Bryostatin 1/ionomycin (B/I) ex vivo stimulation preferentially activates L-selectinlow tumor-sensitized lymphocytes

Cynthia S. Chin1,3, Catriona H.T. Miller2, Laura Graham1, Maryam Parviz1,4, Sharline Zacur1, Bina Patel1, Angie Duong1 and Harry D. Bear1,2

1 Division of Surgical Oncology, Department of Surgery and 2 Department of Microbiology and Immunology, Virginia Commonwealth University Medical College of Virginia, Richmond, VA 23298, USA
3 Present address: Saint Vincent's Hospital and Medical Center, New York City, NY 10011, USA
4 Present address: Cancer Care Northwest, Spokane, Washington, 99204, USA

Correspondence to: H. D. Bear; E-mail: hdbear{at}vcu.edu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 References
 
We have shown that tumor vaccine-sensitized draining lymph node (vDLN) cells activated ex vivo with bryostatin and ionomycin (B/I) were capable of inducing antigen-specific regression of a murine mammary tumor, 4T07. vDLN cells not activated with B/I were ineffective. We hypothesized that B/I selectively activates tumor-sensitized (CD62Llow) lymphocytes, to account for the highly potent and tumor-specific activity. We hypothesized that CD8+ CD62Llow cells may be preferentially activated by B/I treatment, infiltrate the tumors and mediate tumor regression in mice. 4T07-IL2 tumor cells were injected into one hind footpad of BALB/c mice. Ten days later, vDLN were harvested and separated based on CD62L expression. After separation, cells were activated with B/I, expanded with IL2 (40 IU/ml) for 10 days, and adoptively transferred to 4T07 tumor bearing mice. Naive mice were also treated with different subsets of T cells and later were challenged with 4T07 tumor cells. To test in vitro responses to antigen, expanded lymphocytes were cultured either alone or with irradiated 4T07 tumor cells. Supernatants were harvested after 24 h and tested by ELISA for IFN-{gamma}. The importance of the host immune response was tested by AIT into 4T07-bearing nude athymic mice. Host mice were depleted in vivo of CD4 or CD8 T cells after vDLN AIT to ascertain the mediators of tumor regression. In order to track B/I activated vDLN cells, they were prestained with CFSE prior to adoptive transfer into tumor-bearing hosts. At various time points, tumors, spleens and lymph nodes of host mice were harvested, dual stained for activation marker expression and analyzed by flow cytometry. CD62Llow cells expanded 12-fold more than CD62Lhigh lymphocytes during the 10 day culture period. Supernatant from CD62Llow cells + 4T07 cultures contained 33-fold more IFN-{gamma} than supernatant from CD62Lhigh cells + 4T07 cultures (843.9 pg/ml ± 135.8 vs 25.89 pg/ml ± 0.01). Adoptive transfer of CD62Llow lymphocytes induced complete tumor regressions in all mice, while tumors regressed in only 17% of mice treated with CD62Lhigh lymphocytes. Naive mice that received B/I-activated CD62Llow cells were protected from future tumor challenges, while mice given CD62Lhigh cells did not exhibit the same resistance to tumor growth. Tumors in nude host mice regressed after AIT treatment. In vivo depletion of CD4 T cells after AIT did not inhibit tumor regression, but CD8 T cell depletion abrogated tumor regression. vDLN cells tracked preferentially to tumor draining lymph nodes and proliferated in vivo, persisting for at least 21 days, and were 95% CD44+ and 39% CD69+. Bryostatin 1 and ionomycin, by increasing PKC activity and intracellular calcium, respectively, mimic intracellular signals that result in T cell activation. CD62Llow cells are preferentially activated by B/I, leading to a highly effective anti-tumor T cell population.

Keywords: adoptive immunotherapy, bryostatin, ionomycin, L-selectin, 4T07


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 References
 
We have developed a novel technique to activate tumor antigen or vaccine-sensitized draining lymph node (vDLN) cells pharmacologically, using bryostatin 1 and ionomycin (B/I) (1,2). Bryostatin 1, derived from a marine bryozoan, Bugula neritina, activates protein kinase C (PKC) (35), but the specific isoforms and downstream cellular target(s) are not currently known (6,7). Ionomycin, a calcium ionophore, increases intracellular calcium (8). This combination of B/I mimics an important T cell activation pathway (3,8). We have shown that tumor vaccine-sensitized DLN cells activated ex vivo with B/I were capable of inducing antigen-specific regression of established murine mammary tumors, 4T07 (2). Conversely, the same vDLN cells not activated with B/I were ineffective for treatment of tumor-bearing animals after adoptive transfer. It was not clear how a non-specific method of T cell activation could induce anti-tumor T cells with antigen-specific activity, but we hypothesized that antigen-sensitized T cells might be activated preferentially by B/I. Sensitization of T cells induces down-regulation of a lymph node homing receptor, L-selectin (CD62L), which may promote T-cell trafficking to other locations (9). Generally, the loss of surface expression of CD62L (CD62Llow) has been considered a marker for active effector cells, while cells with high level CD62L expression (CD62Lhigh) are associated with a naive phenotype (10). Others have recently shown that CD62Llow cells account for anti-tumor activity in lymph node cells activated with anti-CD3 antibody (11,12). We hypothesized that low expression of CD62L in tumor-vaccine DLN might serve as a marker for tumor-antigen sensitized T lymphocytes and that these cells might be preferentially activated by B/I treatment, producing a population of highly potent, tumor-specific lymphocytes.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 References
 
