Suppression of T-Cell Expansion by Melanoma is Exerted on Resting Cells
- First Online:
- Cite this article as:
- Russ, A.J., Wentworth, L., Xu, K. et al. Ann Surg Oncol (2011) 18: 3848. doi:10.1245/s10434-011-1667-6
- 89 Views
Immunotherapeutic cancer protocols often rely on the ability to promote proliferative expansion of tumor-specific T-cell, but the influence of cancer on in vivo T-cell expansion remains largely undefined.
The ability of control and B16F10 melanoma-bearing C57BL/6 mice to expand lymphocytic choriomeningitis virus antigen-specific T-cell populations in response to acute viral infection was compared by using flow cytometric assays of splenocytes.
The ability to expand virus-specific CD8+ and CD4+ T-cells was globally and markedly suppressed in tumor-bearing mice. Expanded cytotoxic T lymphocytes (CTLs) retained in vivo and in vitro functionality, suggesting that melanoma growth did not induce T-cell anergy. The magnitude of suppressed proliferative expansion was proportional to the extent of tumor burden. Melanoma-induced suppression of CTL expansion was correlated with upregulated apoptotic activity and hampered the induction of memory precursor effector cells. Adoptive transfer of resting LCMV antigen-specific T-cells before or after tumor establishment demonstrated that a critical period of in vivo exposure of resting T-cells to growing melanoma was responsible for the induction of suppressed expansion. This suppression was durable; surgical resection of melanoma after in vivo exposure to resting T-cells but before antigenic stimulation did not restore full expansion.
These data suggest that growing melanoma tumors exert a global, antigen-independent influence on resting T-cells that fundamentally reprograms their ability to undergo proliferative expansion in response to subsequent antigenic stimulation. This finding may have direct implications for T-cell-based immunotherapeutic strategies.
Efforts to mobilize the immune system against cancer have focused on CD8+ cytotoxic T lymphocytes (CTLs) for their ability to mediate antigen-specific tumor killing.1 Immunotherapeutic strategies to infuse and expand tumor-specific CTLs in vivo have shown great promise, but their overall clinical efficacy has been limited.2,3 Investigative attempts to explain and overcome these shortcomings have focused on the undesirable influences of cancer-induced immune suppression,4–9 but the specific influence of cancer on proliferative CTL expansion has not been fully explored.
In this report, we describe a novel quantitative animal model with which we can confirm and measure the suppressive influence of melanoma on proliferative T-cell expansion in vivo. By measuring the ability of immunocompetent C57BL/6 mice to expand antigen-specific T-cell populations in response to lymphocytic choriomeningitis virus (LCMV) infection, we demonstrate that melanoma growth nonspecifically suppresses antigen-driven T-cell expansion. The magnitude of this T-cell suppression is proportional to the extent of tumor burden. By modulating the duration of exposure of resting T-cells to growing tumor, we demonstrate that this suppression is exerted not at the moment of T-cell activation by antigen, but before antigen encounter; exposure of resting T-cells to melanoma weakens their ability to undergo proliferative expansion in response to future antigenic stimulation.
Materials and Methods
Seven- to 8-week-old female C57BL/6/Ly5.2 mice (Taconic, Hudson, NY) and C57BL/6/Ly5.1 background P14 TCR transgenic mice with TCR specificity for the Db-restricted LCMV antigen peptide GP33 10 (obtained from M. Suresh) were maintained in pathogen-free conditions and handled in strict accordance with the guidelines of the University of Wisconsin and William S. Middleton Memorial VA Hospital Animal Care and Use Committees.
