31

Journal of Immunological Methods, 139 (1991) 31-40 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100157U

JIM 05906

An appropriate in vitro culture condition for the induction of human TCR'y8 ÷ cells by heat-killed bacteria Shuhji Seki 1, T o r u A b o 1, Hiroshi Sakihara 1, K e i t a r o Sugiura 1, A k i h i r o K a n n o t, H i d e m i Rikiishi 1, T a k a y u k i M a s u d a 2 a n d K a t s u o K u m a g a i 1 I Department of Microbiology, Tohoku University School of Dentistry, and : Second Department of Pathology, Tohoku University School of Medicine, Sendai 980, Japan (Received 26 November 1990, revised received 17 January 1991, accepted 18 January 1991)

TCRy~ + cells proliferated when MNC were stimulated with various heat-killed bacteria. We investigated here the culture conditions for their maximum proliferation. MNC were cultured for 6 days with Streptococcus pyogenes, and for 3 days with T cell mitogens, PHA and anti-CD3 mAb, in medium supplemented with various concentrations (0.05-50%) of human sera. TCR,/6 + and TCR'/8-CD2+3 double negative cells induced by Str. pyogenes required high concentrations of sera ( > 6%) for their proliferation. Moreover, increased sera (up to 50%) greatly augmented their proliferation. On the other hand, TCRafl + cell proliferation induced by T cell mitogens was supported by a small concentration (even 0.1%) of the sera, and the addition of high concentrations of sera ( > 6%) somewhat suppressed responses. Similar serum requirement patterns were evident for the induction of cytotoxic cells. These results clearly demonstrated the existence of an appropriate culture condition for the proliferation of TCR78 + cells induced in vitro. Key words: TCRT~ + cell; Bacterial stimulation; Serum requirement

Introduction

In recent studies, a new lineage of lymphocytes having y,8-T cell antigen receptors (TCR,/8), but not a, fl-T cell receptors (TCRafl), has been demonstrated (Saito et al., 1984; Hayday et al., 1985). TCR-/8 + ceils have CD2 and CD3 antigens, but the majority lack both CD4 and CD8 antigens, i.e., one of the double negative (DN) lymphocytes

Correspondence to: T. Abo, Department of Microbiology, Tohoku University School of Dentistry, Seiryo-machi 4-1, Sendai 980, Japan. Abbreviations: DN, double negative; CSF, cerebrospinal fluids.

(Lanier and Weiss, 1986; Crispe et al., 1987). TCRy8 + cells appear early in thymic ontogeny and become a minority in the thymus at birth (Raulet et al., 1985; Pardoll et al., 1987; Haynes et al., 1988). Although TCRy6 ÷ cells are a small population in various lymphoid tissues, a significant number are distributed in the epithelia of the skin and the small intestine in man and animals (Borst et al., 1987; Bonneville et al., 1988; Goodman and Lefrancois, 1988; Koning et al., 1987; Kuziel et al., 1987). In terms of their unique tissue localization and distinct structure of TCR gene product, they are considered to have functions distinct from those of TCRa/3 + cells, e.g., the elimination o f microbially infected cells or atypical cells generated in vivo. We have recently dem-

32 onstrated that D N cells including TCR78 + and T C R T 6 - C D 2 + 3 - cells were induced when human blood mononuclear cell.s (MNC) were stimulated with heat-killed bacteria (Abo et al., 1990). Interestingly, they generated in an IL-2 less-dependent manner from lymphocytes lacking the apparent TCR3,6 expression. On the other hand, it is well established that T C R a f l + T cells proliferate in an IL-2-dependent manner when M N C are stimulated with polyclonal T cell mitogens, P H A and anti-CD3 monoclonal antibody (mAb) (Abo et al., 1984). In the course of these TCRT~ + cell proliferation studies, we saw that the induction levels of TCR76 + cells stimulated with bacteria increased in proportion to the increased concentrations of sera added to the medium. Hence, we investigated the culture conditions for their m a x i m u m proliferation. TCRa/3 + cells induced by T cell mitogens could proliferate in the medium supplemented with a very small quantity of sera, and the addition of a large amount of sera somewhat suppressed the responses. On the other hand, the proliferation of TCRT6 + cells induced by Streptococcus (Str.) pyogenes required the addition of a large amount of sera, without such suppression as seen in TCRa/3 + cell proliferation. In our most recent studies in humans and mice, we demonstrated that TCRT6 + cells preferentially exist and proliferate in the sinusoids (serum-rich area) of the liver (Seki et al., 1990). The present study serves not only to demonstrate the appropriate in vitro culture conditions for the TCRT6 + cell induction system, but also suggest why TCRT~ + cells and T C R a f l + cells have a distinct proliferation sites in the tissues.

