The development of intraepithelial and Peyer's patch lymphocyte sub-types in the small intestine of newborn rats.

Small intestinal intraepithelial (IEL) and Peyer's patch (PPL) lymphocytes of newborn rats have been studied in histological sections and in isolated cell suspensions. Initially IEL numbers were low compared with older animals, and fewer cells were labelled by the monoclonal antibody markers used. At birth, 41% of IEL expressed receptors for MRC OX8 (T suppressor marker) yet lacked receptors for W3/13 (pan-T marker) which was present on only 1% of these cells. IEL with receptors for W3/25 (T helper marker) were not seen until 2 weeks of age. Granulated cells (GIEL) accounted for only 19% of IEL at birth. The numbers and relative proportions of these lymphocyte sub-types were still not mature at the time of weaning. In the first week of life there was a short lived upsurge in numbers of GIEL and MRC OX8+ IEL. The relative distribution of PPL sub-types was mature 4 weeks after birth, but the morphology of this tissue did not appear to be well differentiated until 1 week later.     

Mucosal lymphocytes in the rat small intestine: phenotypical characterization in situ.

Lymphocyte subpopulations in the intestinal mucosa of the rat were quantified in situ and compared with data obtained by other authors using enzymic or mechanical methods (Selby et al., 1984; Gibson et al., 1985) in order to assess any selective loss of cell types or contamination with lamina propria lymphocytes after these enzymic or mechanical isolation procedures. Intraepithelial lymphocytes (IEL) were predominantly of the suppressor/cytotoxic phenotype (MRC OX-8) and nearly all cells bore the pan T marker W3/13. About 10% of the IEL phenotypically belonged to the T-helper (W3/25) lineage. MRC OX-19, the rat equivalent of the mouse Lyt-1 antigen, was present on about 15% of the IEL. The main subpopulation of lamina propria T lymphocytes (T-LPL) showed a T-helper phenotype and a smaller subpopulation a suppressor/cytotoxic phenotype. Practically all T-LPL expressed the pan T marker W3/13, and half of these T cells were MRC OX-19+. The results proved to be in agreement with the data obtained after enzymic or mechanical isolation procedures and indicate that proportional contamination with LPL is not likely to occur with these methods.     

Methodology for isolation and phenotypic characterization of feline small intestinal leukocytes

Critical assessment of intestinal immune responses requires the ability to characterize leukocytes from different anatomic locations as leukocytes from inductive sites such as Peyer's patches and lymphoid follicles vary significantly from their effector counterparts, intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL). This study describes (1) methods developed to isolate specific intestinal leukocyte populations with high yield and purity, (2) difficulties encountered in establishing a panel of monoclonal antibodies to assess phenotype, and (3) the phenotypic characterization of effector and inductive sites in the feline small intestine. We found that the phenotypic distribution of feline intestinal leukocytes was similar to that found in other species such as humans, macaques and mice. The majority of IEL were CD5(+) T-cells with less than 7% B-cells. CD8(+) T-cells comprised approximately 60% of the IEL with roughly half displaying CD8alphaalpha homodimers. Approximately 10% of IEL were CD4(+) T-cells. In the LPL, CD4(+) T-cells predominated at 42%, with 33% CD8(+) T-cells and 10% B-cells. As would be expected, B-cells predominated in Peyer's patches with 40% B-cells, 28% CD4(+) T-cells and 20% CD8(+) T-cells. Increased MHCII expression was found in the Peyer's patches as compared to the IEL and LPL. B7.1 expression was significantly higher in mucosal leukocyte populations as compared to organized lymphoid tissue in the periphery with expression detected on 65% of IEL and 53% of LPL. Plasma cells were found in all regions of small intestine examined with greater numbers in lamina propria and Peyer's patches. Lymphoblasts were only identified in inductive tissue. In general, no differences were found between the phenotype of mucosal leukocyte populations from specific pathogen free or random source cats. However, the percentage of CD4(+) CD25(+) T-cells was significantly greater in both IEL and LPL from random source animals. This study provides techniques and a baseline from which future studies of the feline intestinal immune system can be conducted.     

