CHS.
For sensitization 16, 2,4-dinitrofluorobenzene (DNFB; Sigma-Aldrich) (0.5%) in a vehicle of 4:1 acetone in olive oil was applied to shaved abdominal skin on days 0 and 1. On day 5, the right ear was treated with 0.25% DNFB (10 µl on each side), and the left ear was treated with the vehicle. Swelling was measured before and 18–24 h after treatment using an engineer's micrometer.

Langerhans Cell Migration and Function.
Epidermal Langerhans cells (LCs) were isolated from ears and characterized by flow cytometry, or were studied in epidermal sheets 17. LC migration, and the antigen presentation function of migrated LCs in a mixed lymphocyte reaction assay, were assessed according to published procedures 1718.

Lymphocyte Activation, Expansion, and Polarization In Vivo.
Mice were sensitized with 0.5% DNFB on their ears (see Fig. 5) or shaved abdomens (see Fig. 6) on day 0 and day 1. On day 4, the mice were killed, their draining PLNs (cervical, for ear sensitization; inguinal for abdominal sensitization) were removed and were used to prepare a single cell suspension. In some experiments (see Fig. 6), the cells were cultured for 4 h at 37°C in 10 µg/ml brefeldin A, 50 ng/ml phorbol myristic acetate, and 500 ng/ml ionomycin. Lymphocytes were phenotyped using monoclonal Abs specific for cell surface markers (CD4, CD8, CD19, CD44), or by staining for intracellular IFN- or IL-4 19.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Activated T cell expansion in the draining LNs after DNFB sensitization. Mice were sensitized on day 0 and day 1 and their draining LNs were removed and analyzed on day 4 (Materials and Methods). Data are average numbers (± SE), determined for between four and six mice in each group, derived from three independent experiments. Statistical analyses were done as described in the legend to Fig. 1.

Splenic B cells were used as antigen-presenting cells for in vitro cytokine polarization procedures. Splenic leukocytes isolated with a Lympholyte M (Cedarlane) gradient were depleted of T cells by Thy 1.2 staining followed by complement-mediated lysis. The remaining cells (>93% CD19-positive) were washed with CTCM, incubated with 50 µg/ml mitomycin C for 20 min at 37°C, and then washed to remove all mitomycin C before addition to cytokine polarization cultures.

Cytokine polarization was accomplished by coculturing CD4⁺ (Th1) or CD8⁺ (Tc1) lymphocytes (10⁶) with 4 x 10⁶ syngeneic splenic B cells in dishes coated with anti-CD3 (mAb 2C11). Cells were cocultured for 48–72 h as follows. Th1: 50 U/ml IL-2, 1,000 U/ml IL-12 (R&D Systems) and 10 µg/ml anti–IL-4 (NALE; BD PharMingen). Th2: 50 U/ml IL-2, 10 ng/ml IL-4 (Genzyme), 10 µg/ml anti–IFN- (NALE; BD PharMingen). Tc1: 50 U/ml IL-2, 2,000 U/ml IL-12, and 25 µg/ml anti–IL-4. Tc2: 50 U/ml IL-2, 30 ng/ml IL-4, 50 µg/ml anti–IFN-.

Cells were then washed, resuspended in media containing the same cytokine cocktail, and expanded in culture for at least three additional days. Intracellular cytokine expression was evaluated by culturing cells at a concentration of 10⁶ cells/ml in CTCM containing 50 ng/ml phorbol myristic acetate, 500 ng/ml ionomycin, and 10 µg/ ml brefeldin A for 4.0 h at 37°C.

Ab Staining for Flow Cytometry.
Cells were stained on ice in PBS with 3% FCS or in staining medium (SM; DMEM plus 0.1% BSA and 0.1% sodium azide). Cells were incubated with Fc block (1:100) for 5 min, then washed and stained for 10 min with one or more primary mAbs. Fluorochrome-based secondary reagents (SA-Cy-Chrome, SA-PE or SA-FITC) were applied after washing Ab-stained cells. P-selectin IgM and E-selectin-IgM chimeras were used as described previously 6. For simultaneous identification of cell surface antigens and intracellular cytokines, lymphocytes were stained for surface antigens using directly conjugated mAbs, washed once with SM, and fixed with Cytofix/Cytoperm solution (100 µl; BD PharMingen) for 20 min on ice. The cells were then washed and resuspended in mAbs prediluted in 1x Perm/Wash (BD PharMingen), incubated for 20 min, washed twice with Perm/Wash, resuspended in SM, and subjected to flow cytometry.

