Helper-Independent, L-Selectinlow CD8+ T Cells with Broad Anti-Tumor Efficacy Are Naturally Sensitized During Tumor Progression This information is current as of September 30, 2021. Liaomin Peng, Jørgen Kjaergaard, Gregory E. Plautz, David E. Weng, Suyu Shu and Peter A. Cohen J Immunol 2000; 165:5738-5749; ; doi: 10.4049/jimmunol.165.10.5738 http://www.jimmunol.org/content/165/10/5738 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Helper-Independent, L-Selectinlow CD8؉ T Cells with Broad Anti-Tumor Efficacy Are Naturally Sensitized During Tumor Progression1

Liaomin Peng,* Jørgen Kjaergaard,* Gregory E. Plautz,2* David E. Weng,† Suyu Shu, and Peter A. Cohen3†*

We recently reported that the CD4؉ subset with low L-selectin expression (CD62Llow) in tumor-draining lymph nodes (TDLN) can be culture activated and adoptively transferred to eradicate established pulmonary and intracranial tumors in syngeneic mice, even without coadministration of IL-2. We have extended these studies to characterize the small subset of L-selectinlow CD8؉ T cells naturally present in TDLN of mice bearing weakly immunogenic tumors. Isolated L-selectinlow CD8؉

T cells displayed the functional phenotype of helper-independent T cells, and when adoptively transferred could consistently Downloaded from eradicate, like L-selectinlow CD4؉ T cells, both established pulmonary and intracranial tumors without coadministration of -exogenous IL-2. Whereas adoptively transferred L-selectinlow CD4؉ T cells were more potent on a cell number basis for eradi cating 3-day intracranial and s.c. tumors, L-selectinlow CD8؉ T cells were more potent against advanced (10-day) pulmonary metastases. Although the presence of CD4؉ T cells enhanced generation of L-selectinlow CD8؉ effector T cells, the latter could also -be obtained from CD4 knockout mice or normal mice in vivo depleted of CD4؉ T cells before tumor sensitization. Culture low ؉ activated L-selectin CD8 T cells did not lyse relevant tumor targets in vitro, but secreted IFN-␥ and GM-CSF when specifically http://www.jimmunol.org/ stimulated with relevant tumor preparations. These data indicate that even without specific vaccine maneuvers, progressive tumor growth leads to independent sensitization of both CD4؉ and CD8؉ anti-tumor T cells in TDLN, phenotypically L-selectinlow at the time of harvest, each of which requires only culture activation to unmask highly potent stand-alone effector function. The Journal of Immunology, 2000, 165: 5738–5749.

doptive immunotherapy of established mouse tumors is culture activation of TDLN T cells has yielded anti-tumor T cells commonly performed by transferring T cells obtained that can be adoptively transferred to cure syngeneic mice of tumors from the tumor-draining lymph nodes (TDLN)4 or tu- established at all tested anatomic locations, including pulmonary, A by guest on September 30, 2021 mor-infiltrating of syngeneic tumor-bearing mice. s.c., intracranial, and i.p. (14–16, 59). Furthermore, their efficacy Before such adoptive transfer, T cells are typically culture acti- is highly evident even without coadministration of exogenous IL-2 vated to correct tumor-induced signaling abnormalities as well as (14–16). to provide numeric expansion (1–6). Many culture techniques his- The improved ability to unmask and preserve potent anti-tumor torically employed for this purpose have yielded anti-tumor T cells effector T cells in vitro has enabled more precise phenotypic char- that displayed limited or no therapeutic efficacy against weakly acterization of those T cell subpopulations naturally sensitized as immunogenic tumors unless exogenous rIL-2 was coadministered a consequence of tumor growth. For example, highly potent, nat- during treatment. Furthermore, such cultured T cells often lacked urally sensitized, pre-effector T cells are concentrated within the therapeutic efficacy against established extrapulmonary tumors TDLN T cell subset displaying low or absent surface expression of even when exogenous rIL-2 was coadministered (7–13). In con- L-selectin (CD62L), a peripheral lymph node-homing trast, the inclusion of anti-CD3 or bacterial superantigen during whose down-regulation during sensitization may promote T cell trafficking to other locations (17, 18). Anti-CD3 culture activation low *Center for Surgery Research and †Department of Hematology and Medical Oncol- and adoptive transfer of the isolated L-selectin T cell subset has ogy, Cleveland Clinic Foundation, Cleveland, OH 44195 proved therapeutically far superior to adoptive transfer of L-se- Received for publication December 7, 1999. Accepted for publication August lectinhigh (L-selectinhigh) or unfractionated TDLN T cells, illus- 16, 2000. trating the potential value of eliminating irrelevant and/or suppres- The costs of publication of this article were defrayed in part by the payment of page sor T subpopulations during culture activation and adoptive charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. therapy (14, 15). low 1 This work was supported in part by grants from the National Cancer Institute To optimize adoptive immunotherapy with L-selectin TDLN- (CA78263 and CA67324). derived T cells, it is essential to characterize the relative contri- 2 Current address: Department of Pediatric Oncology, Yale University Medical Cen- butions of CD4ϩ and CD8ϩ T cells to tumor rejection. The ob- ter, 333 Cedar Street, LMP4087, New Haven, CT 06510. served contributions of each subset are highly dose dependent and 3 Address correspondence and reprint requests to Dr. Peter A. Cohen, Center for are furthermore influenced by the relative proportions of CD4ϩ Surgery Research, FF50, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleve- ϩ land, OH 44195. E-mail address: [email protected] and CD8 T cells present during culture activation and adoptive ϩ 4 Abbreviations used in this paper: TDLN, tumor-draining lymph node(s); CD62Llow, transfer. For example, TDLN CD4 T cells appear to play mainly L-selectin low; CD62Lhigh, L-selectin high; HIT cell(s), helper-independent T cell(s); a helper role in tumor rejection when numerically dominated by B6 mice, C57BL/6 mice; KO, knockout; PI, propidium iodide; R␣M, rat anti-mouse; ϩ low ␣ TDLN CD8 T cells (19). However, the small L-selectin subset G R, goat anti-rat; CM, complete medium; TAM, tumor-associated macrophages; ϩ LAK, lymphokine-activated killer. of CD4 TDLN T cells can be isolated, culture activated, and

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 5739 adoptively transferred to reject established pulmonary and intra- Sensitization and fractionation of syngeneic tumor-draining cranial tumors even without coadministered CD8ϩ T cells or ex- lymph node cells ogenous IL-2 (14). Given the remarkable “stand-alone” therapeutic ϫ 6 low ϩ B6 mice or BALB/c mice were inoculated s.c. with 1.5 10 MCA-205 potential of purified L-selectin CD4 TDLN T cells, we have or CT-26 tumor cells, respectively, in both flanks. Twelve (MCA-205) or endeavored to better characterize the even smaller subpopulation 9 (CT-26) days later, inguinal TDLN were harvested, and single-cell sus- of L-selectinlow CD8ϩ T cells that is also detected in freshly har- pensions were prepared mechanically by teasing with needles and pressing vested TDLN. Our initial testing of the L-selectinlow CD8ϩ sub- tissue fragments with the blunt end of a 10-ml plastic syringe (15). Mouse T cell enrichment columns containing G␣R-Ig Ab-coated glass beads population failed to reveal therapeutic potency against intracranial (R&D Systems (Minneapolis, MN) and Cytovac Technologies (Edmonton, tumors (14), leading us to investigate alternative isolation tech- Canada)) were used to isolate individual TDLN subpopulations. The man- niques to improve recoveries for more thorough analysis and ther- ufacturers’ protocols was followed, except that TDLN cells were preincu- apeutic dose escalations. We present the first evidence that the bated for 20 min at 4°C in concentrations of anti-L-selectin (CD62L), low ϩ anti-CD4 and/or anti-CD8 ascites pretitrated for efficacy (1/10,000, L-selectin CD8 T cell subset possesses a stand-alone curative 2ϩ 2ϩ ϩ 1/1,000, and 1/1,000, respectively), washed in Ca /Mg -free HBSS, potency with several similarities to that displayed by the CD4 then applied to the columns to isolate L-selectinlow T cells (unfractionated ϩ subset. Because such therapeutically potent L-selectinlow CD8 T for CD4ϩ and CD8ϩ), L-selectinlow CD4ϩ T cells, or L-selectinlow CD8ϩ cells can be obtained from CD4 knockout mice or from normal T cells. Ninety to 95% of effluent cells were strongly CD3pos by direct mice variously depleted of CD4ϩ T cells, it appears that their fluorescent analysis, and effluent cells that had been preincubated with anti-CD4 or anti-CD8 were quantitatively depleted of these populations initial sensitization as well as their effector action has a capacity (see Fig. 1). In some experiments, T cells were negatively immunoselected for true helper independence. for L-selectin at the beginning of culture, then negatively immunoselected

