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The Journal of Immunology, 2006, 177: 3082-3088.
Copyright © 2006 by The American Association of Immunologists, Inc.

Membrane-Associated TGF-beta1 Inhibits Human Memory T Cell Signaling in Malignant and Nonmalignant Inflammatory Microenvironments1

Lori Broderick and Richard B. Bankert2

Department of Microbiology and Immunology, State University of New York, Buffalo, NY 14214


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TGF-beta1 is present on cells derived from the microenvironment of human lung tumors and nonmalignant inflammatory tissues. We establish that this cell-associated cytokine mediates hyporesponsiveness of the memory T cells in these microenvironments in situ by blocking TCR signaling. T cells derived from these tissues failed to translocate NF-{kappa}B to the nucleus in response to CD3 + CD28 cross-linking. This nonresponsiveness was reversed by an anti-TGF-beta1-neutralizing Ab. Refractoriness of the memory T cells to TCR activation was also reversed by the removal of TGF-beta1 by briefly pulsing the cells in a low pH buffer. Addition of exogenous TGF-beta1 to eluted T cells re-established their nonresponsive state. Neither TGF-beta1, anti-TGF-beta1 Ab, nor low pH affected TCR signaling potential of peripheral blood T cells. We conclude that TGF-beta1 mediates a physiologically relevant regulatory mechanism, selective for memory T cells present in the tumor microenvironment and nonmalignant chronic inflammatory tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Transforming growth factor-beta1 plays a critical role in maintaining T cell homeostasis. Both in vitro and in vivo studies have demonstrated a role for TGF-beta1 in the activation of T cells, development of effector functions, and prevention of overwhelming inflammation. For example, disruption of the TGF-beta signaling pathway in T cells results in T cell-mediated autoimmune disease (1, 2), and complete absence of TGF-beta1, as shown in knockout mice, leads to the development of multifocal organ infiltration of inflammatory cells and premature death of the animals (3, 4, 5). In humans, TGF-beta1 has been shown in vitro to inhibit IL-2-induced proliferation of T cells (6, 7). Because TGF-beta1 has been shown to be produced by many different human tumors, it has been suggested that the immunosuppressive effects of this cytokine may contribute to the failure of tumor-associated T cells to control tumor progression and to explain how tumors escape immune recognition and eradication despite vaccination-induced tumor-specific T cell responses (8, 9, 10). A recent report established that the in vitro pulsing of human tumor-specific memory T cells with exogenous TGF-beta1 prevented the acquisition of effector function and blocked the function of fully activated memory T cells (11). Collectively, these results suggest that TGF-beta1 plays a critical role in regulating T cell functions in the tumor microenvironment. However, there are no experimental data yet that establish a suppressive mechanism or a physiologically relevant role for TGF-beta1 in regulating T cell function in the microenvironment of human tumors, i.e., demonstrating that endogenous TGF-beta1 in the tumor microenvironment is responsible for the hyporesponsiveness of tumor-associated T cells to stimulation via their TCR.

A majority of the T cells isolated from human non-small cell lung tumors have a phenotype of effector memory cells (12). These tumor-associated memory T cells, as well as memory T cells derived from the chronic inflammatory microenvironments of rheumatoid arthritic joints and nasal polyps of hyperplastic rhinosinusitis, were found to be refractory to activation induced by the Ab cross-linking of CD3 and CD28 as monitored at the single-cell level by the nuclear translocation of NF-{kappa}B (13). In contrast, T cells (with an activated or memory phenotype) derived from the peripheral blood of cancer patients or patients with chronic inflammatory conditions were found to translocate NF-{kappa}B in response to TCR triggering, suggesting that this T cell quiescence was unique to memory T cells in the tumor and inflammatory tissue microenvironments. However, the refractoriness of the memory T cells to TCR stimulation appears to be a nonpermanent state that can be reversed by IL-12 (13). Furthermore, using a human lung tumor xenograft model in SCID mice, it was demonstrated in vivo that local and sustained release of IL-12 into the xenograft reactivated the human effector memory T cells to proliferate, secrete IFN-{gamma}, and eradicate tumor cells (12, 14). We concluded from these studies that the nonresponsiveness of the memory T cells was reversible and most likely dependent upon normal regulatory signals present within and common to the tumor and inflammatory tissue microenvironments.

