Microenvironment and Immunology Activated STAT 5 Promotes Long-Lived Cytotoxic CD 8 þ T Cells That Induce Regression of AutochthonousMelanoma

Immunotherapy based on adoptive transfer of tumor antigen-specific CD8þ T cell (TC) is generally limited by poor in vivo expansion and tumor infiltration. In this study, we report that activated STAT5 transcription factors (STAT5CA) confer high efficiency on CD8þ effector T cells (eTC) for host colonization after adoptive transfer. Engineered expression of STAT5CA in antigen-experienced TCs with poor replicative potential was also sufficient to convert them into long-lived antigen-responsive eTCs. In transplanted mastocytomaor melanoma-bearing hosts, STAT5CA greatly enhanced the ability of eTCs to accumulate in tumors, become activated by tumor antigens, and to express the cytolytic factor granzyme B. Taken together, these properties contributed to an increase in tumor regression by STAT5CA-transduced, as compared with untransduced, TCs including when the latter control cells were combined with infusion of interleukin (IL)-2/anti–IL-2 complexes. In tumors arising in the autochthonous TiRP transgenic model of melanoma associated with systemic chronic inflammation, endogenous CD8þ TCs were nonfunctional. In this setting, adoptive transfer of STAT5CA-transduced TCs produced superior antitumor effects compared with nontransduced TCs. Our findings imply that STAT5CA expression can render TCs resistant to the immunosuppressive environment of melanoma tumors, enhancing their ability to home to tumors and to maintain high granzyme B expression, as well as their capacity to stimulate granzyme B expression in endogenous TCs. Cancer Res; 72(1); 76–87. 2011 AACR.


Introduction
The discovery of tumor antigens recognized by autologous T cells (TC) in patients with melanoma has led to clinical protocols for adoptive transfer of tumor antigen-specific TCs.The efficacy of this treatment remains poor (1)(2)(3), for several reasons.To eliminate tumor cells, naive CD8 þ TCs must differentiate into effector TCs (eTC) acquiring lytic enzyme-containing granules and the capacity to secrete cytokines.However, tumor antigen-specific TCs may undergo incomplete differentiation (4) or be tolerized upon encounter with tumor antigen (5).During prolonged antigen contact within tumors, CD8 þ TCs may become functionally impaired and subsequently deleted.Methods to enhance in vivo maintenance and function of transferred eTCs are consequently required.TC therapy should aim at transferring long-lived antitumor eTCs (6) with (i) decreased thresholds for TCR signaling and enhanced ability to proliferate in response to antigen alone, thus bypassing costimulation requirement for activation, (ii) increased cytolytic activity, (iii) adequate expression of adhesion molecules or chemokine receptors to allow migration to peripheral tumorinvaded tissues, and (iv) increased resistance to tumor-derived immunosuppressive molecules.Both avidity of TCR stimulation and signals from the interleukin (IL)-2R affect differentiation of fully competent CD8 þ eTCs (7).These results support use of IL-2 as adjuvant to increase reactivity of CD8 þ eTCs, as tumor antigen are generally poorly immunogenic.However, IL-2 contributes to expansion/function of CD4 þ CD25 þ T regulatory cells with immunosuppressive properties (8), so alternative approaches may improve in vivo expansion and function of CD8 þ eTCs.
Cell programming by manipulation of transcription factors is under investigation in a wide variety of biological areas.Terminally differentiated cells are usually limited in their proliferative capacity (9), a characteristic also applying to CD8 þ TCs (10).Transcription factor manipulation succeeded in promoting macrophage cell differentiation while preserving self-renewal capacity (11).Genetic modification of TCs for use in adoptive transfer has been limited to a small number of genes.Therefore, modifying "master switch" genes such as transcription factor-rather than genes encoding effector molecules-might globally enhance TC functions.
CD8 þ eTC function and maintenance of memory CD8 þ TCs capable of self-renewal are regulated by cytokine receptors sharing the gc chain, including IL-2, IL-7, and IL-15.STAT5 is a transcription factor activated downstream of these cytokine receptors upon JAK3-mediated phosphorylation and dimerization.The effect of IL-2 on expression of genes involved in CD8 þ eTC functions could be mimicked by expressing a constitutively active form of STAT5 (STAT5CA; ref. 12).STAT5CA was also shown to promote hematopoietic stem cell self-renewal (13).