Adoptive Transfer of 5-Chloromethylfluorescein Diacetate–labeled Activated Effector Lymphocytes.
Wild-type (WT) mice served as recipients. WT, Fuc-TIV^–/–, and Fuc-TVII^–/– mice were used as donors; Fuc-TIV^–/–/Fuc-TVII^–/– mice could not be used as donors because it was not possible to isolate sufficient numbers of lymphocytes from the DNFB-activated PLNs to allow adoptive transfer (see Fig. 5). Donors and recipients were sensitized with 0.5% DNFB on their shaved abdomens on day 0 and day 1. Donors were killed on day 4, their draining PLNs were removed, single cell suspensions were prepared, and cells were labeled with 5 µM 5-chloromethylfluorescein diacetate (CMFDA; Molecular Probes) 6. Anesthetized recipients received 20 x 10⁶ labeled cells by tail vein injection. Recipients were immediately challenged with 0.25% DNFB on the right ear, and treated with vehicle on the left ear. Ear swelling was measured 24 h later, the recipients were killed, and the dorsal portion of the epidermis and dermis from each ear were removed. Single cell suspensions of the ear tissue were prepared using a published protocol 20, and assessed for CMFDA⁺ by flow cytometry. Vehicle-treated ears from sensitized recipients served as a control for nonspecific trafficking of adoptively transferred CMFDA-labeled lymphocytes. Ears from sensitized and challenged mice, not injected with CMFDA-labeled cells, were analyzed in parallel to determine background fluorescence. Dead cells were excluded from the analyses using propidium iodide staining, and using appropriate forward and side scatter profiles. The ear swelling response and the influx of recipient-derived CD45⁺ leukocytes in the hapten-treated ears were equivalent in all recipients regardless of the genotype of donor cells injected, confirming that the recipients sustained a normal response to antigenic challenge, and indicating that adoptive transfer did not alter the course of the CHS response.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CHS Is Attenuated in Fuc-TVII^–/– Mice, and Absent in Fuc-TIV^–/–/Fuc-TVII^–/– Mice.
Fuc-TIV^–/– mice sustain a slight decrement, relative to WT mice, in ear swelling after sensitization and challenge with the hapten DNFB, but this decrement does not reach statistical significance (Fig. 1). By contrast, Fuc-TVII^–/– mice exhibit a significant reduction in response to DNFB sensitization and challenge (Fig. 1). Fuc-TVII^–/– mice retain a significant response, however, and this is clearly Fuc-TIV dependent, as the doubly deficient mice do not respond to sensitization and challenge with DNFB (Fig. 1). Fuc-TVII therefore makes a prominent contribution to the immune response to a cutaneous antigen. The contribution by Fuc-TIV is subtle when Fuc-TVII expression is normal, and Fuc-TIV accounts for virtually all the response retained in the absence of Fuc-TVII.

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Figure 1 CHS. Mice were sensitized with 0.5% DNFB on the abdomen on day 0 and day 1, then challenged on the ear on day 4 with 0.25% DNFB on the ear (Sensitization and Challenge). Swelling in response to nonspecific irritation by DNFB was measured after a single application of 0.25% DNFB to nonsensitized mice (Challenge). The numbers of mice used to generate each experimental data point are shown below the histogram, and are derived from two independent experiments. Mean ear swelling (± SE) is plotted. Vertical P values derive from a Student's t test evaluation of ear swelling with or without sensitization in mice of the same genotype. Differences in ear swelling between genotypes after sensitization and challenge were analyzed using the Kruskal-Wallis test and post-hoc Mann-Whitney U test. Differences of statistical significance are noted with asterisks.