for CD4 at the end of 5 days of culture. Downloaded from Materials and Methods Anti-CD3/IL-2 activation of TDLN Mice Effluent TDLN cells were activated on 24-well plates precoated with the 6 Female C57BL/6N (B6) and BALB/c mice were purchased from the Bi- anti-CD3 mAb as described previously (14). Each well contained 4 ϫ 10 6 ologic Testing Branch, Frederick Cancer Research and Developmental cells in 2 ml of complete medium (CM), as well as 2 ϫ 10 irradiated (3000 Center, National Cancer Institute (Frederick, MD). They were maintained rad) freshly harvested splenocytes from normal syngeneic mice. CM con- in a specific pathogen-free environment and were used at the age of 8–10 sisted of RPMI 1640 supplemented with 10% heat-inactivated FCS, 0.1 http://www.jimmunol.org/ wk. In addition, B6 background CD4 knockout (CD4KO) and CD8a knock- mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM fresh out (CD8KO) mice (JR2269 and JR2665, respectively) were obtained from L-glutamine, 100 mg/ml streptomycin, 100 U/ml penicillin, 50 mg/ml gen- The Jackson Laboratory (Bar Harbor, ME). tamicin, 0.5 mg/ml Fungizone (all from Life Technologies, Grand Island, NY), and 5 ϫ 105 M 2-ME (Sigma). After 2 days of incubation at 37°C in Tumors 5% CO2, activated cells were suspended in 4 U/ml of human rIL-2 (Chiron, Emeryville, CA) at 1–2 ϫ 105/ml and cultured in 24-well plates or gas- The MCA 205 and 203 fibrosarcomas, syngeneic to B6 mice, were origi- permeable culture bags (Baxter Healthcare, Deerfield, IL) for 3 days. Cells nally induced with 3-methylcholanthrene (14). The tumors have been were harvested, washed, and resuspended in HBSS for adoptive immuno- maintained in vivo by serial s.c. transplantation of thawed cryopreserved therapy or in CM for ELISA or 51Cr release assays (14). mince in B6 mice and were used within the eighth transplantation gener- ation. Single-cell suspensions were prepared from solid tumors by diges- Adoptive immunotherapy by guest on September 30, 2021 tion with a mixture of 0.1% collagenase, 0.01% DNase, and 2.5 U/ml B6 mice were inoculated intracranially in the right hemisphere with 1 ϫ hyaluronidase (Sigma, St. Louis, MO) for2hatroom temperature as 105 syngeneic tumor cells in 10 ␮l of HBSS to establish brain metastases previously described (14). The CT-26 colon adenocarcinoma, syngeneic to (14) or with 1. 5 ϫ 106 tumor cells in 50 ␮l of HBSS under the midline BALB/c mice (20, 21), was provided by Gary Nabel (Vaccine Research ventral skin to establish s.c. tumors (16). Three days after tumor inocula- Center, National Institutes of Health, Bethesda, MD) and was similarly tion, mice received sublethal total body irradiation (500 rad), followed by maintained by serial s.c. transplantation in BALB/c mice. infusion of anti-CD3-activated syngeneic effector T cells suspended in 1.0 ml of HBSS through the tail vein. Mice followed for evidence of intrace- mAbs and flow cytometry rebral tumor progression were monitored for survival with an end point of Hybridomas producing mAb against murine CD4 (GK1.5), CD8 (2.43), cure or preterminal neurologic symptoms (14). Mice with established s.c. and L-selectin (MEL-14) were obtained from the American Type Culture tumors were evaluated by serial caliper measurements and euthanized Ͼ 2 Collection (Manassas, VA) and were used to prepare Ab-rich ascites fluid when the product of two perpendicular dimensions was 300 mm (16). (15). PE- and or FITC-conjugated rat anti-mouse (R␣M) reagents to CD3, The therapeutic efficacy of effector cells was also assessed in the treatment of metastases in the lung. In this model, mice were inoculated i.v. with 3 ϫ CD4, CD8, CD45RB (B220), MAC3, NK1.1, and L-selectin (CD62L) as 5 well as subclass-matched control Ab and FITC-conjugated goat anti-rat 10 tumor cells suspended in 1.0 ml of HBSS to establish pulmonary me- (G␣R) and mouse anti-rat (M␣R) Ab were purchased from PharMingen tastases. Three or 10 days later, mice received 500 rad, then anti-CD3- (San Diego, CA). Cells to be analyzed by direct immunofluorescence were activated syngeneic effector T were given i.v. in 1.0 ml of HBSS through FcR blocked at 4°C for 20 min with 1 ␮g of unconjugated R␣M-CD32 the tail vein. On day 18 (3-day model) or day 11 (10-day model) after T cell (24G2, PharMingen) and 10 ␮g of unconjugated normal mouse IgG (Jack- inoculation, all mice were sacrificed for enumeration of tumor nodules on 2ϩ 2ϩ the surface of the lung, as previously described (14). son ImmunoResearch, West Grove, PA) in FACS buffer (Ca /Mg -free Ϫ HBSS containing 5% FCS and 0.02% sodium azide), then exposed to con- In some experiments, on days 2 and 7 of adoptive transfer, mice jugated Ab. Cells to be analyzed by indirect immunofluorescence because received 1 ml of a 1/4 dilution of GK1.5 ascites or a 1/10 dilution of 2.43 of their prior incubation with unconjugated R␣M-CD62L, R␣M-CD4, ascites i.v. to deplete CD4 or CD8 T cells, respectively. Quantitative in and/or R␣M-CD8 were, depending on the assay, FcR blocked with 10 ␮g vivo depletion was confirmed by splenocyte analysis of sacrificed sentinel of unconjugated mouse IgG and/or exposed to additional unconjugated mice, as described previously (16). R␣M-CD62L, -CD4, and/or -CD8. Samples were washed and counter- Cytokine assays stained either with FITC-G␣R IgG or FITC-M␣R mAb, then washed in FACS buffer. In some cases cells were counterblocked with 1 ␮g of un- A total of 2 ϫ 106 culture-activated T cells derived from MCA-205 TDLN conjugated R␣M-CD32 to block FcR and saturate cell-bound FITC-G␣R were exposed to 5 ϫ 105 irradiated (5000 rad) stimulator cells, the latter Ab, then stained with PE-R␣M-CD4 or -CD8, washed in FACS buffer, and consisting of s.c. passaged, freshly harvested, and enzymatically digested resuspended for analysis. Cells were finally washed and resuspended in 0.5 tumors (MCA-205 or MCA-203); in vitro passed stroma-free tumor cell ml of FACS buffer with 0.8 ␮g/ml PI for immediate analysis or were fixed line (H-12 derivitization of MCA-205); or tumor-associated macrophages in FACS buffer with 1% added paraformaldehyde for deferred analysis. (TAM). To generate TAM, single-cell suspensions of s.c. passaged, enzy- Fixed or unfixed samples were subjected to three- or two-color analysis on matically digested MCA-205 were plated onto glass petri dishes at a den- a FACScan flow microfluorometer (Becton Dickinson, Sunnyvale, CA). sity of 80 ϫ 106 cells/10-cm plate in CM. The cell suspension was incu- The viable cell region was equally well delineated by combined forward bated for 45 min at 37°C, then nonadherent cells were vigorously washed scatter and PI exclusion or combined forward and side scatter properties. away and discarded. The adherent cells were harvested by trypsinization 5740 HELPER-INDEPENDENT L-SELECTINlow ANTI-TUMOR CD8ϩ T CELLS

