Immunohistochemical Detection of Sialyl LeX Antigen on Mucosal Langerhans Cells of Human Oral Mucosa following Neuraminidase Pretreatment

Histochemical assessment of selected carbohydrate sequences on Langerhans cells of human oral mucosa was made by combined use of enzyme digestion and immunostain-ing with monoclonal antibodies against specific carbohydrate structures. In both frozen sections and epithelial sheets without the enzyme pretreatment, mucosal Langerhans cells, identified by positive staining with anti-CD1a and HLA-DR antibodies, did not express any carbohydrate antigens on their surface. In contrast, following neuraminidase pretreatment of both types of material, the fucosylated type 2 chain (Le^X) became detectable on Langerhans cells, indicating that sialic acid is the terminal residue of this sequence. Other enzymes were ineffective in this apparent unmasking, and the staining patterns of the other related carbohydrate sequences (Le^y. Le^a, Le^b) remained unaffected by pretreatment with any of the enzymes used. These findings suggest that the mucosal Langerhans cells possess a unique carbohydrate chain, the sialyl fucosylated type 2 sequence (sialyl Le^X antigen).     

Immunohistochemical Detection of Sialyl LeX Antigen on Mucosal Langerhans Cells of Human Oral Mucosa following Neuraminidase Pretreatment

Histochemical assessment of selected carbohydrate sequences on Langerhans cells of human oral mucosa was made by combined use of enzyme digestion and immunostain-ing with monoclonal antibodies against specific carbohydrate structures. In both frozen sections and epithelial sheets without the enzyme pretreatment, mucosal Langerhans cells, identified by positive staining with anti-CD1a and HLA-DR antibodies, did not express any carbohydrate antigens on their surface. In contrast, following neuraminidase pretreatment of both types of material, the fucosylated type 2 chain (Le^X) became detectable on Langerhans cells, indicating that sialic acid is the terminal residue of this sequence. Other enzymes were ineffective in this apparent unmasking, and the staining patterns of the other related carbohydrate sequences (Le^y. Le^a, Le^b) remained unaffected by pretreatment with any of the enzymes used. These findings suggest that the mucosal Langerhans cells possess a unique carbohydrate chain, the sialyl fucosylated type 2 sequence (sialyl Le^X antigen).     

A novel monoclonal antibody, RM-4, specifically recognizes rat macrophages and dendritic cells in formalin-fixed, paraffin- embedded tissues

Summary An anti-rat macrophage/dendritic cell monoclonal antibody, RM-4, was produced using a homogenate of silica-induced lung granulomas of rat as immunogen. Immunohistochemistry demonstrated that RM-4 was specific for macrophage and dendritic cell populations residing in various organs and tissues. It did not react with any cells other than macrophage/dendritic cells. In the double staining of the spleen, RM-4-positive macrophages showed wider distribution than those of the four other anti-rat macrophage monoclonal antibodies compared. The immunoreactivity of RM-4 was well preserved not only in frozen sections but also in formalin-fixed, paraffin-embedded tissues. The isotype of the monoclonal antibody was IgG₁ kappa and its antigen molecular weight was 46 kDa. Immunoelectron microscopy revealed positive reaction products for RM-4 on the membrane of endosomes and lysosomes in macrophages and epidermal Langerhans cells. Reaction intensity increased after thioglycolate elicitation or endocytosis regardless of ingested materials. From these data, it is concluded that RM-4 recognizes a membrane protein of endolysomes in macrophages and dendritic cells. The antigen may play a role in endolysosomal processing. RM-4 is considered to be a useful tool not only for identifying macrophage/dendritic cells both in frozen and paraffin-embedded tissues, but also for evaluating their endolysosomal processing. This revised version was published online in November 2006 with corrections to the Cover Date.     

Differentiation of Langerhans Cells from Interdigitating Cells Using CD1a and S-100 Protein Antibodies

The present study shows that Langerhans cells can be differentiated from Interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.     