View larger version (122K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Distribution of epidermal LCs. Epidermal sheets were isolated from the dorsal portion of the ear of WT and Fuc-T–deficient mice, stained with DEC-205, and examined by fluorescence microscopy (Materials and Methods; original magnification: x500).

View larger version (57K): [in this window] [in a new window] [Download PPT slide]   Figure 3 LC migration. FITC was applied to ear pinnae, and draining cervical nodes were removed 18–24 h later. Single cell suspensions were made from the PLNs, were stained with Abs specific for MHC class II and CD86, and were then subjected to flow cytometry analysis (Materials and Methods). (A) Quantitation LCs recently migrated to PLNs. The number of FITC+, MHC class IIbright cells in the draining PLNs were calculated for each genotype. Data are mean values of LCs per draining PLN (± SE) obtained in four independent experiments. Mean values are not significantly different as judged by the Kruskal-Wallis test (P = 0.06). (B) FACS® analysis of LCs recently migrated to a draining PLN. Representative two-parameter histograms of viable (based on forward and side scatter parameters) MHC class IIbright cells. The number in the upper right hand quadrant is the percentage of MHC class IIbright cells that are both FITC+ and CD86+.

WT and Fuc-TIV^–/– PLNs do not differ in total cellularity, and have essentially identical numbers of CD19⁺ B cells, CD3⁺ T cells (Fig. 4 A), and T cells with a memory/effector (CD4⁺CD44^highCD45RB^low) or naive (CD4⁺ CD44^lowCD45RB^high) phenotype (2930; Fig. 4 A). These observations indicate that any contribution by Fuc-TIV to maintaining homeostasis in lymphocyte numbers in PLNs is too subtle to detect with these methods, or is fully masked by Fuc-TVII–dependent processes.

View larger version (54K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Quantitative and phenotypic analysis of peripheral node and blood lymphocytes in WT and Fuc-T–deficient mice. Lymphocytes isolated from peripheral nodes (A) or blood leukocytes (B) were stained with Abs against the antigens indicated below each panel, and were subjected to flow cytometry analysis (Materials and Methods). The number of cells of each indicated phenotype was calculated from the fraction of cells with the indicated cell surface phenotype. Data are average numbers (± SE) determined for 7 to 10 mice of each genotype. Statistical analyses were done as described in the legend to Fig. 1.

The partial reduction in B cells, naive T cells, and memory/effector T cells in Fuc-TVII^–/– PLNs implies a residual ability to recruit such cells to these organs, and aligns with the Fuc-TIV–dependent residual L-selectin ligand expression and lymphocyte homing activities assigned by analysis of Fuc-TIV^–/–/Fuc-TVII^–/– mice 8. Indeed, in Fuc-TIV^–/–/VII^–/– nodes, a 95% reduction in cellularity, relative to WT PLNs (Fig. 4 A), is accounted for by a 91% decrease in B lymphocyte content, profound deficiencies of CD4⁺ and CD8⁺ populations (decreased 97 and 92%, respectively; Fig. 4), selective losses of CD4⁺ and CD8⁺ cells of the naive phenotype (reductions of 97 and 96%, respectively), and significant decrements in memory/effector cells (Fig. 4 A).

Reduced accumulation of B lymphocytes and naive or memory/effector T cells are not accounted for by decreases in the number available for recruitment, as essentially equivalent numbers of these cells are present in the peripheral blood of WT and Fuc-TIV^–/–/Fuc-TVII^–/– mice (Fig. 4 B), nor by deficiency of L-selectin, as this molecule is expressed at normal levels on all three Fuc-T–deficient strains 68. By contrast, Fuc-TVII contributes prominently but incompletely to the synthesis of L-selectin ligands on PLN HEVs, and to lymphocyte homing 6. Partial reductions in the accumulation of B and T cells in Fuc-TVII^–/– mice are thus likely consequent to partial deficits in L-selectin–dependent lymphocyte homing processes that recruit these cells to PLNs 2831. We also infer that the profound decrement in B lymphocyte content and nearly complete absence of naive and memory/effector T cells in Fuc-TIV^–/–/VII^–/– PLNs result from the loss of Fuc-TIV–dependent L-selectin ligands that contribute to residual L-selectin–dependent recruitment in Fuc-TVII^–/– mice 8.

