The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jensen, S. M.
Right arrow Articles by Fox, B. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jensen, S. M.
Right arrow Articles by Fox, B. A.
The Journal of Immunology, 2003, 170: 2014-2021.
Copyright © 2003 by The American Association of Immunologists

Regression of a Mammary Adenocarcinoma in STAT6-/- Mice Is Dependent on the Presence of STAT6-Reactive T Cells 1

Shawn M. Jensen*,{ddagger}, Sybren L. Meijer*, Robert A. Kurt§, Walter J. Urba{dagger}, Hong-Ming Hu* and Bernard A. Fox2,*,{ddagger}

* Laboratory of Molecular and Tumor Immunology and {dagger} Clinical Research, Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Providence Portland Medical Center, Portland, OR 97213; {ddagger} Departments of Molecular Microbiology and Immunology, and Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239; and § Department of Biology, Lafayette College, Easton, PA 18042


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Polarization of the immune response toward a type 1 cytokine profile has been posited to be associated with a therapeutic antitumor immune response. STAT6-/- mice are unable to generate a type 2 immune response, and instead mount an enhanced type 1 response. STAT6-/- mice are significantly more resistant to 4T1, a mammary adenocarinoma cell line, resisting a 10-fold higher tumor dose compared with wild-type (wt) BALB/c mice. An analysis of the T cells from tumor-bearing STAT6-/- mice revealed that they contained a population primed by a peptide (STAT6531–539) of the STAT6 protein expressed in 4T1. The adoptive transfer of T cells from STAT6531–539-vaccinated STAT6-/- mice significantly reduced the number of 4T1 pulmonary metastases in recipient mice. Additionally, the role of these STAT6531–539-reactive T cells against s.c. 4T1 tumor challenge was determined by tumor-challenging wt BALB/c mice reconstituted with STAT6-/- bone marrow, thereby assessing whether a polarized type 1 immune response in the absence of STAT6-reactive T cells was sufficient to reject a 4T1 tumor challenge. T cells from the STAT6-/- bone marrow chimeras failed to recognize the STAT6531–539, and these mice proved to be as susceptible as wt BALB/c mice to 4T1 challenge. This demonstrated that the absence of STAT6531–539-reactive T cells correlated with the inability to reject 4T1 challenge. Additionally, these data emphasize that the enhanced ability to mount a type 1-polarized immune response is inconsequential if a sufficient antitumor immune response is not primed by the tumor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The theory of immune surveillance proposes that the immune system is responsible for the detection and elimination of malignant cells at their inception (1). Thus, the occurrence of tumors within individuals argues that occasionally a malignant cell escapes immune surveillance and progresses to form a tumor. Many mechanisms have been proposed to explain why this might occur, including: the lack of tumor Ags or their presentation (2), insufficient costimulation (3), or the secretion of immunosuppressive factors (4). It is also possible that the tumor environment might skew the immune response away from a therapeutic response toward a nontherapeutic response, a process known as immune deviation (5). Previous work has demonstrated that type 1-polarized immune responses correlated with a therapeutic tumor immune response, whereas type 2-polarized immune responses were markedly less therapeutic or nontherapeutic (6, 7). Additionally, it was shown that the therapeutic type 2 response was nontherapeutic if type 2-polarized T cells were transferred into IFN-{gamma} knockout mice (8). This demonstrated that the type 1 cytokine, IFN-{gamma}, derived from the host must play a role in type 2-mediated antitumor responses.

The commitment of T cells to either a type 1 or type 2 pathway is dependent on many factors, including the strength of the Ag (9), type of costimulation (10, 11), and the cytokine environment in which the T cells undergo activation and differentiation (12, 13). IL-4 drives the development of type 2 cells and inhibits type 1 cells. The mechanism by which IL-4 drives the development of a type 2 response is dependent on STAT6 (14, 15). Activation of STAT6 by IL-4 leads to dimerization and translocation to the nucleus, where it enhances the transcription of IL-4-inducible genes, including Gata3 and c-maf (16, 17, 18). The essential role of STAT6 in the polarization of type 2 T cells is supported by data that demonstrate that T cells from STAT6-/- mice fail to develop a type 2 phenotype in conditions that favor type 2 differentiation (19, 20, 21). Furthermore, STAT6-/- mice bred on the Leishmania-susceptible BALB/c background are resistant to Leishmania infection, which is consistent with differentiation of T cells toward a type 1 phenotype (22).

Various groups have used STAT6-/- mice in tumor models to evaluate immune deviation as a possible mechanism of tumor immune suppression. It was shown that STAT6-/- mice rejected a variant of the mastocytoma P815 at a dose that normally grows progressively in wild-type (wt)3 DBA/Z mice (23). Another group has shown that s.c. growth of the mammary adenocarcinoma, 4T1, was delayed in STAT6-/- mice as compared with wt BALB/c mice (24). This group also showed that the number of spontaneous lung metastases from the primary site of this highly metastatic tumor was reduced in STAT6-/- mice. These results supported the hypothesis that polarization toward a type 1 response led to an enhanced antitumor immune response. However, an alternative explanation for the rejection of 4T1 in STAT6-/- mice is that the T cell repertoire of STAT6-/- mice contains STAT6-reactive T cells that could recognize STAT6 peptide epitopes presented by 4T1. In this study, we report that 4T1 primes T cells in STAT6-/- mice that recognize 4T1 as well as a STAT6 peptide epitope. Using bone marrow (BM) from STAT6-/- mice to reconstitute irradiated wt BALB/c mice, we produced mice whose immune system was predisposed to a strong type 1 response, but that lacked STAT6-reactive T cells in their T cell repertoire. This model enabled us to determine whether the rejection of 4T1 by STAT6-/- mice was due to either a strong type 1 response or the combination of the strong type 1 response against a strong foreign Ag (STAT6).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female BALB/cJ and C.129S2-STAT6tm1Gru (19), STAT6-/- mice on a BALB/c background, were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free environment. Recognized principles of laboratory animal care were followed (Guide for the Care and Use of Laboratory Animals, National Research Council, 1996), and all animal protocols were approved by the Earle A. Chiles Research Institute Animal Care and Use Committee.

