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T Lymphocyte Responses to Mycobacterial Antigen in AIDS Patients with Disseminated Mycobacterium avium-Mycobacterium intracellulare Infection
T Lymphocyte Responses to Mycobacterial Antigen in AIDS Patients with Disseminated Mycobacterium avium-Mycobacterlum intracellu/are Infection* Henry
~
Murray, M.D.; Donna A Scavuzzo, M.D.; Sotiros D. Chaparas;
and Richard B. Roberts, M.D.
Patients with acquired immunodeficiency syndrome (AIDS) who have Mf/cobtJcterium amum-Mflcobacterium intracellulare (MAl) infection typically have widely disseminated disease, often fail to respond to multi-drug chemotherapeutic regimens, and show little or DO inftammatory tissue response. To determine if this clinicopathologic state correlates with in vitro lymphocyte responses to speci8c antigen, peripheral blood mononuclear cells from 18 patients with AIDS who had MAl bacillemia were stimulated with either particulate (heat-killed bacille Calmette Gu~rin [BCG]) or soluble (M intracellulare) mycobacterial antigens. In comparison to reactive cells from healthy control subjects testing positive with purified protein derivative of tuberculin (PPD) or from MAI-colonized (non-AIDS) control subjects, cells from 16 (89 percent)
patients with AIDS essentially failed to show any antigeninduced proliferative activity or secretion of 'V-interferon; however, in two patients, antigen-stimulated proliferation or "V-interferon production was modest but within the range of responses of normal healthy control subjects. Thus, although an occasional patient with AIDS can develop disseminated MAl infection despite the presence of antigen-reactive cells in vitro, most MAI-infectE!d patients with AIDS display a striking defect in responsiveness to both particulate and soluble mycobacterial antigens. Since treatment with 'V-interferon activates the mononuclear phagocyte in vivo, these results suggest a rationale for a trial of 'V-interferon therapy in patients with AIDS who have disseminated MAl infection.
1981, disseminated infections caused by the Mycobacterium avium-M ycobacterium intracellulare complex (MAl) were rare and occurred almost exclusively in patients receiving prolonged and intensive immunosuppressive therapy; however, with the advent of the acquired immunodeficiency syndrome (AIDS), MAl infections have become commonplace, 1-9 reflecting the prominence in AIDS of infections caused by pathogens against which a successful host defense is primarily T cell-dependent. 10 In patients with AIDS, MAl infection is often accompanied by persistent bacillemia and positive cultures from multiple sites, and most patients either fail to respond or promptly relapse despite treatment with a four-drug or five-drug regimen. 1-9 Upon organ biopsy or at autopsy, characteristic findings in these patients include little or no inflammatory or granulomatous changes and overwhelming numbers of intracellular mycobacteria in the tissues.r" Thus, both clinical and histopathologic observations strongly suggest the absence of any effective T cell-dependent, macrophage-mediated immune
response to MAl in patients with AIDS. In this report, we provide the in vitro correlate for these clinicopathologic findings by demonstrating that in response to specific mycobacterial antigen, T cells from virtually all patients with AIDS who have disseminated MAl infection display a striking defect in both proliferative activity and the capacity to secrete 'Yinterferon, a key macrophage-activating lymphokine. 1G-12
~or to
*From the Division of Infectious Diseases, Cornell University Medical College, New York, and the Mycobacterial and Fungal Antigens Branch, Division of Bacterial Products, Bureau of Biologics, Food and Drug Administration, US Department of Health, Eaucation, and Welf8re, Bethesda. Supported by research grants from the National Institutes of Health (CA 35018, AI 21510, and AI 21917)and the New York State AIDS Institute (AO 129). Manuscript received June 15; revision accepted October 2. Reprint requem: Dr: Murray, 1300 YorkAvenue, New York 10021
922
MATERIALS AND METHODS
lbtients and C ontrol Subjects The 18 patients studied fulfilled the standard criteria of the Center for Disease Control for the diagnosis of AIDS, 10 and all had positive blood cultures for MAl at the time of study or within the previous four weeks. The ages of the patients ranged from 24 to 49 years, and 16 were homosexual men. Of the two women studied, one was an intravenous drug abuser, and the other was a recipient of a transfusion. Infection with MAl was the first manifestation of AIDS in two patients; the others had developed one or more opportunistic infections (n = 14) or Kaposi's sarcoma (n = 2) 1 to 19 months prior to the diagnosis of disseminated MAl infection. The four positive control subjects consisted of (a) two healthy individuals testing positive with purified protein derivative of tuberculin (PPD), and (b) two non-AIDS patients with chronic obstructive pulmonary disease and positive sputum cultures for MAl presumed to reflect respiratory tract colonization. Three healthy PPD-negative laboratory personnel served as negative control subjects.
