T cell reactivity to penicillin: Phenotypic analysis of in vitro activated cell subsets

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34. Mygind N, Viner AS, Jackman N: Histology of nasal mucosa in normals and in patients with perennial rhinitis. Rhinology 12:131, 1074 35. Wasserman SI: The mast cell and synovial inflammation. Arthritis Rhcum 27:841, 1984 36. Ihle JN, Keller J, Oroszlan S, Henderson LE, Copeland TD, Fitch F, Prystowsky MB, Goldwasser E, Schroder JW, Palaszynski E, Dy M, Label B: Biologic properties of homogenous interlukin-3. J Immunol 131:282, 1983 37. Sredni B, Friedman MM, Bland CE, Metcalfe DD: Ultrastructural biochemical and functional characteristics of histamine containing cells cloned from mouse bone marrow: tentative identification as mucosal mast cells. J Immunol 131:915, 1983

In vivo evidence

for mast cell degranulation

38. Nabel G, Galli SJ, Dvorak HF, Cantor H: Inducer T lymphocytes synthesise a factor that stimulates proliferation of cloned mast cells. Nature 291:332, 1981 39. Haig DM, McKee TA, Jarrett EEE, Woodbury R, Miller HRP: Generation of mucosal mast cells is stimulated in vitro by factor:; derived from T cells of helminth-injected cats. Nature 300:188, 1982 40. Morrisey JF, Carswell F: The fate of inhaled antigen within the respiratory tract of sensitised and unsensitised rats. Monogr Allergy 18:226, 1983 41. Newson B, Dahlstrom A, Enerback L, Ahlman H: Suggestive evidence for a direct innervation of mucosal mast cells. Neuroscience 10:5565, 1983

T cell reactivity to penicillin: analysis of in vitro activated

Phenotypic cell subsets

Marianne Koponen, Ph.D., Werner J. Pichler, M.D., and Alain L. de Week, M.D. Bern, Switzerland Patients with penicillin allergy demonstrate a T cell prolyerative response after in vitro stimulation with penicillin G (Pen G) and other /I-lactam antibiotics. To understand better penicillin-allergic reactions, T cell subset stimulation with Pen G was studied and compared with other soluble (tetanus toxoid and puri$ed protein derivitive [PPD]) and membrane-bound viral (injuenza A and Epstein-Barr viruses) antigens. A double jluorescence method for flow cytometry was used to evaluate the activated cells simultaneously by pyronin Y staining of RNA and by indirect immunofluorescence of cell surface T4, T8, or Leu 8 antigens. The antigens used stimulated mainly the T4’ subset(>90%), whereas the nu.nber of activated T8 cells was slightly increased only in Pen G- and influenza A-triggered cultures (5% to 15%). Leu 8 antigen was used to analyze more precisely the activated T4’ cells. Pen G and injuenza A and EpsteinBarr viruses stimulated both T4’, Leu 8’ (>.50% of activated cells, inducers for suppressor cells), and T4’, Leu 8- (helpers for B cells) subsets, whereas PPD activated mainly T4’, Leu 8 subpopulations. These results indicate that penicillin-allergic patients with skin symptoms demonstrate a T cell subset stimulation that resembles more the reaction versus viral antigens (membrane incorporated) than to soluble antigens like PPD. These results suggest that Pen G is presented to T cells like viral proteins and might thus cause allergic reactions resembling skin symptoms observed in viral diseases. (J ALLERGYCLINIMMUNOL 78.645-52, 1986.)

Allergic reactions to p-lactam antibiotics can cause clinically different symptoms, such as anaphylaxis, pruritus, exanthema, vasculitis, interstitial nephritis, hemolytic anemia, etc.’ Although the anaphylactic re-

Abbreviations used

EB V: Epstein-Barr virus FITC: Fluorescein isothiocyanate PBL: Peripheral blood lymphocytes PPD:

From the Institute of Clinical Immunology, Inselspital, Bern, Switzerland. Supported by Swiss National Foundation Grant No. 3-543-0.83. Received for publication Oct. 11, 1985. Accepted for publication March 19, 1986. Reprint requests: W. J. Pichler, M.D., Institute of Clinical Immunology. Inselspital, 3010 Bern, Switzerland.