In the present study, we demonstrate that a membrane-associated TGF-beta1 is responsible for the blockade in the TCR signaling pathway of memory T cells that exist in an anergic state within the microenvironments of human non-small cell lung tumors and nonmalignant inflammatory tissues. These results suggest that TGF-beta1, in an autocrine or paracrine fashion, mediates a normal, regulatory mechanism that is specific for memory T cells persisting in tumors and at sites of inflammation, and that it has the potential to prevent their uncontrolled activation in the face of continuous Ag stimulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Patient samples

Primary non-small cell lung cancer tissue was obtained from the Tissue Procurement Facility of Roswell Park Cancer Institute, Veterans Administration Medical Center Pathology Laboratory, and Kenmore Mercy Hospital Pathology Laboratory. Tissue was transported in DMEM/F12 to preserve until time of cell isolation. A histological diagnosis for each tumor specimen received was obtained anonymously. In some cases, patient peripheral blood and draining lymph nodes were also obtained with the tumor specimen. Nasal polyps were obtained from DeGraff Memorial Hospital and transported in DMEM/F12. In some cases, patient peripheral blood was also obtained with the polyp sample. Synovial fluid was obtained from patients with rheumatoid arthritis by arthrocentesis (Erie County Medical Center), and, in some cases, a sample of patient peripheral blood was also received. All specimens were obtained under sterile conditions and through institutional review board-approved protocols.

Cell isolation

Single-cell suspensions were derived from primary tumor tissue and draining lymph nodes by mechanical disruption of the tissue using a Teflon policeman to gently force cells through size 50 mesh. Cell suspensions were applied to a Ficoll-PaquePLUS gradient (Amersham Biosciences), and the resulting interface was washed once in RPMI 1640 supplemented with 10% FCS. Leukocytes were isolated from whole blood by diluting 1/1 in medium and applying to a Ficoll-PaquePLUS gradient. As with the tissue suspensions, the interface was washed once in medium. To promote adhesion of monocytes, cells were incubated overnight before plate activation.

Treatment of cells with low pH buffer

Single-cell suspensions of normal donor PBL or patient tissue samples were applied to a Ficoll gradient to obtain the interface cells, which were frozen for long-term storage in liquid nitrogen. The cells were thawed from storage and washed in complete medium. One-half of the cells were treated for 30 or 60 s with NaCl/citrate (pH 4.0), and then washed and returned to complete medium for overnight incubation before plate activation.

Cytokine and Ab treatment of isolated cells

Leukocytes were incubated at 37°C/5% CO2 with 10 ng/ml human rTGF-beta1 (rhTGF-beta1; R&D Systems) or 1000 U/ml rhIFN-{alpha} (PBL Biomedical Laboratories) overnight, then washed once in complete medium and prepared for plate activation via TCR cross-linking. A neutralizing murine mAb to human TGF-beta1 was purchased from R&D Systems and incubated with leukocytes overnight at 1 µg/ml. Isotype control experiments were completed concurrently at the same concentrations.

TCR activation of isolated cells

Abs were immobilized on 24-well plates by incubating 20 µg/ml anti-CD3 (OKT3) and 10 µg/ml purified anti-CD28 (Caltag Laboratories) in PBS, at 4°C overnight. Leukocytes were incubated in coated plates at 37°C/5% CO2 for 1 h (unless otherwise indicated), then washed once in PBS to terminate activation.

Preparation of Alcian blue coverslips

Alcian blue 8 GX dye was prepared as a 1% solution in distilled water and filtered through a Whatman 1 filter overnight. Filtered solution was stored at 4°C. To coat coverslips, ~30 glass coverslips (22 x 22 mm) were washed in 95% ethanol in a 400-ml beaker for 1–2 min with gentle swirling three times. Coverslips were rinsed of all ethanol with deionized distilled water. Coverslips were heated in the Alcian blue solution for 10 min at low heat with occasional vigorous swirling. Following heating period, coverslips were washed with deionized distilled water to remove excess Alcian blue. Coverslips were allowed to dry overnight in ceramic racks and stored in a covered container to protect from dust (15).

