key: cord-0008978-yizj6n99
authors: Kimura, Yuki; Sakai, Tetsu; Takeuchi, Makoto; Matsumoto, Yoshihiro; Watanabe, Kouichi; Yuuki, Masakatsu; Takada, Toshihiko; Yoshikai, Yasunobu
title: An unique CD4(+)CD8(+) intestinal intraepithelial Lymphocyte Specific for DnaK (Escherichia coli HSP70) may be selected by intestinal microflora of rats
date: 2011-11-01
journal: Immunobiology
DOI: 10.1016/s0171-2985(97)80071-x
sha: f19a43478d9c3a05bc698846594f3b5cb70d89bc
doc_id: 8978
cord_uid: yizj6n99

We have previously shown an age-associated increase in unique CD4+CD8+ intestinal intraepithelial lymphocytes (i-IEL) in rats. To elucidate the potential causes of the increase in CD4+CD8+ i-IEL with age, we analyzed the specificity of the CD4+CD8+ i-IEL and influence of intestinal microflora on the increase in this subset in aged rats. The purified CD4+CD8+ i-IEL proliferated in response to DnaK [Escherichia colt (E. coli) HSP70] in the presence of mitomycin-c (MMC)-treated syngeneic, spleen cells. The proportion of CD4+CD8+ T cells in whole i-IEL were significantly increased in aged rats fed commercial (CL-2) diet but not in those fed home-made (purified) diet under conventional condition. No CD4+CD8+ i-IEL were detected in aged rats under germfree condition, irrespective of diet feeding. A larger number of Enterobacteriaceae, especially E. coli, were detected in the intestinal contents and feces from aged rats with CD4+CD8+ i-IEL compared with those from aged rats fed without CD4+CD8+ i-IEL. The unique CD4+CD8+ i-IEL population specific for E. coli HSP may be associated with long term exposure to intestinal E. coli in aged rats.

known to develop along extrathymic pathways, resulting in the generation of an additional T cell repertoire outside the thymus (1) (2) (3) (4) (5) (6) (7) (8) (9) . Furthermore, environmental antigens including intestinal microflora and food antigens are also responsible for the skewed expansion of the T cell repertoire in the periphery, especially in the gut-associated tissues (GALT) (1) .

GALT are composed of non-aggregated components such as i-lEL and lymphocytes in lamina propria, and aggregated components such as Peyer's patch (PP) (10) . The mouse i -lEL consist of heterogeneous populations expressing ThyP or Thyl-, CD8aa or CD8a~ and TCRa~ or TCRyo (5, (11) (12) (13) , whereas the rat i-lEL consist mainly of CD8+ T cells bearing TCRa~ (14, 15) . In both mice and rats, i-lEL also contain the unique CD4+CD8+ lEL bearing TCRa~, which increase in number with age (15, 16) .

i-lEL show non-MHC-restricted cytotoxic activity in a redirected killing assay (17) and help IgA production by B cells through cytokine production (18) , suggesting that these cells may play an important role in host defense against microorganisms as invaders through the intestinal wall (17, (19) (20) (21) (22) (23) (24) . BEAGLEY et al. reported that i-lEL of mice responded to mycobacterial HSP70 but not HSP60 (25) . We previously reported that the proliferation of i-IEL of aged rats significantly proliferated in response to irradiated-syngeneic splenocytes, but those of i -IEL of young rats did not (15) . In a recent report, a proliferative response of whole i-IEL of aged rats was enhanced by bacterial HSP70 in the presence of irradiated-syngeneic splenocytes (26) . These results suggest that a significant fraction of i-IEL in aged rats may be associated with microbial stimulation with highly conserved antigens in the intestines.

In this study, we examined the specificity of unique CD4+CD8+ i-lEL increasing in aged rats under conventional condition and the roles of intestinal microflora for the expansion of this subset in i-IEL. The unique CD4+CD8+ i-lEL bearing TCRa~ were found to respond to HSP70 derived from Escherichia coli (E. coli) in the presence of syngeneic APC. The CD4+CD8+ population expanded in the i-IEL of aged rats in association with the increased number of Enterobacteriaceae, especially E. coli, in the contents of small intestine and feces. The implications of our findings for the influence of the intestinal microflora on the expansion of unique T cell population in i-IEL were discussed.