sera, FCS, and young bovine sera. All of the sera were used after heating at 56 ° C for 30 rain.

Stimulatory agents for lymphocyte proliferation P H A and anti-CD3 m A b were used as representative agents for the induction of TCRafl + cell proliferation. P H A - P (Sigma, U.S.A.) and antiCD3 (IgG2a) antibody (OKT3, Ortho Pharmaceutical, U.S.A.) were used at concentrations of 0.05% and 1 / t g / m l , respectively. In these cultures, 1 x 106/ml M N C (0.2 ml) were incubated with the stimuli at 37°C for 3 days in a plastic microculture plate with 96 flat-bottomed wells in a CO 2 incubator. As previously described, the bacterial agent for the induction of TCR78 + cells was heat-killed Str. pyogenes (final concentration of 10 /~g/ml) (Abo et al., 1990). Here, 2 × 106/ml M N C (0.2 ml) were incubated for 6 days in a microculture plate.

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Materials and methods

Cell preparations Blood M N C were isolated from the heparinized blood of healthy adult donors by Ficoll-Isopaque gradient centrifugation (Abo et al., 1986, 1990). Isolated M N C were washed three times with a serum-free medium and finally suspended in a R P M I 1640 medium containing the indicated concentrations of h u m a n serum or others. The sera with which the medium was supplemented in the present study included human adult and neonatal

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Fig. 1. Distinct serum requirements among different lymphocyte-proliferation responses. MNC derived from three healthy donors were stimulated with (A) Str. pyogenes, and (B) PHA and anti-CD3 mAb. All cultures were performed in a RPMI 1640 medium supplemented with various concentrations (from 0.05 to 50%) of pooled aUogeneicsera. Culture conditions and 3H-TdR incorporation assay are described in the methods section. A representative result from three experimentsis shown.

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Fig. 2. A comparison of the surface phenotypes between Str. pyogenes-stimulatedlymphoblasts in 1% and 50% sera-supplementation. MNC derived from three healthy donors were cultured with Str. pyogenes for 6 days and then analyzed as to their phenotypes by two-color flow cytometryafter staining with fluoresceinated mAbs. A: 1% sera; B: 50% sera. A representative result from the three experiments is shown. Short arrows indicate TcR78+ cells and long arrow shows TCR78- CD2 + 3- cells.

DNA synthesis assay To test the proliferative responses of lymphocytes, [3H]-Thymidine (3H-TdR) incorporation into D N A was assayed as described previously (Abo et at., 1990). 16 h before termination of the culture, cells in each well of the microplate were pulsed with 0.5 #iCi of 3H-TdR. All experiments were performed in triplicate cultures.

Immunofluorescence test The phenotype of lymphoblasts induced was analyzed by two-color flow cytometry after staining the cells with fluoresceinated mAbs (Abo et al., 1990). FITC-conjugated aliquots of mAbs, anti-CD2 (Leu-5b), anti-CD3 (Leu-4) and anti-

TCR-1 (specific for an a / f l chain framework determinant) and biotin-conjugated aliquots of antiCD3 antibody were purchased from Becton-Dickinson Co., U.S.A. A biotin-conjugated anti-TCR~l1 antibody (specific for a 8 chain framework determinant and representing a pan-TCRT~l ÷ cell marker) was obtained from T cell Science, U.S.A. A biotin-conjugated antibody was developed with phycoerythrin (PE)-conjugated avidin. The fluorescent positive cells were enumerated by an FACScan analyzer (Becton-Dickinson Co.) (Abo et al., 1990). 10 4 cells were analyzed.

