- Open Access
Adoptive immunotherapy of cancer with polyclonal, 108-fold hyperexpanded, CD4+ and CD8+ T cells
© Wang et al; licensee BioMed Central Ltd. 2004
- Received: 12 October 2004
- Accepted: 26 November 2004
- Published: 26 November 2004
T cell-mediated cancer immunotherapy is dose dependent and optimally requires participation of antigen-specific CD4+ and CD8+ T cells. Here, we isolated tumor-sensitized T cells and activated them in vitro using conditions that led to greater than 108-fold numerical hyperexpansion of either the CD4+ or CD8+ subset while retaining their capacity for in vivo therapeutic efficacy. Murine tumor-draining lymph node (TDLN) cells were segregated to purify the CD62Llow subset, or the CD4+ subset thereof. Cells were then propagated through multiple cycles of anti-CD3 activation with IL-2 + IL-7 for the CD8+ subset, or IL-7 + IL-23 for the CD4+ subset. A broad repertoire of TCR Vβ families was maintained throughout hyperexpansion, which was similar to the starting population. Adoptive transfer of hyper-expanded CD8+ T cells eliminated established pulmonary metastases, in an immunologically specific fashion without the requirement for adjunct IL-2. Hyper-expanded CD4+ T cells cured established tumors in intracranial or subcutaneous sites that were not susceptible to CD8+ T cells alone. Because accessibility and antigen presentation within metastases varies according to anatomic site, maintenance of a broad repertoire of both CD4+ and CD8+ T effector cells will augment the overall systemic efficacy of adoptive immunotherapy.
- Tumor Antigen
- Pulmonary Metastasis
- Effector Function
- Adoptive Transfer
- Total Body Irradiation
Cancer immunotherapy, using T lymphocytes that recognize tumor-specific antigens, holds great promise. Advantageous features include: exquisite specificity for targeted antigens, thereby sparing normal tissues, and the ability of effector T cells to traffic to tumor in all anatomic locations. Although most effector T cells are subject to activation-induced cell death (AICD), a memory response is established leading to sustained protection . Despite the theoretical appeal of T cell-mediated immunotherapy, clinically relevant benefits have been documented in only a small subset of human cancer patients who present with metastatic disease [2–5]. Several factors contribute to the poor host immune response including defective Antigen Presenting Cell (APC) function in cancer patients, and production of immunosuppressive substances by tumors [6–8].
Cognizant of these features, many preclinical studies of active immunotherapy have used a vaccination/challenge scheme to avoid tumor-induced immunosuppression or have alternatively treated hosts with minimal tumor burdens. Several human clinical trials have similarly focused on hosts with minimal residual disease in order to define the magnitude and characteristics of the immune response. These studies have clearly established that immune responses are successfully generated in vaccinated cancer patients. However, the frequency of responding T cells is typically less than one percent even after multiple cycles of vaccination [9–14]. In contrast, the immune response to pathogens generates a tremendous amplification of reactive T cells [15–17]. In the clinical setting, relatively little is yet known about the magnitude of proliferation of individual precursor cells (burst size) as they mature into effector cells, or the flux between lymphoid tissue, peripheral blood, and tumor sites. This results in ambiguity about the optimal time and site to quantify the immune response. Likewise, analysis of apoptosis of effector cells is likely to be important [18, 19]. These gaps in fundamental knowledge have made it difficult to identify components of active immunotherapy that could be enhanced to boost the aggregate immune response to a therapeutic level.
