Phenotype, effector function, and tissue localization of PD-1-expressing human follicular helper T cell subsets
© Wang et al; licensee BioMed Central Ltd. 2011
Received: 3 May 2011
Accepted: 13 September 2011
Published: 13 September 2011
It is well established that PD-1 is expressed by follicular T cells but its function in regulation of human T helper cells has been unclear. We investigated the expression modality and function of PD-1 expressed by human T cells specialized in helping B cells.
We found that PD-1-expressing T cells are heterogeneous in PD-1 expression. We identified three different PD-1-expressing memory T cell subsets (i.e. PD-1low (+), PD-1medium (++), and PD-1high (+++) cells). PD-1+++ T cells expressed CXCR5 and CXCR4 and were localized in the rim of germinal centers. PD-1+ or PD-1++ cells expressed CCR7 and were present mainly in the T cell area or other parts of the B cell follicles. Utilizing a novel antigen density-dependent magnetic sorting (ADD-MS) method, we isolated the three T cell subsets for functional characterization. The germinal center-located PD-1+++ T cells were most efficient in helping B cells and in producing IL-21 and CXCL13. Other PD-1-expressing T cells, enriched with Th1 and Th17 cells, were less efficient than PD-1+++ T cells in these capacities. PD-1+++ T cells highly expressed Ki-67 and therefore appear active in cell activation and proliferation in vivo. IL-2 is a cytokine important for proliferation and survival of the PD-1+++ T cells. In contrast, IL-21, while a major effector cytokine produced by the PD-1-expressing T helper cells, had no function in generation, survival, or proliferation of the PD-1-expressing helper T cells at least in vitro. PD-1 triggering has a suppressive effect on the proliferation and B cell-helping function of PD-1+++ germinal center T cells.
Our results revealed the phenotype and effector function of PD-1-expressing T helper cell subsets and indicate that PD-1 restrains the B cell-helping function of germinal center-localized T cells to prevent excessive antibody response.
Programmed death-1 (PD-1 or also called CD279) is a member of the CD28 family costimulatory molecules [1, 2]. Unlike CD28, PD-1 has two intracellular tyrosine signaling motifs (immunoreceptor tyrosine inhibition motif and immunoreceptor tyrosine-based switch motif)  and recruits intracellular phosphatase SHP2 (SRC homology 2 domain-containing protein tyrosine phosphatase 2) that dephosphorylates and deactivates downstream signal transducers [4, 5]. PD-1 is expressed by a number of immune cell types including activated T cells, B cells, dendritic cells, monocytes, and mast cells in mice. As the ligands for PD-1, PD-L1 (CD274/B7-H1) and PD-L2 (CD273/B7-DC) have been identified [6, 7].
In general, engagement of PD-1 by PD-L1 or PD-L2 inhibits TCR-mediated T cell proliferation and cytokine production [8, 9], indicating that the cross-linking of PD-1 by its ligands leads to down-regulation of T cell responses in a manner somewhat similar to the effect of CTLA4 stimulation. PD-1-deficient mice are prone to develop autoimmune diseases such as autoantibody formation, dilated cardiomyopathy, acute type I diabetes, and bilateral hydronephrosis [10, 11]. In humans, single nucleotide polymorphisms in the PD-1 gene are linked to a number of autoimmune diseases including lupus, rheumatoid arthritis, Graves' disease, type I diabetes, multiple sclerosis, ankylosing spondylitis, and myocardial infarction [12–18]. In mice, blocking of PD-1 exacerbated a lupus-like nephritis . Also, triggering of PD-1 suppressed rheumatoid arthritic symptoms . While PD-1 and its ligands are thought to function to promote immune tolerance, it was also reported that mice deficient in PD and their ligands had fewer long-lived plasma cells, suggesting a certain positive role of PD-1 in regulation of humoral immunity in mice .
PD-1 is highly expressed by a subset of T cells in the germinal centers (GC) [22–25]. In contrast, most human B cells do not express PD-1 . Additionally, PD-1 is preferentially expressed on exhausted CD8+ T cells during chronic viral infection [26–29]. Although the suppressive function of PD-1 on CD8+ T cells has been studied extensively, the phenotype and role of PD-1-expressing CD4+ T helper cells in regulation of humoral immune responses have been unclear. We investigated the phenotype and function of PD-1-expressing T helper cells in human tonsils and the function of PD-1 in regulation of these T cells. Our study revealed that PD-1-expressing human helper T cells are heterogeneous in PD-1 expression, chemotactic response, tissue localization, cytokine response, and effector function. Moreover, triggering of PD-1 can restrain the B cell-helping function of the PD-1high (+++) T cells.
PD-1-expressing T helper cells are heterogeneous in PD-1 expression and tissue localization in human tonsils
We investigated the localization of the PD-1-expresing T cells. The PD-1+++ cells that expressed PD-1 at the highest level were localized in the outer rim of GC adjacent to the mantle zone (Figure 1D-a). In contrast, PD-1 dim (+/++) cells were frequently found in either the center of GC or interfollicular areas (IFA; Figure 1D-a and 1b). PD-1+++ T cells were localized close to IgD+ B cells in the mantle zone (Figure 1D-c and 1d). Some PD-1-expressing T cells were found even inside the mantle zone (Figure 1D-e). There was no clear association of the sites of PD-1-expressing T cells and dendritic cells (Figure 1D-f). Both small and large germinal centers had PD-1-expressing T cells (Figure 1D-g and 1h). The numbers and location of these T cells in various primary and secondary follicles demonstrate that PD-1 high (+++) T cells are the germinal center T cells, while PD-1 dim (+/++) T cells are in the T cell area or mantle zone.
