We have used HSCA-2, an mAb that recognizes a sialic acid-dependent epitope on the low molecular mass (∼115-kDa) glycoform of CD43 that is expressed in resting T and NK cells, to examine the expression characteristics and stimulatory functions of CD43 in human CD4+ memory T cells. Having previously reported that the memory cells that respond to recall Ags in a CD4+CD45RO+ T cell population almost all belong to a subset whose surface CD43 expression levels are elevated, we now find that exposing these same memory T cells to HSCA-2 mAb markedly increases their proliferative responsiveness to recall Ags. We think it unlikely that this increase in responsiveness is a result of CD43-mediated monocyte activation, especially given that the HSCA-2 mAb differs from all previously used CD43 mAbs in having no obvious binding specificity for monocyte CD43. Predictably, treatment with HSCA-2 mAb did not lead to significant recall responses in CD4+CD45RO+ T cells, whose CD43 expression levels were similar to or lower than those of naive cells. Other experiments indicated that the HSCA-2 mAb was capable of enhancing the proliferative responsiveness of CD4+ memory T cells that had been exposed to polyclonal stimulation by monocyte-bound CD3 mAb and could also act in synergy with CD28 mAb to enhance the responsiveness of CD4+ T cells to CD3 stimulation. Taken together, these findings suggest that the CD43 molecules expressed on CD4+ memory T cells may be capable of enhancing the costimulatory signaling and hence providing accessory functions to TCR-mediated activation processes.

CD43 (leukosialin) is a highly glycosylated transmembrane protein that is expressed in all hemopoietic cells except resting mature B cells and erythrocytes (1, 2). All CD43 molecules possess extracellular domains that consist of multiple O-linked carbohydrate chains (3) and show considerable m.w. heterogeneity due to differential glycosylation (4). Even within T cell populations there are at least two glycoforms of CD43 that are recognized by different mAbs. The lighter of these glycoforms has a molecular mass of 95–115 kDa, whereas that of that heavier one is between 130 and 135 kDa (5, 6). Interestingly, the heavier glycoform contains core 2 O-glycans, appears to be up-regulated during T cell activation (5, 7), and can be used to distinguish between memory and effector CD8+ T cells in mice (8, 9). It is not yet clear exactly what CD43 does in T cells, however, especially in view of the continued existence of a number of unresolved controversies about its roles in such key processes as cell adhesion, cell death, and costimulation in TCR signaling (10).

It has, for instance, been claimed that CD43 molecules may have antiadhesive as well as proadhesive functions in T cell trafficking (9, 11, 12, 13, 14). It has also been claimed that up-regulation of CD43 expression can have a negative effect on activation-induced cell death of T cells (15), and that Ab-mediated cross-linking of CD43 induces apoptosis of Jurkat T cells (16). CD43 also appears to play a role in T cell activation, but precisely what role remains unclear. There are, for example, Ab cross-linking experiments involving CD43 that suggest that it may have a costimulatory role in vitro (17) and act in association with the phosphorylation of signal-transducing molecules in T cell activation (18, 19, 20); other experiments using CD43-deficient mice suggest, on the contrary, that CD43 has either a negative regulatory role as a steric barrier (21) or possibly even no significant role at all (22) in T cell activation. There are several recent reports suggesting that molecular complexes between CD43 and cytoskeletal adaptor proteins are probably excluded from the immunological synapse in T cell activation in vitro (23, 24, 25) and in vivo (26). Some of these reports include suggestions that this relocalization of CD43 is necessary for activation-induced cytokine production (23, 27), although there is at least one recent study that comes to precisely the opposite conclusion (28). The functional significance of redistribution of CD43 in T cell activation is therefore very unclear. Nevertheless, given that CD43 expression levels appear to be considerably elevated in memory T cell populations in humans (29, 30, 31) and mice (15), it seems reasonable to assume that CD43 has an important part to play in one or more aspects of the memory T cell responses.

We have recently described how HSCA-2, a novel CD43 mAb, can be used for the classification of human CD4+CD45RO+ memory T cells into three subsets on the basis of differences in their CD43 expression (31). In this classification, cells of the first of the three subsets (the M1 subset) express elevated levels of CD43, whereas cells of the M2 subset express CD43 levels similar to those of naive cells, and cells of the M3 subset express reduced CD43 levels. We also found that the M1 subset contains the highest proportion of recall Ag-reactive precursors and secretes substantially more IFN-γ and IL-4. The majority of effector memory T cells (CCR7) (32) are assumed to belong to this subset (31). However, as ∼70% of the cells in the M1 subset express CCR7, the subset may also contain central memory T cells. The M2 subset cells are less mature memory cells that retain longer telomeres than do cells of the M1 and M3 subsets, and their memory functionality (including recall Ag reactivity) appears to be marginal (31). The M3 subset consists of cells that are anergic to TCR-mediated stimuli and prone to apoptosis (31). As the level of CD43 expression is correlated with recall Ag reactivity, it is possible that CD43 molecules will prove to have some accessory role in the activation of human CD4+ memory T cells.

In this paper we describe immunological properties and expression characteristics of the CD43 molecules that are recognized by HSCA-2 mAb. We go on to examine the functional properties of these molecules in the proliferative responses of CD4+ memory T cells. The results described in this report demonstrate that the HSCA-2 mAb specifically recognizes a neuraminidase-sensitive epitope of a low molecular mass glycoform (115 kDa) of CD43 that is predominantly expressed in lymphoid populations. It is also suggested that the CD43 glycoform recognized by HSCA-2 mAbs could play an accessory part in the recall Ag-specific responses of mature CD4+ memory T cells (i.e., M1 subset cells). HSCA-2 mAb has therefore proven to be a useful molecular probe for both the classification and the functional analysis of human CD4+ memory T cells. The implications of our work for the involvement of CD43-mediated stimulatory signaling in the activation of CD4+ T cells are discussed.

The HSCA-2 hybridoma is a product of the fusion of NS1 mouse myeloma cells with splenocytes from BALB/c mice immunized by injection of human KG-1 cells (31). Immunization, fusion, selection, and cloning protocols were essentially as described previously (33). Hybridoma supernatants were initially screened for reactivity with KG-1 cells by indirect immunofluorescence. The HSCA-2 hybridoma was selected for further study because of its unique specificity of reactivity with PBMC and cord blood CD34+ stem cells. Isotype characterization showed that the HSCA-2 mAb was of the IgG1 subclass. Ascites fluid was obtained from SCID mice injected with the HSCA-2 hybridoma. After purification from ascites fluid by DE52 ion exchange chromatography, HSCA-2 mAb was labeled with FITC (Sigma-Aldrich, St. Louis, MO) for flow cytometry. Fab of HSCA-2 mAb were prepared by digestion with papain (34). This mAb was filed for participation in the Eighth International Workshop and Conference on Human Leukocyte Differentiation Ags (to be held in Adelaide, Australia).