Rat monocytes in a model of combined injury express the OX8 antigen

We have analyzed peripheral blood mononuclear cell preparations from a rat model of combined injury (CI) [whole-body irradiation (500 cGy 60Co) followed by a thermal injury (20% body surface area, dorsal, scald burn)] for the expression of OX8 antigens. Ficoll-separated mononuclear fractions were labeled with monoclonal antibodies MRC OX8, MRC OX19, W3/13 HLK, or W3/25 for flow cytometric analysis. Combined-injury trauma resulted in decreased mononuclear cells to 6% of normal. This effect was due to the rapid decrease in radiosensitive lymphocytes from 83% to 10%. The relative numbers of monocytes increased from a normal 13% to 70% at day 4 after CI. Labeling of cells with OX8 after CI shifted to a population which was significantly larger in volume than normal lymphocytes. At the same time the mean fluorescence intensity of OX8-positive cells was considerably reduced. With the use of a F(ab) fragment of OX8 as a probe, these results could be partially explained as unspecific binding of the whole molecule of OX8 to Fc receptors expressed by activated monocytes. But double-labeling and cell-sorting experiments also revealed the expression of OX8 antigens by a subset of OX8+/OX19- monocytes after CI.     

The proportion of Th1 cells, which prevail in gut mucosa, is decreased in inflammatory bowel syndrome

T lymphocytes and their cytokines have an important role in the regulation of immune responses in the gut and in the pathogenesis of intestinal inflammation such as in Crohn's disease. The aim of this study was to analyse the Th1/Th2 cytokine profile (IFN-gamma, IL-2, IL-4 and IL-10) in intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) in Crohn's disease (CD) and ulcerative colitis (UC) in relation to healthy controls (C). Colonic and ileal biopsy specimens were obtained from controls (n = 13) and patients with CD (n = 32). Colonic biopsies were obtained from patients with UC (n = 11). Intracytoplasmic IFN-gamma, IL-2, IL-4 and IL-10 were determined by flow cytometry after PMA-ionomycin stimulation in IEL and LPL. In colonic LPL, a significant proportional decrease of IFN-gamma and IL-2 producing CD3+ cells was observed in patients with CD and UC compared to controls. In ileal LPL, a similar tendency was found although differences were not significant. In IEL no differences in cytokine profiles could be observed. Flow cytometric analysis of intracytoplasmic cytokines at single cell level showed a proportional decrease of IFN-gamma and IL-2 producing T cells in colonic lamina propria in patients with inflammatory bowel disease.     

T-cell receptor-gamma delta association with lymphocyte populations in sheep intestinal mucosa.

T cells expressing T-cell receptor (TcR)-gamma delta and CD8 represent a significant population in mouse and chicken intra-epithelial lymphocytes (IEL) but represent a minor population in human IEL. We examined the TcR-gamma delta usage and co-expression of CD5, CD4, CD8 and major histocompatibility complex (MHC) class II on isolated sheep IEL and lamina propria lymphocytes (LPL), and compared them with the TcR-gamma delta + cells in peripheral blood, intestinal lymph and jejunal Peyer's patches (PP). There were a number of notable differences. TcR-gamma delta + cells comprised 18% of IEL and 10% of LPL. Among the population of TcR-gamma delta + IEL, 24% were CD8+ and 54% were CD5+, which contrasts with the TcR-gamma delta + cells in blood and intestinal lymph that were universally CD5+ CD4- CD8-. A notable feature of the IEL was the presence of distinct CD8+ and TcR-gamma delta + populations that lacked CD5. Also a high percentage of IEL and LPL were CD2+ and MHC class II+. Analysis of the expression of MHC class II on T-cell subsets, as an indicator of activation, showed that 60-95% of the various IEL and LPL subsets were MHC class II+ compared with only 5-40% in jejunal PP, lymph nodes, spleen and blood. Therefore, it is possible that the circulating TcR-gamma delta + and CD8+ cells that localize in the gut epithelium might become activated and stop the expression of CD5 under the influence of the local microenvironment. These cells appear not to emigrate while still expressing the TcR-gamma delta + (CD8+) CD5- MHC class II+ phenotype. Our data, together with those from other studies, show that there is much heterogeneity in the use of TcR-gamma delta and accessory T-cell molecules by IEL.     