PD-1-expressing T cells differentially express secondary lymphoid tissue-homing chemokine receptors
Next, we examined the activity of the chemokine receptors expressed by the PD-1-expressing T cells (Figure 2B). PD-1+++ T cells were highly responsive to CXCL13 but poorly migrated to CCL19. PD-1+/++ T cells were responsive to both CXCL13 and CCL19 at moderate levels. PD-1+++ T cells were more responsive to CXCL12 than PD-1++ T cells. Overall the chemotactic responses were in line with the expression levels of chemokine receptors.
Because the high expression of CXCR4 by PD-1+++ T cells and their localization in the rim of GC, we examined the in situ expression of CXCL12 at protein level (Figure 2C). It was found that CXCL12 was expressed by the stromal cells in the mantle zone and throughout the interfollicular area but not significantly within GC. It was notable that the site of CXCL12-expression in the mantle zone was found right next to the sites of PD-1+++ T cell localization (Figure 2C). Thus, it appears that PD-1+++ T cells localize to the outer rim of GC adjacent to the mantle zone because of the combined chemotactic force of CXCL12 and CXCL13 in the absence of the chemotactic influence of CCL19/CCL21.
PD-1-expressing T cells have distinct surface antigen and cytokine production phenotypes
We further investigated the cytokine production capacity and co-stimulation receptor expression property of the PD-1-expressing T cells. All of the three PD-1-expressing T cell subsets were able to produce IL-2 and TNF-α (Figure 3B). PD-1- T cells were largely of naïve T cells and included some cells capable of producing IL-2 (~12%) and TNFα (7%). However, the PD-1-expressing T cells were heterogeneous in production of IFN-γ and IL-17: PD-1+++ cells included few Th1 or Th17 cells, while PD-1+ T cells contained most Th1 and Th17 cells, suggesting that most polarized effector T cells belong to the PD-1+ T group (Figure 3B). PD-1+++ T cells contain a small number (~5%) of IL-4 or IL-10-producing T cells. IL-5 or IL-13 producers were hardly detected. PD-1+++ T cells highly expressed co-stimulator receptors such as OX40 and ICOS (Figure 3C). Also, intracellular CD40L was expressed more highly by PD-1+++ T cells than PD-1+ or PD-1++ cells.
The PD-1-expressing T cells are heterogeneous in production of CXCL13, cell proliferation and survival
Roles of IL-2 and IL-7 in survival and proliferation of PD-1-expressing T cells
Lack of function for IL-21 or IL-6 in maintenance or generation of PD-1+++ T cells in vitro
IL-6 and IL-21 are implicated in generation of follicular helper T cells in mice [31–33]. We examined the impact of IL-6 and IL-21 on the stability of the PD-1-expresing T cells. For this, the T cell subsets were cultured for 6-7 days in the indicated conditions, and expression of CXCR5, CCR7, and PD-1 was examined. Naïve T cells gained some expression of CXCR5 but they did not lose CCR7. PD-1+, PD-1++, and PD-1+++ T cells maintained the expression of CXCR5, CCR7 and PD-1 throughout the culture period (Figure 6C and 6D). IL-6 and IL-21 had no effect on the stability of the PD-1-expresing T cells in terms of expression of PD-1, CXCR5 and CCR7.
We consider that expression of CXCR5, PD-1, CXCL13, and IL21; and loss of CCR7 are the features of mature follicular helper T cells that can effectively help B cells and are localized in the GC [30, 33–37]. We examined if IL-6 and/or IL-21 has any role in generation of the PD-1-expressing CXCR5+ B cell-helping T cells. We observed that these cytokines did not promote the expression of CXCR5, IL-21, or CXCL13 by antigen-primed human naïve T cells at the mRNA (Figure 6E) and protein level (not shown).
PD-1 triggering restrains the B cell-helping ability of PD-1+++ T cells in a cell-specific manner
We investigated the phenotype and function of PD-1-expressing T helper cell subsets in human tonsils. Unexpectedly, PD-1 is expressed by all memory, but not naïve, T cells in tonsils. The PD-1-expressing T cells, however, are heterogeneous in the PD-1-expression level, trafficking receptor phenotype, tissue localization, and effector function in helping B cells. PD-1+++ T cells can stimulate GC B cells for generation of plasma B cells within the GC, while PD-1dim (+/++) T cells are enriched with Th1 and Th17 cells, reside in the mantle zone of GC or interfollicular area, and have weaker B cell-helping activity. Interestingly, PD-1 triggering limits the activity of PD-1+++ T cells in helping B cells.
Expression of several receptors such as CXCR5, CXCR4 and CCR7 is the major determining factor for localization of lymphocytes in lymphoid tissues that are divided into T and B cell areas including the interfollicular area, GC, and mantle zone. Coordinated expression of the three receptors regulates the exact microanatomical positioning of lymphocytes in mice [36, 39]. This is because the chemokine ligands that bind the receptors are differentially expressed in the T cell area (CCL19 and CCL21, CCR7 ligands), the B cell area (CXCL13, the CXCR5 ligand), and certain areas of GC/interfollicular area (CXCL12, the CXCR4 ligand) [40–43]. The expression patterns of CXCL12 in mice and humans are somewhat different from each other. In mice CXCL12 is more expressed in the GC dark zone , while it is expressed at mRNA level by specialized reticulum cells that surround GC  or at the protein level in the mantle zone and interfollicular area in human tonsils as shown in this study. The chemokine receptor phenotypes of PD-1 expressing T cell subsets are in line with the expression sites of the three chemokines. PD-1+++ T cells are CXCR4++ CXCR5++ CCR7low and reside in the rim of GC adjacent to the mantle zone. PD-1++ T cells are CXCR4+ CXCR5+ CCR7low, which would put them elsewhere in GC. The CXCL12 expressed in the mantle zone would attract PD-1+++ T cells but these cells also highly express CXCR5 to stay within the GC close to the mantle zone. PD-1++ T cells express CXCR5 but not CCR7 and stay within the GC. They express CXCR4 at a reduced level and, thus, are more scattered throughout GC instead of localized to the rim of GC. PD-1+ T cells express both CXCR5 and CCR7 at a moderate level and would stay in the peri/interfollicular area outside of GC as the result of the balanced chemoattraction between CXCL13 and CCR7 ligands (CCL19 and 21).
