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The Journal of Immunology, 2007, 178: 4051-4061.
Copyright © 2007 by The American Association of Immunologists, Inc.

Follicular Lymphoma Intratumoral CD4+CD25+GITR+ Regulatory T Cells Potently Suppress CD3/CD28-Costimulated Autologous and Allogeneic CD8+CD25– and CD4+CD25– T Cells1

Shannon P. Hilchey*, Asit De*,{ddagger}, Lisa M. Rimsza§, Richard B. Bankert and Steven H. Bernstein2,*,{dagger}

* James P. Wilmot Cancer Center, {dagger} Lymphoma Biology Program, {ddagger} Department of Surgery, University of Rochester Medical Center, Rochester, NY 14642; § Department of Pathology, University of Arizona, Tucson, AZ 85724; and Department of Microbiology and Immunology, State University of New York, Buffalo, NY 14214


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulatory T cells (TR) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor-reactive effector T cells. In this study, we demonstrate that follicular lymphoma (FL)-infiltrating CD8+ and CD4+ T cells are hyporesponsive to CD3/CD28 costimulation. We further identify a population of FL-infiltrating CD4+CD25+GITR+ TR that are significantly overrepresented within FL nodes (FLN) compared with that seen in normal (nonmalignant, nonlymphoid hyperplastic) or reactive (nonmalignant, lymphoid hyperplastic) nodes. These TR actively suppress both the proliferation of autologous nodal CD8+CD25– and CD4+CD25– T cells, as well as cytokine production (IFN-{gamma}, TNF-{alpha} and IL-2), after CD3/CD28 costimulation. Removal of these cells in vitro by CD25+ magnetic bead depletion restores both the proliferation and cytokine production of the remaining T cells, demonstrating that FLN T cell hyporesponsiveness is reversible. In addition to suppressing autologous nodal T cells, these TR are also capable of suppressing the proliferation of allogeneic CD8+CD25– and CD4+CD25– T cells from normal lymph nodes as well as normal donor PBL, regardless of very robust stimulation of the target cells with plate-bound anti-CD3 and anti-CD28 Abs. The allogeneic suppression is not reciprocal, as equivalent numbers of CD25+FOXP3+ cells derived from either normal lymph nodes or PBL are not capable of suppressing allogeneic CD8+CD25– and CD4+CD25– T cells, suggesting that FLN TR are more suppressive than those derived from nonmalignant sources. Lastly, we demonstrate that inhibition of TGF-beta signaling partially restores FLN T cell proliferation suggesting a mechanistic role for TGF-beta in FLN TR-mediated suppression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Follicular lymphomas (FL)3 are the second most common type of non-Hodgkin’s lymphoma seen in adults. Despite an indolent natural history, with median survival ranging from 7 to 10 years, this is essentially an incurable disease. Newer treatment options for patients with this disease, including the use of rituximab, a chimeric mAb directed against CD20, may however, for the first time in decades, improve the natural history and overall survival of patients with FL. The proposed antitumor mechanisms of action of rituximab include activation of Ab-dependent cellular cytotoxicity, complement-mediated lysis, and direct induction of apoptosis (1). In addition, a "vaccinal effect" of rituximab has been proposed whereby induction of lymphoma cell death results in the elicitation of an active antilymphoma T cell response (1, 2). Although this has not yet been proven to exist in vivo, it would explain a number of clinical findings seen with rituximab, including that the antitumor activity of rituximab can continue to persist for months, a finding that cannot be explained solely by the pharmacokinetics of the drug. In addition, active vaccination of FL patients with Id vaccines has been shown to elicit cellular immune responses and has clinical activity in FL patients (3, 4, 5). The efficacy of such personalized vaccines is now being studied in three randomized phase II clinical trials. Taken together, immunotherapeutic approaches for the treatment of FL represent a promising new area of investigation.

For such immunotherapeutic approaches, however, the elicitation of a cellular antitumor response alone may not be sufficiently robust and durable to result in a clinically relevant response, as T cells must persist in a functionally active state and mediate their effector function within an often immunosuppressive tumor microenvironment. Indeed, the importance of the tumor microenvironment has recently been highlighted by the findings that the molecular features of the tumor-infiltrating nonmalignant immune cells of FL are highly predictive of patient survival (6, 7). A gene expression signature denoted immune response 1 was predictive of favorable survival and includes several T cell and monocyte-restricted genes. In contrast, an immune response 2 expression signature was predictive of a poor outcome and includes genes highly expressed in monocytes, dendritic cells, or both, but not T cells. Taken together, T cells likely play a critical role in both the immunobiology of lymphomas as well as potentially mediating the activity of newer immunological-based therapies. However, it has been demonstrated that CD3+ T cells infiltrating involved FL nodes (FLN) tend to be hyporesponsive to TCR stimulation and this lack of immune responsiveness has been hypothesized to correlate with disease progression by escape from immune surveillance (8). One potential mechanism to account for this T cell hyporesponsiveness may be due, at least in part, to the infiltration of involved FLN by suppressive CD4+CD25+ regulatory T cells (TR).

Naturally occurring CD4+CD25+ TR have been identified as playing a crucial role in the suppression of autoimmune responses and are thought to arise as a distinct lineage from the thymus (9, 10). In addition, other T cells, such as IL-10-producing T regulatory type 1 (Tr1) cells and TGF-beta-producing Th3 cells, can be induced from uncommitted peripheral CD4+ T cells and similarly suppress T cell responses (11, 12). The study of TR, particularly in the context of active immune responses, has been challenging due to the lack of surface molecules truly unique to TR. However, there are several markers that have been shown to be constitutively expressed by TR and thus have been useful for their study. These markers include high-level expression of CD25, CTLA-4, glucocorticoid-induced TNFR-related protein (GITR), and neurophilin (13, 14, 15, 16, 17, 18, 19). In addition, the transcription factor Foxp3/FOXP3 (mouse/human) has been shown to be specifically expressed in TR (20, 21, 22, 23) and crucial to their development in vivo (22). The role of TR in tumor progression has been suggested and, indeed, TR have been shown to be increased in the tumor microenvironment, or peripheral blood (PB), of patients with numerous cancers including lung, ovarian, pancreatic, breast, gastrointestinal, esophageal, melanoma, B cell chronic lymphocytic leukemia, and Hodgkin’s lymphoma (24, 25, 26, 27, 28, 29, 30, 31, 32, 33). It has also recently been reported that CD4+CD25+ TR infiltrate malignant B non-Hodgkin’s lymphoma (B-NHL) nodes of various histologies and are capable of suppressing proliferation and cytokine production of autologous infiltrating CD4+CD25– cells (34). As both CD4+ helper and CD8+ effector T cells are likely to play a crucial role in the elicitation of an antitumor immune response, additional mechanistic information regarding suppression of T cells, including CD8+ effector T cells, is critical both to our understanding of FL biology as well as to the development of novel immunotherapeutic approaches to treatment.

