Phenotype, functions and fate of adoptively transferred tumor draining lymphocytes activated ex vivo in mice with an aggressive weakly immunogenic mammary carcinoma
© Miller et al; licensee BioMed Central Ltd. 2010
Received: 11 January 2010
Accepted: 4 November 2010
Published: 4 November 2010
Regression of established tumors can be induced by adoptive immunotherapy with tumor draining lymph node lymphocytes activated with bryostatin and ionomycin. We hypothesized that tumor regression is mediated by a subset of the transferred T lymphocytes, which selectively infiltrate the tumor draining lymph nodes and proliferate in vivo.
Adoptive transfer of B/I activated tumor draining lymphocytes induces regression of advanced 4T1 tumors, and depletion of CD8, but not CD4 T cells, abrogated tumor regression in mice. The predominant mediators of tumor regression are CD8+ and derived from CD62L- T cells. Transferred lymphocytes reached their peak concentration (10.5%) in the spleen 3 days after adoptive transfer and then rapidly declined. Adoptively transferred cells preferentially migrated to and/or proliferated in the tumor draining lymph nodes, peaking at day 5 (10.3%) and remained up to day 28. CFSE-stained cells were seen in tumors, also peaking at day 5 (2.1%). Bryostatin and ionomycin-activated cells proliferated vigorously in vivo, with 10 generations evident in the tumor draining lymph nodes on day 3. CFSE-stained cells found in the tumor draining lymph nodes on day 3 were 30% CD8+, 72% CD4+, 95% CD44+, and 39% CD69+. Pre-treatment of recipient mice with cyclophosphamide dramatically increased the number of interferon-gamma producing cells.
Adoptively transferred CD8+ CD62Llow T cells are the principal mediators of tumor regression, and host T cells are not required. These cells infiltrate 4T1 tumors, track preferentially to tumor draining lymph nodes, have an activated phenotype, and proliferate in vivo. Cyclophosphamide pre-treatment augments the anti-tumor effect by increasing the proliferation of interferon-gamma producing cells in the adoptive host.
Conventional therapies for cancer, including surgery, radiation and chemotherapeutic agents, are often ineffective at controlling the growth and spread of tumors. The immune system can potentially eliminate cancerous cells, as demonstrated by studies of numerous animal models and a few clinical trials [1, 2]. In most cases, it is thought that anti-tumor effects are mediated by cytotoxic T lymphocytes (CTL), which recognize MHC class I-peptide complexes on the tumor cell surface . Monoclonal antibodies, cytokines, and pharmacological methods have been used successfully in mouse models to activate lymphocytes isolated from tumors or tumor draining lymph nodes (DLN), which can then be adoptively transferred to tumor bearing hosts and cause regression of established tumors [4–15]. Adoptive immunotherapy (AIT), or the adoptive transfer of antigen-sensitized T cells activated and/or expanded in vitro continues to receive attention [16–23].
We have shown that in vitro treatment with bryostatin and ionomycin (B/I) selectively activates antigen-sensitized tumor draining lymph node (tDLN) lymphocytes [19–22]. Bryostatin 1 is a macrocyclic lactone derived from Bugula neritina, a marine invertebrate. Bryostatin activates protein kinase C [23–26] and ionomycin increases intracellular calcium . Together, these mimic signaling through the CD3/TcR complex and lead to activation and proliferation of T cells [24, 27].
Previous research in our lab has shown that adoptive transfer of B/I activated tumor draining lymphocytes can cure subcutaneous tumors and visceral metastases in murine hosts and establish long-term immunity, without evidence of autoimmunity. In the 4T1 mammary carcinoma model, we have shown that B/I selectively activates CD62L- or sensitized T cells and that the anti-tumor activity resides in the CD62L- fraction of lymphocytes obtained from donor lymph nodes; only the CD62L- subset proliferates after B/I activation and has anti-tumor activity . CD62L or L-selectin is an adhesion molecule important in T cell homing to lymph nodes and is down-regulated after T cells are activated and differentiate into their effector or effector memory (TEM) phenotypes [29, 30]. Thus, because of this selective activation of antigen-sensitized T cells from the vaccinated donor mice, B/I stimulated DLN lymphocytes have tumor antigen specific activity in vivo, despite the non-specific stimulus used to promote their growth.
We hypothesized that B/I activated T cells mediate tumor regression primarily by CD8+ T cell mediated functions and may establish T cell memory in the adoptive host by proliferating in vivo. B/I activated cells were characterized prior to adoptive transfer, and the most active subsets of cells identified by depletion or separation of phenotypically distinct subsets of T cells prior to AIT. By AIT using CFSE-labeled cells, we were also able to determine the trafficking patterns and measure the in vivo proliferation of B/I-activated T cells in tumor-bearing hosts.
Virus-free BALB/c mice (Charles River Laboratories, Cambridge, MA) were used between 8 and 12 weeks of age, caged in groups of 6 or fewer, and provided food and water ad libitum. Nude athymic BALB/c mice (National Cancer Institute, Bethesda, MD) were used to produce hybridoma ascites. All guidelines of the Virginia Commonwealth University Institutional Animal Care and Use Committee, which conform to the American Association for Accreditation of Laboratory Animal Care and the U.S. Department of Agriculture recommendations for the care and humane experimental use of animals, were followed.
Tumor cell lines and Hybridomas
4T1 mammary tumor cell line was kindly provided by Dr. Jane Tsai at the Michigan Cancer Foundation, Detroit, Michigan. Cells were maintained in Dulbecco's Modified Essential Medium (DMEM) with 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma, St. Louis MO) (modified DMEM). Meth A 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 × 10-5 M 2-mercaptoethanol (Sigma). Tumor cells were harvested for inoculation of mice with 0.05% trypsin-EDTA (Fisher, Pittsburgh, PA). 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.
Monoclonal Antibody production
Anti-CD4 monoclonal antibody (mAb) and anti-CD8 mAb were produced as ascites fluid from pristane-primed nude mice injected with their respective hybridomas.
