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The Journal of Immunology, 2004, 173: 222-229.
Copyright © 2004 by The American Association of Immunologists

Antigen Distribution Drives Programmed Antitumor CD8 Cell Migration and Determines Its Efficiency 1

Alexandre Boissonnas, Christophe Combadiere, Elise Lavergne, Maud Maho, Catherine Blanc, Patrice Debré and Behazine Combadiere2

Laboratoire d’Immunologie Cellulaire, Institut National de la Santé et de la Recherche Médicale, Unité 543, Faculté de Médecine Pitié-Salpêtrière, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Understanding both the role of tumor Ag in CD8 cell differentiation and the reasons that CD8 cells may work inefficiently is crucial for therapeutic approaches in cancer. We studied OT-1 CD8 cell responses in vivo in a differential Ag-distribution model that used EG-7, the EL-4 thymoma transfected with OVA. On their initial Ag encounter, OT-1 CD8 cells underwent programmed expansion in the lymph nodes, where they acquired the ability to migrate to the encapsulated tumor site after ≥4 divisions, without continuous antigenic stimulation. This short antigenic stimulation was sufficient to induce the migration differentiation program, which included modulation of chemokine receptor mRNA expression and down-regulation of CD62L. Moreover, Ag quantity determined the behavior of the OT-1 CD8 cells, including their effector functions and sensitivity to apoptosis. Thus, the initial Ag encounter drives the programmed cell migration potencies, but neither effector functions nor cell death can occur without continuous TCR triggering.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Current models of CTLs hold that they undergo programmed development early after Ag encounter; the population expands and then acquires memory cell properties (1, 2, 3). Although longer CTL interaction with Ag does not increase their proliferation rate, CD8 and CD4 lymphocytes require a sufficient level of activation and a complete differentiation program to become fit and thus avoid abortive proliferation (4, 5). Ahmed and colleagues (6) have proposed that memory T cell differentiation is also programmed by the first week after immunization. The contraction phase, including deletion of Ag-specific T cells, also appears to proceed regardless of in vivo Ag persistence (7), as does CD8 production of TNF; in contrast, both CD8 and CD4 cells require continuous Ag stimulation to produce such effector cytokines as IFN-{gamma} (8, 9, 10). Mathematical models indicate that the characteristics of programmed cell proliferation cannot be completely defined by the initial Ag encounter: this proliferation augments after re-exposure to Ag (11). In addition, although CTL response is completely determined upon priming, it can be modulated by extrinsic factors, such as homeostatic cytokines (12, 13). This summary reveals the patchiness of our understanding of the role of Ag in programmed CD8 differentiation. Because most of these models were studied with infectious organisms, such as Listeria monocytogenes or lymphocytic choriomeningitis virus, for which Ag distribution is highly systemic, they do not consider variations in local Ag distribution. In tumor models, the precise localization of Ag plays a critical role in CTL induction, which depends on a sufficient amount of tumor Ag reaching the secondary lymphoid organs (SLOs) 3 early enough and long enough (14). Ag concentration may be lower in SLOs even when it is very high at the tumor site. Furthermore, diffusely invading systemic tumors delete CTLs. Thus, it is the Ag dose in the SLOs that determines whether T cells remain ignorant, are activated, or become tolerized (15, 16). Precise definitions of the kinetics of CD8 antitumor activation, proliferation, migration, and differentiation are crucial in improving the efficiency of tumor rejection. We sought to determine whether Ag distribution, specifically that observed in encapsulated tumors, determines programmed CD8 cell responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Wild-type C57BL/6 (B6) females (6–10 wk of age) were obtained from Elevage Janvier (Le Genest, Saint Isle, France). The OT-1 strain is transgenic for the TCR V{alpha}2V{beta}5 specific for the peptide OVA257–264 (SIINFEKL) and restricted to H2-Kb on RAG2–/– background (generous gift from C. Reis e Sousa, Imperial Cancer Research Fund, London, U.K.). The OT-1 mice were bred, and all strains were housed at the Nouvelle Animalerie Commune of Pitié-Salpêtrière. All experiments complied with French legislation and guidelines for animal research.

Tumor cell lines

All cell lines were purchased from the American Type Culture Collection (Manassas, VA). The dimethylbenzanthracene-induced lymphoma, EL-4, and its chicken OVA-expressing derivative, EG-7, were maintained in RPMI 1640 (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% heat-inactivated FCS (Seromed, Munich, Germany), 2 mM L-glutamine, 1000 U/ml penicillin, 1 mg/ml streptomycin, 250 ng/ml amphotericin B (Invitrogen Life Technologies), and 3 µM {beta}2-ME (Sigma-Aldrich, St. Louis, MO). The EG-7 cell line was maintained with 0.4 mg/ml G418 (neomycin).