In the present study, we directly compare T cell infiltrates from FLN, and other B-NHL subtypes with: 1) normal lymph nodes (NLN), obtained from patients undergoing vascular surgery whereby lymph nodes were removed to gain access to vascular structures and 2) reactive lymph nodes (RLN), from patients with unexplained adenopathy whereby lymph nodes were removed for diagnostic purposes and found to be reactive lymphoid hyperplasia rather than malignant. We show that CD8+ as well as CD4+ T cells infiltrating FLN are hyporesponsive, as determined by proliferation, to the very robust stimulation provided by plate-bound anti-CD3 and anti-CD28 mAbs. In contrast, both CD8+ and CD4+ T cells from NLN and RLN exhibit similar proliferative responses upon TCR engagement. In addition, we show that there is a higher proportion of CD4+CD25+GITR+ T cells that infiltrate FLN as compared with that seen in either NLN or RLN. These FL-infiltrating TR suppress both proliferation as well as the cytokine production (IFN-{gamma}, TNF-{alpha}, and IL-2) of CD8+CD25– and CD4+CD25– T cells stimulated with anti-CD3 and anti-CD28 Abs. Furthermore, in vitro depletion of CD25+ T cells from FLN heavily infiltrated with CD4+CD25+GITR+ TR restores both proliferation and cytokine production of autologous infiltrating CD8+CD25– and CD4+CD25– T cells in response to CD3/CD28 costimulation. These data demonstrate that both CD8+ effector and CD4+ Th cell hyporesponsiveness in FLN can be reversed by removal of infiltrating TR. We further demonstrate that in contrast to what has been shown for TR-infiltrating lung tumors (25), FLN TR can potently suppress the proliferation of normal allogeneic T cells (both nodal and PB) stimulated with plate-bound anti-CD3 and anti-CD28 mAbs. Indeed, our findings further suggest that a qualitative difference may exist between TR-infiltrating malignant FLN compared with TR-infiltrating NLN, and those derived from normal donor PB, as the FL TR suppress autologous and allogeneic T cell proliferation to a greater extent than that of the NLN and PBL TR, when comparing equivalent numbers of FOXP3+ cells. Finally, our data demonstrate that FLN TR suppression of T cell proliferation is mediated in part by TGF-beta signaling. As such, modulation of TGF-beta may be a clinically viable approach to attenuate TR-mediated T cell suppression, which would likely augment the efficacy of immunotherapeutic approaches for patients with FL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs for flow cytometry

The following fluorochrome-conjugated Abs were used for surface staining: CD3-FITC, CD4-allophycocyanin-Cy7, CD4-PerCP-Cy5.5, CD8-PE-Cy7, CD25-allophycocyanin, CD25-PE (BD Pharmingen), and GITR-PE (R&D Systems). An FITC-labeled human FOXP3 staining kit was purchased from eBioscience.

Patient samples

Primary lymph node (malignant and nonmalignant reactive) or reactive tonsil biopsy tissues were obtained from 30 different patients undergoing routine biopsy or tonsillectomy, under an Institutional Review Board (IRB)-approved protocol. NLN (nonmalignant, nonreactive) were obtained from 13 different patients undergoing vascular surgery during which time obstructive lymph nodes are removed and normally discarded, under an IRB-approved protocol. Normal donor PB was obtained from separate individuals under an IRB-approved protocol. Biopsy tissues were obtained from the Strong Memorial Hospital Surgical Pathology Laboratory and maintained in sterile specimen containers on ice until processing. An additional 5 FL patient samples were obtained from Dr. L. Rimsza (Department of Pathology, University of Arizona, Tucson, AZ), under an IRB-approved protocol. A histological diagnosis for each specimen received was obtained anonymously.

Cell isolation and separation

Lymph node and tonsil biopsy tissues were mechanically separated, minced, and passed through a 70-µm nylon mesh cell strainer under sterile conditions. The resultant single-cell suspensions were washed with RPMI 1640 medium, counted, and cryopreserved for future analysis. Whole blood was obtained from normal donors by venipuncture and PBL were isolated from the whole blood using Ficoll-Paque Plus according to the manufacturer’s instructions (Amersham Biosciences). The resultant lymphocytes were cryopreserved for future analysis. Depletion of CD25+ cells was performed using MACS CD25+ MicroBeads (Miltenyi Biotec), according to the manufacturer’s cell depletion instructions, on freshly thawed single-cell suspensions. Depletion was checked by FACS analysis (see Fig. 3).


Figure 3
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  		FIGURE 3. FACS analysis of CD25+ depleted/enriched cells. Single-cell suspensions of FLN were depleted of CD25+ cells using MACS CD25+ MicroBeads (see Materials and Methods). Cells before (Unseparated) and after CD25 depletion (Separated CD25– and Separated CD25+) were surface stained for FACS analysis (see Materials and Methods). Shown are the results of separation of the FLN01 single-cell suspension. Left panels, Cells before CD25 depletion (Unseparated); middle panels, the CD25-depleted fraction (Separated CD25–); and right panels, the CD25+-enriched fraction (Separated CD25+). Upper and lower panels correspond to the CD3+CD4+ and CD3+CD8+ T cell gates, respectively. Plotted is the intensity of CD25 vs GITR.

 
Flow cytometry

Cryopreserved samples were thawed, washed once in PBS containing 1% heat-inactivated FBS, incubated on ice with specific fluorochrome-conjugated Abs, and acquired on an LSR-II Flow Cytometer (BD Biosciences) with data analysis using FlowJo version 6.3.4 software (Tree Star). All specimens analyzed underwent only one freeze-thaw cycle and a fresh sample was used for each experiment. An analysis of several tonsil preparations revealed no significant difference in the flow cytometric data comparing cells freshly prepared and acquired vs that obtained after a single freeze-thaw cycle (data not shown).

CFSE labeling

Cryopreserved samples were thawed, washed in RPMI 1640 medium with 10% FBS, and either CFSE labeled immediately (unseparated) or CD25+ MicroBead separated as described above, followed by CFSE labeling. To CFSE label, separated or unseparated cells were washed once in PBS containing 0.5% BSA and resuspended at 1 x 107 cells/ml in prewarmed (37°C) PBS with 0.5% BSA. Freshly prepared CFSE (Molecular Probes) was added to a final concentration of 10 µM and the cells were incubated for 10 min at 37°C. Excess CFSE was quenched by adding 10 volumes of ice-cold RPMI 1640 medium containing 10% FBS and incubating the cells for 5 min on ice. CFSE-labeled cells were then washed three times with RPMI 1640 medium containing 10% FBS and cultured with or without stimulation as below.