Draining lymph node sensitization
Donor mice were vaccinated in the left hind footpad with 1 × 106 4T1 cells. Ten days after footpad vaccination, popliteal tumor draining lymph nodes (tDLN) were harvested under sterile conditions.
Lymphocyte activation and in vitro expansion
DLN's were harvested and dispersed into a single cell suspension in complete RPMI media at 1 × 106 cells/ml. The cells were activated by incubation with 5 nM bryostatin 1 (provided by the National Cancer Institute, Bethesda, MD) and 10 nM ionomycin (Calbiochem, San Diego, CA) (B/I), and 80 U/ml of rIL-2 (Chiron, Emeryville, CA) at 37°C for 18 hours. Cells were washed three times with warm complete RPMI and resuspended at 1-2 × 106 cells/ml with 40 U/ml of rIL-2. The cells were allowed to proliferate in culture for an additional 7 days and were split every 2-3 days in order to maintain 1-2 × 106 cells/ml concentration.
Host mice were inoculated in the left flank with 2.5 × 104 - 5 × 104 4T1 cells (2.5 × 104 for 7-10 day tumors, 5 × 104 for 4 day tumors). One day prior to AIT, mice were pretreated with cyclophosphamide (CYP),100 mg/kg IP(Mead Johnson, Princeton, NJ). On day 4, 7 or 10, the B/I activated and expanded DLN lymphocytes were washed twice in serum free medium (RPMI 1640) and injected intravenously (IV) in 0.5 ml into host mice. No systemic cytokines were administered.
CFSE staining and analysis
Prior to adoptive immunotherapy, B/I activated DLN lymphocytes were stained with 50 μM CFDA-SE (Molecular Probes, Eugene, OR) in PBS at a concentration of 75 million cells/ml for 15 minutes. Cells were washed with warm media and incubated at 37°C for 30 minutes to allow processing by intracellular proteases. CFSE-stained lymphocytes were injected IV into CYP-treated or untreated mice bearing 4T1 tumors.
Cells isolated from spleen, tumor, inguinal tDLN, and cLN of control or treated mice at various time points were stained with a panel of antibodies and analyzed by dual color flow cytometry for CFSE and surface marker expression on an ELITE Beckman Coulter flow cytometer. Fluorescently labeled Abs directed against the following markers were obtained from Pharmingen (San Diego, CA): Pan-DX5(DX5), CD4 (GK1.5), CD8 (53-6.7), CD44 (IM7), CD62L (MEL-14), and CD69 (H1.2F3). Appropriate isotype controls were used in all cases. Generations of proliferation detected by CFSE fluorescence were analyzed using ModFit LT (Verity Software House, Topsham, Maine) and acceptable fits were determined by reduced Chi-Squared values.
In vitro T cell subset depletion experiments
For in vitro depletion studies, DLN were first incubated with antibody (1:100) for 30 minutes, washed, and then incubated with rabbit complement (C') (Accurate Chemical) at 37°C for 30 minutes. The efficacy of the depletion was tested by flow cytometry. To determine the CD62L phenotype of the sensitized T cell precursors that were activated by B/I to become anti-tumor effectors or of the cells after B/I activation that mediated anti-tumor effects in adoptive hosts, DLN cells before and/or after activation with B/I were separated into CD62L- and CD62L+ subsets using magnetic bead separation (EasySep, Stem Cell Technologies). Flow cytometry was used to verify fractionation.
In all AIT experiments, tumor growth was monitored with biweekly measurements of perpendicular diameters. Results are reported as the mean tumor area ± standard error (SE). When the tumor area was greater than 100 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.
Cytokine release assays
Interferon-γ (IFN-γ) release from tumor sensitized, fresh or B/I activated and expanded lymphocytes in response to stimulation with irradiated 4T1 and irradiated Meth A for 24 hours was assayed using ELIspot assays from Pharmingen (San Diego, CA).
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, N.C.). In vivo experiments included at least six mice per group and were repeated at least twice. A p < 0.05 was used throughout to determine significant differences.
Adoptive transfer of tDLN activated by B/I and expanded in vitro induces regression of 10 day 4T1 tumors
CD8+ cells are the predominant mediators of tumor regression
Host T cells are not required for tumor regression
To confirm our hypothesis that host T cells play little or no role in tumor regression after adoptive transfer, athymic nude mice with 4 day 4T1 tumors were treated with CYP alone or CYP + B/I activated 4T1 draining lymphocytes, with or without exogenous IL-2 (7500 U i.p. on days 0 - 3 after AIT). Adoptive transfer of B/I activated tDLN was effective at inducing 4T1 tumor regression in nude mice pre-treated with CYP but IL-2 was neither beneficial nor required(data not shown, [F(2,13) = 11.289, P = 0.0014]). This result indicates that host T cells are neither required for tumor regression.
B/I activated tDLN demonstrate increased expression of message for cytolytic mediators
B/I activated tDLN release IFN-γ in response to tumor challenge
We and others have previously noted a strong correlation between interferon- IFN-γ production and tumor regression in other tumor models [31–37]. BALB/c mice were either untreated, vaccinated with irradiated 4T1, treated with CYP alone, or treated with CYP + AIT with B/I activated 4T1 tDLN lymphocytes. Spleens were harvested 5 days after adoptive transfer, and pooled splenocytes from 3 mice in each treatment group were co-cultured with irradiated 4T1 or unrelated MethA sarcoma for 24 hours on an IFN-γ ELISpot plate. As shown in Figure 3b, numbers of IFN-γ producing cells were greatest in response to 4T1 in mice treated with CYP + AIT. A small number of cells in each of the other groups also produced IFN-γ in response to 4T1, which may be explained by sensitization to 4T1 in these tumor-bearing hosts. There was no IFN- γ response to Meth A.