Mycobacterial Antigens Viable, Pasteur-strain bacille Calmette-Cuerin (BCG) was obT Lymphocyte Responses to Mycobacterial Antigen in AIDS (Murray at III)
MI Antigen
HKBCG 50
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Con A 25
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10
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cultures). For -V-interferon generation.S" mononuclear cells (1.5X1OS per 0.5 ml) were stimulated for 48 hours with either concanavalin A (15..,glml),JOHKBCG (0.5,5.0, and 50x IOS/ml), or MI antigen (1:10, 1:50, and 1:100 dilutions). The resulting culture supernatants were tested for -V-interferon activity using a radioimmunoassay test kit (Centocor) which specifically measures -v-interferon." Gamma-interferon activity was measured against a National Institutes of Health (NIH) standard for -v-interferon (Gg 23-901-530, Biological Response Modifien Program) and is expressed as NIH units. ll The results in Figures 1 and 2 indicate optimal (peak) responses to the various concentrations of the two antigens used. For most control subjects and patients, optimal proliferative and -vinterferon-producing responses were achieved using 5 to 10x lOS OnCG organisms per milliliter and a 1:100 dilution of MI antigen (not shown).
I
Lymphocyte Enumeration, Proliferation, and -v-Interferon Generation Peripheral blood helper T cells were identified by the use of the monoclonal antibodies, OKT4 and OKT8, in a standard indirect immunofluorescence assay.1O Peripheral blood mononuclear cells were separated by the Ficoll-Hypaque technique and cultivated at 3rC in 95 percent air with 5 percent carbon dioxide in RPMI 1640 medium containing antibiotics and 15 percent heat-inactivated normal heterologous human serum." Proliferative activity was determined using standard techmques" by the incorporation of tritiated-thymidine in biplicate cultures ofO.5x1()5 cells after three days of stimulation with concanavalin A (ConA) (15tJ-glml)lO or after six days of stimulation with either HKBCG (1,10, and 100 X lOS/ml) or various dilutions of MI antigen (1:25, 1:50, 1:100, and 1:500). Results are expressed as net counts per minute (counts per minute of stimulated cultures minus counts per minute of unstimulated
E
400
<,
:::> FIGURE 2. Gamma-interferon production by peripheral blood mononuclear cells after stimulation with antigens or mitogen. Horizontal bars indicate mean values. Results indicate values for 14 to 18 determinations for the four positive control subjects (group A), four determinations for the three negative control subjects (group B), and 13 to 16 patients with AIDS (group C), each tested once. For group C only, dot resting on horizontal axis indicates number (n) of patients for whom responses were undetectable. Lower limits of normal responses in group A were 22 units/ml (HKBCG), 14 unitslml (MI antigen), and 200 unitslml (concanavalin A).
c
.2 ~
a.
200
>--. I
z
LL.