Unconjugated CD28 mAb (clone CD28.2) (35), used for T cell culture, was purchased from Coulter-Immunotech (Marseilles, France). Unconjugated and FITC-conjugated CD43 mAbs, DFT-1 (1), L10 (36), and 1G10 (37), were obtained from Coulter-Immunotech, Caltag Laboratories (Burlingame, CA), and BD PharMingen (San Diego, CA), respectively. PE-labeled CD4, CD8, CD14, CD19, and CD56 mAbs and PerCP-labeled CD4 and CD8 mAbs were purchased from BD Biosciences (San Jose, CA). PE-labeled CD45RO mAb was obtained from Caltag Laboratories.

Total RNA of KG-1 cells was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA). First-strand cDNA primed with oligo(dT)30 was synthesized using SuperScript II reverse transcriptase (Invitrogen). CD43 cDNA was PCR-amplified with the primers 5′-ctcttgctcctgcctgtttgc-3′ and 5′-catggtggtgggtgcctgtaa-3′ using Advantage cDNA polymerase mix (Clontech Laboratories, Palo Alto, CA) and cloned into pCR2.1 TA cloning vector (Invitrogen). The sequence-verified clone was recloned into the EcoRI site of pIRESneo (Clontech Laboratories) and designated pIRESneo hCD43. Subsequently, 30 μg of pIRESneo hCD43 or pIRESneo (negative control) was electroporated into 2 × 106 HeLa cells in 200 μl of PBS byGenePulser (Bio-Rad, Hercules, CA) at 0.7 kV and 25 μF. Cells were treated with 1 mg/ml G418 for 2 wk. Drug-resistant colonies were selected and expanded to confirm CD43 expression by flow cytometry. HeLa transfectant cells expressing high levels of CD43 were isolated by a cell sorter for additional experiments.

For direct immunofluorescence of cultured cell lines, 2 × 105 CD43- or mock-transfected HeLa and KG-1 cells were stained with 1 μg of FITC-labeled HSCA-2, DFT-1, and MOPC21 mAbs for 45 min on ice. For the analyses of CD43 glycoepitopes, 2 × 106 KG-1 cells were treated with neuraminidase (0.1 U/ml in PBS) for 30 min at 37°C. For competitive inhibition, KG-1 cells were pretreated with various amounts of HSCA-2 or DFT-1 mAbs (12.5–200 μg/ml) for 1 h on ice and then stained with FITC-labeled HSCA-2 and DFT-1 mAbs for 45 min. FACScan (BD Biosciences) was used for flow cytometric analyses.

For flow cytometry of human blood cells, PBMCs from healthy adult volunteers (n = 6) and cord blood mononuclear cells (n = 3) were isolated by density centrifugation in Ficoll-Hypaque (density, 1.077 g/ml; ICN Biomedical, Aurora, OH). Granulocytes were isolated by double-layered density centrifugation in Ficoll-Hypaque (density, 1.077 and 1.119 g/ml; Wako Pure Chemical, Osaka, Japan) according to the manufacturer’s instructions. For isolation of monocytes, CD14+ cells were purified from PBMCs by positive enrichment using autoMACS (Miltenyi Biotec, Bergish Gladbach, Germany) according to the manufacturer’s instructions. Enriched monocytes also were used for immunoprecipitation and proliferation assay, as described below.

For single-color analysis of purified monocytes and granulocytes, cells were stained with FITC-labeled CD43 mAbs and analyzed by flow cytometry with a gate in a region for monocytes or granulocyte fractions on the forward and side light scatter profiles. For two-color analysis of lymphocytes, PBMCs were stained with PE-labeled CD4, CD8, CD19, and CD56 in combination with FITC-labeled CD43 mAbs. Cord blood mononuclear cells were stained with FITC-labeled CD43 and PE-labeled CD34 mAbs. For triple-color analysis, PBMCs were stained with FITC-labeled CD43, PE-conjugated CD45RO, and PerCP-labeled CD4 mAbs. CD4+ lymphocytes were gated on forward/side scatter and PerCP fluorescence. The proportions of CD4+ CD45RO cells (RO subset) and CD4+ CD45RO+ cells expressing high (M1 subset), intermediate (M2), and low (M3) levels of CD43 were measured by flow cytometry with FACScan (see Fig. 4).

FIGURE 4.

Flow cytometric analyses of CD43 expression in CD4+ T cell subsets. Expression of CD43 detected by either HSCA-2 (left) or DFT-1 (right) mAbs in combination with CD45RO in CD4+ T cells analyzed by triple-color immunofluorescence. Four different subsets were defined within CD4+T cells: CD45RO+ cells expressing higher (M1), intermediate (M2), and lower (M3) levels of CD43, and CD45RO (RO) cells in each CD43 mAb. In each donor a window for the M2 subset was set in a region where the CD43 level detected by each CD43 mAb was from approximately one-half to 2-fold the mean CD43 intensity for RO cells. Results are representative of six donors.

FIGURE 4.

Flow cytometric analyses of CD43 expression in CD4+ T cell subsets. Expression of CD43 detected by either HSCA-2 (left) or DFT-1 (right) mAbs in combination with CD45RO in CD4+ T cells analyzed by triple-color immunofluorescence. Four different subsets were defined within CD4+T cells: CD45RO+ cells expressing higher (M1), intermediate (M2), and lower (M3) levels of CD43, and CD45RO (RO) cells in each CD43 mAb. In each donor a window for the M2 subset was set in a region where the CD43 level detected by each CD43 mAb was from approximately one-half to 2-fold the mean CD43 intensity for RO cells. Results are representative of six donors.

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For preparation of activated CD4+ T cells, MACS-purified CD4+ T cells were stimulated with immobilized anti-CD3 mAb (OKT-3) in the presence of IL-2 (10 ng/ml) for 4 days in RPMI 1640 supplemented with 10% FCS. Immobilized CD3 mAb was prepared by binding OKT3 mAb (10 μg/ml in sodium bicarbonate buffer, pH 9.6) in 24-well plates at room temperature for 2 h, then washing the plates with RPMI 1640 supplemented with 10% FCS.

For isolation of the four CD4+ T cell subsets, M1, M2, M3, and CD45RO, CD4+ cells were purified by negative enrichment using MACS as described previously (31). MACS-purified CD4+ T cells were stained with FITC-labeled HSCA-2 and PE-labeled CD45RO mAbs. After incubation with propidium iodine at 10 μg/ml for 15 min to gate out dead cells, CD4+ T cells in the four subsets were sorted by a single laser cell sorter (FACStar; BD Biosciences). During cell sorting, stained and sorted cell suspensions were maintained at 4°C by a cooling circulation system.