Human CD8+ intraepithelial T lymphocytes are mainly CD45RA-RB+ and show increased co-expression of CD45R0 in celiac disease

Expression of various CD45 isoforms (RA, RB and R0) on CD3+, CD4+ and CD8+ intraepithelial and lamina propria T cells was examined in situ by a three-color immunofluorescence technique in jejunal biopsy specimens from 32 patients with celiac disease and 18 controls. The median percentage of CD3+ intraepithelial lymphocytes (IEL) that expressed CD45R0 increased from 52% in controls to 69% in untreated celiac disease (p less than 0.01). Furthermore, the percentages of CD4+ and CD8+ IEL strongly positive for CD45R0 rose respectively from 94% and 24% in controls to 100% and 55% in untreated celiac disease. Conversely, CD45R0 was strongly expressed on most CD3+ lamina propria lymphocytes (LPL) both in control (81%) and diseased (77%-81%) mucosa. A variable fraction of the intraepithelial and lamina propria CD3+ T cells expressed mainly CD45RB (controls, 46% and 20%, respectively; celiac disease, 29% and 15%). Only 2% IEL and 4% LPL were positive for CD45RA. Expression of different CD45R isoforms thus identified three distinct CD8+ T cell subsets in human intestinal mucosa. In addition, our results suggested that antigen-primed CD8+CD45R0+ memory cells accumulate in the jejunal epithelium of patients with untreated celiac disease.     

T lymphocytes of the human colonic mucosa: functional and phenotypic analysis.

Normal human colonic lymphocyte populations were isolated for both phenotypic analysis by double-label immunofluorescence and assessment for regulatory effects on Ig production by co-culture with responder cells from colonic mucosa and peripheral blood. Mean CD4:CD8 ratios for colonic intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) were comparable to values obtained from tissue sections. IEL alone did not produce Ig in vitro and were without effect on Ig production when co-cultured with LPL. However, T-enriched LPL had a marked helper effect for T-depleted LPL. Maximal help was for IgA production, increasing with numbers of T-enriched cells. In colonic LPL T-depleted and T-enriched co-cultures, pokeweed mitogen (PWM) had no significant effect. By contrast, in co-cultures of T-enriched and T-depleted peripheral blood mononuclear cells, Ig production was PWM-dependent. In all experiments with colonic mucosal responder cells, IgG production was low. The effects of unfractionated colonic biopsy lymphocytes on T-depleted peripheral blood mononuclear cells were additive for IgM production and synergistic for IgA synthesis, although almost no IgG was produced. Moreover, PWM had helper effects for IgM, but was suppressive for IgA production. These data suggest that colonic mucosal regulatory cells reside in the lamina propria, and predominantly provide help for IgA and IgM synthesis. The data further suggest the existence of a pre-stimulated IgA-specific T helper cell population.     

Human intestinal lamina propria and intraepithelial lymphocytes express receptors specific for chemokines induced by inflammation

To determine which chemokine receptors might be involved in T lymphocyte localization to the intestinal mucosa, we examined receptor expression on human intestinal lamina propria lymphocytes (LPL), intraepithelial lymphocytes (IEL) and CD45RO+beta7hi gut homing peripheral blood lymphocytes (PBL). Virtually all LPL and IEL expressed CXCR3 and CCR5, receptors that have been associated with Th1(Tc1)/Th0 lymphocytes, while CCR3 and CCR4, receptors associated with Th2 (Tc2)lymphocytes, CCR7, CXCR1 and CXCR2 were not expressed. CXCR3 and CCR5 receptors were functional, as LPL and IEL migrated to their respective ligands I-TAC and RANTES. In addition, most alphaEbeta7- LPL and IEL expressed high levels of CCR2. While the majority of CD45RO(-)beta7hi PBL also expressed CXCR3 and CCR5, a proportion of these cells were CXCR3- and/or CCR5- and some expressed CCR4 and/or CCR7, indicating that lymphocytes recruited to the intestinal mucosa represent a subset of these cells. In summary, our results show that LPL and IEL within the normal intestine express a specific and similar array of chemokine receptors whose ligands are constitutively expressed in the intestinal mucosa and whose expression is up-regulated during intestinal inflammation. These results support the view that CXCR3, CCR5 and CCR2 may play an important role in lymphocyte localization within the intestinal mucosa.     