Hapten-dependent Lymphocyte Expansion In Vivo Requires Fuc-TIV and Fuc-TVII.
To determine how each Fuc-T may contribute to hapten-dependent generation of memory/effector T cells in vivo, we examined lymphocyte expansion (reflecting increases in recruitment [32, 33] and proliferation 34), activation, and differentiation in PLNs draining the site of cutaneous DNFB sensitization. Sensitization elicited large and significant increases in cellularity in WT, Fuc-TIV^–/–, and Fuc-TVII^–/– PLNs, relative to mice treated with vehicle only (Fig. 5 A). A significant increase was also observed in Fuc-TIV^–/–/VII^–/– PLNs after sensitization, but the absolute number of cells in vehicle-treated mice or after sensitization was extremely low (Fig. 5 A). CD19⁺ B lymphocytes contribute substantially to the expanded cell populations in the PLNs, as do CD3⁺ T cells (Table ). T and B cells account for >90% of the cells in the nodes, except in doubly deficient mice, where the relative larger fraction of non-B and non-T cells is accounted for by stromal cell elements, DCs, and Gr-1⁺ myeloid lineage cells (data not shown).

Fuc-T Deficiency Yields Decrements in Accumulation of IFN-–expressing CD4 and CD8⁺ Lymphocytes In Vivo, but Is Permissive for Normal Th1 and Tc1 Polarization In Vitro.
Th1 and Tc1 lymphocytes, defined in part by their characteristic IFN- expression properties 35, are generated in draining PLNs after antigen sensitization, traffic via the circulation to the skin, and contribute to the cutaneous inflammatory response during elicitation/challenge 353637. Th1 and Tc1 progenitors most likely derive from naive T lymphocytes recruited to PLNs primarily by L-selectin–dependent mechanisms (discussed in reference 11). As Fuc-TIV and Fuc-TVII contribute to L-selectin–dependent lymphocyte recruitment 68, we sought to determine if their deficiency altered the accumulation or generation of such IFN-–expressing lymphocytes in a way that contributes to defective CHS in the Fuc-T–deficient mice. The numbers of these cells were equivalently low in the PLNs of nonsensitized WT and all Fuc-T–deficient strains (Fig. 6). Significant increases in these cells are observed in WT and Fuc-TIV^–/– mice after DNFB sensitization (Fig. 6A and Fig. B). By contrast, in Fuc-TVII^–/– mice and the doubly deficient mice, sensitization did not significantly increase the number of such IFN-–positive CD4⁺ or CD8⁺ lymphocytes. Graded, genotype-specific decrements in the absolute number of these cells recovered from the PLNs of the sensitized mice were also observed, and are most apparent and significant in the doubly deficient mice (Fig. 6). These decrements imply that Fuc-TIV and Fuc-TVII contribute independently to hapten-dependent accumulation of Th1 and Tc1 cells in vivo. To exclude the possibility that the Fuc-Ts also or instead contribute to properties intrinsic to Th1 and Tc1 precursors that allow them to proceed through the polarization process, WT- and Fuc-T–deficient naive T cells were characterized for their ability to differentiate into Th1 or Tc1 cells in vitro. These experiments show that naive CD4⁺ or CD8⁺ T cells isolated from each Fuc-T null strain respond to cytokine polarization by differentiating into cells with a Th1 (IFN-⁺IL-4^–) or Tc1 (IFN-⁺IL-4^–) phenotype (Fig. 7 A and 8 A). These observations imply that defective generation of Th1 and Tc1 cells in Fuc-TVII^–/– and Fuc-TIV^–/–/VII^–/– mice in response to haptenic sensitization (Fig. 6) is due to a deficiency in the number of naive Th1 cell and Tc1 cell progenitors available for cytokine-dependent polarization, and not to an intrinsic differentiation defect. In vitro polarization towards the Th2 (CD4⁺IFN-^–IL-4⁺) and Tc2 (CD8⁺IFN-^–IL-4⁺) phenotypes is also retained in the absence of Fuc-TIV and/or Fuc-TVII (data not shown), indicating that neither enzyme is required for these alternative differentiation pathways.

