Clin. exp. Immunol. (1990) 80, 206-212

T cell reactivity to the purified mycobacterial antigens p65 and p70 in leprosy patients and their household contacts E. ADAMS, R. J. GARSIA, L. HELLQVIST, P. HOLT* & A. BASTEN Centenary Institute for Cancer Medicine Cell Biology, Sydney, New South Wales, and * Thursday Island Hospital, Thursday Island, Queensland, Australia

and

(Acceptedfor publication 23 November 1989)

SUMMARY T cell reactivity to the 70 and 65 kD (p70 and p65) protein antigens derived from Mycobacterium bovis BCG strain was studied by measuring the proliferative responses of peripheral blood mononuclear cells from members of an isolated Aboriginal community resident in the Torres Straits islands. In the nine index leprosy cases the pattern of responsiveness to the purified antigens paralleled that to whole sonicates from M. leprae and BCG. In the 40 contacts of the index cases, a high correlation was observed between the responses to p70 and p65 as well as to the crude sonicates. Significant T cell responses to the purified antigens, as well as the crude sonicates, were obtained with cells from the majority of contacts. Limiting dilution analysis of precursor frequencies in the contacts confirmed the immunogenicity of the purified antigens and excluded both a mitogenic component and the presence of suppressor cells in those moderate or low responders whose blood contained sufficient precursors to be tested. p70 appeared to be more potent in stimulating a proliferative response than p65 at equivalent protein concentrations. No correlation between responder status to either antigen and disease type was detected in families. These findings provide confirmation of the importance of p70 and p65 as major T cell immunogens in man and indicate that they are both potential candidates for inclusion in a bivalent vaccine for leprosy and tuberculosis.

Keywords leprosy mycobacterial antigens T cell responses

INTRODUCTION The ability to detect a self-limiting immune response to Mycobacterium leprae in exposed individuals remains difficult despite recent advances in serological assays based on PGL-1 and its synthetic analogues (Cho, Chatterjee & Brennan, 1986). Thus most serological tests fail to detect patients with tuberculoid disease who have a low antigen load and make little antibody to the mycobacteria (Bloom, 1986). In endemic areas positive responses are obtained in some individuals without overt clinical disease; however, it is unclear whether they will progress to lepromatous leprosy or become immune at a later stage in life, and requires long term follow up (Baumgart et al., 1987). Assays which measure T cell responses to antigen are thought to indicate not only exposure but development of immunity and may therefore be a more reliable guide than serological assays to self-limiting infections. Furthermore, as the deficit in lepromatous disease appears to be a lack of T cell reactivity specifically to M. leprae, cellular testing of exposed individuals may detect undiagnosed disease by virtue of an absent response to the mycobacteria. Given the availability of Correspondence: Dr Elizabeth Adams, Centenary Institute for Cancer Medicine and Cell Biology, Sydney, NSW 2006, Australia.

purified antigens, investigations of populations from endemic areas should now include a comparison of T cell responses to them as well as to crude sonicates of whole organisms. Since 1983, 12 cases of leprosy have been diagnosed in seven inter-related families from an isolated Aboriginal community in the Torres Straits, north of Australia. These patients and their household and school contacts have been tested periodically for serum antibodies to PGL-1 and more recently the synthetic analogue dBSA. It was logical therefore to extend this study to encompass measurements of T cell reactivity to purified antigens and sonicates of mycobacteria in both patients and their contacts.

MATERIAL AND METHODS

Antigens M. bovis BCG (Commonwealth Serum Laboratories, Melbourne, Australia) and M. leprae (World Health Organisation IMMLEP Program, Dr R. J. Rees, London, UK) soluble antigen preparations were obtained by sonication (Britton et al., 1985). The antigens p65 and p70 were derived from BCG sonicate by affinity chromatography using the monoclonal antibodies L22 and L7 respectively. Their purity was confirmed on immunoblots as described previously (Britton et al., 1986,

206

Mycobacterial responses in leprosy patients and their contacts

207

Table 1. Proliferative responses in leprosy patients

Proliferative response (mean d/min x 10-3+s.e.m.) Patient no. 633 642 643 691 646 641 702 734 735

Type of

leprosy

M. Leprae

BCG

p65

p70

LL LL LL LL BL NT rT TT TT

0 1-6+1-0 0-8+0-9 08+06 47 7+6-9 83+20 04+04 198+1 3 207+23

0 1-2+0-3 2-6+1 8 34+03 43 0+9-4 52+13 05+04 96+49 475+11-5

0 0 0 0 25 3+1 9 19+08 0 1+0 1 0 16-2+1-6

0 0 0 0 17 4+6-2 39+16 0 145+25 542+55

LL, lepromatous leprosy; BL, borderline lepromatous; NT, neuritic tuberculoid; TT, tuberculoid.