~
RESULTS
Control Responses to Mycobacterial Antigen As indicated in Figures 1 and 2, mononuclear cells from the PPD-positive and MAI-colonized controls (group A) readily proliferated and secreted 'Y-inter-
HKBCG
MI Antigen
•
250
• • •
100
I
2000
200
150
-I-
• • • •
1000
100
50
I A
8
C
•
500
I A
•
•
•
1500
I
•
I 0
Con A 2500
•
300
-0 ::J
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C
B
tained from the 'Irudeau Mycobacterial Collection ("fMC lOll), and organisms were heat-killed (HK) by autoclave. J3 Mycobacterium intraceUulare (MI) (MAl complex serotype 14)antigen was prepared using organisms actively growing in Longs synthetic medium as described previously," Washed organisms were sonicated and then centrifuged, and the supernatant was used as antigen. 14
500
FIGURE 1. Proliferative activity (tritiated-thymidine incorporation) for peripheral blood mononuclear cells after stimulation with antigens or mitogen. Horizontal bar» indicate mean values. Results indicate values for six to ten determinations for the four healthy positive control subjects (group A), one determination for each for the healthy negative control subjects (group B), and eight to nine patients with AIDS (group C), each tested once. For group Conly, dot resting on horizontal axis indicates number (n) of patients for whom responses were undetectable. Lower limits of nonnal responses in group A were 7,095 counts per minute (cpm) (OKBCG), 2,820 counts per minute (MI antigen), and 2,167 counts per minute (concanavalin A).
8
C
0
I
•
•
T • •
I A
• B
CHEST I 93 I 5 I MAY, 1988
•••
(n=4)
C 923
feron in response to stimulation with both HKBCG and MI antigen; however, these responses were quite variable. Cells from PPD-negative control subjects (group B) showed little or no proliferation or 'V-interferon generation, indicating the specificity of the responses of the positive control subjects.
Patients with AIDS As a group, mononuclear cells from the 18 patients with AIDS who had MAl bacillemia (group C) displayed essentially no response to any concentration of either mycobacterial antigen, and mean values for proliferative activity and 'V-interferon production were equal to or lower than those of the PPD-negative control subjects (Fig 1 and 2); however, seven of 13 patients with AIDS secreted normal levels of 'V-interferon in response to stimulation with concanavalin A consistent with our prior observations using nonspecific mitogens.f-" Analysis of data from individual patients showed that in response to HKBCG, no patient with AIDS who was tested displayed either proliferative activity or 'V-interferon production in the normal range (eg, ~7,095 counts per minute or ~25 units/ml; see legends to Fig 1 and 2); and in both assays, the responses of most patients with AIDS were barely detectable. Individual responses to MI antigen were largely similar; however, cells from one (9 percent) of 11 tested patients showed normal proliferative activity (4,483 counts per minute) and one (7 percent) of 15 patients showed 'V-interferon production (17 unitslml) which was in the normal range (eg, ~14 unitslml) (see legends to Fig 1 and 2). To test responses to another specific but nonmycobacterial antigen, cells from eight ofthe patients with AIDS who were seropositive to cytomegalovirus (serum complement fixation titer ~1:811) were stimulated with cytomegalovirus antigen." Seven of the eight showed no proliferation «500 counts per minute) or 'V-interferon production «10 units/ml) in response to this viral antigen (data not shown), whereas cells from healthy seropositive control subjects show 10,500 counts per minute or more and 100 unitslml or more, respectively,'' One patient's cells displayed low but measurable values to cytomegalovirus antigen, and he was the one patient with AIDS who showed normal MI antigen-induced proliferative activity.