Tumor Cell Lines and Virus
B16F10, a poorly immunogenic melanoma cell line derived from C57BL/6 mice, was maintained in RPMI-1640 medium (Mediatech, Herndon, VA) supplemented with 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 2 mM of l-glutamine (Life Technologies, Inc., Grand Island, NY). Single inocula of 106 B16F10 cells suspended in serum-free RPMI1640 media were injected subcutaneously into mice. Mice were infected with 2x105 PFU of Armstrong strain LCMV by intraperitoneal injection. Levels of LCMV in explanted tissues were quantified using Vero cell-based plaque assays.11
Splenocytes were harvested from P14 mice and 103 to 104 CD8+ GP33-specific T-cells (Ly5.1+) were adoptively transferred into C57BL/6/Ly5.2 mice via tail vein injection.
MHC class I tetramers loaded with various LCMV antigen peptides were prepared as previously described.12 Single cell splenocyte suspensions were stained with APC-labeled MHC class I (Db) tetramers loaded with class I-restricted LCMV epitope peptides, PE-labeled anti-CD8, and FITC-labeled anti-CD44 antibodies. Anti-CD62L, anti-CD127, anti-Ly5.1, and anti-granzyme B antibodies also were used. Freshly harvested splenocytes (106 cells/well) were stimulated with or without LCMV epitope peptides (0.1 μg/mL) in the presence of brefeldin A and human recombinant IL-2 (10 U/well) at 37°C for 5 h in flat-bottomed 96-well plates. Cells were stained with anti-CD8 and anti-CD62L antibodies, and then permeabilized and stained for intracellular cytokines using anti-IFN-γ, anti-TNF-α, and anti-IL-2 antibodies using the Cytofix/Cytoperm kit (BD Biosciences, San Diego, CA). Stained cells were acquired on a FACSCalibur flow cytometer (BD Biosciences) and resulting data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR). All reagents and antibodies were purchased from BD Biosciences with the exception of anti-CD127 (eBioscience, Inc., San Diego, CA) and anti-granzyme B antibodies (Invitrogen, Inc., Carlsbad, CA).
Data were analyzed by using the SPSS 16.0 statistical analysis package (SPSS, Inc., Chicago, IL). Groups were compared by using the two-tailed Student’s t test, and significance was defined as p < 0.05. Comparisons between tumor mass and T-cell expansion were performed by using Pearson correlation coefficient.
Antigen-driven T-cell Expansion is Suppressed in the Presence of Melanoma
C57BL/6 mice were inoculated with media or 106 B16F10 cells on day 0 and were infected with LCMV on day 10. Splenocytes were harvested on day 18 (postinfection day 8, the point of maximal LCMV-specific CTL expansion).12 Tumor-bearing mice exhibited localized subcutaneous tumors without metastases or evidence of systemic illness, alterations in behavior, or weight loss on day 18 (data not shown). Tumor growth was comparable between uninfected and infected mice, and no inhibition of tumor growth was seen after LCMV infection (data not shown).
Magnitude of Suppression of T-cell Expansion is Proportional to Extent of Melanoma Burden
Melanoma Does Not Alter the Timing of T-cell Expansion
The kinetics of T-cell activation involve an expansion phase of rapid antigen-driven proliferation, which ultimately results in antigen clearance followed by a contraction phase in which CTL populations involute through apoptotic cell death. A subset of memory precursor effector T-cells (MPEC), identifiable by high expression of IL-7R-α (CD127) and low expression of the senescence marker KLRG-1, survive and differentiate into mature memory T-cells capable of responding rapidly to antigen challenge. Eventual memory T-cell population size is directly proportional to clonal burst size at the time of maximal proliferative expansion.12,13 After LCMV infection, peak CTL expansion and viral clearance occur on postinfection day 8.12 To determine whether suppression of T-cell expansion by melanoma was accompanied by alterations in the timing of expansion and contraction, control, and tumor-bearing mice were sacrificed on days 16, 18, and 20 (postinfection days 6, 8, and 10). Timing of proliferative expansion and apoptotic contraction was similar between control and tumor-bearing mice, but fewer LCMV-specific T-cells were present in tumor-bearing mice on days 18 and 20 (Supplementary Fig. 1a, b). Proliferative activity among LCMV-specific T-cell populations (as measured by expression of the proliferation marker Ki-67) was similar (Supplementary Fig. 1c, d), but significantly more apoptotic activity (as measured by Annexin Vhigh expression) was seen in tumor-bearing mice on day 18 (Supplementary Fig. 1e, f). Concordant with the lower peak of CTL expansion in tumor-bearing mice, we identified smaller CD127high/KLRGlow MPEC populations among LCMV-specific T-cells in tumor-bearing mice on day 20 (Supplementary Fig. 1g). Thus, tumor-induced suppression of antigen-driven T-cell expansion is associated with augmented apoptotic activity, and may ultimately result in attenuated immunological memory.