Adoptive immunotherapy is another approach to cancer immunotherapy that circumvents some of the limitations of active immunotherapy. Animal tumor models have convincingly demonstrated that hosts bearing progressively growing weakly immunogenic tumors nevertheless generate sensitized T cells in TDLN . Antigen-sensitization causes T cells to downregulate expression of L-selectin (CD62L) providing a convenient phenotypic marker for segregation of primed T cells from the majority of irrelevant T cells [21–23]. Our previous studies have demonstrated that ex vivo activation of purified CD62Llow T cells from TDLNs generates potent effector CD4+ and CD8+ T cells that can mediate regression of advanced tumors in every tested anatomic location [24–26]. The high potency of such cells permitted brief 5-day activation and limited numerical amplification (10-fold) to supply sufficient quantities of cells for the previous mechanistic analysis of the anti-tumor response. Importantly, these experiments demonstrated that there is tight dose dependence, oftentimes with even a three-fold reduction in the number of transferred cells accounting for a difference between minimal treatment effect and complete cure. The relative efficacy of CD4+ versus CD8+ effector cells also varies considerably between pulmonary metastases and intracranial (i.c.) or subcutaneous (s.c.) tumors . This indicates that maintenance of CD4+ as well as CD8+ tumor-reactive effector T cells would be required for optimal adoptive immunotherapy against disseminated metastatic disease.
We investigated whether we could overcome quantitative limitations associated with active immunotherapy through extensive numerical expansion of effector cells. In a previous study, we determined that in vitro activation of tumor-sensitized L-selectinlow precursors with anti-CD3 mAb and high concentrations of IL-2 (100 U/ml) induced rapid proliferation of CD8+ effector cells . Adoptive transfer of such cells cured established tumors in recipients. However, these culture conditions led to maximal proliferation in 9 days with subsequent decline in cell numbers thus limiting the total expansion to approximately 103-fold. In this report, we define ex vivo activation conditions that permit numerical expansion of either CD4+ or CD8+ effector T cells to greater than 108-fold while retaining their high therapeutic potency and preserving a broad T cell receptor (TCR) repertoire.
Mice and tumors
Female C57BL6N (B6) mice were purchased from the biologic Testing Branch, National Cancer Institute (Frederick, MD). They were maintained in a specific pathogen-free environment according to National Institute of Health guidelines. Mice were used for experiments at 8–10 weeks of age. The MCA 205 and MCA 207 fibrosarcomas, syngeneic to B6 mice were serially passaged in vivo s.c. as described previously .
Preparation and culture activation of TDLN CD62Llow cells
Tumors were established by s.c. flank inoculation of 1.5 × 106 MCA 205 cells and 12 days later the TDLNs were removed and mechanically disrupted to obtain a single cell suspension. The TDLN cells were incubated with 100 μl anti-CD62L microbeads per 108 cells and applied to MACS columns (Miltenyi Biotech, Auburn CA) and the flow through fraction was collected. For CD4+ hyperexpansion, the CD62Llow subset was depleted of CD8+ cells by MACS on day 0 and day 36 of culture activation. CD62Llow cells, containing approximately 50% TCR+ and 50% B220+ subsets, were suspended in complete medium (CM) and incubated for 2 days at 4 × 106 per well in 24 well culture plates coated with anti-CD3 (145-2C11) as previously described . Activated cells were washed, counted, and suspended at 0.5 × 105/ml in CM with IL-2 (4 U/ml) (Chiron Corp. Emeryville, CA), with or without rmIL-7 (10 ng/ml) or rhIL-23 (2 ng/ml) (each from R&D Systems, Minneapolis, MN) and then diluted to 105/ml on day 5 of activation. On days 9 and 15, the cell concentration was adjusted to 2 × 105/ml. For experiments with two cycles of anti-CD3 stimulation, T cells were incubated with immobilized anti-CD3 for 14 hrs on day 15 and used for adoptive therapy on day 23. For long-term expansion, cultures were maintained for 23 days after the initial anti-CD3 stimulation in CM with the indicated combination of IL-2 (4 U/ml), IL-7 (10 ng/ml), and IL-23 (2 ng/ml) and then were stimulated with anti-CD3 for 14 hrs on day 23 and every 7 days thereafter.