Animal models

B6 mice received s.c. injections of 200,000 tumor cells in 100 µl of PBS in the right flank. Tumor size was measured three times a week with a caliper, and tumor volume was estimated with the following formula: width x length x (width + length)/2. Mice were sacrificed when tumor volume reached ~10,000 mm3. EL-4 and EG-7 tumors grew at similar rates. OT-1 CD8 cells were isolated from lymph nodes (LNs), and 5 million CFSE-labeled cells were adoptively transferred i.v. Nearly all (>95%) of these cells were CD8+, V{beta}5+, CD44low, and CD62Lhigh. Naive CD8 T lymphocytes were labeled with 5- (and 6)-CFSE (5 µM) (Molecular Probes, Leiden, The Netherlands) as previously described (17). Mice were sacrificed at different times after the transfer for collection of the right mesenteric (nondraining), right inguinal (also described as tumor-proximal), and right axillary (tumor-distal) LNs, as well as spleens and tumors. Cell suspensions were prepared as previously described (17). In some experiments, CD8 cells were purified with magnetic beads and a MACS separation column, according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany), and transferred to new B6 recipient mice.

Flow cytometry

Cell surface Ags were characterized with a standard staining method. Briefly, cells were incubated with 4 µg/ml appropriate fluorochrome-conjugated mAbs for 20 min at room temperature, washed in 1x PBS, and fixed for 15 min at room temperature in 300 µl of 4% paraformaldehyde. The mAbs, all from BD PharMingen (Le Pont de Claix, France), were as follows: PE-conjugated anti-mouse V{beta}5 (clone MR9-4), PE-conjugated anti-mouse CD44 (clone IM7), PerCP-cyanine 5.5 (PerCP-Cy5.5)-conjugated anti-mouse CD8 (clone Ly-2), biotin-conjugated anti-mouse CD62L (clone MEL-14) plus allophycocyanin-streptavidin, and allophycocyanin-conjugated annexin V. Cell suspensions were incubated ex vivo for 3 h at 37°C with brefeldin A at 5 µg/ml, and then washed and permeabilized with 1x PBS-2% FCS-0.1% saponin before intracellular staining with allophycocyanin-conjugated anti-mouse IFN-{gamma}. Cells were then run for four-color fluorescence staining on a cytofluorometer (FACSCalibur; BD Biosciences, Mountain View, CA) and analyzed with CellQuest software.

Reverse transcriptase-multiplex PCR

Total RNA was isolated from sorted CD8 T lymphocytes. Three different CD8+V{beta}5+CFSE+ cell populations were sorted with the FACSVantage SE cell sorter (BD Biosciences): cells that had already divided at least once, tumor-infiltrating cells, and naive cells. The latter were purified as described below (Miltenyi Biotec). The positive fractions contained 98% CD8+ cells. Total RNA was extracted with the QIAamp RNA Blood mini-kit (Qiagen, Courtaboeuf, France), and cDNA was generated with SuperScript II RNase H (Invitrogen Life Technologies) and the standard protocol.

The mRNA expression of several CD8 chemokine receptor genes was analyzed with kits for detecting mouse CXCR and CCR receptors (BioSource International, Camarillo, CA), as previously described (18). The kits were designed to detect the expression of mouse CXCR1 and CXCR2, CXCR3, CXCR4, and CXCR5 for the CXCR genes; CCR1, CCR2, CCR3, CCR4, and CCR5 for the CCR genes; and GAPDH genes. PCR products were analyzed in a 2% agarose gel electrophoresis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Kinetics of CD8 cell proliferation and tumor infiltration

To evaluate the efficiency and kinetics of antitumor CTL activity in vivo, we conducted experiments involving the adoptive transfer of OVA-specific CD8 naive cells into B6 mice, which then developed solid s.c. OVA-expressing EL-4 (EG-7) tumors. We monitored tumor growth for several weeks, measuring tumor volume with a caliper, as described in Materials and Methods. Solid tumors were detectable ~10 days after the EG-7 injection and grew exponentially in the mice (70% of those injected) used for additional experiments (Fig. 1a). Anti-OVA TCR transgenic (OT-I) CD8 lymphocytes were adoptively transferred when the tumor reached 500-1000 mm3. The OT-1 lymphocytes induced a complete regression of the tumor within 3–7 days after cell transfer (p = 0.03 at day 5; p = 0.006 at day 7). Tumor rejection remained stable for the 15 days that followed. However, similar naive OT-1 cells transferred into hosts with a tumor >2000 mm3 did not reject it (Fig. 1b). Partial tumor regression (30%; p = 0.08 at day 5, p = 0.06 at day 7) was observed 5 days after transfer, but tumor growth then resumed. These findings indicate that tumor size determines the efficiency of CTL response. No Ag-specific CTLs were detectable on day 10 posttransfer (Fig. 1b).