For proliferative response to recall Ags, PBMCs (5 × 104 cells/well) in 96-well, flat-bottom plastic plates were stimulated with tuberculosis purified protein derivative (PPD;3 Connaught Laboratories, Ontario, Canada) or tetanus toxoid (TT; Calbiochem, La Jolla, CA) at 5 μg/ml. For total and subset CD4+ T cells, T cells (5 × 104 cells/well) were stimulated with these recall Ags in the presence of autologous monocytes (2.5 × 104 cells/well) that were previously isolated using autoMACS with anti-CD14 Ab (Miltenyi Biotec) and irradiated with x-ray at 30 Gy. The culture medium used for this assay was RPMI 1640 supplemented with 10% human serum. For proliferative responses to anti-CD3 mAb, total CD4+ T cells or sorted subset T cells were stimulated with various concentrations of soluble CD3 (OKT-3) mAb (0.0001–1 μg/ml) in the presence of autologous monocytes. The culture medium used for this assay was RPMI 1640 medium supplemented with 10% FCS.

The effects of CD43 (0.05–5 μg/ml) and CD28 mAbs (1 μg/ml) on cell proliferation were evaluated. Proliferation was measured on day 3 for CD3 mAb and on day 5 for PPD by adding [3H]thymidine (NEN, Boston, MA) at 1 μCi/well during the last 16 h of culture. All cultures were set up in triplicate.

Cells used for immunoprecipitation were KG-1 cells, PBMCs, MACS-purified CD14+ monocytes, PBMCs depleted with CD14+ monocytes, MACS-purified CD4+ T cells, and activated CD4+ T cells. Cells (5 × 106-1 × 107) were labeled at the cell surface by lactoperoxidase-catalyzed iodination as described previously (38). Radioiodinated cells were lysed with extraction buffer (0.5% Nonidet P-40, 10 mM Tris-HCl, 0.15 M NaCl, 1 mM PMSF, and 0.02% NaN3) for 10 min on ice. The mixture was centrifuged at 27,000 × g at 4°C for 20 min, and the supernatant was collected. Immunoprecipitations were performed by incubating radiolabeled cell lysate with 10 μg of HSCA-2, DFT-1, or MOPC21 mAbs for 1 h on ice, then adding a 20 μl-packed volume of protein G-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ), and further incubating the mixture for 30 min. Immunoprecipitates were washed four times in extraction buffer and solubilized in reducing Laemmli sample buffer subjected to SDS-PAGE (7.5% gel). After fixing and drying, the gels were autoradiographed at −80°C using x-ray film. Prestained SDS-PAGE standards were obtained from Bio-Rad (Hercules, CA).

HSCA-2 mAb resembles the reference CD43 mAb DFT-1 in binding to HeLa cells stably transfected with CD43+ cDNA, but not to their mock-transfected (CD43) counterparts (Fig. 1,A). HSCA-2 mAb also resembles the DFT-1 mAb in recognizing a neuraminidase-sensitive epitope on KG-1 cells (Fig. 1,B). However, although the binding of HSCA-2 mAb to KG-1 cells was all but completely blocked by DFT-1 mAb, the binding of DFT-1 mAb was only ∼90% blocked by HSCA-2 mAb even at the highest HSCA-2 mAb concentration (Fig. 1,C). These findings may imply that the binding affinity of HSCA-2 mAb is lower than that of DFT-1 mAb. The HSCA-2 mAb immunoprecipitated a surface protein of ∼115 kDa in KG-1 cells, whereas DFT-1 mAb immunoprecipitated both the115-kDa protein and a minor protein with a higher molecular mass of ∼125 kDa (Fig. 2 A). Immunoprecipitation and blocking experiments with another characterized CD43 mAb, 1G10, confirmed the results with the DFT-1 mAb (data not shown). These results suggest that HSCA-2 mAb reacts with a sialic acid-dependent epitope on the 115-kDa CD43 glycoform in KG-1 cells, but not with its equivalent on the 125-kDa glycoform.

FIGURE 1.

Flow cytometric analyses of CD43 expression in CD43+ HeLa transfectant and KG-1 cells with HSCA-2 and DFT-1 mAbs. A, Direct immunofluorescence staining of mock-transfected (upper panel) and CD43-transfected HeLa cells (lower panel) with FITC-labeled HSCA-2 (thick line), DFT-1 (thin line), and control IgG1 (broken line) mAbs. B, Effect of neuraminidase treatment on the expression of CD43 in KG-1 cells. Nontreated (thick line) and treated (thin line) KG-1 cells were stained with FITC-labeled HSCA-2 (upper panel), DFT-1 (lower panel) mAbs. Treated cells also were stained with control IgG1 (broken line). C, Blocking of the binding of FITC-labeled HSCA-2 (left) and DFT-1 (right) mAbs to KG-1 cells by excess amounts of HSCA-2, DFT-1, or MOPC21 (IgG1 control) mAbs.

FIGURE 1.

Flow cytometric analyses of CD43 expression in CD43+ HeLa transfectant and KG-1 cells with HSCA-2 and DFT-1 mAbs. A, Direct immunofluorescence staining of mock-transfected (upper panel) and CD43-transfected HeLa cells (lower panel) with FITC-labeled HSCA-2 (thick line), DFT-1 (thin line), and control IgG1 (broken line) mAbs. B, Effect of neuraminidase treatment on the expression of CD43 in KG-1 cells. Nontreated (thick line) and treated (thin line) KG-1 cells were stained with FITC-labeled HSCA-2 (upper panel), DFT-1 (lower panel) mAbs. Treated cells also were stained with control IgG1 (broken line). C, Blocking of the binding of FITC-labeled HSCA-2 (left) and DFT-1 (right) mAbs to KG-1 cells by excess amounts of HSCA-2, DFT-1, or MOPC21 (IgG1 control) mAbs.

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FIGURE 2.

Immunoprecipitation of 125I-labeled surface proteins from various types of white blood cells with HSCA-2, DFT-1, and MOPC21 (IgG1 control) mAbs. A, Immunoprecipitation of KG-1 cells and totalPBMCs. B, Immunoprecipitation of CD14, CD14+, CD4+, and activated CD4+ cells; 105-, 115-, 125-, and 135-kDa protein bands are indicated by an asterisk, arrows, open triangles, and closed triangles, respectively.

FIGURE 2.

Immunoprecipitation of 125I-labeled surface proteins from various types of white blood cells with HSCA-2, DFT-1, and MOPC21 (IgG1 control) mAbs. A, Immunoprecipitation of KG-1 cells and totalPBMCs. B, Immunoprecipitation of CD14, CD14+, CD4+, and activated CD4+ cells; 105-, 115-, 125-, and 135-kDa protein bands are indicated by an asterisk, arrows, open triangles, and closed triangles, respectively.