Tumor cell lines

The 4T1 is a 6-thioguanine-resistant cell line that was selected from a tumor cell line derived from a single spontaneously arising mammary tumor in a BALB/c3H mouse (provided by S. Ostrand-Rosenberg, University of Maryland Baltimore County, Baltimore, MD). The colon carcinoma cell line, CT26, was provided by G. Plautz (University of Michigan, Ann Arbor, MI); the renal cell carcinoma, Renca, was provided by B. Wiltrout (Frederick Cancer Research and Development Center, Frederick, MD); and the mammary adenocarcinoma cell line, EMT6, was provided by E. T. Akporiaye (University of Arizona, Tucson, AZ). Each of these tumors was derived from BALB/c mice. The C57BL/6 melanoma cell line, B16BL6-D5 (D5), was provided by S. Shu (Cleveland Clinic, Cleveland, OH). All cell lines were maintained in complete medium (CM) comprising the following: RPMI 1640 (BioWhittaker, Walkersville, MD) containing 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, and 50 µg/ml gentamicin sulfate. This was further supplemented with 50 µM 2-ME (Aldrich, Milwaukee, WI) and 10% (v/v) FBS (Life Technologies, Grand Island, NY).

Immunoprecipitation and Western blot of STAT6

Tumor cell lines or splenocytes were lysed in cell lysis buffer (250 mM NaCl, 25 mM Tris-HCl, 5 mM EDTA, 1% Nonidet P-40, freshly added protease inhibitors). STAT6 Ab (M-20; Santa Cruz Biotechnology, Santa Cruz, CA) and protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden) were added to the total cell lysate and incubated overnight at 4°C. Samples were run on SDS gel and Western blotted using STAT6 Ab. Western blots were developed using chemiluminescent Pierce SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL).

Peptide loading of STAT6-/- splenocytes

Potential H-2d-binding STAT6 peptide sequences were determined using a computer program (www-bimas.cit.nih.gov/molbio/hla_bind/) that ranks 9-mer peptides based on a predicted t1/2 of dissociation to H-2d class I molecules. The analysis was based on coefficient tables deduced from the published literature by K. C. Parker (25). Based on their predicted binding scores to H-2Kd, three different STAT6 peptides, STAT618–26 (LYVDFPQRL), STAT6129–137 (KFTTPLGRL), and STAT6531–539 (SYWSDRLII), were synthesized. The SV40 large T Ag peptide containing residues 499–507 (DYLDGSVKV), LTA499–507, was also synthesized. All peptides were synthesized and purchased from Research Genetics (Huntsville, AL).

Spleens from STAT6-/- mice were collected and mechanically disrupted to create single cell suspensions. RBCs were lysed using ACK lysing buffer (BioWhittaker), and splenocytes were resuspended in 2 ml of CM (106 cells/ml) with 20 ng/ml peptide and incubated at 37°C for 1 h. Peptide-pulsed splenocytes were washed in CM and used as stimulators in cytokine release assays.

Tumor or peptide vaccination and activation of vaccine-draining lymph node cells

Tumor cell cultures were trypsinized (Bio Whittaker) and washed twice in HBSS (BioWhittaker). A total of 7.5 x 105 4T1 tumor cells were injected s.c. into both axillary regions and hind flanks of BALB/c and STAT6-/- mice. Eight days following vaccination, the superficial inguinal lymph nodes and axillary lymph nodes draining the four vaccination sites were harvested, and single cell suspensions were resuspended at 2 x 106 cells/ml in CM and cultured in 24-well plates with 50 µl of a 1/40 dilution of 2c11 ascites (anti-CD3). After 2 days of activation, the cells were harvested and expanded in CM containing 60 IU/ml of human rIL-2 (Chiron, Emeryville, CA) at a starting cell density of 1.25 x 105 cells/ml in 300 ml gas-permeable tissue culture bags (Nexell Therapeutics, Irvine, CA). After 3 days, effector T cells were harvested and used in cytokine release assays, intracellular staining assays, or adoptive immunotherapy.

BALB/c and STAT6-/- mice were injected s.c. with 25 µg of either STAT6531–539 or LTA499–508 emulsified in CFA. The mice received a second and third s.c. injection of peptide in IFA in the opposite flank at 14-day intervals. Ten days following the last peptide injection, lymph nodes draining the injection sites were obtained and activated and expanded as described for tumor vaccine-draining lymph node (TVDLN) cells.

Adoptive immunotherapy

Experimental pulmonary metastases were established by i.v. inoculation of STAT6-/- mice with 2 x 105 4T1 tumor cells. Three days after metastases were established, effector T cells were adoptively transferred i.v. Starting on the day of T cell infusion, mice received 90,000 IU of IL-2 i.p. daily for 3 days. Animals were sacrificed 12 days following tumor inoculation, the lungs were resected and fixed in Fekete’s solution, and the number of pulmonary metastases were enumerated by visual inspection.

Cytokine release assay

After in vitro activation and expansion, effector T cells were washed and incubated alone or were stimulated with 4T1, CT26, Renca, D5, peptide-pulsed STAT6-/- splenocytes, or plate-bound anti-CD3. Effector T cells (2 x 106/well) were cultured with tumor cells (2 x 105/well) or peptide-pulsed STAT6-/- splenocytes (2 x 106/well) in 2 ml in 24-well plates. Supernatants were recovered 14 h after stimulation, and IFN-{gamma} or IL-4 concentration was measured in duplicate by ELISA using commercially available reagents (BD PharMingen, San Diego, CA). The concentration of cytokine in the supernatant was determined by regression analysis.