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  		FIGURE 1. Tumor size determines the efficiency of antitumor CD8 responses. Tumor volume was measured as described in Materials and Methods. OT-1 cells from LNs were adoptively transferred on day 0: 15 days after tumor transplants, mean tumor size 592 mm3, dashed line (a); 22 days after tumor transplants, mean tumor size, 2289 mm3 (b). Tumors of the mice receiving the OT-1 cell transfers ({circ}) were then compared with those of control mice (•). Each data point represents the mean tumor volume of 10 mice and was representative of three separate experiments. The difference in tumor growth between the groups was assessed with the Student t test: *, p < 0.05; **, p < 0.01.

 
All subsequent investigations of the role of tumor Ag in antitumor response used the first model (tumor size, 500-1000 mm3) (Fig. 1a). We evaluated the percentage of CD8+V{beta}5+ cells in LNs, spleens, and tumors in these mice compared with those that did not receive OVA-specific CD8 lymphocytes (Fig. 2). Two days after transfer, CD8+V{beta}5+ cells accounted for 2–3% of the total CD8 T cells in the LNs, a percentage that increased significantly thereafter, reaching 6.5% on day 3 (p < 0.05) and remaining significantly higher on days 4 and 6 than at day 2 after transfer (p < 0.05) (Fig. 2, upper panel). The percentage of CD8+V{beta}5+ cells in the spleen did not increase significantly after cell transfer; this result suggests that they proliferate at a lower rate there, or turn over rapidly, or are redistributed (Fig. 2, middle panel). Interestingly, CD8+V{beta}5+ infiltrating cells were not detectable at the tumor site until 3 days after cell transfer (Fig. 2, lower panel). Tumor regression was associated with the infiltration of CD8+V{beta}5+ cells into the tumor. The percentage of CD8+V{beta}5+ cells decreased by day 6 and was correlated with tumor disappearance (Figs. 1a and 2, lower panel). The time (3–5 days) between cell transfer and tumor regression may thus reflect the need for naive CD8 T cells to proliferate and differentiate into cytotoxic effector cells.



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  		FIGURE 2. Tumor-specific CD8 cell distribution in SLOs and tumor. Flow cytometry was used to evaluate the percentage of transferred CD8+V{beta}5+ cells in the total CD8+ cell population of the LNs, spleens, and tumors at different days posttransfer. The figure represents this percentage after subtraction of the endogenous CD8+V{beta}5+ cells (corresponding to 13% of total CD8 cells in LNs, 10% in spleens, and 21.5% in tumors) in the control population (B6 mice without transferred OT-1 cells). All statistical analyses used the Student t test: *, p < 0.05; **, p < 0.01. ns, Nonsignificant.

 
Because tumor Ag determines Ag-specific T cell response, we evaluated the localization of tumor cells in vivo. We injected GFP-expressing EG-7 cell lines s.c. under the same conditions as for the other injections and then assessed the percentage, if any, of GFP+ cells in different organs (Fig. 3). When the s.c. tumor was 500-1000 mm3 in volume, a tiny percentage of such cells was found in most of the tumor-proximal (right inguinal) (0.45%) and tumor-distal (right axillary) LNs (0.46%), and the lungs (0.16%), but none was found in the spleen, mesenteric LNs, or liver. These results do not reflect the processed form of tumor Ag, nor do they indicate the amount of Ag distributed in vivo; they are, however, highly suggestive of unequal tumor Ag distribution in different organs during tumor growth.



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  		FIGURE 3. GFP-expressing EG-7 tumor invasion. GFP+ stable EG-7 transfectants were injected into B6 mice. Data represented GFP+ events evaluated by flow cytometric analyses in several mouse organs when the tumor size reached 600 mm3.