Mice
Virus-free BALB/c mice (Charles River Laboratories, Cambridge, MA) were used between 8 and 12 weeks of age, caged in groups of six or fewer, and provided with food and water ad libitum. Nude athymic BALB/c mice (National Cancer Institute, Bethesda, MD) were used for some experiments. All guidelines at Virginia Commonwealth University, which conform to the American Association for Accreditation of Laboratory Animal Care and the US Department of Agriculture recommendations for the care and humane experimental use of animals, were followed.

Tumor cell lines and hybridomas
The 4T07 mammary tumor, its IL2 transduced counterpart (4T07-IL2) and the less immunogenic variant 4T1 were kindly provided by Dr Jane Tsai at the Michigan Cancer Foundation, Detroit, MI. Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% heat-inactivated fetal calf serum (FCS, Hyclone, Logan, UT), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma, St Louis MO). G418 (Sigma) 600 µg/ml was added to 4T07-IL2 cultures to maintain selective pressure in favor of the transfected tumor cells. MethA sarcoma, an unrelated tumor cell line (ATCC, Rockville, MD) was maintained in RPMI 1640 medium with 10% heat-inactivated FCS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES buffer and 5 x 10–5 M 2-mercaptoethanol (Sigma) (complete RPMI). Hybridomas [GK1.5 (anti-CD4), 2.43 (anti-CD8)] were obtained from ATCC and grown in complete RPMI. All cells were incubated in 250 ml T-flasks (PGC, Gaithersburg, MD) at 37°C in humidified air with 5% CO2. Tumor cells were harvested for passage or for inoculation of mice with 0.05% trypsin–EDTA (Fisher, Pittsburgh, PA).

Sensitization of draining lymph nodes
Mice were inoculated in one hind footpad with 1 x 106 viable 4T07-IL2 cells. The expression of the IL2 gene in 4T07 leads to complete regression of 4T07-IL2 tumors by the fifteenth day after inoculation (data not shown). Mice were euthanized by CO2 inhalation 10 days after vaccination and the ipsilateral popliteal draining lymph node(s) (vDLN) were harvested under sterile conditions.

Lymphocyte preparation and sorting
Immediately after harvest, lymph nodes were dispersed into single cell suspensions, washed and resuspended in complete RPMI at 7 x 106 cells/ml. The vDLN cell suspension was then enriched for T cells by passage through a nylon wool column. After enrichment, one quarter of the T-lymphocytes were set aside as ‘unsorted’ vDLN T cells. The remaining vDLN cells were separated based on expression of the CD62L surface marker. This separation was achieved using an unlabeled rat anti-mouse CD62L monoclonal antibody (MEL-14, Pharmingen, San Diego, CA), panning, and immunomagnetic beads (Dynabeads, Dynal Biotech, Lake Success, NY). After harvesting and separation, there were three groups of T lymphocytes: unsorted, CD62Llow and CD62Lhigh. Effectiveness of the separation was determined using flow cytometry. Cells in each group were either incubated with unlabelled MEL-14 antibody and phycoerythin (PE)-conjugated anti-rat antibody and analyzed by flow cytometry. Any previous labeling of CD62L-expressing lymphocytes with MEL-14 antibody during separation would result in positive staining with the PE-conjugated anti-rat antibody.

Pharmacologic activation and expansion of lymphocytes in culture
The three groups of lymphocytes—unsorted, CD62Llow and CD62Lhigh—were incubated in complete RPMI for 18 h with 5 nM bryostatin 1 (kindly provided by the National Cancer Institute, Bethesda, MD), 1 µM ionomycin (Calbiochem, San Diego, CA) and 80 IU/ml rIL-2 (Chiron, Emeryville, CA) at 37°C in humidified air with 5% CO2 (B/I-activated). After incubation, cells were washed three times with warm (37°C) RPMI 1640 medium and cultured for up to 10 days in complete RPMI with 40 IU/ml of rIL-2 in 50 ml T-flasks. Cells were split to 1 x 106 cells/ml and refed with medium and 40 IU/ml rIL-2 every other day. After 10 days of expansion, cells were washed and resuspended in plain RPMI for use in either in vitro assays or in vivo treatment of tumor-bearing hosts.

Flow cytometry
Unsorted, CD62Llow and CD62Lhigh lymphocytes were stained both before and after B/I with a panel of antibodies (as follows) and analyzed on an ELITE Beckman Coulter flow cytometer. Fluorescently labeled antibodies directed against the following markers were obtained from Pharmingen (San Diego, CA): CD4 (GK1.5), CD8 (53–6.7), CD44 (IM7), CD62L (MEL-14) and CD69 (H1.2F3). Appropriate isotype controls were used in all cases.

