Clin. exp. Immunol. (1991) 83, 505-509

ADONIS

000991049100092R

Human T cell responses to fi-galactosidase J. D. OXLEY, R. H. BROOKES, L. S. RAYFIELD & P. S. SHEPHERD Department of Immunology, United Medical and Dental Schools of Guy's and St Thomas' Hospitals, Guy's Campus, London, England

(Acceptedfor publication 9 October 1990)

SUMMARY The peripheral blood of most normal individuals has been shown to contain T cells that respond to f-galactosidase (fl-Gal), presumably as a result of natural priming. Three T cell clones (clones 1,2,4) specific for f-Gal were isolated from peripheral blood mononuclear cells (PBMC) after pretreatment with leucine methyl ester (LeuOMe); a fourth clone from the same individual was isolated from untreated cells. All four clones were CD4+ CD8- aBTcR+ and clone I was additionally shown to be cytotoxic. Epstein-Barr virus (EBV) transformed B cell lines were derived from LeuOMe-treated or untreated PBMC and used to study the efficiency of presentation of f-Gal to one of the clones. The results indicated that B cells transformed after LeuOMe treatment presented #-Gal at lower concentrations than untreated controls. f-Gal would therefore appear to be a highly suitable model antigen for studies of immunoregulation in humans.

Keywords fi-galactosidase T cell clones leucine methyl ester antigen presentation

INTRODUCTION T cell responses to a wide variety of antigens have been studied in animal model systems. One of these is the bacterial antigen, Pgalactosidase (fl-Gal), which has been used to investigate the interactions between B, T helper and T suppressor cells in mice by immunizing with different fragments of the antigen in the presence of Freund's adjuvant (Manca et al., 1985; Kryzch, Fowler & Sercarz, 1985; Shivakumar, Sercarz & Krzych, 1989). It was found that some fragments promoted T cell help while others favoured suppression, strengthening the notion that distinct epitopes on an antigen may regulate the immune response (Shivakumar et al., 1989). As far as we are aware, T lymphocyte responses to f-Gal have not been previously reported in man. Our finding of high titres of antibody to fl-Gal in human serum (unpublished) led us to hypothesize that f-Gal might be a suitable model antigen for studies of immunoregulation in man and for the generation of antigen-specific T and B lymphocyte lines or clones. f-Gal has several advantages over other model antigens, for example tetanus toxoid, in that any response would be the result of natural, as opposed to artificial immunization. The antigen is also easy to obtain and has been characterized immunologically in animal systems. Treatment of peripheral blood mononuclear cells (PBMC) with the lysosomotropic agent leucine methyl ester (LeuOMe) prior to infecting them with Epstein-Barr virus (EBV) has been

shown by Ohlin et al. (1989) to increase their proliferation and rate of antibody secretion in comparison to untreated controls. The condensation product of LeuOMe produced by monocytes is also known to be toxic to cytotoxic T lymphocytes and natural killer (NK) cells (Thiele & Lipsky, 1986a). We have therefore investigated the potential of f-Gal as a model antigen in a human system and examined the effects of LeuOMe on the generation of fl-Gal-specific B and T lymphocytes. MATERIALS AND METHODS Tissue culture medium The standard medium used was RPMI 1640 (074-0810ON; GIBCO, Paisley, UK) supplemented with 20 mg/ml of sodium hydrogen carbonate (10247; BDH, Poole, UK), I mm sodium pyruvate (16-820-49, Flow Labs., Irvine, UK), 100 U/ml of penicillin and 100 yg/ml streptomycin (043-05070H; GIBco) and 2-4 mg/ml HEPES (44285; BDH) (pH 7-3). This was filtersterilized before use. Antigens fl-Gal (G-6008, Sigma, Poole, UK), and bovine serum albumin (BSA; A-4503, Sigma), were aliquoted at 1 mg/ml in standard medium and stored frozen at - 20°C. Interleukin-2

Lymphocult-T (TLF; 811020, Biotest, Frankfurt, Germany), a lectin-free supernatant produced by phytohaemagglutinin (PHA) stimulated human T lymphocytes, was used as a source of interleukin-2 (IL-2) (stock solution 200 U/ml).

Correspondence: P. S. Shepherd, Department of Immunology, 3rd Floor, Medical School, UMDS, Guy's Campus, London Bridge, London, SEI 9RT, UK.