1987). Streptokinase-streptodornase (SKSD) was purchased from Lederle (Cyanamid Australia, Sydney, Australia); it was dialysed against phosphate-buffered saline (PBS), pH 7 4, to remove preservatives and served as an unrelated control antigen. Peripheral blood samples Blood samples were obtained from nine leprosy patients and 41 household and school contacts who were Aboriginal residents of the Torres Straits islands (Queensland). Additional samples were collected from four members of a family with no known exposure to leprosy, a single case of tuberculosis and two contacts as well as from two members of the field survey team; the latter two samples served as controls for the collection and transport conditions. Blood was collected into preservative-free heparin, 10 U/ml (Fisons, Sydney, Australia) diluted with an equal volume of McCoy's medium (Flow Laboratories, Sydney, Australia) and maintained at room temperature, including during shipping by air to Sydney. The time delay between collection and sample processing was approximately 30 h. On receipt in Sydney the mononuclear cells (PBM) were separated on a Ficoll-Hypaque gradient (Boyum, 1968), stored in RPMI containing 20% fetal calf serum (FCS), 10% DMSO and frozen in liquid nitrogen until used in proliferative assays. McCoy's medium was selected as the transport medium since it has been shown to be the most suitable for preservation of cell surface markers and lymphocyte function when samples are maintained at ambient temperatures (Opelz & Terasaki, 1974). Preliminary studies on cells from BCG-vaccinated volunteers which were left unseparated for 48 h indicated that the cells maintained the capacity to proliferate in response to antigenic stimulation (data not shown). Proliferative assay Frozen PBM were thawed, washed twice in RPMI 1640 (Flow Laboratories), containing 5% heat-inactivated FCS, 0 85 g/l NaHCO3, 4 67 g/l HEPES buffer, 10 000 U/l penicillin, 10 mg/l streptomycin, 50 mg/l gentamycin and 2 mM L-glutamine. After checking viability by Trypan blue exclusion, the cell suspensions were adjusted to 106 viable cells/ml in RPMI containing 20% heat-inactivated human A serum. In most of the samples viability was approximately 90% with a recovery rate of 80%.

Two-hundred microlitre aliquots were dispensed into the wells of flat-bottomed 96-well microtitre plates (Flow Laboratories) and antigens were added to give final concentrations of between 0-1 and 10 Mg/ml. The cultures were incubated for 6 days at 370C in 5% CO2 in air and were pulsed with 3H-thymidine, 0 5 yuCi/ well (Amersham International, Amersham, UK) for a further 16 h, when the amount of incorporated isotope was measured by liquid scintigraphy on a rack beta counter (LKB, Pharmacia, Sydney, Australia). Limiting dilution assays Experiments were performed on selected samples to estimate the frequency of antigen reactive T cells. The cells were thawed, washed and separated into E+ (T cell) and E- (B cell) enriched populations by rosetting with AET-treated sheep red blood cells (SRBC) (Saxon, Feldhaus & Robbins, 1976). E- cells were irradiated with 2500 rads from a 6OCobalt source and served as antigen presenting cells. Cultures were established in RPMI medium containing 10% heat-inactivated human A serum, and the E+ cells were titrated to give final concentrations ranging from 2 x 104 to 5 x 102/well. Antigens (M. leprae or BCG sonicates, p65 and p7O) were added at a final concentration of 10 yg/ml and the cells dispensed in 20-pl volumes into Microtest plates (Nunc, Roskilde, Denmark), which were inverted, placed in humid chambers and incubated for 9 days. On day 6, 5 yl of medium were added to compensate for evaporation. Sixteen hours before the cultures were terminated, 1 Ml of 3H-TdR, 0 1 pCi/well, was added, and the plates were harvested onto glass fibre paper using a Titertek microharvester (Flow Laboratories). Twenty replicate cultures for each T cell concentration were established. Positive cultures were scored in antigen-stimulated wells when the 3H-TdR incorporation exceeded the value of the mean + 3 s.d. for the corresponding unstimulated cultures. Serum antibodies to disaccharide BSA (dBSA) Antibody levels to dBSA were measured using a modification of the WHO protocol (Douglas et al., 1986). Briefly, microtitre plates (Nunc Immuno 1) were coated with 50 p1 of dBSA (0 16 ug/ml in carbonate-bicarbonate buffer, pH 9 6) for 16 h at 370C. After blocking with PBS containing 1% BSA and 1% Tween 20 (PBST) the wells were incubated with serum samples (50 pl) diluted 1:300 in PBST containing 10% goat serum at 37°C for 1