Significance of S-100 protein immunostaining in the immunohistological analysis of normal and neoplastic lymphoid tissues--an appraisal

S-100 protein is a heterogeneous fraction of dimeric polypeptides (alpha and beta subunits) that can exist in different combination forms within the various tissues. Concerning the S-100 protein immunodetection within lymphoid tissue, the heterogeneity of the S-100 antigen, the tissue quality (frozen or paraffin-embedded after treatment with different fixatives) and the treatment of the tissue with different immunostaining methods and antibodies of different nature, all make for inconsistent results obtained in the immunohistological studies reported in the literature. Most of the S-100-positive cells of the lymphoreticular system are dendritic cells involved in the immune response (interdigitating reticulum cells, Langerhans cells, and follicular dendritic reticulum cells), other S-100-positive cells belonging to the mononuclear/phagocytic system. S-100 protein immunostaining may be used as a helpful immunohistological diagnostic clue to certain malignancies of the immune system (follicular center cell lymphomas) on the basis of their specifically related dendritic cell microenvironment. In addition to monoclonal antibodies for the immunophenotypic characterization of dendritic cells and macrophages and to enzyme reactions, the combined use of anti-S-100 antibodies specific for each of the S-100 protein subunits, tested with sensitive procedures, would be a very useful tool in the attempt to classify the proliferative disorders of dendritic cells and macrophages.     

朗格汉斯细胞与病毒感染

朗格汉斯细胞位于黏膜状组织和皮肤分层鳞状上皮,是高度专职的抗原呈递细胞家族成员,也是表皮中唯一的树突细胞。其作为一种皮肤免疫细胞,在摄取、加工处理和呈递抗原及诱导 T 细胞反应等方面发挥着巨大作用。机体皮肤或黏膜在遭遇不同病原微生物入侵时,朗格汉斯细胞与病原体的相互作用及引起后续免疫反应的机制存在差异。本文就朗格汉斯细胞的生物学功能及其在一些病毒感染中的作用进行综述。     

Differentiation of Langerhans Cells from Interdigitating Cells Using CD1a and S-100 Protein Antibodies

The present study shows that Langerhans cells can be differentiated from Interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.     

Induction of epithelial migration of lymphocytes by intercellular adhesion molecule-1 in a rat model of oral mucosal graft-versus-host disease

To elucidate the involvement of intercellular adhesion molecule-1 (ICAM-1) in the migration of lymphocytes to the oral mucosal epithelium in a rat model of acute graft-versus-host disease (AGVHD), we investigated (1) ICAM-1 and major histocompatibility complex (MHC) class II expression by keratinocytes (KCs) and their role in the epithelial infiltration of CD8+ cells, (2) the tissue expression of interferon-γ (IFN-γ) mRNA and expression of IFN-γ receptor by KCs, and (3) the ability of KCs to direct CD8+ cells into the epithelial layers. We classified the oral mucosal lesions into three consecutive temporal phases on the basis of increased epithelial ICAM-1 expression: basal- (phase I), parabasal- (phase II), and pan-epithelial except for the cornified cell layer (phase III). Basal ICAM-1 expression by KCs preceded that of MHC class II molecules, infiltration of CD8+ cells and epithelial histological changes. Tissue expression of IFN-γ mRNA and expression of IFN-γ receptor on KCs evidenced by immunohistochemistry were detected in early lesions (phase I), indicating that locally produced IFN-γ induced ICAM-1 expression by KCs. CD8+ cells were bound to KCs in frozen sections of epithelial lesions, whereas no lymphocyte attachment was observed in normal KC. Adherence could be inhibited by pretreating CD8+ cells with lymphocyte function-associated antigen-1 (LFA-1) antibody and/or by pretreating sections with ICAM-1 antibody. Our data suggest that in the early phase of acute oral mucosal GVHD, the induction of ICAM-1 expression on KCs leads to the migration of CD8+ cells into the epithelium and that this is mediated in part by the ICAM-1/LFA-1 pathway.     