 
Tumor-induced cell proliferation and localization in vivo

We also evaluated the kinetics and localization of the OVA-specific CD8 cell response in this model. After adoptively transferring CFSE-labeled OT-1 naive CD8 cells into B6 recipient mice with EG-7 growing tumor, we analyzed in vivo cell division in the LNs, spleen, and tumor (Fig. 4). OVA-specific CD8 cells started proliferating in the LNs on day 2, when they underwent four divisions. The OVA-specific CD8 cells continued to proliferate at a very high rate in LNs, with three additional cell divisions on day 3, for a total of 7. To assess whether proliferation differed in draining and nondraining LNs, we analyzed three types of LNs separately: tumor-proximal, tumor-distal, and mesenteric (Fig. 4). Only the two first types were draining LNs. We found that the percentage of dividing cells was higher in the draining (70–77% dividing cells) than nondraining (49% dividing cells) LNs (Fig. 4). Interestingly, proliferation started later in the spleen (Fig. 4), where subsequent generations were not detectable until day 3. Moreover, the spleen contained relatively few cells from the earliest generations (first to third) at any time. These results suggest either that cells proliferate faster in the spleen, or that dividing cells migrate from LNs to the spleen. The slight increase of CD8+V{beta}5+ cells in the spleen favors the latter hypothesis, as does the decreased detectability of GFP+ cells. Moreover, although all dividing cells in the tumor-distal and tumor-proximal LNs were CD69+, those in the mesenteric LNs and the spleen remained CD69– (data not shown). The efficiency of cell division and activation was thus correlated with the distribution of Ag, and most of the dividing cells came from draining LNs.



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  		FIGURE 4. In vivo OVA-specific CD8 cell proliferation. CFSE-labeled OT-1 cells from total LNs (tumor proximal, tumor distal, and mesenteric) (a) or individualized LNs, spleens, and tumors (b) were analyzed by flow cytometry. Dot plots represent V{beta}5+CFSE+ gated on CD8+ cells from days 1 to 3 after cell transfer into B6 mice with growing EG-7 or EL-4 tumors. The percentages of dividing cells are indicated. Results are representative of five different experiments.

 
OVA-specific CD8 cells were not detectable at the tumor site on day 1 or day 2, although cells that had undergone at least four divisions appeared in the tumor on day 3. At no time point did we find any naive cells in the tumor. These results indicate that naive CD8 cells do not migrate to the tumor inflammatory site; they further suggest that CD8 cells that infiltrate tumors divide in SLOs before migrating to the tumor site. We were able to assess the Ag specificity of CD8 proliferation by adoptively transferring OVA-specific CD8 naive cells into recipient B6 mice with growing EL-4 s.c. tumors. Fig. 4a shows that OT-1 CD8 cells did not proliferate on day 3 or thereafter (data not shown) and thus demonstrates the Ag specificity of CD8 cell proliferation even in an inflammatory environment. Furthermore, we observed no infiltration of OT-1 naive CD8 cells into EL-4 tumors. Taken together, these results show that Ag-specific CD8 cells proliferate mostly in the LNs and then migrate after several (more than four) divisions to the spleen and into the inflammatory site.

Ag distribution determined outcome: effector functions or cell death

The final stage in the developmental program of Ag-specific CD8 cells is their acquisition of effector functions, although apoptosis may prevent this outcome. After finding that naive T CD8 cells activated and expanded in LNs, migrated to the tumor site, and caused rejection of tumors that were sufficiently small at the time of the adoptive transfer, we sought to determine whether CTL effector functions were also compartmentalized. Because tumor regression evokes potent cytotoxic activity, we tested the ability of OT-1 cells to produce IFN-{gamma}. Ex vivo analyses of transferred CD8 cells showed that 35% of the tumor-infiltrating lymphocytes (TILs) spontaneously produced IFN-{gamma} within the first 3 h ex vivo (Fig. 5a). Surprisingly, spontaneous IFN-{gamma} production was not detected in Ag-specific CD8 cells from the LNs or spleen (Fig. 5a). In addition, its ex vivo production by CD8 cells was correlated with TCR down-regulation at the tumor site, where it was lower than in the LNs or spleen (Fig. 5b). This suggests high in vivo antigenic stimulation at the tumor site. Overall, these results show that the induction of effector function is compartmentalized.



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  		FIGURE 5. Spontaneous ex vivo production of IFN-{gamma} by OVA-specific CD8 cells. a, Spontaneous ex vivo IFN-{gamma} production by CD8+V{beta}5+ CFSE cells (3-h brefeldin A) was detected in LNs, spleen, and tumor on day 3 after transfer in B6 mice with growing EG-7 tumors. The percentages of IFN-{gamma}+ cells are indicated. b, Down-regulation of V{beta}5 surface expression on CD8+CFSE+ cells in the tumor (solid histogram) compared with the spleen (gray line). Results are representative of three different experiments.