We here investigate long-term behavior of CD8 þ eTCs expressing this active STAT5CA.We show that STAT5CA expression in CD8 þ TCs favors acquisition of a phenotype reminiscent of effector memory TCs while maintaining the increased potential for antigen recall responses associated with central memory TCs.We also evaluate the potential of STAT5CA-expressing CD8 þ eTCs for antitumor responses upon adoptive transfer in tumor-bearing hosts.We used genetically engineered TiRP mice (14,15) in which inducible tumor development recapitulates key aspects of human melanoma.In these mice, melanocyte-specific deletion of Ink4a/Arf is associated with a gain-of-function mutation of H-Ras and expression of mouse cancer germline gene P1A.We show that STAT5CA-expressing CD8 þ eTCs infiltrate autochthonous melanomas and remain functional in the immunosuppressive environment (15) of those tumors.Compared with unmanipulated CD8 þ eTCs, STAT5CA-expressing CD8 þ eTCs develop higher cytolytic activity against antigen-expressing tumors, associated with strong Tc1 (IFN-gþ) responses upon restimulation.Infusion of STAT5CA-expressing CD8 þ eTCs induces tumor regression more efficiently than infusion of CD8 þ eTCs, alone or in combination with IL-2/IL-2 monoclonal antibody (mAb) complexes.STAT5CA-mediated reprogramming applied at different stages of CD8 þ TC differentiation was also efficient at boosting cytotoxic activity and tissue-migratory properties of antigenexperienced TCs.

Cell preparation
TCs were prepared from lymph nodes or spleen using standard procedures.For analysis of tissue-infiltrating TCs, donor mice were anesthetized and perfused with PBS.Livers were dispersed and passed over Ficoll-Paque (Amersham Biosciences AB).For solid tumor-infiltrating leukocytes (TIL), tissues were cut in small pieces with the GentleMacs Dissociator (Myltenyi Biotech), incubated for 40 minutes in medium containing Collagenase I (200 m/mL) and DNAse 1 (16 mg/mL) before loading over Ficoll-Paque.

Preparation and administration of cytokine complexes
Recombinant human IL-2 (TECIN, Roche, received from Linda Sherman, Scripps Research Institute, La Jolla, CA) and MAB602 mouse anti-Human IL-2 (R&D system) were incubated at 1-to-1 molar ratio.Mice received intravenous injections (daily for the first 3 days and every other day until the end of experiments) of complexes containing 4 mg IL-2 þ 20 mg MAB602 (referred to as IL-2c).
Comparative analysis also showed increased migration of TCRP1A eTC-STAT5CA in nonlymphoid tissues [liver, lung Lys-eGFP mice expressing eGFP in myeloid cells only were used as GFP tolerant hosts.Percentages of GFP þ eTCs among total gated CD8 þ TCs (corresponding exclusively to transferred eTCs) are reported in lymph node and spleen (C and D), lung and liver (C).E, activation or migration markers (as indicated) are reported for splenocytes from Rag-1 À/À B10.D2 mice 42 days after transfer of 10 6 : TCRP1A eTC-STAT5CA (blue line; gated CD8 þ GFP þ ) or untransduced TCRP1A eTCs (green line; gated CD8 þ ); TCRP1A naive CD8 þ TCs are shown in gray.For GzmB, the MFI ratio between anti-GzmB and isotype control (not shown) staining is reported on gated CD8 þ eTC splenocytes.Representative of 5 experiments.LN, lymph node.