T cell stimulation and proliferation assay

Abs were immobilized on 48-well tissue-culture plates by incubating 20 µg/ml anti-CD3 (OKT3) and 10 µg/ml anti-CD28 (CD28.2) in PBS overnight at 4°C (eBioscience). Excess Ab was removed by washing the wells with PBS. CFSE-labeled cells were added at 2.5 x 106 cells/well in 0.5 ml of complete RPMI 1640 medium containing 10% FBS and incubated at 37°C and 5% CO2. For CD25+ cell add back experiments, 5 x 105 unlabeled cells from the CD25+ MicroBead fraction, or in select experiments 5 x 105 unlabeled CD25-depleted cells (CD25–), were added back to 2.5 x 106 CFSE-labeled CD25-depleted cells (CD25–) per well. Cells were harvested on day 5 and culture supernatants collected and stored at –20°C until cytokine analysis. Cells were washed once in PBS containing 1% FBS and surface stained with fluorochrome-conjugated anti-CD8 and anti-CD4 Abs. Surface-stained, CFSE-labeled cells were acquired on an LSR-II Flow Cytometer (BD Biosciences) and CFSE intensity for the CD8+ or CD4+ cells was analyzed using FlowJo software version 6.3.4 (Tree Star). For cell add back studies, the CFSE+ cells, both CFSEBRIGHT and CFSEDIM, but not CFSE– cells, were gated on to distinguish between cells that have reduced CFSE as a result of proliferation (CFSEDIM), from the unlabeled (CFSE–) cells added back to the coculture assay (see Fig. 4). In select TGF-beta inhibition experiments, unseparated single-cell suspensions were labeled with CFSE as above and pretreated with the indicated doses of recombinant latency associated peptide of TGF-beta1 (rLAP) or neutralizing anti-TGF-beta1 mAb (R&D Systems) for 2 h at 4°C. Pretreated cells were then added to anti-CD3- and anti-CD28-coated wells and assayed for proliferation by FACS as above.


Figure 4
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  		FIGURE 4. Gating strategy used to exclude unlabeled CD25+-enriched cells from analysis of coculture experiment data. Single-cell suspensions of FLN were depleted of CD25+ cells using MACS CD25+ MicroBeads (see Materials and Methods). CD25-depleted cells (CD25–) were labeled with CFSE and unstimulated or stimulated with plate-bound anti-CD3 and anti-CD28 mAbs. For coculture experiments, the CFSE-labeled CD25– cells were stimulated with plate-bound Abs in the presence of unlabeled CD25+-enriched cells. Cells were harvested after 5 days and surface stained for CD8+ and CD4+ and subjected to FACS analysis. A, Gating strategy used to eliminate unlabeled CD25+-enriched cells from analysis. CFSEBRIGHT and CFSEDIM but not CFSE– cells were gated on for analysis, as indicated, to differentiate between proliferating cells (CFSEDIM) and unlabeled (CFSE–) CD25+-enriched cells added back to the cocultures. B, Cells were gated based upon CD8+ and CD4+ expression, as well as CFSE signal as defined above (A), and the percentage of CFSEDIM cells for each of these subsets was used to determine cellular proliferation. Shown is a representative experiment for patient FLN01. Plotted is CFSE intensity vs cell number.

 
FOXP3 analysis

CD25 MicroBead-separated lymphocytes isolated from normal donor PBL, involved FLN, and NLN were first stained for surface expression of CD25 and CD4 using specific Abs or respective isotype controls. Cells were washed, fixed, permeabilized, and then stained with anti-FOXP3 Ab according to the manufacturer’s instruction (eBioscience). Stained cells were then analyzed by flow cytometry as above.

Cytokine analysis

Culture supernatants obtained from the T cell stimulation and proliferation assays were analyzed at the Roswell Park Cancer Institute Laboratory (Flow Cytometry, Buffalo, NY) for IFN-{gamma}, TNF-{alpha}, and IL-2 levels using a multiplex assay and Luminex 100 analyzer. The lower limits of detection in picograms per milliliter (determined using cytokine standards) were 20, 2.3, and 6.7 for IFN-{gamma}, TNF-{alpha}, and IL-2, respectively.

Statistical analysis

The significance of the results was determined using the Student t test with p < 0.05 considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Follicular lymphoma intratumoral CD8+ and CD4+ T cells are hyporesponsive to combined TCR stimulation/CD28 costimulation

We first assessed T cell function in NLN, RLN, and FLN by evaluating the proliferation of CFSE-labeled cells upon stimulation with plate-bound anti-CD3 and anti-CD28 mAbs. This technique allows us to examine proliferative responses of discrete T cell subsets to very robust stimulation in the context of an unseparated mononuclear cell population, as opposed to examining proliferative responses to a T cell-enriched population, as has been done previously (8, 34). The percentage of CD8+ and CD4+ T cells shifting from CFSEBRIGHT to CFSEDIM (indicative of the distribution of CFSE among daughter cells as a result of proliferation), of pre- and poststimulated NLN, RLN and FLN single-cell suspensions is shown in Fig. 1A. Fig. 1B shows the composite proliferation data for CD8+ T cells from seven NLN, five RLN, and seven FLN samples. Whereas there is a significant percentage of CD8+ NLN and RLN T cells that proliferate after stimulation (p < 0.05 relative to the unstimulated controls), there is no significant proliferation of FLN CD8+ T cells after stimulation. Similar results are shown for CD4+ T cells from the same samples (Fig. 1C). These results illustrate that the CD8+ and CD4+ T cells for all of the FLN examined are hyporesponsive to CD3/CD28 costimulation while both the NLN and RLN CD8+ and CD4+ T cells exhibit significant and similar responsiveness to TCR engagement.


Figure 1
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  		FIGURE 1. Intratumoral CD8+ and CD4+ T cells are hyporesponsive to CD3/CD28 costimulation. Single-cell suspensions of NLN, RLN, or FLN were labeled with CFSE and left unstimulated or were stimulated with plate-bound anti-CD3 and anti-CD28 mAbs for 5 days. Cells were harvested and surfaced stained for CD8+ and CD4+ and subjected to FACS analysis. Viable cells (determined by 4',6'-diamidino-2-phenylindole exclusion) were gated based upon CD8+ and CD4+ expression and the percentage of cells expressing CFSEDIM for each of these subsets was used to determine cellular proliferation. A, Shown are representative experiments for NLN (donor NLN03), RLN (donor RLN01), and FLN (patient FLN01). Plotted is CFSE intensity vs cell number for CD8+ and CD4+ gated cells, as indicated. Summary of the proliferation data of B (CD8+) and C (CD4+ T cells of NLN (n = 7), RLN (n = 5), and FLN (n = 7)) is shown. Plotted is tissue type vs percent cells proliferating with (*) indicating statistical significance relative to the matched, unstimulated controls (p < 0.05).