 
To determine whether the Ag itself causes this selective induction of IFN-{gamma} production or whether the T cells present in SLOs are incompletely differentiated effector cells, CD8 cells from the LNs and spleen were stimulated in vitro with fresh EG-7 or EL-4 tumor cells for 16 h and tested for IFN-{gamma} production (Fig. 6a). We found that IFN-{gamma} was produced only by the effector T cells (7 and 6% of all Ag-specific CD8 cells in the LNs and spleen, respectively) after at least four divisions (Fig. 6a). We also observed a massive down-regulation of TCR after in vitro antigenic stimulation of CD8 cells from the LNs or spleen (Fig. 6b), similar to that observed in vivo at the tumor site (Fig. 5). These findings indicate that the amount of Ag in each compartment determines CTL effector functions in vivo.



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  		FIGURE 6. Ag dose determines whether effector functions or AICD ensue. a, LN cells and splenocytes were restimulated in vitro at day 3 with fresh EG-7 cells or EL-4 cells for 16 h. IFN-{gamma} production was detected. The percentages of IFN-{gamma}-positive cells are indicated. b, Down-regulation of V{beta}5 surface expression of CD8+CFSE+ cells in the LNs and spleen was detected after in vitro EG-7 restimulation (solid histogram) and EL-4 restimulation (gray line). c, Cell death in the LNs and spleen was evaluated with annexin V staining, 16 h after in vitro EG-7 or EL-4 restimulation. The percentages of annexin V-positive and -negative cells are indicated. Results are representative of three different experiments.

 
Other studies (16) suggest that high Ag doses in SLOs lead to the development of T cell tolerance. Accordingly, we examined the susceptibility to cell death of Ag-specific CD8 T cells in the LNs and spleen upon TCR re-engagement with high doses of Ag: we used annexin V staining to analyze the death rate and found that many more CD8 cells restimulated with EG7 died within 24 h (39 and 30% of the CD8 cells isolated, respectively, from the LNs and spleen) than did those restimulated with EL-4 (12 and 16%) (Fig. 6c). Thus, dividing cells in SLOs were highly sensitive to apoptosis upon TCR re-engagement. When differentiating T CD8 cells re-encounter high Ag doses, activation-induced cell death (AICD) may follow. This hypothesis is supported by the result above—arrested then renewed tumor growth after adoptive transfer of OT-1 cells (Fig. 1b). Large quantities of Ag thus lead to T cell tolerance. In contrast, we did not observe annexin V+ cells among the CD8 TILs. This finding suggests that in vivo the detectable level of CD8 cell death at the tumor site is low (data not shown). We cannot rule out the possibility that dying cells are rapidly eliminated in vivo. Moreover, Ag-specific CD8 cells in peripheral tissues are more resistant to apoptosis than those in SLOs (19). Overall, the dose and distribution of Ag in the different compartments determine effector functions and cell death in vivo.

Ag-induced CTL differentiation in vivo

After finding that Ag distribution played a role in the compartmentalization of the CD8 response, we examined whether it influenced T cell migration from SLOs to the tumor site. To look at the CTL differentiation phenotype and its relationship to cell proliferation and migration to the tumor site, we studied the expression of CD62L and CD44 by CFSE-labeled CD8 cells during their proliferation and migration (Fig. 7). T cell activation and differentiation (D) involved up-regulation of CD44 and down-regulation of CD62L, in comparison to the levels of naive CD8 cells (N), as Fig. 7a shows. In the LNs and spleen, the dividing cells were mainly CD44high. The up-regulation of CD44 was followed by a slight down-regulation of CD62L in the LNs and spleen, mainly for cells that had divided at least four times (Fig. 7b). Interestingly, the OVA-specific CD8 cells collected at the tumor site were mainly CD44high (65%) and CD62Llow (84%). These results show that the cells that infiltrated the tumor were those able to leave the SLOs after the down-regulation of L-selectin expression (20, 21).



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  		FIGURE 7. Tumor Ag-induced concomitant CD8 differentiation and proliferation. a, Histogram analyses of naive (N) and activated/differentiated (D) OVA-specific CD8 cells for CD44 and CD62L expression in LNs. b, Dot plots represent CD44 and CD62L expression in the cell generations in the LNs, spleen, and tumor on day 3 after transfer. Cells were gated on CFSE+CD8+V{beta}5+. The percentage of cells in each generation is indicated. Results are representative of three independent experiments.