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The particular CD43 epitopes recognized by the HSCA-2 or DFT-1 mAb (hereafter abbreviated to CD43(HSCA-2) or CD43(DFT-1)) in normal lymphoid and myeloid cell populations were analyzed by two-color flow cytometry (Fig. 3). The CD43(HSCA-2) and CD43(DFT-1) epitopes were expressed at similar levels in CD4+ and CD8+ T cell populations. Neither HSCA-2 mAb nor DFT-1 mAb reacted with resting CD19+ B cells, whereas they both bound reasonably strongly to either PWM-activated or EBV-transformed B cells (data not shown). The majority of CD56+ NK cells expressed both CD43(HSCA-2) and CD43(DFT-1) epitopes at high levels, whereas there was relatively little of the CD43(HSCA-2) epitope in the minor subpopulation of NK cells. DFT-1 mAb was found to bind quite strongly to both purified monocytes and granulocytes, whereas binding by the HSCA-2 mAb was weak enough to be described as nonspecific, as judged by the results of immunoprecipitation analyses (see Fig. 2 B). We did not detect any increase in the level of either CD43(HSCA-2) or CD43(DFT-1) in cultured monocytes, even after the addition of LPS (data not shown). The HSCA-2 and DFT-1 mAbs both reacted quite strongly with cord blood CD34+ cells (data not shown). Two previously used CD43 mAbs, 1G10 and L10, appeared to share the cell type specificities of the DFT-1 mAb (data not shown).

FIGURE 3.

Flow cytometric analyses of CD43 expression in peripheral blood cells. For two-color analyses, PBMCs were stained with FITC-labeled HSCA-2 (thick line), DFT-1 (thin line), and control IgG1 (broken line) mAbs in combination with PE-labeled CD4, CD8, CD19, and CD56 mAbs. Monocytes and granulocytes isolated by MACS with CD14 mAb and density centrifugation, respectively, were singly stained with FITC-labeled mAbs. Results are representative of seven different donors.

FIGURE 3.

Flow cytometric analyses of CD43 expression in peripheral blood cells. For two-color analyses, PBMCs were stained with FITC-labeled HSCA-2 (thick line), DFT-1 (thin line), and control IgG1 (broken line) mAbs in combination with PE-labeled CD4, CD8, CD19, and CD56 mAbs. Monocytes and granulocytes isolated by MACS with CD14 mAb and density centrifugation, respectively, were singly stained with FITC-labeled mAbs. Results are representative of seven different donors.

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The results of experiments involving immunoprecipitation of 125I-labeled surface proteins from PBMCs, CD14 lymphoid cells, and CD4+ T cells with CD43 mAbs revealed that the HSCA-2 mAb recognizes only 115-kDa proteins, and whereas the DFT-1 mAb also reacts with 115-kDa proteins it can interact with a second minor, but higher molecular mass (∼125 kDa), protein as well (Fig. 2); these findings mirror our previous findings with KG-1 cells (see above). Other findings include the fact that HSCA-2 mAb failed to immunoprecipitate any specific proteins from CD14+ monocytes, unlike the DFT-1 mAb, which reacted with a 135-kDa protein (Fig. 2,B). In tests with activated CD4+ T cells, the DFT-1 mAb immunoprecipitated both the 135-kDa protein and a minor protein with lower molecular mass of 105 kDa, unlike the HSCA-2 mAb, which did not react with proteins of either of these sizes (Fig. 2 B). Both the HSCA-2 and DFT-1 mAbs appeared to specifically immunoprecipitate several common, low molecular mass (25- to 40-kDa) proteins in activated CD4+ T cells (data not shown).

As shown in Fig. 4 (left), the CD4+CD45RO+ cell population can be divided into three distinct subsets (M1, M2, and M3) on the basis of their CD43(HSCA-2) expression levels; this confirms our previous findings (31). We therefore tried to define the same three subsets on the basis of their CD43(DFT-1) expression levels (Fig. 4, right); interestingly, the proportions of the M1 subset detected with the DFT-1 and HSCA-2 mAbs were not significantly different (Table I), whereas the proportion of the M2 subset defined by the DFT-1 mAb was significantly larger when defined by HSCA-2 mAb,and the proportion of the M3 subset defined by the DFT-1 mAb was significantly smaller than when defined by HSCA-2 mAb. We observed similar subset percentages when the 1G10 and L10 mAbs were used in place of the DFT-1 mAb (Table I). These results indicate that the low levels of CD43(HSCA-2) expression that typify the M3 population do not affect the ability of M3 cells to express other CD43 epitopes.

Table I.

CD4 memory T cell subsets defined by four different CD43 mAbs

CD43 mAbSubsets (% in total CD44 T cellsa)
M1M2M3
HSCA-2 19.9 ± 4.4b 21.8 ± 8.0 5.7 ± 3.0 
DFT-1 19.0 ± 3.7 25.3 ± 9.4c 3.0 ± 1.4c 
L10 20.5 ± 4.3 23.6 ± 8.7c 2.6 ± 1.4c 
1G10 19.0 ± 4.1 24.5 ± 8.2c 2.8 ± 1.8c 
CD43 mAbSubsets (% in total CD44 T cellsa)
M1M2M3
HSCA-2 19.9 ± 4.4b 21.8 ± 8.0 5.7 ± 3.0 
DFT-1 19.0 ± 3.7 25.3 ± 9.4c 3.0 ± 1.4c 
L10 20.5 ± 4.3 23.6 ± 8.7c 2.6 ± 1.4c 
1G10 19.0 ± 4.1 24.5 ± 8.2c 2.8 ± 1.8c 
a

The percentage of each subset in total CD4+ T cells was determined by three-color flow cytometry, as shown in Fig. 4 A.

b

Average ± SD (n = 6)

c

Value was significantly larger or smaller than that of HSCA-2 mAb by Wilcoxon signed rank test (p < 0.05).

To analyze possible accessory functions of CD43(HSCA-2) in memory T cells, we first examined the effects of exposure to HSCA-2 mAb on the recall Ag-induced proliferation of totalPBMCs in culture. As shown in Fig. 5, HSCA-2 mAb seemed to dose-dependently accelerate the proliferation of PPD Ag-stimulated PBMCs, but had no comparable effect on their proliferation in the absence of PPD. The three traditionally used anti-CD43 mAbs (DFT-1 (Fig. 5,B), and 1G10 and L10 (data not shown)) also proved at least as effective as the HSCA-2 mAb in accelerating PPD-stimulated proliferation of PBMCs. As the majority of PPD-reactive cells appear to be CD4+ T cells (data not shown), we examined the effects of the addition of a combination of HSCA-2 mAb and CD28 mAb on the responses of MACS-purified CD4+ T cells in the presence of autologous monocytes (Fig. 6). The CD28 mAb that we chose to use in this experiment was the CD28.2 clone, which is capable of strongly costimulating polyclonal T cell responses to plastic- or monocyte-bound CD3 mAb, but (if anything) inhibits Ag-specific responses (39). Thus, whereas HSCA-2 mAb led to a significantly enhanced PPD response in CD4+ T cells, the addition of CD28 mAb led to it being slightly inhibited. We also examined the effect of the HSCA-2 mAb on the TT-dependent proliferative response of CD4+ T cells and found that it had an enhancing effect (Fig. 6).

FIGURE 5.