 
Increased proportions of CD3+CD4+CD25BRIGHT and CD3+CD4+CD25+GITR+ T cells infiltrate B-NHL nodes

Although the CD25 marker alone is not sufficient to distinguish TR from activated T cells, the CD3+CD4+CD25BRIGHT population has been a better discriminator of TR. To evaluate the infiltration of the FLN by putative CD3+CD4+CD25BRIGHT TR, we characterized the phenotype of single-cell suspensions by FACS using the gating strategy shown in Fig. 2A. As shown in Fig. 2B, the FLN contain a higher percentage of CD4+ T cells that are CD25BRIGHT as compared with that of the NLN (21.49 ± 15.09% as compared with 3.65 ± 1.45%, respectively; p < 0.01). Whereas the diffuse large cell lymphoma (DLCL) and other B-NHL nodes also contain a slightly higher percent of CD4+CD25BRIGHT T cells as compared with that of the NLN, these differences are not statistically significant (Fig. 2B; p > 0.05). The percentage of CD4+CD25BRIGHT T cells in the tonsil were significantly higher than that of the NLN (9.68 ± 4.10% vs 3.65 ± 1.4%; p < 0.01), however, these were not of the same magnitude as that of the FLN (Fig. 2B).


Figure 2
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  		FIGURE 2. Elevated numbers of CD3+CD4+CD25BRIGHT and CD3+CD4+CD25+GITR+ T cells infiltrate involved B-NHL nodes. Single-cell suspensions from NLN (n = 13), RLN (n = 5), tonsil (TN; n = 8), FLN (n = 11), DLCL (n = 4), and other B-NHL node (n = 5; including: 1 marginal zone B cell lymphoma, 1 small lymphocytic lymphoma, 1 anaplastic large cell lymphoma, and 2 marginal zone lymphomas) biopsies were subjected to FACS analysis (see Materials and Methods). A, Representative dot plot showing CD25 and GITR expression on FLN01 CD4+ T cells. The dash-lined box corresponds to the CD25BRIGHT gate, with the indicated frequency of CD3+CD4+CD25BRIGHT cells displayed as a percentage of CD3+CD4+CD25+ T cells. Plotted is the intensity of CD25 vs GITR. B, Frequency of CD3+CD4+CD25BRIGHT infiltrating cells displayed as a percent CD3+CD4+CD25+ T cells. C, Frequency of CD3+CD4+CD25+GITR+ infiltrating cells displayed as a percent of CD3+CD4+ T cells. Plotted in B and C is tissue-type vs percent-positive cells with (*) indicating statistical significance relative to NLN (**, p < 0.01 and ***, p < 0.001).

 
To further differentiate phenotypically between activated effector T cells that express CD25 and the putative TR, we also examined the expression of GITR. GITR expression has been linked to TR, both phenotypically and functionally (17, 18, 35, 36). As shown in Fig. 2C, the percentage of CD4+CD25+GITR+ T cells is indeed higher in the B-NHL samples as compared with that of the other tissues. Specifically, the FLN, DLCL, and other B-NHL cells demonstrate an increase in the numbers of CD4+CD25+GITR+ T cells, 9.86 ± 6.70%, 5.81 ± 7.77%, and 1.97 ± 0.98%, respectively, relative to that observed in the NLN, 0.70 ± 0.29%. Despite the observed increase in infiltration of these cells for all of the different B-NHL types examined here, only the FLN demonstrate an increase that is statistically significant relative to that of the NLN (p < 0.001). In addition, both the RLN and tonsil exhibit a slight increase in the numbers of infiltrating CD4+CD25+GITR+ cells, 1.40 ± 1.04% and 2.76 ± 1.05%, respectively, relative to the NLN, 0.70 ± 0.29%, however, only the increase observed for the tonsil is statistically significant relative to that of the NLN (p < 0.001). Although CD25 and GITR are not necessarily exclusive to TR (37, 38, 39, 40, 41, 42), combining the CD4+CD25BRIGHT and CD4+CD25+GITR+ expression data detailed herein shows that, phenotypically, FLN contain elevated numbers of putative TR as compared with that of either NLN or even inflamed secondary lymphoid tissues (lymph node or tonsil).

FLN-infiltrating CD25+ cells inhibit the proliferation of autologous FLN CD8+ and CD4+ T cells

We next determined whether the functional hyporesponsiveness we have shown for both CD8+ and CD4+ T cell populations in FLN is due, in part, to TR suppression. As described above (Fig. 2, B and C), the FLN tend to be infiltrated by significant numbers of putative TR. In particular, patient samples FLN01, FLN02, FLN03, and FLN04 demonstrate very significant degrees of infiltration by CD4+CD25BRIGHT and CD4+CD25+GITR+ T cells (Fig. 2A). In addition, all of the FLN samples examined herein (including these four samples) are similarly hyporesponsive to TCR engagement (Fig. 1). To determine whether T cell hyporesponsiveness is due to TR suppression: 1) CD25+ cells from the single-cell suspensions were depleted; 2) CD8+CD25– and CD4+CD25– T cell proliferation was then assessed in the CD25+-depleted fractions after CD3/CD28 costimulation using CFSE-labeled cells as above; and 3) proliferation was again assessed after adding back the CD25+-enriched cell fraction which was not stained with CSFE. As shown in Fig. 3, using the MicroBead technique, the separated CD25– cells have been significantly depleted of CD25BRIGHT cells.

CFSE+ cells, both CFSEBRIGHT and CFSEDIM, but not CFSE– cells (which are the CD25+-enriched cells in the add-back experiments), were gated as shown in Fig. 4 and the percentage of CFSEDIM cells was used as a measure of cellular proliferation. Before depletion, CD8+ T cells from the FLN01, FLN02, FLN03, and FLN04 demonstrate only minimal (nonsignificant) increases in proliferation, to 5.45, 6.41, 4.29, and 2.78% proliferating cells, respectively, after stimulation (shown in Fig. 1). Upon CD25 depletion, however, the percentage of CD8+CD25– T cells demonstrating proliferation increases to 32.1, 22.2, 26.1, and 15.8% proliferating cells for FLN01, FLN02, FLN03, and FLN04, respectively (Fig. 5A). When the CD25+-enriched populations are added back, the poststimulation proliferation decreases to 7.55, 3.23, 7.28, and 12.3% proliferating cells for FLN01, FLN02, FLN03, and FLN04, respectively (Fig. 5A). A similar pattern is shown for the CD4+ T cells of these same samples (Fig. 5B) whereby depletion of the CD25+ cells restores CD4+ T cell proliferation after stimulation, which is again suppressed with the subsequent add back of the CD25+-enriched cells (Fig. 5B). A summary of the composite data (Fig. 5C) demonstrates that CD25 depletion of the FLN results in a statistically significant increase in proliferation of both CD8+CD25– and CD4+CD25– T cells (p < 0.05) upon CD3/CD28 costimulation (comparing CD25-depleted and -stimulated cells to that of the CD25-depleted and -unstimulated controls). In addition, the data show that the FLN CD25+-enriched cells markedly diminishes the proliferation of CD3/CD28-costimulated autologous nodal CD8+CD25– and CD4+CD25– T cells to levels similar to, and not statistically different from, that observed before stimulation (p > 0.05) thus demonstrating that the CD25+-enriched fractions from the FLN contain functionally suppressive cells.