B/I activation leads to increased expression of memory markers by tumor draining lymphocytes
There have recently been reports in the literature that adoptive transfer of central memory or early effector phenotype cells (which are CD62Lhigh) is more effective against tumor than adoptive transfer of effector memory or late effector phenotype (CD62Llow ) T cells [38, 39]. Over the course of expansion after B/I activation, we observed upregulation of CD62L by previously CD62L- cells; 70% of the adoptively transferred cells in the B/I-activated cultured tDLN cells were CD62L+. In addition, we have previously observed in B/I activated cells minimal levels of cytotoxic activity and high IFN-γ production, which are also consistent with a central memory/early effector phenotype. Therefore, we investigated the hypothesis that activation with B/I could skew the phenotype of activated T cells towards a central memory or early effector phenotype, and that these cells were largely responsible for their anti-tumor efficacy.
Phenotype of CD62L--enriched T cells from tDLN, before and after B/I pulse and expansion in IL-2
% of CD8 cells which express:
Before and after B/I activation and expansion, anti-tumor activity resides predominantly in the CD62L- subset
Adoptively transferred cells persist in the tumor-bearing host, accumulating preferentially in the tumor draining lymph nodes
In viv o trafficking and accumulation over time of adoptively transferred cells in tumor-bearing mice
Absolute numbers of adoptively transferred cells in lymph nodes of tumor-bearing mice
4.6 × 105
3.9 × 105
2.8 × 105
2.1 × 105
9.1 × 104
2.3 × 10 3
1.8 × 104
6.4 × 104
CD8+ T cells infiltrate 4T1 tumors after AIT treatment
Day 1: Control
Day 4: Control
Day 7: Control
Day 11: Control
The trafficking and/or proliferation of adoptively transferred cells in the host is tumor specific
B/I activated tDLN track preferentially to 4T1 tDLN and persist
Adoptively transferred cells have an activated phenotype in the tumor bearing host (TBH)
Adoptively transferred cells proliferate in the tumor bearing host, with more generations seen in the tumor draining lymph nodes than in the contrateral nodes
Proliferation in vivo was dependent upon B/I activation before adoptive transfer
How does CYP augment the anti-tumor effect of AIT with B/I-activated lymphocytes?
We observed slightly greater proliferation of adoptively transferred CFSE-labelled cells in the tDLN of CYP pretreated hosts compared to similar hosts not treated with CYP. In CYP pretreated hosts, 9.38% of cells were in generation 6 and 5.26% of cells in generation 7 on day 3, versus 6.63% in generation 6 and 2.5% in generation 7 in mice not treated with CYP. The numbers of adoptively transferred cells in the tDLN of CYP-treated or untreated hosts were roughly similar on days 3 and 7 after adoptive transfer. The number of CFSE+ cells declined by day 10 in the tDLN of un-treated hosts (5 × 104 cells vs 3 × 105 cells in CYP pre-treated hosts). CYP pretreatment, therefore, modestly enhanced the proliferation of adoptively transferred cells and the persistence of these cells at later time points in the tDLN.
In this and previous studies, we have demonstrated that B/I activation of tDLN cells produces a population of cells with potent anti-tumor activity against established tumors up to 10 days after inoculation. Even tDLN harvested from donors 20 days post-tumor inoculation, from a metastatic disease state and possibly immunosuppressive milieu, are capable of inducing tumor regression after B/I activation and adoptive transfer into tumor bearing hosts. 14 days after tumor inoculation, 80% of 4T1-tumor bearing mice have measurable metastatic disease, and by 18 days, 100% have lung metastases . We had previously shown that B/I preferentially activated a subset of tDLN cells that were initially CD62L- and that these cells accounted for all of the subsequently developed anti-tumor activity . In the present studies, we have further characterized the post-activation phenotype and the in vivo trafficking, and proliferation of the anti-tumor effector cells generated by B/I activation and expansion in culture. The most potent anti-tumor effector cells were highly activated CD8+ T cells which remained CD62L- after B/I activation and expansion. These cells also respond to tumor antigen by specific release of IFN-γ, which we and others have shown correlates with anti-tumor activity [31–37].
Recently, there have been a number of reports that either central memory and/or early effector T cells are the most effective cell types for AIT [38, 39]. Conversion of adoptively transferred cells to the central memory phenotype has also been observed in adoptive hosts . TCM cells or early effector cells, as opposed to fully differentiated effector or effector memory (TEM) cells, have been found to have a greater ability to home to lymphoid tissue via expression of CD62L and CCR7, produce IL-2, proliferate more rapidly in response to antigen and cytokines, and then differentiate into effector cells [41–43]. TEM cells lack lymph node homing receptors, exhibit direct (ex vivo) cytotoxic activity, do not produce IL-2, and are found in non-lymphoid tissues [44–46]. Thus, TCM cells, despite a lower level of immediate effector functions, may be more effective for AIT, because they produce a larger number of anti-tumor cells for a longer period of time. As T cells are repeatedly stimulated, they differentiate from early effector to effector to late effector memory cells. They also downregulate receptors for homeostatic cytokines, and upregulate pro-apoptotic molecules.
We found that after 18 hour B/I activation and 7 days of expansion in IL-2, the initially CD62L- effector cell population harvested from donor mice had largely up-regulated the expression of CD62L (up to 68% CD62L+ ). We also observed up-regulation of memory markers CD27, CD44, CD69, and CD127. Furthermore, 76.4% of the adoptively transferred cells that were isolated from the adoptive hosts' tDLN 3 days after AIT were CD62L+, which suggested that activation with B/I may stimulate sensitized or CD62L- TEM (CD62L- CCR7low, CD27+, BCL-2 hi) or effector T cells to shift to a CD62L+ TCM phenotype, which we initially supposed would be largely responsible for the anti-tumor effects we have observed.