Susceptibility of Naïve Resting T-Cells to Melanoma-Induced Suppression of Antigen-Driven Expansion is Dependent on Timing of Adoptive Transfer
Exposure of Resting T-Cells to Melanoma is Sufficient for Induction of Suppressed Antigen-Driven Expansion
Surgical Resection of Established Melanoma does not Completely Reverse Suppression Antigen-Driven T-cell Expansion
Immunotherapeutic strategies to augment antitumor immune responses have employed tumor antigen-specific CTLs for their ability to mediate immunological tumor rejection.1 Experimental animal models and clinical trials of adoptive T-cell immunotherapy in which tumor-specific CTLs are introduced into subjects with cancer, then expanded in vivo via antigenic stimulation, suggest that adjunctive immunomodulatory strategies, such as host lymphodepletion or cytokine-mediated immune stimulation, may be needed for adoptively transferred CTLs to achieve their potential efficacy.2,3 However, persistent shortcomings of T-cell–based cancer immunotherapies continue to motivate efforts to fully understand the influence of cancer on T-cell physiology.
In this study, we utilized a quantitative readout of T-cell expansion (proliferation of clonal LCMV epitope-specific CTL populations) to demonstrate that activation-induced T-cell expansion is weakened in the presence of melanoma. The absence of antigenic overlap between B16F10 and LCMV suggests that this melanoma-induced suppression of T-cell expansion is not antigen-specific. Indeed, a similar degree of suppression was seen for all clonal populations tested, regardless of the antigenicity of their target LCMV epitopes. This is compatible with recent observations of weakened CTL expansion in response to gp96-Ig-expressing vaccines in the presence of tumor,14,15 and suggests that cancers exert a globally suppressive influence on T-cell proliferation that is not restricted to tumor-antigen specific T-cells alone. The linear relationship between tumor burden and extent of suppression is in agreement with heightened deficits in cellular immunity observed among mice bearing bulky tumors and inverse relationships observed between tumor burden and responsiveness to T-cell vaccination strategies.15,16 These findings underscore the possibility that the optimal window of opportunity for effective clinical strategies in cancer immunotherapy may be in the setting of minimal disease.17
The kinetics and magnitude of activation-induced T-cell expansion and subsequent contraction are a function of antigenic stimulation and the resulting balance between cycle progression and apoptosis. The timing of LCMV-driven expansion and contraction did not differ between control and tumor-bearing mice. Analysis of explanted liver and lung tissue confirmed that both groups were capable of viremic clearance 8 days after LCMV infection (data not shown); thus, differences in T-cell expansion were not a result of differences in viral exposure. Rather, analysis of proliferative and apoptotic activity at various time points after LCMV infection suggest that the suppression of T-cell expansion in the presence of melanoma may be causally related to a pro-apoptotic milieu. Similar phenomena have been described among tumor-infiltrating lymphocytes, whose propensity toward apoptotic cell death in vitro is associated with downregulation of the antiapoptotic proteins Bcl-xL and Bcl-2.18 An eventual consequence of acute T-cell expansion in response to antigen is the induction of long-lived memory T-cells arising from MPEC populations.19 Because of the finite life expectancy of tumor-bearing mice, we were unable to measure mature memory T-cells in tumor-bearing mice at appropriate time points (>50 days) after LCMV clearance. However, the presence of fewer MPECs on postinfection day 10 in tumor-bearing mice suggests that melanoma-induced suppression of acute T-cell expansion may have durable implications throughout the longitudinal spectrum of activated T-cell homeostasis.