An interesting function of human GC-T cells is to produce CXCL13 , which is thought to attract CXCR5+ T cells, B cells, dendritic cells, and follicular dendritic cells to form and expand GC. Interestingly, the CXCL13 production ability of PD-1-expressing T cells is positively associated with their PD-1 expression level. We found that the GC-localized PD-1+++ T cells are highly efficient in production of CXCL13. Other features of these PD-1-expressing GC-T cells include expression of LFA3, CD40L, ICOS, OX40, and CD10. LFA3 is important for cell-cell interaction, and thus, is likely to play a role in their interaction with other cells for cell activation and effector function . ICOS, CD40L and OX40 are co-stimulatory receptors which would be involved in activating target cells (e.g. B cells) or the T cells themselves. We observed that the ICOS and CD40L signals are required for the optimal B cell-helping activity of PD-1+++ T cells. The function of CD10 in GC is unclear at this time but it is likely that PD-1+++ T cells would be a normal counterpart of CD10+ angioimmunoblastic T-cell lymphoma in human patients [47, 48].
It has been determined previously that GC-T cells are prone to cell death and do not proliferate well . Some even called the cells anergic . The dilemma in calling them "anergic" is that many T cells in GCs are active in cell cycling  and they are functionally active in producing IL21 and CXCL13 and helping B cells. These functions cannot be performed, at least by definition, by anergic T cells. Moreover, PD-1+++ T cells, although as suggested before to be apoptotic and non-proliferative, can be driven to survive and proliferate in response to IL-2. IL-2 and IL-7 can rescue the T cells from cell death. Active proliferation of PD-1+++ T cells in vivo is supported by expression of Ki-67 by many PD-1+++ cells in tonsils. These results suggest that the PD-1+++ GC-T cells are actively propagating in the GC environment in response to antigen presenting cells and cytokines. We noted also that the PD-1+++ T cells were highly stable and did not lose their phenotype in expression of PD-1 and CXCR5 and in B cell-helping function upon subsequent antigen stimulation.
It is still controversial what directly regulates the generation of fully committed B cell-helping T cells, which express IL21, CXCL13, CXCR5 and PD-1 but not CCR7. ICOS, IL-21, and IL-6 are implicated in increasing the number of CXCR5-expressing T cells or B cell-helping T cells in mice [32, 33, 51]. As internal transcription factors, Bcl6, c-Maf, STAT3 and BATF1 are implicated in regulating the number of CXCR5+ or B cell-helping T cells in vivo [51, 52]. IL-12, a cytokine for Th1 cells, can increase the CXCR5 and IL21 expression by human T cells . Also, IL-4 can increase the CXCR5 expression by naïve T cells in response to activation by dendritic cells . We found in this study that IL6 and IL-21 lack the ability to induce the fully differentiated GC-T cells in vitro. One should note that CXCR5 induction is a spontaneous event following T cell priming in vivo , and is not sufficient by itself to define fully committed B cell-helping effector T cells. Also, IL21 is widely produced by different T cell types, and, thus, expression of IL21 alone would not be sufficient to define fully differentiated GC-T cells. In this regard, to date, none really established a reproducible in vitro system to induce a stable B cell-helping effector T cell lineage with a GC tropism (PD-1+++ CXCR5+ CCR7-). Our results point out that there are several subsets of CXCR5+ T helper cells with distinct phenotype and cell function. "Follicular T helper cells," defined by CXCR5 expression alone, is an ambiguous term, and these T cells are actually comprised of heterogeneous T helper subsets. Additional phenotypes such as effector function, tissue localization and expression of additional antigens such as CCR7 should all be considered in characterization of the diverse B cell-helping T cells.
A key question would be "what is the function of PD-1, highly expressed by GC-T cells?" It has been established that mice deficient with PD-1 expression are prone to develop various autoimmune diseases [10, 11]. Some of these autoimmune diseases are induced, in part, by autoantibodies produced by B cells, a process promoted by GC-T cells. Thus, we hypothesized that PD-1 has the function of down-regulating the T-cell-dependent B cell responses. To test this hypothesis, we stimulated the PD-1 by PD-L2-Fc protein and observed that the PD-1+++ T cell-dependent antibody response was decreased. Also, observed was suppressed proliferation of PD-1+++ T cells by the PD-1 triggering. However, the production capacity of CXCL13 was not altered. These results suggest that PD-1 can limit the magnitude of GC-T cell response. The degree of suppression following PD-1 triggering was moderate which suggests that PD-1 triggering would not completely shut down the GC-T cell response. Hyper-activity of PD-1+++ T cells would cause chronic inflammation or autoimmune diseases. PD-1 appears to function to restrain the function of PD-1+++ T cells possibly to prevent the aberrant humoral immune responses.