Lymphocyte Counts Peripheral blood T cell subsets were enumerated for all 18 patients, and each showed grossly abnormal values. The absolute number ofT4 + (helper) cells was markedly reduced below the lower limit of normal (3281cu mm)," ten patients had 2 to 25 T4 + cells per cubic millimeter; six had 3O/cu mm to 70/cu mm, and two had > l00/cu mm (llO/cu mm and 174/cu mm). The mean number ofT4 + cells per cubic millimeter for the 924
group with AIDS was 40 ± 11 vs 853 ± 53 for healthy individuals." In addition, in all 18 patients, the T4fr8 cell ratio was less than 0.55 (normal range, 0.84 to 3.4), with a mean of 0.15±0.04 (normal, 1.6±0.1).1l Although deficient numbers of T4 + cells probably explained the failure to respond to mycobacterial antigen,15,17 there was no apparent correlation between T4 + cell number and function. The two patients whose cells displayed either normal proliferative or 'V-interferon-generating activity had 35 and 18 T4 + cells per cubic millimeter, respectively; and, conversely, the two patients with more than 100 T4 + cells per cubic millimeter both showed less than 500 counts per minute and less than 5 units/ml in the proliferation and 'V-interferon assays, respectively. DISCUSSION
The failure of peripheral blood mononuclear cells to respond to stimulation with a previously encountered, specific microbial antigen is characteristic of patients with fully established AIDS and opportunistic infections. 10,11.18,19 In such patients, this defect has been demonstrated using a number of diverse microbial antigens (tetanus toxoid; cytomegalovirus; Herpes simplex virus; Toxoplasma gondii; Candida'?11.18,19) in assays which have measured T cell proliferation, interleukin 2 secretion, and 'V-interferon production. 1O,11.18,19 On the basis of the results of this study in which cells from 16 (89 percent) of18 patients with MAl bacillemia showed essentially no response, both particulate and soluble mycobacterial antigen can now be added to the list of specific microbial antigens to which T cells from infected patients with AIDS typically fail to respond; however, cells from two patients showed either proliferative or 'V-interferon-generating responses, which, although modest, were considered to be normal, given the broad range of control responses. While there is little doubt that successful host defense against mycobacteria is primarily dependent on T cells,1O-13 the results with these latter two patients indicate that an occasional patient with AIDS may nevertheless develop disseminated MAl infection despite antigen-reactive cells in vitro; however; in such patients the level of T cell reactivity as judged by 'Vinterferon secretion is low, and if the direct macrophage-activating effects of'V-interferonlO,12.i4 are important in defense against mycobacteria.f''" this level is still apparently inadequate to control or suppress systemic MAl infection. To test this possibility, we have proceeded to initiate a trial of recombinant 'Vinterferon therapy in patients with AIDS in whom MAl bacillemia persists despite a multi-drug chemotherapeutic regimen." Given that (1) AIDS mononuclear phagocytes including alveolar macrophages are fully responsive to recombinant 'V-interferon in vitro;lO,12.28 (2) the recombinant 'V-interferon-stimulated T Lymphocyte Responses to Mycobacterial Antigen In AIDS (Murray et aI)
monocyte can act against mycobacteria," (3) experimental M intraceUulare infection responds in vioo; to treatment with recombinant -y-interferon;15 (4) exogenous recombinant -y-interferon activates the human monocyte in vioo (patients with cancel;· lepromatous leprosy, i4 and AIDS),· including the induction of activity against mycobacteria (M lepr~); and, finally, (5) as demonstrated herein, that AIDS T cells fail to secrete mycobacterial antigen-induced -yinterferon, such a trial appears to be warranted. REFERENCES
1 Zalowski ~ Fligiel S, Berlin G~ Johnson BL Jr. Disseminated Mycobt.u:terium aoium-intrtJceUulare infection in homosexual men dying of acquired immunodeficiency syndrome. JAMA 1982; 248:2980 2 Sohn CC, Schroff RW, Kliewer KE, Lebel DM, Fligiel S. Disseminated Mycobacterium aoium-intracellulare infection in homosexual men with acquired cell-mediated immunodeficiency: a histologic and immunologic study of two cases. Am J Clin Pathol 1983; 79:247 3 Green JB, Sidhu GS, Lewin S, Levine JF, Masur H, Simberkoff MS, et all Mycobacterium avium-intraceUulare: a cause of disseminated life-threatening infection in homosexuals and drug abusers. Ann Intern Med 1982; 97:539 4 Macher AM, Kovacs]A, Gill ~ Roberts GD, Ames J, Park CH, et all Bacteremia due to Mycobt.u:terium aoium-intraceUulare in the acquired immunodeficiency syndrome. Ann Intern Med 1983; 99:782 5 Cohen RJ, Sameszuk MK, Busch D, Lagios