A major fraction of human intraepithelial lymphocytes simultaneously expresses the gamma/delta T cell receptor, the CD8 accessory molecule and preferentially uses the V delta 1 gene segment

The frequency of T cell receptor (TcR) type and the variable gene segment expression in human intraepithelial lymphocytes (IEL) from the large intestinal mucosa were studied by flow cytometry and immunohistochemistry, and compared to those in peripheral blood lymphocytes (PBL) - or lamina propria lymphocytes (LPL). Employing anti-gamma/delta TcR and anti-alpha/beta TcR monoclonal antibodies (mAb), flow cytometric analysis revealed that a large fraction of IEL (37%) are gamma/delta T cells, whereas within LPL and PBL gamma/delta T cells comprise a minor population (4.6% and 3.8% respectively). At these sites the number of gamma/delta T cells labeled with anti-CD8 mAb were 58.3% (IEL), 43.3% (LPL) and 24.4% (PBL). In situ staining of serial sections of large intestine confirmed these results. Hence, these data suggest a selective accumulation of CD8+ gamma/delta T cells in the human epithelium of the large intestine. Furthermore, analysis of gamma/delta TcR bearing IEL+ disclosed a marked preponderance of cells using the V delta 1 gene segment, whereas gamma/delta TcR+ PBL preferentially express V delta 2. Strikingly, the majority of these V delta 1+ IEL bear the CD8 molecule on their surface. These results are taken as evidence for a selective localization of V delta 1+ CD8+ gamma/delta T cells in the epithelium of the large intestine.     

Lymphocyte subpopulations in the human small intestine. The findings in normal mucosa and in the mucosa of patients with adult coeliac disease

Lymphocyte subpopulations in human small intestinal mucosa have been studied using an immunofluorescence technique on tissue sections. In the normal intestine, the majority of intraepithelial lymphocytes (IEL) were of suppressor-cytotoxic phenotype (HuTLA+ UCHTI+ OKT8+ OKT4-; 84%). Only one-third of these OKT8+IEL reacted with anti-Leu-1, and antibody directed towards a 67,000 dalton antigen found on peripheral blood T cells. IEL failed to express the activation antigen, Tac, and also lacked detectable C3b receptor (C3RTO5-). The remaining T IEL, as well as the predominant lamina propria T lymphocytes (LPL), were OKT4+ OKT8-, helper type T cells. Most of the lamina propria OKT8+ cells were also Leu-1-. In patients with adult coeliac disease, the proportions of OKT8+ and OKT4+ lymphocytes in the epithelium were not altered. However, the proportion of OKT8+ Leu-1+TIEL was significantly increased (56 vs 32%; P less than 0.02). IEL were also HLA-DR-, Tac- and C3RTO5-. The proportion of OKT8+ cells in the lamina propria was slightly, but significantly, increased (40 vs 32%; P less than 0.005). Mucosal findings in treated patients did not differ from normal. Lymphocytes with the phenotype of natural killer cells (HNK-1) were rarely found in normal or diseased mucosa. No alterations in the proportions of circulating T lymphocytes or their subsets were found in patients with coeliac disease. These findings illustrate the heterogeneity of lymphocyte subpopulations in normal and in diseased small intestinal mucosa. The changes found in adult coeliac disease may reflect the increased traffic of IEL into the epithelium.     

Bovine gut-associated lymphoid tissue--morphologic and functional studies. I. Isolation and characterization of leukocytes from the epithelium and lamina propria of bovine small intestine

A successful technique for the isolation of highly pure suspensions of viable leukocytes from the small intestine of cattle is described. Procedures ranging from mechanical mincing to enzymatic digestion of tissues were compared. The most reliable and reproducible procedure was the sequential treatment of tissues with dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA) in calcium-magnesium-free salt solutions, and collagenase. Two populations of mucosal leukocytes were obtained from the small intestine. One population was derived from within the epithelium (intraepithelial leukocytes, IEL), the second from within the lamina propria (lamina propria leukocytes, LPL). At least 2 X 10(6) viable leukocytes were obtainable from each square centimeter of the intestinal mucosa from either the epithelium or lamina propria. Erythrocyte rosetting and immunofluorescence characterization with conventional antisera and monoclonal antibodies (MAB) demonstrated that IEL were predominantly T cells (60%), with relatively few B cells present (10%), while LPL contained relatively high numbers of B cells (28%) and a reasonable percentage of T cells (45%). Both cell populations proliferate in response to stimulation with T and B cell mitogens. Addition of the thiol compound, 2-mercaptoethanol (2-ME) strongly augmented the mitogenic response of both cell isolates. Human recombinant interleukin-2 (hr-IL-2) in the presence or absence of additional stimuli was found to be able to induce the proliferation of both cell types. These results demonstrate that functional leukocytes can be isolated from the small intestine of cattle, and that they can maintain their responsiveness to both T and B cell mitogens and to exogenous cloned IL-2.     