PD-1-expressing human T helper cells are highly heterogeneous, including PD-1high (+++), PD-1medium (++), and PD-1dim (+) cells in tonsils. Among them, PD-1+++ T cells have the phenotype of the germinal center T helper cells in tissue localization, cellular phenotype and effector function. Triggering of PD-1 restrains the B cell-helping activity and proliferation of PD-1+++ germinal center T cells, supporting the role of PD-1 in promoting tolerance in humoral immunity.
Isolation of T cells expressing PD-1 at different levels using a novel antigen density-dependent magnetic sorting (ADD-MS)
The use of human tonsils for this study has been approved by the institutional review board at Purdue. The specimens, byproducts of surgeries and obtained as pathological specimens without any associated patient information, were exempted from obtaining consent forms by the review board. Human tonsil specimens were obtained from young patients (3-10 yr) undergoing tonsillectomy to relieve obstruction of respiratory passages and improve drainage of the middle ear and had no apparent inflammation. Tonsil mononuclear cells were prepared by density gradient centrifuge on histopaque 1077 (Sigma-Aldrich, St. Louis, MO). T cells were enriched from the mononuclear cells by a sheep red blood cell (SRBC) rosetting method. CD4+ T cells were isolated by the CD4+ T cell isolation kit (Miltenyi Biotec Inc. Auburn, CA). CD25+ (Treg-enriched) T cells were depleted with anti-CD25/magnetic beads to obtain CD4+CD25- cells. Naïve and memory CD4+ T cells were isolated by depleting CD45RO+ and CD45RA+ cells respectively. To isolate the T cell subsets, we developed a novel magnetic sorting method utilizing controlled bead release based on antigen expression density. The advantage of this method is to isolate cells at a relatively high speed using a magnetic sorting method utilizing commercially available magnetic beads (anti-FITC/PE beads, Miltenyi Biotec Inc). This method was more efficient than a flow cytometry method in preparing many cells (1- 5 million sorted cells) within a short time period (5 h). PD-1+/- T cells were isolated by magnetic sorting from the CD4+CD25-CD45RA- cells by magnetically selecting PD-1++/+++ T cells. PD-1+ memory T cells were further isolated from the PD-1+/- T cell fraction by magnetically selecting PD-1-expressing cells. PD-1++/+++ T cells were cultured for 30 min at 37°C in complete RPMI-1640 to release attached beads from PD-1++ cells. PD-1+++ T cells were positively selected from the cultured PD-1+ +/+++ fraction. The negative fraction of this isolation was used as PD-1++ T cells. All of the T cell fractions were highly pure (> 97% based on the expression of CD4 and CD45RO or CD45RA). Figure 4 shows the typical purity of PD-1-expressing T cells isolated by this method. Total CD19+ B cells were isolated by depleting T cells with a SRBC-rosetting method. Naïve B cells were further isolated from the total B cells by positively selecting IgD+ cells (purity > 99%), and CD19+CD38+IgD- GC B cells (purity = ~95%) were isolated by depleting IgD+ cells and positively selecting CD38+ cells as described before .
Expression of trafficking receptors and other antigens by T cells by flow cytometry or real time PCR
The T cells isolated with SRBC-rosetting were stained with antibodies to CCR7 (150503), CXCR4 (44717.111), CXCR5 (51505.111, all from R&D Systems, Minneapolis, MN), or mouse control IgG1 (BioLegend, CA). Cells were further stained with a biotinylated horse anti-mouse IgG (H+L) antibody (Vector Lab, Mountain View) for 20 min, followed by staining with APC-streptavidin (BD Biosciences) and antibodies to PD-1 (eBioJ105), CD45RO (UCHL1), and CD4 (RPA-T4). For surface or intracellular staining, T cells were stained with antibodies to CD4 (RPA-T4), CD45RO (UCHL1), PD-1 (eBioJ105), CD58 (LFA-3, 1C3), CD134 (OX40, ACT35), CD10 (HI10a), CD127 (hIL-7R-M21), CD62L (Dreg 56), CD69 (FN50), ICOS (C398.4A), CD25 (BC96), FOXP3 (259D), Ki-67 (B56), and/or CD154 (TRAP-1). The antibodies to the antigens were purchased from R&D systems, eBioscience, BioLegend, or BD Biosciences (San Jose, CA). Intracellular staining for cytokine production was performed as described previously . Stained cells were acquired on a FACS Canto II. Real time PCR detection was performed on cDNA with a 7500 Sequence Detection System (Applied Biosystems, Foster City, CA) using the SYBR green Master Mix (Applied Biosystems). Primers used were: hIL-21-F (TTC TGC CAG CTC CAG AAG ATG), hIL-21-R (CAC TTC CGT GTG TTC TAG AGG), h-CXCR5-F (GCC AGA GAT TCT CTT CGC CAA), h-CXCR5-R (TGT CCA GGA AGA TGA CGA TGT G), h-CXCL13-F (TCC AAG GTG TTC TGG AGG TC), and h-CXCL13-R (TTT CTT GGA CAA CCA TTC CC).
Frozen sections of tonsils were cold acetone-fixed and stained with monoclonal antibodies to human CD3 (HIT3a), CD4 (RPT-T4), PD-1 (eBioJ105), CD19 (HIB19), CD21 (BU32), CD11c (3.9), and/or IgD (IA6-2). For detection of human CXCL12, the acetone fixed sections were stained with anti-CXCL12 (Clone 79018; R&D systems) and then with biotin-conjugated horse-anti-mouse IgG (H+L) (Vector lab). The sections were further stained with FITC or PE-conjugated streptavidin (eBioscience). After blocking with 10% mouse serum, the sections were stained with an antibody to PD-1. Slides were examined with a microscope equipped with epifluorescence as described previously .