Differentiated antigen-experienced CD8 þ TCs can be reprogrammed by STAT5CA
In experiments presented in Fig. 1B, naive CD8 þ TCs were transduced 20 hours after antigen stimulation.We asked whether STAT5CA expression could reprogram antigen-experienced CD8 þ TCs ex vivo.These cells are more representative of CD8 þ TCs present in tumor-bearing hosts.We adoptively transferred untransduced TCRP1A eTCs into primary hosts, recovered them at late time points (Fig. 2A) and transduced them to express STAT5CA.We next transferred them into secondary hosts (Fig. 2A).Although the efficiency of retroviral infection of Ag-experienced TCRP1A eTCs was low (about 10%; not shown), TCRP1A eTC-STAT5CA were strongly enriched 35 days after the second transfer, suggesting that STAT5CA also conferred a survival advantage at this stage.These cells expressed an effector memory TC phenotype, including GzmB upregulation and migration toward tissues (Fig. 2B).Ex vivo restimulation with P1A þ tumor cells triggered efficient IFN-g secretion by reprogrammed TCRP1A eTC-STAT5CA (Fig. 2C).In addition, TCRP1A eTC-STAT5CA after secondary transfer were as efficient as primary TCRP1A eTC-STAT5CA at eliminating P1A-peptide pulsed targets in 5-hour in vivo cytolytic assays (not shown).Altogether, STAT5CA expression in antigen-experienced CD8 þ TCs seemed to reprogram their migration and functional potential and to enhance their survival upon transfer into congenic hosts.

Tumor antigen-specific intratumor accumulation and reactivation of STAT5CA-expressing CD8 þ eTCs
To evaluate intratumor accumulation of TCRP1A eTCs, we used (bÀactin-LucxTCRP1A) double transgenic mice as donors of TCRP1A CD8 þ TCs.After in vitro activation and transduction to express or not STAT5CA, 10 6 Luc þ TCRP1A eTCs were injected in mastocytomas bearing hosts, and their  in vivo localization was followed by bioluminescence monitoring (Fig. 3).Luc þ TCRP1A eTC-STAT5CA expanded in P1A þ tumor-bearing hosts to a greater extent than Luc þ TCRP1A eTCs and accumulated to a 10-fold higher extent at the tumor site (central abdomen) by day 8 (Fig. 3A and B).Presence of Luc þ TCRP1A eTCs in tumor-draining lymph node was also observed at day 8.Although accumulation of Luc þ TCRP1A eTC-STAT5CA was limited in P1A À tumor-bearing hosts, these TCs were nevertheless present in areas surrounding tumors, without affecting their growth (Supplementary Fig. S3A and C).In tumor-free hosts, in contrast, Luc þ TCRP1A eTC-STAT5CA homed preferentially to lung and liver (Fig. 3A and not shown).
Antigen-specific local reactivation of TCRP1A eTC-STAT5CA was evaluated by injecting them in hosts bearing P1A þ and P1A À mastocytomas on opposite flanks (Supplementary Fig. S4).TCRP1A eTC-STAT5CA have a GzmB hi effector memory TC-like phenotype when transferred in tumor-free hosts (Fig. 1E).Upon transfer in tumor-bearing hosts (Supplementary Fig. S4), TCRP1A eTC-STAT5CA maintained a GzmB hi expression in spleens of the recipients.In addition, an efficient antigen-specific response was triggered in TCRP1A eTC-STAT5CA by P1A þ tumors, as shown by further upregulation of GzmB and CD25, both inside P1A þ tumors and draining lymph node (Supplementary Fig. S4).
Altogether, TCRP1A eTC-STAT5CA were specifically stimulated by P1A þ tumor cells, accumulated to a greater extent than control TCRP1A eTCs, and exhibited a higher cytolytic potential.All these parameters may contribute to efficient control of P1A þ tumor growth.

Regression of transplanted melanomas is more efficiently induced by STAT5CA-expressing than by control CD8 þ eTCs, even when associated with infusions of IL-2 complexes
We next evaluated antitumor responses in immunocompetent hosts (Fig. 4) injected with a melanoma cell line expressing P1A and luciferase (T429-Luc þ ; see Materials and Methods).Mice bearing a solid tumor (see photon emission reported in Fig. 4A, day 0) were infused with either control or STAT5CAexpressing TCRP1A eTCs (10 6 cells).As poor accumulation/ survival of adoptively transferred control TCRP1A eTCs (Fig. 1, Fig. 3) may limit their antitumor potential, we additionally provided infusions of IL-2/IL-2mAb complexes (IL-2c; ref. 25) that were shown to enhance expansion and activity of some antitumor CD8 þ TCs in vivo (26).