IFN-γ and FACS analysis
T cells were stimulated with a single cell suspension of either MCA 205 or MCA 207 tumors at a 1:1 ratio, or with immobilized anti-CD3. Brefeldin A was added after five hours of stimulation and the cells were harvested after 20 hrs and stained for intracellular IFN-γ according to the manufacturers instructions (BD Biosciences, San Diego, CA). FACS analysis was performed using FITC or PE conjugated antibodies or isotype control antibodies (BD Biosciences).
RNA isolation and CDR3 size distribution analysis (TCR spectratyping)
TDLN cells were lysed using TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA was reverse transcribed into cDNA using the SuperScript II RT kit (Invitrogen). cDNA was amplified using PCR with 22 different VB-specific primers paired with a hex-labeled constant region primer which spans the CDR3 region as previously described . CDR3 size distribution analysis was performed by mixing 1.0 μl of hex-labeled PCR amplified cDNAs with 12.0 μl deionized formamide (Sigma) and 0.5 μl size standard (Genescan-400 ROX, ABI 310; Perkin-Elmer, Shelton, CT), heated for 2 minutes at 90°C and chilled on ice prior to analysis. Samples were applied to an ABI 310 sequencer for CDR3 size distribution analysis. Samples were determined to be oligoclonally skewed if the CDR3 size patterns failed to exhibit a Gaussian bell-shaped distribution and were dominated by one or two prominent peaks.
Mice were inoculated with MCA 205 or MCA 207 tumor cells (3 × 105) i.v. to establish pulmonary metastases. Subcutaneous tumors were established by inoculation of 1.5 × 106 cells. Intracranial tumors were established by transcranial inoculation of 105 tumor cells at a depth of 4 mm as previously described . Mice bearing 3-day s.c. or i.c. tumors or 10-day pulmonary metastases were treated with 5 Gy nonmyeloablative total body irradiation (TBI) delivered from a 137Cs irradiator prior to intravenous transfer of the T cells whereas mice with 3-day pulmonary tumors were not irradiated. For pulmonary tumors, mice were euthanized on day 20 post inoculation, the lungs were insufflated with India ink and the number of surface tumor nodules was enumerated using a dissecting microscope. Subcutaneous tumors were measured in two perpendicular dimensions three times per week and mice with progressive tumors were euthanized when the product of dimensions exceeded 200 mm2. Mice bearing intracranial tumors were monitored daily for survival or were euthanized when neurologic symptoms such as decreased grooming and decreased spontaneous movement were apparent.
Treatment groups consisted of five individuals. Analysis of tumor size for s.c. tumors was performed by the Mann-Whitney rank sum test. For pulmonary tumors, a t test was performed on paired samples and p < 0.05 was considered significant. Survival of mice bearing i.c. tumors was compared using the Wilcoxon rank sum test.
Ex vivo stimulation with anti-CD3, IL-2, and IL-7 augments effector cell generation
The morphology of the cells changed from lymphoblastoid to small round cells at day 15 and there was no additional proliferation. IL-7 preserved the viability of cells whereas IL-2 alone could not prevent a 8-fold numerical decline between days 15 to 30. Because the TDLN cells were initially segregated based on phenotype rather than antigen specificity and the anti-CD3 stimulation was antigen-independent, it was not known whether the enhanced proliferation achieved in the presence of IL-7 was due to preferential growth of irrelevant T cells or preservation of tumor-reactive T cells. As demonstrated in figure 1C, there was equivalent therapeutic efficacy against s.c. tumors at day 9 using T cells cultured with IL-2 alone or the combination of IL-2 and IL-7. Regression of established tumors requires efficient trafficking and the dose of 5 × 106 CD62Llow TDLN cells is near the lowest threshold dose required to cure 3-day s.c. MCA 205 tumors [26, 33]. Thus, the addition of IL-7 during in vitro activation augmented the total number of cells but did not substantially diminish per-cell therapeutic efficacy.
