Human CD1-restricted T cell recognition of lipids from pollens

Abstract

Plant pollens are an important source of environmental antigens that stimulate allergic responses. In addition to acting as vehicles for foreign protein antigens, they contain lipids that incorporate saturated and unsaturated fatty acids, which are necessary in the reproduction of higher plants. The CD1 family of nonpolymorphic major histocompatibility complex-related molecules is highly conserved in mammals, and has been shown to present microbial and self lipids to T cells. Here, we provide evidence that pollen lipids may be recognized as antigens by human T cells through a CD1-dependent pathway. Among phospholipids extracted from cypress grains, phosphatidyl-choline and phosphatidyl-ethanolamine were able to stimulate the proliferation of T cells from cypress-sensitive subjects. Recognition of phospholipids involved multiple cell types, mostly CD4(+) T cell receptor for antigen (TCR)alphabeta(+), some CD4(-)CD8(-) TCRgammadelta(+), but rarely Valpha24i(+) natural killer-T cells, and required CD1a(+) and CD1d(+) antigen presenting cell. The responding T cells secreted both interleukin (IL)-4 and interferon-gamma, in some cases IL-10 and transforming growth factor-beta, and could provide help for immunoglobulin E (IgE) production. Responses to pollen phospholipids were maximally evident in blood samples obtained from allergic subjects during pollinating season, uniformly absent in Mycobacterium tuberculosis-exposed health care workers, but occasionally seen in nonallergic subjects. Finally, allergic, but not normal subjects, displayed circulating specific IgE and cutaneous weal and flare reactions to phospholipids.

Figure 1.

Interactions between cypress pollen and CD1d⁺ DCs. Pollen capture in vivo. (a) The pollen grain constituted by a dark blue exine envelope with extruded cytoplasmic material as appear in a BAL, sampled during pollinating season (May-Grünwald-Giemsa; 400×). The cellular infiltrate contains pulmonary DCs, macrophages, and lymphocytes. (b) A BAL cytospin sample, representative of those obtained from controls (n = 10), as it appeared after labeling with anti–human CD1d mAb (clone NOR3.2, working dilution 1:10) and staining with alkaline phosphatase/antialkaline phosphatase technique. Cells with morphology consistent with macrophages and DCs did not show surface or intracellular staining. (c) CD1d⁺ APC in BAL cytospin from an allergic patient representative of all samples obtained from all allergic subjects (n = 15). (d) Pollen grain stained with fluorescein diacetate (0.1 mM); (e) DC stained with lipophylic dye 4-[4-(dihexadecylamino)styryl]-N-methylquinolinium iodine (DiQ; 1 mM); (f) a DiQ-labeled (red) DC after coculture with fluorescein diacetate–labeled (green) pollen grain for 15 min. Labeled fragments of the pollen grain have colocalized with DiQ-labeled membranes, as evidenced by the merged orange color.

Figure 2.

CD1-dependence of pollen capture in vitro. (a) Expression of CD1 receptors on monocyte-derived DCs. (b) Pre-treatment of DCs with anti-human CD1d and anti-CD1a mAbs (20 μg/ml) was able to inhibit adherence of pollen grains to DCs (*, P < 0.005). Results are expressed as mean percentages (±SD) of inhibition with respect to samples incubated with control Ig. (c) Polar PLs, as opposed to neutral lipids or protein extract from cypress pollen, were able to saturate CD1 receptors on DCs at a concentration of 10 μg/ml, thus preventing further grain capture (*, P < 0.005). (d) HeLa CD1d transfectants bound cypress pollen grains and such binding could be specifically inhibited by preincubation with anti-CD1d mAb (*, P < 0.005). For each of the 10 different preparations, 500 cells were counted by each of three independent observers, who were blinded to the identity of the samples.

Figure 3.