Langerhans cell-like dendritic cells in the cornea,tongue and oesophagus of the chicken (Gallus gallus)

Langerhans cells are dendritic leucocytes which reside mainly within stratified squamous epithelia of skin and mucosa. Their visualization requires the use of ATPase histochemistry, electron microscopy for identifying the unique trilaminar cytoplasmic organelles (the Langerhans cell granules or Birbeck granules), and the expression of major histocompatibility complex class II molecules. Following uptake of antigen, Langerhans cells migrate via the afferent lymphatics to the lymph nodes and undergo differentiation from an antigen-processing cell to an antigen-presenting cell. Using the same approach as that employed in previous studies for the identification of chicken epidermal Langerhans cells, we show here the presence of ATPase-positive and major histocompatibility complex class II-positive Langerhans cell-like dendritic cells at the mucosal surface of the eye, tongue and oesophagus of the chicken. Ultrastructurally, these cells qualified as Langerhans cells except that they lack Langerhans cell granules. Thus, as in mammalian skin and mucosa, chicken mucosa contains mucosal dendritic cells with morphological and phenotypical features for the engagement of incoming antigens within epithelium and lamina propria.     

Human dendritic reticulum cells of lymphoid follicles: their antigenic profile and their identification as multinucleated giant cells

The B-dependent areas of human lymphoid tissue contain non-lymphoid, non-phagocytic cells known as dendritic reticulum cells (DRC). These cells can be detected only very occasionally in routinely stained histologic sections. Recently we were able to overcome this limitation by preparing a monoclonal antibody, termed R 4/23, that reacts selectively with DRC. Thus by using an optimized immunoperoxidase method applied to frozen sections, it is possible to detect DRC in situ. To determine the antigenic profile of DRC, serial frozen sections of human tonsils were immunostained with R 4/23 and a large panel of other monoclonal antibodies or conventional antisera. In addition, touch imprints of tonsils and cytocentrifuge slides of cell suspensions with increased concentrations of DRC were immunostained with these reagents. DRC proved to be positive for mu, gamma, alpha, kappa and lambda chains, complement component C3b, C3b receptors, C3d receptors, HLA-A,B,C antigens, human Ia-like antigens, common ALL antigen (cALLa), and antigens that are characteristic of the monocyte/macrophage lineages. DRC did not express delta chains, T cell antigens, or antigens that are expressed on interdigitating reticulum cells (IDC) and Langerhans cells. DRC in touch imprints and suspensions prepared from hyperplastic tonsils were found to be giant cells often with 10 or more nuclei. In certain cases of follicular hyperplasia and of centroblastic-centrocytic lymphoma, DRC with several nuclei were also detectable in situ. These results show that (1) the phenotype of DRC differs from that of all other cell types in lymphoid tissue, (2) this phenotype most nearly resembles that of cells of the monocyte/macrophage series, thus suggesting that DRC are related to these cell lineages, and (3) DRC are multinucleated giant cells.     

Immunohistochemical detection of strain-specific major histocompatibility complex class I antigens in paraffin-embedded rat osteochondral tissue

Summary An immunohistochemical method using formalin-fixed, paraffin wax-embedded sections is described for detecting strain-specific major histocompatibility complex class I antigens in knee-joint tissue from DA and Lewis strains of rat. The fixed osteochondral tissues were additionally decalcified in formic acid before processing for paraffin wax embedding. For immunohistochemistry, two monoclonal antibodies, one specific for DA class I allele RT1Aa and the other for Lewis class I allele RT1A1, were used together with the avidin-biotin immunoperoxidase procedure. It was necessary to use strain-specific normal rat serum as a diluent for the antibodies to suppress cross-strain recognition. DA-specific antibody stained positively only on DA rat sections, not on Lewis rat sections, and Lewis-specific antibody stained positively only on Lewis rat sections, and not on DA. Positive staining was localized in the bone marrow, osteochondral cells and endothelium. We propose that the use of a decalcification medium may have enhanced the immunoreactivity of the tissue. The method described can be used on sections of allografts from the two strains of rat to assess morphologically the extent of cellular replacement of the graft by the host's cells.     