FIGURE 1. Representative FACS analysis demon- strating efficacy of negative immunoselection purifi- cations. TDLN cells were freshly harvested from B6 mice bearing syngeneic MCA-205 tumors, then were unprocessed (undepleted), preincubated with rat anti- mouse L-selectin (R␣M-CD62L) Ab alone (L-selectin depleted), preincubated with R␣M-CD8 and R␣M- CD62L (CD8/L-selectin depleted), or preincubated with R␣M-CD4 and R␣M-CD62L (CD4/L-selectin depleted). Each Ab-preincubated group was then ap- plied to glass bead columns precoated with G␣R-Ig Ab (see Materials and Methods), and effluents were further processed in parallel with the undepleted group. Aliquots of each group were variously stained with additional unconjugated Ab (R␣M-CD62L, R␣M-CD4, R␣M-CD8, R␣M-CD3, or control, as shown), counterstained with FITC-M␣R second Ab, then FACS analyzed. A fixed marker (M1) was set for each group to enable quantitation of positively staining subpopulations as well as background staining in the control groups. Displayed (percentage) values in the con- Downloaded from trol histograms indicate the percentage of background staining within the M1 region. Displayed (percentage) values in the other histograms indicate the percentage of cells staining positively for anti-CD4, anti-CD8, or anti- CD3, corrected for control background staining. Percent- ages were not determined for anti-CD62L staining, be- cause a continuum of CD62L expression, rather than http://www.jimmunol.org/ positive or negative expression, was observed.

ϩ ϩ with 0.25% trypsin for 15 min at 37°C, then washed in CM. T cells were CD4 and CD8 subsets (14). Such multistep purifications estab- exposed to these various stimulators or to immobilized anti-CD3 mAb for lished the L-selectinhigh subpopulation’s lack of therapeutic po- 24 h in 2 ml of CM in 24-well plates at 37°C (14). Each stimulator group tency, but also subjected L-selectinlow subpopulations to prolonged was also cultured in the absence of added T cells to enable subsequent correction for background (nonspecific) cytokine production. Supernatants processing before culture. Therefore, in the present studies we by guest on September 30, 2021 were harvested, and the concentrations of IFN-␥, GM-CSF IL-2, IL-4, and used a single-step negative immunoselection procedure to remove IL-10 were measured by ELISA using paired mAb and standards pur- nontherapeutic L-selectinhigh T cells while hastening isolation and chased from PharMingen as described previously (14). culture of highly enriched L-selectinlow T cells. Freshly harvested TDLN cells were preincubated with preti- In vitro cytotoxicity assay trated concentrations of 1) rat anti-mouse L-selectin (R␣M- ␣ ␣ Four-hour 51Cr release assays, and preparation of lymphokine-activated CD62L) Ab alone, 2) R M-CD62L plus R M-CD8 Ab, or 3) killer (LAK) control cells from syngeneic B6 normal splenocytes were R␣M-CD62L plus R␣M-CD4 Ab, then were applied to glass bead performed as described previously (14). The MCA-205 tumor cells (1 ϫ columns precoated with G␣R Ig Ab. Depending on the preincu- 7 51 51 10 ) were labeled with Cr (Na CrO4, 100 mCi; DuPont, Wilmington, ϫ 4 bation cocktail, fractionated subsets emerging in the column ef- DE) at 37°C for 1 h and washed three times in CM. Target cells (1 10 ) low ϩ were incubated with various numbers of effector cells at 37°C in a volume fluents were 1) purified L-selectin T cells (both CD4 and ϩ low ϩ of 0.2 ml of CM for 4 h. The supernatant was collected (Titer-Tek Col- CD8 ), 2) purified L-selectin CD4 T cells, or 3) purified L- lecting System, Flow Laboratories, McLean, VA), and the samples were selectinlow CD8ϩ T cells. Depletion of L-selectinhigh cells was counted in a gamma counter. The percent lysis was calculated as follows: confirmed by FACS analyses. R␣M-CD8 or R␣M-CD4 coated T ϭ Ϫ Ϫ % lysis (experimental cpm spontaneous cpm/maximal cpm spon- Յ taneous cpm) ϫ 100. Nonspecific LAK cells were generated from B6 nor- cells were effectively depleted to 0.5% final contamination in the pos mal spleen cells by incubating 2 ϫ 106 cells/ml in CM containing 1000 column effluent (Fig. 1). In addition, FcR and/or adherent sub- U/ml rIL-2 for 3 days and were used as a cytotoxic effector cell-positive populations such as B cells, myeloid cells, and NK cells were control. efficiently removed by the G␣R-Ig Ab-coated glass beads, as ev- Ͻ Statistical analysis idenced by a 1% contamination of cells staining positively for non-T-lineagemarkersanti-B220(CD45RB),anti-MAC-3,andanti- The significance of differences in numbers of pulmonary metastases be- NK1.1, respectively (not shown). Column effluent cells were tween groups and the survival of mice with intracranial tumors were ana- Ͼ90% CD3ϩ, with Ͻ10% of CD3ϩ cells possessing a lyzed by the exact rank modification of the Wilcoxon rank-sum test. A Ϫ Ϫ ϩ Ϫ Ϫ two-tailed p Ͻ 0.05 ( p1 ϭ 0.025) was considered significant. CD4 CD8 phenotype (Fig. 1). This minor CD3 CD4 CD8 subpopulation was also evident in TDLN not subjected to mAb exposure and column fractionation. Results The observed total content of L-selectinlow CD8ϩ T cells in low Isolation of L-selectin TDLN T cell subsets TDLN was typically half that observed for L-selectinlow CD4ϩ T Our previous studies had combined positive and negative immu- cells due variously to a smaller total CD8ϩ T cell content and/or noselection techniques (e.g., nylon wool passage, panning, and a smaller proportion of CD8ϩ T cells displaying low L-selectin magnetic beading) to purify both L-selectinhigh and L-selectinlow expression (not shown). Nonetheless, adequate numbers of highly TDLN T cell subpopulations, followed by subfractionation of enriched L-selectinlow cells of either the CD4ϩ or the CD8ϩ subset The Journal of Immunology 5741 could be obtained for study purposes by processing sufficient num- lesser therapeutic potency on a cell number basis than the L-se- bers of TDLN. Preparations depleted of CD4ϩ or CD8ϩ T cells lectinlow CD4ϩ subset, both subsets possessed a dose-dependent before culture (Fig. 1) remained consistently depleted when ana- capacity to eradicate intracranial and pulmonary tumors as single- lyzed by FACS at the end of 5-day culture (not shown). Isolated agent therapy without administration of exogenous rIL-2. In addi- effluent subpopulations displayed only a modest proliferative re- tion, adoptive transfer of either 1 ϫ 106 L-selectinlow CD4ϩ or 1 ϫ sponse in anti-CD3/IL-2 culture (typically 3-fold expansion in 5 106 L-selectinlow CD8ϩ TDLN T cells was sufficient to eradicate days), but a consistently remarkable anti-tumor effector activity established 3-day pulmonary metastases (Fig. 2B). (see below). As shown previously for L-selectinlow CD4ϩ T cells (14), tumor rejection by L-selectinlow CD8ϩ T cells was restricted to the rel- ϩ ϩ low Either CD4 or CD8 sensitized L-selectin TDLN T cells evant sensitizing tumor (Fig. 3), consistent with an Ag-dependent, can eradicate 3-day established MCA-205 pulmonary and rather than an LAK-mediated or otherwise Ag-unrestricted mech- intracranial tumors anism of rejection.