Preservation of effector function after anti-CD3 re-stimulation
The relative per-cell potency of re-stimulated cultures on day 23 was compared with the 5-day culture activation approach we have employed in previous studies. The segregated CD62Llow TDLN cells were frozen and one aliquot was thawed and activated for a total of 23 days with anti-CD3 stimulation on days 0–2 and again on day 15. The second aliquot was thawed on culture day 18 of the first aliquot and stimulated with anti-CD3 for 48 hrs and then cultured with IL-2 and IL-7 for an additional 3 days. The two T cell cultures were synchronously harvested and transferred into hosts bearing 3-day s.c. tumors. As demonstrated in Figure 2D, whereas 5 × 106 cells activated for 5 days was curative in 5/5 mice, 5 × 106 cells cultured for 23 days had minimal therapeutic effect. However, a modest increase in the cell dose to 1.5 × 107 cells led to a significant therapeutic effect and at a dose of 4 × 107 cells 2/5 mice were cured. The s.c. tumor model is highly dependent on the presence of tumor-specific CD4+ T cells [36, 37]. The relative decrease in percentage of CD4+ cells from 24% on day 5 of culture to 4% on day 23 may account for some of the differential therapeutic effects. In contrast to the modest difference in per-cell efficacy, there was nearly 1000-fold greater proliferation in the 23-day versus 5-day cultures indicating that the aggregate therapeutic effect was substantially greater following extended culture.
Repetitive anti-CD3 stimulation induces hyper-expansion of CD8+ effectors
There are immunologic scenarios that demonstrate exhaustion of the effector response leading to failure of immunologic control of infection or tumor [38–40]. By contrast, selection and extensive propagation of T cell clones indicates that T cells can undergo massive proliferation yet retain antigen-specific function. To assess whether there is an intrinsic limit to the retention of in vivo effector function of CD62Llow cells, they were stimulated with anti-CD3 followed by IL-2 and IL-7 for 23 days. Starting on day 23, the T cells were activated with anti-CD3 every 7 days. The time course between the initial and subsequent anti-CD3 stimulations was chosen based on evidence that T cells undergo changes in gene expression, phenotype, and function over a twenty-day time course in the transition from naïve to memory cells .
At day 50, the cultures were >99% TCR+ and CD8+ indicating preferential expansion or survival of CD8+ cells under the conditions employed. As shown in Figure 3B, adoptive transfer of 2 × 107 cells to hosts with 3-day MCA205 pulmonary metastases eliminated tumors and 6 × 106 cells was the threshold dose for complete response whereas 2 × 106 cells were subtherapeutic. In addition, there was no response against the antigenically distinct MCA207 tumor. In an independent experiment, the dose of T cells required to completely eliminate 3-day pulmonary metastases was 2 × 106 indicating some inter-experimental variability in per-cell efficacy. Because of the critical role of CD4+ T cells for therapy of s.c. or i.c. tumors it was not anticipated that the hyperexpanded CD8+ cultures would mediate complete regression of tumors at these anatomic sites. Indeed, there was substantially less efficacy against 3-day s.c.tumors. Adoptive transfer of 4 × 107 cells showed a trend toward response (P = 0.061) with 1/5 mice cured in only one of two identically designed experiments (Figure 3C). In addition, a dose of 2 × 107 cells was subtherapeutic against 3-day i.c. tumors (Figure 3D).
Despite the rapid proliferation of CD8+ T cells in vitro, there was no evidence of lymphoid hyperplasia when the mice were sacrificed to enumerate lung metastases 17 days after adoptive transfer. Moreover, there was no evidence of lymphoproliferative disease even when the hyperexpanded T cells were transferred into 5Gy TBI hosts bearing i.c. or s.c. tumors that had transient lymphodepletion of host cells. Thus, despite extensive proliferation in vitro the T cells did not demonstrate any evidence of transformation.