Laryngeal papillomas: local cellular immune response, keratinization and viral antigen

Various parameters of the local cellular response have been studied in 16 laryngeal papillomas from ten patients with recurrent papillomas as well as normal control laryngeal and tracheal tissue by indirect immunofluorescence on frozen sections using monoclonal antibodies specific for T-cell subsets, Langerhans cells (LC) and HLA-DR antigens. Keratinization was investigated with a monoclonal antibody KL1 recognizing an acidic 56.5 Kd keratin, which is a marker of suprabasal cells in stratified squamous epithelium and is absent from the basal layer. The presence of viral antigen was detected with a rabbit antiserum raised against SDS-dissociated purified virus. A mild inflammatory response was observed in most biopsies. Cytotoxic/suppressor T-cells were the predominant cells found in the lesions. Compared with normal epithelium, the number of LC was dramatically reduced in the papillomatous epithelium. High densities of HLA-DR-positive cells were found mainly in the corium. The keratinization process was disturbed in most specimens in that both basal and suprabasal compartments reacted positively with the KL1 monoclonal antibody. Viral antigen was present in the nucleus of very occasional epithelial cells in some samples.     

Nasal-associated lymphoid tissue: phenotypic and functional evidence for the primary role of peripheral node addressin in naive lymphocyte adhesion to high endothelial venules in a mucosal site.

Nasal-associated lymphoid tissue (NALT), a mucosal inductive site for the upper respiratory tract, is important for the development of mucosal immunity locally and distally to intranasally introduced Ag. To more fully understand the induction of nasal mucosal immunity, we investigated the addressins that allow for lymphocyte trafficking to this tissue. To investigate the addressins responsible for naive lymphocyte binding, immunofluorescent and immunoperoxidase staining of frozen NALT sections were performed using anti-mucosal addressin cell adhesion molecule-1 (MAdCAM-1), anti-peripheral node addressin (PNAd), and anti-VCAM-1 mAbs. All NALT high endothelial venules (HEV) expressed PNAd, either associated with MAdCAM-1 or alone, whereas NALT follicular dendritic cells expressed both MAdCAM-1 and VCAM-1. These expression profiles were distinct from those of the gut mucosal inductive site, Peyer's patches (PP). The functionality of NALT HEV was determined using a Stamper-Woodruff ex vivo assay. The anti-L-selectin MEL-14 mAb blocked >90% of naive lymphocyte binding to NALT HEV, whereas the anti-MAdCAM-1 mAb, which blocks almost all naive lymphocyte binding to PP, minimally blocked binding to NALT HEV. NALT lymphocytes exhibited a unique L-selectin expression profile, differing from both PP and peripheral lymph nodes. Finally, NALT HEV were found in increased amounts in the B cell zones, unlike PP HEV. These results suggest that NALT is distinct from the intestinal PP, that initial naive lymphocyte binding to NALT HEV involves predominantly L-selectin and PNAd rather than alpha4beta7-MAdCAM-1 interactions, and that MAdCAM-1 and VCAM-1 expressed by NALT follicular dendritic cells may play an important role in lymphocyte recruitment and retention.     

Immunohistochemical detection of Langerhans cells in dental granulomas and radicular cysts

Objective Dental granulomas (DGs) and radicular cysts (RCs) are chronic periapical lesions frequently involving the jaws. Langerhans cells (LCs) are dendritic cells responsible for the presentation of antigens to T lymphocytes. This study examined the expression of LCs in DG and RCs by immunohistochemical staining. Study Design Eighteen cases of DGs and 26 cases of RCs were analyzed using anti-CD1a marker. Results CD1a-labeled LCs were observed in 11.1% of DGs and in 69.2% of RCs, showing a significant correlation (P < 0.0001; Fisher’s test). In DGs, LCs were only observed in granulation tissue, showing discrete immunostaining density. In RCs, LCs exhibited both a round and a dendritic shape in all epithelial layers. Although a correlation was observed between immunostaining density and epithelial thickness, as well as between immunostaining and inflammatory intensity, the differences were not significant in radicular cysts. Conclusion Langerhans cells provide important insight into the immunopathogenesis of chronic periapical lesions.     