View larger version (62K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Selectin ligand expression by Th1 cells in WT and Fuc-T–deficient mice. CD4+ T cells were isolated from mesenteric nodes and cultured in vitro under Th1 polarization conditions (Materials and Methods). Cells were then subjected to flow cytometry analysis to confirm Th1 polarization by assessing expression of intracellular IFN- and IL-4 (A), or to assess selectin ligand expression using P-selectin-IgM or E-selectin-IgM chimeras (B). Divalent calcium dependence observed for binding of the P-selectin-IgM chimera (B, bottom) was also observed for the E-selectin-IgM chimera (data not shown). Data shown are representative of two or more independent experiments. Vertical and horizontal lines in the histograms define the fluorescence intensities below which 99% of the cells fall when analyzed using a nonbinding isotype control for AntiCD3, or observed with either selectin chimera used in the presence of 5 mM EDTA.

View larger version (60K): [in this window] [in a new window] [Download PPT slide]   Figure 8 Selectin ligand expression by Tc1 cells in WT and Fuc-T–deficient mice. CD8+ T cells were isolated from mesenteric nodes and cultured in vitro under Tc1 polarization conditions (Materials and Methods). Cells were then subjected to flow cytometry analysis to confirm Tc1 polarization by assessing expression of intracellular IFN- and IL-4 (A), or to assess selectin ligand expression using P-selectin-IgM or E-selectin-IgM chimeras (B). Divalent calcium dependence observed for binding of the P-selectin-IgM chimera (B, bottom) was also observed for the E-selectin-IgM chimera (data not shown). Data shown are representative of two or more independent experiments.

View larger version (83K): [in this window] [in a new window] [Download PPT slide]   Figure 9 Recruitment of activated T cells into inflamed skin in WT and Fuc-T–deficient mice. Lymphocytes were isolated from the PLNs of mice previously sensitized with DNFB, and were used in flow cytometry analyses (A–C), or were labeled with CMFDA and used in adoptive transfer experiments (D) (Materials and Methods). (A) The fraction of activated (CD44high) lymphocytes (mean ± SE) determined by flow cytometry. (B) Expression of P-selectin ligands or the Fuc-TVII locus (reported by EGFP) by flow cytometry in WT, Fuc-TVII–/–, and Fuc-TVII(-EGFP/-EGFP) mice. The top pair of representative two parameter flow cytometry histograms show CD44 expression level versus P-selectin-IgM chimera binding in lymphocytes prepared from DNFB-sensitized WT or Fuc-TVII–/– mice. The bottom pair of representative two parameter flow cytometry histograms show CD44 expression level versus green fluorescence in lymphocytes prepared from DNFB-sensitized WT or Fuc-TVII(-EGFP/-EGFP) mice. Numbers in the top right and top left quadrants are the percentage of all viable cells in the node that fall within that quadrant. Schematic depictions of the WT Fuc-TVII locus (reference 6), the null Fuc-TVII locus (reference 6), and the Fuc-TVII null/EGFP knock-in locus (Materials and Methods) in Fuc-TVII(-EGFP/-EGFP) mice are at the bottom. DNA and protein sequences in the region encompassing the initiation codon of the Fuc-TVII-EGFP fusion protein are above the Fuc-TVII null/EGFP knock-in schematic. (C) The fraction of CD44high cells in WT, Fuc-TIV–/–, or Fuc-TVII–/– cells that are positive for E-selectin-IgM or P-selectin-IgM chimera binding (mean ± SE) determined by flow cytometry (black bars), or the fraction of Fuc-TVII(-EGFP/-EGFP) cells (see B) that are green fluorescent (dashed box overlying the Fuc-TVII–/– bars; mean ± SE). (D) Adoptive transfer. 20 x 106 CMFDA-labeled cells were administered by tail vein injection to DNFB sensitized WT recipients. Recipients were immediately challenged with DNFB on one ear, and were treated with vehicle on the other ear. 24 h later, the number of CMFDA-positive donor cells in the dermis and epidermis combined, for each ear, was enumerated by flow cytometry (mean ± SE). Differences in CMFDA+ cell accumulation between genotypes were analyzed as in Fig. 1.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Several distinct (1,3)fucosyltransferases can construct sialylated, fucosylated molecules capable of contributing to the activities of counter-receptors for E-, P-, and L-selectins 12. Their expression patterns and gene ablation studies implicate two (Fuc-TVII and/or Fuc-TIV) in the physiological control of selectin ligand activity on neutrophils, monocytes, and HEVs 678. However, a role for Fuc-TVII in determining expression of Th1 and Tc1 cell E- and P-selectin ligands has been inferred only through correlative associations between its expression and selectin ligand display, and a requirement for either Fuc-TVII or Fuc-TIV in this process has not been established. Furthermore, requirements have not been established for either enzyme in the control of selectin ligand–dependent LC recruitment and migration, in effector T cell trafficking in vivo, or generally in the support of cell-mediated immunity in vivo. We address each of these issues by analyzing the components of the CHS response in mice deficient in Fuc-TIV, Fuc-TVII, and both Fuc-TIV and Fuc-TVII. These analyses disclose that both fucosyltransferases contribute essentially to the adaptive immune response by regulating selectin-dependent T lymphocyte migration.