Previous experiments demonstrated a dose-dependent ability of low ϩ low ϩ Purified L-selectin CD8 TDLN T cells are more effective L-selectin CD4 TDLN T cells to eradicate established intra- low ϩ cranial tumors when 1–2 ϫ 106 cells were administered as adop- than purified L-selectin TDLN CD4 T cells for the tive therapy after culture activation (14). The same ability was eradication of advanced (10-day) MCA-205 pulmonary tumors consistently demonstrated in the present experiments by culture- Although, compared with purified L-selectinlow CD4ϩ T cells, activated L-selectinlow CD4ϩ TDLN T cells enriched by single- larger numbers of L-selectinlow CD8ϩ T cells were required to step negative immunoselection columns (Fig. 2A). eradicate established 3-day intracranial tumors, the opposite effi- Downloaded from The therapeutic effects of similarly prepared L-selectinlow cacy profile was observed for advanced (10-day) pulmonary me- CD8ϩ T cells also proved dose dependent, and administration of tastases. MCA-205-sensitized, purified, L-selectinlow T cells un- 5 ϫ 106 culture-activated L-selectinlow CD8ϩ T cells proved suf- fractionated with regard to CD4ϩ and CD8ϩ, purified ficient to eradicate established 3-day intracranial tumors (Fig. 2A). L-selectinlow CD4ϩ T cells, and purified L-selectinlow CD8ϩ T This was a considerably higher dose of L-selectinlow CD8ϩ T cells cells were each culture-activated and adoptively transferred to treat than had previously been available for study (14). Therefore, al- established 10-day MCA-205 pulmonary metastases (Fig. 4). http://www.jimmunol.org/ though culture-activated L-selectinlow CD8ϩ T cells displayed Although combined CD4ϩ and CD8ϩ L-selectinlow T cells proved by guest on September 30, 2021

FIGURE 2. Treatment of day 3 intracranial and pulmonary metastases with L-selectinlow TDLN T cell subsets. L-selectinlow T cells unfractionated for CD4 and CD8, L-selectinlow CD4ϩ T cells, and L- selectinlow CD8ϩ T cells were each prepared from freshly harvested TDLN cells of B6 mice bearing syngeneic 12-day MCA-205 tumors as described in Materials and Methods. Following 5-day culture activation with anti-CD3/IL-2, each T cell group was harvested and administered to sublethally irra- diated (500 rad) B6 mice bearing day 3 intracranial (A) or pulmonary (B) metastases as described in Materials and Methods. A, Long term survival of mice with intracranial MCA-205 treated with 1 ϫ 106 (1E6) L-selectinlow T cells unfractionated for CD4 and CD8 (L-Sel- All), 1E6 L-selectinlow CD4ϩ T cells (L-Sel- CD4), or 1E6, 2E6, or 5E6 L-selectinlow CD8ϩ T cells (L-Sel- CD8). Histori- cally, mice surviving symptom free at day 60 are cured in this model. B, Enumeration of pulmonary metastases (each dot represents a mouse) in mice sacrificed 18 days after treatment with 1E6 L-se- lectinlow T cells unfractionated for CD4 and CD8 (all), L-selectinlow CD4ϩ T cells, or L-selectinlow CD8ϩ T cells. 5742 HELPER-INDEPENDENT L-SELECTINlow ANTI-TUMOR CD8ϩ T CELLS

selectinlow T cells culture-activated from either day 9 or day 12 MCA-205 sensitized TDLN (Fig. 5 and not shown). Such L-se- lectinlow T cells were unfractionated with regard to CD4ϩ and CD8ϩ, with each mouse receiving ϳ1 ϫ 106 CD4ϩ and 4 ϫ 106 CD8ϩ T cells at adoptive transfer. The observed rejection con- sisted of an initial 1- to 2-wk phase of attenuated tumor growth, followed by a 1- to 2-wk phase of objective tumor regression (Fig. 5, A–C). In vivo mAb depletion studies demonstrated that either CD4ϩ or CD8ϩ L-selectinlow T cells alone were competent to sustain the initial phase of growth attenuation, whereas subsequent tumor regression failed to occur with either CD4ϩ or CD8ϩ cell depletion (Fig. 5A). In the absence of such in vivo mAb depletion, complete rejec- tion of established 3-day s.c. tumors was also eventually achieved FIGURE 3. Successful treatment of day 3 pulmonary metastases re- by adoptive transfer of as few as 1 ϫ 106 L-selectinlow CD4ϩ T ϩ quires specifically sensitized L-selectinlow TDLN CD8 T cells. L-select- cells alone (Fig. 5B). However, a protracted initial phase of atten- low ϩ in CD8 T cells were isolated and culture activated as described in Fig. uated tumor growth delayed objective regression by 1–3 wk, de- 2 from the day 12 TDLN of MCA-205-bearing or MCA-207-bearing B6 pending on the L-selectinlow CD4ϩ T cell dose, compared with mice. Sublethally irradiated (500 rad) B6 mice bearing day 3 pulmonary ϩ ϩ low therapy with combined (CD4 plus CD8 ) L-selectin T cells. Downloaded from metastases (3 Day MCA-205 tumors or 3 Day MCA-207 tumors, received ϫ 6 Such delayed rejection appeared to require the late recruitment of either no therapy (No adoptive transfer) or 1 10 specifically sensitized ϩ low ϩ host CD8 T cells, because, as shown in Fig. 5A, mice depleted of L-selectin CD8 T cells (205-sensitized T cells or 207-sensitized T ϩ cells). This figure shows enumeration of pulmonary metastases (each dot CD8 cells failed to achieve such rejection despite receiving an low ϩ represents a mouse) in mice sacrificed 18 days after treatment. otherwise curative dose of L-selectin CD4 T cells. Mice receiving purified L-selectinlow CD8ϩ T cells alone expe-

rienced an initial phase of tumor growth attenuation that was not http://www.jimmunol.org/ the most potent on a cell number basis for eradicating established significantly different from that observed following treatment with 10-day pulmonary metastases, purified L-selectinlow CD8ϩ T cells combined (CD4ϩ plus CD8ϩ) L-selectinlow T cells (Fig. 5, A and were significantly more potent than purified L-selectinlow CD4ϩ T C). In addition, several mice receiving the highest dose of purified cells and, moreover, were effective as single-component therapy with- L-selectinlow CD8ϩ T cells experienced early (rather than delayed) out coadministration of exogenous rIL-2 (Fig. 4). L-selectinlow CD8ϩ complete tumor regression (Fig. 5D). Except for these infrequent T cells remained therapeutically more potent than L-selectinlow CD4ϩ cures, no objective tumor regression, early or delayed, was ob- T cells regardless of whether each subset was isolated before or served in mice receiving purified L-selectinlow CD8ϩ T cells after culture activation (not shown). alone, although all such treated mice experienced significant de- lays in tumor progression compared with untreated mice (Fig. 5, A by guest on September 30, 2021 low ϩ Adoptively transferred purified L-selectin CD4 TDLN T and C). cells are more effective than purified L-selectinlow TDLN CD8ϩ T cells for the eradication of established MCA-205 s.c. tumors, Anti-tumor sensitization of CD4ϩ and CD8ϩ TDLN T cells can but are CD8ϩ dependent occur in the complete absence of either subset Established s.c. tumors display a relatively low susceptibility to Huang et al. demonstrated that CD8ϩ as well as CD4ϩ T cell adoptive immunotherapy (16), and trafficking studies have dem- sensitization to tumor Ag is initially mediated by cross-priming onstrated the low initial accumulation of T cells in s.c. tumors host APC rather than by direct contact with tumor cells (22). Host compared with pulmonary or intracranial tumors (17). Nonethe- APC are capable of processing and presenting exogenous Ag in an less, 3-day established MCA-205 s.c. tumors were completely re- MHC class I-restricted context to CD8ϩ T cells, but such CD8ϩ jected within 3–4 wk following adoptive transfer of 5 ϫ 106 L- sensitization may require or can be enhanced by temporally linked