Germfree Wistar male rats and conventional Wistar male rats (supplied by Wistar Inst. PA, USA) were maintained in our laboratory and fed commercial diet (CL-2) obtained from Crea Japan (Tokyo, Japan) or home-made diet which were made in our laboratory (27) . The composition of the diets are shown in Tables 1 and 2. The germfree rats had been produced by our own technique and maintained for more than 40 generations in this laboratory (28) . They were maintained in stainless isolators. Their germfree status was checked as described by WAGNER et al. (29) . 

The following monoclonal antibodies (mAbs) were used in this work: PE-conjugated W3125 (anti-rat CD4 mAb), FITC-conjugated OX-8 (anti-rat CD8 mAb), FITC-conjugated OX-19 (anti-rat CDS mAb), and FITC-conjugated OX-12 (anti-rat 1C chain mAb) were purchased from Serotec (Oxford, UK). R73 (anti-rat TCRa~ mAb) was kindly provided by Dr. T. HONIG (Universitat Wiirzburg) (30) and was conjugated with FITC or biotin according to standard procedure.

Biotin-conjugated lF4 (anti-rat CD3 mAb) was kindly provided by Dr. T. TANAKA (the Tokyo Metropolitan Institute of Medical Science) (31, 32) . Secondary antibodies were PE-conjugated anti-mouse IgM, biotinylated anti-mouse IgM and biotinylated anti-mouse IgG purchased from Caltag Laboratories (San Francisco, CA, USA). Peroxidase-conjugated goat anti-mouse (F(ab')2 fragments of IgG was obtained from Amersham International pic. (Amersham, UK). Streptavidin-PE and streptavidin-DuoCHROM was obtained from Becton Dickinson (San Jose, CA, USA). Streptavidin-RED613 was purchased from Gibco BRL (Gaithersburg, MD, USA).

i-lEL were prepared according to a modification of a method of CERF-BENSUSSAN et al. (33) . Briefly, small intestine was flushed with SO ml of sterile phosphate-buffered saline (PBS), fat and PPs were removed. The intestine was opened longitudinally, cut into 5 mm-long pieces, and then washed in PBS. The pieces were stirred in 199 medium (GIBCO, Gland Island, NY, USA) supplemented with 20% Nu serum (Collaborative Research Inc., Bedford, MA, USA) and 1 mmollL dithioerythritol (Sigma Chemical, Co., St. Louis, MO, USA) at 37°C for 30 min and then centrifuged [Kubota model 5700 (Tokyo, Japan), x 150 g, 4°C,S min]. The pieces were resuspended in RPM I 1640 (GIBCO) supplemented with 20% Nu serum and agitated at 37°C for 20 min, and then debris were removed. The resultant soup was then filtered through a glass wool column. This filtration procedure removed a large proportion of the dead cells and debris. This glass wool column-passed cell suspension was centrifuged, resuspended in 8 ml of 44% Percoll (Sigma), and layered on 5 ml of 67.5% of Percoll. The gradient was centrifuged at x 700 g at 20°C for 20 min (Kubota model 5700). Lymphocytes at the interface were harvested, and washed twice with Hanks-balanced salt solution (HBSS). The CD4-CD8+ or CD4+CD8+ i-lEL were sorted with EPICS® Elite ESP (COULTER Corp., Miami, FL, USA) after stained with FITC-conjugated OX-8 and PE-conjugated W3125. The purity of CD4-CD8+ i-IEL or CD4+CD8+ i-IEL was 99.3% or 97.5%, respectively as accessed by two color flowcytometric (FCM) analysis. The syngeneic spleen was homogenized with HBSS using glass slides and then filtered by cotton gauze to remove cell debris.

For two-color FCM analysis, cells were stained with FITC-conjugated mAb and PE-conjugated mAb or biotinylated mAb followed by streptavidin-PE. For three color FCM analysis, cells were stained with FITC-conjugated mAb, PE-conjugated mAb and biotinylated mAb followed by streptavidin-DuoCHROM or streptavidin-RED613. The cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA). 

Immunohistochemical analysis was performed by a peroxidase staining technique as previously described (13) . Briefly, intestine was promptly fixed in periodate-lysine 4% paraformaldehyde for 6 h, frozen in OCT compound (Miles Pharmaceutical, Naperville, IL, USA) and sectioned at 6 mm on a cryostat. For single staining, the sections were incubated with anti-CD4 mAb or anti-CDS mAb washed and incubated with peroxidase-conjugated goat anti-mouse F(ab')2 fragments of IgG and then reacted with 0.25% diaminobenzine (DAB) containing 0.5% hydrogen peroxide. For double staining with anti-CD4 and anti-CDS mAb, the sections were incubated with anti-CD4 mAb followed by peroxidase-conjugated anti-mouse IgG, washed and reacted with 0.02% 4-chloro-1-naphtol containing 0.5% hydrogen peroxide. The stain sections were washed with 0.1 mol!L glycine (pH 2.2). The sections were incubated with anti-CDS mAb, washed and stained with DAB.