Electron microscopy after immunostaining M N C were fixed with 4% paraformaldehyde in

34

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Fig. 3 (continued on next page) a cacodylate buffer (pH 7.2) using microwave irradiation (Seki et al., 1990). They were embedded in an optimal cutting temperature compound. Frozen sections were cut and stained with biotinylated anti-TCRS1 antibody (T cell Science, U.S.A.) and left overnight. After washing in phosphate-buffered saline, the sections were incubated with peroxidase labelled avidin and reacted with 3,3'-diaminobenzidine. After postfixation with osmium tetroxide, these specimens were embedded in Epon 812-filled gelatin capsules. The ultrathin sections were stained with lead citrate. Cytotoxicity assay

Cytotoxicity was examined by a specific 51Crrelease assay of 4 h incubation. The target cell used in this study was a NK-resistant, human fibroblast cell line, WISH. The method and formula for calculating specific cytolysis were previously described (Abo et al., 1986).

Results L y m p h o c y t e proliferation stimulated with bacteria occurs in the medium with high serum concentration

In this experiment, we used a RPMI 1640 medium supplemented with various concentrations of human pooled allogeneic sera to induce lymphocyte proliferation responses (Fig. 1). The lymphocyte-proliferation induced by the stimulation of Str. pyogenes required high concentrations of sera ( > 6%) in the medium for maximum proliferation (Fig. 1A). Moreover, the increased sera (up to 50%) in the medium augmented proliferation. In this case, the cultures were performed for 6 days to induce their maximum proliferation. On the other hand, anti-CD3- and PHA-induced T lymphocyte proliferations were supported by a small quantity of the sera (even 0.1%) in the medium (Fig. 1B). The addition of high concentrations of sera somewhat suppressed the re-

35

Fig. 3. Morphologyof TCRT6+ cells, a: TCRT8~ cells before the stimulation; b: TCR't~+ cells after the stimulation culture. Human blood MNC were stimulated with Str. pyogenes for 6 days in medium supplemented with 20% human pooled sera and analyzed by electron microscopyin conjunctionwith immunostaining of TCRS1 antibody.

sponses to anti-CD3 ( > 3% of sera) and P H A ( > 6% of sera). The latter responses were induced for 3 days for maximum lymphocyte proliferation. Although the results shown in the figure were produced by using allogeneic pooled sera, autologous serum from reproduced the results. Neonatal human sera also produced the same results as adult sera for both proliferation responses (data not shown). We did not experience a significant spontaneous growth of M N C in the control culture at any applied concentrations of sera.

Phenotypic characterization of the stimulated lymphocytes We then investigated the p h e n o t y p e of lymphocytes stimulated with P H A and Str. pyogenes. It was demonstrated that more than 90% of lymphoblasts stimulated with P H A in the

medium supplemented with both 1 and 3% sera were TCRafl + and CD3 + as identified with the TCR-1 and anti-CD3 mAb (data not shown). Almost all of these lymphoblasts (n = 4) express either CD4 ( 5 0 . 2 _ 4.3%) or CD8 (39.6 ___6.7%). On the other hand, the lymphoblasts stimulated with Str. pyogenes showed a mixture of unique lymphocyte subsets, i.e., TCRT8 + cells, TCRTSC D 2 + 3 - cells, and TCRafl ÷ cells (Fig. 2). Furthermore, the culture with 50% serum supplementation induced an apparent increase in proportion of both TCRT8 + (indicated with short arrows) and T C R T S - C D 2 + 3 - (indicated with long arrows) D N cells than the culture using 1% serum. It was concluded that both the maximum cell proliferation response and the maximum induction of TCRT~ + and TCRTS- CD2 + 3 - D N cells occurred in the medium supplemented with high