Immunofluorescence staining for confocal microscopy

Following activation, cells were affixed to Alcian blue coverslips and incubated in a humid chamber. Cells were stained for visualization by immunofluorescence confocal microscopy using a protocol adapted from Schooley et al. (13, 16). Briefly, cells were stained for extracellular expression of CD3 using a purified mouse anti-human Ab (BD Biosciences) and goat anti-mouse AlexaFluor 568 (Molecular Probes) as the secondary Ab for detection. Following extracellular staining, cells were fixed in 2% paraformaldehyde with 0.1% Triton X-100, then incubated in blocking/permeabilization buffer (PBS with 5% normal goat serum + 0.1% Triton X-100) for 1 h. Cells were incubated with 2 µg/ml purified anti-NF-{kappa}B relA (Santa Cruz Biotechnology) or anti-NF-AT1 (Upstate Biotechnology) for 1 h, then washed in blocking buffer (PBS with 5% normal goat serum). Cells were incubated for 30 min with goat anti-rabbit AlexaFluor 488 (Molecular Probes) and TO-PRO-3 (Molecular Probes), then washed successively twice with blocking buffer and twice with PBS before mounting the coverslips on glass slides with VECTASHIELD Mounting Medium (Vector Laboratories). Cells were visualized in 0.5-µm sections using a Bio-Rad MRC 1024 3-channel Laser Scanning Confocal Imaging System with Krypton-Argon laser on Nikon Optiphot 2 under a x60 oil immersion objective (Confocal Microscopy and 3-Dimensional Imaging Facility, State University of New York). Percentages were determined by examining at least 50–100 CD3+ T cells under each treatment condition, with slides read blindly. Statistical significance was determined by applying Student’s t test, and p values <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Previous work from our laboratory revealed that the majority of T cells within the microenvironment of lung tumors and nonmalignant chronic inflammatory tissues were memory T cells that were anergic due to a blockade in the TCR signaling pathway (12, 13). These findings led to the hypothesis that memory T cells present within tumor and chronic inflammatory microenvironments were maintained in a nonresponsive state by a common regulatory mechanism. The data presented in this study sustain this hypothesis and establish that membrane-associated TGF-beta1 is responsible for the inhibition of the TCR signaling pathway that occurs upstream of NF-{kappa}B and NF-AT activation.

Low pH stripping reverses the TCR-signaling blockade in tumor-associated memory T cells

Inflammatory leukocytes in tissues respond to a wide variety of signals by virtue of biologically active molecules (either soluble, cell associated, or immobilized on an extracellular matrix) binding to cell surface receptors. To determine whether such a regulatory molecule (either bound directly to memory T cells or to T-associated leukocytes) was responsible for the TCR-signaling blockade, leukocytes derived from human lung tumor biopsy tissues were incubated briefly in a low pH NaCl/citrate buffer (pH 4.0) to elute the putative suppressor molecule from the surface of the cells. The mixed population of cells was allowed to recover in culture overnight, and then the T cells in this population were stimulated for 1 h with TCR cross-linking Abs, before evaluating the cells for evidence of activation using immunofluorescence confocal microscopy (13). The images shown in Fig. 1 reflect the analysis of NF- {kappa}B translocation in tumor-associated memory T cells at the single-cell level. Cells affixed to the Alcian blue coverslips are identified by nuclear staining (Fig. 1, A, D, G, and J, blue). Individual T cells are then specifically identified by the punctate CD3 staining on the cell membrane (Fig. 1, B, E, H, and K, red). NF-{kappa}B and NF-AT are located primarily in the cytosol and within 1 h translocate to the nucleus following the activation of T cells derived from the peripheral blood of normal donors or from cancer patients (13). Under the same activation conditions, little or no translocation of NF-{kappa}B is observed in T cells derived from the tumor microenvironment. In the Tricolor-merged image of the noneluted tumor-associated T cells (Fig. 1C), the majority of the NF-{kappa}B (green fluorescence, arrow) is located in the narrow rim of cytoplasm at the periphery, despite having been in the presence of immobilized Abs to CD3 and CD28 for 1 h. In stark contrast, however, the CD3+ T cells first pulsed at low pH for 30 s and then activated demonstrate that the majority of the NF-{kappa}B is diffusely present throughout the nucleus of the T cells, indicating a response to TCR cross-linking (Fig. 1F). Therefore, a brief, 30-s treatment with low pH buffer reversed the anergy of the tumor-associated T cells to TCR stimulation and resulted in nuclear translocation of NF-{kappa}B. As expected, NF-{kappa}B is present in the nucleus of peripheral blood T cells following activation (Fig. 1I), and it was determined that the low pH elution had no effect upon the redistribution of NF-{kappa}B into the nucleus following activation of these T cells (Fig. 1L). When quantified, the elution pretreatment results in a statistically significant increase in the ability of the tumor-derived T cells to respond to TCR signals. The data shown in Fig. 2 demonstrate that, in cells derived from multiple tumors, the percentage of CD3+ T cells with nuclear NF-{kappa}B following cross-linking of CD3 and CD28 increased significantly if pretreated briefly with a low pH buffer (p < 0.001). Furthermore, the percentage of tumor-derived T cells in which NF-{kappa}B translocated into the nucleus in response to TCR cross-linking is similar to the levels reached by normal donor peripheral blood T cells without elution (as shown in Fig. 2). We conclude that pretreatment of memory T cells derived from human lung tumors with a low pH eluting buffer releases the cells from their quiescent state and makes them responsive to TCR cross-linking signals. The data in Fig. 2 also confirm that the low pH elution had no effect upon the response of T cells derived from the peripheral blood to the activation signal.