Intracellular cytokine staining

After in vitro activation and expansion, effector T cells (2 x 106) were washed and incubated alone or stimulated with 4T1 (2 x 105) or peptide-pulsed STAT6-/- splenocytes (2 x 106) in 2 ml of CM in 24-well plates. After 1 h, 1 µl GolgiPlug (BD PharMingen) was added to each well. After 16 h of in vitro stimulation, cells were harvested and stained with FITC-labeled anti-CD8 Ab (BD PharMingen), CyChrome-labeled anti-CD3 Ab (BD PharMingen), and isotype controls (BD PharMingen) for 30 min at 4°C. The cells were washed and permeabilized by incubation with Cytofix/Cytoperm solution (BD PharMingen), for 20 min at 4°C. After washing and resuspension in Perm/wash solution (BD PharMingen), intracellular cytokine staining with PE-labeled anti-IFN-{gamma} Ab (BD PharMingen) was performed for 20 min at 4°C. Cells were washed twice with Perm/wash solution, resuspended in FACS buffer, and analyzed on a BD Bioscience FACSCalibur (San Jose, CA). A total of 150,000 gated events based on CD3 expression were collected and analyzed for expression of CD8 and IFN-{gamma}.

BM reconstitution

BALB/c or STAT6-/- BM was collected by flushing both femurs and tibias with a 27-gauge needle containing CM. CD4+ and CD8+ T cells were depleted by incubating the BM cells with mouse anti-CD4 (L3T4) Microbeads (20 µl/107 cells) and mouse anti-CD8a (Ly-2) Microbeads (20 µl/107 cells) (Miltenyi Biotec, Auburn, CA) for 15 min at 4°C. Cells were washed in CM and passed over magnetic MACS column. The flow through (CD4- CD8- cells) was collected and washed in HBSS twice. A total of 107 CD4- CD8- BM cells were transferred i.v. to BALB/c or STAT6-/- mice that had been irradiated (700 cGy). Mice were allowed 6–8 wk to reconstitute their immune system with the BM graft before experiments were performed.

Tumor challenge

BALB/c, STAT6-/-, or BM-reconstituted mice were injected s.c. in the hind flank with 104 or 105 4T1 cells (TD100 = 104). Tumor growth was determined by multiplying the measured perpendicular diameters of the tumor. Mice were sacrificed when multiplied diameters equaled 150 mm2.

Statistical analyses

Data were analyzed using Student’s t test for unequal variances (Microsoft Excel, Redmond, WA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
STAT6-/- mice reject 4T1 tumor challenge

It has been reported that growth of the mammary adenocarcinoma, 4T1, is slowed in STAT6-/- mice as compared with wt BALB/c mice (24). This observation was confirmed by our lab; s.c. challenge with 104 or 105 4T1 cells led to progressive tumor growth in all wt BALB/c mice, but after initial tumor formation 4T1 was subsequently rejected by nearly all STAT6-/- mice (Fig. 1). Titration of the dose of 4T1 tumor cells showed that 80% of STAT6-/- mice were capable of rejecting a tumor dose that was 1 log higher than the tumor dose that resulted in tumor growth in 100% of wt BALB/c mice.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 1. STAT6-/- mice reject 4T1 tumor challenge. STAT6-/- mice (lower panels) or wt BALB/c mice (upper panels) were challenged s.c. in the left flank with either 104 (left panels) or 105 (right panels) 4T1 tumor cells. Tumor size was determined by multiplying the longest tumor diameter by the corresponding perpendicular diameter.

 
STAT6 expression in 4T1

STAT6 was reported to be expressed in mammary tissue (26). To determine whether STAT6 was expressed by 4T1, detergent-soluble cellular lysates were immunoprecipitated with anti-STAT6 Ab (Fig. 2A). Splenocytes from wt BALB/c mice, STAT4-/- mice, as well as 4T1 and Renca tumor cells expressed the 102-kDa protein, whereas STAT6 was undetectable in STAT6-/- splenocytes. The BALB/c colon and mammary carcinoma cell lines, CT26 and EMT6, as well as D5, a C57BL/6 melanoma, produced STAT6 protein (Fig. 2B).



View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 2. STAT6 was expressed in all tumor cell lines tested, including 4T1. Detergent-soluble cellular lysates from wt BALB/c, STAT4-/-, or STAT6-/- splenocytes (A) or tumor cells (A and B) were immunoprecipitated with anti-STAT6 Ab, subjected to SDS-PAGE, and immunoblotted with anti-STAT6 Ab.

 
The 4T1 vaccination primes STAT6531–539 peptide-specific T cells

The identification of STAT6 protein in 4T1 tumor cells raised the possibility that tumor rejection in STAT6-/- mice could be due to endogenous processing of STAT6 protein, resulting in the presentation of peptides that stimulate STAT6-specific T cells in STAT6-/- mice. It has been shown that the immune response responsible for the rejection of 4T1 in STAT6-/- mice was dependent on the presence of CD8+ cells (24). Therefore, we used a computer algorithm to determine theoretical binding affinities of STAT6 peptides for the following MHC class I alleles: H-2Kd, H-2Ld, and H-2Dd. A panel of peptides predicted to have high or medium binding affinities to H-2Kd was selected from the list of possible STAT6 peptides. To determine whether 4T1 could prime STAT6-specific T cells in STAT6-/- mice, both wt BALB/c and STAT6-/- mice were vaccinated with 4T1, and the TVDLN were removed 8 days later. TVDLN cells were stimulated in vitro with soluble anti-CD3 Ab for 48 h, followed by expansion in low dose IL-2 (60 IU) for 3 days. This method of polyclonal activation has been shown to support the acquisition of Ag-specific effector function by recently primed T cells (27, 28, 29). After polyclonal activation, these effector T cells were assayed for their ability to respond to tumor cells or peptide-pulsed STAT6-/- splenocytes. T cells generated from 4T1-vaccinated STAT6-/- mice secreted significantly (p < 0.05) more IFN-{gamma} than T cells from 4T1-vaccinated wt BALB/c mice when stimulated with either 4T1 or CT26 tumor cells (Fig. 3A). This cross-reactivity was also observed against RENCA and EMT6 (data not shown), suggesting that all of these tumors share an Ag, possibly a peptide from STAT6 because they all express STAT6. Induction of IFN-{gamma} was restricted to H-2d tumor cells because effector T cells generated from 4T1-vaccinated STAT6-/- mice did not respond to D5, a STAT6+, H-2b tumor. We determined whether T cells from 4T1-vaccinated STAT6-/- mice could respond to any of the panel of H-2Kd-binding STAT6 peptides and observed very strong responses to the STAT6 peptide comprising residues 531–539 (STAT6531–539). T cells from 4T1-vaccinated wt BALB/c mice were unable to recognize this peptide. Three other H-2Kd-binding peptides, STAT618–26, STAT6129–137, and LTA499–508 (30), were unable to stimulate IFN-{gamma} secretion from these T cells, documenting the specificity of this response. The frequency of CD8+ T cells that secreted IFN-{gamma} in response to 4T1 and STAT6531–539 was also much greater in 4T1-vaccinated STAT6-/- mice than in 4T1-vaccinated wt BALB/c mice (Fig. 3B). These data show that vaccination with 4T1 can prime STAT6531–539 peptide-specific T cells and that these T cells are present only in the T cell repertoire of STAT6-/- mice and not wt BALB/c mice.