PY: SI: ‘IT: HLA: Pen G:

Purified

protein derivative

Pyronin Y Stimulation index Tetanustoxoid Human leukocyte antigens Penicillin G 545

J. ALLERGY

646 Koponen et al.

TABLE I. Lymphocyte nonallergic

proliferative

response

Ampicillin

Control subjects ( IO) P. G. G. S.-i. M. T.l

0.8-f 7.9 28.8 16.5

1.2110.5 39.2 30.0

B. T. P. M. J. N. U. T. H. M. U. B.S

13.3 43.6 35.5 10.5 8.5 77.3 65.0 10.1

4.3 16.8 37.1 17.5 9.3

J. H.

antibiotics

in penicillin-allergic

and

donors

Pen G

M. M.i

to different

CLIN. IMMUNOL. OCTOBER 1986

Clamoxyl

Floxapen

Erythomycin

Tetracyclin

lndomethacin

1.3t 36.0 23.3

0.9

0.6 0.9 0.7 1.2 I.1

6.0 13.3 0.7 2.2 42.7 3.3

5.1

1.5

1.1

Final concentrationof Pen G was 1000 p&ml. Ampicillin, clamoxyl, and floxapen were used at a final concentrationof SO0pgiml. Final concentrationsof erythromycin, tetracyclin, and indomethacinwere 10 kg/ml. Cells were cultured for 6 days. *St = cpm with antigenicpm without antigen. Unstimulated cultures had 590 to 1590cpm. :-Mean of SID of 10 donors: SI between 0.34 to 1.20 for Pen G; 0.42 to 2.50 for ampicillin; 0.73 to 2.40 for clamoxyl. fPatients chosenfor further experiments.

action can in most cases be related to an IgE-mediated mechanism, patients with exanthematous skin reactions to penicillin do not always have a positive whealand-flare reaction to penicilloyl skin tests or penicilloyl-specific IgE in RAST. However, often, an enhanced proliferative response can be observed in penicillin-stimulated cultures in vitro,‘. 3 suggesting that a T cell reaction to penicillin takes place and may contribute to the skin symptoms, which indeed often appear days after treatment. In this study the T cell activation to penicillin and other antigens in vitro was studied by use of a double-fluorescence method to identify the stimulated T cell subsets. The activation was determined by the increased RNA content (PY staining) and the T cell subset by FITC-labeled T4, T8, and Leu 8 antibodies .4The Leu 8 antibody dissects further the T4 subset into T4+, Leu 8- cells, which act as helper cells for B cell maturation5 and T4+ and Leu 8” cells, so-called suppressor-inducer cells6 With this phenotypic analysis, interesting differences in the in vitro stimulation between Pen G and soluble (TT and PPD) or membrane-bound (influenza virus and EBV) antigens could be observed as Pen G preferentially stimulated T4’, Leu 8’ cells. MATERIAL Donors

AND METHODS

Patients with typical histories of penicillin allergy and high SIs in the lymphocyte transformation test were selected for detailed analyses. Three patients had mainly skin symptoms (maculopapular exanthems), and one patient had an

immedial:e asthmatic reaction. None of the four individuals had IgE antibodies in penicillin-specific RAST, and all patients had negative or low antipenicillin IgG antibodies. Ten control subjects who had received penicillin without an adverse reaction previously were evaluated in lymphocyte transformation tests as well. Donors used for in vitro stimulations with tuberculin PPD were previously found to be skin test positive. Cells used for TT stimulations were from donors immunized with TT 1 to 4 weeks before the tests were performed.

Cell preparation Human peripheral blood mononuclearcells were diluted 1: 1 wiih Hanks’ balanced salt solution and isolated from heparinized blood by Ficoll-Ronpacon (Pharmacia Fine Chemicals, Uppsala, Sweden, and Nyegaard & Co. A/S, Oslo, Norway) gradients (30 minutes at 800 x g). Leukocytes at the serumiFicol1 interface were removed and washed twice in Hank’s balanced salt solution and resuspended in the culture medium (see below).

Medium The medium used was RPM1 1640 (Gibco Biocult, Glasgow, Scotland) containing 2 mmol/L of L-glutamine, 10 mmol/L of N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic lcid, gentamycin, (50 kg/ml), and 20% autologous plasm.3 or AB serum. For penicillin G stimulations, cells were cultured without gentamycin.