Marked tumor regression was observed in mice receiving TCRP1A eTC-STAT5CA (Fig. 4A and B).Analysis of TILs at day 11 revealed the presence of CD8 þ TCs mostly composed of transferred TCRP1A eTC-STAT5CA (Fig. 4C-E).In contrast, in mice receiving control TCRP1A eTC, tumors did not regress (Fig. 4A and B), and these eTCs hardly invaded the tumor mass (Fig. 4C-E), whereas endogenous CD8 þ TCs were present among TILs.IL-2c treatment for 10 days did not increase tumor infiltration by transferred TCRP1A eTCs (Fig. 4C-E).However, as previously described (26), IL-2c treatment increased numbers of both natural killer cells and endogenous CD8 þ TCs in the spleen (not shown), as well as among TILs (Fig. 4E).
STAT5CA-expressing TCRP1A TILs expressed high levels of GzmB in comparison with endogenous CD8 þ TILs or to TCRP1A eTC TILs from mice that received IL-2c infusions (Fig. 5A-C).Comparison of GzmB expression by endogenous CD8 þ TILs showed marked increase in TCRP1A eTC-STAT5CA-injected and limited but significant increase in TCRP1A eTC/IL-2c-injected mice (Fig. 5A-C; Supplementary Fig. S5A for statistics).Interestingly, endogenous as well as TCRP1A eTC-STAT5CA and TCRP1A eTC/IL-2c CD8 þ TILs expressed high levels of the inhibitory receptor PD-1 (Fig. 5D-F), but only TCRP1A eTC-STAT5CA CD8 þ TILs responded efficiently to recall restimulation (IFN-g secretion and CD107a exposure; Fig. 5G-I).These data suggest that IL-2c promoted proliferation of CD8 þ TCs and slightly increased their expression of GzmB in TILs and in splenic CD8 þ TCs (Supplementary Fig. S5B) but did not induce intratumor accumulation of infused TCRP1A eTCs.Accordingly, although tumor regression in recipients infused with TCRP1A eTCs and IL-2c was more pronounced than in mice receiving only TCRP1A eTCs (Fig. 4A and B), it was significantly weaker than in counterparts receiving TCRP1A eTC-STAT5CA.In the latter case, a significant increase in GzmB expression was observed in endogenous TILs in absence of IL-2c (Fig. 5A, Supplementary Fig. S5A), suggesting that tumor regression induced by TCRP1A eTC-STAT5CA and/ or activation of TCRP1A eTC-STAT5CA also favored activation of surrounding CD8 þ TILs.
When following the long-term fate of TCRP1A eTC-STAT5CA in mice that rejected a T429-Lucþ melanoma, we detected TCRP1A eTC-STAT5CA in the peripheral blood of mice killed for analysis at day 70.No pathologic signs and no evidence for tumor escape variants were observed in these mice (data not shown).

STAT5CA-expressing CD8 þ eTCs induced regression of autochthonous mouse melanoma
The stroma of naturally occurring tumors may both impede access of TCs to tumors (27) and promote immunosuppression through complex cytokine/chemokine secretion and recruitment of suppressive cells (28,29).This situation is not fully reproduced in transplanted tumors.In the inducible TiRP model of melanoma expressing the P1A-encoded tumor antigen, we recently reported (15) that endogenous CD8 þ TILs expressed a GzmB low phenotype and showed suppressed functions.TiRP mice developing aggressive induced melanoma tumors in a Rag-1 À/À B10.D2 background were injected with either 10 6 untransduced TCRP1A eTCs or TCRP1A eTC-STAT5CA.In the 9 mice receiving TCRP1A eTC-STAT5CA, we observed a very rapid and extensive tumor necrosis (Fig. 6A and B).None of the mice (n ¼ 7) treated with TCRP1A eTCs showed tumor regression (Fig. 6B).Both analyses by cytometry (Fig. 6) and by immunostaining (not shown) showed tumor infiltration by TCRP1A eTC-STAT5CA with preserved GzmB hi expression, IFN-g production, and CD107a exposure upon ex vivo restimulation (Fig. 6C).In comparison, tumor infiltration by untransduced TCRP1A eTCs was very limited (Fig. 6D).In this latter case, injection of increased cell numbers (7 Â 10 6 ) led to higher tumor infiltration, but those TILs maintained a low GzmB expression (Fig. 6E) and failed to produce IFN-g (Supplementary Fig. S6).