505

J. D. Oxley et al.

506

Cell separation Venous blood was obtained from healthy volunteers and defibrinated. PBMC were obtained by density sedimentation on Ficoll-Histopaque 1077 (1077-1, Sigma). The serum was removed and the cells washed twice in medium before being resuspended at 2 x 106 cells/ml in medium supplemented with 10% autologous serum. Treatment with LeuOMe PBMC were suspended at 107 cells/ml in serum-free medium, containing 2 5 mm of freshly prepared LeuOMe (L-9000, Sigma), for 40 min at room temperature. The cells were then washed three times in medium supplemented with 10% fetal calf serum (FCS; S-OOOla, Sera Lab, Sussex, UK) and were recounted.

Transformation of B cells with EBV Normal or LeuOMe-treated PBMC (Fig. 1) were resuspended at 107 cells/ml and transformed with EBV (strain M81) according to the method ofJohnston et al. (1990). Once established, the lymphoblastoid cells were expanded and continuously passaged in flasks (163371; Nunc, Roskilde, Denmark). Proliferation assays PBMC (105/well) were cultured in round-bottomed 96-well microtitre plates (I -63320; Nunc) together with antigen at a final concentration of 1-25 pg/ml in a volume of 200 p1. The plates were incubated at 370C in a humidified 5% CO2 atmosphere for 3-7 days. On days 3, 5 and 7 the plates were pulsed with 20 p1 of 3H-thymidine (TRA120; Amersham International, Amersham, UK; 9 25 KBq-culture) and 4 h later the wells were harvested onto glass fibre and counted in an LKB (Bromma, Sweden) fspectrometer. Isolation of T cell lines and clones specific for f-Gal Two T cell lines were established from normal or LeuOMetreated PBMC of a single donor (Fig. 1). Treated or untreated cells (0 75 x 106 cells) were cultured in 24-well plates (76-063-04, Flow Labs.) together with f-Gal (5 pg/ml) and 0 5 x 106 irradiated (40 Gy) autologous PBMC in a final volume of 2 ml. Cultures were restimulated weekly by adding fresh irradiated autologous PBMC (106 cells) and f-Gal (5 pg/ml). TLF (5% v/v) was included in the culture medium from the second restimulation (i.e. week 3 of culture), and at this stage the cells were cloned at 0-3 cells/well in Terasaki plates (1-63118A, Nunc; final volume 20 p1) and 0-5 cells/well or I cell/well in round-bottomed

Peripheral blood LeuOMe

LeuOMe

untreated

treated

EBV transform

T line

EBV Leut

Tclone 3

T line

Tclones 1,2,4

EBV transform

EBV Leu+

Fig. 1. The isolation of LeuOMe-treated and untreated EBV-transformed B cell lines and T cell clones from peripheral blood.

microtitre plates (final volume 200 p1). Irradiated autologous PBMC (2 x 104 or 2 x 105 cells for Terasaki or microtitre plates, respectively) were included with TLF (10%) and fl-Gal (25 pg/ ml). After 6 days of culture 10 u1 of TLF were added to wells showing growth. As the numbers of cells increased they were transferred to, and restimulated in round-bottomed microtitre plates, then in flat-bottomed plates (1-67008, Nunc) and finally in 24-well plates. Antigen specificity was examined at the end of a 7-day cycle by culturing 104 line or clone cells in roundbottomed wells with 105 irradiated autologous PBMC and antigen. The cultures were harvested on day 3 following an overnight pulse with 3H-thymidine.

Cytofluorimetric analysis T cell phenotype was analysed according to the method of Fortune & Lehner (1988) by staining with murine monoclonal antibodies to leucocyte common antigen (LCA, CD45), CD3 (OKT3), CD4 (OKT4), CD8 (OKT8) and the af T cell receptor (TcR) (7770; Becton Dickinson, Mountain View, CA) followed by an FITC-conjugated goat anti-mouse IgG (9031, Becton Dickinson). The presence of the yc TcR was investigated by means of a direct FITC conjugate (TA2061; T Cell Sciences, Cambridge, MA). Cytofluorimetric analysis was performed using a FACScan (Becton Dickinson).