FIGURE 4. Treatment of 10 day pulmonary metas- tases with L-selectinlow TDLN T cell subsets. Synge- neic C57BL/6 (B6) mice were injected with viable MCA-205 cells and TDLN harvested on day 12. L-se- lectinlow TDLN T cells containing both CD4ϩ and CD8ϩ cells (CD4ϩCD8) as well as individual L-select- inlow CD4ϩ and CD8ϩ subsets (CD4 and CD8) were isolated for 5-day culture activation with anti- CD3/rIL-2, as described in Materials and Methods. Each cultured T cell group was harvested and adop- tively transferred into syngeneic mice with 10 day es- tablished MCA-205 pulmonary metastases. Recipient mice received 500 rad before adoptive transfer. Mice received 1 ϫ 106 1E6, 3E6, or 6E6 T cells. The ordinate shows the number of pulmonary metastases observed in mice sacrificed 11 days after adoptive therapy. Each point represents a single mouse (five mice per treatment group). The Journal of Immunology 5743 Downloaded from http://www.jimmunol.org/

FIGURE 5. Treatment of 3-day s.c. metastases with L-selectinlow TDLN T cell subsets. Syngeneic C57BL/6 (B6) mice were injected with viable MCA-205 cells, and TDLN was harvested on day 9 to prepare purified L-selectinlow T cells, unfractionated for CD4 and CD8, purified L-selectinlow CD4ϩ ϩ T cells, and/or purified L-selectinlow CD8 T cells. On day 5 after anti-CD3/IL-2 culture activation, T cell groups were harvested and adoptively transferred by guest on September 30, 2021 to treat syngeneic mice with 3-day established s.c. tumors (see Materials and Methods). Recipient mice received 500 rad before adoptive transfer. Subsequent growth of the s.c. tumors was serially evaluated. The ordinate displays the tumor area determined by two perpendicular caliper measurements; the abscissa displays the day following tumor inoculation. Symbols on each line display the average tumor measurement of each treatment group at particular time points (five mice per treatment group, with the SD displayed, except for D, where each line represents an individual mouse). Data are shown in the log scale to facilitate visualization of differences between groups at early time points. A, Adoptive therapy with Ab depletion. Mice received no adoptive therapy or 5 ϫ 106 (5E6) L-selectinlow T cells unfractionated for CD4 and CD8; in addition, groups receiving T cells received no in vivo mAb depletion (L-selectin low), concomitant in vivo CD4 mAb depletion (L-selectin low & anti-CD4), or concomitant in vivo CD8 mAb depletion (L-selectin low & anti-CD8). On day 12, group A1 vs A2: p1 ϭ 0.0002; A2 vs A3: p1 ϭ 0.0779; A3 vs A4: p1 ϭ 0.222. B and C, CD4ϩ and CD8ϩ dose titrations. Each treatment group received adoptive transfer of L-selectinlow T cells unfractionated for CD4 and CD8 (L-selectin low (CD4 ϩ CD8)), purified L-selectinlow CD4ϩ T cells (L-selectin low CD4), and/or purified L-selectinlow CD8ϩ T cells (L-selectin low CD8) at the doses shown in the legends. No in vivo Ab depletions were performed. On day 12, group C1 vs C2: p1 Ͻ 0.0001; C2 vs C3: p1 ϭ 0.7404; C2 vs C4: p1 ϭ 0.2598. On day 38, C1 vs C3: p1 ϭ 0.0021; C1 vs C4: p1 ϭ 0.0001. D, Divergent treatment outcomes for individual group C3 mice displayed; each line represents a single mouse. interactions between APC and CD4ϩ T cells (23–25). We there- by the copresence of TDLN CD4ϩ T cells (23–25). In studies fore examined whether sensitization of L-selectinlow CD8ϩ pre- performed primarily in normal syngeneic B6 mice, CD8ϩ T cells effector T cells in tumor-bearing mice required the presence of host were depleted of CD4ϩ T cells at various experimental stages (ei- CD4ϩ T cells. ther by in vivo depletion with anti-CD4 during sensitization or We inoculated syngeneic CD4KO and CD8KO mice with MC- adoptive therapy or by in vitro CD4ϩ depletion using negative 205 sarcoma cells s.c., then 12 days later harvested TDLN T cells immunoselection immediately before or after culture activation, see for a single immunoselection procedure to remove L-selectinhigh T Materials and Methods); in parallel, TDLN T cells were prepared cells (see Materials and Methods). Cells were culture activated by from syngeneic CD4KO mice. In each instance, effectiveness of CD4 anti-CD3/IL-2 treatment and were evaluated in adoptive therapy depletion was confirmed by FACS analyses (see Materials and experiments. Methods). Sufficient cells were prepared under each experimental Adoptive transfer of either 2 ϫ 106 L-selectinlow CD4ϩ or L- condition to permit treatment of individual B6 tumor-bearing mice selectinlow CD8ϩ T cells sensitized in MCA-205-bearing syngeneic with 1 ϫ 106 T cells. knockout mice eradicated established pulmonary or intracranial For the treatment of 3-day pulmonary metastases, 1 ϫ 106 adop- MCA-205 tumors when adoptively transferred into tumor-bearing tively transferred CD4-depleted L-selectinlow CD8ϩ T cells were normal B6 mice (Figs. 6), demonstrating that either subset was as effective as 1 ϫ 106 CD4-intact L-selectinlow T cells (81% effectively sensitized in the absence of the other. CD8ϩ T cells and 19% CD4ϩ T cells), except in the case of L- Nonetheless, it remained possible that sensitization and/or acti- selectinlow CD8ϩ T cells prepared from knockout mice (Fig. 7A). vation of L-selectinlow CD8ϩ effector function could be enhanced As previously observed and anticipated (Fig. 2A) (14), adoptive 5744 HELPER-INDEPENDENT L-SELECTINlow ANTI-TUMOR CD8ϩ T CELLS

even though stimulation with anti-CD3 resulted in IL-2 production (not shown) (14). Furthermore, L-selectinlow CD8ϩ T cells did not lyse the relevant tumor targets in standard 4-h 51Cr release assays (Fig. 9). In additional experiments, the reactivity of MCA-205 sensitized TDLN T cells was assayed using either purified MHC class IIϪ tumor cells or enriched MHC class IIϩ TAM as stimulators. Cul- ture-activated L-selectinlow CD4ϩ T cells released IFN-␥ upon contact with MHC class IIϩ TAM, but not upon contact with the MHC class II-nonexpressing tumor cells. In contrast, culture acti- vated L-selectinlow CD8ϩ T cells released IFN-␥ upon contact with either relevant TAM or MHC class I-expressing tumor cells (Fig. 8C).

Generation of L-selectinlow CD8ϩ TDLN T cells with highly potent effector activity is not tumor or mouse strain restricted To confirm that the sensitization of highly potent pre-effector L- selectinlow CD8 T cells was not confined to a single tumor model ϩ or mouse strain, L-selectin down-regulated CD8 T cells were Downloaded from isolated and culture activated from the TDLN of syngeneic BALB/c mice bearing the colonic adenocarcinoma CT-26. Isolated L-selectinlow CD8ϩ T cells or L-selectinlow CD4ϩ T cells acti- vated by anti-CD3/IL-2 treatment displayed marked therapeutic potency against CT-26 in adoptive transfer experiments even with-