Altogether, TCRP1A eTC-STAT5CA showed higher potential to infiltrate autochthonous mouse melanomas and maintained a GzmB hi expression in an immunosuppressive context.

Discussion
We here report that STAT5CA-transduced CD8 þ eTCs present many properties described to positively impact efficacy of adoptive TC therapy for solid tumors (30).In particular, they show high specific cytolytic potential, strong Tc1 recall responses, and migration in tissues in a manner similar to effector memory TCs (CD44 hi CD62L lo ).In addition, STAT5CA-expressing CD8 þ eTCs showed central memory characteristics of long-term survival and capacity to self-renew.At the molecular level, the combination of these properties seems to be associated with concomitant high expression of transcription factor T-Bet, characterizing effector TCs, and Eomes, as in central memory TCs (Grange and colleagues; in preparation).
Although not constitutively activated, STAT5CA-expressing eTCs were capable of enhanced secondary responses.This may be due to their increased expression of transcripts encoding effector molecules (Grange and colleagues; in preparation) B10.D2 mice developing an induced melanoma received 10 6 TCRP1A eTC-STAT5CA (A-C) or 10 6 (B, D) or 7 Â 10 6 (E) untransduced TCRP1A eTCs.A, example of tumor regression in a TiRP mouse treated with 10 6 TCRP1A eTC-STAT5CA.B, mice bearing amelanotic-induced melanomas were infused with 10 6 untransduced (7 mice) or STAT5CA-transduced (9 mice) TCRP1A eTCs.Tumors were measured on the day of TC transfer (d0) and 7 days later.Ratios of tumor volumes, calculated as (l 2 Â L)/2, are shown for individual mice.In the first group, 2 mice died before day 7.In the second group, the initial tumor size was not recorded for one mouse.Statistical analysis was done with a t test using GraphPad and 2-tailed P. ÃÃ , P ¼ 0.002.C and D, 11 or (E) 7 days after adoptive transfer, splenocytes and TILs were stained directly for CD8 and GzmB.GzmB expression and MFI values are shown for gated CD8 þ TCs.A fraction of cells was stimulated for 4 hours with anti-CD3 presented by FcR-bearing P1A À tumors and stained for CD8/CD107a/IFN-g (bottom panels in C and D); CD107a versus IFN-g plots are shown for CD8 þ TCs with % reported in each quadrant.
allowing rapid recall responses.Moreover, expression of STAT5CA in antigen-experienced CD8 þ TCs endowed with poor replicative potential converted them into long-lived eTCs.These reprogrammed eTCs also showed increased capacity for tissue infiltration and responses upon antigen recall, extending the potential application of the approach with particular relevance for tumor-bearing hosts.
In TiRP mice, developing autochthonous induced melanomas are infiltrated by PD-1 þ GzmB À nonfunctional endogenous CD8 þ TILs associated with systemic chronic inflammation (15), akin to that detected in a subset of melanoma patients (31).These mice also presented defects in lymph node and splenic T cell zone stroma associated with impaired recruitment of naive TCs (32), stressing the importance of tissue-migratory properties for TCs in adoptive immunotherapy.Our ultimate goal was to evaluate the ability of STAT5CA-expressing CD8 þ TCs to infiltrate these autochthonous melanomas and to resist their immunosuppressive effects.However, given the asynchrony of melanoma development within a cohort of treated TiRP mice and their limited availability, we first used transplanted P1A þ mastocytomas and derived P1A À variants to establish the tumor antigen specificity of the eTC-STAT5CA antitumor effects.We also used a P1A þ melanoma line established from a TiRP-induced melanoma for the comparison of protocols involving adoptive transfers with STAT5CA-expressing or untransduced CD8 þ eTCs with or without Il-2c.