Cytotoxicity assay T cell clones were tested for cytotoxicity in a standard 4-h 51Cr release assay using autologous lymphoblastoid cells generated after LeuOMe pretreatment as targets. Cytotoxic potential was measured by including PHA (6 pg/ml, L9132; Sigma) in the assay and specific cytotoxicity was assessed on lymphoblastoid cells prepulsed with f-Gal (25 pg/ml) for 24 h before the assay. Briefly, clone or line cells were titrated in duplicate in roundbottomed microtitre plates to give effector: target ratios of 20: 1 to 2-5: 1, in RPMI medium+5% FCS. Target cells (5 x 103) labelled with 5'Cr (Rayfield, Brent & Rodeck, 1981) were added to each well to give 200 p1 final volume and incubated at 37°C in a humidified 5% CO2 atmosphere. After 4 h, 100 p1 of supernatant were removed for gamma counting (80 000 Gamma Sample Counter, LKB). Maximum release was estimated by adding 10% Triton X100 (T-7003; Sigma) in place of effectors. Specific lysis was calculated using the formula: Experimental - Spontaneous release 100 Maximum -Spontaneous release

RESULTS Proliferation of normal PBMC to f-Gal In order to determine the optimal conditions for proliferation responses to f-Gal, PBMC from two donors were cultured with different concentration of f-Gal for 3, 5 and 7 days. Figure 2 shows that the kinetics of the response were those expected of a recall antigen in that the response was detectable by day 5 and maximal by day 7. Nine further donors (seven women, two men; age range 22-47 years) were screened for responsiveness to fiGal; on day 6 at a dose of 25 pg/ml the mean stimulation index+s.d. was 16+8 with a mean A d/min+s.d. of 7628 + 3963. Thus, proliferative responses to fl-Gal were clearly and consistently evoked in normal individuals. To investigate the nature of the T cell response to P-Gal we next attempted to generate antigen-specific T cell lines.

Human T cell responses to fi-galactosidase (a)

Table 1. Antigen specificity assays on T cell lines

I

Line Leu± (Mean d/min ±1 s.d.)

(Mean d/min ± 1 s.d.)

72 + 20 2524 + 667 13 654+ 1623 9347 + 452 6210+ 1058 3979 + 935 2754+ 1320 10057+ 1111 222 + 271 12418 +699

4558 + 740 17443+ 1102 13 672 + 212 7339 +423 4682 +241 4681 ± 813 7227+704 41 + 14 2938 +1462

F+Med F+L F+ L + 15/3-Gal

F+L+5fl-Gal F+L+ 1/3-Gal F + L + 0-2f-Gal F+L+BSA F+L+TLF L+M L+TLF

d/min x 10-3

(b) I Tr li

507

El

67+17

Leu+ or Leu- T cell lines (L) were incubated with irradiated autologous feeders (F), medium (M), fl-Gal (02- 15 pg/ml), BSA (25 pg/ ml) or TLF (20%) and pulsed on day 3.

777771 m

Line Leu-

I

777 .77 77H (n=2)

m 0

2 d/min

3 x

4

5

10-3

Fig. 2. Proliferation measured by 3H-thymidine incorporation on days 3 (U), 5 (-) and 7 (0) from two donors (a, b). I, PBMC (105 cells/well)+ standard medium; II, PBMC + 15 yg/ml /3-Gal; III, PBMC + 5 Mg/ml /3-Gal; IV, PBMC + 1 Mg/ml /3-Gal; 4-h pulse with 3H-thymidine. Counts expressed as mean d/min + 1 s.d.

1V

Antigen presentation by EBV-transformed B cells Two lymphoblastoid cell lines were generated from the same individual as used to obtain the cell lines and clones. One was isolated following LeuOMe treatment (designated EBV Leu+) and one without (EBV Leu-). Culture supernatants from the EBV Leu+ line were found to contain IgM antibodies to fl-Gal

c)

(n=3)

m

3-a

ZJu-

zm =lI 7~L

5

15

i0

20 0 5 d/min x 10-3

(n=2)

(b)

Generation of T cell lines and clones specific for f-Gal Two T cell lines were derived from a single individual whose proliferation data are shown in Fig. 2b. The effect of establishing lines from LeuOMe-treated cells or untreated (conventional) cells was also compared. Treatment with LeuOMe depletes CD8+ T cells, macrophages and NK cells selectively (Thiele & Lipsky, 1986a, 1986b) and it was thought that such treatment may increase the efficiency of generating T cell lines and influence either the phenotype or function. In the event, T cell lines specific for /3-Gal were readily established from both the treated and untreated PBMC (Table 1). Cytofluorimetric analysis revealed both the lines to be CD3+, CD4+ and CD8-. Four clones were isolated from the lines and then characterized further. Three clones were derived from the LeuOMe-treated line; numbers I and 2 had been obtained at I cell/well and clone 4 was obtained by an initial cloning at 3 cells/well followed by 0 5 cells/well; clone 3 was derived from the untreated line at 0-3 cells/well. The responses of the clones to f-Gal are shown in Fig. 3. Phenotypically they were CD3+, CD4+, CD8- and a#+ ybTcR.

