in and labeled apoptotic cells using tunnel. Apoptotic cells were most abundant in the periphery of EG7 tumors at the end of the early phase (). The number of tunnel-positive cells was counted in sequential sections and plotted as a function of the distance from the surface of the tumor. As shown in , tunnel-positive cells accumulated in the periphery of the EG7 tumors. Reduced accumulation of apoptotic cells was detected in the EL4 tumors. The low numbers of tunnel-positive cells detected in the periphery of EL4 are most likely caused by the endogenous immune response. We conclude that apoptotic tumor cells accumulate in the periphery of the tumor, consistent with the peripheral infiltration of the CTLs evidenced in .
Figure 7. Apoptotic cells after tumor T cell infiltration. Tunnel assay after adoptive transfer of OVA-specific OT1 T cells in EG7 or MCA-OVA tumor-bearing mice. (A) Typical epifluorescence images (900 × 670 μm, right and left; 225 × 167 (more ...)
To extend our conclusions about the role of antigen in tumor infiltration to another tumor model, we used a fibrosarcoma tumor cell line, MCA-101, expressing (MCA-OVA) or not expressing (MCA) recombinant OVA. OT1-GFP cells adoptively transferred to tumor-bearing mice induce the rejection of MCA-OVA, but not MCA-tumors, with a kinetic similar to that of EG7 rejection (unpublished data). OT1 cells were recruited to both tumors, although the recruitment to the OVA-expressing tumors was higher compared with the control MCA tumors (Fig. S8). Similar to the thymoma model, OT1-GFP cells infiltrated the periphery of MCA tumors independently of antigen expression (, bottom left). OVA expression, however, was required for deep tumor infiltration at days 5–6 (, bottom right). Similar to the thymoma model, at days 3–4 tunnel-positive apoptotic tumor cells accumulated in the tumor periphery of antigen-expressing tumors (). We conclude that deep tumor infiltration, but not peripheral infiltration, requires antigen expression by fibrosarcoma tumor cells.