Accumulation of TCRP1A eTC-STAT5CA in P1A þ tumors probably resulted from antigen-induced expansion as well as a TC-intrinsic propensity to migrate to tissues/tumors because TCRP1A eTC-STAT5CA were also concentrated to some extent in P1A À mastocytomas.This accumulation was about 3.5-fold lower (evaluation by bioluminescence in Fig. 4B and C) in P1A À as compared with P1A þ mastocytomas, but it was almost 80-fold higher than that of control-transduced TCRP1A eTC-GFP in P1A À mastocytomas.The presence of TCRP1A eTC-STAT5CA was not associated with regression of P1A À tumors, however, in agreement with the TC specificity.
Targeted destruction of malignancies by adoptive transfer of antitumor CD8 þ eTCs has been combined with IL-2 infusions to support survival and proliferation of eTCs in cancer patients (6,33,34).Nevertheless, in vivo maintenance of CD8 þ eTCs was hardly increased (6), and IL-2R-driven signals seemed to generate terminally differentiated eTCs that rapidly became senescent (10,35,36).In addition, as IL-2 contributes to expansion/function of CD4 þ CD25 þ regulatory TCs with immunosuppressive properties (37), substitutes such as use of IL-2c have been developed (25).In mice inoculated with a P1A þ melanoma, IL-2c treatment improved CD8 þ eTCs survival.However, IL-2c did not promote migration of infused antitumor CD8 þ eTCs inside tumors and had limited effects on GzmB expression.In contrast, STAT5CA expression induced stable phenotypic and migratory properties and survival of CD8 þ eTCs.The difference between STAT5CA and IL-2c treatment for modulating homing properties and survival of the transferred eTCs may depend on the capacity of STAT5CA to concomitantly mimic signals elicited by IL-7 and IL-15, as well as IL-2 (38).In addition, although they expressed PD-1 inhibitory receptors to the same extent as endogenous TCs or untransduced TCRP1A eTCs, TCRP1A eTC-STAT5CA were found responsive to secondary stimulation.Ligation of PD-1 is thought to induce its phosphorylation and to increase its association with the SHP-2/SHP-1 phosphatases that in turn dampen TCR signaling.However, this negative PD-1mediated signal can be overcome through cytokine receptor signaling, particularly cytokines that activate STAT5 (39).Additional analysis is required to establish the molecular bases maintaining responsiveness of TCRP1A eTC-STAT5CA.
Tumor regression induced by TCRP1A eTC-STAT5CA revealed that those TCs remained active in the face of tumor-derived immunosuppression.In TiRP mice, a state of Th2/Th17-oriented chronic inflammation develops that resembles, in part, the Th2-dominant chronic inflammation observed in advanced melanoma patients (31).Consistent with detection of cytokines capable of activating STAT3 (40), nuclear phospho-STAT3 in tumor cells and in some infiltrating CD45 leukocytes was observed in the TiRP model (15).In CD8 þ TCs, STAT3 deletion improved their tumor-induced proliferation and infiltration within tumors (41).STAT3 and STAT5 have been reported to compete for binding on a similar DNA consensus sequence on the IL-17 promoter (42).Whether this competition is a mechanism for resistance to immunosuppression of STAT5CA-expressing CD8 þ eTCs will be further investigated.
In conclusion, STAT5CA expression contributed to optimize antitumor activity of CD8 þ eTCs, with increased intratumor accumulation and strong specific Tc1 recall responses.This result suggests consideration of use of STAT5CA-transduced CD8 þ TCs for adoptive immunotherapy.This approach here required the use of retroviruses.Recent clinical data with retrovirus-engineered TCs for cancer and HIV patients indicate that retroviral gene transfer in mature TCs is safe (43).This conclusion is supported by studies showing resistance of mature TCs to oncogene transformation (44).In addition, no transformation of STAT5CA-transduced CD8 þ TCs was observed in this and in a previous study (45).Coupling of STAT5CA transduction with that of suicide genes such as thymidine kinase will allow further security for adoptive therapy.Finally, the survival advantage of STAT5CA-expressing cells over their control counterparts suggests that STAT5CA transduction of CD8 þ TCs specific for some poorly immunogenic tumor antigen might allow their enrichment in vitro and render them efficient for adoptive immunotherapy.The fact that antigen experienced and polyclonal CD8 þ TCs can be reprogrammed by STAT5CA expression could thus enhance the number of tumor antigen that may be targeted in adoptive therapy.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

Figure 2 .