Figure 1
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  		FIGURE 1. TCR cross-linking induced nuclear translocation of NF-{kappa}B in tumor-associated T cells following low pH elution. Tumor-associated T cells from non-small cell lung tumor biopsy samples before or following a 30-s low pH treatment were applied to positively charged Alcian blue coverslips and stained with a nuclear dye, TO-PRO-3 (A, D, G, and J). From the mixed leukocyte population, T cells were specifically identified by membrane staining of CD3 (B, E, H, and K). The difference in intracellular location of proteins following TCR triggering is demonstrated in the Tricolor-merged images of the nuclei (blue), CD3 (red), and NF-{kappa}B (green, C, F, I, and L). The arrow points to NF-{kappa}B located in the periphery in unstimulated tumor-associated T cells (C). The arrowhead points to the diffuse nuclear staining of NF-{kappa}B induced in the CD3+ T cells by TCR cross-linking following low pH elution (F). Prestimulation elution did not affect the ability of normal donor peripheral blood T cells to translocate NF-{kappa}B to the nucleus (arrowheads, I and L). Representative images are shown.

 

Figure 2
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  		FIGURE 2. Low pH treatment of tumor-derived T cells results in responsiveness to TCR stimulation. T cells derived from the tumor microenvironment or normal donor peripheral blood T cells were evaluated by confocal microscopy for their ability to be activated with Abs to the TCR, with and without prior treatment with a low pH elution buffer. The cells were stained with Abs to CD3 and NF-{kappa}B and evaluated at the single-cell level for nuclear translocation of NF-{kappa}B by immunofluorescence confocal microscopy. Compared with noneluted T cells, the tumor-derived T cells that were pre-eluted significantly translocated NF-{kappa}B to the nucleus after a 1-h incubation with immobilized Abs to CD3 + CD28, while the elution had no effect on peripheral blood T cells. Pretreatment with a low pH buffer had no effect on the response to TCR cross-linking in normal donor peripheral blood T cells. Percentages were obtained by counting at least 100 CD3+ cells per condition, expressed as an average with error bars representing SD. *, p < 0.001 compared with noneluted T cells under the same conditions.

 
TGF-beta1 reinstates suppression to TCR triggering in tumor-associated T cells

Our previous finding that the anergy of the tumor-associated T cells was reversed by IL-12 (13) and that IL-12 significantly decreased TGF-beta1 and TGF-beta1-activating proteins (decorin and thrombospondin) in the tumor microenvironment (14) led us to speculate that the molecule being released by the low pH elution was TGF-beta1 and that this molecule was responsible for the T cell anergic state. To test this hypothesis, tumor-associated T cells from human non-small cell lung cancer tissues were pulsed for 30 s with the low pH NaCl/citrate buffer. The cells were returned to physiological pH and incubated with rhTGF-beta1 overnight. Following the incubation, the cells were stimulated for 1 h with immobilized cross-linking Abs to the TCR. As shown in Fig. 3, incubation with TGF-beta1 appears to reinstate the suppression to TCR cross-linking that had been reversed by the low pH elution (p < 0.001), whereas treatment of eluted cells with an irrelevant protein, BSA, did not suppress. Incubation with TGF-beta1 alone, i.e., without elution, did not negatively or positively affect the ability of the tumor-associated T cells to translocate NF-{kappa}B into the nucleus in response to TCR cross-linking. It should also be noted that incubation with TGF-beta1 did not affect surface expression of CD3, as visualized by immunofluorescence confocal microscopy. Furthermore, neither the 30-s low pH elution treatment nor the incubation with TGF-beta1 affected nuclear localization of NF-{kappa}B in CD3-negative, non-T cells (our unpublished results).


Figure 3
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  		FIGURE 3. TGF-beta1 induces suppression in tumor-associated T cells made responsive to TCR stimuli by low pH elution. Tumor-associated T cells were treated with or without a 30-s low pH elution before overnight incubation. Cells were treated with 10 ng/ml rhTGF-beta1 during the overnight incubation, as indicated above. Cells that were not treated with TGF-beta1 were incubated with BSA. The following day, the cells were stimulated for 1 h with Abs to CD3 and CD28 and immunofluorescently stained. Translocation of NF-{kappa}B was evaluated at the single-cell level by confocal microscopy. Percentages were obtained by counting at least 100 CD3+ cells per condition, expressed as an average of three independent experiments with error bars representing SD. *, Indicates p = 0.01, comparing eluted cells vs noneluted cells post-TCR stimulation. **, Indicates p = 0.006, comparing eluted cells + TGF-beta1 vs eluted-only cells post-TCR stimulation.