Monoclonal

antibodies

Monoclonal antibodies were purchased from Ortho Pharmaceutical Corp., Raritan, N. Y. (OKT4 and OKT8); Biotest, Frankfurt/Main, Federal Republic of Germany (Clonab

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T cell reactivity

--

Type of clinical

reaction

None Exanthem Exanthem Exanthem. serum sickness-like reaction, myalgia, arthralgia, fever Exanthem Exanthem Exanthem, angioedema Exanthem, angioedema Exanthem, erythema nodosum Asthmatic reaction Exanthem Exanthem -T4 and T8); and Becton-Dickinson, (Leu 8).

Soluble

Rutherford, N. Y.

antigens

Tuberculin PPD was from Statens Serum Institute. Tuberculin Department, Copenhagen, Denmark. TT (20 times flocculation units per milliliter) was obtained from Swiss Serum Institute, Bern, Switzerland. Pen G (kalium salt) was from Glaxo Laboratories, Ltd., Greenford, Middlesex, England. Ampicillin (trihydrate) was from Fluka, Buchs, Switzerland. Clamoxyl and floxypen were from Beecham Research Laboratory, Brentford, England. Erythromycin was from Lilly Research Laboratory, Windlesham, Surrey, England. Tetracycline (HCI) was from Pfizer, Brussels, Belgium. Indomethacin was from Merck Sharp & Dohme Research Laboratory, Rahway, N. J.

Viruses Transforming EBV was strain B 95-8. Influenza type A virus was HK (A/Hong Kong/8168x-JI), HK/A/-H3N2, from Dr. L. Nelson, Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Md., and was propagated in hen eggs.

Stimulation

with

soluble

antigens

PBL, 10’. in 1 ml of culture medium, were distributed in the tissue-culture tubes (Falcon Labware, No. 3033, Oxnard, Calif.) and supplemented with a selected dilution of antigen in culture medium. The final concentrations used were for TT 0.2 or 0.4 Lf units per milliliter; PPD, IO, 1, or 0.1 kg/ml; and Pen G, 1000 kg/ml. Cultures were incubated at 37” C in 95% air, 5% CO, humid atmosphere. After 4 to 7 days the cells were analyzed in the cytofuorograph.

In vitro stimulation

with

viruses

Fresh PBL at the concentration of 3 x 10” cells per milliliter were exposed to influenza A virus (1: 150 to 1: 500

to penicillin

647

dilution of allantonic fluid) in medium without plasma for 90 minutes at 37” C. The cells were pelleted, washed twice, and ther incubated with 12 x 10” uninfected autologous PBL in tissue-culture tubes at the final concentration of I.5 X I Ohcells per milliliter in complete medium. The EBV supernatant at a I :5 final dilution was added at 1.5 > 10” PBL in 0.5 ml for 3 hours at 37” C. Cells were washed and grown with complete medium. After 7 to 11 days in culture, cells were analyzed in the cytofluorograph.

Proliferation

assay

The proliferative response to different antigens was measured by ‘H-thymidine incorporation in micro- or macrocultures. In brief, 2 x 10’ cells in 0.1 ml of culture medium were placed in flat-bottom microtiter plates (Falcon Labware, No. 3070) and supplemented with 0.1 ml of antigen at a proper concentration; 50 l.~l of methyl-‘H-thymidine (;!O pCi/ml, specific activity of 5 Ci!mmol/L Amersham Radiochemical, Amersham, England) was added 5 hours before harvesting. For the macroculture. IO” or I .5 X 10’ cells per milliliter were incubated with antigen or after virus immunization in tissue-culture tubes; 100 p,l of radiolabeled thymidine was added 5 hours before harvesting. Cultures were harvested with a semiautomatic multifold cell harvester (Skatron, Lierbyen, Norway), and the incorporated radioactivity was determined in a liquid scintillation (counter (LSSOOO, Beckmann Instruments, Inc., Palo Altc. Calif.).

Acridine

orange staining

Acridine orange staining’ was used to measure the number of activated cells. The fluorescence of individual cells, stained with acridine orange, was measured in a cytofluorograph 4,302 (Bio/Physics Systems, Inc., Mahopac, N. Y.). Green (DNA) and red (RNA) fluorescence was measured, and the sgnals were recorded by a multichannel analyzer. The total cell number of each culture was counted with the cytofluorograph.