Acceleration of proliferative responses of PBMCs to PPD by stimulation with CD43 mAbs. A, Time courses of PPD responses.PBMCs were stimulated with (solid line) or without (broken line) PPD (5 μg/ml) in the presence (•) or the absence (○) of HSCA-2 mAb (5 μg/ml). B, Dose responses of CD43 mAbs. PBMCs were stimulated with (solid line) or without (broken line) PPD (5 μg/ml) in the presence of HSCA-2 (•), DFT-1 (▵), and MOPC21 (○) at the various concentrations. Proliferation was measured on day 5 by adding [3H]thymidine during the last 16 h of culture. Results are representative of five donors.

FIGURE 5.

Acceleration of proliferative responses of PBMCs to PPD by stimulation with CD43 mAbs. A, Time courses of PPD responses.PBMCs were stimulated with (solid line) or without (broken line) PPD (5 μg/ml) in the presence (•) or the absence (○) of HSCA-2 mAb (5 μg/ml). B, Dose responses of CD43 mAbs. PBMCs were stimulated with (solid line) or without (broken line) PPD (5 μg/ml) in the presence of HSCA-2 (•), DFT-1 (▵), and MOPC21 (○) at the various concentrations. Proliferation was measured on day 5 by adding [3H]thymidine during the last 16 h of culture. Results are representative of five donors.

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FIGURE 6.

Effects of HSCA-2 and CD28 mAbs on the proliferative responses of CD4 T cells to recall Ags. MACS-purified CD4 T cells were stimulated with PPD or TT in the presence of autologous CD14+ APC. HSCA-2 (5 μg/ml) and/or CD28 (1 μg/ml) mAbs were added to the culture. Proliferation was measured on day 5 (PPD) or day 7 (TT) by adding [3H]thymidine during the last 16 h of culture. Results were expressed as the mean cpm ± SD and are representative of three donors.

FIGURE 6.

Effects of HSCA-2 and CD28 mAbs on the proliferative responses of CD4 T cells to recall Ags. MACS-purified CD4 T cells were stimulated with PPD or TT in the presence of autologous CD14+ APC. HSCA-2 (5 μg/ml) and/or CD28 (1 μg/ml) mAbs were added to the culture. Proliferation was measured on day 5 (PPD) or day 7 (TT) by adding [3H]thymidine during the last 16 h of culture. Results were expressed as the mean cpm ± SD and are representative of three donors.

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Next we examined the effects of the addition of CD28 and HSCA-2 mAbs on recall responses in each of the three CD4+ memory T cell subsets as defined by their separation in a cell sorter on the basis of their CD43(HSCA-2) expression levels (Fig. 4). As shown in Fig. 7,A, only the M1 subset cells appeared to be capable of responding to PPD; this confirms our previous findings (31). The results of our experiments with the M1 subset mirrored our findings with unseparated CD4+ T cell populations, in that the PPD response of M1 subset cells was significantly and dose-dependently enhanced by the addition of HSCA-2 mAb (Fig. 7, B and C), but was inhibited, rather than enhanced, in the presence of the CD28 mAb (Fig. 7,A). The PPD responses of the M2 and M3 subset cells were virtually unaffected by the addition of the HSCA-2 mAb (Fig. 7, B and C). This did not surprise us, given that the M2 and M3 subsets appeared to contain a relatively very small PPD-reactive precursor component (31). The stimulatory effect of the FITC-HSCA-2 mAb that remains attached to cells after its use in the course of their separation was negligible, given that the pretreatment of unseparated CD4+ T cells with FITC-HSCA-2 mAb did not significantly alter their subsequent response to PPD, whereas addition of the mAb to cultures of both FITC-HSCA-2 mAb-pretreated and nontreated CD4+ T cells increased their responses to PPD to very much the same extent (Fig. 7 B). Taken together, the above results indicate that CD43(HSCA-2) is capable of acting as an accessory molecule in the Ag-specific recall response of mature CD4+ memory T cells.

FIGURE 7.

Effects of CD28 and HSCA-2 mAbs on the proliferative responses of CD4 T cell subsets to PPD. A–C, MACS-purified CD4+ T cells were stained with FITC-HSCA-2 and PE-CD45RO mAbs (see Fig. 4,A, left) and thereafter sorted by FACS into the indicated subsets. Each subset cells and total CD4+ T cells were stimulated with PPD in the presence of autologous CD14+ APC. CD28 (1 μg/ml; A) or HSCA-2 (5 μg/ml; B) mAbs were added to the culture. Total CD4+ T cells pretreated with FITC-HSCA-2 mAb also were examined for possible effects of immunofluorescence staining with HSCA-2 mAb on the PPD response (B). M2+M3, Cell populations sorted by gating the combined region of the M2 and M3 subsets (Fig. 4 A, left). Dose responses of HSCA-2 mAb for M1 subset cells and a combined cell population of M2 and M3 subsets were examined (C). Proliferation was measured on day 5 by adding [3H]thymidine during the last 16 h of culture. Results were expressed as the mean cpm ± SD and are representative of three donors.

FIGURE 7.

Effects of CD28 and HSCA-2 mAbs on the proliferative responses of CD4 T cell subsets to PPD. A–C, MACS-purified CD4+ T cells were stained with FITC-HSCA-2 and PE-CD45RO mAbs (see Fig. 4,A, left) and thereafter sorted by FACS into the indicated subsets. Each subset cells and total CD4+ T cells were stimulated with PPD in the presence of autologous CD14+ APC. CD28 (1 μg/ml; A) or HSCA-2 (5 μg/ml; B) mAbs were added to the culture. Total CD4+ T cells pretreated with FITC-HSCA-2 mAb also were examined for possible effects of immunofluorescence staining with HSCA-2 mAb on the PPD response (B). M2+M3, Cell populations sorted by gating the combined region of the M2 and M3 subsets (Fig. 4 A, left). Dose responses of HSCA-2 mAb for M1 subset cells and a combined cell population of M2 and M3 subsets were examined (C). Proliferation was measured on day 5 by adding [3H]thymidine during the last 16 h of culture. Results were expressed as the mean cpm ± SD and are representative of three donors.

Close modal

To determine whether CD43(HSCA-2) can play an accessory role in the polyclonal activation of T cells, we examined the possible effects of the HSCA-2 mAb on the proliferative response of CD4+ T cells to monocyte-bound CD3 mAb (Fig. 8). We found that the HSCA-2 mAb did have an effect, but that it was only marginally costimulatory at lower (0.001–0.1 μg/ml) CD3 mAb concentrations and that its effectiveness disappeared at the highest concentration tested (1 μg/ml). Interestingly, the results shown in Fig. 8,A provide convincing evidence that the CD28/HSCA-2 mAb combination had a synergistic effect on the CD3 mAb-mediated polyclonal response (Fig. 8 A). A similar synergistic effect on the CD3-mediated response was observed when the mAbs involved were DFT-1 and CD28 (data not shown).

FIGURE 8.