 
Previous work by our laboratory has demonstrated that NF-{kappa}B is not the only transcription factor downstream of the TCR that is nonresponsive to receptor cross-linking by immobilized Abs to CD3 and CD28 in tumor-associated T cells (13). In this study, it was determined that NF-AT translocation is also not observed in these T cells in response to receptor cross-linking. To evaluate whether the response to rhTGF-beta1 was due to a direct effect on the NF-{kappa}B portion of the TCR-signaling cascade, the nuclear translocation of NF-AT was also examined. Tumor-associated T cells were incubated overnight with rhTGF-beta1 following a brief pulse with a low pH buffer and then stimulated for 1 h with TCR cross-linking Abs the next morning. Translocation events in T cells were evaluated at the single-cell level by immunofluorescence confocal microscopy. Similar to the results described for NF-{kappa}B, the translocation of NF-AT in response to TCR stimuli was enhanced by the low pH elution, and this enhancement was reversed when the cells were subsequently incubated with TGF-beta1 (our unpublished results).

In addition to TGF-beta, other cytokines have been postulated to play a role in maintaining memory T cells in the tumor microenvironment, including IFN-{alpha}. To determine whether the suppression induced by rhTGF-beta1 in eluted T cells was specific to TGF-beta1 or could be induced by other cytokines present in the tumor microenvironment, similar experiments were conducted using IFN-{alpha}. Tumor-associated T cells were derived from human lung tumor biopsy samples and treated briefly with a low pH buffer. The eluted T cells were allowed to recover overnight in complete medium with or without the addition of IFN-{alpha}. The T cells were stimulated with immobilized Abs to CD3 and CD28 the following morning and immunofluorescently stained for evaluation of NF-{kappa}B translocation by confocal microscopy. In contrast to the results demonstrated for TGF-beta1, incubation with IFN-{alpha} did not induce suppression in the low pH eluted tumor-associated T cells (our unpublished results). These findings further support a specific role for TGF-beta in the suppression of the tumor-associated T cells to respond to TCR-induced signals.

To determine whether the observed suppression induced by TGF-beta1 treatment was specific to tumor-associated T cells, normal donor peripheral blood T cells were incubated with increasing concentrations of rhTGF-beta1, ranging from 0 to 10 ng/ml. As shown in Fig. 4, TGF-beta1 had no effect on the ability of T cells derived from the peripheral blood of a normal donor to translocate NF-{kappa}B in response to TCR stimulation, even at maximum concentrations. These results suggest that the TGF-beta1-induced suppression is specific for T cells in the tumor microenvironment or for T cells with a memory cell phenotype, as the tumor-associated T cells have been shown to display (12).


Figure 4
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  		FIGURE 4. TGF-beta1 has no effect on the ability of normal donor peripheral blood T cells to respond to TCR stimuli. T cells derived from the peripheral blood of a normal donor were cultured with or without rhTGF-beta1 overnight, as indicated above. Cells that were not treated with TGF-beta1 were incubated with BSA. Following the overnight incubation, the cells were stimulated for 1 h with Abs to CD3 and CD28 and immunofluorescently stained for evaluation of NF-{kappa}B translocation to the nucleus by confocal microscopy.

 
Although the data presented in Figs. 3 and 4 establish the capacity of TGF-beta1 to selectively inhibit tumor-associated memory T cells, they do not prove that the reversal of their anergy by the low pH pulse is due to the release of TGF-beta from the cell surface, nor do they establish a physiologically relevant role of endogenous TGF-beta1 in regulating the T cells in the tumor microenvironment.

Reversal of the anergy of tumor-associated T cells with TGF-beta1-neutralizing Ab