Figure 5
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  		FIGURE 5. Intratumoral CD25+-enriched cells from FLN actively inhibit the proliferation of autologous nodal CD8+ and CD4+ T cells. Single-cell suspensions from FLN (shown in Fig. 1 to be hyporesponsive to CD3/CD28 costimulation) were depleted of CD25+ cells as described. CD25-depleted cells (CD25–) were labeled with CFSE and left unstimulated or stimulated with plate-bound anti-CD3 and anti-CD28 mAbs in the presence of unlabeled autologous CD25+-enriched cells. Cells were harvested after 5 days and surface stained for CD8+ and CD4+ and subjected to FACS analysis. Shown is the proliferation of the CD8+ (A) or CD4+ (B) gated cells cocultured with or without autologous CD25+-enriched cells using the gating strategy described in Fig. 4. C, Summary of the coculture proliferation data (n = 5). Plotted in A and B are individual tissue vs percent cells proliferating. Plotted in C is T cell subset vs percent cells proliferating with (*) indicating statistical significance relative to the unstimulated control (p < 0.05).

 
We next addressed whether the suppression observed upon the add back of the CD25+-enriched cells to the cocultures is simply due to cell crowding, rather than to actual functional suppression, by adding back to the FLN02 CFSE-labeled CD25– cells unlabeled autologous CD25– cells during the proliferation assay (as opposed to unlabeled autologous CD25+-enriched cells). Whereas add back of FLN02 non-CFSE-labeled CD25+ cells significantly decreased the percentage of CD8+CD25– and CD4+CD25– T cells proliferating after stimulation (Fig. 5, A and B), the addition of an equivalent number of unlabeled autologous CD25– cells had no significant effect on the percentage of CD8+CD25– and CD4+CD25– T cells proliferating (data not shown). Therefore, the suppression seen with the add back of CD25+-enriched cells is due to the presence of functionally suppressive TR cells rather than to a nonspecific effect of cell crowding.

FLN-infiltrating CD25+ cells inhibit cytokine production of autologous FLN T cells

In addition to examining proliferative responses, we also examined cytokine levels produced in the culture supernatants during the CD25 depletion/proliferation experiments described above. We analyzed levels of soluble IFN-{gamma}, TNF-{alpha}, and IL-2 using a multiplex assay for the FLN01 and FLN03 cultures. The unseparated cells (before CD25 depletion) from FLN01 and FLN03 produce little or undetectable amounts of soluble IFN-{gamma} or TNF-{alpha} (Fig. 6, A and B, respectively), either before or post-CD3/CD28 costimulation. Upon CD25 depletion (CD25 separated), cells from these samples also produce very little IFN-{gamma} and TNF-{alpha} before stimulation. However, stimulation of the CD25-depleted FLN01 and FLN03 cells (CD3/CD28 costimulation) results in a 10- and 40-fold increase in IFN-{gamma}, respectively, and a 15- and 5-fold increase in TNF-{alpha}, respectively, as compared with that produced by the stimulated cells before separation (Fig. 6, A and B). Similar to what was seen with proliferation, add back of the CD25+-enriched population decreased the poststimulation levels of IFN-{gamma} and TNF-{alpha} to undetectable levels (Fig. 6, A and B, respectively). We also evaluated the effect of CD25+ cell depletion on levels of soluble IL-2 (Fig. 6C). Similar to what was seen for IFN-{gamma} and TNF-{alpha}, CD25+ cell depletion resulted in a 2- to 4-fold increase in the levels of IL-2 relative to that produced by the unseparated cells. In addition, add back of the CD25+ cells resulted in a decrease in detectable IL-2 levels (Fig. 6C). However, in contrast to what was seen with IFN-{gamma} and TNF-{alpha}, stimulation of either unseparated or CD25+-depleted cell populations did not result in any further increase in IL-2 levels (Fig. 6C). Taken together, our data show that FL CD25+ T cells significantly suppress CD3/CD28 costimulation induced production of IFN-{gamma} and TNF-{alpha} as well as suppresses constitutive production of IL-2 from autologous nodal CD25– T cells.


Figure 6
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  		FIGURE 6. Intratumoral CD25+-enriched cells from FLN inhibit cytokine production from autologous nodal T cells. Single-cell suspensions from FLN were depleted of CD25+ cells as described. CD25-depleted cells (CD25–) were either unstimulated or stimulated with plate-bound anti-CD3 and anti-CD28 mAbs in the presence of autologous CD25+-enriched cells. Culture supernatants were harvested after 5 days and analyzed for soluble amounts of IFN-{gamma} (A), TNF-{alpha} (B), and IL-2 (C) using a multiplex assay. Plotted is coculture/stimulation conditions vs cytokine amount (picograms per milliliter). The lower limits of detection for the IFN-{gamma}, TNF-{alpha}, and IL-2 assays were 20, 2.3, and 6.7 pg/ml, respectively.

 
FLN-infiltrating CD25+ cells inhibit proliferation of TCR-stimulated/CD28-costimulated allogeneic CD8+CD25– and CD4+CD25– NLN and normal PB T cells

Whereas TR have previously been shown to inhibit alloreactivity (42, 43), TR derived from lung tumors were able to suppress autologous but not allogeneic PB-derived T cells stimulated with plate-bound anti-CD3 and anti-CD28 Abs (25). As such, we next determined whether the FLN-derived CD25+ TR, unlike lung tumor-derived TR, could suppress allogeneic T cells from either NLN or normal donor PBL, vigorously stimulated with plate-bound anti-CD3 and anti-CD28 mAbs. As is evident in Fig. 7, stimulation of CD25– cells from either NLN (donors NLN01 and NLN02) or normal donor PBL (donor PBL01) results in significant proliferation of both CD8+CD25– and CD4+CD25– T cells. Addition of unlabeled autologous normal nodal CD25+-enriched cells to the NLN01 and NLN02 CD25– cells results in a modest decrease in the proliferation of both CD8+CD25– and CD4+CD25– T cells (Fig. 7, A and B). However, the addition of autologous normal PBL01-derived CD25+-enriched cells to the stimulated CD25– PBL01 does not result in a significant decrease in CD8+CD25– or CD4+CD25– T cell proliferation (Fig. 7C), as is consistent with previous studies using T cell targets vigorously stimulated with anti-CD3/CD28 (44). To assess whether the FL-infiltrating CD25+-enriched cells can suppress allogeneic CD8+CD25– or CD4+CD25– T cells, we added unlabeled allogeneic FLN01 or FLN03 CD25+-enriched cells (an equivalent number of cells as was used for the autologous CD25+-enriched cell add-back experiments) to the CFSE-labeled NLN or normal donor PBL CD25– cells and assessed proliferation upon stimulation. As shown in Fig. 7, A and B, addition of FLN01 or FLN03 CD25+-enriched cells, respectively, to the stimulated allogeneic CD25– NLN01 or NLN02 cells, respectively, results in a marked decrease in both CD8+CD25– and CD4+CD25– T cell proliferation. In both instances, normal nodal CD8+CD25– and CD4+CD25– T cell proliferation is inhibited to a greater extent by allogeneic FLN-derived CD25+ cells as compared with that of autologous normal nodal CD25+ cells (Fig. 7, A and B). Lastly, although unlabeled autologous CD25+-enriched cells from PBL01 are unable to noticeably suppress proliferation of stimulated autologous CD8+CD25– or CD4+CD25– T cells, the allogeneic FLN03 CD25+ cells are able to significantly decrease proliferation of the PBL01 T cells (Fig. 7C).