E. Adams et al.

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Table 2. Limiting dilution analysis of the frequency of M. leprae and BCG-reactive T cells in the blood of leprosy contacts M. leprae

BCG

M. leprae Contact no.

d/min

1/f

r

d/min

1/f

r

Low

701 718 644 710 739 717 706

1495 2286 4082 7269 7877 9325 9662

1/15 376 1/18 652 1/24011 1/10274 1/5643 1/13632 1/10082

703 708 692 709 732

15 867 16216 17746 20913 29756

1/7361 1/6210 1/5966 1/7511

-0 97 -0 96

679 1223 3530 8103 5836 5596 13474 4099 10028 24905 14915 77414

1/9990 1/9234 1/7076 1/5475 1/5653 1/24219 1/9016 1/8263 1/10029 1/11930 1/4082

-0 98 -0 93 -0 83 -0-98 -0 94 -0 93 -0 96

Moderate

-0-99 -0-98 -0 95 -0 98 -0 95 -0-99 -0-97 -0 97 -0 94

-0 97 -0 95 -099 -0 64

731 694 693

32808 35347 62 144

1/6064

-0-98

1/6414

-0 98

45680 56810 53 510

1/3695 1/4669 1/4989

-0 95 -0 74 -0 94

Responder status

High

Table 3. Limiting dilution analysis of the frequency of p65 and p70 reactive T cells in the blood of leprosy contacts P70

P65

Responder

Contact no.

d/min

1/f

r

d/min

1/f

r

Low

718 739 706

3302 4631 3268

-0 99 -0 96 -0 91

1015 8913 4255

1/77 802 1/10940

-0 86 -0 99

Moderate

703 708 709 711 732

0 8210 16736 7691 19322

1/34052 1/9578 1/24682 1/13 340

-0 98

1/46485

-091

1/7253 1/13 310

-0 84 -0 94

1353 11171 18334 32019 80954

-0 98 -0-94

731 693

10072 44283

1/10682 1/31 720 1/20 532 1/20 128

status

High

NT

1/8733

-0 99

35 563 40529

-0 95 -0 98

NT, not tested.

h. They were then washed in PBST and 50 p1 of peroxidaseconjugated anti-human IgM (Kirkgaarde and Perry Laboratories, Gaithersburg, MD) diluted 1:4000 in PBST/10% goat serum were added to each well for 1 h at 370C. After five further washes in PBST, 50 pl of O-phenyldiamine (Sigma, St Louis, MO), 1 mg/ml in citrate phosphate buffer was added and incubation continued for 20 min in the dark. The reaction was stopped by addition of 50 yl 2 5 N H2SO4 and the absorbance was read at 492 nm on a Titertek Multiskan (Flow Laboratories). Positive control samples from a pool of untreated lepromatous patients (LSP4) were included on each plate as was a negative sample from a healthy laboratory worker. Results were expressed as a percentage of the value for the positive control.

Statistical analysis Statistical analysis was performed on limiting dilution assays according to the method of Lefkovits & Waldmann (1979). By plotting the natural logarithm of the fraction of negative wells against the number of cells plated per well it was possible to calculate the frequency of antigen-reactive cells. Linear regression analysis was performed and the probability that the data conformed to Poisson distribution was determined for each plot.