During the afferent phase of CHS, LCs are required for antigen acquisition and processing, for delivery of processed antigen to naive T cells in draining LNs, and for events leading to T cell activation and expansion 212242. Selectin ligands are indirectly implicated in recruitment of LC precursors from the blood, as blood-derived DCs express E- and P-selectin ligands 1523, engage in selectin-dependent tethering and rolling on endothelium in noninflamed skin, and can be recruited to inflamed cutaneous sites 15. LCs themselves express the sialyl Lewis X determinant 14 and cutaneous lymphocyte antigen (CLA; reference 24), surrogate markers for selectin ligand activity 2. However, we find normal numbers of resident epidermal LC in the absence of Fuc-TIV and/or Fuc-TVII, and observe that emigration and the general function of LCs are intact in the absence of Fuc-TIV or Fuc-TVII. A possible modest reduction in LC emigration is observed in Fuc-TIV^–/–/Fuc-TVII^–/– mice. This could be a direct result of selectin ligand loss by the LCs, or an indirect consequence of the perturbed PLN microenvironment in these mice, and may contribute modestly to the CHS defect in these mice. Notwithstanding this caveat about LC migration in the doubly deficient mice, these results imply that any contribution by selectin ligands to populating noninflamed epidermis with LCs, or to LC function generally, must be determined by other fucosyltransferases, or by selectin ligands that are fucosyltransferase independent. Whether either enzyme controls DC recruitment in the context of cutaneous inflammation remains to be determined.

In Fuc-TVII^–/– mice, and in Fuc-TIV^–/–/Fuc-TVII^–/– mice, substantial, or profound deficiencies are observed, respectively, in the PLN content of B cells, memory T cells, and naive T cells. Fuc-TIV and Fuc-TVII collaboratively contribute to the synthesis of L-selectin ligands on PLN HEVs, and to lymphocyte homing to PLNs 68. L-selectin is required for homing of naive T cells to PLNs 2628, contributes to B cell homing 31, and in humans is implicated in the homing of CCR7⁺ central memory T cells 43. The numbers of peripheral blood B cells, naive T cells, and memory T cells available for homing are normal in Fuc-TVII^–/– and Fuc-TIV^–/–/Fuc-TVII^–/– mice, and their L-selectin expression levels are normal 68. These considerations infer that defects in HEV-borne L-selectin ligands, and corresponding defects in L-selectin–dependent lymphocyte adhesion and homing thus account for the paucity of such cells in these animals. Reductions in accumulation of activated CD4⁺ and CD8⁺ T cells, including Th1 and Tc1 cells, in Fuc-TVII^–/– and Fuc-TIV^–/–/VII^–/– PLNs in response to DNFB sensitization are clearly not consequent to an intrinsic defect in the ability to become activated or cytokine polarized, but instead likely reflect a quantitative deficiency of cells available to respond to this hapten. Such quantitative reductions in cells available for antigen-dependent activation and proliferation are reminiscent of L-selectin^–/– mice 252627, and of mice treated with Abs to L-selectin 28, where CHS defects are assigned to an inability to populate PLNs with naive T cells. The partial and nearly complete deficiencies, respectively, in the content of PLN naive T cells in Fuc-TVII^–/– or doubly deficient mice likely contribute to their partial or complete defects in CHS response by severely (Fuc-TVII^–/– mice) or profoundly diminishing (doubly deficient mice) the number of hapten-specific memory T cells generated during sensitization that are available for distribution to the skin and other nonlymphoid tissues 374445.