Figure 7
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  		FIGURE 7. Intratumoral CD25+-enriched cells from FL actively inhibit the proliferation of allogeneic nodal and allogeneic PB CD8+CD25– and CD4+CD25– T cells. NLN cells and normal donor PBL were depleted of endogenous CD25+ cells as described. The resultant CD25– cells were labeled with CFSE and either left unstimulated or were stimulated with plate-bound anti-CD3 and anti-CD28 mAbs in the presence of unlabeled autologous or allogeneic CD25+-enriched cells derived from FLN (FLN01 or FLN03). CFSE-positive, CD8+ and CD4+ cells, were gated, as described in Fig. 4, to determine the percentage of cellular proliferation. A, NLN01 CD25– cells cocultured with autologous or allogeneic FLN01 CD25+-enriched cells. B, NLN02 CD25– cells cocultured with autologous or allogeneic FLN03 CD25+-enriched cells. C, PBL01 CD25– cells cocultured with autologous or allogeneic FLN03 CD25+-enriched cells. D, FLN03 CD25– cells cocultured with autologous, allogeneic NLN02, or allogeneic PBL01 CD25+-enriched cells. Plotted is T cell subset vs percent of cells proliferating.

 
To determine whether the enhanced suppression observed for the FLN CD25+-enriched cells is a function of the allogeneic suppression assay or due to an increase in suppressive activity of the FLN CD25+ cells themselves, we also determined whether proliferation of FLN03-infiltrating CD8+CD25– and CD4+CD25– T cells were similarly suppressed by the addition of allogeneic CD25+-enriched cells derived from normal nodes (NLN02) or normal PB (PBL01). As is evident from Fig. 7D, CD25– T cells from FLN03 (both CD8+CD25– and CD4+CD25– T cells) proliferate in response to CD3/CD28 costimulation. Upon addition of autologous FLN03 CD25+-enriched cells to the FLN03 CD25– cells, the percentage of CD8+CD25– and CD4+CD25– T cells proliferating poststimulation decreases significantly. However, the addition of an equivalent number of allogeneic CD25+-enriched cells from either NLN02 or PBL01 does not significantly reduce the FLN03 CD25– T cell proliferation (Fig. 7D). Our data show that the enhanced allogeneic suppression exhibited by the FLN-derived CD25+-enriched cells is characteristic of the cells themselves and not a function of the allogeneic suppression assay.

The question then arises, whether there is a true qualitative difference between the suppressive capacity of the FLN-derived TR vs those derived from normal donors, or is there a quantitative difference in the numbers of "true" TR between the CD25+-enriched populations. To address this question, we examined the FOXP3 expression within the CD25+-enriched cell fractions to determine whether there is an increased percentage of FOXP3+ cells within the FLN CD25+ fractions vs those from NLN and normal PBL. In this regard, the CD25+-enriched cells from FLN01 contained 60% FOXP3+ cells, while NLN02 and PBL01 contain 23 and 16% respectively (Fig. 8A). This could therefore account for our initial findings of increased suppressive capacity of the CD25+-enriched cells from the FLN. To directly compare suppressive capacity of the TR between the FLN and NLN or normal donor PBL, we again setup the coculture assays, as described above, except we added back equivalent numbers of FOXP3+ cells to the CFSE-labeled CD25– cells and once again assessed proliferation upon stimulation. As shown in Fig. 8B, addition of FLN01-derived FOXP3+ cells results in significant suppression of PBL01 CD8+CD25– T cell proliferation. Interestingly, addition of an equivalent number of PBL01 FOXP3+ cells to the assay does not result in a significant decrease in proliferation. Furthermore, addition of an equivalent number of PBL01 FOXP3+ cells to the allogeneic FLN01 CD25– cells also does not result in suppression, while addition of autologous FLN01 FOXP3+ cells significantly inhibits FLN01 CD8+CD25– T cell proliferation. Thus, on a 1:1 FOXP3+ cell basis, the FLN01-derived TR cells are qualitatively more suppressive than those derived from normal donor PBL01. To address the question of whether the difference in suppressive capacity is the result of anatomical localization of the CD25+ cells (PBL vs lymph node), we performed a similar experiment normalizing the numbers of FOXP3+ cells added back to coculture assays derived from either malignant or nonmalignant lymph nodes (FLN05 vs NLN02). Interestingly, the CD25+-enriched cells derived from FLN05 contained only 9% FOXP3+ cells, however, as is evident from Fig. 8C, and similar to what was observed with FLN01, the FLN05 CD25+ cells were significantly more suppressive on a 1:1 FOXP3+ cell basis as compared with those derived from NLN02. For example, addition of 10-fold fewer FLN05 FOXP3+ cells results in a level of suppression that is still greater than that observed with the autologous NLN02 FOXP3+ cells (Fig. 8C). We have also observed similar enhanced suppressive capacity of the FLN CD25+ cells (which are comprised of >95% CD3+ T cells) as compared with that of NLN or PBL when using T cell-enriched targets (by negative selection with T cell enrichment columns normally resulting in populations of >98% CD3+ T cells), thus ruling out the possibility that accessory cells (i.e., APCs, macrophages, NK cells), or the lymphoma cells themselves are significantly contributing to the enhanced suppressive capacity of the FLN-derived CD25+ cells (data not shown). As such, our results indicate that the CD25+ cell population derived from FLN often contain a higher relative proportion of FOXP3+ cells as compared with that of either the normal donor PBL or NLN CD25+ cells examined, but also, on a 1:1 FOXP3+ cell basis, regardless of the extent of FOXP3+ cell infiltration, the FLN cells are significantly more suppressive than the cells derived from either normal donor PBL or NLN.