out coadministration of exogenous IL-2 or the absent T cell subset http://www.jimmunol.org/ (Fig. 10). FIGURE 6. Treatment of day 3 pulmonary and intracranial tumors with TDLN T cell subsets derived from knockout mice. CD4KO or CD8KO mice were inoculated with syngeneic MCA-205, and 12 days later TDLN were Discussion harvested. TDLN cells were either unfractionated or subselected for L- Previous characterization of the highly potent effector T cells ac- selectinlow cells in vitro, culture activated for 5 days (anti-CD3/IL-2), then tivated from TDLN T cells by anti-CD3 or superantigen treatment administered to sublethally irradiated (500 rad) B6 mice bearing day 3 revealed that their therapeutic activity involved participation of ϩ ϩ pulmonary (A) or intracranial (B) metastases as described in Materials and both CD8 T cells and CD4 T cells (16, 19). It was subsequently Methods and Fig. 2. A, Mice with 3-day pulmonary metastases were treated demonstrated that the culture-activated subset of L-selectinlow with no cells (control) or 2E6 cells from CD4KO or CD8KO mice sub- CD4ϩ T cells could even be adoptively transferred as single agent by guest on September 30, 2021 low selected for L-selectin before culture activation. B, Same treatment therapy to achieve ablative tumor rejections regardless of whether groups as in A. the sensitizing tumor cells themselves expressed MHC class II molecules (14). The dramatic stand-alone therapeutic potency of this L-selectinlow CD4ϩ subset has tentatively been ascribed to the ϩ transfer of 1 ϫ 106 L-selectinlow CD8 T cells did not cure mice superior ability of L-selectin-down-regulated T cells to traffick into with 3-day intracranial tumors, but did significantly prolong sur- tumors at all tested anatomic locations (17) coupled with the CD4ϩ vival regardless of the CD4 depletion mode, except in the case of T cell’s capacity to recognize tumor Ag presented by MHC class ϩ L-selectinlow CD8 T cells prepared from CD4KO mice (Fig. 7B). IIϩ host APC resident within tumors (9, 14, 17, 26, 27). The mech- However, among all CD4-depleted treatment groups, significantly anisms by which adoptively transferred L-selectinlow CD4ϩ T ϩ longer survival was observed when CD4 T cells were present in cells mediate successful rejection of MHC class II-nonexpressing ϩ vivo during sensitization as well as when TDLN CD4 T cells tumors are under study and may variously depend upon cytokine- were included during culture activation. Nonetheless, as shown induced or anti-angiogenic effects, contact-mediated ϩ above, even L-selectinlow CD8 T cells that had been CD4 de- (e.g., FAS/APO-1-ligand) apoptosis, recruitment of CD8ϩ CTL, pleted before sensitization or culture activation could cure estab- and/or recruitment of Ag-unrestricted accessory cells such as tu- lished intracranial tumors when adoptively transferred in sufficient moricidal macrophages and LAK cells (reviewed in Ref. 9). numbers (Figs. 2A and 6B). We hypothesized that L-selectinlow CD8ϩ T cells might also

ϩ possess potent anti-tumor activity, coupling the superior trafficking In vitro correlates of the anti-tumor CD8 T cell response capacity of L-selectinlow T cells (17) with a capacity for direct Following culture activation with anti-CD3/IL-2, CD4ϩ and interactions with MHC class I-expressing tumor cells (Fig. 8). The CD8ϩ L-selectinlow T cells isolated from TDLN each displayed a present report confirms that isolated L-selectinlow CD8ϩ TDLN T similar capacity to produce cytokines in vitro. Both L-selectinlow cells display a remarkable therapeutic potential following anti- CD4ϩ and L-selectinlow CD8ϩ T cells consistently produced high CD3/IL-2 activation, which is far superior in potency to anti-tumor concentrations of IFN-␥ during in vitro exposure to the sensitizing CD8ϩ T cells derived from TDLN T cells or tumor-infiltrating tumor, and such IFN-␥ production was specific for the sensitizing lymphocytes by previous culture techniques. Optimally prepared, tumor (Fig. 8A), corresponding to the anti-tumor specificity also anti-CD3/IL-2-activated L-selectinlow CD8 provided consistently demonstrated during adoptive therapy (Fig. 3). In addition, specific curative stand-alone adoptive therapy against relevant tumors im- GM-CSF, but not TNF-␣, IL-4, or IL-10 production was observed planted in either lung or brain and significantly attenuated s.c. following in vitro exposure to the sensitizing tumor (Fig. 8B and tumor progression, with no requirement for coadministration of not shown). Specific IL-2 production was usually not demonstrable exogenous IL-2. Culture-activated L-selectinlow CD8ϩ T cells when these T cells were stimulated with sensitizing tumor cells, from TDLN developed greater effector activity when L-selectinlow The Journal of Immunology 5745 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 7. Treatment of day 3 intracranial and pulmonary metastases with TDLN T cell subsets under various CD4-depleting conditions. CD4KO and normal B6 mice were inoculated with syngeneic MCA-205; some B6 mice were additionally in vivo depleted of CD4 cells by treatment with R␣M-CD4 as described in Materials and Methods. Twelve days after tumor inoculation TDLN were harvested. All groups of TDLN cells were subselected for L-selectinlow cells before culture activation; in addition, some groups were negatively immunoselected for CD4ϩ cells (see Materials and Methods) in vitro before culture or 5 days later at the time of culture harvest. Following 5 days of culture activation with anti-CD3/IL-2, harvested cells were adoptively transferred into syngeneic, sublethally irradiated (500 rad) mice bearing either 3-day pulmonary (A) or 3-day intracranial (B) MCA-205 tumors (see Fig. 3 and Materials and Methods). All mice received 1 ϫ 106 harvested TDLN T cells. Some groups of adoptively transferred tumor-bearing mice were also in vivo-depleted of CD4ϩ cells by treatment with R␣M-CD4 as described in Materials and Methods. Therapy was evaluated as described in Figs. 2 and 6. A, control (received no TDLN T cells); B, TDLN T cells harvested from MCA-205-sensitized CD4KO mice; C, TDLN T cells harvested from MCA- 205-sensitized normal B6 mice that had been in vivo depleted of CD4ϩ cells; D, TDLN T cells harvested from MCA-205-sensitized normal B6 mice that were in vitro depleted of CD4ϩ cells before culture activation; E, TDLN T cells harvested from MCA-205-sensitized normal B6 mice that were in vitro depleted of CD4 cells following culture activation; F, TDLN T cells harvested from MCA-205-sensitized normal B6 mice that were adoptively transferred into mice in vivo-depleted of CD4ϩ cells; G, TDLN T cells harvested from MCA-205-sensitized normal B6 mice that were not CD4 depleted during culture activation or during adoptive therapy. A, Statistical comparisons by Wilcoxon rank-sum test. All treatment groups displayed significantly decreased pulmonary metastases compared with untreated mice (A vs B: p1 ϭ 0.008; A vs C: p1 ϭ 0.0001; A vs D: p1 ϭ 0.008; A vs E: p1 ϭ 0.0001; A vs F: p1 ϭ 0.0001; A vs G: p1 ϭ 008). Among treatment groups, only T cells prepared from knockout mice (group B) displayed significantly different efficacy compared with non-CD4-depleted mice (G vs B: p1 ϭ 0.008; G vs C: p1 ϭ 0.31; G vs D: p1 ϭ 0.69; G vs E: p1 ϭ 0.31; G vs F: p1 ϭ 0.31). B, Statistical comparisons by exact rank modification of the Wilcoxon rank-sum test All treatment groups except B displayed significantly prolonged survival compared with untreated mice with intracranial tumors (A vs B: p1 ϭ 0.222; A vs C: p1 ϭ 0.008; A vs D: p1 ϭ 0.02; A vs E: p1 ϭ 0.008; A vs F: p1 ϭ 0.008; A vs G: p1 ϭ 0.008). This trend held for all treated B6 whether treated before (C and D) or after (E and F) culture activation with anti-CD4 (A vs C plus D: p1 ϭ 0.01; A vs E plus F: p1 ϭ 0.005; A vs C, D, E, and F: p1 ϭ 0.01). Survival was significantly different in groups CD4 depleted before culture compared with the non-CD4-depleted group (G vs B: p1 ϭ 0.008; G vs C: p1 ϭ 0.008; G vs D: p1 ϭ 0.05; G vs C plus D: p1 ϭ 0.002), but there was no statistically significant difference between the nondepleted group and groups CD4 depleted after culture activation (G vs E: p1 ϭ 0.17; G vs F: p1 ϭ 0.17; G vs E plus F: p1 ϭ 0.06). In addition, comparison of preculture and postculture CD4-depleted groups displayed a trend toward a significant difference in survival (C plus D vs E plus F: p1 ϭ 0.03).