The contents in the small intestine and feces were collected from rats fed CL-2 diet or homemade diet. They were homogenized in 10 ml of sterile PBS. The homogenates were diluted serial with PBS and spread on agar medium plates (Eiken Chemical Co., Ltd., Tokyo, Japan) for Enterobacteriaceae, and on to LBS agar medium plates (BBL Microbiology Systems, Cockeysville, MD, USA) supplemented with S giL Lab Lemco powder (Oxoid Ltd., Basingstoke, UK), 1.5 giL sodium acetate trihydrate and 3.7 giL acetate for Lactobacilli. The agar plates were incubated at 37°C for 24 h, and the LBS agar plates were incubated anaerobically with an anaerobic system (GasPak Anaerobic System; BBL Microbiology Systems) at 37°C for 4S h. The colonies on the plates were finally enumerated (35) .

Two-way ANOVA (36) was used to analyze the main effects (diet, breeding condition) and the interaction (diet x breeding condition). The analyses were performed by using a statistics package (Mascot; Yukkum, Tokyo, Japan). Data were analyzed by Student's t test. Differences of p < 0.05 were considered to be significant. All experiments were performed at least twice.

Proliferative responses of (04+(08+ i-IEL from aged rats to OnaK (E. coli HSP70) We have previously reported that i-lEL from aged F344 rats but not from young rats respond to syngeneic spleen cells (15) , and NAKAMURA et al. has recently reported that the proliferation of the i-IEL from aged Lewis rats was enhanced by mycobacterial HSPs (26) . To confirm these earlier findings, we first examined the proliferative responses of whole i-IEL from 6 month-old Wistar rats against E. coli HSP70 (DnaK) in the presence of syngeneic antigen presenting cells (APC). Consistent with our previous report with F344 rats (15) , the i-lEL from the aged Wistar rats showed proliferative response in the presence of MMCtreated syngeneic splenocytes, whereas such response was not detected in the i-IEL of young (8 week-old) Wistar rats (Fig. la) . The proliferative response of CD4+CD8+ i-IEL respond to E. coli hsp 70 . 555 the aged rats i-IEL was significantly enhanced (p < 0.05) in response E. coli HSP70 (Fig. 1 b) , but those of the young rats did not respond to the HSP70. To further determine which cell population in the i-IEL from aged rats was able to respond to the bacterial HSP70, we purified CD4-CD8+ i-IEL and CD4+CD8+ i-IEL from aged rats by using EPICS® Elite ESP of COULTER cell sorting system. The purity of each subset was 99.3% for CD4-CD8+ i-IEL or 97.5% for CD4+CD8+ i-IEL (Fig. 2a) . As shown in Figure 2b , the purified CD4+CD8+ i-IEL responded to syngeneic spleen cells and much more vigorously to HSP70 in the presence of syngeneic spleen cells, whereas CD4-CD8+ i-IEL showed little, if any, proliferation in response to HSP70 with syngeneic spleen cells.

These results indicate that CD4+CD8+ i-lEL of aged rats can respond to E. coli HSP70 in the presence of syngeneic APC.

Comparison of appearance of CD4+CD8+ i-IEL among aged rats fed CL-2 diet or home-made diet under conventional conditions or germfree conditions i-lEL were recovered from 6 month-old rats bred under conventional conditions, which were fed commercial CL-2 diet or home-made diet. After staining with various mAbs, the i-IEL were examined by FCM analysis. A representative FCM profile from 5 rats of each group was shown in Figure 3 and the data were summarized in Table 3 . In 6-month old rats bred under conventional conditions, which were fed CL-2, a much larger proportion of CD4+CD8+ i-IEL were detected (Fig. 3a, Table 2 , p < 0.005). Although most of the i-IEL expressed TCRa~ in these rats, an appreciable number of CD3+ TCRa~i-IEL corresponding to TCRyb i-IEL were detected in the i-IEL (Fig. 3b) . The CD3-TCRa~i-IEL were also detected at an appreciable level in the rats. The CD3-TCRa~-CD4-CD8-i-IEL did not belong to B cells because they did not express surface 19 as accessed by staining with anti-IgK chain mAb (data not shown). The CD4+CD8+ cells were reported to be detected, albeit only a few, in the GALT including PP and lamina propria in mice (10) . To confirm that the CD4+CD8+ i-IEL were really located within the epithelium, we carried out immunohistochemical analysis of the small intestine of aged conventional rats. Two color staining using naphtol and DAB revealed that CD4+ i-lEL coexpressed CD8 in these rats (Fig. 4a) . The CD4+CD8+ i-lEL were detected at the intraepithelium and preferentially located at the lateral surface in the longitudinal sections of villi from the terminal ileum, whereas these CD4+CD8+ i-lEL were rare in the crypts or dome epithelium overlying PP (Fig. 4b, 4c , and data not shown).