I

I

II

(d

15

25

35

)3)

z I

,,,

0

5

'1,,,,

15

25 0 d/min x 10-3

5

10

15

20

Fig. 3. Antigen specificity assays on four T cell clones (clones 1-4: a-d, respectively). Proliferation measured by 3H-thymidine incorporation on day 3 (18-h pulse); counts expressed as mean d/min + 1 s.d. I, F; II, F+C; III, F+C+15 ug/ml #-Gal; IV, F+C+5 ug/ml /3-Gal; V, F+C+1 jg/ml /-Gal; VI, F+C+BSA; VII, F+C+TLF; VIII,C+TLF. n, number of experiments; F, irradiated feeders at 105 cells/well; C, T cell clone cells at 104 cells/well; BSA, bovine serum albumin at 25 pg/ml; TLF used at 20%.

and tetanus toxoid, but not thyroglobulin, in a solid-phase ELISA (data not shown), indicating the presence of antigenspecific B cells. By contrast, the EBV Leu- line did not secrete detectable levels of anti-f-Gal or anti-tetanus toxoid. These lines were then compared for their capacity to present antigen to the fl-Gal-specific T cell clones in the absence of other antigenpresenting cells. Figure 4 shows the response of clone 3. It can be seen that at higher concentrations (25 pg/ml) the lines were

J. D. Oxley et al.

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targets even in the absence of PHA, suggesting recognition of processed antigen. The T cell line from which clone 1 was isolated (Leu+) was weakly cytotoxic and none of the other clones were lytic.

DISCUSSION

0/

CI

0

5

I

10

15

20

25

13-Gal (pLg/ml) Fig. 4. Antigen presentation by EBV Leu+ ( ...) and EBV Leu- lines ( ) to T cell clone 3. EBV Leu+ and EBV Leu- lines were preincubated for 24 h with different concentrations of fl-Gal, irradiated (80 Gy), and then added to the wells at 105 cells/well. T cell clone 3 was used at 104 cells/well. T cell proliferation was measured by 3H-thymidine incorporation on day 3 (18-h pulse). Results of one representative experiment expressed as mean stimulation indices + I s.d.

similarly effective at presenting #-Gal but that the EBV Leu+ line was more effective at lower concentrations of f-Gal. Lysis of lymphoblastoid cells by a fl-Gal-specific clone The clones were screened for cytotoxic activity using the EBV Leu+ lymphoblastoid cells as targets. One set of target cells was pulsed with f-Gal (25 ,ug/ml) for 24 h prior to 5'Cr labelling, the other set was incubated with medium as a control. Cytotoxicity of the clones was assessed on each target in the presence or absence of PHA. It can be seen from Table 2 that clone I killed targets in the presence of PHA, indicating the lytic function of this clone. Most interestingly, the clone killed P-Gal-pulsed

P-Gal has been the antigen of choice in two recent studies looking at T cell responses. In the first the gene for #-Gal has been inserted into BALB/c-derived tumour cells, then class Irestricted fl-Gal-specific T cell lines have been used to study cytotoxicity against these tumour cells (Rammensee, Schild & Theopold, 1989). It was found that the T cells recognized fragments of f-Gal whether expressed as intracellular, membrane-inserted, or secreted products. The second study employed different cleavage fragments of f-Gal to show that T cell responsivenes (either help or suppression), was dependent on the nature of the fragment used to induce the response and concluded that the processing of antigens determines which T cells are activated (Shivakumar et al., 1989). Both these studies were carried out in mice and there have been no equivalent studies in humans. The results reported here show that humans are naturally primed to fl-Gal in vivo. The most likely route of sensitization is via the gut since organisms synthesizing fl-Gal are a normal constituent of the gut flora; however, the presence of IgG class serum antibodies, in addition to the circulating T cells, strongly suggests that f-Gal has entered the systemic immune system. fGal can be regarded as a convenient antigen for use in human studies, since there is no necessity for artificial immunization and its structure at the protein and gene level has been determined (Fowler & Zabin, 1977). Although we have not examined the capacity of cleavage fragments to stimulate T cells, our results suggest that suppression is not a dominant feature of the natural human response. Whether cleavage fragments might reveal subtle differences in the balance between help and suppression remains to be determined. Both T cell clones and EBV-transformed B lymphocytes expressing specificity for f-Gal have been isolated here and used to investigate some in vitro effects of LeuOMe. LeuOMe is a lysosomotropic agent which readily enters the lysosomes of monocytes, tissue macrophages and cytolytic cells (Goldman &