 
Ag-induced chemokine receptor expression during CD8 cell differentiation

Because of the importance of the chemokine/chemokine receptor network in CTL trafficking (22), we next tested whether specific migration to the tumor site was associated with a specific chemokine expression profile: we analyzed the expression of chemokine receptor mRNA in three sorted cell populations: naive OT-1 CD8 lymphocytes (N), dividing OT-1 CD8 cells (D) (cell generations 2 through 6 were determined by CFSE dilution), and Ag-specific CD8 TILs (E) (Fig. 8). Each population expressed a different pattern of chemokine receptor mRNA (Fig. 8). Naive CD8 cells from the OT-1 transgenic mice expressed CCR5, CCR2, CCR9, CCR7, CXCR4, and CXCR1/-2. In vivo dividing cells also expressed CXCR3, CXCR4, CCR5, CCR2, and CCR7 mRNA. The expression of CXCR3, CXCR4, and CCR7 mRNA was down-regulated in TILs, but CD8 cells did express CCR5 and CCR2 mRNA. We also found that the tumor environment expressed mRNA for most of the chemokine receptor ligands, a finding that reflects extreme inflammation (data not shown). These results indicate that the ability to leave SLOs and migrate into the tumor may be controlled primarily by modulation of the chemokine receptor expression profile, because most of these ligands were expressed at the inflammatory site.



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  		FIGURE 8. Chemokine receptor expression during Ag-induced T cell differentiation. a, Cell populations were sorted: naive OT-1 CD8 cells (N), dividing cells (D), and effector cells (E) were isolated on day 3 after adoptive transfer. b, Multiplex-PCR for chemokine receptors was performed for each sorted fraction and corresponded to a pool of 10 mice.

 
Ag-induced programmed cell migration

Programmed T cell homing after Ag activation is currently a major issue in this field. Induction of specific homing is reported to depend on the lymphoid tissues where activation occurs (23), and dendritic cells appear to be responsible for the selective imprinting of T cell homing during activation (24). We investigated whether Ag activation was sufficient to program cell migration into an inflammatory environment in the absence of Ag at the tumor site.

To determine whether in vivo activation programs cell proliferation and migration into the tumor site regardless of Ag presence, we conducted the experiment outlined in Fig. 9a. On day 2 posttransfer (D2), purified CFSE-labeled OT-1 CD8 cells were collected from the LNs of B6 recipient mice with growing EG-7 or EL-4 tumors. Purified total Ag-primed or naive CD8 cells were then transferred into B6 mice with growing EL-4 tumors. At 24 h after the second adoptive transfer, that is, 3 days after priming (D3), we assessed CFSE dilution in the CD8+V{beta}5+ cells.



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  		FIGURE 9. Cell migration is programmed by initial Ag encounter. a, Diagram of double adoptive transfer experiments: CFSE-labeled OT-1 CD8 cells were transferred into mice with growing EG-7 or EL-4 tumors; 2 days later, LNs were collected, and total CD8+ cells were purified with magnetic beads and transferred into mice with growing EL-4 tumors; 24 h after second transfer, OVA-specific CD8+V{beta}5+ cells were analyzed for proliferation in LNs and migration. b, Ag-induced programmed proliferation. Histograms represent OVA-specific CD8+ proliferation in LNs at day 2 (D2) before second transfer (thin line) and 24 h after second transfer (bold line). Upper panel, OVA-specific CD8 cells from B6 mice with EG-7 tumors, purified on D2. Lower panel, OVA-specific CD8 cells from B6 mice with EL-4 tumors. c, Ag-induced programmed migration. Percentage of OVA-specific CD8 TILs on day 3 of double transfer: EG-7 in vivo priming ({blacksquare}) or EL-4 in vivo priming ({square}) (n = 5 mice). d, CFSE-labeled OT-1 CD8 cells were activated ({blacksquare}) or not ({square}) in vitro with 0.5 µM OVA (SIINFEKL) (16 h) and then transferred into B6 recipient mice with growing EL-4 tumors. On day 3 posttransfer, the percentage of OVA-specific CD8+V{beta}5+ cells in the total CD8 cell population was analyzed by flow cytometry in the LNs, spleens, and tumors. Data are the mean of three different experiments. Endogenous CD8+V{beta}5+ cells have been subtracted. Statistical analyses used the Student t test: *, p < 0.05; **, p < 0.01.