Figure 8
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  		FIGURE 8. Intratumoral FOXP3+ cells from FLN exhibit enhanced suppressive capacity, as compared with those derived from NLN or normal donor PBL. A, Single-cell suspensions of normal donor PBL, NLN, and FLN were separated using MACS CD25+ MicroBeads and the CD25+-enriched fractions were stained for surface CD4+ and intracellular FOXP3+ as described. Shown is a representative experiment for samples PBL01, NLN02, and FLN01. Plotted is the intensity of CD4 vs FOXP3. B, FLN01 (left) or PBL01 (right) CD25– cells cocultured with autologous or allogeneic CD25+ cells, normalized to the numbers of FOXP3+ cells, resulting in a 2:1 CD25– target cell:FOXP3+ cell ratio. Plotted is T cell subset vs percent cells proliferating. C, NLN02 CD25– cells cocultured with autologous or allogeneic FLN05 CD25+ cells, normalized to numbers of FOXP3+ cells, at the indicated CD25– target cell:FOXP3+ cell ratios.

 
TGF-beta blockade partially abrogates the suppressive capacity of FLN-infiltrating CD25+ cells

The exact mechanisms by which TR cells exert their suppressive effects remain poorly defined. However, TGF-beta signaling has been implicated in a number of murine and human models (45, 46). As such, we examined the effects of blocking TGF-beta on the T cell hyporesponsiveness observed within the unseparated FLN single-cell suspensions. Addition of rLAP of TGF-beta1, a peptide that can block TGF-beta activity by directly inhibiting active soluble and/or cell-bound TGF-beta or by blocking the association of latent TGF-beta with activating molecules (47, 48, 49), to the unseparated single-cell suspensions from either FLN01 or FLN05 results in a dose-dependent increase in proliferation of the both CD8+ T cells (Fig. 9) as well as CD4+ T cells (data not shown), although not to levels observed upon CD25+ cell depletion (Fig. 9, far right bars). Interestingly, addition of 100 µg/ml neutralizing anti-TGF-beta mAb has no effect on the proliferation of either CD8+ (Fig. 9) or CD4+ (data not shown) T cell proliferation. This is consistent with results reported by Nakamura et al. (45) who demonstrated that neutralizing anti-TGF-beta Abs are not as effective at abrogating TGF-beta-mediated TR suppression as is the addition of rLAP, which is presumably inhibiting both the active and nonactive forms of TGF-beta. As such, our results indicate that TGF-beta may play a mechanistic role in the suppressive capacity of FL-infiltrating TR cells.


Figure 9
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  		FIGURE 9. Intratumoral FL TR suppression can be partially abrogated through inhibition of TGF-beta1. CD8+ T cell proliferation was assessed within unseparated CFSE-labeled FLN01 (A) or FLN05 (B) single-cell suspensions treated in vitro with the indicated amounts of rLAP or neutralizing anti-TGF-beta1 mAb. Plotted is the in vitro treatment (micrograms per milliliter of rLAP or neutralizing anti-TGF-beta1 Ab) as compared with CD25+ depletion vs percent cells proliferating. Nearly identical results were obtained analyzing the CD4+ T cell subsets (data not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Given the central role that both CD8+ and CD4+ T cells play in tumor immunity and immune surveillance, it is likely that the T cell hyporesponsiveness we demonstrate that occurs in FL contributes to tumor progression. Indeed, there are multiple lines of evidence to suggest that the host immune response plays a critical role in lymphoma progression and response to treatment. For example, gene expression profiling defines discrete subsets of FL, each having a different natural history, based on the expression of immune response genes from the infiltrating immune effector cells (6, 7). Moreover, the degree of lymphoma T cell infiltration has prognostic significance (50, 51). Recent studies also suggest that the lymphoma-associated macrophage content is an independent predictor of survival in FL, with those patients whose tumor has a high lymphoma-associated macrophage, having a worse prognosis (52). In addition, the clinical observation that patients with FL can undergo spontaneous regression, lends further support to the role of the immune response in lymphoma progression. Indeed, evaluation of "host cell infiltrates" in nodal biopsies of FL patients who have had undergone spontaneous regression demonstrate the presence of significantly more Th cells than that seen in biopsies of FL patients with progressive disease (53). In addition, using in vitro T cell cultivation techniques, it has been shown that there exists FL-infiltrating T cells that display antitumor specificity (54). Indeed, the augmentation of naturally occurring antitumor T cells, through ex vivo cell expansion, is being tested clinically (55). Finally, promising clinical results are being seen in studies of Id vaccination for FL, with evidence of the elicitation of peripheral antitumor CD8+ and CD4+ T cell responses (3, 4, 5). Taken together, we would anticipate that the restoration of intratumoral CD8+ and CD4+ T cell responsiveness may in and of itself lead to tumor regression and would also likely augment the efficacy of such immunotherapeutic strategies as Id vaccination and possibly rituximab by augmenting the potential "vaccinal" effect of this agent as described earlier.

Our findings of increased numbers of phenotypically identifiable TR-infiltrating FLN, as compared with that seen in either NLN or RLN, suggests a possible mechanism for the CD8+ and CD4+ T cell hyporesponsiveness that we demonstrate herein. To our knowledge, this is the first demonstration that FL-associated TR are capable of functionally suppressing the proliferation of CD3/CD28-costimulated autologous CD8+CD25– T cells. Furthermore, we also confirm the findings of Yang et al. (34) that such TR suppress the proliferation of autologous CD4+CD25– T cells and extend the observation to show that these TR suppress CD4+CD25– (and CD8+CD25–) T cells potently stimulated through direct TCR engagement (using plate-bound anti-CD3/anti-CD28 Ab stimulation) rather than with PHA. In addition to showing that removal of CD25+ cells in vitro restores CD8+ and CD4+ T cell proliferative responses, we also show that such removal restores IFN-{gamma} and TNF-{alpha} production upon T cell stimulation. CD25+ cell removal also results in an increase in IL-2 production by both unstimulated and stimulated T cells. This is of interest in that one potential mechanism of suppression by TR is the competition for IL-2 between TR cells and effector T cells, as the TR express the high-affinity heterochimeric IL-2R complex (56, 57, 58). As such, when the TR are depleted, there is more IL-2 available. Addition of IL-2 to the unseparated and stimulated FLN T cells did not, however, consistently restore T cell responsiveness (data not shown), suggesting that this may not be a dominant mechanism of TR suppression within the context of FL. That stimulation of CD25– cells in the absence of CD25+ cells does not result in a significant production of IL-2 was likely due to the fact that cytokine levels were assessed after 5 days of stimulation at which time much of the soluble IL-2 produced by the CD25–-stimulated cells was most likely sequestered by the activated T cells within these cultures.