By transferring splenocytes from LCMV-naïve P14 TCR transgenic mice into control or tumor-bearing mice before or after tumor inoculation, we tested whether suppressed expansion in tumor-bearing mice was mediated before or at the time of antigenic stimulation. The observation of normal LCMV-driven expansion among P14 T-cells adoptively transferred after tumor establishment but immediately before viral exposure suggests that the 9 days of in vivo exposure of T-cells to growing melanoma in our model was necessary for the induction of suppressed expansion. Expansion of endogenous non-P14 polyclonal T-cells was suppressed in tumor-bearing animals regardless of the timing of adoptive transfer; therefore, it is unlikely that cotransferred populations of cells (e.g., mature dendritic cells) were responsible for allowing P14 T-cells transferred into tumor-bearing mice on day 9 to have expanded normally. Recapitulating tumor exposure by harvesting donor cells from tumor-bearing P14 mice 9 days after tumor inoculation restored the suppressed phenotype. Interestingly, this relatively brief exposure was sufficient to suppress expansion, as P14 T-cells transferred from tumor-bearing donors into tumor-free recipients immediately before LCMV infection remained suppressed (although not to the extent seen when transferred into tumor-bearing recipients). Resection of established tumors 8 days after melanoma inoculation (2 days before LCMV infection) was incapable of fully restoring normal T-cell expansion, and suppression among mice bearing resected tumors was less than that seen in melanoma-bearing mice that did not undergo resection. These parallel observations suggest that melanoma-induced inhibition of antigen-driven T-cell expansion is at least partly mediated by intrinsic suppression of resting T-cells occurring before antigenic stimulation.
Our observations suggest a causal relationship between melanoma growth and suppressed T-cell expansion. The mechanisms by which melanoma growth alters the ability of resting T-cells to undergo activation-induced expansion remain to be determined. The presence of similar numbers of resting P14 TCR transgenic T-cells in control versus tumor-bearing mice and the fact that tumors did not fundamentally alter baseline CD4+ and CD8+ T-cell populations in uninfected mice (data not shown) suggest that this suppression is not mediated by a reduction in CTL precursor frequency. Downregulated expression of the TCR signaling protein CD3ζ, which could impair TCR-mediated proliferation, has been observed among tumor-infiltrating lymphocytes.6,7 We were unable to identify evidence that expanded activated CTLs were functionally anergic in tumor-bearing mice, as evidenced by their ability to elaborate inflammatory cytokines in response to in vitro antigenic stimulation. Others also have observed restoration of functional activity of CTLs harvested from tumor-bearing using in vitro stimulation assays.15,20 However, the presence of similar levels of granzyme B expression among activated T-cells harvested directly ex vivo from control and tumor-bearing mice suggests that CTLs remained functionally competent in melanoma-bearing mice. Thus, the dominant consequence of melanoma on T-cells in our model is suppression of numbers, not functional activity.
These data offer evidence that growing melanoma tumors exert a globally suppressive influence on resting T-cells that intrinsically weakens their ability to undergo maximal proliferative expansion in response to antigenic stimulation. This phenomenon may have clinical implications for T-cell–based adoptive immunotherapy strategies designed to induce in vivo expansion of tumor antigen-specific CTL populations. Ongoing work in our laboratory seeks to elucidate the mechanisms underlying this suppression and identify strategies by which this clinically undesirable phenomenon may be overcome.
This work was supported by grant support from the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Science Research and Development Service, Career Development Award (CDA-2), American College of Surgeons Faculty Research Fellowship, and Central Surgical Association Foundation Grant to CSC.
Conflicts of interest
There are no potential conflicts of interest; the contents of this work do not represent the views of the Department of Veterans Affairs or the United States Government.