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 3. Vaccination with 4T1 primes STAT6531–539-specific T cells in STAT6-/- mice. A, STAT6-/- mice (filled bars) or wt BALB/c mice (open bars) were vaccinated with 4T1, and 8 days later TVDLN were collected. TVDLN were stimulated with soluble anti-CD3 for 2 days and expanded in IL-2 (60 IU) for 3 days to generate effector T cells. Effector T cells were stimulated for 14 h with tumor targets or peptide-pulsed (20 ng/ml) STAT6-/- splenocytes; supernatants were collected; and IFN-{gamma} concentration was measured in duplicate by ELISA. Data presented are the mean of three independent experiments (±SE). *, p < 0.05. B, Effector T cells were stimulated with 4T1 or peptide-pulsed (20 ng/ml) STAT6-/- splenocytes for 8 h. Cells were collected and stained with anti-CD3 CyChrome mAb and anti-CD8 FITC mAb, and intracellular staining was performed with anti-IFN-{gamma} mAb. Panels show CD8 FITC (x-axis) and IFN-{gamma} (y-axis) on CD3+ cells (gated). Number in upper right of each panel represents the percentage of CD8+ cells expressing IFN-{gamma}.

 
STAT6531–539 peptide vaccination primes STAT6-/- T cells that recognize 4T1

To demonstrate that STAT6531–539 was an epitope presented by 4T1, we determined whether STAT6531–539-specific T cells from wt BALB/c or STAT6-/- mice could recognize 4T1 following vaccination with STAT6531–539. Lymph node cells draining the peptide vaccination site were activated with soluble anti-CD3 and expanded in low dose IL-2, as described for TVDLN. Expanded effector T cells were assayed for tumor-specific IFN-{gamma} secretion. STAT6531–539 vaccination primed T cells in STAT6-/- mice that responded to STAT6531–539-pulsed STAT6-/- splenocytes (Fig. 4). STAT6531–539 vaccination of wt BALB/c mice also primed T cells that responded to STAT6531–539, but these T cells secreted significantly lower levels of IFN-{gamma} than T cells from STAT6-/- mice. Only T cells from STAT6-/- mice vaccinated with STAT6531–539 responded to 4T1 (p < 0.005). These T cells also responded to the STAT6+, H-2d renal cell tumor, Renca. T cells from STAT6-/- mice vaccinated with LTA499–508 secreted background levels of IFN-{gamma} in response to 4T1, demonstrating that the ability to respond to 4T1 was dependent on vaccination with STAT6531–539. Although wt BALB/c mice vaccinated with STAT6531–539 mounted a low level response to STAT6531–539-pulsed STAT6-/- splenocytes, they did not respond to 4T1 any better than T cells from LTA499–508-vaccinated wt BALB/c mice. These data show that 4T1 presents an epitope that can be recognized by STAT6531–539 peptide-specific T cells generated in STAT6-/- mice.



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 4. STAT6531–539 vaccination of STAT6-/- mice primes 4T1-reactive T cells. The wt BALB/c and STAT6-/- mice were injected s.c. with 25 µg of either STAT6531–539 or LTA499–508 emulsified in CFA. Mice received a second and third s.c. injection of peptide in IFA in the opposite flank at 14-day intervals. Ten days following the last peptide injection, lymph nodes draining the injection sites were collected, and lymphocytes were polyclonally stimulated with soluble anti-CD3 for 2 days and expanded in IL-2 (60 IU) for 3 days to generate effector T cells. Effector T cells were stimulated for 14 h with the indicated tumor targets or peptide-pulsed (20 ng/ml) STAT6-/- splenocytes; supernatants were collected; and IFN-{gamma} concentration was measured in duplicate by ELISA. Data presented are the mean of two independent experiments (±SE). *, p < 0.005.

 
To determine their therapeutic potential, effector T cells from peptide-vaccinated mice were adoptively transferred into STAT6-/- recipient mice that had 3-day 4T1 pulmonary metastases. STAT6-/- recipient mice that received effector T cells from STAT6531–539-vaccinated STAT6-/- mice had a significant reduction (p < 0.002) in the number of 4T1 metastases compared with mice that received effector T cells from STAT6531–539-vaccinated wt BALB/c mice (Table I). These data demonstrate that STAT6531–539 peptide-specific T cells generated in STAT6-/- mice not only respond to 4T1 in vitro, but are also sufficient to cause the regression of 4T1 in vivo.