Oral administration of a bacterial immunomodulator enhances murine intestinal lamina propria and Peyer's patch lymphocyte traffic to the lung: possible implications for infectious disease prophylaxis and therapy

LW50020, a bacterial immunomodulator, is a preparation consistingof seven bacteria, commonly causing respiratory disease. Whengiven orally, LW50020 has been shown to enhance the host defenseof the respiratory tract. Intestinal lamina propria lymphocytes(LPL), Peyer's patch lymphocytes (PPL), and splenocytes fromBALB/c mice gavaged either with LW50020 or carrier alone wereisolated, labeled with either H33342, a supravital nuclear fluorochrome,or ⁵¹Cr, and injected i.v. into untreated, age-matched BALB/cmice. Two hours later, spleen, liver, lung, kidneys, Peyer'spatch, and mesenteric lymph nodes of the recipients were harvestedand screened for the presence of labeled cells. LPL from micegavaged with carrier only (controls) migrated preferentiallyto the lung, PPL equally well to the lung, and the spleen andsplenocytes were found mostly in the spleen. LPL and PPL fromLW50020-treated mice were found in significantly larger numbersin the lungs of recipients than LPL and PPL from control animals.Both labeling techniques gave roughly the same results. Sixty-fiveper cent of LPL in the lung were Thy-1.2⁺ and 20% B cells. Thesefindings should contribute to the understanding of parametersnecessary for the assessment of the mode of action and efficacyof immunomodulation and vaccination via the mucosa-associatedlymphoid tissue.     

痢疾杆菌口服免疫小鼠肠道相关淋巴组织中CD45R~+和CD45RB~+细胞动态变化的观察

为了解肠道相关淋巴组织（GALT）对痢疾杆菌口服免疫的应答状况,分离了Shigella fiexneri四次免疫后Balb/c 小鼠牌（SP）、肠系膜淋巴结（MLN）、Peyer’s结（PP）、固有膜（LP）和上皮内（IE）淋巴细胞,对末次免疫后1～13d内免疫组与对照组GALT中的CD45R~+和CD45RB~+细胞群作了FACS分析。结果免疫后起初7d IE和LPCD45R~+细胞增加而MLN和PP CD45R~+细胞减少。CD45RB~+LPL在起初4d增加,而CD45RB~+IEL总是低于对照。CD45RB~(lo)/CD45RB~(hi)比例在SP和MLN中几乎无变化,但在PP中先降至最低后上升并超过对照。LP中主要为CD45RBD~(lo),IE中主要为CD45RB~(hi)。这些结果在一定程度上反映了粘膜淋巴细胞CD45表达的组织特异性及在痢疾杆菌免疫后B细胞、记忆细胞和辅助细胞的功能状态和变化趋势。     

IL-4 down-regulates the responsiveness of human intraepithelial lymphocytes

IL-4 is an important regulator of intestinal inflammation, yet little is known regarding its action on intestinal lymphocytes. Intestinal lymphocytes were isolated from jejunal mucosa of patients undergoing gastric bypass operations for morbid obesity. The impact of IL-4 was measured by its effects on proliferation using ³H-thymidine incorporation, IL-2 production using the CTLL assay, and IL-2 receptor generation using immunofluorescence. The production of IL-4 was measured by ELISA. IL-4 significantly inhibited the proliferation of intraepithelial lymphocytes (IEL) to a variety of stimuli as well as the development of lymphokine-activated killer (LAK) cells. In contrast, it had no effect on the proliferation of CD8⁺ T cells from peripheral blood. The inhibitory effect of IL-4 on IEL did not occur during activation, since IL-2 production and receptor expression were not altered. Rather, it occurred during cell cycling, since over 50% inhibition resulted whether IL-4 was added at the initiation of an IL-2-stimulated culture or after 24 or 48 h incubation. IL-4 was secreted by activated lamina propria lymphocytes (LPL) but not by IEL. IL-4,produced by activated LPL, may enter the epithelial compartment and down-regulate responsiveness of IEL.     