To further characterize the CD4+CD8+ i-IEL, three color staining analysis with anti-CD4 mAb, anti-CD8 mAb, and anti-CDS mAb was carried out for lymph nodes (LN) and IEL from aged rats fed CL-2 diet under conventional conditions. As shown in Figure 5 , only a few CD4-CD8+ i-IEL expressed CDS, which is a pan T cell marker, but all CD4+CD8+ i-IEL expressed much the same intensity of CDS as the peripheral mature T cells, suggesting that the lineage of CD4+CD8+ i-IEL may differ from that of CD4-CD8+ i-IEL. The phenotype of i-IEL from aged rats fed home-made diet under conventional conditions were found to be quite different from those fed CL-2. The i-IEL consisted of a large number of CD4-CD8+ cells and appreciable numbers of CD4-CD8-cells and CD4+CD8-cells but very few, if any, CD4+CD8+ cells (Fig. 3a) . Thus, these results suggest that the composition of a diet may deeply influence the increase in the CD4+CD8+ i-IEL with age.

To elucidate whether diets affect directly or indirectly the expansion of CD4+CD8+ i-IEL, we examined cell surface markers on i-IEL from aged rats fed CL-2 diet or home-made diet which were bred under germfree conditions. Representative FCM profile from data of five rats of each groups was shown is Figure 6 and the data were summarized in Table 3 . i-IEL in aged germfree rats, irrespective of home-made or CL-2 diet feeding, consisted of a large number of CD4-CD8+ cells, CD4-CD8-cells and only a few CD4+ cells but no CD4+CD8+ cells. These results suggest that intestinal microflora play important roles in expansion of CD4+CD8+ i-1EL in aged rats fed CL-2 and exclude the possibility for direct stimulation of antigens in CL-2 diet to expand the CD4+CD8+ i-IEL.

Comparison of intestinal microflora between aged rats fed CL-2 diet and those fed home-made diet

To determine the effects of diets on microflora, bacterial counts of the contents in the small intestine and feces were examined in three aged rats fed CL-2 diet or home-made diet under conventional conditions. There was no significant difference in total bacterial counts both in the contents and feces between aged rats fed CL-2 diet and those fed home-made diet. However, the number of Enterobacteriaceae, especially E. coli, in contents and feces of rats fed CL-2 diet were approximately 100-fold higher than those in rats fed home-made diet (p < 0.05), while fewer H2S-producing bacteria were detected in rats fed CL-2 diet (p < 0.05, Table 4 ). The counts of the other bacterial group present in the rats fed CL-2 diet were much the same as those in rats fed home-made diet. 

We have obtained evidence that the unique CD4+CD8+ i-IEL from aged Wistar rats are able to respond to E. coli HSP70 in the presence of syngeneic APC. This extends the scope of an earlier finding of NAKAMURA et al. that whole i-IEL from aged Lewis rats recognize purified protein derivative (PPD) including mycobacterial HSP70 presented by syngeneic APC (26) . Our present data suggest that the responders to the bacterial HSP are a CD4+CD8+ i-IEL subset, which are increased in aged rats of various strains. Furthermore, this unique subset is able to respond to HSP70 derived from E. coli, which is one of major members in intestinal microflora. CD4+CD8+ i-IEL may expand with age through long term exposure to the bacterial HSPs.