Evaluation of in vitro cytokine release responses
Expanded unsorted, CD62Llow and CD62Lhigh lymphocytes were cultured in 24-well plates either alone or with irradiated 4T07 tumor cells (ratio of lymphocytes to tumor cells, 10:1). Supernatant samples were harvested at 24, 48 and 72 h time points. ELISA assay kits (R&D Systems, Minneapolis, MN) for IFN-{gamma} and IL-4 were used to assay the supernatant fluids for cytokine concentrations.

Treatment of established tumors
Mice were inoculated subcutaneously (s.c.) into their shaven left flanks with 3 x 105 wild-type 4T07 cells in 0.05 ml DMEM. Three or nine days later, some mice were given cyclophosphamide (CYP, 100 mg/kg) intraperitoneally (IP). Unsorted, CD62Llow or CD62Lhigh lymphocytes (10 x 106) expanded in culture for adoptive immunotherapy (AIT) and resuspended in 0.5 ml of RPMI, were infused intravenously (IV) 24 h later (i.e. on day 4 or day 10). To analyze long-term memory effects of AIT, mice in which complete tumor regression had occurred after AIT were inoculated in the right flank with 3 x 105 wild-type 4T07 cells in 0.05 ml DMEM 1 month later.

Protection experiments with AIT
Naive BALB/c mice received either no cells or unsorted, CD62Llow or CD62Lhigh vDLN lymphocytes that had been activated with B/I and expanded in culture. One month after adoptive transfer, treated mice and age-matched controls were inoculated in their shaven left flanks with 3 x 105 wild-type 4T07 or with one million unrelated MethA sarcoma cells in 0.05 ml DMEM.

In vivo T cell subset depletion experiments
Mice (n = 6) were injected with 3 x 105 4T07 tumors. Three days later, all but one group was pretreated with CYP injection. On the fourth day, mice received an IV infusion of 6 x 106 B/I activated vDLN cells. Animals were depleted of CD4+ or CD8+ cells by IV injection of 250 µl of anti-CD4 (GK1.5 hybridoma) or anti-CD8 (2.43 hybridoma) ascitic fluid 1 h, 5 days and 10 days after AIT. Flow cytometry was used to verify depletion.

Tumor measurements
In all experiments, tumor growth was monitored with biweekly measurements of perpendicular diameters. Data shown are products of these diameters. When the tumor area was greater than 144 mm2 or if a mouse appeared ill, the animal was euthanized by CO2 inhalation. Complete tumor regression was defined as the absence of a measurable tumor on three consecutive measurements.

Immunohistochemistry
4T1 tumors were excised from the flanks of mice 1, 4, 7 and 11 days after adoptive transfer, placed in OCT embedding fluid and frozen in liquid nitrogen. Frozen tissues were cut into 6 µm sections and fixed in acetone at –20°C for 2 min on slides. Slides were dried, blocked with 5% rabbit serum and stained with purified rat monoclonal antibodies, anti-CD4 and anti-CD8{alpha} or BSA as an unstained control. Slides were then incubated with a secondary antibody, biotinylated anti-rat IgG. Stained slides were visualized using the Vectastain ABC kit system (Vector Laboratories, Burlingame, CA) and counterstained with hemotoxylin. Stained slides were assigned scores according to the percentage of cells stained, + = 1–15% stained; ++ = 16–50% stained; and +++ for >50% stained.

CFSE staining and analysis
Prior to adoptive immunotherapy, B/I activated 4T07-IL2 vDLN lymphocytes were stained with 50 µM CFDA-SE (Molecular Probes, Eugene, OR) in PBS at a concentration of 75 million cells/ml for 15 min. Cells were washed with warm medium and incubated at 37°C for 30 min to allow processing by intracellular proteases. 50 million CFSE-stained lymphocytes in 500 µl were injected intravenously into cyclophosphamide pre-treated 10 day 4T1 (25 000 cell inoculum) tumor-bearing hosts. Cells were isolated from the spleen, tumor, tumor draining lymph node and contralateral lymph node, stained for CD4, CD8, CD44 and CD69 expression, and analyzed by dual color flow cytometry for CFSE and surface marker expression. Generations of proliferation were analyzed using ModFit LT (Verity Software House, Topsham, Maine).

Statistical analysis
Differences in tumor growth were assessed by analysis of variance (ANOVA) and Tukey–Kramer honestly significant difference test (Tukey's HSD) using JMPIN software (SAS Institute Inc., Cary, NC). Results of tumor growth are presented as the means ± SE of tumor area in each treatment group. In vivo experiments included at least six mice per group and were repeated at least twice. An {alpha} < 0.05 was used throughout to determine significant differences.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 References
 
Expansion of lymphocytes based on CD62L surface expression
The first hypothesis to be tested was that bryostatin and ionomycin selectively activate lymphocytes previously sensitized to tumor antigen. We used the CD62Llow phenotype as a marker for tumor-stimulated T lymphocytes and therefore monitored the expansion of CD62Llow lymphocytes compared to that of CD62Lhigh and unsorted lymphocytes after stimulation with B/I. Through the use of flow cytometry, it was verified that the immunomagnetic separation process produced a T cell population that was >95% pure for CD62Llow lymphocytes. We found that after B/I-activation and 11 days in culture, expansion of the CD62Llow population was 12-fold that of the CD62Lhigh subset (Fig. 1) and >5-fold that of the unsorted cells.