In this regard, the mechanism/s by which TR in general suppress T cell function remains unclear. There has been much controversy over the role of TGF-beta in mediating the suppressive effects of TR. TR can secrete TGF-beta and/or express cell surface TGF-beta, depending on the potency and type of stimulation through the TCR (45). Similar to other studies, using a high dose of a TGF-beta-neutralizing Ab we were unable to restore any proliferation of FL T cells. It has recently been suggested, however, that this may be due to the fact that such Abs may not block the ability of latent TGF-beta to bind to sites where TGF-beta is activated and/or block the ability of TGF-beta to bind to its receptor after it is activated. Therefore, it was proposed that rLAP may be more useful as it can act on TGF-beta before it is activated. Using this approach, Nakamura et al. (45) have shown TGF-beta to play a critical role in mediating CD4+CD25+ TR suppression. When we add rLAP to unseparated FL T cells we find a dose-dependent increase in T cell proliferation, although not to the extent seen with CD25+ cell depletion. This suggests that TGF-beta is contributing to the T cell suppression by the TR. These findings further suggest that inhibition of TGF-beta, such as that seen using small molecule inhibitors of TGF-betaRI kinase, may have therapeutic potential alone or in combination with vaccination strategies in the treatment of lymphoma.

It is not surprising, particularly given the robust stimulation used in our system, that TGF-beta blockade would not completely restore T cell proliferation and that other mechanisms of suppression are also operational. For example, Yang et al. (34) have suggested a potential role for programmed death 1 (PD-1) signaling in the mechanisms by which B-NHL-derived CD4+CD25+ TR may suppress infiltrating CD4+CD25– T cells. In this regard, we have also seen higher expression of PD-1 on FL-infiltrating CD8+ and CD4+ T cells, as compared with that of NLN or RLN (data not shown), however, in our studies we were unable to abrogate TR suppression through addition of blocking anti-PD-1 Abs as demonstrated by Yang et al. (34). The differences between our results and those reported by Yang et al. (34) may be due to technical difference between the in vitro suppression assays used by our laboratory as compared with theirs, such as stimulation using plate-bound Abs vs stimulation using PHA, respectively.

Although CD4+CD25+ TR have been shown to block allogeneic stimulation of T cell proliferation, as well as prevent allograft rejection in both in vitro and in vivo models (43, 59), the strength of the stimulus provided in our assays, plate-bound anti-CD3 and anti-CD28 Abs, has been shown to be sufficient to overcome the suppressive effects of murine TR (60, 61), autologous human TR (44), as well as prevent suppression of allogeneic T cells by TR derived from lung tumors (24). In contrast, FLN-derived CD25+ TR are capable of suppressing autologous and allogeneic T cells stimulated with plate-bound anti-CD3 and anti-CD28 Abs. In fact, our data further show that FL-derived CD25+ cells are more potent suppressors of autologous and allogeneic T cell proliferation then are NLN and normal PBL-derived CD25+ cells. Initially, we hypothesized that the differences in suppression among equal numbers of CD25+ cells derived from FLN, NLN, and PB were due to differences in the proportion of such cells that were true FOXP3+ TR. Indeed, although such differences did exist, when equivalent numbers of FOXP3+ cells were used in the coculture experiments, the FL FOXP3+ TR were significantly more suppressive than those derived from NLN or PB. To our knowledge, this is the first data to suggest that a qualitative difference exists between the CD4+CD25+FOXP3+ cells derived from malignant FL nodes compared with that derived from NLN or normal donor PBL, with the FL-derived TR having stronger suppressive effects. However, our studies do not rule out the possibility that the differences in the degree of suppression between the malignant vs normal CD25+ cells may be due to differences in other suppressor T cell populations.

Finally, whereas others have compared T cell reactivity from malignant and reactive nodes to that of normal donor PBL, to our knowledge, this is the first direct comparison of T cell reactivity (through CD3/CD28 costimulation) between malignant (FL) and RLN compared with that of NLN T cells. Indeed, Agrawal et al. (8) concluded that the hyporesponsiveness of FL T cells was due, in part, to chronic inflammation, as T cells from RLN were hyporesponsive relative to that of PBL T cells. However, in our study FL-derived T cells were not at all responsive to stimulation, whereas T cells from RLN and NLN were similarly responsive (and less responsive that PB T cells), suggesting that the lowered responsiveness of RLN T cells may not be due to inflammation as suggested by Agrawal et al. (8).

In summary, our studies show that: 1) infiltrating FL TR functionally suppress both intratumoral CD8+ and CD4+ T cells independent of any contribution that accessory cells, and/or lymphoma cells, may have on T cell suppression; 2) such suppression is, in part, TGF-beta mediated; and 3) there appears to be a qualitative difference in the potency of CD4+CD25+FOXP3+ cells derived from malignant nodes compared with that derived from normal nodes or PB. Our data further suggest that strategies designed to either inhibit TR function, such as using small molecule inhibitors that block TGF-beta signaling, or eliminate intratumoral TR altogether, such as using ontak, a fusion protein combining diphtheria toxin and IL-2 that targets cells expressing the IL-2R, may have therapeutic benefits for patients with FL. In addition, inhibiting or eliminating TR before Id vaccination, or other immunotherapeutic approaches, such as rituximab treatment, may enhance the efficacy of such therapies. This is particularly important in light of recent studies with a murine lymphoma model that demonstrated that vaccination against a model tumor Ag could elicit an Ag-specific TR response (62). Lastly, a more thorough understanding of the molecular differences that relate to the functional differences between malignant and normal TR that are seen in this study will provide further insight into mechanisms of immune dysregulation in cancer.


   Acknowledgments
 
We thank Drs. Tim Mosmann and Alexandra Livingstone for stimulating and critical discussions. We also thank Drs. Craig Jordan and Timothy Bushnell from the University of Rochester’s Center for Pediatric and Biomedical Research Flow Laboratory for their flow cytometry assistance and support.


   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 a Leukemia and Lymphoma Society Translational Research Grant, a James P. Wilmot Cancer Center Scholar Award, University of Rochester Human Immunology Core Pilot Project Grant 5-29712, and U.S. Public Health Service Grants R01-CA 10897 and R01-GM 65237. Back

2 Address correspondence and reprint requests to Dr. Steven H. Bernstein, Lymphoma Biology Program, James P. Wilmot Cancer Center, University of Rochester Medical Center, 601 Elmwood Avenue, Box 704, Rochester, NY 14642. E-mail address: Steven_Bernstein{at}urmc.rochester.edu Back

3 Abbreviations used in this paper: FL, follicular lymphoma; FLN, FL node; TR, regulatory T cell; GITR, glucocorticoid-induced TNFR-related protein; NLN, normal lymph node; RLN, reactive lymph node; rLAP, recombinant latency associated peptide of TGF-beta1; DLCL, diffuse large cell lymphoma; B-NHL, B non-Hodgkin’s lymphoma; PD.1, programmed death 1. Back

Received for publication June 22, 2006. Accepted for publication January 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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