RESULTS of Proliferative responses index leprosy cases Of the nine index cases tested for T cell reactivity to mycobacter-

Mycobacterial responses in leprosy patients and their contacts M Ieprae

15 _

10_ 5_

'0'0 15 _

BCG 10 _

0

0

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I, *^ |

I

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15 _

p65

_-

z 10

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we

me

0

ft

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g,Io 20

30 A

I, d/min

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40 x

60

70

80

10-3

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keprae,

ial antigens four had lepromatous disease, one borderline lepromatous disease, one neuritic tuberculoid disease and three tuberculoid disease (Table 1). One of the lepromatous cases (633) did not respond to the control antigen SKSD as well as to the mycobacterial products and should be considered a technical failure even though the cells were viable when thawed. In the remaining three lepromatous patients there was a negligible response to M. leprae and only weak responses to BCG sonicate while all of them failed to proliferate to the purified antigens. By contrast, cells from the borderline lepromatous case responded well to all antigens. Two of the three tuberculoid patients also demonstrated intact T cell reactivity but a selective failure to respond to p65 was observed in the third case (734). The single neuritic case had low responses to all antigens. The poor responses to BCG as well as M. leprae by cells from lepromatous patients are in accordance with data from a larger group of leprosy patients resident in Sydney who have been studied over the past 2 years (data not shown).

Proliferative responses of contacts The proliferative response to mycobacterial antigens was measured in 41 close contacts of the leprosy cases. BCG vaccination was documented in 25 of them (61%), and three others had mantoux scars. One subject failed to respond to any of the antigens including SKSD and was excluded from further analysis. Despite some inter-subject variation, analysis of the responses to BCG and M. leprae revealed that they could be

209

ranked according to their magnitude into: low, < 10 000 A d/ min; moderate/intermediate, 10-30 000 A d/min; and high responders, > 30 000 A d/min (Fig. 1). Low responsiveness was not associated with a lack of vaccination, as similar proportions of non-vaccinees were found in each response category. As expected, proliferation to the purified antigens was lower than to the crude sonicates. Indeed a significant proportion failed to respond at all (35% to p65 and 25% to p70). In general, when the response to sonicated antigen was below a A d/min of 5000, there was no response to the purified antigens. This was most noticeable for p65, and is interesting in view of its wider distribution than p70 in mycobacterial and non-mycobacterial bacterial species (Young et al., 1987) and suggests that at equivalent protein concentrations p70 may be more immunogenic for T cells. The responses of cells from family members with no known household contact with leprosy were varied but all four were positive for M. leprae sonicate, once again confirming that at least part of the response to M. leprae can be explained in terms of cross-reactive epitopes on other mycobacteria including BCG, since they had been BCG vaccinated. Correlation between proliferative responses to purified and sonicated antigens in contacts An estimate of the relative potency of the purified antigens as T cell immunogens can be obtained by carrying out a correlation analysis of the proliferative responses to them versus the whole sonicates which contain a broad spectrum of antigens (Ivanyi et al., 1988). In order to minimize variations related to disease category, the analysis was performed on the data from the contacts and the results from patients were excluded. The findings shown in Fig. 2 illustrate two important points: first, the degree of correlation between responses to purified antigens and sonicates was remarkably high, consistent with the suggestion that p70 and p65 are both important T cell immunogens; and second, despite the fact that the antigens were derived from BCG the correlation with M. leprae reactivity was comparable to that of BCG reactivity for each antigen. When the response to p70 was compared with the control antigen SKSD, a much lower correlation coefficient (r=0 50) was obtained (data not shown).

Analysis of T cell reactivity within families The finding that a significant proportion of contacts as well as patients with lepromatous leprosy failed to respond either to whole sonicates or purified T cell immunogens suggested that there might be a genetic association with low responder status, particularly as the families from the Torres Straits are interrelated. Although all members of some of them could not be tested, sufficient members were available from five families to permit analysis of responder status. There was no apparent correlation between the type of disease within a family and the magnitude of the response in contacts. Furthermore, lepromatous cases were present in both high and low responder families as were tuberculoid cases. Two particular families illustrate these conclusions: the first included two members with lepromatous disease and a borderline lepromatous case, while the healthy contacts were high responders. The second consisted of two multicase interrelated families which contained both lepromatous (633 and 689) and tuberculoid (734 and 735) cases, the remaining members tested being mainly low responders. In

E. Adams et al.

210 100

to0 K C

100

r = 0-68

80 _

80

601"

60

r=0-89 0

.E S0 0.