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Fig. 1. Ex vivo expansion curve of 4T07-IL2 sensitized DLN cells after separation based on CD62L surface expression and B/I activation. Fold-expansion of different subsets of cells after activation with B/I during 11 days in culture. CD62Llow population of lymphocytes expanded 12-fold more than the CD62Lhigh subset.

 
Characterization of CD62L populations
Lymphocyte populations were stained for flow cytometry both before separation and B/I activation and after separation and B/I expansion for expression of CD44 and CD69 activation markers, as well as CD4 and CD8. As shown in Table 1, all three populations had roughly equal CD4+ (57–62%) and CD8+ (23–24%) populations prior to B/I activation. After B/I activation, the CD62Llow population had a lower proportion of CD4+ and a higher proportion of CD8+ cells as compared to the CD62Lhigh cells, with the unsorted CD4+ and CD8+ populations being intermediate between them. The CD62Llow population had a higher proportion of CD69+ cells (16%) than unsorted (9%) or CD62Lhigh (7%) cells before B/I expansion. All populations before B/I activation had roughly equal CD44 expression levels (80–84%). After B/I activation, CD44 expression decreased in the CD62Llow (70%) and unsorted populations (76%), but increased in the CD62Lhigh population (95%). CD69 expression declined slightly in the CD62Llow and unsorted populations after B/I, while increasing slightly in the CD62Lhigh population. Even though the percentage of CD62Llow cells expressing activation markers declined somewhat after B/I activation, the CD62Llow cells undergo ~92-fold expansion, compared to 7-fold in the CD62Lhigh population, leading to far greater numbers of CD62Llow cells.


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Table 1. Characterization of CD62L sorted populations by T lymphocyte subset and activation marker expression before and after B/I expansion

 
Ex vivo cytokine response to tumor cells
After separation, B/I-activation and expansion, lymphocytes from each subset and from the unsorted group were cultured either alone or with irradiated 4T07 tumor cells. The CD62Llow and CD62Lhigh subsets differ in their Type 1 cytokine response to tumor cells (Fig. 2). Supernatant from CD62Llow cells + 4T07 cultures contained 33-fold more IFN-{gamma} than supernatant from CD62Lhigh cells + 4T07 cultures (843.9 pg/ml ± 135.8 vs 25.89 pg/ml ± 0.01). These B/I-activated and expanded lymphocyte subsets produced no appreciable IFN-{gamma} in the absence of tumor cell stimulation. None of these cells produced significant amounts of IL-4, as detected by ELISA, with or without tumor antigen stimulation (Fig. 3). We have previously shown (13) that IFN-{gamma} production by 4T07-IL2 tumor draining lymphocytes activated ex vivo with bryostatin and ionomycin react specifically to 4T1 and 4T07 tumors, but not to the unrelated MethA sarcoma cell line.



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Fig. 2. Ex vivo IFN-{gamma} production. The production of IFN-{gamma} by unsorted (A), CD62Llow (B), or CD62Lhigh (C) lymphocytes activated with B/I, cultured in IL2 for 10 days and then washed and cultured alone or with irradiated 4T07 tumor cells. IFN-{gamma} levels in supernatant fluids are charted in pg/ml over days in restimulation culture. Supernatant from CD62Llow cells + 4T07 cultures at 48 h contained 33-fold more IFN-{gamma} than supernatant from CD62Lhigh cells + 4T07 cultures (843.9 pg/ml ± 135.8 vs 25.89 pg/ml ± 0.01). Cells cultured alone did not make any appreciable amounts of IFN-{gamma}.

 


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Fig. 3. Ex vivo IL-4 production. The production of IL-4 by unsorted (A), CD62Llow (B), or CD62Lhigh (C) lymphocytes alone or after stimulation with 4T07 tumor cells are charted in pg/ml over days in restimulation culture. There was no significant difference in IL-4 production among the three groups of lymphocytes.

 
Treatment of established 4T07 flank tumors
Mice with 4 day established 4T07 flank tumors were either left untreated or treated with adoptively transferred CD62Llow, CD62Lhigh or unsorted lymphocytes. All mice were pretreated with cyclophosphamide (100 mg/kg) 24 h earlier. Adoptive transfer of unsorted or CD62Llow lymphocytes induced complete tumor regressions in all mice (six out of six), while none of the mice (zero out of six) treated with CD62Lhigh lymphocytes experienced tumor regression (Fig. 4). The tumor growth curve of the CD62Llow group was significantly different from the growth curves of both the untreated and CD62Lhigh groups. There was no significant difference between the untreated and CD62Lhigh growth curves. Adoptive transfer of B/I activated vaccine draining lymphocytes was also capable of inducing the regression of 10 day 4T07 tumors (Fig. 5), which may be argued to be a more clinically relevant model.