Analysis of binding of mannosides in relation to Langerin (CD207) in Langerhans cells of normal and transformed epithelia

Tandem-repeat C-type lectins (pattern-recognition receptors) with specificity for mannosides are intimately involved in antigen recognition, uptake, routing and presentation in macrophages and dendritic cells. In Langerhans cells, Langerin (CD207), a type-II transmembrane protein with a single C-type carbohydrate recognition domain attached to a heptad repeat in the neck region, which is likely to establish oligomers with an -coiled-coil stalk, has been implicated in endocytosis and the formation of Birbeck granules. The structure of Langerin harbours essential motifs for Ca²⁺-binding and sugar accommodation. Lectin activity has previously been inferred by diminished antibody binding to cells in the presence of the glycan ligand mannan. In view of the complexity of the C-type lectin/lectin-like network, it is unclear what role Langerin plays for Langerhans cells in binding mannosides. In order to reveal in frozen tissue sections to what extent mannose-binding activity co-localizes with Langerin, we have used a synthetic marker, i.e. a neoglycoprotein carrying mannose maxiclusters, as a histochemical ligand, and computer-assisted fluorescence monitoring in a double-labelling procedure. Mannoside-binding capacity was detected in normal epithelial cells. Double labelling ensured the unambiguous assessment of the binding of the neoglycoprotein in Langerhans cells. Light-microscopically, its localization profile resembled the pattern of immunohistochemical detection of Langerin. This result has implications for suggesting rigorous controls in histochemical analysis of this cell type, because binding of kit reagents, i.e. mannose-rich glycoproteins horseradish peroxidase or avidin, to Langerin (or a spatially closely associated lectin) could yield false-positive signals. To show that recognition of carbohydrate ligands in dendritic cells is not restricted to mannose clusters, we have also documented binding of carrier-immobilized histo-blood group A trisaccharide, a ligand of galectin-3, which was not affected by the presence of a blocking antibody to Langerin. Remarkably, access to the carbohydrate recognition domain of Langerin appeared to be impaired in proliferatively active environments (malignancies, hair follicles), indicating presence of an endogenous ligand with high affinity to saturate the C-type lectin under these conditions.     

Immunohistochemical recognition of human follicular dendritic cells (FDCs) in routinely processed paraffin sections.

A number of monoclonal antibodies (MAbs) that recognize human follicular dendritic cells (FDCs) have been identified. Although some of them have already been applied individually in routine immunolabeling using formalin-fixed paraffin sections for diagnostic and experimental purposes, many antibodies are still employed only for immunolabeling using cryostat sections or particularly processed sections because they have been thought unsuitable for routine sections. A comprehensive examination re-evaluating their suitability in paraffin sections has not been reported. Accordingly, there is limited ability to examine the immunopathological contribution or diagnostic value of FDCs using routinely processed specimens or archived materials. In this study a broad panel of antibodies was systematically applied to the immunolabeling of paraffin sections of reactive tonsils or lymph nodes, in combination with advanced antigen retrieval (AR) techniques. Several antibodies, including Ki-M4p, X-11, 12B1, CNA.42, 1F8/BU32 (anti-CD21), BU38/1B12 (anti-CD23), Ber-MAC-DRC/To5 (anti-CD35), 1.4C3 (anti-CD106), NGFR5 (anti-nerve growth factor receptor p75), IIH6 (anti-CD55), 55K-2 (anti-fascin), and anti-S100 protein alpha-chain, were found to label FDCs in routine sections when combined with suitable AR techniques. Our results are easily adaptable for routine practice and provided useful suggestions concerning the immunopathological behavior and diversity of the particular cells.     

Quantification of vascular density using a semiautomated technique for immunostained specimens

OBJECTIVE: To develop a semiautomated, quantitative techniquefor the assessment of vascular density in immunohistochemically stained tissue sections using diaminobenzidine tetrahydrochloride (DAB) and hematoxylin as chromagens. STUDY DESIGN: A semiautomated thresholding technique was developed to quantitate vascular density in tissue sections stained with anti-CD31 (1 degrees antibody). The immunohistochemically stained specimens were digitally imaged using a 24-bit color camera. The blue component of the RGB image was segmented using a variable high-pass filter. After thresholding, the segmented areas (CD31 positive) were quantified and vascular density determined. The validity of the method was verified by calculating the precision of the technique using the coefficient of repeatability and by quantifying its agreement with manual analysis according to the Bland-Altman approach. RESULTS: Vascular endothelial cells were specifically selected using anti-CD31 as the primary antibody and the appropriate horseradish peroxidase-conjugated secondary antibody. Utilizing the semiautomated thresholding technique, the separation of DAB-stained tissuefrom non-DAB-stained tissue was achieved. The method developed possesses a low coefficient of repeatability (0.49%), agrees well with manual assessment (mean difference = -0.29 +/- 0.92%), is highly automated and is user friendly. CONCLUSION: A novel semiautomated technique for the quantification of vascular density was developed. This technique provides a method for reproducible measurement of immunostaining procedures (immunohistochemistry, immunocytochemistry and in situ hybridization) utilizing immunoperoxidase techniques with DAB as a chromagen.     