Synergistic effects of HSCA-2 and CD28 mAbs on the proliferative responses of CD4+ T cells to monocyte-bound CD3 mAb. Total CD4+ T cells (A) and CD4+ T cell subset cells (B) were stimulated with various concentrations (A) or 0.1 μg/ml (B) of CD3 mAb in the presence of autologous CD14+ monocytes. The effects of HSCA-2 (5 μg/ml) and CD28 (1 μg/ml) mAbs on the CD3 responses were tested by single or combined use of these mAbs. Proliferation was measured on day 3 by adding [3H]thymidine during the last 16 h of culture. Results were expressed as the mean cpm ± SD and are representative of three donors.

FIGURE 8.

Synergistic effects of HSCA-2 and CD28 mAbs on the proliferative responses of CD4+ T cells to monocyte-bound CD3 mAb. Total CD4+ T cells (A) and CD4+ T cell subset cells (B) were stimulated with various concentrations (A) or 0.1 μg/ml (B) of CD3 mAb in the presence of autologous CD14+ monocytes. The effects of HSCA-2 (5 μg/ml) and CD28 (1 μg/ml) mAbs on the CD3 responses were tested by single or combined use of these mAbs. Proliferation was measured on day 3 by adding [3H]thymidine during the last 16 h of culture. Results were expressed as the mean cpm ± SD and are representative of three donors.

Close modal

Cells of two of the three CD4+ memory T cell subsets (M1 and M2) responded strongly to monocyte-bound CD3 mAb, whereas M3 subset cells did not (Fig. 8 B). These findings are in agreement with those in our original report (31). The HSCA-2 mAb was almost as effective as CD28 mAb in their enhancement of polyclonal responses in M1 and M2 subset cells, but had no such effect in M3 subset cells. There were marginal synergistic effects on polyclonal responses when M1 and M2 subset cells were cotreated with the HSCA-2 and CD28 mAbs, although a much more obvious synergistic effect became evident when we used RO naive subset cells instead.

When the Fab portion of the HSCA-2 mAb was used instead of intact Ab, we could see no indication of either an enhanced PPD-mediated stimulatory response or a synergistic interaction involving the CD28 mAb and CD3 mAb polyclonal responses (data not shown).

HSCA-2 mAb specifically recognizes a neuraminidase-sensitive epitope on the low molecular mass (115-kDa) glycoform of the CD43 molecule that is predominantly expressed in lymphoid cells, including resting T and NK cells. By contrast, all previously described CD43 mAbs (including the DFT-1 mAb) react strongly or even very strongly with a larger (135-kDa) CD43 glycoform that is expressed in myeloid cells such as monocytes and granulocytes. Importantly, the HSCA-2 mAb does not appear to recognize the 135-kDa glycoform and hence binds only marginally, if at all, to myeloid cells; it also does not immunoprecipitate a third high molecular mass (125-kDa) CD43 protein that is recognized by the DFT-1mAb in both KG-1 and CD4+ T cells. Taken together, these findings suggest that the HSCA-2 mAb is specific for a novel glycoepitope on the 115-kDa glycoform of CD43.

Interestingly, HSCA-2 mAb differs from all pre-existing CD43 mAbs in being unable to recognize the high molecular mass CD43 glycoform (135 kDa) that is present on activated CD4+ T cells. Thus, the 135-kDa CD43 glycoform consists of a more fully glycosylated version that is generated in the course of the increase in molecular mass of the 115-kDa CD43 glycoform that occurs during T cell activation (5, 7); it is possible that the HSCA-2 glycoepitope is either lost or masked in the course of this glycosyl modification process. As the molecular mass of the CD43 polypeptide is ∼40 kDa, the 25- to 40-kDa proteins recognized by HSCA-2 mAb in activated T cells could well be degradation forms of CD43, and we have even observed what appeared to be the gradual disappearance of CD43(HSCA-2) epitopes from a subpopulation of activated CD4+CD45RO+ T cells (manuscript in preparation).

We noticed that there were significant differences in the distribution of the CD4 memory T cell subsets depending upon which of the available CD43 mAbs was used in their separation. Thus, for example, the percentages of M2 subset cells that we observed in separations achieved using the HSCA-2 mAb were significantly smaller than those observed in separations using any of the other available mAbs. In the case of the M3 subsets, the percentages were larger with the HSCA-2 mAb than with any of the others. These CD43 mAb-dependent differences in subset distributions may correspond to differences in such subset cell functions as memory vs anergy, but we have yet to explore this possibility in any detail.

In this report we show that HSCA-2 and certain other CD43 mAbs are capable of accelerating both the recall Ag-induced and CD3 mAb-induced proliferation of CD4 memory T cells. There are previous reports indicating that CD43 molecules may also have accessory involvements in T cell activation, such as, for example, in mice, where CD43 mAb appears to costimulate T cell activation during treatment with plastic-bound CD3 mAb and alloantigens (17, 40). In humans, however, there does not appear to be any evidence of CD43 mAb being involved in a costimulatory capacity in the polyclonal activation of T cells (41). In situations involving Ag-specific responses, there is one report that CD43 is necessary for the production of IL-2 in HLA class II-specific human hybridoma T cells (42), but it is important to note that the experimental system used to obtain these data was an unusually artificial one. There are, however, several publications in which it is claimed that a number of CD43 mAbs can stimulate Ag-independent human T cell proliferation in a multicomponent test system that requires the presence of CD43-stimulated monocytes (43, 44, 45, 46). Thus, the results described in this study appear to provide the first evidence that T cell-determined CD43 may help to stimulate the Ag-specific proliferative responses of freshly isolated T cells in humans. In the case of our new mAb (HSCA-2), we can exclude any involvement of CD43-mediated monocyte stimulation in T cell activation. The reason why HSCA-2 mAb differs from all previously used CD43 mAbs in this important way is that it appears to lack reactivity to the 135-kDa CD43 glycoform expressed in monocytes.

Given the above consideration, it seems reasonable to assume that CD43 plays a part in some of the cell signaling events that are likely to be involved in memory T cell activation. Up-regulated expression of CD43 in M1 subset cells may cause an increase in activation signaling in concert with other up-regulated costimulatory molecules such as CD28 (31). Our observation that CD43 and CD28 mAbs act synergistically to stimulate the polyclonal response of CD4+ T cells to anti-CD3 mAb may indicate that both CD43 and CD28 have an accessory signaling role in the induction of the polyclonal response. It has previously been reported that T cell activation through CD43 cross-linking in humans induces serine phosphorylation of Cbl proteins and tyrosine phosphorylation of Vav (19, 20). It has also been reported that one of the CD28-determined costimulatory signaling processes is mediated by tyrosine phosphorylation of Vav1, which is, in turn, negatively regulated by Cbl-b (47, 48, 49). Thus, although the precise molecular mechanisms underlying the costimulatory effects of CD43 remain to be determined, it is possible that both CD28 and CD43 are capable of synergistically enhancing the activation of CD4+ memory T cells by a mechanism involving the common signaling pathway.

We grateful to Dr. Donald MacPhee for his valuable suggestions, to Mika Yamaoka for her excellent assistance with FACS analysis, and to Mika Yonezawa and Jeffrey Hart for manuscript preparation.