However, we found instead that adoptive transfer of B/I activated and expanded CD62L- cells (separated after expansion in culture) were most effective at mediating tumor regression, and that the CD62L+ fraction had little or no anti-tumor activity. There are a few explanations for these somewhat unexpected results and how it differs from previous reports. For example, this result may reflect enrichment of tumor antigen specific Treg cells in the CD62L+ subset, as recently reported . Alternatively, the CD62L- cells present after B/I activation and expansion may be analogous to secondary response derived memory cells, which are CD62L-. Recent literature suggests a distinct phenotype for T memory cells derived from primary versus those from secondary immune responses [48–52]. CD8 T cells undergoing a secondary response expand more rapidly and divide at a faster rate than in a primary response . In the Listeria monocytogenes (LM), Lymphocytic Choriomeningitis Virus (LCMV), and TcR transgenic models, it has been found that secondary immune responses produced memory CD8 T cells which are slow to convert to TCM, as measured by both CD62L expression and antigen induced IL-2 production . Primary and secondary response memory CD8 T cells have equal proliferative capacities in respect to numbers of generations, but secondary memory CD8 T cells remain CD62Llow, in contrast to the CD62Lhigh memory CD8 cells resulting from primary responses. Moreover, secondary CD62Llow memory cells are more effective against virulent LM infection and have increased cytolytic activity . The CD62L- T cells harvested from culture after B/I activation and expansion may be more analogous to memory cells derived from a secondary response, which our protocol mimics by re-activation of antigen-sensitized T cells with B/I. This contrasts with the TEM or TCM cells generated by stimulating naïve pmel-1 TcR transgenic T cells with antigen and cytokines in vitro[38, 39]. These approaches would generate TCM CD8 cells of the primary type, with the secondary immune response not occurring until after adoptive transfer into the tumor bearing host and subsequent in vivo vaccination with fowlpox vaccine encoding hgp100 [38, 39]. In our model, the primary immune response occurs in vivo in the tDLNs of the donor mice, and the secondary immune response occurs in vitro upon B/I activation of the tDLN. Our model may be more analagous with the clinical situation, in which sensitized PBMCs, tDLN, or vDLN are isolated from cancer patients, activated and expanded in vitro, and secondary memory T cells are then adoptively transferred into patients. Thus, B/I may have the advantage of generating secondary type T memory cells for adoptive transfer.
To further characterize the nature of the anti-tumor effector cells and their CD62L phenotype, tDLN lymphocytes were separated by CD62L phenotype both before and after B/I expansion. The most potent anti-tumor cells were the (CD62L- →CD62L- ) cells, which were capable of inducing tumor regression when as few as 375,000 B/I activated cells were transferred and even when double the normal tumor cell inoculum was used to establish the tumors. Studies with CFSE labeled cells showed that the adoptively transferred (CD62L- →CD62L- ) subset, despite the lack of CD62L, were able to traffic to tDLN after adoptive transfer, proliferated extensively in the adoptive hosts, and maintained their lack of CD62L expression (data not shown). H The (CD62L- →CD62L+ ) subset was less effective than (CD62L- →CD62L- ) cells at inducing tumor regression, especially at higher tumor inocula. Surprisingly, (CD62L+ →CD62L- ) cells were capable of delaying tumor progression. We previously had not seen any evidence of anti-tumor activity from initially CD62L+ subsets. These (CD62L+ →CD62L- ) cells may be tumor specific early effector or memory cells when harvested from donor mice and then may acquire an effector phenotype after exposure to B/I. The existence of these cells may have been masked in previous experiments in which separations were done either before or after B/I activation. When the CD62L separation was carried out only before B/I activation and expansion, they would have been vastly outnumbered by the ineffective (CD62L+ →CD62L+ ) subset and possibly suppressed by any Treg (CD4+ CD25+) cells also present in that fraction. When CD62L separation was carried out only after B/I activation and expansion, these cells would have been in the CD62L- subset. We have repeated these experiments with similar results, in both the B16 melanoma and B16-OVA models, indicating that this is not a tumor model specific result (data not shown).
Adoptively transferred cells were shown to persist and/or proliferate preferentially in hosts bearing the relevant tumor and accumulated preferentially in tumor draining lymph nodes. The decline in adoptively transferred cells in the spleens after day 3 is consistent with trafficking of these cells to sites of tumor antigen concentration. The low number of CFSE+ cells seen in the tumors may reflect proliferation of the cells beyond the limits of dye detection; after generation 10-12, CFSE becomes too dilute to be readily detected. Using immunohistochemical staining of tumors (not shown), both CD4+ and CD8+ cells were observed to infiltrate into tumors, although CD4+ cells do not appear to be required for tumor regression. Although some of these infiltrating cells could be of host origin, we have found that AIT is effective in nude mice [not shown and ], suggesting that host T cells are also not required.
In our model, the increased numbers of adoptively transferred cells in the tDLN suggest that specific uptake and/or proliferation of lymphocytes occurred at the site of antigen and antigen-presenting cells. However, it is not possible to tell how much of the difference in the numbers of cells at the tDLN vs the cLN is due to specific trafficking and accumulation at the site of the tDLN and how much is due to increased proliferation in the tDLN. In fact, we did also observe increased proliferation of the adoptively transferred cells in the tDLN as compared to the cLN, especially on day 3. However, in the absence of specific tumor antigen, adoptively transferred cells did not persist in the host LN. Thus, accumulation of adoptively transferred lymphocytes in the tDLN seems to be specific for tumor antigen, and not merely a result of non-specific inflammation.
In contrast to our trafficking results, others have reported that trafficking and accumulation of infused T cells in TBH mice was indiscriminate, without preference for tumor-draining LN versus other LN [38, 39]. It is important to note, however, that after infusion of T cells in those experiments, the adoptive hosts received systemic antigen, in the form of a recombinant fowlpox vaccine, as well as exogenous cytokines. Both of these would be expected to result in stimulation and proliferation of the adoptively transferred cells throughout the adoptive host [38, 39]. Furthermore, the TcR transgenic T cells that were infused in that model recognize a self antigen that would be expected to be present throughout the host, not only at the site of tumor growth or the tDLN.