Double fluorescence Simultaneous staining of RNA and cell-surface antigens was performed according to the procedure described by Shapiro.” Briefly, PY (Sigma Chemical Co., St. Louis. MO.) was used to stain RNA. PY at the final concentration of 4 FmoliL was added in 1 ml cultures, incubated 45 minutes at 37” C, centrifuged at 1000 X g for 5 minutes, and resuspended in phosphate-buffered saline supplemented with 3% fetal calf serum and 0.1% NaN, at 4” C containing 4 p,mol/L of PY (used throughout). The antibody (5 )~l/lO~ cells) used was added to the cells and incubated 30 minutes at 4” C. Cells were washed three times with medium. To evaluate indirect immunofluorescence, fluorescein-conjugated go& antimouse immunoglobulin F(ab,) fragment (Cappel L.aboratories, Malvem, Pa.) was added and incubated 30 minutes in the dark at 4” C. Cells were washed and analy.zed with an Ortho 50H cytofluorograph. Both fluorescein and PY fluorescence were excited by a 488 nm Argon ion laser and were simultaneously distinguished ac-

648

Koponen

J. ALLERGY

et al.

activated

TL positive

0

5

cells

activated

7

6 Days

0 in

T8 positive

Em

6

CLIN. IMMUNOL. OCTOBER 1986

cells

7

culture

FIG. 1. Kinetic of T4 and T8 cell activation of PBL by penicillin G in three different penicillinallergic blood donors; 1 x lo6 cells were cultured with 1 m#g/ml of penicillin G for 5, 6, and 7 days. T cell activation was determined by PY staining that allowed the simultaneous demonstration of T4 and T8 markers by FITC-labeled antibodies. Perc:entage of activated cells in controls (o - - - - o) of each donor were for T4 between 2% and 3% and for T8 between 1% and 3%. Patient M. T. (O-O); patient U. 6. (+-t); patient G. S. (A---A.).

cording to their emission maxima of 525 nm and 580 nm, respectively. Data were collected in a 512 channel distribution device and stored in a PDP 11 computer (Adcomp AG , Dietikon, Switzerland). By evaluating the contour plots obtained with unstimulated cells, windows could be placed that allowed the calculation of cell numberssimultaneously having an increased RNA content and a specific cell surface marker. RESULTS Proliferative

response

to p-lactam

antibiotics

The PBL of patients with penicillin allergy demonstrated on day 6 an increased incorporation of 3Hthymidine in the lymphocyte transformation test when the PBL were exposed in vitro to B-lactam antibiotics, Pen G, ampicillin, clamoxyl, or floxapen, whereas there was no response to other antibiotics to which the patients were e.xposed (Table I). No increased SIs were obtained with individuals who were not allergic to Pen G. The result is expressed as an SI calculated according to the formula: sI = cpm (+ antigen) cpm ( - antigen) The test is considered positive when the SI was >3. The concentrations of drugs used in the test were found to be optimal for all patients in preliminary experiments (results not presented). T cell subset activation

with

Pen G

The kinetics of ‘T cell subset activation of penicillinallergic patients with Pen G was followed after 5, 6,

and 7 da:ys in culture (Fig. 1). The percentage of activated T4 cells increased with time in all patients. On day 5 the percentage of activated T4 cells was between 18 and 45. The percentage of activated T8 cells stayed low but was constantly 1% to 5% more than background. After 7 days the culture conditions were suboptimal, and the rate of proliferation started to decrealse (results not presented). Cells from four patients chosen for the further analysis demonstrate a high incorporation of ‘H-thymidine in Pen G-stimulated cultures (Table 11). The total number of activated cells (PY’) was between 20% and 40%, depending on the donor. In all donors about 85% to 90% of the activated cells belonged to the T4 cell subset. Only a few activated T8 cells (10% to 15%, background deducted) were observed. A clear increase in the absolute numbers of T4’ cells in Pen G-activated cultures confirms the main activation of T4 cell subset. A weak increase in the absolute number of T8’ cells correlates with the low number of activated T8 cells. T cell stubset activation and influenza A virus

with

PPD, TT, EBV

Soluble antigens PPD and TT elicited a maximal response with about 40% activated cells on day 5 (Table III). More than 95% of activated cells belonged to the T4 cell subset. There were none or very few activated T8 cells. The doubling of absolute number of T4 cells also confirms the main T4 cell subset activation with PPD and IT. The maximal responses in viral stimulations were reached after 8 to 11 days. In the EBV-stimulated