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Fig. 4. Induction levels of natural cytotoxicity dependent on serum concentration. MNC derived from three healthy donors were stimulated with (A) Str. pyogenes for 6 days and were stimulated with (B) PHA for 3 days, in the medium supplemented with 1-50% sera. These stimulated lymphocytes were used as the effector cells for the cytotoxicity assay against WISH cells. The cell recoveries of 1 x 106 M]'qC culture were ranged 0.4-0.6, 0.8-1.2 and 1.2-1.8 ( x 106) in the control culture, Str. pyogenes culture and PHA culture, respectively. The effector to target cell ratio was 20 : 1 and the medium of assay system was RPMI 1640 medium supplemented with 5% pooled allogeneic sera. A representative result from the three experiments is shown.

c o n c e n t r a t i o n s of sera. If b l o o d M N C were s t i m u l a t e d with b a c t e r i a in the m e d i u m with 50% sera, m a n y blastic l y m p h o c y t e s were shown to a p p e a r b y the analysis of their light scatter. If the analysis of T C R T 6 + cells was g a t e d to the p o p u l a tion with large light scatter, the p e r c e n t a g e s of T C R 7 8 + cells were significantly increased.

Morphology of TCR78 + cells T h e m o r p h o l o g y of TCR'y6 + cells b e f o r e a n d after s t i m u l a t i o n with Str. pyogenes was investig a t e d b y electron m i c r o s c o p y in c o n j u n c t i o n with i m m u n o s t a i n i n g b y the TCRS1 a n t i b o d y (Fig. 3). A l l TCR-y6 + cells b e f o r e s t i m u l a t i o n were shown to b e resting small l y m p h o c y t e s ( > 99%) (Fig. 3 a ) . O n the o t h e r h a n d , T C R 7 8 + cells i n d u c e d b y s t i m u l a t i o n h a d a l y m p h o b l a s t i c a p p e a r a n c e with a few e l e c t r o n - d e n s e granules (Fig. 3b). I n the s t i m u l a t e d culture on d a y 6, TCR'y8 + cells consisted of resting TCR'y6 + a n d l y m p h o b l a s t i c

T C R T 6 + cells ( a p p r o x i m a t e l y 1"1), d e s p i t e indiv i d u a l variations.

Induction levels of natural cytotoxicity depending upon serum concentration Since b o t h P H A - i n d u c e d T C R a f l + cell res p o n s e a n d Str. pyogenes-induced T C R 7 8 + cell r e s p o n s e were a c c o m p a n i e d b y p o t e n t n o n - M H C restricted cytotoxicity, we then e x a m i n e d the serum effect o n the i n d u c t i o n levels of n a t u r a l cytotoxicity (Fig. 4). Here, the target cells for c y t o t o x i c i t y used a n N K - r e s i s t a n t cell line, W I S H . T h e l y m p h o b l a s t s i n d u c e d b y the s t i m u l a t i o n o f Str. pyogenes s h o w e d i n c r e a s e d c y t o t o x i c i t y in p r o p o r tion to the i n c r e a s e d a m o u n t of serum in the m e d i u m (Fig. 4A). O n the o t h e r h a n d , the inc r e a s e d s e r u m c o n c e n t r a t i o n in the m e d i u m also s u p p r e s s e d the i n d u c t i o n o f n a t u r a l c y t o t o x i c i t y in the T C R a f l + cell r e s p o n s e i n d u c e d b y P H A (Fig.

37

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4B). The unstimulated lymphocytes used as a control experiment showed no cytotoxic activity in any of the added serum concentrations added.