View this table:
[in this window]
[in a new window]
  		Table I. STAT6531–539 vaccination primed T cells that were therapeutic against experimental 4T1 pulmonary metastases

 
Peptide-specific T cells from BM chimeras fail to reject 4T1 tumor challenge

Because STAT6531–539 peptide-specific T cells primed in STAT6-/- mice responded to 4T1 in vitro and eliminated experimental pulmonary metastases in vivo, it raised the question whether the rejection of s.c. 4T1 challenge in STAT6-/- mice was due to the type 1-polarized immune response of STAT6-/- mice or the presence of STAT6531–539-reactive T cells. To investigate this question, STAT6-/- BM chimeras were generated by injecting STAT6-/- BM into irradiated wt BALB/c hosts (STAT6-/-BM->wt). These mice would still generate predominantly a polarized type 1 response, but could no longer develop STAT6-reactive T cells because they were either deleted within the wt thymus or peripherally tolerized to avoid autoimmunity. The wt BALB/c BM into wt BALB/c chimeras (wt BM->wt) and STAT6-/- BM into STAT6-/- chimeras (STAT6-/-BM->STAT6-/-) were also produced as controls for the reconstitution. To verify that the BM compartment was reconstituted with donor BM, splenocytes were stimulated with anti-CD3, and the supernatants were assayed for IL-4 production by ELISA. Previous work has demonstrated that STAT6-/- lymphocytes secrete significantly less IL-4 than wt lymphocytes when stimulated with anti-CD3 (19, 31). Both STAT6-/-BM->STAT6-/- mice and STAT6-/-BM->wt mice made significantly less IL-4 than wt BM->wt mice, demonstrating that the BM grafts were successful (Fig. 5A). Subcutaneous tumor challenges with as few as 104 4T1 cells caused progressively growing tumors in 10 of 10 wt BALB/c mice as well as 10 of 10 of wt BM->wt mice (Fig. 5B). As expected, 8 of 9 STAT6-/- mice rejected the 4T1 tumor challenge at 104 tumor cells. The STAT6-/-BM->STAT6-/- mice behaved similar to STAT6-/- mice, and 8 of 10 were able to reject 4T1 tumor challenge. In contrast, STAT6-/-BM->wt mice were unable to reject a 4T1 tumor challenge; all 10 mice developed progressively growing tumors.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 5. Irradiated wt BALB/c mice reconstituted with STAT6-/- BM did not reject 4T1 tumor challenge. A, Splenocytes from the indicated BM chimeras were stimulated for 24 h with plate-bound anti-CD3 mAb. Supernatants were collected, and IL-4 concentration was measured in duplicate by ELISA. B, BM chimeras were challenged s.c. in the left flank with 104 4T1 tumor cells. Tumor size was determined by multiplying the longest tumor diameter by the corresponding perpendicular diameter. Numbers in the upper right of each panel represent the number of mice without measurable tumor at 50 days.

 
Considering our data and previous reports that the regression of 4T1 in STAT6-/- mice is dependent on CD8+ cells (24), it appeared that it was the loss of STAT6-reactive T cells in STAT6-/-BM->wt mice that explained the growth of 4T1 in these mice. To address whether T cells from STAT6-/-BM->wt mice responded to 4T1, we collected tumor-draining lymph node cells from the reconstituted mice, and activated and expanded the cells to generate effector T cells. Effector T cells from wt BALB/c mice and wt BM->wt mice did not respond to stimulation by 4T1 (Fig. 6). Effector T cells from STAT6-/- mice as well as effector T cells from STAT6-/-BM->STAT6-/- mice responded to 4T1 by secreting significantly higher amounts of IFN-{gamma} than the other groups (p < 0.03). Effector T cells from STAT6-/-BM->STAT6-/- mice responded less to the 4T1 stimulation than effector T cells from STAT6-/- mice; however, the difference was not statistically significant. In contrast, effector T cells from STAT6-/-BM->wt mice did not respond to stimulation by 4T1 and exhibited levels of IFN-{gamma} secretion similar to wt BALB/c mice and wt BM->wt mice. This demonstrated that the 4T1-specific T cells that are present in STAT6-/-BM->STAT6-/- mice have been either centrally deleted from the T cell repertoire, or are nonresponsive in the periphery of the wt mouse. As expected, effector T cells from STAT6-/-BM->wt mice also failed to respond to the STAT6531–539 peptide, unlike effector T cells from STAT6-/-BM->STAT6-/- mice. These data demonstrate that the loss of STAT6531–539-reactive T cells correlated with the loss of reactivity to 4T1, the consequence of which is the progressive growth of 4T1 in vivo. This is consistent with the conclusion that STAT6-/- mice reject 4T1 because T cells recognize STAT6 as a foreign Ag rather than because of the preference toward developing a type 1 response.



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 6. T cells from wt BALB/c mice reconstituted with STAT6-/- BM did not respond to 4T1 or STAT6531–539-pulsed splenocytes. The 4T1 TVDLN cells from wt BALB/c, STAT6-/-, or BM chimera mice were collected 8 days after vaccination. TVDLN cells were stimulated with soluble anti-CD3 for 2 days and expanded in IL-2 (60 IU) for 3 days to generate effector T cells. Effector T cells were stimulated for 14 h with tumor targets or peptide-pulsed (20 ng/ml) STAT6-/- splenocytes. Supernatants were collected, and IFN-{gamma} concentration was measured in duplicate by ELISA. Data are presented as the mean of three independent experiments (±SE). *, p < 0.03 (STAT6-/- or STAT6-/- BM->STAT6-/-) compared with wt, wt BM->wt, or STAT6-/- BM->wt against 4T1, CT26, or STAT6531–539 targets.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
STAT6-/- mice may be an important model system to examine the effect of a polarized type 1 immune response on tumor growth. Previous investigators using STAT6-/- mice have concluded that the deletion of the STAT6 gene, which facilitated the development of potent antitumor immunity, did so via enhancement of type 1 immune responses (23, 24). We observed the same findings; however, when T cells from 4T1-vaccinated STAT6-/- mice were adoptively transfered into recipient wt BALB/c mice, we noticed signs reminiscent of graft-vs-host disease (data not shown). Necropsy of these mice showed inflammation of the small and large intestines, suggesting that the transferred cells were responding to a tissue Ag located in the gastrointestinal tract of wt BALB/c mice. These observations led us to test the hypothesis that STAT6 was not only the Ag recognized by T cells mediating the graft-vs-host response, but more importantly, the Ag responsible for the regression of 4T1 observed in STAT6-/- mice. The work presented in this study documents the presence of T cells that recognize the STAT6531–539 peptide in 4T1-vaccinated STAT6-/- mice. When the response against other H-2Kd-binding STAT6 peptides was examined, no reactivity was observed. In BM chimeras (wt mice reconstituted with STAT6-/- BM), the absence of STAT6531–539-reactive T cells resulted in mice that were unable to reject the 4T1 challenge, even though the reconstituting STAT6-/- T cells should be predisposed toward a type 1 phenotype. From these experiments, it is clear that the rejection of 4T1 tumor cells by STAT6-/- mice is dependent on STAT6531–539-reactive T cells. However, it is not clear whether prevention of tumor growth in STAT6-/- mice is solely due to the presence of STAT6531–539-reactive T cells, or whether it is the combination of STAT6531–539-reactive T cells and the strong polarization toward a type 1 phenotype that is necessary for tumor rejection. Previous data have shown that the growth of naturally progressing mouse mammary tumor virus-induced tumors is delayed in STAT6-/- mice compared with wt mice (32). Because these tumors arose endogenously within the mice, the complicating issue of STAT6 priming STAT6-reactive T cells in Stat6-/- mice is avoided. Nonetheless, tumors in both wt and STAT6-/- mice should express foreign mouse mammary tumor virus Ags, suggesting that the type 1-polarized response of STAT6-/- mice is beneficial compared with wt mice when both immune systems are primed with a strong foreign Ag.