类风湿关节炎前后肠道T淋巴细胞的分布变化

目的探讨在类风湿关节炎条件下大鼠小肠(十二指肠、空肠、回肠)上皮内T淋巴细胞(intraepithelial lymphocyte,IEL)、固有层T淋巴细胞(lamina propria lymphocyte,LPL)的变化规律。方法通过免疫组织化学染色方法,观察正常组和致病组肠管黏膜的T淋巴细胞的变化规律,然后分别对IEL和LPL计数,并进行统计学分析。结果大鼠致病组肠道各部分(十二指肠、空肠、回肠)上皮层和固有层可见T淋巴细胞数量增多。结论通过免疫组织化学方法可见在类风湿关节炎条件下,肠道黏膜的上皮层和固有层均可见T淋巴细胞的分布增多,肠道各段的IEL、LPL可能在黏膜免疫应答中起着关键的生物学作用。     

Seasonal changes in the intestinal immune system of hibernating ground squirrels

Hibernation is associated with a prolonged fast (5-8 mo) which has the potential to affect intestinal immunity. We examined several aspects of the intestinal immune system in summer (non-hibernating) and hibernating ground squirrels. Peyer's patches were largely unaffected by hibernation, but numbers of intraepithelial lymphocytes (IEL) and lamina propria leukocytes (LPL) were greater in hibernators compared with summer. Hibernator IEL were less mature as demonstrated by low numbers of cells expressing activation-associated markers and co-receptors. Compared with summer, the percentage of B cells was higher and percentage of T cells was lower in the hibernator LPL. Hibernation was associated with greater mucosal levels of IFN-gamma, TNF-alpha, IL-10 and IL-4, but IL-6 and TGF-beta were unchanged. Mucosal IgA levels were greater in entrance and torpid hibernators compared with summer. The results suggest that modifications of the intestinal immune system during hibernation may help preserve gut integrity throughout the winter fast.     

CD28+ intraepithelial lymphocytes with long telomeres are recruited within the inflamed ileal mucosa in Crohn disease.

Crohn disease is a chronic inflammatory bowel disease that involves all the intestine but predominantly alters the ileum. The disease largely depends on T cells, but the biologic role of intestinal intraepithelial lymphocytes (IEL) in transmural inflammation remains poorly characterized. To address this issue, a comparison of IEL and lamina propria lymphocytes (LPL) isolated from the uninvolved and the inflamed ileal mucosa of Crohn disease patients was performed. More CD8+ IEL (26% versus 8%) from the inflamed ileal mucosa expressed the CD28 receptor and the CD11a integrin than IEL from the uninvolved ileal mucosa, which were mostly CD28-. IEL had longer telomeres in the inflamed than in the uninvolved areas and a TCR Vbeta repertoire more similar to circulating T cells, suggesting that the increased proportion of CD28+ TCRalphabeta+ IEL within the inflamed mucosa is more likely due to recruited lymphocytes from the periphery that populate the epithelial layer than to the acquisition of the CD28 molecule by activated resident lymphocytes. In the uninvolved ileal mucosa, IEL from Crohn disease patients had shorter telomeric lengths than IEL from control patients, suggesting that they have been chronically stimulated. Such perturbation of the IEL population within the ileal mucosa could contribute to the inflammation in Crohn disease.     

A comparison of membrane markers on rat cytotoxic cells.

The antigenic characteristics of cytotoxic T cells (CTL) and cells mediating antibody-dependent cellular cytotoxicity (ADCC) were defined using monoclonal antibodies W3/13, W3/25 and MRC OX8, and compared with the phenotype of natural killer (NK) cells previously defined by these antibodies. CTL, ADCC effector cells and NK cells were also tested for expression of Ia antigen using MRC OX6 monoclonal antibody. The fluorescence-activated cell sorter was used to separate effector populations into antigen-positive and antigen-negative subsets, and the cytotoxicity of the resultant lymphocyte fractions was then assessed using 6 hr 51Cr release assay and a quantitative method of analysis based on consideration of target cell lysis as an enzyme substrate reaction. CTL, ADCC effector cells and NK cells were W3/25 negative and OX6 negative. CTL were positive for W3/13 and OX8 antibody with no cytotoxicity in the W3/13 negative and OX8 negative fractions. In contrast, ADCC effector cells showed heterogeneity of staining with W3/13 and OX8 antibodies, with 50% cytotoxic activity in the W3/13 negative fraction, and 20-30% cytotoxic activity in the OX8 negative fraction. In parallel experiments, NK cells and ADCC effector cells showed identical marker heterogeneity when tested with W3/13 and OX8 antibodies.