Failure of TCRy8 + cell induction by the supplementation of FCS and human cerebrospinal fluids (CSF) In the experiments described thus far, we demonstrated that the induction of TCRy8 + and TCRafl + cells showed distinct human serum requirements. We further examined whether or not FCS and human CSF were effective in inducing their proliferation (Fig. 5). The main purposes of these experiments were to examine (i) whether xenogeneic serum, FCS, which is the most popular sera used for medium supplementation, was also effective for the present TCR78 + cell induction and (ii) whether one of the human body fluids, CSF, that contains only a low protein-concentration like other extra-cellular body fluids such as transudate, lymph fluid and ascitic fluid, was effective for TCRT/J + cell induction. The supplementation of the medium with FCS was effective for the proliferation of lymphocytes stimulated

with both PHA and the anti-CD3 antibody, showing less efficacy with high serum-dose suppression (Fig. 5A). On the other hand, the supplementation with FCS did not significantly induce the proliferation of lymphocytes stimulated by Str. pyogenes. The same results were produced by using several different sources of FCS, and even when young calf serum was used instead of FCS (data not shown). CSF, which contains only a low concentration of protein ( < 0.5 m g / m l ) in comparison with serum ( > 35 m g / m l ) , induced the proliferation of lymphocytes stimulated with PHA, but did not induce prominent proliferation of lymphocytes stimulated with the anti-CD3 antibody (Fig. 5B). More importantly, CSF did not induce proliferation of lymphocytes stimulated with Str. pyogenes at all.

Discussion

TCRafl + cells induced by the polyclonal T cell rnitogens, PHA and anti-CD3 mAb, proliferated

38 in medium supplemented with a very small quantity of sera, and the addition of a large amount of sera somewhat suppressed the responses. On the other hand, the proliferation of TCRy8 + cells induced by the stimulation of Str. pyogenes required the addition of a large amount of sera. As shown in the results from the studies on D N A synthesis, phenotypic characterization and cytotoxicity, the induction levels of TCRy8 + cells were extremely dependent on the amount of serum added to the medium. For the appropriate induction of TCRy8 + cells, medium containing more than 20% serum was required. We have recently revealed that TCRy8 + CD2 + 3 + and T C R y 6 - CD2 + 3 - cells were induced when human blood M N C were cultured with various bacterial stimulations (Abo et al., 1990). In contrast to the hitherto described proliferation of TCRT8 + cells in the presence of exogenous IL-2, the TCRy8 + cells stimulated with bacteria were generated in an IL-2 less-dependent manner. The prominent induction of TCRy8 + cells was mainly produced through the use of G r a m ( + ) bacteria such as Str. pyogenes, Staphylococcus (Sta.) aureus and Str. faecalis (Abo et al., 1990). The stimulatory component of bacteria was determined to be a substance with a molecular weight of 25-26 kDa composed of the sonicated material of these bacteria. Recently, it was demonstrated that a purified protein derivative ( P P D ) / h e a t shock protein (HSP) of Mycobacterium tuberculosis was one of the molecules to specifically interact with TCRy6 + cells (Janis et al., 1989; Haregewoin et al., 1989; O'Brien et al., 1989). However, with respect to the IL-2 dependency, the requirement of prior sensitization, and the physicochemical properties, the PPD-induced lymphocyte proliferation response was clearly different from the TCRy8 + cell proliferation response induced by using whole bacterial particles (Abo et al., 1990). In the phenotypic characterization study, it was demonstrated that the increased amount of sera added to the medium not only augmented the proliferation of lymphocytes, but also increased the proportion of TCRy8 + cells generated. This was also true in TCRy8 CD2 + 3 - cell proliferation. In the bacterial stimulation, both TCRy6 + and T C R y 6 - C D 2 + 3 - cells were induced as predominant cells. It is conceivable that these cells