If endogenous TGF-beta1 bound to the surface of cells in the tumor microenvironment were responsible for the observed T cell anergy, a blockade of TGF-beta1 with a function-blocking Ab would be expected to reverse the T cell nonresponsiveness to TCR-activating signals. Tumor-associated T cells derived from tumor biopsy samples were incubated overnight with a neutralizing Ab to TGF-beta1 and stimulated for 1 h the following day with immobilized Abs to CD3 and CD28. As shown in Fig. 5, tumor-associated CD3+ T cells that were incubated overnight with a neutralizing Ab to TGF-beta1 translocate NF-{kappa}B into the nucleus following TCR cross-linking, whereas T cells that were not incubated with the anti-TGF-beta1 Ab or were incubated with an isotype control Ab fail to translocate NF-{kappa}B into the nucleus. To determine whether this effect was specific to tumor-associated T cells, the neutralizing Ab to TGF-beta1 was incubated with T cells derived from the peripheral blood of a cancer patient. This treatment had no effect, either positive or negative, on the translocation of NF-{kappa}B into the nucleus in response to TCR stimulation via immobilized Abs to CD3 and CD28 in these peripheral blood T cells (our unpublished results). We conclude that TGF-beta1 constitutively present in the tumor microenvironment and bound to the cell surface is responsible for maintaining the tumor-associated T cells in a quiescent state.


Figure 5
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  		FIGURE 5. Neutralization of TGF-beta1 alleviates suppression in tumor-associated T cells to TCR cross-linking stimuli. Tumor-associated T cells were treated with 1 µg/ml anti-TGF-beta1 during the overnight incubation, as indicated above. The following day, the cells were stimulated for 1 h with Abs to CD3 and CD28 and immunofluorescently stained. Translocation of NF-{kappa}B was evaluated at the single-cell level by confocal microscopy. Percentages were obtained by counting at least 100 CD3+ cells per condition, expressed as an average with error bars representing SD. *, Indicates p < 0.005, comparing anti-TGF-beta1-treated cells vs nontreated cells post-TCR stimulation.

 
T cells derived from other chronic inflammatory microenvironments can respond to TCR stimuli following a low pH shock

We previously demonstrated that T cells derived from the tumor microenvironment were not unique in their inability to respond to TCR cross-linking, because rather similar results were observed in T cells derived from two additional chronic inflammatory microenvironments, namely nasal polyp tissue from patients with chronic hyperplastic rhinosinusitis with nasal polyposis and synovial fluid from patients with rheumatoid arthritis (12, 13). T cells isolated from human nasal polyps and from rheumatic synovial fluid were found to possess a memory cell phenotype similar to the T cells isolated from human non-small cell lung tumors, namely CD45RO+, CD3+ T cells with expression of CXCR3 and CD28 and lacking CD62L, CD27, and CD45RA (13). As described for human non-small cell lung tumor T cells, this phenotype is consistent with that of an effector memory T cell (12, 13). To determine whether the anergic state of these T cells could also be reversed by low pH treatment, T cells derived from each of these chronic inflammatory microenvironments were treated for 30 s with the NaCl/citrate buffer (pH 4.0) and incubated overnight. Following the low pH pulse and then the overnight incubation, T cells derived from the nasal polyp microenvironment and from the synovial fluid were able to transduce signals through the TCR, similar to T cells derived from the tumor microenvironment (Fig. 6 and our unpublished results). However, as observed in the T cells from the peripheral blood of healthy donors, the activation potential of T cells derived from the peripheral blood of the nasal polyp-bearing patient was not affected by the low pH treatment (our unpublished results). Together with the data from the tumor-derived T cells, these results provide additional evidence that the lack of responsiveness of T cells in chronic inflammatory microenvironments is likely to be a common regulatory mechanism, rather than a tumor-induced suppression of associated leukocytes.


Figure 6
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  		FIGURE 6. Anergic T cells derived from chronic inflammatory microenvironments can respond to TCR triggering following low pH elution. T cells isolated from synovial fluid were evaluated for their ability to be activated by Abs to the TCR with and without low pH elution using immunofluorescence confocal microscopy. Percentages were obtained by counting at least 100 CD3+ cells per condition, expressed as an average with error bars representing SD. *, Indicates p < 0.05, comparing eluted cells vs noneluted cells post-TCR stimulation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It has been established in this study that cells present in the microenvironment of human non-small cell lung tumors and cells derived from nonmalignant chronic inflammatory tissues bind TGF-beta1 to their cell surface, which is responsible for the inability of memory T cells to respond to a TCR signal. The TGF-beta1-mediated suppression is durable as it persists in T cells that have been incubated overnight in culture, but the suppression is reversible either with the blockade of TGF-beta1 with neutralizing Abs or the stripping of cells with a brief pulse in a low pH buffer. Although it is possible that the low pH stripping results in the release of TGF-beta from the surface of cells, we do not yet have definitive proof of this. Attempts to re-establish the hyporesponsiveness in the low pH eluted cells by adding back the neutralized acid eluates have not yet been successful, but Western blots of pooled and concentrated eluates have revealed a very faint band that comigrates with a standard TGF-beta1-positive control. And, the addition of TGF-beta1 is able to re-establish the hyporesponsiveness in the low pH eluted T cells. Previous studies have also shown that the TCR nonresponsiveness can be reversed by a brief pulse of anergic memory T cells with IL-12 (13). The suppression also appears to be selective for memory T cells derived from the microenvironments of the human lung tumor and chronic inflammatory tissues because T cells derived from the peripheral blood of normal donors or cancer patients are fully responsive to TCR signals, and neither exogenous TGF-beta1 nor Abs to TGF-beta1 have any effect upon the ability of normal peripheral blood T cells to transduce a signal following the cross-linking of CD3 and CD28. Moreover, whereas memory T cells derived from non-small cell lung tumors are hyporesponsive to TCR signaling, the T cells derived from the tumor-draining lymph nodes remain fully responsive (13). The selective inhibition of TCR signaling in memory T cells may explain how these cells are able to persist in the tumor microenvironment and other chronic inflammatory peripheral tissues. In the absence of such a regulatory mechanism, one would expect that these memory cells, in the face of continuous Ag stimulation, would be driven to senescence or activation-induced cell death (17). Such regulation may also be required to attenuate the immune response to protect the host from substantial bystander damage to adjacent tissues or organs.