M. Occult infections with Mycobacterium avium-intraceUulare in bone marrow biopsy specimens from patients with AIDS. N Eng} J Med 1983; 308:1475 6 Strom RL, Groninger Iffi AIDS with Mycobacterium aviumintrtJceUulare lesions resembling those of Whipple's disease. N Eng} J Med 1983; 309:1323 7 Roth BI, Owen RL, Keren DF: AIDS with Mycobt.u:terium avium-intraceUulare lesions resembling those of Whipple disease. N Eng} J Med 1983; 309:1324 8 Masur H. Mycobacterium aoium-intraceUulare: another scourge fOr individuals with the acquired immunodeficiency syndrome. JAMA 1982; 248:3013 9 Wong B, Edwards FF, Kiehn TE, Whimbey E, Donnelly H, Bernard EM, et all Continuous high-grade Mycobacterium aoium-intraceUulare bacteremia in patients with the acquired immunode6ciency syndrome. Am J Med 1985; 78:35 10 Murray ~ Rubin B~ Masur H, Roberts RB. Impaired production of lymphokines and immune (gamma) interferon in the acquired immunodeficiency syndrome. N Eng} J Med 1984; 310:883 11 Murray Hillman JK, Rubin B~ Kelly CD, l)'ler LM, Donnelly OM, et all Patients at risk fOr AIDS-related opportunistic infections: clinical manifestations and impaired gamma interferon production. N Engl J Med 1985; 313:1504 12 Murray aw Gellene RA, Libby OM, Rothermel CD, Rubin BY. Activation of tissue macrophages from AIDS patients: in vitro response of AIDS alveolar macrophages to lymphokines and gamma interferon. J Immunoll985; 135:2374 13 Murray H~ Cohn ZA. Machrophage oxygen-dependent anti-
s
aw
14 15 16
17 18
19
20 21 22 23 24
25
26
microbial activity: 3. enhanced oxidative metabolism as an expression ofmacrophage activation. J Exp Med 1980; 152:1596 Chapparas SD. Antigenic relationships among mycobacterial species studied by modi6ed-rocket and crossed immunoelectrophoresis. Rev Infect Dis 1981; 3:934 Cunningham AL, Merigan Te. Leu-3 + T cells produce linterferon in patients with recurrent herpes labialis. J Immunol 1984; 132:197 Biondi A, Roach ]A, Schlossman SF, Todd RF: Phenotypic characterization of human T lymphocyte populations producing macrophage-activating factor (MAF) lymphokines. J Immunol 1984; 133:281 Kelly CD, Welte K, Murray H~ Antigen-induced interferongamma production: differential dependence on interleukin 2 secretion and receptor activity. J Immunoll987; 139:2325 Epstein IS, Frederick WR, Rook AH, Jackson L, Manischewitz JF, Mayner RE, et all Selective defects in cytomegalovirus and mitogen-induced lymphocyte proliferation and interferon release in patients with acquired immunodeficiency syndrome. J Infect Dis 1985; 152:727 Lane HC, Depper JM, Greene WC, Whalen G, Waldman TA, Fauci AS. Qualitative analysis of immune function in patients with the acquired immunodeficiency syndrome: evidence fOr selective defect in soluble antigen recognition. N Eng} J Med 1985; 313:79 North RJ. Importance of thymus-derived lymphocytes in cellmediated immunity to infection. Cell Immunoll973; 7:166 Lefford MJ. 1i'ansfer of adoptive immunity to tuberculosis in mice. Infect Immun 1975; 11:1174 Orme 1M, Collins FM. Protection against Mycobacterium tuberculoriB infection by adoptive immunotherapy. J Exp Med 1983; 158:74 Orme 1M, Collins FM. Crossprotection against nontuberculous mycobacterial infections by Mycobacterium tuberculoriB memory immune T lymphocytes. J Exp Med 1986; 163:203 Nathan CIf: Kaplan G, Lewis WR, Nusrat A, Witmer MD, Sherwin SA, et ale Local and systemic effects of low doses of recombinant interferon-l after intradermal injection in patients with lepromatous leprosy. N Engl J Med 1986; 315:6 Edwards CK, Hedegaard HB, Zlotnik A, Gangadharam PH, Johnston RD, Pabst MJ. Chronic infection due to Mycobacterium .ntracellulare in mice: association with macrophage release of prostaglandin E. and reversal by injection of indomethacin, muramyl dipeptide, or interferon-l. J Immunoll986; 136:1820 Rook CAW, Steele J, Fraher L, Barker S, KamaliR, 01\iordan J. Vitamin 0 3, gamma interferon, and control of proliferation of Mycobacterium tuberculoaU by human monocytes. Immunology
1986;57:159
v
27 Masur H, Tuazon C, Gill Grimes G, Baird B, Fauci AS, et ale Effect of combined clofazimine and ansamycin therapy on Mycobacterium avium-Mycobacterium intrace"ulare bacteremia in patients with AIDS. J Infect Dis 1987; 155:127 28 Murray H~ Scavuzzo 0, Jacobs JL, Kaplan MR, Libby OM, Roberts RB. In vitro and in vivo activation of human mononuclear phagocytes by gamma interferon: studies with normal and AIDS monocytes. J Immunoll987; 138:2462 29 Nathan CF, Kaplan G, Lewis WR, Nusrat A, Witmer MD, Sherwin SA, et aleAdministration of recombinant interferon-l to cancer patients enhances monocyte secretion ofhydrogen peroxide. PNAS USA 1985; 82:8686
CHEST" 93 , 5 I MAY. 1888
125
~
Murray, M.D.; Donna A Scavuzzo, M.D.; Sotiros D. Chaparas;
and Richard B. Roberts, M.D.