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Fig. 4. Treatment of established 4 day 4T07 tumor either with nothing or with CYP + AIT using either unsorted, CD62Llow or CD62Lhigh lymphocytes. Mice with established 3 day tumors either were left untreated or were treated with CYP followed by adoptive transfer of 10 x 106 cells of either CD62Llow, CD62Lhigh or unsorted lymphocytes which had been activated with B/I and expanded. Mean tumor areas ± SE are charted over time. Treatment with unsorted or CD62Llow lymphocytes induced complete tumor regressions in 100% of the mice in this group. None of the mice treated with CD62Lhigh lymphocytes had complete tumor regression. The unsorted and CD62Llow groups were significantly different from both the untreated and CD62Lhigh groups [F(3,20) = 17.6143, P < 0.0001]. Numbers to the right indicate the number of mice with complete tumor regression per total number of mice in each group.

 


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Fig. 5. Treatment of established 10 day 4T07 tumor with CYP + AIT of unsorted lymphocytes. Mice with established 10 day tumors were either left untreated, treated with cyclophosphamide (CYP) or with CYP (day 9) followed by adoptive transfer (day 10) of 10 x 106 unsorted lymphocytes. Mean tumor areas ± SE are charted over time. Adoptive transfer of B/I activated tumor vaccine draining lymphocytes induced complete tumor regressions in 83% (five out of six) of the mice in this group. The CYP + AIT group was significantly different from both the untreated and CYP only groups [F(2,15) = 5.0470, P = 0.0211]. Numbers to the right indicate the number of mice with complete tumor regression per total number of mice in each group.

 
Immunologic memory and resistance to 4T07 challenge
Mice in which tumors had regressed after AIT with either unsorted or CD62Llow B/I-activated vDLN cells were rechallenged with 4T07 tumor 1 month after complete regression of the initial tumor. Both groups were completely resistant to tumor growth (Fig. 6). In contrast, age-matched controls exhibited progressive 4T07 flank tumor growth. Thus, AIT with CD62Llow purified lymphocytes induces primary tumor regression as well as conferring long-term resistance to tumor growth.



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Fig. 6. Rechallenge of mice previously treated successfully with AIT. Those mice cured of 4T07 tumors by AIT with either unsorted or CD62Llow lymphocytes were inoculated into their contralateral flank with 3 x 105 wild-type 4T07 cells 1 month after complete regression of the initial tumor. 100% of the mice in both groups were resistant to tumor growth. All age-matched untreated mice, however, exhibited progressive tumor growth. The unsorted and CD62Llow groups were significantly different from the untreated group [F(2,15) = 41.5802, P = 0.0001]. Numbers to the right indicate the number of mice with complete tumor regression per total number of mice in each group.

 
To differentiate between long-term memory induced by the adoptively transferred lymphocytes versus host immune sensitization to tumor antigen, we adoptively transferred B/I-activated T lymphocyte subsets into naive mice. Naive mice, 1 month after adoptive transfer of either CD62Llow or unsorted lymphocytes, were resistant to growth of a 4T07 tumor (Fig. 7), but not to the unrelated MethA sarcoma (Fig. 8). Those mice that received equal numbers of B/I-activated CD62Lhigh lymphocytes were not resistant to tumor growth; in fact the tumor growth curve in mice treated with CD62Lhigh cells was not significantly different from that in the untreated age-matched controls.



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Fig. 7. Adoptive transfer of unsorted or T cell subset lymphocytes into naive mice. Naive BALB/c mice received IV infusion of 10 x 106 cells of either unsorted, CD62Llow, or CD62Lhigh vDLN lymphocytes that were activated with B/I and expanded in culture. One month later, these mice and age-matched controls were inoculated with 4T07 tumor cells into their flank. After a short period of tumor growth, 100% of the mice in the unsorted or CD62Llow group exhibited complete tumor regression. Mice in the CD62Lhigh group did not have any tumor regressions, and growth curves in these mice were not significantly different from controls. However, the unsorted and CD62Llow groups were significantly different than both the untreated and CD62Lhigh groups [F(3,20) = 15.93, P = 0.0001]. Numbers to the right indicate the number of mice with complete tumor regression per total number of mice in each group.

 


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Fig. 8. Adoptive transfer of 4T07-IL2 vaccine draining lymphocytes does not protect against MethA sarcoma tumors. Naive BALB/c mice received IV infusion of 7 x 106 unsorted vDLN lymphocytes activated ex vivo with B/I and expanded. One month later, these mice and age matched controls were inoculated with 3 x 105 4T07 cells or 1 x 106 MethA cells. After 30 days, all of the 4T07 tumors in mice pretreated by adoptive transfer had regressed, while only 2/6 of the MethA tumors treated by AIT regressed. One of six untreated MethA tumors also regressed. Numbers to the right indicate the number of mice with complete tumor regression per total number of mice in each group.