Detection of guinea pig macrophages by a new CD68 monoclonal antibody, PM-1K

A new monoclonal antibody, PM-1K, was raised against 24-h cultured human peritoneal macrophages. In immunohistochemical assays, PM-1K recognized freshly isolated blood monocytes and most tissue macrophages as well as myeloid dendritic cells such as Langerhans cells and interdigitating cells. The molecular size of the antigen recognized by PM-1K was determined to be 110 kD by means of immunoaffinity purification. Because this affinity-purified antigen recognized by PM-1K was also recognized by anti-CD68 antibodies, it is believed to be one of the heterogeneous molecules of the CD68 antigen. Analysis showed interspecies reactivity of PM-1K with macrophages from guinea pigs, pigs, bovine species, and monkeys. Among these macrophages, those of the guinea pig reacted strongly with PM-1K. Patterns of PM-1K immunostaining in guinea pig tissues were similar to those found in human tissues. Studies with the immunoelectron microscope revealed reaction products of PM-1K in the cytoplasm, especially around endosomes. Since only a few antibodies are available to label guinea pig macrophages, PM-1K is considered to be one of the most suitable antibodies to examine macrophages in experimental guinea pig models.     

Increased occurrence of PGP 9.5-immunoreactive epidermal Langerhans cells in rat plantar skin after sciatic nerve injury

This study examines the occurrence and distribution of epidermal dendritic cells (DCs) in cryostate sections from plantar skin in normal rats and in rats with a crush injury or neurotomy and suture of the sciatic nerve. The dendritic cells were visualized with antibodies against protein-gene product 9.5 (PGP 9.5). Counts under the fluorescence microscope showed that the occurrence of dendritic cells is increased and that the proportion of dendritic cells in the basal layer is elevated 3 months after sciatic neurotomy and suture but not after a crush lesion. The countings also revealed that the number of cells is elevated as soon as 1 week after neurotomy and suture. Labelling with specific antibodies showed that the dendritic cells examined represent Langerhans cells (LCs). These observations show that there is a neural influence on the occurrence and distribution of PGP 9.5-immunoreactive epidermal Langerhans cells. Whether this influence is direct or indirect remains to be ascertained.     

Accumulation of mucosal T lymphocytes around epithelial cells after in vitro infection with Cryptosporidium parvum.

We had previously shown that ileal intraepithelial lymphocytes isolated from calves with cryptosporidiosis include significantly increased numbers of CD8+ T lymphocytes and activated CD4+ cells. These increases could result from redistribution of resident mucosal lymphocytes or from homing of peripheral T cells to ileal mucosa. To determine whether resident mucosal lymphocytes can redistribute to Cryptosporidium parvum-infected epithelium, oocysts were inoculated in vitro onto ileum explants taken from 1-2-wk-old noninfected calves. After 24 hr of incubation, the explants were collected and frozen in liquid nitrogen. Immunohistochemical analysis of T-lymphocyte subpopulations was performed on sections, and labeled lymphocytes adjacent to villous epithelial cells were counted. Compared with uninoculated explants, there was a statistically significant increase in the number of CD8+ T lymphocytes per 100 epithelial cells in oocyst-inoculated tissue. In addition, there were increased numbers of CD4+ T cells and activated (CD25+) lymphocytes adjacent to C. parvum-infected epithelium. These results show that resident mucosal T lymphocytes can accumulate at the epithelium during C. parvum infection.