Figure 2. STAT5CA expression promotes effector functions and tissue homing independent of the activation state of TCs.A-C, untransduced TCRP1A eTCs from primary Rag-1 À/À B10.D2 hosts were recovered from lymph nodes and spleens at d95 after transfer pooled from 2 mice (A).Enriched CD8 þ TCs were in vitro activated with P1A 35-43 peptide for 20 hours to trigger proliferation and retrovirally transduced.After additional 48 hours in culture, they were injected in secondary Rag-1 À/À B10.D2 hosts.Thirty-five days later, second recipients were analyzed spleen (A-C) and liver (A); CD8 versus GFP dot plots are shown in A (right).B, expression of markers (as indicated) and GzmB on gated CD8 þ eTC splenocytes; the MFI ratio between GzmB and isotype control (not shown) is reported on gated CD8 þ TC.C, a fraction of splenocytes were activated for 4 hours with P1A þ P815 or P1A À P1.204 tumor cells, after which intracellular IFN-g staining was done (shown on gated CD8 TCs).Representative of 3 experiments.LN, lymph node.

Figure 3 .
Figure 3. Higher intratumor accumulation of TCRP1A eTC-STAT5CA than control TCRP1A eTCs when transferred in hosts inoculated with P1A þ mastocytoma.A and B, Rag-1 À/À B10.D2 mice were untreated or inoculated subcutaneously with 10 6 P1A þ P815 or P1A À P1.204 mastocytoma cells.Naive CD8 þ TCs from (b-actin-LucxTCRP1A) mice were used for retroviral infection.Luc þ TCRP1A eTC-STAT5CA (blue in B and C) and Luc þ TCRP1A eTC-GFP sorted for GFP expression (green in B and C, diamonds in C) or nontransduced (green in B and C, squares in C) Luc þ TCRP1A eTC (10 6 cells each) were injected in tumor-free (2 mice) or tumor-bearing hosts.Two experiments were conducted.Bioluminescence was recorded at days 1, 3, and 8 after transfer.A, a representative image is shown for each experimental group.B and C, CD8 TCs accumulation in tumor areas is reported as number of photons/mm 2 S on a defined surface.Mean values AE SD are reported at day 1 to 8 for P1A þ (left graph) and P1A À (right graph; B) and as values for individual mice on day 8 (C).Statistical analyses were done with an unpaired t test (GraphPad).Two-tailed P: Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001.

Figure 4 .
Figure 4. Higher intratumor accumulation and tumor regression by TCRP1A eTC-STAT5CA than control TCRP1A eTCs AE IL-2c infusions in hosts inoculated with a P1A þ melanoma.Lys-eGFP CD45.1 B10.D2 mice were inoculated subcutaneously with 10 6 P1A þ Luc þ melanoma (T429-Lucþ) cells.Mice with solid tumors (day 0 in A) received 10 6 TCRP1A eTC-STAT5CA (A-E; n ¼ 7), or untransduced TCRP1A eTCs alone (A-C; n ¼ 7), or in combination with IL-2c infusions (A-D; n ¼ 4).A and B, tumor development was measured by counting photons emitted by luciferase-expressing tumors on a defined skin surface (mm 2 S).Values between day 0 (time of adoptive transfer) and day 11 (time of analysis) are reported in A as means AE SD for each group.For each mouse (B), photon emission is also reported as % of initial tumor photon emission, which is a ratio between day 0 (normalized to 100%) and day 11.C-E, eleven days after TC transfer, TILs were stained directly for CD8, CD45.1 (in C, host: CD45.1þ;TCRP1A eTCs: CD45.2 are CD45.1-negative).D and E, ratios between transferred and endogenous CD8 þ TILs (D) and absolute numbers of transferred and endogenous TILs (E) were evaluated from fluorescenceactivated cell sorting analysis.Statistical analyses were done with a t test as in Fig.3.In E, 2 by 2 comparisons of the different groups gave significant differences ( ÃÃ ) as indicated.All other comparisons had P > 0.05 (ns).