Patients with acquired immunodeficiency syndrome (AIDS) who have Mf/cobtJcterium amum-Mflcobacterium intracellulare (MAl) infection typically have widely disseminated disease, often fail to respond to multi-drug chemotherapeutic regimens, and show little or DO inftammatory tissue response. To determine if this clinicopathologic state correlates with in vitro lymphocyte responses to speci8c antigen, peripheral blood mononuclear cells from 18 patients with AIDS who had MAl bacillemia were stimulated with either particulate (heat-killed bacille Calmette Gu~rin [BCG]) or soluble (M intracellulare) mycobacterial antigens. In comparison to reactive cells from healthy control subjects testing positive with purified protein derivative of tuberculin (PPD) or from MAI-colonized (non-AIDS) control subjects, cells from 16 (89 percent)
patients with AIDS essentially failed to show any antigeninduced proliferative activity or secretion of 'V-interferon; however, in two patients, antigen-stimulated proliferation or "V-interferon production was modest but within the range of responses of normal healthy control subjects. Thus, although an occasional patient with AIDS can develop disseminated MAl infection despite the presence of antigen-reactive cells in vitro, most MAI-infectE!d patients with AIDS display a striking defect in responsiveness to both particulate and soluble mycobacterial antigens. Since treatment with 'V-interferon activates the mononuclear phagocyte in vivo, these results suggest a rationale for a trial of 'V-interferon therapy in patients with AIDS who have disseminated MAl infection.
1981, disseminated infections caused by the Mycobacterium avium-M ycobacterium intracellulare complex (MAl) were rare and occurred almost exclusively in patients receiving prolonged and intensive immunosuppressive therapy; however, with the advent of the acquired immunodeficiency syndrome (AIDS), MAl infections have become commonplace, 1-9 reflecting the prominence in AIDS of infections caused by pathogens against which a successful host defense is primarily T cell-dependent. 10 In patients with AIDS, MAl infection is often accompanied by persistent bacillemia and positive cultures from multiple sites, and most patients either fail to respond or promptly relapse despite treatment with a four-drug or five-drug regimen. 1-9 Upon organ biopsy or at autopsy, characteristic findings in these patients include little or no inflammatory or granulomatous changes and overwhelming numbers of intracellular mycobacteria in the tissues.r" Thus, both clinical and histopathologic observations strongly suggest the absence of any effective T cell-dependent, macrophage-mediated immune
response to MAl in patients with AIDS. In this report, we provide the in vitro correlate for these clinicopathologic findings by demonstrating that in response to specific mycobacterial antigen, T cells from virtually all patients with AIDS who have disseminated MAl infection display a striking defect in both proliferative activity and the capacity to secrete 'Yinterferon, a key macrophage-activating lymphokine. 1G-12
~or to
*From the Division of Infectious Diseases, Cornell University Medical College, New York, and the Mycobacterial and Fungal Antigens Branch, Division of Bacterial Products, Bureau of Biologics, Food and Drug Administration, US Department of Health, Eaucation, and Welf8re, Bethesda. Supported by research grants from the National Institutes of Health (CA 35018, AI 21510, and AI 21917)and the New York State AIDS Institute (AO 129). Manuscript received June 15; revision accepted October 2. Reprint requem: Dr: Murray, 1300 YorkAvenue, New York 10021
922
MATERIALS AND METHODS