VOLUME NUMBER

78 4, PART 1

T cell reactivity

TABLE II. T cell subset activation --

of PBL of penicillin-allergic

patients

%Activated cells Patient

to penicillin

649

with Pen G

%Total

Absolute number* (W5)

cells

CPM

%PY’

T4+

T8’

T4’

T8+

T4’

3.384 -+ 349

2.0 35.0

2.0 32.0

1.0 4.0

60.0 67.0

15.0 11.0

15.6

2.0 2.5

2.0 23.0

2.0 19.0

2.0 5.0

59.0 69.0

25.0 24.0

7.7 12.3

3.0 4.4

T8+

M. M. Control Pen Gt

46.718

-r- 5.974

7.6

G. S. Control Pen Gi M. T. Control Pen GO U. B. Control

Pen G§

NIX NDS

1.0

1.0

21.0

68.0 73.0

22.0 19.0

2.5

24.0

2.0 3.0

7.7

k 5.524

11.9

3.1

2.350 k 362 77.563 +- 3.217

2.0 19.0

1.0 15.0

2.0 4.0

62.0 65.0

24.0 22.0

7.8 11.6

3.0 3.9

2.544 + 452 81.523

ND = not done. *Absolute numbers of T4 and T8 positive cells were calculated from the perter tage of immunofluorescence number of viable cells per culture, measured by acridine orange. tSeven days of cultures. SOn day 6, control 540 cpm; Pen G 12’000 cpm. ISix days of cultures.

TABLE III. T cell subset activation

of PBL with Pen G, PPD, l7, EB, and influenza %Activated cells

Antigen

CPM

-

%Total

positivecells andthe totat

A viruses

cells

Absolute number (1O-5)

%PY’

T4’

T8’

T4+

T8+

T4’

T8’

Control* Pen G*,t

2.760 t 550 68.601 2 19.054

2.0 25.0

2.0 22.0

2.0 4.0

62.0 69.0

22.0 19.0

7.7 12.9

2.6 3.5

Control*

2.059 +- 787 44.880 2 1.838

3.0 35.0

3.0 33.0

3.0 3.0

64.0 74.0

22.0 18.0

7.5 13.5

3.2 3.3

Control* TTS,$

990 + 89 101.895 +- 3.686

3.0 44.0

2.0 41.0

3.0 3.0

60.0 72.0

24.0 16.0

7.7 15.s

3.2 3.3

Control*

1.128 k 184 194.789 k 3.728

4.0 36.0

3.0 34.0

3.0 3.0

61.0 73.0

19.0

7.6

11.0

13.6

2.4 2.1

4.0 40.0

3.0 34.0

3.0 6.0

56.0 72.0

28.0 19.0

6.2 13.8

2.9 3.1

PPDS,S

EBVS.11 Controlll Influenza

A$,#

2.150 49.000

*Mean of 4 different penicillin-allergic patients. +Six to seven days of cultures; donors M. M., G. S., M. T., and U. B *Mean of two different donors. 9Five days of cultures; donors K. W. and W. P. lITen to eleven days of cultures; H. S. and B. S. ‘IOnly single values. #Eight to nine days of cultures; H. M. and J. R.

cultures, about 40% of the cells was activated on day 10 to 11. From these activated cells, >95% belonged to the T4 cell subset. The absolute number of T4’ cells was doubled compared to the control culture. The number of activated TS cells stayed near the back-

ground level, and the absolute number of T8+ cells was below the control value. In influenza A virus stimulated cultures, about 40% of the lymphocytes was activated on day 8 to 9; 90% belonged to the T4 cell subset, and 10% belonged to the T8 subset.

650

Koponen

et al.

J. ALLERGY

absolute number of Leu 8+ cells was slightly higher than the control value. Thus, mainly T4’, Leu 8cells appear to be stimulated. The results presented in Table IV with differential response pattern were well reproducible and consistent among different donors.

P

//

/

I’ / /’

P’

//

. /I

DISCUSSION

/I

0’

/I / I I

,

,

,.’