Biochemical characterization of PLs extracted from cypress pollen. (a) Bidimensional TLC of PLs. The main species represented were phosphatidyl-choline (PC), phosphatidyl-ethanolamine (PE), phosphatidyl-serine (PS), phosphatidyl-inositol (PI), and free phosphatidic acid (PAc). (b) Electrospray ionization MS analysis of purified PC and PE from cypress pollen. (c) In vitro [³H]thymidine incorporation by T cell lines to pollen lipids or control protein (previously defatted) cypress pollen extract. (d) CD1 restriction of PC and PE recognition. Mean values ± SD of single PC- and PE-specific T cell lines representative of all tested in triplicate.

Figure 4.

Specificity of antigen recognition by PL-specific CD4⁺ αβ ⁺ or γδ ⁺ T cell clones. (a) Effect of fixation on PL presentation by CD1d-transfected HeLa cells. (b) IL-4 production by T cell clones after in vitro pulsing with cypress-derived or synthetic PLs. Note: for CD4⁺αβ⁺ clones, data are referred to C2, F1, and P3, for CD4⁺Vδ1⁺ clones data are referred to S7 and EA6, whereas for CD4⁺αβ⁺CD161⁺ data are referred to DD6 clone. All illustrated in Table I.

Figure 5.

Functional characteristics of selected PL-specific T cell clones. For cytokine production by T cell clones and proliferation assays, mitomycin C treated HeLa cells transfected with CD1a (for clones Q5 and 3F9) or CD1d (for clone N3) were used as APCs. Stimulating antigens consisted of 10 μg/ml of cypress, brain, liver, and egg PL mixtures (PC or PE), as well as of synthetic PL species. Secreted IL-4, IL-10, TGF-β1, and IFN-γ (mean ± SD of pg/ml in the supernatants of triplicate cultures) were measured after 48 h of culture by ELISA. [³H]thymidine incorporation (mean cpm ± SD of triplicate cultures) after 5 d of culture was measured by scintillation counting. See Table I for CD1 restriction.

Figure 6.

Response to cypress pollen PLs in allergic subjects is specific and shows seasonal variability. (a) Correlation over a 1-yr period between atmospheric levels of cypress pollen (absolute counts, referred to the right y axis) in the region inhabited by the study subjects and the frequency of PL-specific T cell lines (percentage of responding T cell lines calculated over the total number obtained, referred to the left y axis). (b) Mean values (±SD) of proliferative responses of PBMC to stimulation with cypress antigens. A clear response (P < 0.005) was seen in allergic, as compared with normal controls or M. tuberculosis exposed subjects. (c) CD1a- and CD1d-restricted proliferative response in T cell lines of allergic subjects (top) toward pollen-derived PLs (10 μg/ml; filled square), mycolic acid stimulation (10 μg/ml; shaded square), or medium alone (open square). The bottom panel shows the responses of T cell lines derived from M. tuberculosis– exposed subjects. One representative experiment of all performed in 14 patients and 10 controls is depicted.

Figure 7.

IgE reactivity toward pollen PLs. (a) Two representative experiments of in vitro IgE production by autologous B cells cocultured with CD4⁺ TCRαβ⁺ cell clones, expressing or not the CD161 molecule, and stimulated with cypress PE or synthetic PLs. Filled bars refer to clones from allergic subjects (clones B1.5 and FA8 αβ⁺CD161⁻ and clone DD6 αβ⁺CD161⁺, as reported in Table I), open bars to those of normal controls (clone NC-FP5 αβ⁺CD161⁻ and clone NC-A2 αβ⁺CD161⁺, as reported in Table II). *, P < 0.001 with respect to unstimulated, 16:0/16:0 PE-stimulated or normal control clones. **, P < 0.05 with respect to unstimulated cultures. (b) In the left panel is the average of mean wheal and flare reactions to cypress-derived PLs in study population. The right panel represents the circulating PE-specific IgE (mean OD at 405 nm) as detected by ELISA assay. NC, normal controls.