 
Effector cells of anti-tumor activity
To test whether the anti-tumor activity of AIT treatment is mediated by host T cells or the adoptively transferred T cells, athymic nude mice with 4 day 4T07 tumors were treated with unsorted vaccine draining lymphocytes. Adoptive transfer of B/I activated vDLN was effective at inducing 4T07 tumor regression in nude mice (Fig. 9), indicating that host immune T cells are not required for tumor regression, whether or not exogenous IL2 is also given (data not shown). IL2 and cyclophosphamide treatment alone had no significant anti-tumor effect.



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Fig. 9. Adoptive transfer of unsorted lymphocytes into nude mice. Athymic nude mice with established 4 day 4T07 flank tumors were either left untreated, received cyclophosphamide (CYP) or CYP and AIT with unsorted B/I activated vDLN cells. AIT treatment induced regression of 5/6 4T07 tumors, while untreated and CYP + IL2 treatment groups showed no tumor regression. The AIT treated group was significantly different from the untreated and CYP + IL2 groups [F(2,13) = 11.289, P = 0.0014].

 
To differentiate the roles of CD4 and CD8 T cell subsets in inducing tumor regression, BALB/c mice bearing 4 day tumors and treated with AIT using B/I-activated vDLN cells were then either untreated, or received anti-CD4 (GK1.5) or anti-CD8 (2.43) ascites fluid IV to deplete those cell populations after AIT. In mice depleted of the CD8 subset, 4T07 tumor growth was slowed, but did not regress completely in any mouse (Fig. 10). In mice depleted of CD4+ cells after AIT, 4T07 tumors still regressed, indicating that CD4 cells are not essential for tumor regression. This suggests that while CD4 cells have some anti-tumor activity, they cannot eradicate tumors and that CD8 cells are required for optimal anti-tumor effect.



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Fig. 10. In vivo depletion of CD4 and CD8 subsets after adoptive transfer. Mice with established 4 day 4T07 flank tumors were either left untreated, received cyclophosphamide (CYP) only or CYP and AIT with unsorted B/I activated vDLN cells. After AIT, mice received either no antibody, anti-CD4 (GK1.5) or anti-CD8 (2.43) by IV injection 1 h, 5 days and 10 days after AIT. After a short period of tumor growth, all of the tumors in the AIT and the CD4 depleted AIT group regressed completely. AIT + depletion of CD8+ cells results in slowing of tumor growth, but no complete regressions. The AIT and CD4 depleted AIT groups were significantly different from the untreated group, the CYP only group and the CD8 depleted AIT groups [F(4,24) = 32.45, P < 0.0001]. Numbers to the right indicate the number of mice with complete tumor regression per total number of mice in each group.

 
In vivo trafficking of adoptively transferred cells
The degree of CD4+ and CD8+ T cell subset infiltration into untreated, CYP treated and CYP and vDLN AIT treated 4T1 tumors was assessed in tumor sections by immunohistochemistry at 1, 4, 7 and 11 days after adoptive transfer. It was not possible to assess the amount of macrophage or NK cell infiltration of 4T1 tumors, because the tumor cells stained with MAC-3 and asialo-GM antibodies. Tumor infiltration was scored according to the percentage of stained cells as compared to unstained controls. Slides were categorized into four groups: unstained (–), 1–15% stained (+), 16–50% stained (++) and >50% stained (+++). As shown in Table 2, tumors from mice treated with AIT using vDLN cells showed infiltration of CD8+ T cells (16–50%) on day 1 after AIT and CD8+ cells persisted at levels <15% until the last time point checked (day 11). We did not observe infiltration of untreated or CYP treated tumors by CD8+ T cells at any of the time points. AIT + CYP treated tumors had high levels of CD4 infiltrate at day 1 (16–50%), which peaked on day 4 (>50%), declined to <15% on day 7 and peaked again on day 11 (>50%). In CYP alone treated tumors, CD4+ cells were not observed until day 4, peaking at >50% and then declined to <15% on day 7 and persisting at that level until day 11. Untreated or control tumors had CD4+ infiltrate <15% from days 1 to 7 after AIT, with levels peaking at 16–50% on day 11. Tumors treated with B/I activated vDLN cells had a high degree of CD4 and CD8 infiltration early on (days 1 and 4 after AIT). Lower numbers of CD4+ cells are seen in untreated and CYP treated tumors, with levels peaking at later time points, and their presence was not associated with tumor regression. These results reinforce the association of CD8+ T cells with tumor regression that we observed in our in vivo depletion experiments.