In our model, proliferation of the adoptively transferred cells in the host was also dependent upon prior B/I activation; we did not observe proliferation of unactivated tDLN lymphocytes after adoptive transfer, even in CYP-pretreated hosts. Thus, the proliferation seen in our protocol is a result of B/I activation, and not merely homeostatic proliferation. One of the key potential advantages of B/I activation of tDLN is the ability of these cells to continue proliferating in adoptive hosts after AIT, without the need for exogenous cytokines or antigen vaccination. Interestingly, at the time these cells are harvested from culture, proliferation in vitro is generally declining, but apparently accelerates again in vivo.
In previous studies, we have shown that adoptively transferred B/I activated lymphocytes were more effective at inducing tumor regression in combination with CYP pre-treatment. The effectiveness of B/I activated lymphocytes with CYP pre-treatment and the lack of requirement for exogenous cytokine therapy is a major advantage of our method of activating tDLN. Several different mechanisms may account for CYP mediated modulation of the immune system. Several studies suggest that CYP causes a breakdown of regulatory mechanisms by removal of a suppressor cell population [54–58]. In early studies, mice that were pre-treated with CYP 2-3 days in advance of sensitization exhibited enhanced contact sensitivity or delayed type hypersensitivity [58–60]. Recent data suggest that CYP may selectively inhibit or deplete CD4+ CD25+ Treg cells [61–65]. CYP may also enhance AIT by temporarily inhibiting tumor growth, by creating "space" for T cell growth, or by a relative increase in cytokines that stimulate T cell proliferation .
As noted earlier, in the absence of B/I activation, adoptively transferred cells did not proliferate in CYP pre-treated hosts, indicating that CYP alone does not lead to homeostatic proliferation of adoptively transferred cells and that B/I activation was required for the proliferation of these cells in vivo. B/I activated lymphocytes, on the other hand, proliferated in the adoptive hosts with or without CYP pre-treatment. Arguably, slightly more proliferation was seen in CYP pre-treated hosts, but what was more striking was the increase in tumor antigen specific IFN-γ-producing cells in CYP pre-treated hosts compared to untreated hosts. Pre-treatment with CYP before AIT, it would appear, increases the resulting number of IFN-γ producing cells, possibly from the increase in availability of homeostatic cytokines or the removal of suppressive Treg populations.
B/I activation of tDLN generates large numbers of potent anti-tumor effector tells, as demonstrated by tumor regression in vivo and production of IFN-γ in vitro. These cells have the ability to traffic to the tumor draining lymph nodes, to proliferate extensively in vivo and to mediate tumor regression. Pre-treatment with CYP enhances the efficacy of adoptive immunotherapy, by increasing the number of resulting IFN-γ producing cells. The predominant mediators of anti-tumor activity are CD8+ T cells which are intially CD62L- when harvested from tDLN donors and remain CD62L- after B/I activation and expansion.
The results reported here will be used to distinguish more precisely the most effective anti-tumor populations in the tDLN and to explore methods of generating more of these cells. B/I activation is unique in that it is capable of selectively activating only sensitized T cells, appears to generate highly effective T cells of a secondary memory phenotype, and perhaps for that reason, does not require exogenous cytokines to be administered to the adoptive tumor-bearing host. This could avoid much of the toxicity associated with some AIT regimens. We are currently exploring the use of alternate γ-chain cytokines instead of IL-2 to program even more effectively the phenotypic development of B/I activated tDLN to a T memory phenotype, while decreasing the potential for expanding Treg cells.
analysis of variance
bryostatin and ionomycin
Carboxyfluorescein Diacetate Succinimidyl Ester
carboxyfluoroscein succinimidyl ester
contralateral lymph node
cytotoxic T lymphocytes
draining lymph node
Lymphocytic Choriomeningitis Virus
peripheral blood mononuclear cells
tumor bearing host
central memory T cells
T cell receptor
tumor draining lymph node
effector memory T cells
T regulatory cells
- Tukey's HSD:
Tukey-Kramer honestly significant difference test.
Flow Cytometry supported in part by NIH Grant P30 CA16059. Research was supported by R01 CA48075 and T32 A1074707-12 grants from the NIH, and DHHS. Additional support provided by A.D. Williams Grant # 6-40438.
- Saleh F, Renno W, Klepacek I, Ibrahim G, Dashti H, Asfar S, Behbehani A, Al-Sayer H, Dashti A: Direct evidence on the immune-mediated spontaneous regression of human cancer: an incentive for pharmaceutical companies to develop a novel anti-cancer vaccine. Curr Pharm Des. 2005, 11: 3531-3543. 10.2174/138161205774414556.View ArticlePubMedGoogle Scholar