Recruitment of hapten-primed T lymphocytes to the cutaneous site of hapten challenge also provides a primary contribution to the CHS response. Recruitment of activated T cells to inflammatory sites requires E- and P-selectins expressed at such sites and selectin ligands expressed by Th1 and Tc1 cells 383940414647. Prior studies correlate inducible expression of Fuc-TVII with differentiation of T cells to Th1 or a Tc1 phenotype and their expression of E- and/or P-selectin ligands 9111213, though a requirement for Fuc-TVII in control of selectin ligand expression in these polarized cell types, in vitro, or in vivo, or in T cell trafficking to inflamed sites in vivo was not addressed. Our observations demonstrate that Fuc-TVII is an essential concomitant to normal E- and P-selectin ligand expression by Th1 and Tc1 cells generated in vitro or in vivo, and is required for normal recruitment of hapten-sensitized T cells to cutaneous sites of hapten challenge. These defects will thus contribute to the faulty CHS response in Fuc-TVII^–/– mice by diminishing recruitment of memory/effector T cells, at least in the context of haptenic challenge. The CMFDA-labeled cells used in the adoptive transfer experiments (Fig. 9) are largely activated CD4⁺ and CD8⁺ T cells (Fig. 5). Nonetheless, as unfractionated LN suspensions were used in these studies, additional work will be necessary to define the efficiency with which each cell type traffics in this model, and how Fuc-TVII deficiency may diminish or delete such trafficking. Absence of T cell selectin ligand expression in these mice may also contribute to their defective CHS responses by disallowing dissemination of hapten-specific memory T cells to skin and other peripheral tissues after hapten sensitization, although a requirement for the selectins and their ligands in distributing antigen-specific memory cells to peripheral tissues 4445 remains to be established.

Inducible expression of Fuc-TIV is inconsistently observed in association with Th1 polarization 913. Our observations indicate that Fuc-TIV deficiency in the context of a normal Fuc-TVII locus does not detectably diminish Th1 and Tc1 cell selectin ligand expression or trafficking. It remains to be determined if Fuc-TIV directs residual E- or P-selectin ligand expression by activated, cytokine-polarized T cells, as it does in neutrophils 8, if this accounts for the small amount of trafficking by Fuc-TVII^–/– donor cells to the cutaneous site of hapten challenge in our adoptive transfer experiments, and if this in turn permits the residual CHS response observed in Fuc-TVII^–/– mice.

Neutrophil recruitment is also required for the CHS response 48, in part by promoting recruitment of CD8⁺ T cells after hapten challenge 1937, and presumably by release of inflammatory mediators. Recruitment of neutrophils to the skin in response to nonspecific irritation is normal in Fuc-TIV^–/– mice, partially defective in Fuc-TVII^–/– mice, and fully deleted in Fuc-TIV^–/–/Fuc-TVII^–/– mice 8, indicating that defective neutrophil recruitment may also contribute to Fuc-TIV– and Fuc-TVII–dependent defects in the CHS response. However, the relative roles played by defective neutrophil and T cell recruitment in the efferent phase of CHS in these mice, as in mice with defects in the selectins or PSGL-1 46474950, are not addressed by these observations and remain to be defined.