The 4T1 appears to prime the wt immune system insufficiently, as evidenced by the low levels of IFN-{gamma} secreted by TVDLN cells from 4T1-vaccinated wt mice when stimulated with 4T1. Because the question of immune deviation is relevant only after T cells have been primed, the significance of immune deviation to regression of 4T1 is questionable. However, previous studies suggest that 4T1 is immunogenic because the adoptive transfer of effector T cells generated from 4T1-vaccinated wt mice reduced the number of experimental pulmonary metastases in recipient mice (data not shown). These data suggest that either the frequency of wt T cells primed by 4T1 is not sufficient to cause regression of s.c. 4T1 challenge or the wt T cells do not differentiate in vivo into effector T cells in the 4T1-challenged animal. Therefore, additional mechanisms apart from immune deviation were responsible for the inability of wt BALB/c mice reconstituted with STAT6-/- BM to reject 4T1.

It is also possible that 4T1 might actively suppress the immune response through immunosuppressive factors. Other groups have observed that 4T1 secretes significant amounts of TGF-{beta}, and that blocking TGF-{beta} secretion by 4T1 enhanced the immune response primed by 4T1 (33, 34). Increased IFN-{gamma} production by tumor-draining lymph node cells was observed when mice were vaccinated with 4T1 transduced with an antisense TGF-{beta} transgene (33). The role of TGF-{beta} as an antiproliferative factor for T cells appears to work primarily through inhibition of IL-2 production (35) and inhibition of intracellular activators of T cell differentiation pathways (36, 37, 38). If TGF-{beta} secreted by 4T1 was responsible for the inhibition of proliferation of 4T1-specific T cells in wt BALB/c mice, our data suggest it was unable to exert the same inhibition in STAT6-/- mice. High avidity interactions between STAT6531–539-specific T cells and STAT6531–539:H-2Kd complexes could induce strong proliferative responses characterized by IL-2 secretion and expression of high affinity IL-2R (39, 40) that override the inhibition exerted by TGF-{beta}. Further studies using STAT6-/-BM->wt mice that include strategies that block TGF-{beta} will attempt to examine the role of TGF-{beta} in the initial 4T1-primed proliferation of T cells. More importantly, if the initial priming to tumor Ags can be augmented either by blocking immunosuppressive tumor factors or through enhancing the priming event, then the benefits of a polarized type 1 immune response can be investigated in greater detail.

Note added in proof:

Recent work published after this article was submitted has provided additional clarity to the 4T1 tumor model in STAT6-/- mice. The important role of the type 1 cytokine, IFN-{gamma}, was demonstrated by the inability of STAT6-/-IFN-{gamma}-/- mice to survive long term against a 4T1 tumor challenge (41). This was in contrast to STAT6-/- mice, where >80% of the mice survived for longer than 150 days. Taken together, these data indicate that 4T1 regression in STAT6-/- mice is dependent on both STAT6531-539-reactive T cells and their strong polarization toward a type 1 phenotype.


   Footnotes
 
1 This work was supported by grants from Department of Defense, DAMD17-99-1-9179, and the Chiles Foundation. Back

2 Address correspondence and reprint requests to Dr. Bernard A. Fox, 4805 NE Glisan Street, Portland, OR 97213. E-mail address: foxb{at}foxlab.org Back

3 Abbreviations used in this paper: wt, wild type; BM, bone marrow; CM, complete medium; LTA, SV40 large T Ag peptide; TVDLN, tumor vaccine-draining lymph node. Back