- Saleh FH, Crotty KA, Hersey P, Menzies SW: Primary melanoma tumour regression associated with an immune response to the tumour-associated antigen melan-A/MART-1. Int J Cancer. 2001, 94: 551-557. 10.1002/ijc.1491.View ArticlePubMedGoogle Scholar
- Boon T, Cerottini JC, Van den Eynde B, Van der Bruggen P, Van Pel A: Tumor antigens recognized by T lymphocytes. Annu Rev Immunol. 1994, 12: 337-365. 10.1146/annurev.iy.12.040194.002005.View ArticlePubMedGoogle Scholar
- Chang AE, Li Q, Jiang G, Sayre DM, Braun TM, Redman BG: Phase II trial of autologous tumor vaccination, anti-CD3-activated vaccine-primed lymphocytes, and interleukin-2 in stage IV renal cell cancer. J Clin Oncol. 2003, 21: 884-890. 10.1200/JCO.2003.08.023.View ArticlePubMedGoogle Scholar
- Cohen PA, Peng LM, Kjaergaard J, Plautz GE, Finke JH, Koski GK, Czerniecki BJ, Shu SY: T-cell adoptive therapy of tumors: Mechanisms of improved therapeutic performance. Crit Rev Immunol. 2001, 21: 215-248.PubMedGoogle Scholar
- Crossland KD, Lee VK, Chen W, Riddell SR, Greenberg PD, Cheever MA: T cells from tumor-immune mice nonspecifically expanded in vitro with anti-CD3 plus IL-2 retain specific function in vitro and can eradicate disseminated leukemia in vivo. J Immunol. 1991, 146: 4414-4420.PubMedGoogle Scholar
- Dudley ME, Rosenberg SA: Adoptive-cell-transfer therapy for the treatment of patients with cancer. Nat Rev Cancer. 2003, 3: 666-675. 10.1038/nrc1167.PubMed CentralView ArticlePubMedGoogle Scholar
- Goedegebuure PS, Douville LM, Li H, Richmond GC, Schoof DD, Scavone M, Eberlein TJ: Adoptive immunotherapy with tumor-infiltrating lymphocytes and interleukin-2 in patients with metastatic malignant melanoma and renal cell carcinoma: A pilot study. J Clin Oncol. 1995, 13: 1939-1949.PubMedGoogle Scholar
- Harada M, Okamoto T, Omoto K, Tamada K, Takenoyama M, Hirashima C, Ito O, Kimura G, Nomoto K: Specific immunotherapy with tumour-draining lymph node cells cultured with both anti-CD3 and anti-CD28 monoclonal antibodies. Immunol. 1996, 87: 447-453. 10.1046/j.1365-2567.1996.487568.x.View ArticleGoogle Scholar
- Morse MA, Clay TM, Lyerly HK: Current status of adoptive immunotherapy of malignancies. Expert Opin Biol Ther. 2002, 2: 237-247. 10.1517/14712518.104.22.168.View ArticlePubMedGoogle Scholar
- Nijhuis EWP, Wiel-van Kemenade EVD, Figdor CG, Van Lier RAW: Activation and expansion of tumour-infiltrating lymphocytes by anti-CD3 and anti-CD28 monoclonal antibodies. Cancer Immunol Immunother. 1990, 32: 245-250. 10.1007/BF01741708.View ArticlePubMedGoogle Scholar
- Plautz GE, Cohen PA, Shu S: Considerations on clinical use of T cell immunotherapy for cancer. Arch. Immunol Ther Exp (Warsz). 2003, 51: 245-257.Google Scholar
- Ribas A, Butterfield LH, Glaspy JA, Economou JS: Current developments in cancer vaccines and cellular immunotherapy. J Clin Oncol. 2003, 21: 2415-2432. 10.1200/JCO.2003.06.041.View ArticlePubMedGoogle Scholar
- Schoof DD, Selleck CM, Massaro AF, Jung SE, Eberlein TJ: Activation of human tumor-infiltrating lymphocytes by monoclonal antibodies directed to the CD3 complex. Cancer Res. 1990, 50: 1138-1143.PubMedGoogle Scholar
- Yoshizawa H, Chang AE, Shu S: Specific adoptive immunotherapy mediated by tumor-draining lymph node cells sequentially activated with anti-CD3 and IL-2. J Immunol. 1991, 147: 729-737.PubMedGoogle Scholar
- Alexander JP, Kudoh S, Melsop KA, Hamilton TA, Edinger MG, Tubbs RR, Sica D, Tuason L, Klein E, Bukowski RM: T-cells infiltrating renal cell carcinoma display a poor proliferative response even though they can produce interleukin 2 and express interleukin 2 receptors. Cancer Res. 1993, 53: 1380-1387.PubMedGoogle Scholar
- Correa MR, Ochoa AC, Ghosh P, Mizoguchi H, Harvey L, Longo DL: Sequential development of structural and functional alterations in T cells from tumor-bearing mice. J Immunol. 1997, 158: 5292-5296.PubMedGoogle Scholar
- Danna EA, Sinha P, Gilbert M, Clements VK, Pulaski BA, Ostrand-Rosenberg S: Surgical removal of primary tumor reverses tumor-induced immunosuppression despite the presence of metastatic disease. Cancer Res. 2004, 64: 2205-2211. 10.1158/0008-5472.CAN-03-2646.View ArticlePubMedGoogle Scholar
- Tuttle TM, Inge TH, Bethke KP, McCrady CW, Pettit GR, Bear HD: Activation and growth of murine tumor-specific T-cells which have in vivo activity with bryostatin 1. Cancer Res. 1992, 52: 548-553.PubMedGoogle Scholar
- Tuttle TM, Bethke KP, Inge TH, McCrady CW, Pettit GR, Bear HD: Bryostatin 1-activated T cells can traffic and mediate tumor regression. J Surg Res. 1992, 52: 543-548. 10.1016/0022-4804(92)90126-K.View ArticlePubMedGoogle Scholar
- Tuttle TM, McCrady CW, Inge TH, Salour M, Bear HD: γ-interferon plays a key role in T-cell-induced tumor regression. Cancer Res. 1993, 53: 833-839.PubMedGoogle Scholar
- Tuttle TM, Fleming MF, Hogg PS, Inge TH, Bear HD: Low-dose cyclophosphamide overcomes metastasis-induced immunosuppression. Ann Surg Oncol. 1994, 1: 53-58. 10.1007/BF02303541.View ArticlePubMedGoogle Scholar
- Yee C: Adoptive T cell therapy--immune monitoring and MHC multimers. Clin Immunol. 2003, 106: 5-9. 10.1016/S1521-6616(02)00015-3.View ArticlePubMedGoogle Scholar