Table 2. Specific lysis (0/°) of autologous EBV-transformed B cell line by f-Gal specific T cell line and clones

Effector

E:T

20:1 Clone Clone1I

10:1 5:1

Pulsed with f-Gal +medium

Not pulsed +medium

Pulsed with f-Gal +PHA

Not pulsed +PHA

354 29 8

-57 -4 3

319 20 8 110

201 16 1 5-2 56

18-2

23-9

-3-2

25:1

161

-64

Clone 2

10:1

1.9

-4 5

29

Clone3

10:1 10:1

11 55

-10 -39

01

Leu+ line

73

-3 5 -34 34

Background release was 13% and 22% for the fl-Gal-pulsed and unpulsed targets, respectively. Data from one representative assay.

Human T cell responses to /3-galactosidase Kaplan, 1973). Inside the lysosomes, LeuOMe is metabolized to form free leucine which diffuses from the organelles at a much slower rate than LeuOMe enters. This results in the swelling and rupture of the lysosomes and cell death. LeuOMe has been shown to destroy all monocytes and macrophages (Thiele & Lipsky, 1985a, 1985b), and its condensation product leucylleucine methyl ester, which is released from monocytes is toxic for NK cells and cytotoxic T lymphocytes (Thiele & Lipsky, 1986a, 1986b). In this study, LeuOMe did not alter the character of the T cell lines generated or their ease of isolation from peripheral blood. However, LeuOMe pretreatment might be applied to the isolation of CD4+ T cells which are at a low frequency or overriden by suppressive effects. It was of interest that one of the clones was clearly cytotoxic, a characteristic revealed both by non-specific activation through PHA (Lanier & Phillips, 1986) and after specific pulsing of target cells with antigen (Table 2). The phenomenon of antigen-specific CD4+ cytotoxic T cells is not new, and it has been postulated that as many as 90% of CD4+ peripheral blood T cells may be lytic if activated through CD3 (Hayward, Boylston & Beverley, 1988). This demonstration adds further evidence, albeit circumstantial, that some CD4+ T cells may have an immunoregulatory influence through the killing of antigen-presenting, and probably antigen-specific B lymphocytes (Shinohara et al., 1989). The CD4+ cytotoxic f-Gal-specific clone was isolated from a cell population treated with LeuOMe and this deserves comment. It has been reported that human T cell CD4+ clones may acquire cytotoxicity during prolonged culture (Fleischer, 1984), but antigen specificity is not usually a feature of this cytotoxicity. The cytotoxicity displayed by our CD4+ clone may have developed early on in culture or been spared following LeuOMe treatment. We are unable to distinguish between these two possibilities. LeuOMe was also used in the selection of a B lymphoblastoid cell line. We confirm here the finding that LeuOMe results in increased secretion of antibody from EBV-transformed B cells, reported by Ohlin et al. (1989). This selection method generated a lymphoblastoid cell line which presented antigen more efficiently than a line derived from untreated cells. Although the exact mechanism of the selection process is not fully understood, the method increases the probability of obtaining lymphoblastoid cells with defined specificity. It should be noted that the efficiency of the EBV Leu+ line compared to the EBV Leu- line in presenting antigen was 10fold (Fig. 4) and not 1000-fold as in the case of cloned antigen specific lymphoblastoid cells presenting to T cell clones (Lanzavecchia, 1987). This presumably reflects the lower frequency of fl-Gal-specific B lymphocytes in the polyclonal EBV Leu+ line. Nevertheless, isolation of an antigen-specific EBV-transformed B cell line should be facilitated by the inclusion of a LeuOMe selection step. Our results show that f-Gal is a suitable antigen for studies of human T cell function. Additionally, they demonstrate that LeuOMe treatment may have certain advantages in the establishment of antigen-specific lymphoblastoid cell lines in the study of antigen presentation to T cell clones.