lbtients and C ontrol Subjects The 18 patients studied fulfilled the standard criteria of the Center for Disease Control for the diagnosis of AIDS, 10 and all had positive blood cultures for MAl at the time of study or within the previous four weeks. The ages of the patients ranged from 24 to 49 years, and 16 were homosexual men. Of the two women studied, one was an intravenous drug abuser, and the other was a recipient of a transfusion. Infection with MAl was the first manifestation of AIDS in two patients; the others had developed one or more opportunistic infections (n = 14) or Kaposi's sarcoma (n = 2) 1 to 19 months prior to the diagnosis of disseminated MAl infection. The four positive control subjects consisted of (a) two healthy individuals testing positive with purified protein derivative of tuberculin (PPD), and (b) two non-AIDS patients with chronic obstructive pulmonary disease and positive sputum cultures for MAl presumed to reflect respiratory tract colonization. Three healthy PPD-negative laboratory personnel served as negative control subjects.
Mycobacterial Antigens Viable, Pasteur-strain bacille Calmette-Cuerin (BCG) was obT Lymphocyte Responses to Mycobacterial Antigen in AIDS (Murray at III)
MI Antigen
HKBCG 50
•
40
• •
ttl
0
)(
E
25
~
-
30
>
0
Q)
>
20
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~ (5
10
~
a..
•
10
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15
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0
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•
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Con A 25
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8
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• • •
10
• • • • •
5
•
•
0
15
8
0
C
• •
•• A
cultures). For -V-interferon generation.S" mononuclear cells (1.5X1OS per 0.5 ml) were stimulated for 48 hours with either concanavalin A (15..,glml),JOHKBCG (0.5,5.0, and 50x IOS/ml), or MI antigen (1:10, 1:50, and 1:100 dilutions). The resulting culture supernatants were tested for -V-interferon activity using a radioimmunoassay test kit (Centocor) which specifically measures -v-interferon." Gamma-interferon activity was measured against a National Institutes of Health (NIH) standard for -v-interferon (Gg 23-901-530, Biological Response Modifien Program) and is expressed as NIH units. ll The results in Figures 1 and 2 indicate optimal (peak) responses to the various concentrations of the two antigens used. For most control subjects and patients, optimal proliferative and -vinterferon-producing responses were achieved using 5 to 10x lOS OnCG organisms per milliliter and a 1:100 dilution of MI antigen (not shown).
I
Lymphocyte Enumeration, Proliferation, and -v-Interferon Generation Peripheral blood helper T cells were identified by the use of the monoclonal antibodies, OKT4 and OKT8, in a standard indirect immunofluorescence assay.1O Peripheral blood mononuclear cells were separated by the Ficoll-Hypaque technique and cultivated at 3rC in 95 percent air with 5 percent carbon dioxide in RPMI 1640 medium containing antibiotics and 15 percent heat-inactivated normal heterologous human serum." Proliferative activity was determined using standard techmques" by the incorporation of tritiated-thymidine in biplicate cultures ofO.5x1()5 cells after three days of stimulation with concanavalin A (ConA) (15tJ-glml)lO or after six days of stimulation with either HKBCG (1,10, and 100 X lOS/ml) or various dilutions of MI antigen (1:25, 1:50, 1:100, and 1:500). Results are expressed as net counts per minute (counts per minute of stimulated cultures minus counts per minute of unstimulated
E
400
<,
:::> FIGURE 2. Gamma-interferon production by peripheral blood mononuclear cells after stimulation with antigens or mitogen. Horizontal bars indicate mean values. Results indicate values for 14 to 18 determinations for the four positive control subjects (group A), four determinations for the three negative control subjects (group B), and 13 to 16 patients with AIDS (group C), each tested once. For group C only, dot resting on horizontal axis indicates number (n) of patients for whom responses were undetectable. Lower limits of normal responses in group A were 22 units/ml (HKBCG), 14 unitslml (MI antigen), and 200 unitslml (concanavalin A).
c
.2 ~
a.