 
We found that OVA-activated CD8 cells maintained their proliferation abilities (47% dividing cells at D2, and 94% at D3) in vivo even in the absence of continuous Ag stimulation (Fig. 9b, upper panel), whereas the unprimed CD8 cells (B6 mice with EL-4 growing tumor) did not proliferate at all (2% dividing cells at D2 and 5% at D3) (Fig. 9b, lower panel). Similar results were observed when CFSE-labeled CD8 cells were cultured in vitro after in vivo Ag priming (data not shown). These results demonstrate for the first time in a s.c. localized tumor model that an initial Ag encounter in SLOs programs CD8 cells to proliferate, even in the absence of continuous Ag stimulation. We also analyzed the infiltration of OT-1 CD8 cells into the EL-4 tumor after in vivo priming and found that 36% of CD8 TILs were OVA-specific CD8 cells. No significant infiltration was detectable after transfer of naive OT-1 cells (<0.5%) (Fig. 9c). These results show that the initial in vivo Ag encounter in the SLOs programs OT-1 CD8 cells to migrate to a s.c. localized tumor.

Similar results were observed when CD8 cells were primed by Ag in vitro. CFSE-labeled CD8 cells were adoptively transferred into B6 recipient mice with growing EL-4 tumors. We observed a programmed cell expansion of OVA-specific CD8 cells in the LNs and spleen (Ag-stimulated CD8 cells: 11% of the CD8 cells in LNs, 17% of the CD8 cells in the spleen; unstimulated CD8 cells: 1–2% of the CD8 cells at day 3) (Fig. 9d). In addition, OVA-stimulated cells infiltrated the tumor at 3 days after transfer (56.5% OVA-specific CD8 cells), whereas unstimulated cells did not migrate there (<0.3% OVA-specific CD8 cells) (Fig. 9d). These results together indicate that a short initial encounter with Ag programs CD8 cells to migrate, regardless of continuous antigenic stimulation in vivo.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Many studies (1, 2, 3) show that both proliferation and differentiation in models of systemic infection are programmed to begin after the first Ag encounters. An interesting characteristic of tumor models is that Ag is distributed in several compartments at various concentrations. This model enabled us to study the compartmentalization of the CD8 T cell response and the influence of Ag encounter during its different stages. A brief in vivo antigenic stimulation was sufficient to induce a complete program of migration to inflamed tissues. Ag did not have to be present in the inflammatory environment for activated CD8 T cells to migrate there. We observed activation and proliferation in the LNs during the first 2 days after transfer, together with differentiation into effector T cells; migration started on the third day. These results are consistent with the kinetics of T cell priming by dendritic cells reported by Mempel et al. (25).

We showed that Ag compartmentalization influences CD8 response. CD8 cells followed a differentiation program after their initial Ag encounter in SLOs, but did not produce IFN-{gamma} until they reached the inflammatory site. Models of viral and bacterial infection have yielded similar observations (26). One hypothesis is that effector functions are not induced until complete maturation of CD8 cells, when they accumulate in the inflamed tissue (27, 28). Another hypothesis proposes that a sufficient quantity of Ag is necessary to elicit effector functions (10). We lean toward the latter, because adding Ag to the proliferating cells induced IFN-{gamma} production by the cells that had divided enough times. The ability to produce IFN-{gamma} is known to require at least four cell divisions (29), although one study reports that this production can occur during the S phase of the first cell cycle (30). The level of TCR expression on CD8 cells may reflect differences in the antigenic stimulation of the tumor site compared with the LNs. Our results are consistent with those from other groups and suggest that the induction of effector functions requires multiple TCR engagements (8, 31). Thus, higher-dose Ag encounters at the tumor site, reflected by intense TCR down-regulation, induce effector functions including IFN-{gamma} production and cytotoxicity. However, we also found that TCR re-engagement of CD8 cells in SLOs induced cell death. This suggests that the amount of Ag in each compartment of the immune response controls the behavior of CD8 cells, as previously shown in vitro for CD4 T cell behavior (32). A previous study by Shrikant and Mescher (33) showed that the delivery of OT-1 cells before injection of a large quantity of tumor cells leads to T cell activation and a short period of tumor growth inhibition. However, CD8+ cells continue to ignore tumors when there is an insufficient amount of tumor Ag to activate the T cells (14). Similarly, we found no T cell proliferation in the mice with very small tumors (data not shown). In our model, adoptive transfer of OT-1 naive cells concomitantly with the EG-7 tumor cell line did not induce any detectable proliferation on day 4 (data not shown) and thus suggests that antitumor immune response depends on the amount of tumor Ag in vivo. However, response can be increased by peptide-pulsed APCs, as others have shown (34).