- Cantrell D: T cell antigen receptor signal transduction pathways. Annu Rev Immunol. 1996, 14: 259-274. 10.1146/annurev.immunol.14.1.259.View ArticlePubMedGoogle Scholar
- Kazanietz MG, Lewin NE, Gao F, Pettit GR, Blumberg PM: Binding of [26-3H]bryostatin 1 and analogs to calcium- dependent and calcium-independent protein kinase C isozymes. Mol Pharmacol. 1994, 46: 374-379.PubMedGoogle Scholar
- Pettit GR, Herald SL, Doubek DL, Arnold E, Clardy J: Isolation and structure of bryostatin 1. J Am Chem Soc. 1982, 104: 6846-6848. 10.1021/ja00388a092.View ArticleGoogle Scholar
- Chatila T, Silverman L, Miller R, Geha R: Mechanisms of T cell activation by the calcium ionophore ionomycin. J Immunol. 1989, 143: 1283-1289.PubMedGoogle Scholar
- Chin CS, Miller CH, Graham L, Parviz M, Zacur S, Patel B, Duong A, Bear HD: Bryostatin 1/ionomycin (B/I) ex vivo stimulation preferentially activates L-selectinlow tumor-sensitized lymphocytes. Int Immunol. 2004, 16: 1283-1294. 10.1093/intimm/dxh130.View ArticlePubMedGoogle Scholar
- Kagamu H, Shu SY: Purification of L-selectinlow cells promotes the generation of highly potent CD4 antitumor effector T lymphocytes. J Immunol. 1998, 160: 3444-3452.PubMedGoogle Scholar
- Kjaergaard J, Shu SY: Tumor infiltration by adoptively transferred T cells is independent of immunologic specificity but requires down-regulation of L-selectin expression. J Immunol. 1999, 163: 751-759.PubMedGoogle Scholar
- Parviz M, Chin CS, Graham LJ, Miller C, Lee C, George K, Bear HD: Successful adoptive immunotherapy with vaccine-sensitized T cells, despite no effect with vaccination alone in a weakly immunogenic tumor model. Cancer Immunol Immunother. 2003, 52: 739-750. 10.1007/s00262-003-0405-8.View ArticlePubMedGoogle Scholar
- Aruga A, Aruga E, Tanigawa K, Bishop DK, Sondak VK, Chang AE: Type 1 versus type 2 cytokine release by Vb T cell subpopulations determines the in vivo antitumor reactivity: IL-10 mediates a suppressive role. J Immunol. 1997, 159: 664-673.PubMedGoogle Scholar
- Lowes MA, Bishop GA, Crotty K, Barnetson RS, Halliday GM: T helper 1 cytokine mRNA is increased in spontaneously regressing primary melanomas. J Invest Dermatol. 1997, 108: 914-919. 10.1111/1523-1747.ep12292705.View ArticlePubMedGoogle Scholar
- Tsung K, Meko JB, Peplinski GR, Tsung TL, Norton JA: IL-12 induces T helper-1 directed antitumor responses. J Immunol. 1997, 158: 3359-3365.PubMedGoogle Scholar
- Winter H, Hu HM, McClain K, Urba WJ, Fox BA: Immunotherapy of melanoma: A dichotomy in the requirement for IFN-γ in vaccine-induced antitumor immunity versus adoptive immunotherapy. J Immunol. 2001, 166: 7370-7380.View ArticlePubMedGoogle Scholar
- Zitvogel L, Mayordomo JI, Tjandrawan T, Deleo AB, Clarke MR, Lotze MT, Storkus WJ: Therapy of murine tumors with tumor peptide-pulsed dendritic cells: Dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines. J Exp Med. 1996, 183: 87-97. 10.1084/jem.183.1.87.View ArticlePubMedGoogle Scholar
- Hu HM, Urba WJ, Fox BA: Gene-modified tumor vaccine with therapeutic potential shifts tumor-specific T cell response from type 2 to type 1 cytokine profile. J Immunol. 1998, 161: 3033-3041.PubMedGoogle Scholar
- Gattinoni L, Klebanoff CA, Palmer DC, Wrzesinski C, Kerstann K, Yu Z, Finkelstein SE, Theoret MR, Rosenberg SA, Restifo NP: Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8(+) T cells. J Clin Invest. 2005, 115: 1616-1626. 10.1172/JCI24480.PubMed CentralView ArticlePubMedGoogle Scholar
- Klebanoff CA, Gattinoni L, Torabi-Parizi P, Kerstann K, Cardones AR, Finkelstein SE, Palmer DC, Antony PA, Hwang ST, Rosenberg SA, Waldmann TA, Restifo NP: Central memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells. Proc Natl Acad Sci USA. 2005, 102: 9571-9576. 10.1073/pnas.0503726102.PubMed CentralView ArticlePubMedGoogle Scholar
- Wrzesinski C, Restifo NP: Less is more: lymphodepletion followed by hematopoietic stem cell transplant augments adoptive T-cell-based anti-tumor immunotherapy. Curr Opin Immunol. 2005, 17: 195-201. 10.1016/j.coi.2005.02.002.PubMed CentralView ArticlePubMedGoogle Scholar
- Wherry EJ, Ahmed R: Memory CD8 T-cell differentiation during viral infection. J Virol. 2004, 78: 5535-5545. 10.1128/JVI.78.11.5535-5545.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Wherry EJ, Teichgraber V, Becker TC, Masopust D, Kaech SM, Antia R, Von Andrian UH, Ahmed R: Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat Immunol. 2003, 4: 225-234. 10.1038/ni889.View ArticlePubMedGoogle Scholar
- Sallusto F, Geginat J, Lanzavecchia A: Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu Rev Immunol. 2004, 22: 745-63. 10.1146/annurev.immunol.22.012703.104702.View ArticlePubMedGoogle Scholar
- Lanzavecchia A, Sallusto F: Understanding the generation and function of memory T cell subsets. Curr Opin Immunol. 2005, 17: 326-332. 10.1016/j.coi.2005.04.010.View ArticlePubMedGoogle Scholar
- Roberts AD, Ely KH, Woodland DL: Differential contributions of central and effector memory T cells to recall responses. J Exp Med. 2005, 202: 123-133. 10.1084/jem.20050137.PubMed CentralView ArticlePubMedGoogle Scholar