6’ -J

0'

I, L

5

I 6

I 7

LO

Days in culture FIG. 2. Kinetic of Leu 8 antigen expression (----) and percentage of PY positive ceils ( - - - - ) in penicillin G (a-01 (donor U. 6.) and PPD (o-o) (donor K. W.)activated cultures; 1 x lo6 cells were cultured with 1 mg/ml of penicillin G for 5, 6, and 7 days and with 10 kg/ml of PPD for 4, 5, and 7 days.

Expression

CLIN. IMMUNOL. OCTOBER 1986

of Leu 8 antigen

The kinetics of Leu 8 antigen expression was studied in Pen G- and PPD-stimulated cultures (Fig. 2). The percentage of PY+ cells increased from day 4 to 7 in both cultures. However, the percentage of activated Leu 8’ cells was increasing (from 7% to 36%) only in Pen G-stimulated cultures but stayed constant (6% to 7%) in PPD-triggered cultures. The results presented in Table IV are from optimally activated cultures (25% to 60% PY+ cells). The number of Leu Xi cells in unstimulated cultures was between 60% and 70%. Mainly, in all four antigen stimulations, the T4 cell subset was activated. This implies that the activated Leu 8’ cells belong also to the T4 subset. The percentage of activated Leu 8+ cells was highest in Pen G (69% of antigen-activated cells)EBV (55% of antigen-activated cells)- and influenza A (7 1% of antigen-activated cells)-triggered cultures. In these cultures, also, the absolute number of Leu 8’ cells was doubled, which confirms clearly T4’, Leu 8’ subset activation. The lowest number of activated Leu 8’ cells was observed in PPD-stimulated cultures (10% of antigenactivated cells) in which, also, the absolute number of Leu 8 + cells was decreased compared to the control value. In this case, mainly the T4’, Leu 8- subpopulation appears to be activated. ‘IT induced a higher number of activated Leu 8’ cells (35% of antigen-activated cells) than PPD. The

The lymphocyte transformation test to Pen G is a well established method to investigate penicillin-allergic patients, whereby high SIs (33) are found in penicillin-allergic individuals or in patients”. ’ after short-term treatment with large doses of penicillin. We selected four patients (three with skin symptoms and one with asthmatic reactions) with high SIs to analyze further the T cell reactivity to Pen G in vitro. The cell activation was analyzed by the presence of increased RNA levels visualized by PY staining. This correlates excellently with interleukin-2 receptor expression and ‘H-thymidine uptake.” Additional staining of membrane components by FITC-labeled antibodie:j allowed the characterization of the stimulated T cell subset. Because the way antigen is presented determines which T cell subset is stimulated,“’ the analysis of T cell subset reactivity to antigen may allow conclusions of how antigen is recognized. Our results revealed that >50% from Pen G-activated T4’ cells belonged to T4’ and Leu 8 + subclasses that was quite similar to T cell subset stimulation elicited by viral infections but differed from the stimulations induced by soluble antigen PPD. In all antigen-driven cultures, mainly T4 cells were activated. which makes it necessary to use an additional antibody to dissect this functionally heterogeneous T4 cell subset. Gatenby et a1.5reported that Leu 3’ (T4+), Leu 8- subset had, mainly, a helper effect for antibody production. Furthermore, Damle et a1.6 demonstrated that Leu 3 + (T4’), Leu 8 + lymphocytes induced Leu 2’ (T8+) cells to become suppressor effecters in a PPD-driven proliferation system, and Kansas et al. ” demonstrated that both Leu 3+ (T4+), Leu 8+, and Leu 3’, Leu 8- cells were capable of amplifying the development of allospecific T killer cells. The data obtained in the antigen-driven culture agree well with our knowledge about the respective immune reactions to these antigens in vivo. PPD stimulated, mainly, the T4+, Leu 8- subset that might be responsible for the typical delayed-type hypersensitivity of tuberculin reaction. TT-activated T4’, Leu 8 cells might act as helpers for B cells. The immune reactions for EBV and influenza virus differed from the T cell subset stimulation elicited by PPD and TT antigens, since >50% of virus-stimulated cells were found to be T4’, Leu 8’. Because this T cell subset act as suppressor-inducer cells, it is possible that their