CD4ϩ T cells were also present during sensitization and/or culture in the absence of TDLN CD4ϩ T cells, they could consistently activation (Fig. 7). Nonetheless, even when L-selectinlow CD8ϩ T eradicate both established pulmonary and intracranial tumors when cells were sensitized, culture activated, and adoptively transferred administered in sufficient numbers (Figs. 6 and 7). 5746 HELPER-INDEPENDENT L-SELECTINlow ANTI-TUMOR CD8ϩ T CELLS Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 8. Cytokine production by L-selectinlow T cells and L-selectinlow T cell subsets. A and B, MCA-205-sensitized CD4ϩCD62L low and CD8ϩCD62L low T cells were cultured alone (no tumor) or with relevant MCA-205 digest (205 digest) or irrelevant MCA-207 digest (207 digest), then 24-h supernatants were assayed for specific IFN-␥ (A) or GM-CSF (B) production, displayed in nanograms per milliliter (106 T cells) per 24 h C, L-selectinlow T cells unfractionated for CD4ϩ and CD8ϩ (All CD62L low), purified L-selectinlow CD4ϩ T cells (CD4ϩCD62L low), and purified L- selectinlow CD8ϩ T cells (CD8ϩCD62L low) were each prepared from MCA-205-sensitized TDLN. At the end of 5-day culture activation with anti-CD3/ IL-2 treatment, T cell groups were harvested and were cocultured with irradiated stimulator cells: irrelevant MCA-203 whole cell digest (T cell ϩ; 203 digest), relevant MCA-205 whole cell digest (T cell ϩ 205 digest), H-12 cell line (stroma-free, MCA-205-derived; T cellϩH12), or MCA-205 tumor-associated macrophages from MCA-205 whole cell digest (T cell ϩ 205 TAM). Twenty-four-hour supernatants were assayed by ELISA; displayed is IFN-␥ production in nanograms per 106 T cells per 24 h. In this assay, production by T cells or tumor cells alone was below the limits of assay detection (Ͻ25 pg).

It is therefore apparent that L-selectinlow CD8ϩ TDLN T cells The mechanism(s) by which L-selectinlow anti-tumor CD8ϩ T display therapeutic characteristics of helper-independent T (HIT) cells achieve helper independence are under study and may include cells during both affector and effector limbs of the anti-tumor im- their ability to achieve high expression of CD40 ligand even in the mune response (4, 28, 29). Although CD8ϩ HIT cells have long presence of tumor (our manuscript in preparation), leading to su- been recognized to represent a naturally occurring subset of CD8ϩ perior APC conditioning (37). L-selectinlow anti-tumor CD8ϩ HIT T cells (4, 28–34), before the present study they had been identi- cells share this property and additional characteristics with the L- fied only in rare animal tumor models in which the tumors ex- selectinlow anti-tumor CD4ϩ T cells subset. Each subset is thera- pressed very strong viral Ag (4, 28, 29) and in experimentally peutically active against tumors at multiple anatomic locations as contrived transgenic models (35, 36). In contrast, the therapeuti- stand-alone adoptive therapy without IL-2 coadministration. Thus, cally potent L-selectinlow CD8ϩ HIT cells described in the present each appears to possess the anatomically unrestricted, broad traf- study were spontaneously sensitized in syngeneic mice bearing ficking capacity that is the signature property of activated L-se- progressive, weakly immunogenic tumors. The failure to identify lectinlow TDLN T cells (9, 14, 17, 26). Each subpopulation pro- CD8ϩ HIT cells previously in these long-studied tumor models duces IFN-␥ and GM-CSF in vitro following contact with the was probably a consequence of their typically minimal presence in relevant sensitizing tumor target (Fig. 8), but with a typical ab- TDLN. In addition, because culture activation of CD8ϩ HIT cells sence of specific IL-2, TNF-␣, IL-4, and IL-10 production or direct appears to be enhanced by the copresence of CD4ϩ T cells (Fig. 6), target lysis (Fig. 9). Despite this highly circumscribed in vitro it is possible that earlier culture methods that routinely failed to response to tumor contact, both CD4ϩ and CD8ϩ L-selectinlow T sustain CD4ϩ T cells (9) were also suboptimal for promoting cells retain the capacity for more diverse cytokine production, in- CD8ϩ HIT cells. cluding IL-2, as evidenced with anti-CD3 restimulation at the end The Journal of Immunology 5747

FIGURE 10. Therapeutically potent L-selectinlow CD4ϩ and CD8ϩ T cells are also present in TDLN of mice bearing CT-26. Syngeneic BALB/c mice were inoculated with CT26 (colonic adenocarcinoma), and TDLN FIGURE 9. Tumor cytolysis by culture-activated T cells from MCA- were harvested 9 days later. TDLN were fractionated by negative immu- 205-sensitized TDLN. The cytolytic activities of L-selectinlow CD4ϩ noselection before culture activation to generate L-selectinlow T cells (L- (CD4ϩ/L-Sellow) and L-selectinlow CD8ϩ (CD8ϩ/L-Sellow) TDLN T cells Sellow; unfractionated for CD4ϩ and CD8ϩ). L-selectinlow CD4ϩ T cells following culture activation are compared with that of syngeneic LAK cells (L-Sellow CD4) and L-selectinlow CD8ϩ T cells (L-Sellow CD8) were then (LAK) generated from normal B6 splenocytes. All effector groups were culture activated for 5 days with anti-CD3/IL-2, harvested and used to treat assayed against 51Cr-labeled MCA-205 whole-cell tumor digests in a stan- BALB/c mice i.v. injected 3 days previously with CT-26 to generate pul- Downloaded from dard 4-h 51Cr release assay. The abscissa shows the E:T cell ratio; the monary metastases. Each treatment group received 1 ϫ 106 L-selectinlow ordinate shows the percent cytolysis, calculated by the percentage of spe- culture-activated T cells in adoptive transfer, except for the control group. cific 51Cr release. Pulmonary metastases were enumerated on day 18 as described in Mate- rials and Methods. of culture (14). It is also likely that L-selectinlow CD8ϩ T cells