Received for publication October 10, 2002. Accepted for publication December 9, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Burnet, F. M.. 1970. The concept of immunological surveillance. Prog. Exp. Tumor Res. 13:1.[Medline]
  2. Gilboa, E.. 1999. How tumors escape immune destruction and what we can do about it. Cancer Immunol. Immunother. 48:382.[Medline]
  3. Abken, H., A. Hombach, C. Heuser, K. Kronfeld, B. Seliger. 2002. Tuning tumor-specific T-cell activation: a matter of costimulation?. Trends Immunol. 23:240.[Medline]
  4. Antonia, S. J., M. Extermann, R. A. Flavell. 1998. Immunologic nonresponsiveness to tumors. Crit. Rev. Oncog. 9:35.[Medline]
  5. Rocken, M., E. M. Shevach. 1996. Immune deviation: the third dimension of nondeletional T cell tolerance. Immunol. Rev. 149:175.[Medline]
  6. Hu, H. M., W. J. Urba, B. A. Fox. 1998. Gene-modified tumor vaccine with therapeutic potential shifts tumor-specific T cell response from a type 2 to a type 1 cytokine profile. J. Immunol. 161:3033.[Abstract/Free Full Text]
  7. Dobrzanski, M. J., J. B. Reome, R. W. Dutton. 1999. Therapeutic effects of tumor-reactive type 1 and type 2 CD8+ T cell subpopulations in established pulmonary metastases. J. Immunol. 162:6671.[Abstract/Free Full Text]
  8. Dobrzanski, M. J., J. B. Reome, R. W. Dutton. 2001. Role of effector cell-derived IL-4, IL-5, and perforin in early and late stages of type 2 CD8 effector cell-mediated tumor rejection. J. Immunol. 167:424.[Abstract/Free Full Text]
  9. Rogers, P. R., M. Croft. 2000. CD28, Ox-40, LFA-1, and CD4 modulation of Th1/Th2 differentiation is directly dependent on the dose of antigen. J. Immunol. 164:2955.[Abstract/Free Full Text]
  10. Rulifson, I. C., A. I. Sperling, P. E. Fields, F. W. Fitch, J. A. Bluestone. 1997. CD28 costimulation promotes the production of Th2 cytokines. J. Immunol. 158:658.[Abstract]
  11. Salomon, B., J. A. Bluestone. 1998. LFA-1 interaction with ICAM-1 and ICAM-2 regulates Th2 cytokine production. J. Immunol. 161:5138.[Abstract/Free Full Text]
  12. Nakamura, T., R. K. Lee, S. Y. Nam, E. R. Podack, K. Bottomly, R. A. Flavell. 1997. Roles of IL-4 and IFN-{gamma} in stabilizing the T helper cell type 1 and 2 phenotype. J. Immunol. 158:2648.[Abstract]
  13. Allen, J. E., R. M. Maizels. 1997. Th1-Th2: reliable paradigm or dangerous dogma?. Immunol. Today 18:387.[Medline]
  14. Hou, J., U. Schindler, W. J. Henzel, T. C. Ho, M. Brasseur, S. L. McKnight. 1994. An interleukin-4-induced transcription factor: IL-4 Stat. Science 265:1701.[Abstract/Free Full Text]
  15. Quelle, F. W., K. Shimoda, W. Thierfelder, C. Fischer, A. Kim, S. M. Ruben, J. L. Cleveland, J. H. Pierce, A. D. Keegan, K. Nelms, et al 1995. Cloning of murine Stat6 and human Stat6, Stat proteins that are tyrosine phosphorylated in responses to IL-4 and IL-3 but are not required for mitogenesis. Mol. Cell. Biol. 15:3336.[Abstract]
  16. Kurata, H., H. J. Lee, A. O’Garra, N. Arai. 1999. Ectopic expression of activated Stat6 induces the expression of Th2-specific cytokines and transcription factors in developing Th1 cells. Immunity 11:677.[Medline]
  17. Mikita, T., M. Kurama, U. Schindler. 1998. Synergistic activation of the germline {epsilon} promoter mediated by Stat6 and C/EBP {beta}. J. Immunol. 161:1822.[Abstract/Free Full Text]
  18. Mikita, T., C. Daniel, P. Wu, U. Schindler. 1998. Mutational analysis of the STAT6 SH2 domain. J. Biol. Chem. 273:17634.[Abstract/Free Full Text]
  19. Kaplan, M. H., U. Schindler, S. T. Smiley, M. J. Grusby. 1996. Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. Immunity 4:313.[Medline]
  20. Takeda, K., T. Tanaka, W. Shi, M. Matsumoto, M. Minami, S. Kashiwamura, K. Nakanishi, N. Yoshida, T. Kishimoto, S. Akira. 1996. Essential role of Stat6 in IL-4 signalling. Nature 380:627.[Medline]
  21. Shimoda, K., J. van Deursen, M. Y. Sangster, S. R. Sarawar, R. T. Carson, R. A. Tripp, C. Chu, F. W. Quelle, T. Nosaka, D. A. Vignali, et al 1996. Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene. Nature 380:630.[Medline]
  22. Stamm, L. M., A. Raisanen-Sokolowski, M. Okano, M. E. Russell, J. R. David, A. R. Satoskar. 1998. Mice with STAT6-targeted gene disruption develop a Th1 response and control cutaneous leishmaniasis. J. Immunol. 161:6180.[Abstract/Free Full Text]
  23. Kacha, A. K., F. Fallarino, M. A. Markiewicz, T. F. Gajewski. 2000. Cutting edge: spontaneous rejection of poorly immunogenic P1.HTR tumors by Stat6-deficient mice. J. Immunol. 165:6024.[Abstract/Free Full Text]
  24. Ostrand-Rosenberg, S., M. J. Grusby, V. K. Clements. 2000. Cutting edge: STAT6-deficient mice have enhanced tumor immunity to primary and metastatic mammary carcinoma. J. Immunol. 165:6015.[Abstract/Free Full Text]
  25. Parker, K. C., M. A. Bednarek, J. E. Coligan. 1994. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J. Immunol. 152:163.[Abstract]
  26. Watson, C. J.. 2001. Stat transcription factors in mammary gland development and tumorigenesis. J. Mammary Gland Biol. Neoplasia 6:115.[Medline]
  27. Yoshizawa, H., A. E. Chang, S. Y. Shu. 1992. Cellular interactions in effector cell generation and tumor regression mediated by anti-CD3/interleukin 2-activated tumor-draining lymph node cells. Cancer Res. 52:1129.[Abstract/Free Full Text]
  28. Yoshizawa, H., A. E. Chang, S. Shu. 1991. Specific adoptive immunotherapy mediated by tumor-draining lymph node cells sequentially activated with anti-CD3 and IL-2. J. Immunol. 147:729.[Abstract]
  29. Yoshizawa, H., K. Sakai, A. E. Chang, S. Y. Shu. 1991. Activation by anti-CD3 of tumor-draining lymph node cells for specific adoptive immunotherapy. Cell. Immunol. 134:473.[Medline]
  30. Newmaster, R. S., L. M. Mylin, T. M. Fu, S. S. Tevethia. 1998. Role of a subdominant H-2Kd-restricted SV40 tumor antigen cytotoxic T lymphocyte epitope in tumor rejection. Virology 244:427.[Medline]
  31. Zhang, S., N. W. Lukacs, V. A. Lawless, S. L. Kunkel, M. H. Kaplan. 2000. Cutting edge: differential expression of chemokines in Th1 and Th2 cells is dependent on Stat6 but not Stat4. J. Immunol. 165:10.[Abstract/Free Full Text]
  32. Czarneski, J., J. Meyers, T. Peng, V. Abraham, R. Mick, S. R. Ross. 2001. Interleukin-4 up-regulates mouse mammary tumor virus expression yet is not required for in vivo virus spread. J. Virol. 75:11886.[Abstract/Free Full Text]
  33. Wu, R. S., J. J. Kobie, D. G. Besselsen, T. C. Fong, V. D. Mack, J. A. McEarchern, E. T. Akporiaye. 2001. Comparative analysis of IFN-{gamma} B7.1 and antisense TGF-{beta} gene transfer on the tumorigenicity of a poorly immunogenic metastatic mammary carcinoma. Cancer Immunol. Immunother. 50:229.[Medline]
  34. Muraoka, R. S., N. Dumont, C. A. Ritter, T. C. Dugger, D. M. Brantley, J. Chen, E. Easterly, L. R. Roebuck, S. Ryan, P. J. Gotwals, et al 2002. Blockade of TGF-{beta} inhibits mammary tumor cell viability, migration, and metastases. J. Clin. Invest. 109:1551.[Medline]
  35. Gorelik, L., R. A. Flavell. 2002. Transforming growth factor-{beta} in T-cell biology. Nat. Rev. Immunol. 2:46.[Medline]
  36. Heath, V. L., E. E. Murphy, C. Crain, M. G. Tomlinson, A. O’Garra. 2000. TGF-{beta}1 down-regulates Th2 development and results in decreased IL-4-induced STAT6 activation and GATA-3 expression. Eur. J. Immunol. 30:2639.[Medline]
  37. Gorelik, L., P. E. Fields, R. A. Flavell. 2000. Cutting edge: TGF-{beta} inhibits Th type 2 development through inhibition of GATA-3 expression. J. Immunol. 165:4773.[Abstract/Free Full Text]
  38. Gorelik, L., S. Constant, R. A. Flavell. 2002. Mechanism of transforming growth factor {beta}-induced inhibition of T helper type 1 differentiation. J. Exp. Med. 195:1499.[Abstract/Free Full Text]
  39. Heath, W. R., L. Kjer-Nielsen, M. W. Hoffmann. 1993. Avidity for antigen can influence the helper dependence of CD8+ T lymphocytes. J. Immunol. 151:5993.[Abstract]
  40. Girgis, L., M. M. Davis, B. Fazakas de St Groth. 1999. The avidity spectrum of T cell receptor interactions accounts for T cell anergy in a double transgenic model. J. Exp. Med. 189:265.[Abstract/Free Full Text]
  41. Ostrand-Rosenberg, S., V. K. Clements, M. Terabe, J. M. Park, J. A. Berzofsky, S. K. Dissanayake. 2002. Resistance to metastatic disease in STAT6-deficient mice requires hemopoietic and nonhemopoietic cells and is IFN-{gamma} dependent. J. Immunol. 169:5769.