may be classified into a closely related population despite their distinct T C R y 8 expression, since they share the same phenotype as DN, CD4 8 - cells. The induction levels of non-MHC-restricted cytotoxicity also increased depending on the amount of sera supplementing the medium. The morphology of both resting and activated TCRy6 + cells was clearly demonstrated in the present study. In contrast to N K cells (Abo et al., 1986), the vast majority of resting TCRy6 + cells lacked visible electron-dense granules in their cytoplasm. However, the activated TCRy6 + cells had a lymphoblastic appearance and acquired cytoplasmic electron-dense granules. This morphology of activated T C R y 6 + cells was very similar to that of activated N K cells in the presence of IL-2 (i.e., lymphokine-activated killer cells derived from N K cells) (Abo et al., 1983). In a recent study, we have demonstrated that such activated TCRy~ + cells preferentially exist in vivo in the sinusoids of the liver in humans and mice (Seki et al., 1990). It is very difficult to determine precisely what components of serum modulate the proliferation response of TCRcq~ + and TCRy8 + cells observed here. It is, however, apparent that some of the supporting components of sera for T C R a B + cells may consist of albumin, transferrin, insulin and unknown growth factors, since the TCRa/~ + cells proliferated in the serum-free medium commercially available (data not shown). Thus, the serumfree medium contains such supplements. On the other hand, some of the candidates for the suppression factor of TCRa/3 + cells in the serum are lipoproteins for PHA-induced responses (Morse et al., 1977) and y-globulins for anti-CD3-induced response (Looney et al., 1984). Indeed, we also observed considerable suppression of each response by lipoproteins or y-globulins. In this regard, we examined whether anti-CD3 mAb induced a significant proportion of TCRy8 + cells in the medium supplemented with 50% serum. However, this was not the case. No cell proliferative response itself occurred, probably because of a high y-globulin concentration. In contrast to TCRcq~ + cell responses, the induction of TCRy8 + cell proliferation-response was not produced in the serum-free medium commercially available (data not shown). Moreover, supplementation with

39

FCS, calf serum and human cerebrospinal fluid could not substitute for the human adult sera. Here, human neonatal sera were also effective for TCR'~3 + cell induction at almost the same level as human adult sera. We have recently examined whether some of the human purified serum-components, including albumin, y-globulin, lipoprotein fractions, fibronectin and laminin, could be substituted for human sera in induction. None of them, alone or in combination, could be substituted for the sera. Only the components of MW > 50,000 in human sera were effective for the induction (unpublished observation). Finally, although one of the body fluids, human cerebrospinal fluid, was effective for TCRa/3 + cell proliferation, it was not effective at all for TCR73 + cell induction. The same result was produced by using another transudate, pericardiac fluid, that contains only low concentrations of protein components ( < 0.5 m g / m l ) (Seki et al., unpublished observation). It is conceivable that some of the components with a high protein concentration ( > 35 m g / m l ) in the human serum, but not in the transudate, are effective for the induction of TCR76 + cells. We have recently demonstrated that there is an accumulation of activated TCR76 + cells in human and murine hepatic sinusoids (Seki et al., 1990). Furthermore, freshly isolated hepatic TCR'y3 + lymphocytes from mice spontaneously proliferate in vitro (Seki et al., 1990). It was also demonstrated that TCR73 + cells exist in splenic sinusoids of humans and chickens (Bucy et al., 1988, 1989; Falini et al., 1989). Although TCR73 + cells preferentially reside in the epithelia of the skin and intestine, the proliferating sites of TCR-/3 + cells might be the serum-rich sinusoid area, especially after birth. Therefore, the present results seem to reflect the important physiological behavior of TCR'/3 + cells in vivo. It is probable that TCRafl + cells may proliferate in the solid area of lymphoid organs, where such lymphocytes are exposed to the transudate of body fluids. On the other hand, TCR'~3 + cells may proliferate in the serum-rich sinusoid area and may occasionally proliferate in inflammatory sites where such lymphocytes are exposed to abundant proteins such as in bacterial infections and arthritis of joints. In any case, the present serum-dependent induction system for

TCR73 + cell proliferation is a useful tool for further investigation of this population.

Acknowledgments We thank Miss Noriko Akaishi and Miss Mariko Haraya for their expert editorial assistance.

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An appropriate in vitro culture condition for the induction of human TCR gamma delta + cells by heat-killed bacteria.

TCR gamma delta + cells proliferated when MNC were stimulated with various heat-killed bacteria. We investigated here the culture conditions for their...
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