Hyperexpanded CD4+ T effector cells mediate regression of i.c. and s.c. tumors
The CD4+ T cells maintained in IL-7 plus IL-23 were subjected to continued repetitive anti-CD3 restimulation every 7 days starting on day 56. These conditions led to exclusive proliferation of CD4+ T resulting in 1.2 × 108-fold total proliferation by day 85 of culture. Despite extensive in vitro proliferation in response to antigen-independent stimulation for 85 days, 1.5 × 107 CD4 cells retained efficacy against 3-day s.c.tumors, with 3/5 mice achieving complete tumor regression (P = 0.019 versus control) (Figure 4D). As previously demonstrated, CD8+ cells synchronously cultured in the presence of IL-2 plus IL-7 for 50 days and expanded to 108-fold demonstrated minimal efficacy against subcutaneous tumors (P = 0.39 versus control) with 1/5 mice achieving complete tumor regression.
Hyperexpanded cultures retain IFN-γ producing cells
These experiments demonstrate that T cells, sensitized to tumor antigens in vivo, can be activated in vitro under conditions that promote hyperexpansion of either the CD4+ or CD8+ subset while retaining their potent therapeutic efficacy against established tumors. A notable feature of in vitro activation is that it permits selection and enrichment of a minor subset of tumor-reactive precursor cells. The mechanism of antigen sensitization of T cells through cross-priming by APC within draining LNs provides a convenient localized anatomic source that is already highly enriched. When coupled with physical segregation based on phenotypic characteristics that distinguish between antigen-stimulated versus naïve T cells, enrichment to nearly 40% of tumor-specific T cells was achieved. One important aspect of this strategy for selection and enrichment is that it does not require pre-existing knowledge of the immunogenic tumor antigens and does not require freshly acquired T cells to exhibit effector function. These conditions have relevance for many clinical situations where tumor antigens are not yet described or where unique tumor antigens may be immunodominant. Moreover, signaling defects have been observed in freshly acquired T cells from tumor-bearing hosts that might impede segregation based on functional properties [42–44].
Starting with a highly enriched population of T cells we were able to use a powerful, yet antigen-independent, stimulus such as anti-CD3 mAb that preserved the initial TCR repertoire diversity. Interestingly, anti-CD28 stimulation was not required for this experimental model, presumably because the T cells had already received co-stimulation during APC-mediated priming in vivo. The principal advantage to in vitro activation is that the culture conditions can be adapted to optimize proliferation of distinct subsets of responding cells. It is important to note that anti-CD3 activation in the absence of exogenous cytokine support did not lead to T cell proliferation even among CD4+ cells. The low cell density may have prevented secreted cytokines from reaching a critical threshold concentration. Moreover, IL-7, produced by non-hematopoetic cells and IL-23, produced by APC, mandated an exogenous source of these cytokines for in vitro culture activation. The combination of IL-2 and IL-7 provided rapid proliferation of CD8+ T cells and preserved their viability after completion of the initial mitogenic burst. The reason this combination was effective is that IL-7 receptor α chain is constitutively expressed on naïve and memory T cells but is downregulated on activated T cells [45, 46]. By contrast, the IL-2 receptor α chain is reciprocally expressed on activated cells in a transient manner. Thus, the combination of these two cytokines ensured continuous mitogenic signal transduction. IL-7 is crucial for development and homeostasis of T cells and is markedly increased following lymphodepletion. Therefore, there is considerable interest in employing lymphodepletion as a strategy to augment active as well as adoptive immunotherapy . Likewise, exogenous IL-2 has been administered in the context of tumor antigen vaccination as well as in nearly every clinical application of adoptive transfer to provide helper function . However, in addition to their mitogenic effects on antigen-stimulated T cells, systemic production of IL-7 or systemic administration of IL-2 has effects on irrelevant T lymphocytes, other hematopoetic cells, and the vasculature. The inability to target cytokine support specifically to the relevant T cells limits the effectiveness of in vivo cytokine administration. More importantly, we have clearly documented that adjunctive IL-2 inhibits trafficking of adoptively transferred T cells into intracranial or subcutaneous tumors . By contrast, cytokine stimulation can be targeted specifically to effector cells under optimal conditions in vitro without adverse systemic effects on the host. Future experiments to adjust the sequence, and concentration of supplemental cytokines using more sophisticated schedules than employed here might provide superior effector function.