We also demonstrate in the present study that diets influence the selection of CD4+CD8+ i-IEL in aged rats under conventional conditions. The CD4+CD8+ i-IEL were detected in aged rats fed CL-2 diet but hardly detected in aged conventional rats fed home-made diet. Two possibilities can be brought forward to explain the difference between aged rats fed CL-2 diet and those fed home-made diet. The first possibility is that CL-2 may contain antigens capable of directly stimulating CD4 +CD8+ i-IEL. The second possibility is that the different diets may cause differences in the composition of the microflora, which may affect the selection of CD4+CD8+ i-IEL. Our results with aged germfree rats, fed a CL-2 diet or home-made diets suggest that stimulation with intestinal microflora, rather than antigens present in a CL-2 diet, is important for the expansion of the CD4+CD8+ i-IEL. Interestingly, aged rats fed on a CL-2 diet contained a large number of E. coli in microflora, which may express DnaK E. coli HSP70. Taken together, these results suggest that an unique CD4+CD8+ i-IEL population may be selected by HSP70 through long term exposure to intestinal E. coli. So far, we do not know why rats who fed home-made contained fewer Enterobacteriaceae but a larger number of H2S-producing bacteria in their microflora as compared with those fed on a CL-2 diet. Major differences in the composition of these two diets were the amount of vitamins and amino acids. The differences in various compositions of diets and the mutual interaction of microflora may be responsible for the different compositions of the microflora between rats fed on a CL-2 diet and those fed home-made diet.

The origin of the unique CD4+CD8+ i-IEL is unknown. Immature CD8+ T cell precursors in the thymus are reported to acquire CD4 during differentiation in vivo and in vitro (37, 38) . Recent studies, which were examined with athymic nude mice, scid mice, and RAG gene knock-out mice repopulated with T celldepleted bone marrow cells, have revealed that a significant fraction of i-IEL can differentiate independently of the thymus (3, 39) . It is assumed that the CD8+ i-JEL may differentiate to CD4+CD8+ i-IEL at the local site of intestine under the influence of stimulation with intestinal microflora. On the other hand, there is evidence that activation and culturing of human CD4+ T cell clones in IL-4 resulted in the acquisition of CD8 due to its de novo synthesis (40) . Therefore, it is alternatively possible that CD4+CD8+ i-IEL may originate from CD4+ cells. IL-4 is known to have an ability to induce expression of Ia on B cells and macrophages. CERF-BENSUSSAN et al. (33) reported that IFN-y but not IL-2 appear to contribute to induction of Ia on intestinal epithelium. Stimulation with intestinal microflora including E. coli HSP70 may activate the i-lEL to produce both la-inducing and CD8-inducing cytokines, resulting in appearance of CD4+CD8+ i-IEL and Ia intestinal epithelium in aged conventional rats fed CL-2 diet.

CD4 molecules are known to play an important role as accessory molecules for T cell recognition to MHC class II molecules (41) . CD4+CD8+ i-IEL may be involved in recognizing antigens in the context of MHC class II expressed on intestinal epithelium. NAKAMURA et al. have suggested that the proliferative response by CD4+CD8+ i-IEL to PPD is also involved in CD8 and MHC class I molecules (26) . Therefore, CD4+CD8+ i-lEL may contain cells responding to the bacterial antigens in a MHC class I-restricted CD8-dependent manner. The cytolytic activity of i-IEL is known to be regulated by externally derived stimuli via a specific functional interaction between i-IEL and gut-associated antigens (21) . Expression of CD8 is reported to be important for anti-CD3 directed cytolytic activity by the CD4+CD8+ T cell clones (37) . The CD4+CD8+ i-lEL have been recently reported to display a high level of cytolytic activity upon TCR triggering (23) . Taken together, the CD4+CD8+ i-IEL may play important roles in the first line of defense against various pathogens in epithelium through recognizing them. And they may be presented by MHC class II in a CD4dependent manner and through their cytolytic activities against MHC class 1restricted antigens. We need further functional analysis for elucidation of the roles of the CD4+CD8+ i-IEL in the mucosal immunity.

In the present study, we demonstrated that purified CD4+CD8+ i-IEL of aged rats proliferated in response to E. coli HSP70 in the presence of APC and an increase number of E. coli were detected in the intestinal content and feces of aged rats. In conclusion, CD4+CD8+ i-IEL specific for E. coli HSP70 are expanding during exposure to microbial flora, especially E. coli that has been in the intestines for a long period during aging.

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This work was supported in part by grants to Y. Y. from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare, and Special Coordination Funds of the Science and Technology Agency of the Japanese Government.The authors thank Drs. T. HONIG and T. TANAKA for kindly providing R73 and Biotin-conjugated 1F4, respectively and also thank Mr. Y. YAMAKAWA for technical assistance in sorting of lymphocytes.