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Table 2. Immunohistochemical analysis of CD4 and CD8 lymphocyte infiltration of tumors left untreated, treated with CYP only, and with AIT and CYP

 
Because of the subjective nature of scoring IHC slides and the inability to differentiate between host and transferred T cells, vDLN cells were stained with CFSE after expansion and prior to AIT. At 1 h, 4, 7 and 10 days after AIT, tumors, tumor DLN (tDLN), contralateral LN (cLN) and spleens were harvested and analyzed by flow cytometry. CFSE stained cells were seen as early as 1 h after AIT in the spleen (1.17%), tumor-DLN (tDLN) (0.17%) and contralateral LN (cLN) (0.17%) (Table 3). CFSE+ cells rapidly reached their peak concentration (6.48%) in the spleen on day 4 after AIT and then declined rapidly. AIT cells tracked preferentially to tDLN, peaking at day 7 (14.67%) vs 7.57% in cLN and remained at high percentages in tDLN up to day 10, even after tumor regression. CFSE-stained cells were also seen in tumors in smaller percentages, peaking at day 4 (1.52%). The small percentages of CFSE+ cells in the tumors could be attributed either to the cells trafficking out of the tumors or to proliferation causing the dye to become diluted and undetectable.


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Table 3. Percentage of CFSE+ cells isolated from host tissues after AIT

 
Adoptively transferred lymphocytes did proliferate in vivo, with nine generations evident in tDLN on day 3 (Fig. 11). In the tDLN on day 3, CFSE-stained cells were 30% CD8+, 4% PAN DX5+ and 72% CD4+. vDLN cells which traffic to the tDLN had an activated phenotype, being 95% CD44+ and 39% CD69+.



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Fig. 11. AIT cell proliferation in tumor draining lymph nodes. vDLN cells were activated with B/I, expanded in culture and then stained with CFSE prior to adoptive transfer into mice with established 10 day 4T1 flank tumors. Three days after AIT, tDLN were harvested and CFSE+ cells were visualized by flow cytometry. Proliferation analysis of CFSE+ cells was done using Modfit LT and showed the presence of nine cell generations (reduced chi-squared = 1.238). Cross hatched peaks represent fitted generations, percentage of cells in each generation is listed after the generation number.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 References
 
We have repeatedly observed tumor-specific regression of various tumors after adoptive transfer of tumor antigen-sensitized lymphocytes activated with B/I (1,2,14). Since this pharmacologic method of activation might be expected to activate T cells non-specifically in a polyclonal fashion, and the number of tumor-specific cells in a given lymph node would be expected to be only a minority of the cells present, the efficacy and specificity of this approach was somewhat surprising. We had shown previously that allospecific CTL precursor frequency increased 11–80-fold after B/I activation and expansion, suggesting that this stimulus might preferentially activate antigen-sensitized cells, but this evidence was indirect (15). Moreover, the mechanism for this apparent selectivity remained unclear. The preferential activation and proliferation of antigen-sensitized T cells after incubation in B/I, which was up to 12-fold greater than CD62Lhigh cells, may well account for this phenomenon.

Ours is not the first report to indicate that the anti-tumor activity of tumor-sensitized T cells resides predominantly in certain subsets of cells, including those characterized by low CD62L expression (9,11). In these previous reports, however, there was only minimal difference between the proliferation of cells expressing high or low levels of CD62L after activation with anti-CD3 monoclonal antibody and culture in IL2. The magnitude of difference in the ex vivo expansion after B/I-activation seen here has not been reported after anti-CD3 antibody stimulation (11,12). The markedly increased expansion of the purified CD62Llow lymphocytes over unsorted or CD62Lhigh cells after pharmacologic activation is a unique finding.

Qualitative differences in proliferative and cytokine release responses to antigen or anti-receptor antibody stimulation between naive and memory cells have been described previously, but have focused primarily on differences in peptide dosage, affinity, kinetics of response and requirements for costimulation (1622). What makes the observations reported here novel is that differences in proliferation were observed using a pharmacologic method of activation that directly stimulates downstream signal transduction events. Thus, the differences cannot be explained on the basis of cell surface signaling events or costimulation. Rather, our findings suggest that memory T cells are somehow different from naive cells at the level of signal transduction, thus accounting for differing responses to B/I. Experiments to address this question, using T cell receptor (TcR) transgenic mice and looking at differences in PKC isozymes expression and/or translocation are currently in progress.

The CD62Llow purified lymphocytes also demonstrated a greater subsequent Type 1 cytokine response to tumor-antigen than the CD62Lhigh lymphocytes from the same lymph nodes. Similar differences between naive and memory T cells, albeit without a preceding period of activation and culture in IL2, were observed in other comparisons of naive and memory CD4 T cells (16). We have consistently observed a correlation between anti-tumor activity in vivo and IFN-{gamma} release in vitro, as seen here. Others have investigated the role of IFN-{gamma} in tumor regression with mixed results; some have found that immunologic tumor regression is not dependent on this cytokine (2325), while others have found that tumor regression relies heavily on the presence of IFN-{gamma} (12,2630). Nevertheless, there is a consistent correlation between these two activities in our hands.