20

1

1

20

60 x M.leproe (d/min 10-3)

50

40

i 80

00

50

r08~~~~~~

r=0-8

I

80

r=0-74 0

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30

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uE 20

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x

I

20 40 60 BCG (d/min x 10-3)

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40

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0

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*

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1o

0

40

0

40

r-

CL

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-

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o1

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I

0

S

0

20

40

M. /eproe (d/min

60 x

10-5)

80

0

20 40 60 BCG (d/min x 10-3)

80

Fig. 2. Linear regression analysis of the proliferative responses in contacts to the purified antigens p65 and p70 with the sonicate antigens M. leprae and BCG. 80 6

0

60 _

x

.E_ "I

q0 40 _ -

S*

0

0

20

0

0

0

- 0

0

0

AO-

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t

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40 60 80 100 LSP4 anti-dBSA (IgM) (%) Fig. 3. Linear regression analysis of the proliferative response in contacts to M. leprae with the serum antibody titre to dBSA.

general, the correlation between responses to sonicates and purified antigens was maintained within families and no genetic traits in unresponsiveness to the individual antigens emerged. T cell precursor frequencies in contacts The frequency of precursor cells specific for sonicated or purified antigens was estimated by limiting dilution analysis. Preliminary experiments using frozen lymphocytes from normal BCG vaccines showed that the addition of interleukin-2 (IL-2) in the hanging drop culture system was unnecessary as the precursor frequencies obtained without IL-2 were in the same range as those reported by Brett, Kingston & Colston (1987). Of 31 samples, set up results were obtained from only 15, presumably because of the fragile nature of the cells which did not remain viable for the prolonged culture period on 9 days. When the precursor frequencies to BCG and M. keprae sonicates were analysed the magnitude of the mean proliferative re-

sponses showed a reasonable correlation with the frequency of antigen-reactive cells although there were individual exceptions to the rule (e.g. 739). The results including analysis of precursor frequencies are shown in Table 2 except for those from experiments where r values were unacceptably low. For low responders (A d/min < 10000), the mean frequency for M. leprae reactive cells was one in 13 952 compared with one in 10 094 for BCG. The higher frequency of antigen-reactive cells to BCG would be expected in a population where the majority had been vaccinated. For moderate responders (10-30 000 d/min; n = 4) the mean values were reversed, M. leprae-reactive cells being more frequent (one in 6829) than those to BCG (one in 8576). Only three high responders were tested, but as expected yielded the highest frequencies of antigen reactive cells (means values for BCG one in 4451; M. leprae one in 6230). When the purified antigens were substituted for the whole sonicates in the limiting dilution assay (Table 3), the precursor frequencies were significantly lower, as expected. Indeed, in about half the experiments the frequency could not be quantified, due to too few positive cultures even with a high T cell input. Trivial explanations can be effectively excluded; for example, unresponsiveness cannot be attributed to non-viable cells since positive results were obtained with cells from some individuals in the presence of sonicates. Similarly, the failure to respond in certain cases cannot be explained by differences in antigenic potencies, since the same batches of p65 and p7O were used in both series of experiments. Thus, the lower frequency of antigen reactive cells to p65 and p70 indicated that the T cell reactivity of these antigens reflects their immunogenicity rather than being due to any non-specific mitogenic effect.

Comparison of T cell responses and serum antibody to dBSA Antibody titres to dBSA were measured in the contacts of the index cases as part of an ongoing seroepidemiological study.