Chemotaxis was performed as described previously . Human CXCL12, CCL19 and CXCL13 were purchased from R&D Systems. 5 ×105 CD4+ T cells in 100 μl of chemotaxis medium (RPMI with 0.5% BSA) were placed in each Transwell insert (5 μm pore, 24-well format; Corning Costar Corp., Cambridge, MA), and the Transwell inserts were placed in 24-well plates containing 600 μl of chemotaxis medium (RPMI-1640 with 0.5% BSA) with optimal concentrations of CCL19 (2000 ng/ml), CXCL12 (100 ng/ml) or CXCL13 (3000 ng/ml). Cells were allowed to migrate for 3 h in a 5% CO2 incubator at 37°C. After chemotaxis, the cells that migrated to the lower chambers were harvested and stained with antibodies to CD4 (RPA-T4), PD-1 (eBioJ105 (J105)), and CD45RO (UCHL1). Stained cells were acquired on a FACS Canto II, and specific percent migration after subtraction of the background migration was calculated.
Proliferation, cell survival, differentiation and CXCL13 production
Sorted T cells were cultured in U-bottomed 96-well plates for 5-6 days in the presence of phytohemagglutinin (PHA, 5 μg/ml) or anti-CD3 (5 μg/ml, immobilized) and anti-CD28 (2 μg/ml, soluble) in the presence of hIL-2 (20 U/ml), hIL-7 (20 ng/ml), hIL6 (20 ng/ml), and/or IL21 (50 ng/ml). For assessment of proliferation, cells were further incubated with 1 μCi/well of 3H-thymidine for 8 hours, and 3H-thymidine incorporation was measured by a beta scintillation counter (Packard Top Count Microplate Scintillation Counter, Packard Instruments, Meriden, CT). For the cell survival assay, isolated T cell subsets were cultured for 5 days in RPMI/10% FBS and, then, stained with 7-Amino-actinomycin D (7-AAD; final 0.5 μg/ml) immediately before flow cytometric detection of dead (7-AAD+ or FSClow) cells. The 5-day old culture medium was examined for CXCL13 production with an anti-hCXCL13 ELISA kit (R&D Systems).
Assessment of B cell-helping activity
To cross-link the B cell receptors, isolated B cells were incubated for 2 h at 4°C with sepharose-conjugated rabbit Ab to human Ig μ chain and human Ig (H + L) chain (Irvine Scientific, Santa Ana, CA; mixed 1:1 at 2 μg/ml) and then washed with cold PBS. Sorted T cells and B cells (105 each) were co-cultured in each well of 96-well plates in RPMI1640 medium supplemented with 10% FBS, gentamycin, streptomycin, and penicillin in the presence of Staphylococcal enterotoxin B (SEB; 1 μg/ml, Sigma-Aldrich, St. Louis, MO). When indicated, recombinant human PD-L2-Fc chimera (10 μg/ml, R&D Systems) or control antibodies (mouse IgG1, 11711.11, R&D systems) were added at 10 μg/ml to trigger PD-1.
Student's paired 2-tailed t test was used for statistical analysis when indicated. p values < or = 0.05 were considered significant.
List of abbreviations
Staphylococcal enterotoxin B
We thank SG Kang and JH Lee (Purdue University) for their helpful input. This study was supported, in part, from grants from NIH (R01AI074745, R01DK076616, and R01AI080769), and Crohn's and Colitis Foundation of America to CHK.
- Keir ME, Butte MJ, Freeman GJ, Sharpe AH: PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol. 2008, 26: 677-704. 10.1146/annurev.immunol.26.021607.090331.PubMedView ArticleGoogle Scholar
- Okazaki T, Honjo T: PD-1 and PD-1 ligands: from discovery to clinical application. Int Immunol. 2007, 19: 813-24. 10.1093/intimm/dxm057.PubMedView ArticleGoogle Scholar
- Ishida Y, Agata Y, Shibahara K, Honjo T: Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. Embo J. 1992, 11: 3887-95.PubMedPubMed CentralGoogle Scholar
- Okazaki T, Maeda A, Nishimura H, Kurosaki T, Honjo T: PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine. Proc Natl Acad Sci USA. 2001, 98: 13866-71. 10.1073/pnas.231486598.PubMedPubMed CentralView ArticleGoogle Scholar
- Sathish JG, Johnson KG, Fuller KJ, LeRoy FG, Meyaard L, Sims MJ, Matthews RJ: Constitutive association of SHP-1 with leukocyte-associated Ig-like receptor-1 in human T cells. J Immunol. 2001, 166: 1763-70.PubMedView ArticleGoogle Scholar
- Dong H, Zhu G, Tamada K, Chen L: B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med. 1999, 5: 1365-9. 10.1038/70932.PubMedView ArticleGoogle Scholar
- Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I, Iwai Y, Long AJ, Brown JA, Nunes R, Greenfield EA, Bourque K, Boussiotis VA, Carter LL, Carreno BM, Malenkovich N, Nishimura H, Okazaki T, Honjo T, Sharpe AH, Freeman GJ: PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol. 2001, 2: 261-8. 10.1038/85330.PubMedView ArticleGoogle Scholar
- Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, Fitz LJ, Malenkovich N, Okazaki T, Byrne MC, Horton HF, Fouser L, Carter L, Ling V, Bowman MR, Carreno BM, Collins M, Wood CR, Honjo T: Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000, 192: 1027-34. 10.1084/jem.192.7.1027.PubMedPubMed CentralView ArticleGoogle Scholar
- Carter L, Fouser LA, Jussif J, Fitz L, Deng B, Wood CR, Collins M, Honjo T, Freeman GJ, Carreno BM: PD-1:PD-L inhibitory pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2. Eur J Immunol. 2002, 32: 634-43. 10.1002/1521-4141(200203)32:3<634::AID-IMMU634>3.0.CO;2-9.PubMedView ArticleGoogle Scholar
- Nishimura H, Okazaki T, Tanaka Y, Nakatani K, Hara M, Matsumori A, Sasayama S, Mizoguchi A, Hiai H, Minato N, Honjo T: Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science. 2001, 291: 319-22. 10.1126/science.291.5502.319.PubMedView ArticleGoogle Scholar
- Nishimura H, Nose M, Hiai H, Minato N, Honjo T: Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity. 1999, 11: 141-51. 10.1016/S1074-7613(00)80089-8.PubMedView ArticleGoogle Scholar
- Prokunina L, Castillejo-Lopez C, Oberg F, Gunnarsson I, Berg L, Magnusson V, Brookes AJ, Tentler D, Kristjansdottir H, Grondal G, Bolstad AI, Svenungsson E, Lundberg I, Sturfelt G, Jonssen A, Truedsson L, Lima G, Alcocer-Varela J, Jonsson R, Gyllensten UB, Harley JB, Alarcon-Segovia D, Steinsson K, Alarcon-Riquelme ME: A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans. Nat Genet. 2002, 32: 666-9. 10.1038/ng1020.PubMedView ArticleGoogle Scholar
- Wang SC, Chen YJ, Ou TT, Wu CC, Tsai WC, Liu HW, Yen JH: Programmed death-1 gene polymorphisms in patients with systemic lupus erythematosus in Taiwan. J Clin Immunol. 2006, 26: 506-11. 10.1007/s10875-006-9048-9.PubMedView ArticleGoogle Scholar
- Lin SC, Yen JH, Tsai JJ, Tsai WC, Ou TT, Liu HW, Chen CJ: Association of a programmed death 1 gene polymorphism with the development of rheumatoid arthritis, but not systemic lupus erythematosus. Arthritis Rheum. 2004, 50: 770-5. 10.1002/art.20040.PubMedView ArticleGoogle Scholar
- Nielsen C, Laustrup H, Voss A, Junker P, Husby S, Lillevang ST: A putative regulatory polymorphism in PD-1 is associated with nephropathy in a population-based cohort of systemic lupus erythematosus patients. Lupus. 2004, 13: 510-6. 10.1191/0961203303lu1052oa.PubMedView ArticleGoogle Scholar
- Nielsen C, Hansen D, Husby S, Jacobsen BB, Lillevang ST: Association of a putative regulatory polymorphism in the PD-1 gene with susceptibility to type 1 diabetes. Tissue Antigens. 2003, 62: 492-7. 10.1046/j.1399-0039.2003.00136.x.PubMedView ArticleGoogle Scholar
- Kroner A, Mehling M, Hemmer B, Rieckmann P, Toyka KV, Maurer M, Wiendl H: A PD-1 polymorphism is associated with disease progression in multiple sclerosis. Ann Neurol. 2005, 58: 50-7. 10.1002/ana.20514.PubMedView ArticleGoogle Scholar
- Lee SH, Lee YA, Woo DH, Song R, Park EK, Ryu MH, Kim YH, Kim KS, Hong SJ, Yoo MC, Yang HI: Association of the programmed cell death 1 (PDCD1) gene polymorphism with ankylosing spondylitis in the Korean population. Arthritis Res Ther. 2006, 8: R163-10.1186/ar2071.PubMedPubMed CentralView ArticleGoogle Scholar
- Kasagi S, Kawano S, Okazaki T, Honjo T, Morinobu A, Hatachi S, Shimatani K, Tanaka Y, Minato N, Kumagai S: Anti-programmed cell death 1 antibody reduces CD4+PD-1+ T cells and relieves the lupus-like nephritis of NZB/W F1 mice. J Immunol. 2010, 184: 2337-47. 10.4049/jimmunol.0901652.PubMedView ArticleGoogle Scholar
- Raptopoulou AP, Bertsias G, Makrygiannakis D, Verginis P, Kritikos I, Tzardi M, Klareskog L, Catrina AI, Sidiropoulos P, Boumpas DT: The programmed death 1/programmed death ligand 1 inhibitory pathway is up-regulated in rheumatoid synovium and regulates peripheral T cell responses in human and murine arthritis. Arthritis Rheum. 2010, 62: 1870-80.PubMedGoogle Scholar
- Good-Jacobson KL, Szumilas CG, Chen L, Sharpe AH, Tomayko MM, Shlomchik MJ: PD-1 regulates germinal center B cell survival and the formation and affinity of long-lived plasma cells. Nat Immunol. 2010, 11: 535-42. 10.1038/ni.1877.PubMedPubMed CentralView ArticleGoogle Scholar
- Iwai Y, Okazaki T, Nishimura H, Kawasaki A, Yagita H, Honjo T: Microanatomical localization of PD-1 in human tonsils. Immunol Lett. 2002, 83: 215-20. 10.1016/S0165-2478(02)00088-3.PubMedView ArticleGoogle Scholar
- Wong RM, Scotland RR, Lau RL, Wang C, Korman AJ, Kast WM, Weber JS: Programmed death-1 blockade enhances expansion and functional capacity of human melanoma antigen-specific CTLs. Int Immunol. 2007, 19: 1223-34. 10.1093/intimm/dxm091.PubMedView ArticleGoogle Scholar
- Nam-Cha SH, Roncador G, Sanchez-Verde L, Montes-Moreno S, Acevedo A, Dominguez-Franjo P, Piris MA: PD-1, a follicular T-cell marker useful for recognizing nodular lymphocyte-predominant Hodgkin lymphoma. Am J Surg Pathol. 2008, 32: 1252-7. 10.1097/PAS.0b013e318165b0d6.PubMedView ArticleGoogle Scholar
- Roncador G, Garcia Verdes-Montenegro JF, Tedoldi S, Paterson JC, Klapper W, Ballabio E, Maestre L, Pileri S, Hansmann ML, Piris MA, Mason DY, Marafioti T: Expression of two markers of germinal center T cells (SAP and PD-1) in angioimmunoblastic T-cell lymphoma. Haematologica. 2007, 92: 1059-66. 10.3324/haematol.10864.PubMedView ArticleGoogle Scholar
- Golden-Mason L, Palmer B, Klarquist J, Mengshol JA, Castelblanco N, Rosen HR: Upregulation of PD-1 expression on circulating and intrahepatic hepatitis C virus-specific CD8+ T cells associated with reversible immune dysfunction. J Virol. 2007, 81: 9249-58. 10.1128/JVI.00409-07.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhang JY, Zhang Z, Wang X, Fu JL, Yao J, Jiao Y, Chen L, Zhang H, Wei J, Jin L, Shi M, Gao GF, Wu H, Wang FS: PD-1 up-regulation is correlated with HIV-specific memory CD8+ T-cell exhaustion in typical progressors but not in long-term nonprogressors. Blood. 2007, 109: 4671-8. 10.1182/blood-2006-09-044826.PubMedView ArticleGoogle Scholar
- Urbani S, Amadei B, Tola D, Massari M, Schivazappa S, Missale G, Ferrari C: PD-1 expression in acute hepatitis C virus (HCV) infection is associated with HCV-specific CD8 exhaustion. J Virol. 2006, 80: 11398-403. 10.1128/JVI.01177-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Petrovas C, Casazza JP, Brenchley JM, Price DA, Gostick E, Adams WC, Precopio ML, Schacker T, Roederer M, Douek DC, Koup RA: PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection. J Exp Med. 2006, 203: 2281-92. 10.1084/jem.20061496.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim CH, Lim HW, Kim JR, Rott L, Hillsamer P, Butcher EC: Unique gene expression program of human germinal center T helper cells. Blood. 2004, 104: 1952-1960. 10.1182/blood-2004-03-1206.PubMedView ArticleGoogle Scholar
- Eddahri F, Denanglaire S, Bureau F, Spolski R, Leonard WJ, Leo O, Andris F: Interleukin-6/STAT3 signaling regulates the ability of naive T cells to acquire B-cell help capacities. Blood. 2009, 113: 2426-33. 10.1182/blood-2008-04-154682.PubMedPubMed CentralView ArticleGoogle Scholar
- Vogelzang A, McGuire HM, Yu D, Sprent J, Mackay CR, King C: A fundamental role for interleukin-21 in the generation of T follicular helper cells. Immunity. 2008, 29: 127-37. 10.1016/j.immuni.2008.06.001.PubMedView ArticleGoogle Scholar
- Nurieva RI, Chung Y, Hwang D, Yang XO, Kang HS, Ma L, Wang YH, Watowich SS, Jetten AM, Tian Q, Dong C: Generation of T follicular helper cells is mediated by interleukin-21 but independent of T helper 1, 2, or 17 cell lineages. Immunity. 2008, 29: 138-49. 10.1016/j.immuni.2008.05.009.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim CH, Rott LS, Clark-Lewis I, Campbell DJ, Wu L, Butcher EC: Subspecialization of CXCR5+ T cells: B helper activity is focused in a germinal center-localized subset of CXCR5+ T cells. J Exp Med. 2001, 193: 1373-81. 10.1084/jem.193.12.1373.PubMedPubMed CentralView ArticleGoogle Scholar
- Chtanova T, Tangye SG, Newton R, Frank N, Hodge MR, Rolph MS, Mackay CR: T follicular helper cells express a distinctive transcriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B cells. J Immunol. 2004, 173: 68-78.PubMedView ArticleGoogle Scholar
- Haynes NM, Allen CD, Lesley R, Ansel KM, Killeen N, Cyster JG: Role of CXCR5 and CCR7 in follicular Th cell positioning and appearance of a programmed cell death gene-1high germinal center-associated subpopulation. J Immunol. 2007, 179: 5099-108.PubMedView ArticleGoogle Scholar
- Bryant VL, Ma CS, Avery DT, Li Y, Good KL, Corcoran LM, de Waal Malefyt R, Tangye SG: Cytokine-mediated regulation of human B cell differentiation into Ig-secreting cells: predominant role of IL-21 produced by CXCR5+ T follicular helper cells. J Immunol. 2007, 179: 8180-90.PubMedView ArticleGoogle Scholar
- Butte MJ, Keir ME, Phamduy TB, Sharpe AH, Freeman GJ: Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity. 2007, 27: 111-22. 10.1016/j.immuni.2007.05.016.PubMedPubMed CentralView ArticleGoogle Scholar
- Hardtke S, Ohl L, Forster R: Balanced expression of CXCR5 and CCR7 on follicular T helper cells determines their transient positioning to lymph node follicles and is essential for efficient B-cell help. Blood. 2005, 106: 1924-31. 10.1182/blood-2004-11-4494.PubMedView ArticleGoogle Scholar
- Schaerli P, Willimann K, Lang AB, Lipp M, Loetscher P, Moser B: CXC chemokine receptor 5 expression defines follicular homing T cells with B cell helper function. J Exp Med. 2000, 192: 1553-62. 10.1084/jem.192.11.1553.PubMedPubMed CentralView ArticleGoogle Scholar
- Gunn MD, Tangemann K, Tam C, Cyster JG, Rosen SD, Williams LT: A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc Natl Acad Sci USA. 1998, 95: 258-63. 10.1073/pnas.95.1.258.PubMedPubMed CentralView ArticleGoogle Scholar
- Ngo VN, Tang HL, Cyster JG: Epstein-Barr virus-induced molecule 1 ligand chemokine is expressed by dendritic cells in lymphoid tissues and strongly attracts naive T cells and activated B cells. J Exp Med. 1998, 188: 181-91. 10.1084/jem.188.1.181.PubMedPubMed CentralView ArticleGoogle Scholar
- Ansel KM, McHeyzer-Williams LJ, Ngo VN, McHeyzer-Williams MG, Cyster JG: In vivo-activated CD4 T cells upregulate CXC chemokine receptor 5 and reprogram their response to lymphoid chemokines. J Exp Med. 1999, 190: 1123-34. 10.1084/jem.190.8.1123.PubMedPubMed CentralView ArticleGoogle Scholar
- Allen CD, Ansel KM, Low C, Lesley R, Tamamura H, Fujii N, Cyster JG: Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5. Nat Immunol. 2004, 5: 943-52. 10.1038/ni1100.PubMedView ArticleGoogle Scholar
- Bleul CC, Schultze JL, Springer TA: B lymphocyte chemotaxis regulated in association with microanatomic localization, differentiation state, and B cell receptor engagement. J Exp Med. 1998, 187: 753-62. 10.1084/jem.187.5.753.PubMedPubMed CentralView ArticleGoogle Scholar
- Nielsen M, Gerwien J, Geisler C, Ropke C, Svejgaard A, Odum N: MHC class II ligation induces CD58 (LFA-3)-mediated adhesion in human T cells. Exp Clin Immunogenet. 1998, 15: 61-8. 10.1159/000019055.PubMedView ArticleGoogle Scholar
- Iannitto E, Ferreri AJ, Minardi V, Tripodo C, Kreipe HH: Angioimmunoblastic T-cell lymphoma. Crit Rev Oncol Hematol. 2008Google Scholar
- Dorfman DM, Brown JA, Shahsafaei A, Freeman GJ: Programmed death-1 (PD-1) is a marker of germinal center-associated T cells and angioimmunoblastic T-cell lymphoma. Am J Surg Pathol. 2006, 30: 802-10. 10.1097/01.pas.0000209855.28282.ce.PubMedPubMed CentralView ArticleGoogle Scholar
- Lim HW, Kim CH: Loss of IL-7 receptor alpha on CD4+ T cells defines terminally differentiated B cell-helping effector T cells in a B cell-rich lymphoid tissue. J Immunol. 2007, 179: 7448-56.PubMedView ArticleGoogle Scholar
- Johansson-Lindbom B, Ingvarsson S, Borrebaeck CA: Germinal centers regulate human Th2 development. J Immunol. 2003, 171: 1657-66.PubMedView ArticleGoogle Scholar
- Bauquet AT, Jin H, Paterson AM, Mitsdoerffer M, Ho IC, Sharpe AH, Kuchroo VK: The costimulatory molecule ICOS regulates the expression of c-Maf and IL-21 in the development of follicular T helper cells and TH-17 cells. Nat Immunol. 2009, 10: 167-75. 10.1038/ni.1690.PubMedPubMed CentralView ArticleGoogle Scholar
- Nurieva RI, Chung Y, Martinez GJ, Yang XO, Tanaka S, Matskevitch TD, Wang YH, Dong C: Bcl6 mediates the development of T follicular helper cells. Science. 2009, 325: 1001-5. 10.1126/science.1176676.PubMedPubMed CentralView ArticleGoogle Scholar
- Bubier JA, Sproule TJ, Foreman O, Spolski R, Shaffer DJ, Morse HC, Leonard WJ, Roopenian DC: A critical role for IL-21 receptor signaling in the pathogenesis of systemic lupus erythematosus in BXSB-Yaa mice. Proc Natl Acad Sci USA. 2009, 106: 1518-23. 10.1073/pnas.0807309106.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim CH, Nagata K, Butcher EC: Dendritic cells support sequential reprogramming of chemoattractant receptor profiles during naive to effector T cell differentiation. J Immunol. 2003, 171: 152-8.PubMedView ArticleGoogle Scholar
- Lim HW, Hillsamer P, Banham AH, Kim CH: Cutting Edge: Direct Suppression of B Cells by CD4+CD25+ Regulatory T Cells. J Immunol. 2005, 175: 4180-3.PubMedView ArticleGoogle Scholar
- Kim CH, Kunkel EJ, Boisvert J, Johnston B, Campbell JJ, Genovese MC, Greenberg HB, Butcher EC: Bonzo/CXCR6 expression defines type 1-polarized T-cell subsets with extralymphoid tissue homing potential. J Clin Invest. 2001, 107: 595-601. 10.1172/JCI11902.PubMedPubMed CentralView ArticleGoogle Scholar
- Lim HW, Broxmeyer HE, Kim CH: Regulation of Trafficking Receptor Expression in Human Forkhead Box P3+ Regulatory T Cells. J Immunol. 2006, 177: 840-851.PubMedView ArticleGoogle Scholar