1

This publication is based on research performed at the Radiation Effects Research Foundation (RERF), Hiroshima and Nagasaki, Japan. RERF is a private nonprofit foundation funded equally by the Japanese Ministry of Health, Labor, and Welfare (MHLW) and the U.S. Department of Energy, the latter through the National Academy of Sciences. This work was supported by RERF Research Protocols 1-93 and 4-02 and in part by funds for Research Promotion on AIDS Control from MHLW.

3

Abbreviations used in this paper: PPD, purified protein derivative; TT, tetanus toxoid.

1
Remold-O’Donnell, E..
1995
. CD43 cluster report. S. F. Schlossman, and L. Boumsell, and W. Gilks, and J. M. Harlan, and T. Kishimoto, and C. Morimoto, and J. Ritz, and S. Shaw, and R. Silverstein, and T. Springer, et al eds.
Leucocyte Typing. Vol. V: White Cell Differentiation Antigens
1697
. Oxford University Press, New York.
2
Rosenstein, Y., A. Santana, G. Pedraza-Alva.
1999
. CD43, a molecule with multiple functions.
Immunol. Res.
20
:
89
.
3
Cyster, J. G., D. M. Shotton, A. F. Williams.
1991
. The dimensions of the T lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation.
EMBO J.
10
:
893
.
4
Carlsson, S. R., H. Sasaki, M. Fukuda.
1986
. Structural variations of O-linked oligosaccharides present in leukosialin isolated from erythroid, myeloid, and T-lymphoid cell lines.
J. Biol. Chem.
261
:
12787
.
5
Piller, F., F. Le Deist, K. I. Weinberg, R. Parkman, M. Fukuda.
1991
. Altered O-glycan synthesis in lymphocytes from patients with Wiskott-Aldrich syndrome.
J. Exp. Med.
173
:
1501
.
6
Jones, A. T., B. Federsppiel, L. G. Ellies, M. J. Williams, R. Burgener, V. Duronio, C. A. Smith, F. Takei, H. J. Ziltener.
1994
. Characterization of the activation-associated isoform of CD43 on murine T lymphocytes.
J. Immunol.
153
:
3426
.
7
Piller, F., V. Piller, R. I. Fox, M. Fukuda.
1988
. Human T-lymphocyte activation is associated with changes in O-glycan biosynthesis.
J. Biol. Chem.
263
:
15146
.
8
Harrington, L. E., M. Galvan, L. G. Baum, J. D. Altman, R. Ahmed.
2000
. Differentiating between memory and effector CD8 T cells by altered expression of cell surface O-glycans.
J. Exp. Med.
191
:
1241
.
9
Onami, T. M., L. E. Harrington, M. A. Williams, M. Galvan, C. P. Larsen, T. C. Pearson, N. Manjunath, L. G. Baum, B. D. Pearce, R. Ahmed.
2002
. Dynamic regulation of T cell immunity by CD43.
J. Immunol.
168
:
6022
.
10
Ostberg, J. R., R. K. Barth, J. G. Frelinger.
1998
. The Roman god Janus: a paradigm for the function of CD43.
Immunol. Today
19
:
546
.
11
Manjunath, N., M. Correa, M. Ardman, B. Ardman.
1995
. Negative regulation of T-cell adhesion and activation by CD43.
Nature
377
:
535
.
12
Stockton, B. M., G. Cheng, N. Manjunath, B. Ardman, U. H. von Andrian.
1998
. Negative regulation of T cell homing by CD43.
Immunity
8
:
373
.
13
McEvoy, L. M., H. Sun, J. G. Frelinger, E. C. Butcher.
1997
. Anti-CD43 inhibition of T cell homing.
J. Exp. Med.
185
:
1493
.
14
Woodman, R. C., B. Johnston, M. J. Hickey, D. Teoh, P. Reinhardt, B. Y. Poon, P. Kubes.
1998
. The functional paradox of CD43 in leukocyte recruitment: a study using CD43-deficient mice.
J. Exp. Med.
188
:
2181
.
15
He, Y. W., M. J. Bevan.
1999
. High level expression of CD43 inhibits T cell receptor/CD3-mediated apoptosis.
J. Exp. Med.
190
:
1903
.
16
Brown, T. J., W. W. Shuford, W. C. Wang, S. G. Nadler, T. S. Bailey, H. Marquardt, R. S. Mittler.
1996
. Characterization of a CD43/leukosialin-mediated pathway for inducing apoptosis in human T-lymphoblastoid cells.
J. Biol. Chem.
271
:
27686
.
17
Sperling, A. I., J. M. Green, R. L. Mosley, P. L. Smith, R. J. DiPaolo, J. R. Klein, J. A. Bluestone, C. B. Thompson.
1995
. CD43 is a murine T cell costimulatory receptor that functions independently of CD28.
J. Exp. Med.
182
:
139
.
18
Pedraza-Alva, G., L. B. Merida, S. J. Burakoff, Y. Rosenstein.
1996
. CD43-specific activation of T cells induces association of CD43 to Fyn kinase.
J. Biol. Chem.
271
:
27564
.
19
Pedraza-Alva, G., L. B. Merida, S. J. Burakoff, Y. Rosenstein.
1998
. T cell activation through the CD43 molecule leads to Vav tyrosine phosphorylation and mitogen-activated protein kinase pathway activation.
J. Biol. Chem.
273
:
14218
.
20
Pedraza-Alva, G., S. Sawasdikosol, Y. C. Liu, L. B. Merida, M. E. Cruz-Munoz, F. Oceguera-Yanez, S. J. Burakoff, Y. Rosenstein.
2001
. Regulation of Cbl molecular interactions by the co-receptor molecule CD43 in human T cells.
J. Biol. Chem.
276
:
729
.
21
Thurman, E. C., J. Walker, S. Jayaraman, N. Manjunath, B. Ardman, J. M. Green.
1998
. Regulation of in vitro and in vivo T cell activation by CD43.
Int. Immunol.
10
:
691
.
22
Carlow, D. A., S. Y. Corbel, H. J. Ziltener.
2001
. Absence of CD43 fails to alter T cell development and responsiveness.
J. Immunol.
166
:
256
.
23
Allenspach, E. J., P. Cullinan, J. Tong, Q. Tang, A. G. Tesciuba, J. L. Cannon, S. M. Takahashi, R. Morgan, J. K. Burkhardt, A. I. Sperling.
2001
. ERM-dependent movement of CD43 defines a novel protein complex distal to the immunological synapse.
Immunity
15
:
739
.
24
Delon, J., K. Kaibuchi, R. N. Germain.
2001
. Exclusion of CD43 from the immunological synapse is mediated by phosphorylation-regulated relocation of the cytoskeletal adaptor moesin.
Immunity
15
:
691
.
25
Shaw, A. S..
2001
. FERMing up the synapse.
Immunity
15
:
683
.
26
Stoll, S., J. Delon, T. M. Brotz, R. N. Germain.
2002
. Dynamic imaging of T cell-dendritic cell interactions in lymph nodes.