implement CTL activity following adoptive transfer, because cul- cell-mediated tumor rejection was a delayed process with apparent http://www.jimmunol.org/ ture-activated TDLN T cells obtained from syngeneic perforin dependence upon recruitment of host CD8ϩ T cells (Fig. 5, A and knockout mice display severe anatomic restrictions in their tumor B). Furthermore, undelayed rejection of established MCA-205 s.c. rejection capacity when adoptively transferred into normal tumor- tumors was only observed when CD8ϩ L-selectinlow T cells were bearing hosts (59). included as a component of adoptive therapy (Fig. 5, A and D). It is nonetheless apparent that L-selectinlow CD4ϩ and CD8ϩ These results suggest that CD8ϩ and CD4ϩ L-selectinlow T cell anti-tumor T cells possess significant functional differences, be- subsets can play distinctive and complimentary roles during adop- ginning with different practical requirements for recognizing tumor tive therapy. Whereas the therapeutic efficacy of purified L-selec- Ag. L-selectinlow CD8ϩ T cells sensitized to MCA-205 produced tinlow CD8ϩ T cells varies strongly in proportion to the observed IFN-␥ when exposed to either MHC class I-expressing MCA-205 accumulation efficiencies of T cells at these sites (pulmonary tu- by guest on September 30, 2021 tumor cells or enriched MCA-205 derived TAM (Fig. 8C). In con- mors Ͼ intracranial tumors ϾϾ s.c. tumors) (17), the therapeutic trast, L-selectinlow CD4ϩ T cells sensitized to MCA-205 did not efficacy of purified L-selectinlow CD4ϩ T cells appears to be produce IFN-␥ when exposed to MHC class IIϪ MCA-205 tumor largely independent of such trafficking variances. This may reflect cells, but did produce IFN-␥ when exposed to MHC class IIϩ superior abilities of L-selectinlow CD4ϩ T cells to proliferate in- MAC-205-derived TAM. It is therefore likely that tumor rejection tratumorally, sustain APC conditioning, and/or gradually recruit may be triggered either through direct T cell contact with tumor additional host effector elements, including CD8ϩ T cells (37, 40– cells or, alternatively, by T cell contact with cross-stimulating host 44) (our manuscript in preparation). Nonetheless, purified L-se- APC present within the tumor bed (26, 38, 39). In the MCA-205 lectinlow CD8ϩ T cells displayed greater efficacy than purified L- tumor model, either option is theoretically available for anti-tumor selectinlow CD4ϩ T cells in eradicating advanced (day 10) CD8ϩ T cells, but only the second option is available to anti-tumor pulmonary tumors even without coadministration of exogenous CD4ϩ T cells, because MCA-205 tumor cells in situ do not express rIL-2 (Fig. 4) and also were essential for achieving rapid rejection MHC class II molecules (27). Although it is possible that the re- of MCA-205 s.c. tumors with adoptive therapy (Fig. 5, A and D). activity of CD8ϩ T cells to TAM involves an element of triggering It remains to be determined whether such therapeutic distinctions by contaminant tumor cells, the triggering of anti-tumor CD8ϩ T reflect the L-selectinlow CD8ϩ T cell’s superior capacity to interact cells by cross-priming host APC that process exogenous tumor Ag directly with MHC class Iϩ, MHC class IIϪ tumor cells (Fig. 8C). is a well-accepted phenomenon (22). Given the relative insensitivity of L-selectinlow CD4ϩ T cells to Our studies have repeatedly demonstrated that purified L-selec- trafficking variances and the effector impact of L-selectinlow CD8ϩ tinlow CD8ϩ and CD4ϩ TDLN T cell subsets are not simply in- T cells even against advanced tumors, it is not surprising that these terchangeable as therapy. For example, adoptively transferred L- subsets are often therapeutically superior and even synergistic selectinlow CD4ϩ T cells were relatively more potent on a cell when administered together (Figs. 4 and 5A). mAb depletion ex- number basis for eradicating 3-day intracranial tumors, whereas periments furthermore suggest that purified L-selectinlow CD4ϩ T L-selectinlow CD8ϩ T cells proved more effective against 10-day cells can eventually replicate such synergy during adoptive therapy pulmonary metastases (see Figs. 2 and 4). The most extreme dif- of both s.c. and intracranial tumors by recruiting host anti-tumor ferences in therapeutic performance were observed for established CD8ϩ T cells (Fig. 5A; H. Kagamu and S. Shu, unpublished ob- s.c. tumors, probably reflecting the lower trafficking efficiency of servations). In these experimental models such CD8ϩ recruitment even L-selectinlow T cells into tumors at this anatomic site (17). appears to be appropriately delayed by the host’s exposure to im- Although purified CD4ϩ and CD8ϩ L-selectinlow subsets were munosensitizing sublethal irradiation before adoptive transfer (Fig. each therapeutically active against MCA-205 s.c. tumors, only pu- 5, A and B). Because both CD8-dependent and CD8-independent rified L-selectinlow CD4ϩ T cells provided consistently curative therapeutic effects are observed during subsequent tumor rejection adoptive therapy (Fig. 5). Nonetheless, such L-selectinlow CD4ϩ T (Fig. 5A), it is reasonable to postulate a dual helper and effector 5748 HELPER-INDEPENDENT L-SELECTINlow ANTI-TUMOR CD8ϩ T CELLS role for L-selectinlow CD4ϩ T cells. For example, L-selectinlow the availability of highly potent L-selectinlow pre-effector T cells in CD4ϩ T cell adoptive therapy of s.c. tumors required CD8ϩ T TDLN for purposes of culture activation and adoptive therapy. cells to achieve objective tumor regression, but long-term tumor Furthermore, improved yields may allow better delineation of growth arrest was nonetheless achieved even in the absence of functional differences in the L-selectinϪ and L-selectindim subsets CD8ϩ T cells (Fig. 5A). of L-selectinlow subpopulations. Finally, the heightened stability of In contrast, although adoptive transfer of purified L-selectinlow CD8ϩ HIT cells in culture compared with non-HIT cells (28, 29) CD8ϩ HIT cells could initially attenuate s.c. tumor growth as well may permit their long term numerical expansion with retained as purified CD4ϩ or even combined (CD4ϩ plus CD8ϩ) L-selec- function in vitro despite marginal initial yields and sluggish pro- tinlow T cells, the former’s therapeutic effect was usually unsus- liferation during brief anti-CD3/IL-2 culture activation. Efforts are tained beyond 2 wk. These results suggest that the helper inde- ongoing to define the optimal TCR-stimulating and costimulatory pendence of CD8ϩ HIT cells may be less easily sustained at tumor stimuli to enhance propagation of isolated L-selectinlow TDLN T sites where T cell trafficking is relatively inefficient, as epitomized cells, including repeated coculture with tumor Ag-pulsed dendritic in murine models by established s.c. challenges (17). However, cells (49, 56–58). because Ag availability often causes vaccination strategies to favor ϩ CD8 T cell sensitization (3, 45–47), it is desirable to identify References adjunct treatments that promote sustained effector activity of 1. Liu, J., J. Finke, J. C. Krauss, S. Shu, and G. E. Plautz. 1998. Ex vivo activation CD8ϩ HIT cells when they must be adoptively transferred without of tumor-draining lymph node T cells reverses defects in signal transduction CD4ϩ T cells. The adjunct administration of exogenous rIL-2 for molecules. Cancer Immunol. Immunother. 46:268. 2. Marzo, A. L., R. A. Lake, D. Lo, L. Sherman, A. McWilliam, D. Nelson, this purpose is well precedented. In fact, previously characterized B. W. Robinson, and B. Scott. 1999. Tumor are constitutively presented Downloaded from cultured CD8ϩ HIT cells with specificity for the FBL3 lymphoma in the draining lymph nodes. J. Immunol. 162:5838. 3. Rosenberg, S. A., J. C. Yang, D. J. Schwartzentruber, P. Hwu, F. M. Marincola, were therapeutically effective as adoptive therapy only when ex- S. L. Topalian, N. P. Restifo, M. E. Dudley, S. L. Schwarz, P. J. Spiess, et al. ogenous rIL-2 was coadministered (29, 48). More recently, Shrin- 1998. Immunologic and therapeutic evaluation of a synthetic vaccine for kant and Mescher demonstrated that adoptively transferred OVA- the treatment of patients with metastatic melanoma. Nat. Med. 4:321. ϩ 4. Speiser, D. E., R. Miranda, A. Zakarian, M. F. Bachmann, K. McKall-Faienza, specific CD8 HIT cells from transgenic OT-1 mice could traffick B. Odermatt, D. Hanahan, R. M. Zinkernagel, and P. S. Ohashi. 1997. Self an-

successfully to a peritoneal challenge of EL4-OVA tumor and tigens expressed by solid tumors do not efficiently stimulate naive or activated T http://www.jimmunol.org/ transiently control tumor growth, but spontaneously left the site of cells: implications for immunotherapy. J. Exp. Med. 186:645. 5. Schwartzentruber, D. J., S. S. Hom, R. Dadmarz, D. E. White, J. R. Yannelli, tumor and developed elements of split anergy unless rIL-2 was S. M. Steinberg, S. A. Rosenberg, and S. L. Topalian. 1994. In vitro predictors coadministered (35, 36). Such previous reports demonstrate the of therapeutic response in melanoma patients receiving tumor-infiltrating lym- capacity of adjunct cytokine treatment to provide a satisfactory phocytes and interleukin-2. J. Clin. Oncol. 12:1475. ϩ ϩ 6. Kawakami, Y., S. Eliyahu, C. H. Delgado, P. F. Robbins, K. Sakaguchi, surrogate for CD4 participation during CD8 HIT cell adoptive E. Appella, J. R. Yannelli, G. J. Adema, T. Miki, and S. A. 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