VOLUME NUMBER

T cell reactivity to penicillin

78 4. FART 1

TABLE IV. Expression stimulated cultures

of Leu 8 antigen

in Pen G-, PPD-, TT-, EBV-, and influenza

A virus-

Absolute Antigen

%PY+ cells

WY+

Leu

a+ cells

%Total

Leu 6’ cells

Leu

number

a+ cells

Control Pen G”

2.0 52.0

2.0 36.0

63.0 64.0

7.2 14.0

Control PPDi

3.0 56.0

3.0 6.0

73.0 21.0

7.5 5.4

Control TTS

2.0 40.0

4.0 14.0

65.0 50.0

8.2 10.0

Control EBV$

2.0 40.0

3.0 22.0

65.0 68.0

7.0 13.7

Control

2.0 24.0

4.0 17.0

58.0 67.0

6.0 12.0

Influenzall

651

*Seven days of culture; donor M. T. iFive days of culture; donor K. W. *Five days of culture; donor P. Z. aEleven days of culture; donor H. S. J/Eight days of culture; donor J. R.

stimulation preceeds the T8 cell activation observed in, i.e., EBV-induced infectious mononucleosis in vivo.‘2-‘5 In view of this analysis of stimulated cells in vitro and immune reactions in vivo, it is likely that also the data obtained in penicillin-stimulated cultures are relevant for penicillin-allergic reactions in vivo. Pen G is immunogenic as a hapten that is able to bind to proteins.16 Relatively high concentrations of Pen G are needed to obtain an immune response in vitro, most likely because only a minor part of penicillin degrades to reactive components and is then able to bind to proteins. Some of these proteins to which penicillin binds are glycoproteins of the HLA system, whereby penicillin is even able to block the binding of certain alloantibodies to the respective HLA antigens (Aw32, B15, and B40).“, I8 The immune reaction to penicillin may thus be directed versus “modified” HLA-A, B, and C antigens that is similar to the T cell reactions versus virally infected cells, where viral proteins also appear in association with HLA-A, B, and C.‘9W24 In view of this similarity between “antigen presentation” in Pen G-exposed and EBV/influenza virus-infected cells, it is not surprising to find a stimulation of the same T cell subset, namely, the high number of activated T4+, Leu 8’ cells. T4 + , Leu 8’ cells are inducer cells for suppressor or cytotoxic cells and activate T8’ cells for effector functions, i.e., suppressor or cytotoxic, that are necessary to control viral diseases. In agreement with this concept is our finding that both in virus- and penicillin-

stimulated cultures, a small increment of T8’ cells could be observed. The meaning of the peculiar T cell reactivity to Pen G is open to speculations. Because we used penicillinallergic patients in this study, mainly with skin symptoms, the findings may only be relevant for this subgroup. The similarity to the T cell reaction to penicillin, EBV, and influenza virus is a reminder that penicillin-induced rashes have similar morphologic description as viral diseases (i.e., morbilliform-like rash). F’unctional studies of the stimulated T cells or T cell hnes/clones are underway and may be helpful to understand further the pathophysiologic meaning of the described T cell reaction. REFERENCES 1. van Arsdel PP Jr: Adverse drug reactions. In Middleton E Jr, Reed CA, Ellis EF, editors: Allergy: principles and practice. St.Louis, 1983, The CV Mosby Co, pp 1133-58 2. de Week AL, Spengler H, Geczy AF: Stimulation of penicillinsensitive lymphocytes by antigenic determinants covalently bound to cell membranes. Monogr Allergy 8:120, 1974 3. Vickers MR, Assem ESK: Tests for penicillin allergy in man. I. Carrier effect on response to penicilloyl conjugates. Immunology 26:425, 1974 4. Walker C, Pichler WJ, de Week AL: Different T cell subset stimu ation by IgGl or IgG2a anti-T3 antibodies. Immunobiology (in press) 5. Gatenby PA, Kansas GS, Xian CY, Evans RL, Engleman EG: Dissection of immunoregulatory subpopulations of T-lymphocytes within the helper and suppressor sublineages in man. J Immunol 129:1997, 1982 6. Damle NK, Mohagheghpour N, Engleman EG: Soluble anti-

652

Koponen

et al.

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r Correction By mistake the valuable contributions of Dr. Hugh A. Sampson were not recorded in the “Acknowledgment to Reviewers” section in the December 1985 issue of the JOIJRNAL. Herewith, the Editor expresses his gratitude to Dr. Sampson for his excellent reviews.