Mycobacterial responses in leprosy patients and their contacts When the results of proliferative responses to either sonicates or purified antigens were plotted against antibody titre, no correlation was obtained. Figure 3 shows the plot of the response to M. leprae sonicates versus dBSA which yielded correlation coefficient of r = 0 20. Predictably, some individuals with low antibody titres ( < 30% LSP4) had high proliferative responses while at the other end of the spectrum there were others with high antibody titres and low T cell responses. However, there were isolated individuals with high antibody titres and high T cell reactivity which may reflect self-limiting responses in the presence of high antigen loads. DISCUSSION During the past 5 years, several antigens with defined T cell and/ or B cell epitopes have been isolated from pathogenic mycobacteria, including M. leprae, M. tuberculosis and M. bovis, by immunochemical and molecular techniques (Ivanyi et al., 1988). Among these are two proteins of apparent Mr 65 and 70 kD (p65 and p70), both of which have been shown to stimulate a T cell response in human subjects exposed to M. leprae (Britton et al., 1986, 1987). p65 expresses M. leprae-specific and cross-reactive B cell epitopes, whereas to date only cross-reactive B cell epitopes have been demonstrated on p70 (Britton et al., 1985). The aim ofthe current study was to determine whether these two antigens are major T cell immunogens in humans, and to compare their relative potency. PBM were obtained from patients and contacts belonging to an isolated community on the Torres Straits islands where leprosy is endemic. The community included several large inter-related families and have been subject of a sero-epidemiological study since 1985. As expected, cells from the index cases displayed predicted proliferative responses to M. leprae sonicates across the clinical spectrum with no response being detected in lepromatous patients and a reasonable response in those with tuberculoid disease. Interestingly, despite the fact that most subjects had been vaccinated previously with BCG, proliferation to the BCG sonicate was also reduced at the lepromatous end of the spectrum in contrast to some previous studies (Myrvang et al., 1973) and presumably reflects a lack of reactivity to shared T cell epitopes on M. leprae and BCG (Table 1). A similar pattern of responsiveness was observed with the two purified antigens (p70 and p65) which provided the first indication that they were significant T cell immunogens. The 40 contacts of the index cases could be sub-divided into three groups depending on their responder status to M. leprae sonicates (Fig. 1). The strong correlation in all three groups (r = 0 82) with the response to BCG (data not shown) once again pointed to the major contribution made by cross-reactive epitopes to the overall level of T cell reactivity. The finding of similar precursor cell frequencies in known contacts to the two sonicates and the demonstration of a positive response to M. leprae in non-exposed BCG-vaccinated subjects (data not shown) are also consistent with this conclusion. On comparing the proliferative responses of cells from the contacts to the purified antigens, p7O and p65 to each of the crude sonicates a high correlation was observed in both cases (Fig. 2). When taken in conjuction with the fact that the antigens elicited a significant response in the majority of contacts tested (Fig. 1) these findings confirm their importance as T cell immunogens. By measuring responses to purified antigens in individuals exposed to M.

211

leprae those which are involved in protection may be identified. In relative terms, p70 appeared to be more immunogenic than p65 at equivalent protein concentrations-which is surprising, given the more ubiquitous distribution of p65 in organisms other than pathogenic mycobacteria (Young et al., 1987). The apparent immunogenicity of the two antigens could in theory have been due to a mitogenic component. In practice, however, this possibility was effectively excluded by the lack of response to either antigen in some patients and contacts and the overall correlation between precursor frequencies and responder status (Tables 2,3). Conversely there was no evidence from the shape of the precursor frequency plots to implicate a suppressor cell in any moderate or low responders. Precursor frequencies were measured by the hanging drop technique. Owing to its sensitivity and requirement for very small numbers of cells this method is clearly superior to conventional microculture systems particularly when cell supplies are limiting as they frequently are in field surveys of this kind. Our data suggested that p70 and possibly p65 could be candidates for inclusion in a vaccine against both leprosy and tuberculosis. The apparent lack of correlation between responder status and disease type or HLA haplotype within families provides further support for this conclusion, since the responses to them are unlikely to be restricted to particular HLA-DR alleles. Whether the same will apply to predicted T cell reactive peptides is of course questionable. It remains unclear at this stage what the significance of a positive T cell response to the two antigens is, i.e. whether such a response signifies exposure and immunity or exposure and possible future susceptibility to infections. Although there was no overall correlation with the presence of antibodies to dBSA from M. leprae, a few subjects were identified with high antibody titres and either high or low T cell responses to p65 or, p70, or both. Clearly, such individuals should be followed up with a view of determining which ofthem may subsequently develop of clinical infection.

ACKNOWLEDGMENTS We would like to thank the families of Thursday Island for their participation in this study, and Sister Bedford for collating the family data. The assistance of Manita Datt in the preparation of the manuscript is greatly appreciated. Financial support was received from the Special Research Centre Scheme of the Australian Research Council and the National Health and Medical Research Council of Australia.

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Cambridge. MYRVANG, B., GODAL, T., RIDLEY, D.S., FROLAND, S.S. & SONG, Y.K. (1973) Immune responsiveness to mycobacterium leprae and other mycobacterial antigens throughout the clinical and histopathological spectrum of leprosy. Clin. exp. Immunol. 14, 541. OPELZ, G. & TERASAKI, P.I. (1974) Lymphocyte antigenicity loss with retention of responsiveness. Science, 184, 464. SAXON, A., FELDHAUS, J.L. & ROBBINS, R.A. (1976) Single step separation of human T cells and B cells using AET treated SRBC rosettes. J. immunol. Methods, 12, 285. YOUNG, D.A., IVANYI, J., Cox, J.H. & LAMB, J.R. (1987) The 65 kDa antigen of mycobacteria-a common bacterial protein? Immunol. Today, 8, 215.