These experiments confirm the importance of CD4+ T cells for therapy of tumors in certain anatomic sites, such as the brain and subcutaneous tissue. The slower rate of CD4+ cell proliferation relative to CD8+ cells following the initial anti-CD3 stimulation led to their rapid marginalization in mixed cultures. However, depletion of CD8+ cells and use of cytokine combinations such as IL-7 and IL-23 favored the selective hyperexpansion of CD4+ cells that retained potent in vivo function. IL-23 is a member of the IL-12 family of cytokines and contains the IL-12 p40 subunit that transduces signals through the shared IL-12β1 chain in addition to the unique p19 subunit [50, 51]. The IL-23 receptor is expressed on memory but not naive CD4+ cells, thus it is ideal for previously sensitized LN T cells. Myeloid cells, which are the natural source of IL-23, disappear rapidly in the in vitro cultures mandating an exogenous source. There is not a substantial amount of data on the effects of in vivo IL-23 administration, therefore its utility as a systemically administered adjuvant for T cell adoptive immunotherapy is unclear. However, the related cytokine IL-12 has substantial systemic effects that have limited its clinical use .
Although CD4+ T cells have been investigated as a source of helper function for CD8+ cytolytic cells our previous experiments have clearly established their stand-alone potential against MHC class II negative tumors . CD4+ T cell anti-tumor function is mediated through cross-presentation of specific tumor-antigens by tumor associated APC . As demonstrated, CD4+ cells cultured with IL-23 produced greater levels of INF-γ that would augment antigen presentation. Indeed, addition of tumor-reactive CD4+ cells to tumor digest increases the reactivity of the CD8+ cells. In addition to their autonomous effector functions, CD4+ cells are required to generate a functional CD8+ memory response in vivo [55, 56]. Our recent experiments have demonstrated that adoptive transfer of effector T cells causes tumor destruction and sensitization of a secondary wave of regenerating host T cells . In this regard, IL-23 stimulation of CD4+ cells might be particularly useful because it, unlike IL-12, induces production of the pro-inflammatory cytokine IL-17 . Our observation that such sensitization occurred even in hosts with partial tumor regression indicates that the presence of effector CD4+ T cells and inflammatory conditions of tumor antigen acquisition by host APC are important in perpetuating the anti-tumor response.
Repetitive anti-CD3 stimulation was utilized to drive hyperproliferation of T cells yet the TCR/CD3 complex also activates genetic programs required for effector function. Effector molecules such as Fas, Perforin, and IFN-γ have autoinhibitory as well as paracrine inhibitory effects during culture activation. Viewed purely in operational terms, sequential in vitro activation first under conditions that optimize T cell proliferation, then with conditions that restore effector functions immediately before adoptive transfer, would be advantageous. Future experiments will explore whether it is possible to dissociate proliferative signaling pathways from those mediating effector function through selective transient gene inactivation. The quantitative aspects of hyperexpansion are of less scientific interest but do have some practical implications. The 108-fold extent of proliferation far exceeded what was required to treat the tumor models employed. Moreover, the availability of uniformly primed T cells for mechanistic studies is not numerically limited when using an inbred strain. However these experiments establish an approach to maintain polyclonality and preserve effector function despite extensive antigen-independent proliferation. As such, the quantitative aspects of hyperexpansion may have relevance to certain clinical situations where an autologous source of antigen-primed T cells may be limited and extensive host tumor burden may demand a large number of effector T cells.
This work was supported by a grant from NIH, CA 091981.
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