We, and others, have shown a significant improvement in efficacy of AIT when used in conjunction with CYP (1,2,14,31). Although CYP has a significant adjuvant role in this immunotherapy model, CYP alone was not effective treatment for tumor-bearing mice, causing only slight inhibition of tumor growth (2,14). In addition to a weak direct anti-tumor effect, CYP may also inhibit tumor-induced suppressor cell function, increase Type 1 cytokine responses and increase the proliferation and distribution of adoptively transferred T lymphocytes in the adoptive host (32). This also is currently being studied in a TcR transgenic model.

The adoptive transfer of vDLN lymphocytes enriched for CD62Lhigh T cells appeared to have no significant anti-tumor effect. We have previously shown that the anti-tumor effect of B/I-activated T cells is a tumor-specific response, causing regression only of tumors expressing the same antigens as were used to sensitize the vDLN cells in the donor mice (1,2). Treatment with CD62Llow or unsorted vaccine draining lymph node T lymphocytes were equally successful in inducing complete regression of 4 day established 4T07 tumors in 100% of the mice. However, CD62Llow cells proliferated much more rapidly and extensively than unsorted T cells, producing larger numbers of ‘effector’ cells from the same starting number. Whether this enrichment of antigen-sensitized T cells based on cell surface marker expression prior to activation and expansion would increase the efficiency of this procedure in clinical trials would depend largely on the loss of cells during the enrichment procedure. Detailed cell dosing experiments would be required to clarify this issue but due to the limited numbers of CD62Llow cells, a titration experiment is not feasible. Enrichment prior to B/I activation would probably not be useful, however, since our results indicate that the method of activation itself selectively induces proliferation of the antigen-sensitized T cells of interest.

Recently, others have observed that tumor induced CD62Lhigh suppressor T cells inhibit successful tumor regression by adoptive immunotherapy (33). In at least one experiment, we have observed accelerated tumor growth in host mice treated by CD62Lhigh adoptive immunotherapy as compared to untreated mice (Fig. 7). While it is a possibility that the anti-tumor efficacy of our B/I activated CD62Llow vDLN treatment is partly due to the removal of suppressor CD62Lhigh cells, we observed in vivo proliferation of unsorted vDLN T cells and complete regression of tumors treated with unsorted B/I activated vDLN cells. This would argue against an important suppressive effect of CD62Lhigh cells in this model. Furthermore, Peng et al. (33) observed the effects of CD62Lhigh suppressor cells with adoptive transfer of tDLN harvested from late (day 12) tumors, but not with AIT from earlier (9 day) tumors. Our vDLN were harvested 10 days after injection of IL2 transfected 4T07 tumor cells, which may not induce the development of CD62Lhigh suppressor T cells.

Host T cells do not appear to be required for tumor rejection in this model, as AIT into nude athymic mice was equally effective as in wild-type mice. This is consistent with our previously published results in a sarcoma model using Thy-1 congenic T cell donors and tumor-bearing recipients (34). The anti-tumor activity of the adoptively transferred cells seems to reside predominantly in the CD8+ subset. B/I activated and expanded vDLN cells traffic preferentially to tDLN and persist up to day 10, proliferate rapidly in vivo and have an activated phenotype. Experiments are in progress to delineate more clearly the cell subsets involved in anti-tumor activity and the role of the host immune system.


    Conclusion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 References
 
Bryostatin and ionomycin, by activating PKC and increasing intracellular calcium concentrations, respectively, mimic intracellular signals that result in T cell activation. CD62Llow cells were preferentially activated by B/I, leading to a T cell population that is functionally enriched for tumor antigen-specific clones. As a result, these cells were highly effective in treatment of established tumors as well as conferring long-term immunity against subsequent tumor growth.

The results reported here, which demonstrate a difference in responsiveness to B/I among T cell subsets based on expression of L-selectin, will be used to distinguish more precisely the specific intracellular targets of bryostatin in T cells. Intracellular pathways involving PKC isoforms (6,35), Ras (36,37) and MEK/MAPK (38,39), have been implicated as possible targets for bryostatin. We are currently investigating the specific differences between naive and memory T cells in expression of intracellular signaling molecules that might explain this preferential activation by B/I.


    Acknowledgements
 
Flow cytometry supported in part by NIH grant P30 CA16059. Supported by R01 CA48075 and T32 CA09573 grants from the NCI, NIH and DHHS.


    Abbreviations
 
AIT   adoptive immunotherapy
B/I   bryostatin and ionomycin
CFDA-SE   carboxy-fluorescein diacetate succinimidyl ester
CFSE   carboxy-fluorescein succinimidyl ester
cLN   contralateral lymph node
CTL   cytotoxic T lymphocyte
CYP   cyclophosphamide
IHC   immunohistochemistry
IgG   immunoglobulin G
IFN-{gamma}   interferon-{gamma}
IP   intraperitoneally
IV   intravenously
PKC   protein kinase C
tDLN   tumor draining lymph node
Tukey's HSD   Tukey–Kramer honestly significant difference test
vDLN   vaccine sensitized draining lymph node

    Notes
 
Transmitting editor: S. L. Swain

Received 16 March 2004, accepted 17 June 2004.


    References
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 Introduction
 Methods
 Results
 Discussion
 Conclusion
 References
 

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