Science
296
:
1873
.
27
Cullinan, P., A. I. Sperling, J. K. Burkhardt.
2002
. The distal pole complex: a novel membrane domain distal to the immunological synapse.
Immunol. Rev.
189
:
111
.
28
Savage, N. D., S. L. Kimzey, S. K. Bromley, K. G. Johnson, M. L. Dustin, J. M. Green.
2002
. Polar redistribution of the sialoglycoprotein CD43: implications for T cell function.
J. Immunol.
168
:
3740
.
29
Youseffi-Etemad, R., B. Axelsson.
1996
. Parallel pattern of expression of CD43 and of LFA-1 on the CD45RA+ (naive) and CD45RO+ (memory) subsets of human CD4+ and CD8+ cells: correlation with the aggregative response of the cells to CD43 monoclonal antibodies.
Immunology
87
:
439
.
30
Mukasa, R., T. Homma, T. Ohtsuki, O. Hosono, A. Souta, T. Kitamura, M. Fukuda, S. Watanabe, C. Morimoto.
1999
. Core 2-containing O-glycans on CD43 are preferentially expressed in the memory subset of human CD4 T cells.
Int. Immunol.
11
:
259
.
31
Ohara, T., K. Koyama, Y. Kusunoki, T. Hayashi, N. Tsuyama, Y. Kubo, S. Kyoizumi.
2002
. Memory functions and death proneness in three CD4+CD45RO+ human T cell subsets.
J. Immunol.
169
:
39
.
32
Sallusto, F., D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia.
1999
. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.
Nature
401
:
708
.
33
Kyoizumi, S., M. Akiyama, N. Kouno, K. Kobuke, M. Hakoda, S. L. Jones, M. Yamakido.
1985
. Monoclonal antibodies to human squamous cell carcinoma of the lung and their application to tumor diagnosis.
Cancer Res.
45
:
3274
.
34
Andrew, S. M., J. Titus.
1996
. Fragmentation of immunogloblin G. J. E. Coligan, and A. M. Kruisbeek, and D. H. Margulies, and E. M. Shevach, and W. Strober, eds.
Current Protocols in Immunology
2. 10
. John Wiley & Sons, New York and London.
35
Nunes, J., S. Klasen, M. Ragueneau, C. Pavon, D. Couez, C. Mawas, M. Bagnasco, D. Olive.
1993
. CD28 mAbs with distinct binding properties differ in their ability to induce T cell activation: analysis of early and late activation events.
Int. Immunol.
5
:
311
.
36
Remold-O’Donnell, E., D. M. Kenney, R. Parkman, L. Cairns, B. Savage, F. S. Rosen.
1984
. Characterization of a human lymphocyte surface sialoglycoprotein that is defective in Wiskott-Aldrich syndrome.
J. Exp. Med.
159
:
1705
.
37
Horejsi, V., H. Stockinger.
1997
. CD43 workshop panel report. T. Kishimoto, and H. Kikutani, and A. E. G. Kr. von dem Borne, and S. M. Goyert, and D. Y. Mason, and M. Miyasaka, and L. Moretta, and K. Okumura, and S. Shaw, and T. A. Springer, et al eds.
Leucocyte Typing, Vol. VI: White Cell Differentiation Antigens
494
. Garland, New York and London.
38
Kyoizumi, S., M. Akiyama, Y. Hirai, Y. Kusunoki, K. Tanabe, S. Umeki.
1990
. Spontaneous loss and alteration of antigen receptor expression in mature CD4+ T cells.
J. Exp. Med.
171
:
1981
.
39
Olive, D., C. Cerdan, R. Costello, I. Sielleur, M. Ragueneau, F. Pages, S. Klasen, J. Nunes, J. Imbert.
1995
. CD28 and CTLA-4 cluster report. S. F. Schlossman, and L. Boumsell, and W. Gilks, and J. M. Harlan, and T. Kishimoto, and C. Morimoto, and J. Ritz, and S. Shaw, and R. Silverstein, and T. Springer, et al eds.
Leucocyte Typing, Vol. V: White Cell Differentiation Antigens
360
. Oxford University Press, New York.
40
Walker, J., J. M. Green.
1999
. Structural requirements for CD43 function.
J. Immunol.
162
:
4109
.
41
Tkaczuk, J., T. Al Saati, I. Escargueil-Blanc, A. Salvayre, V. Horejsi, M. Durand, C. de Preval, E. Ohayon, G. Delsol, M. Abbal.
1999
. The CBF.78 monoclonal antibody to human sialophorin has distinct properties giving new insights into the CD43 marker and its activation pathway.
Tissue Antigens
54
:
1
.
42
Park, J. K., Y. J. Rosenstein, E. Remold-O’Donnell, B. E. Bierer, F. S. Rosen, S. J. Burakoff.
1991
. Enhancement of T-cell activation by the CD43 molecule whose expression is defective in Wiskott-Aldrich syndrome.
Nature
350
:
706
.
43
Mentzer, S. J., E. Remold-O’Donnell, M. A. Crimmins, B. E. Bierer, F. S. Rosen, S. J. Burakoff.
1987
. Sialophorin, a surface sialoglycoprotein defective in the Wiskott-Aldrich syndrome, is involved in human T lymphocyte proliferation.
J. Exp. Med.
165
:
1383
.
44
Axelsson, B., R. Youseffi-Etemad, S. Hammarstrom, P. Perlmann.
1988
. Induction of aggregation and enhancement of proliferation and IL-2 secretion in human T cells by antibodies to CD43.
J. Immunol.
141
:
2912
.
45
Nong, Y. H., E. Remold-O’Donnell, T. W. LeBien, H. G. Remold.
1989
. A monoclonal antibody to sialophorin (CD43) induces homotypic adhesion and activation of human monocytes.
J. Exp. Med.
170
:
259
.
46
Alvarado, M., C. Klassen, J. Cerny, V. Horejsi, R. E. Schmidt.
1995
. MEM-59 monoclonal antibody detects a CD43 epitope involved in lymphocyte activation.
Eur. J. Immunol.
25
:
1051
.
47
Chiang, Y. J., H. K. Kole, K. Brown, M. Naramura, S. Fukuhara, R. J. Hu, I. K. Jang, J. S. Gutkind, E. Shevach, H. Gu.
2000
. Cbl-b regulates the CD28 dependence of T-cell activation.
Nature
403
:
216
.
48
Bachmaier, K., C. Krawczyk, I. Kozieradzki, Y. Y. Kong, T. Sasaki, A. Oliveira-dos-Santos, S. Mariathasan, D. Bouchard, A. Wakeham, A. Itie, et al
2000
. Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b.
Nature
403
:
211
.
49
Krawczyk, C., K. Bachmaier, T. Sasaki, G. R. Jones, B. S. Snapper, D. Bouchard, I. Kozieradzki, S. P. Ohashi, W. F. Alt, M. J. Penninger.
2000
. Cbl-b is a negative regulator of receptor clustering and raft aggregation in T cells.
Immunity
13
:
463
.