ACKNOWLEDGMENTS We are grateful to Mr P. Walker for technical advice, to the Department of Rheumatology, Guy's Campus, for the use of their FACScan, and to the Department of Radiotherapy, for irradiation of cells. We are

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indebted to Mrs T. Beck for the typing of the manuscript, and to the Wellcome Trust for financial support. REFERENCES FLEISCHER, B. (1984) Acquisition of specific cytotoxic activity by human T4+ T lymphocytes in culture. Nature, 308, 365. FORTUNE, F. & LEHNER, T. (1988) Phenotypic expression of Vicia villosa binding T cell subsets, as markers of contrasuppressor cells in systemic lupus erythematosus. Clin. exp. Immunol. 74, 100. FOWLER, A.V. & ZABIN, I. (1977) The amino acid sequence of fiGalactosidase of Escherichia coli. Proc. natl Acad. Sci. USA, 74, 1507. GOLDMAN, R. & KAPLAN, A. (1973) Rupture of rat liver lysosomes mediated by L-amino acid esters. Biochem. biophys. Acta, 318, 205. HAYWARD, A., BOYLSTON, A. & BEVERLEY, P. (1988) Lysis of CD3 hybridoma targets by cloned human CD4 lymphocytes. Immunology, 64, 87. JOHNSTON, D.A., KNIGHT, A.M., NAYLOR, B.A., WEDDERBURN, N. & MITCHELL, G.H. (1990) Monoclonal antibodies from Epstein-Barr virus-transformed lymphocytes of common marmosets (Callithrix jacchus) immune to malaria. J. immunol. Methods, 127, 187. KRYZCH, U., FOWLER, A.V. & SERCARZ, E.E. (1985) Repertoires of Tcells directed against a large protein antigen fi-galactosidase. II. Only certain T-helper or T-suppressor cells are relevant in particular regulatory interactions. J. exp. Med. 162, 31 1. LANIER, L.L. & PHILLIPS, J.H. (1986) Evidence for three types of human cytotoxic lymphocyte. Immunol. Today, 7, 132. LANZAVECCHIA, A. (1987) Antigen uptake and accumulation in antigenspecific B cells. Immunol. Rev. 99, 39. MANCA, F., KUNKL, A., FENOGLIO, D., FOWLER, A., SERCARZ, E. & CELADA, F. (1985) Constraints in T-B cooperation related to epitope topology on E. coli /3-galactosidase. I. The fine specificity of T cells dictates the fine specificity of antibodies directed to conformationdependent determinants. Eur. J. Immunol. 15, 345. OHLIN, M., DANIELSSON, L., CARLSSON, R. & BORREBAECK, C.A.K. (1989) The effect of leucyl-leucine methyl ester on proliferation and antibody secretion of EBV-transformed human B-lymphocytes. Immunology, 66, 485. RAMMENSEE, H., SCHILD, H. & THEOPOLD, U. (1989) Protein-specific cytotoxic T lymphocytes. Recognition of transfectants expressing intracellular, membrane-associated or secreted forms of fl-galactosidase. Immunogenetics, 30, 96. RAYFIELD, L.S., BRENT, L. & RODECK, C.H. (1980) Development of cellmediated lympholysis in human fetal blood lymphocytes. Clin. exp. Immunol. 42, 561. SHINOHARA, N., WATANABE, M., SACHS, D.H. & HozuMI, N. (1988) Killing of antigen-reactive B cells by class Il-restricted, soluble antigen-specific CD8 + cytolytic T lymphocytes. Nature, 336, 481. SHIVAKUMAR, S., SERCARZ, E.E. & KRZYCH, U. (1989) The molecular context of determinants within the priming antigen establishes a hierarchy of T cell induction: T cell specificities induced by peptides of fl-galactosidase vs. the whole antigen. Eur. J. Immunol. 19, 681. THIELE, D.L. & LIPSKY, P.E. (1985a) Modulation of human natural killer cell function by L-leucine methyl ester: monocyte dependent depletion from human peripheral blood mononuclear cells. J. Immunol. 134, 786. THIELE, D.L. & LIPSKY, P.E. (1985b) Regulation of cellular function by products of lysosomal enzyme activity: elimination of human natural killer cells by a dipeptide methyl ester generated from L-leucine methyl ester by monocytes or polymorphonuclear leukocytes. Proc. natl Acad. Sci. USA, 82, 2468. THIELE, D.L. & LIPSKY, P.E. (1986a) The immunosuppressive activity of L-leuCyl-L-leucine methyl ester: Selective ablation of cytotoxic lymphocytes and monocytes. J. Immunol. 136, 1038. THIELE, D.L. & LIPSKY, P.E. (1986b) Leu-leu-OMe sensitivity of human activated killer cells: delineation of a distinct class of cytotoxic T lymphocytes capable of lysing tumor targets. J. Immunol. 137, 1399.

Human T cell responses to beta-galactosidase.

The peripheral blood of most normal individuals has been shown to contain T cells that respond to beta-galactosidase (beta-Gal), presumably as a resul...
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