- Tough DF: Deciphering the relationship between central and effector memory CD8+ T cells. Trends Immunol. 2003, 24: 404-407. 10.1016/S1471-4906(03)00169-8.View ArticlePubMedGoogle Scholar
- Hiura T, Kagamu H, Miura S, Ishida A, Tanaka H, Tanaka J, Gejyo F, Yoshizawa H: Both regulatory T cells and antitumor effector T cells are primed in the same draining lymph nodes during tumor progression. J Immunol. 2005, 175: 5058-5066.View ArticlePubMedGoogle Scholar
- Jabbari A, Harty JT: The generation and modulation of antigen-specific memory CD8 T cell responses. J Leukoc Biol. 2006, 80: 16-23. 10.1189/jlb.0206118.View ArticlePubMedGoogle Scholar
- Badovinac VP, Porter BB, Harty JT: Programmed contraction of CD8(+) T cells after infection. Nat Immunol. 2002, 3: 619-626. 10.1038/nrm880.View ArticlePubMedGoogle Scholar
- Barber DL, Wherry EJ, Ahmed R: Cutting edge: rapid in vivo killing by memory CD8 T cells. J Immunol. 2003, 171: 27-31.View ArticlePubMedGoogle Scholar
- Jabbari A, Harty JT: Secondary memory CD8+ T cells are more protective but slower to acquire a central-memory phenotype. J Exp Med. 2006, 203: 919-932. 10.1084/jem.20052237.PubMed CentralView ArticlePubMedGoogle Scholar
- Roberts AD, Woodland DL: Cutting edge: effector memory CD8+ T cells play a prominent role in recall responses to secondary viral infection in the lung. J Immunol. 2004, 172: 6533-6537.View ArticlePubMedGoogle Scholar
- Veiga-Fernandes H, Walter U, Bourgeois C, McLean A, Rocha B: Response of naive and memory CD8+ T cells in antigen stimulation in vivo. Nature Immunology. 2000, 1: 47-53. 10.1038/76907.View ArticlePubMedGoogle Scholar
- Polak L, Geleick H, Turk JL: Reversal by cyclophosphamide of tolerance in contact sensitization. Tolerance induced by prior feeding with DNCB. Immunology. 1975, 28: 939-942.PubMed CentralPubMedGoogle Scholar
- Yasunami R, Bach JF: Anti-suppressor effect of cyclophosphamide on the development of spontaneous diabetes in NOD mice. Eur J Immunol. 1988, 18: 481-484. 10.1002/eji.1830180325.View ArticlePubMedGoogle Scholar
- Mitsuoka A, Baba M, Morikawa S: Enhancement of delayed hypersensitivity by depletion of suppressor T cells with cyclophosphamide in mice. Nature. 1976, 262: 77-78. 10.1038/262077a0.View ArticlePubMedGoogle Scholar
- Rollinghoff M, Starzinski-Powitz A, Pfizenmaier K, Wagner H: Cyclophosphamide-sensitive T lymphocytes suppress the in vivo generation of antigen-specific cytotoxic T lymphocytes. J Exp Med. 1977, 145: 455-459. 10.1084/jem.145.2.455.PubMed CentralView ArticlePubMedGoogle Scholar
- Asherson GL, Ptak W: Contact and delayed hypersensitivity in the mouse. I. Active sensitization and passive transfer. Immunology. 1968, 15: 405-416.PubMed CentralPubMedGoogle Scholar
- Maguire JRHC, Ettore VL: Enhancement of dinitrochlorobenzene (DNCB) contact sensitization by cyclophosphamide in the guinea pig. J Invest Dermatol. 1967, 48: 39-43.PubMedGoogle Scholar
- Sullivan S, Bergstresser PR, Streilein JW: Analysis of dose response of trinitrochlorobenzene contact hypersensitivity induction in mice: pretreatment with cyclophosphamide reveals an optimal sensitizing dose. J Invest Dermatol. 1990, 94: 711-716. 10.1111/1523-1747.ep12876288.View ArticlePubMedGoogle Scholar
- Beyer M, Kochanek M, Darabi K, Popov A, Jensen M, Endl E, Knolle PA, Thomas RK, von Bergwelt-Baildon M, Debey S, Hallek M, Schultze JL: Reduced frequencies and suppressive function of CD4+CD25hi regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine. Blood. 2005, 106: 2018-2025. 10.1182/blood-2005-02-0642.View ArticlePubMedGoogle Scholar
- Lutsiak ME, Semnani RT, De PR, Kashmiri SV, Schlom J, Sabzevari H: Inhibition of CD4(+)25+ T regulatory cell function implicated in enhanced immune response by low-dose cyclophosphamide. Blood. 2005, 105: 2862-2868. 10.1182/blood-2004-06-2410.View ArticlePubMedGoogle Scholar
- Ikezawa Y, Nakazawa M, Tamura C, Takahashi K, Minami M, Ikezawa Z: Cyclophosphamide decreases the number, percentage and the function of CD25+ CD4+ regulatory T cells, which suppress induction of contact hypersensitivity. J Dermatol Sci. 2005, 39: 105-112. 10.1016/j.jdermsci.2005.02.002.View ArticlePubMedGoogle Scholar
- Ghiringhelli F, Larmonier N, Schmitt E, Parcellier A, Cathelin D, Garrido C, Chauffert B, Solary E, Bonnotte B, Martin F: CD4+CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur J Immunol. 2004, 34: 336-344. 10.1002/eji.200324181.View ArticlePubMedGoogle Scholar
- Ercolini AM, Ladle BH, Manning EA, Pfannenenstiel LW, Armstrong TD, Machiels JPH, Bieler JG, Emens LA, Reilly T, Jaffee EM: Recruitment of latent pools of high-avidity CD8+ T cells to the antitumor immune response. J Exp Med. 2007, 201: 1591-1602. 10.1084/jem.20042167.View ArticleGoogle Scholar
- Proietti E, Greco G, Garrone B, Baccarini S, Mauri C, Venditti M, Carlei D, Belardelli F: Importance of cyclophosphamide-induced bystander effect on T cells for a successful tumor eradication in response to adoptive immunotherapy in mice. J Clin Invest. 1998, 101: 429-441. 10.1172/JCI1348.PubMed CentralView ArticlePubMedGoogle Scholar