Although intermediate doses of Ag induce proliferation and migration, high-dose Ag restimulation of proliferating CD8 cells causes AICD. In vivo, the transfer of CD8 cells into mice with larger tumors induced a partial regression of the tumor, followed by its resumed growth. CD8 effector cell migration to the tumor site is thus not sufficient to cause complete tumor regression; it may instead induce other mechanisms such as activation-induced nonresponsiveness (35, 36, 37), rapid contraction of CD8 cells (7), and AICD (38). It is likely that proliferating cells in SLOs are more susceptible to apoptosis induced by high doses than are effector T cell in the periphery (19). One explanation may be that cells in SLOs are cycling, whereas those in the periphery are not (39). Cell cycling combined with IL-2 clearly plays a major role in AICD (40, 41, 42). Thus, large quantities of tumor Ag in SLOs would favor cell death. These findings raise the question of the importance of Ag distribution in the different compartments of the immune system. Efficient tumor rejection thus appears to depend on a very narrow window between immunogenicity and tolerization. Increasing T cell recruitment during this period would favor tumor rejection.

We demonstrated that migration into the tumor occurred only after a certain number of cell divisions, most likely after the cells acquired adequate chemokine receptor and adhesion molecule phenotypes. Ag experience changes the adhesion molecules, chemokine receptors, and memory markers expressed by cells (6, 43, 44, 45). We were able to assess two populations separately: the effector CD62Llow CCR7 mRNA population at the tumor site, that is, the effector memory-like cells, and the CD62Lhigh CCR7 mRNA+ population in the LNs, corresponding to central memory-like cells (46). Migration became possible in our model, as in a previous study (47), after four to seven divisions. Interestingly, CD62L down-regulation occurred mainly in this generation of cells, consistent with the homing of CD62LlowCD44high effector cells observed in the tumor site. Up-regulation of CCR5 and CXCR3 has been described at the cell surface of effector T cells (48). Indeed, dividing cells may become responsive to several chemokines, such as CCL-1, -2, -3, -5, -7, -8, and -13, and CXCL-9, -10, and -11, by expressing high levels of CCR2, CCR5, and CXCR3 mRNA. In contrast, tumor-infiltrating effector T cells have down-regulated most these chemokine receptor messengers. This result suggests negative feedback by the tumor environment, including Ag and high concentration of chemokines, on chemokine receptor expression. Thus, these effector cells may be trapped in the tumor and may not migrate out.

Our most interesting observation was that Ag stimulation induced a program of proliferation and differentiation that determined the ability of CD8 cells to leave the LNs and migrate into peripheral tissue, regardless of Ag presence there. We first showed that Ag-induced programmed cell proliferation occurs in localized tumor models: cell division continues in vivo even in the absence of Ag (1, 2, 3). Our findings enable us to propose a new aspect of this concept: the primary Ag encounter also determines the ability of CTL to migrate into peripheral tissue, regardless of the continuous presence of Ag. It has been recently suggested that Peyer’s patch dendritic cells imprint gut-homing specificity on T cells (24). Our findings reveal that in vitro Ag stimulation of CD8+ cells when there are no APCs from inflamed tissue, is not a prerequisite for tumor homing. However, we cannot rule out the possibility that specific activation by tissue-specific APCs in vivo would improve recruitment into the tumor site.

This new concept raises the question of the specificity of tumor-infiltrating cells. We found that only OT-1 infiltrating cells produced IFN-{gamma} at the tumor site, whereas endogenous cells did not (data not shown). Our observation suggests that the inflammatory environment may induce bystander recruitment of activated T cells. These results bolster the observation of Topham et al. (49) that activated CD8 cells migrate into the lungs during virus infection, regardless of whether specific Ag is present. Our findings thus provide new insights into the specificity of T cell homing and open up an interesting approach for antitumoral strategies.


   Acknowledgments
 
We thank Professor B. Autran for her critical review of the manuscript and Abdelkader Aribi for animal care.


   Footnotes
 
1 A.B. was supported by a grant from Fondation pour la Recherche Médicale. E.L. was supported by a grant from the French Ministry of Research and Technology. Back

2 Address correspondence and reprint requests to Dr. Béhazine Combadière, Laboratoire d’Immunologie Cellulaire, Institut National de la Santé et de la Recherche Médicale, Unité 543, Faculté de Médecine Pitié-Salpêtrière, 91 Boulevard de l’hôpital, 75634 Paris cedex 13, France. E-mail address: combadie{at}ccr.jussieu.fr Back

3 Abbreviations used in this paper: SLO, secondary lymphoid organ; LN, lymph node; TIL, tumor-infiltrating lymphocyte; AICD, activation-induced cell death. Back

Received for publication October 24, 2003. Accepted for publication April 22, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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