Related articles in The JI:

IN THIS ISSUE

The JI 2003 170: 1609-1610. [Full Text]  



This article has been cited by other articles:


Home page
 DevelopmentHome page
W. T. Khaled, E. K. C. Read, S. E. Nicholson, F. O. Baxter, A. J. Brennan, P. J. Came, N. Sprigg, A. N. J. McKenzie, and C. J. Watson
The IL-4/IL-13/Stat6 signalling pathway promotes luminal mammary epithelial cell development
Development, August 1, 2007; 134(15): 2739 - 2750.
[Abstract] [Full Text] [PDF]

Home page
 Ann. Surg. Oncol.Home page
J. A. Norton, M. Li, N. C. Lee, and K. Tsung
Inhibition of Host Signal Transducer and Activator of Transcription Factor 6 Results in Cure With Cyclophosphamide and Interleukin 12 Immunotherapy
Ann. Surg. Oncol., January 1, 2006; 13(1): 118 - 124.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. J. McKallip, M. Nagarkatti, and P. S. Nagarkatti
{Delta}-9-Tetrahydrocannabinol Enhances Breast Cancer Growth and Metastasis by Suppression of the Antitumor Immune Response
J. Immunol., March 15, 2005; 174(6): 3281 - 3289.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. Sinha, V. K. Clements, and S. Ostrand-Rosenberg
Reduction of Myeloid-Derived Suppressor Cells and Induction of M1 Macrophages Facilitate the Rejection of Established Metastatic Disease
J. Immunol., January 15, 2005; 174(2): 636 - 645.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. J. Sussman, R. Parihar, K. Winstead, and F. D. Finkelman
Prolonged Culture of Vaccine-Primed Lymphocytes Results in Decreased Antitumor Killing and Change in Cytokine Secretion
Cancer Res., December 15, 2004; 64(24): 9124 - 9130.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Guiducci, E. Di Carlo, M. Parenza, M. Hitt, M. Giovarelli, P. Musiani, and M. P. Colombo
Intralesional Injection of Adenovirus Encoding CC Chemokine Ligand 16 Inhibits Mammary Tumor Growth and Prevents Metastatic-Induced Death after Surgical Removal of the Treated Primary Tumor
J. Immunol., April 1, 2004; 172(7): 4026 - 4036.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jensen, S. M.
Right arrow Articles by Fox, B. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jensen, S. M.
Right arrow Articles by Fox, B. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS