Rules of chemokine receptor association with T cell polarization in vivo (original) (raw)
Each CKR-defined memory T cell population has a characteristic composition of polarized and nonpolarized subsets. Initially, we investigated the production of Th1 and Th2 cytokines by circulating fresh blood CD4 T cell subsets expressing different CKRs. Expression of CKRs on CD4 T cells characterizes distinct populations of T helper cells. Many members of the CKR family such as CCR2 (28), CCR3 (29–31), CCR4 (22, 32), CCR5 (33, 34), CCR6 (35–37), CXCR3 (38), CXCR5 (39), and CXCR6 (19, 40, 41) are expressed only on memory (CD45RA– or CD45RO+) CD4 T cells (Figure 1a). CCR7 (42) and CCR9 (43–45) are expressed on both naive and memory CD4 T cells (Figure 1a). CCR1 and CXCR4 are not included in this study, since they are expressed by too few (CCR1) or by almost all (CXCR4) blood CD4 T cells (data not shown). Each CKR-expressing memory CD4 T cell population is composed of characteristic frequencies of Th1, Th2, Th0, and Tnp cells (Figure 1, b and c). CXCR3-, CCR5-, and CXCR6-expressing CD4 T cells are enriched for Th1 cells (45–49%, n = 8), although cells expressing these CKRs also contain significant numbers of Th2 cells (1–3% of each population; 30–90% of the Th2 cell frequency in the whole memory population). CCR2+CD4 T cells, while relatively enriched for Th1 cells, contain similar levels of Th2 cells to the total memory CD4 T cell pool. CCR4+ cells are enriched for Th2 cells (9.3%) compared with total memory CD4 T cells (2.8%). Surprisingly, although Th1 cells are relatively depleted among CCR4+ cells, in fact approximately 10% of CCR4+ cells are Th1, as well. CCR3+ cells, a rare subset in blood, contain twice the frequency of Th2 cells as the bulk memory CD4 cell population, but on average contain a similar frequency of Th1 cells. Th0 cells are enriched within CCR2-, CCR5-, CXCR3-, and CXCR6-expressing memory CD4 T cells (Figure 1c).
Profiles of polarized and nonpolarized T cell frequencies in CKR+ memory CD4 T cell populations. (a) Expression of CKRs on total (left, FACS plots) and memory CD4 T cells (right, histogram). Frequencies of Th1 (IFN-γ+ IL-4–), Th2 (IL-4+ IFN-γ–), Th0 (IFN-γ+ IL-4+), or Tnp (IFN-γ– IL-4–) cells in total, memory, or CKR-expressing memory CD4 T cell populations are shown as dot plots (b) and graphs (c). Peripheral blood CD4 T cells were activated by PMA and ionomycin for 4 hours before cytokine analysis. Each CKR+/– population is gated (b and c). Averages from 6–11 different donors are shown.
The majority (77%, n = 9) of blood memory CD4 T cells are nonpolarized Tnp’s, unable to produce IL-4 or IFN-γ. Eighty-eight percent of CXCR5+ or 84% of CCR7+ T cells are Tnp’s (n = 8), levels higher (77%) than that of total memory T cells (Figure 1c). Correlating with their reduced numbers of Th1 cells, CCR4+ T cells also contain many (81%) Tnp’s. Fewer but still considerable numbers of Tnp’s are contained within the CCR2-positive (46%), CCR5-positive (45%), CXCR3-positive (48%), and CXCR6-positive (48%) populations. CCR3-, CCR6-, and CCR9-positive subsets contain similar frequencies of Tnp’s to the total memory CD4 T cells.
CKR expression profiles of Th1, Th2, Th0, and Tnp cells. Precommitted Th1 and (especially) Th2 cells represent only a small fraction of most of these CKR-defined subsets. To characterize polarized effector and nonpolarized memory cells more directly, we therefore identified these minor effector cell populations based on cytokine staining and directly evaluated their expression of the different receptors. Most Th1 cells express CXCR3 (on average 90%) (Figure 2). In contrast, only approximately 50% of Th1 cells express CCR5, only approximately 15% of Th1 cells express CXCR6, and only approximately 42% express CCR2. Almost all (∼95%) Th2 cells express CCR4, while CCR3 marks only approximately 3% of Th2 cells. Many Th0 cells express CXCR3, CCR5, CCR2, CCR4, and CCR7 receptors that are expressed by many Th1 and/or Th2 cells. CCR6 is also expressed on approximately 40% of Th1 and approximately 10% of Th2 cells. Many Tnp’s express CCR7 (76%), CCR4 (34%), CXCR5 (25%), CXCR3 (24%), and CCR6 (24%) in a decreasing order. Notably, CXCR5 is particularly expressed by more Tnp versus Th1, Th2, or Th0 cells. In contrast, CCR2+ T cells are underrepresented in the Tnp versus Th1, Th2, or Th0 cell population.
CKR expression by Th1, Th2, Th0, and Tnp cell populations. Percentage of peripheral blood Th1, Th2, Th0, and nonpolarized cells expressing each CKR was examined by gating on Th1 (IFN-γ+ IL-4–), Th2 (IL-4+ IFN-γ–), Th0 (IFN-γ+ IL-4+), or Tnp (IFN-γ– IL-4–) cells. Averages from at least six different donors with SDs.
The majority of polarized effector T cells are CCR7+. A CRK CCR7 has been described previously as a defining factor for two different memory T cell populations: CCR7+ memory cells were associated with a proliferation reserve population and lacked effector function, while CCR7– cells were equated with immediate effector T cells that produce effector cytokines upon stimulation (42). However, our results show that both CCR7+ and CCR7– T cells contain effector T cells and that there are in fact many more CCR7+ than CCR7– polarized T cells in human blood (Figure 3). Approximately, 30% of CCR7– memory T cells and 15% of CCR7+ memory T cells were IFN-γ producers, and 6% of CCR7– and 3% of CCR7+ memory T cells were IL-4 producers (Figure 3a). In blood, CCR7– memory T cells are relatively rare (24% of memory CD4 T cells) when compared with CCR7+ memory T cells (76% of memory CD4 T cells) (Figure 1a). Therefore, when the absolute number of polarized effector (IL-4 or IFN-γ producers) T cells was calculated based on the frequency of each memory subset in blood (42% ± 8% of CD4 cells are CCR7+, 11% ± 3% are CCR7–; n = 8), there are more CCR7+ IFN-γ (8% of CD4 T cells) than CCR7– IFN-γ (3.5%) producers among CD4 T cells (Figure 3b). Similarly, more CCR7+ (1.6% of CD4 T cells) than CCR7– (0.7%) IL-4 producers are present in the circulation.
Polarized T cell populations are enriched in frequency among CCR7– T cells, but are predominantly CCR7+. (a) The frequencies of IL-4– or IFN-γ–producing cells are shown as percentage of each CKR-positive or -negative subset by gating on naive (CD45RA+), CCR7+, CCR7–, CXCR5+, or CXCR5–CD45RO+ CD4 T cell population. (b) The absolute frequency (%) of IL-4– or IFN-γ–producing cells among CCR7+/– or CXCR5+/–CD45RO+ subsets in blood CD4 T cells. The absolute frequency (%) was obtained by normalizing (i.e., multiplying) the frequencies of IL-4– or IFN-γ–producing cells in each subset (the values in the a) by the frequency of each memory subset in total CD4 T cells (0.42 for CCR7+; 0.11 for CCR7–; 0.1 for CXCR5+; 0.43 for CXCR5–; n = 8). CD4 cells (5 × 104) were acquired for each analysis. Results from four donors are shown.
We also examined the effector frequencies of CXCR5+/– memory CD4 T cell populations for direct comparison with the CCR7+/– populations. CXCR5+ cells are a subset of the CCR7+ population (on average, 26% of CCR7+ memory T cells are CXCR5+, whereas almost no CCR7– T cells express CXCR5; not shown), and CXCR5+ cells are largely defective in production of IL-4 and IFN-γ (9, 10). Fewer polarized effectors were found among CXCR5+ than among CXCR5– or CCR7+/– memory T cell populations. When the absolute number of polarized cells in blood was examined, most effectors were found in CXCR5– but not in CXCR5+ memory T cell population, in contrast to the distribution of polarized cells in CCR7+/– populations (Figure 3). These data suggest (a) that not all, but a subset of the CCR7+ memory T cell population (e.g., CXCR5+ T cells), are really defective in Th1/2 effector cytokine production and (b) that the majority of effector T cells are CCR7+. Therefore, many polarized cells may circulate through central lymphoid tissues as well as tissue sites of inflammation, but these IFN-γ– and IL-4–producing cells are largely independent of the presumed B helper–related T cells expressing the follicle homing–associated CKR CXCR5.
More specific definition of polarized T cells by combinations of CKRs. Leukocyte microenvironmental homing is controlled not by individual receptors, but rather through multistep processes of navigation that require sequential engagement of several chemoattractant (and other) receptors expressed in combination on the cell surface (5). We therefore asked if overlapping patterns of expression of CKRs (especially CXCR3 and CCR4, which are expressed by most Th1 or Th2 cells, respectively, as shown in Figure 2) might more effectively distinguish Th1 versus Th2 cells. To ask whether singly positive or coexpressing subsets display distinctive Th effector associations, we separated the CCR4+CXCR3–, CCR4+CXCR3+, and CCR4–CXCR3+ populations and examined the polarized T helper subset composition of each population (Figure 4). Surprisingly, Th1 cells (representing 37–65%) but almost no Th2 cells are found in the CCR4–CXCR3+ population, while Th2 (up to 10%), but very few Th1 cells (<2%) are CCR4+CXCR3–. Interestingly, the double-positive CCR4+CXCR3+ cells have intermediate levels of Th1 (10–20%) and Th2 (∼2%) cells and contain more Th0 cells (up to 2%) than the other two subsets (Figure 4b). On average, 88% of Th1 cells in blood are in fact CXCR3+CCR4–, and 90% of Th2 cells are CXCR3–CCR4+, so that these combinations of receptors define major subsets of circulating effector-polarized T cells. Thus, analyses of these receptors in combination reveal a more specific phenotype for most Th1 (CXCR3+CCR4–, 88% of Th1) and Th2 (CXCR3–CCR4+, 90% of Th2) cells and show that CXCR3+ Th2 cells and CCR4+ Th1 cells are primarily within a CXCR3+CCR4+ double-positive population, which contains a mixture of Th0, Th1, and Th2 cells.
Polarized CD4 T cell populations defined by CXCR3 and CCR4 in combination. (a) Coexpression of CXCR3 and CCR4, and (b) frequencies of Th1, Th2, and Th0 cells in CCR4+CXCR3–, CCR4+CXCR3+, and CCR4–CXCR3+ populations. Each subset was individually sorted from peripheral blood CD4 T cells for cytokine analyses. (c) Expression of CXCR3 and CCR4 and frequencies of Th1 and Th2 cells in the CLA+/– (skin-homing receptor) memory CD4 T cell subsets. Representative of four independent experiments.
In the context of the multistep processes of cell migration, individual receptors can play different roles in different physiological settings (at different steps). As an example, CCR4 has been implicated in apparently “conflicting” roles as (a) a preferential Th2 cell chemoattractant receptor, and (b) a lymphocyte “homing receptor” for skin involved in vascular recognition during lymphocyte (presumably both Th1 and Th2) recruitment from the blood in diverse settings of cutaneous inflammation (46). To determine if overlapping receptor patterns could help explain this apparent conflict, we analyzed the phenotype of skin-homing memory T cells defined by expression of the CLA. As shown in Figure 4c, many CLA+ CD4 T cells coordinately express CXCR3 and CCR4: as reported previously, almost all CLA+ T cells are CCR4+ (46), but fully half of CLA+ T cells are also CXCR3+ (Figure 4c). Interestingly, compared with CLA–CXCR3+ CD4 T cells (most of which are CCR4–), CLA+CXCR3+ T cells (which are almost all CCR4+) contain reduced numbers of Th1 cells, consistent with the reduction frequency of Th1 cells among CXCR3 cells coexpressing CCR4. The frequency of Th2 cells is also lower among CLA+CXCR3+ than among CLA+CXCR3– cells (even though both populations are predominantly CCR4+). We conclude that many skin-homing T cells display both CXCR3 and CCR4 and that the frequencies of Th1 and Th2 cells in each subset closely correlate with the coexpression pattern of the two receptors as predicted from Figure 4a and b.
CKR association with T cell polarization in inflamed tissues. It is important to ask whether the same rules of CKR association with polarization of circulating blood CD4 T cells would apply for the T cells in extralymphoid tissues, particularly inflamed tissues. To answer this, we examined CKR expression by polarized and nonpolarized T cells in synovial fluids from patients with psoriatic arthritis, a type 1 polarized disease (47). All of the arthritis synovial CD4 T cells were memory cells (CD45RA–CD45RO+; Figure 5b). These CD4 T cells differ from blood T cells in that many more express the CKR CCR5 (71–89%), CXCR3 (68–85%), CXCR6 (22–61%), and CCR2 (33–47%) (n = 3, Figure 5a). Surprisingly, approximately half (48.5%) of the tissue CD4 T cells also express CCR4, a pro-Th2 receptor. Most of these CCR4+ T cells coexpress CXCR3 (Figure 5c). Furthermore, one-third of the synovial CD4 T cells also express CCR7, a CKR proposed by others to define lymphoid tissue–homing lymphocytes excluded from extralymphoid tissues. Small but significant percentages of the CD4 T cells express CXCR5 (4–9.5%) or CCR3 (5–8%). CCR1+ or CCR9+ CD4 T cells are relatively rare (0.5–2.1%) in synovial fluid.
CKR association with T cell polarization in inflammatory tissues (CD4 T cells in psoriatic arthritic synovial fluid). (a) CKR expression by synovial CD4 T cells. (b) CD45RA and CD45RO expression by synovial CD4 T cells. (c) Coexpression of CXCR3 and CCR4 by synovial fluid CD4 T cells. (d) Intracellular IFN-γ and IL-4 production pattern of arthritic synovial CD4 T cells. (e) Frequencies of Th1 (IFN-γ+ IL-4–), Th2 (IL-4+ IFN-γ–), Th0 (IFN-γ+ IL-4+), or Tnp (IFN-γ– IL-4–) cells in CKR-expressing CD4 T cell populations are shown. (f) Expression of CKRs by Th1, Th2, Th0, and Tnp cells in arthritis synovial fluid. Prestained cells with anti-CKR and anti-CD4 were activated by PMA and ionomycin for 4 hours in the presence of monensin before intracellular staining of IFN-γ and IL-4. Representative data from three different experiments are shown.
We next examined the frequency of polarized and nonpolarized T cells in CKR+ CD4 T cell populations in synovial fluid. Many more Th1 and Th0, but fewer Th2, cells are found in arthritic synovial fluid than in blood, confirming that they are mainly type 1 polarized (Figure 5d). CCR2+, CCR3+, CCR5+, CXCR3+, and CXCR6+ CD4 T cell populations contain slightly more than or similar frequencies of Th1 and Th0 cells to the total CD4 T population, while CCR4+, CCR7+, CXCR5+, and CCR6+ populations contain reduced, but significant, numbers of Th1 cells. Synovial CCR4+ T cells do not contain more Th2 cells than other CKR+ subsets (Figure 5e). Expression of CKRs by Th1, Th2, Th0, and Tnp cells are shown in Figure 5f. Although expressed by all subsets, CCR4 is expressed by more Th2, Tnp, and Th0 than Th1 cells. In contrast, CXCR6, CXCR3, CCR5, and CCR2 are expressed by more Th1 and Th0 than Th2 and Tnp cells. As in blood, CXCR5 and CCR7 are expressed by more Tnp than Th1, Th2, and Th0 cells in synovial fluid.
Coexpression of CXCR3 confers polarity toward Th1 on CKR+ populations. To explore further the significance of overlapping CKR profiles in defining fingerprints of specialized effector subsets, we next asked if functionally distinct subsets of CXCR3+ cells could be identified by the presence or absence of additional CKRs. Data presented in Figure 6 show that most Th1 cells that express CCR5, CCR2, CCR6, or CXCR5 actually coexpress CXCR3. Thus, in the absence of CXCR3, neither CCR5, CCR2, CCR6, or CXCR5 expression is associated with a significant frequency of Th1 cells, and none of these receptors predicts Th1 cytokine production by itself. In fact, the percentage of CXCR3+ double-positive cells in these populations (75% in CCR5+, 73% in CCR2+, 48% in CCR6+, 25% in CXCR5+) positively correlates with their polarity toward Th1 within each CKR-defined subset. More importantly, the data lead to interesting and surprising conclusions about the significance of coexpression of each of these receptors with CXCR3. CCR5 and CCR6, respectively, have only marginal positive and negative association with the frequency of Th1 or Th2 cells among the CXCR3+ population, suggesting that these receptors are more likely regulated independently of Th1 cytokine preference (Figure 6, a and c). On the other hand, expression of CCR2 is associated with a decrease rather than an increase in Th1 frequency and a slight increase in Th2 cells among CXCR3+ cells (Figure 6b). CXCR5 is most strongly associated with a decrease in Th1 frequency (Figure 6d).
Dependence of Th1-containing populations on coexpression of CXCR3. Peripheral blood CD4 T cells expressing CXCR3 and/or another CKR among CCR5 (a), CCR2 (b), CCR6 (c), or CXCR5 (d) are shown along with frequencies of Th1 and Th2 cells. Each subset was sorted after staining with Ab’s to CKRs, stimulated with PMA and ionomycin, and examined for production of IL-4 and/or IFN-γ. Results from three to four donors are shown.