The ability of T cells to remain viable for an extended period of time in a functionally inactivated or hyporesponsive state has been well characterized in murine studies and is referred to as adaptive tolerance (18). This T cell anergy is typically initiated in the face of persistent Ag stimulation and requires a continued presence of Ag to maintain the adaptive tolerant state (18). The path by which human memory T cells develop their anergic state remains open to question. Based upon murine studies of memory T cell development in the lung following infection with Listeria monocytogenes (19), we suggest that a similar scenario may exist in the development and persistence of human memory T cells in sites in which there is a continuous presence of Ag. Thus, with the initial development of a tumor, there would be a rapid expansion and homing of effector T cells into the tumor microenvironment, followed by the contraction of these cells that occurs in a tightly regulated fashion even in the face of persistent Ag. Also as is seen in the later stages following a Listeria infection in the lung (19), a small T cell population survives within the tumor environment to initiate the memory cell pool in a preprogrammed manner that is independent of the magnitude of the initial expansion. We predict that this tumor-associated memory T cell pool will be held inactive by TGF-beta1 until there is a change within the microenvironment that is able to reverse the T cell anergy. As indicated above, exogenous IL-12 is able to reverse the nonresponsiveness of tumor-associated T cells both in vitro (13) and in situ (12). Thus, reactivation of tumor-associated T cells could occur under physiological conditions following the release of endogenous IL-12 possibly from dendritic cells or other APC that enter the tumor microenvironment and are activated by the presentation of Ag.

A large number of very diverse immunosuppressive networks have been proposed to explain the apparent dysfunction of tumor-associated lymphocytes with regard to their role of immunosurveillance against cancer (reviewed in Refs. 20 and 21). T cell functional defects have been suggested to be mediated by tumor factors unique to the tumor microenvironment. Examples include loss of signaling molecules, T cell death due to tumor-mediated activation-induced cell death, tumor-derived Fas ligand, tumor-expressing programmed death-1 ligands, T cell clonal exhaustion and/or deletion, suppressive or regulatory DC including IDO+ myeloid DC, B7-H1+ myeloid DC, myeloid suppressor cells, and CD4+CD25+ regulatory T cells (TReg).3 Controversy exists with many of the explanations that have been put forward to explain the dysfunction of tumor-associated T cells, and the growing number of hypotheses indicates that this conundrum is still unresolved. The need to generate a central and viable explanation of the dysfunctional state of tumor-associated lymphocytes is critical, and will help significantly in designing more effective immunotherapeutic strategies. Our results suggest that the nonresponsiveness observed with memory T cells in the human tumor microenvironment is not the result of one or more tumor-induced dysfunctions, but rather a normal regulatory mechanism of memory T cells found in peripheral inflammatory tissues (both tumor and nonmalignant).

Although our data show that the suppression of such memory T cells in the tumor microenvironment is dependent upon cell surface-bound TGF-beta1, it has not yet been determined whether the immunosuppressive cytokine is bound directly to the memory T cell or to another cell type that acts indirectly upon the memory cell to interfere with the TCR signaling. One possible and testable hypothesis is that TGF-beta1 is bound to CD4+CD25+ TReg present within tumors and inflammatory sites that mediate the suppression of memory T cells (22, 23). Consistent with this notion is the finding that murine TReg produce high levels of TGF-beta1 and that TReg suppression is abolished by the presence of neutralizing anti-TGF-beta1 Abs (24). Furthermore, others have reported finding regulatory CD4+CD25+ T cells in human early stage non-small cell lung tumors and late-stage ovarian cancer (25). Our initial attempts to identify a distinct TReg population in human lung tumors have to date been unsuccessful, and others have reported that TGF-beta1 production by TReg is not essential for their suppressive activity (26). Accordingly, further studies are necessary and warranted to determine whether the TGF-beta1-suppressive activity reported in this study is mediated indirectly by a defined suppressor cell population or acting directly by binding to receptors on memory T cells.

Several specific biochemical blocks in signal transduction have been found in murine studies of adaptively tolerized T cells (18). However, the mechanism by which TGF-beta1 blocks the signaling pathway in human memory T cells isolated from tumors and nonmalignant tissues has not been established. The activation of T cells via the TCR increases the surface expression of TGF-beta1 receptor subunit II, thereby providing an opportunity for TGF-beta1 to bind and complete a negative feedback, regulatory circuit (27). Furthermore, maintenance of TGF-beta1 on the surface of CD4+CD25+ TReg without additional stimulation implies that this inhibitory signal can have long-term effects (24). Thus, naive T cells rendered tolerant in such a manner by TGF-beta1 are unable to phosphorylate TCR {zeta}-chain or to activate Zap70, ras, or MAPK, similar to T cells that have been tolerized in vitro by a coregulatory cascade (28). In other murine studies, the ability of TGF-beta1 to inhibit TCR-induced cell proliferation was found to be largely, but not completely dependent upon Smad3 (29). Recent studies have established that exogenous TGF-beta1 discriminantly regulates CD4+ and CD8+ TCR-induced activation by signaling through distinct intracellular pathways that include both a Smad3-dependent and Smad3-independent mechanism (29). In our studies with human memory T cells, we have determined that the TGF-beta1 blockade of the TCR signaling pathway and the reversal of the blockade with IL-12 occur similarly in both CD4+ and CD8+ memory T cells. The block occurs in these cells somewhere upstream of NF-{kappa}B and NF-AT activation and translocation because we previously determined by alternative signaling pathways that this downstream translational machinery remains intact (13). Others have suggested that TGF-beta1 may suppress the effector functions of human tumor-associated memory T cells by its ability to suppress T-bet expression in these cells (11).

The recognition that TGF-beta1 is present and playing a physiologically relevant role in inhibiting the TCR signaling pathway in tumor-associated human memory T cells has potential clinical significance that may be exploited to enhance the ability of these T cells to respond to and eradicate tumor cells in situ. Thus, if one were able to bypass or overcome the immunosuppressive effects of TGF-beta1, it may be possible to reactivate the tumor-associated T cells. Previous studies, both in murine tumors and human tumor xenografts, have established that a local and sustained release of IL-12 into the tumor microenvironment results in an in situ reactivation of tumor-associated T cells that proliferate, secrete IFN-{gamma}, and orchestrate a complete eradication of the tumor (12, 14). Although these earlier studies did not address the mechanism by which IL-12 was able to reactivate the tumor-associated T cells, the ability of IL-12 to reverse the immunosuppressive effects of TGF-beta1 is a possible and likely contributing factor. In murine studies, IL-12 was shown to overcome the suppressive effects of TReg by signaling through the IL-12R expressed on CD25-responder T cells (30). It may be possible to design other strategies for reversing the effects of TGF-beta1 that could be used to enhance the effectiveness of cancer vaccines and other immunotherapeutic strategies for cancer, such as the adoptive transfer of tumor-specific effector T cells.


   Acknowledgments
 
We thank Drs. Hiroshi Takita, Todd Demmy, and Harry Slocum, and the Tissue Procurement Facility at Roswell Park Cancer Institute for providing us with patient tumor samples; Dr. Alan N. Baer for providing synovial fluid samples; Joel M. Bernstein for providing nasal polyp specimens; Dr. Sarah Gaffen for sharing her cytokine stripping protocol; and Sandra Yokota, Jenni Loyall, and Robert Parsons for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by U.S. Public Health Service Grants RO1-CA10897, The John R. Oishei Foundation, the Mary Kay Ash Foundation (to R.B.B.), and National Research Service Award Training Grant AIO7614 (to L.B.). Back

2 Address correspondence and reprint requests to Dr. Richard B. Bankert, Department of Microbiology and Immunology, 138 Farber Hall, State University of New York, 3435 Main Street, Buffalo, NY 14214. E-mail address: rbankert{at}buffalo.edu Back

3 Abbreviation used in this paper: TReg, regulatory T cell. Back

Received for publication February 24, 2006. Accepted for publication June 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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