200
>--. I
z
LL.
~
RESULTS
Control Responses to Mycobacterial Antigen As indicated in Figures 1 and 2, mononuclear cells from the PPD-positive and MAI-colonized controls (group A) readily proliferated and secreted 'Y-inter-
HKBCG
MI Antigen
•
250
• • •
100
I
2000
200
150
-I-
• • • •
1000
100
50
I A
8
C
•
500
I A
•
•
•
1500
I
•
I 0
Con A 2500
•
300
-0 ::J
'"0 0
naS)
C
B
tained from the 'Irudeau Mycobacterial Collection ("fMC lOll), and organisms were heat-killed (HK) by autoclave. J3 Mycobacterium intraceUulare (MI) (MAl complex serotype 14)antigen was prepared using organisms actively growing in Longs synthetic medium as described previously," Washed organisms were sonicated and then centrifuged, and the supernatant was used as antigen. 14
500
FIGURE 1. Proliferative activity (tritiated-thymidine incorporation) for peripheral blood mononuclear cells after stimulation with antigens or mitogen. Horizontal bar» indicate mean values. Results indicate values for six to ten determinations for the four healthy positive control subjects (group A), one determination for each for the healthy negative control subjects (group B), and eight to nine patients with AIDS (group C), each tested once. For group Conly, dot resting on horizontal axis indicates number (n) of patients for whom responses were undetectable. Lower limits of nonnal responses in group A were 7,095 counts per minute (cpm) (OKBCG), 2,820 counts per minute (MI antigen), and 2,167 counts per minute (concanavalin A).
8
C
0
I
•
•
T • •
I A
• B
CHEST I 93 I 5 I MAY, 1988
•••
(n=4)
C 923
feron in response to stimulation with both HKBCG and MI antigen; however, these responses were quite variable. Cells from PPD-negative control subjects (group B) showed little or no proliferation or 'V-interferon generation, indicating the specificity of the responses of the positive control subjects.
Patients with AIDS As a group, mononuclear cells from the 18 patients with AIDS who had MAl bacillemia (group C) displayed essentially no response to any concentration of either mycobacterial antigen, and mean values for proliferative activity and 'V-interferon production were equal to or lower than those of the PPD-negative control subjects (Fig 1 and 2); however, seven of 13 patients with AIDS secreted normal levels of 'V-interferon in response to stimulation with concanavalin A consistent with our prior observations using nonspecific mitogens.f-" Analysis of data from individual patients showed that in response to HKBCG, no patient with AIDS who was tested displayed either proliferative activity or 'V-interferon production in the normal range (eg, ~7,095 counts per minute or ~25 units/ml; see legends to Fig 1 and 2); and in both assays, the responses of most patients with AIDS were barely detectable. Individual responses to MI antigen were largely similar; however, cells from one (9 percent) of 11 tested patients showed normal proliferative activity (4,483 counts per minute) and one (7 percent) of 15 patients showed 'V-interferon production (17 unitslml) which was in the normal range (eg, ~14 unitslml) (see legends to Fig 1 and 2). To test responses to another specific but nonmycobacterial antigen, cells from eight ofthe patients with AIDS who were seropositive to cytomegalovirus (serum complement fixation titer ~1:811) were stimulated with cytomegalovirus antigen." Seven of the eight showed no proliferation «500 counts per minute) or 'V-interferon production «10 units/ml) in response to this viral antigen (data not shown), whereas cells from healthy seropositive control subjects show 10,500 counts per minute or more and 100 unitslml or more, respectively,'' One patient's cells displayed low but measurable values to cytomegalovirus antigen, and he was the one patient with AIDS who showed normal MI antigen-induced proliferative activity.
Lymphocyte Counts Peripheral blood T cell subsets were enumerated for all 18 patients, and each showed grossly abnormal values. The absolute number ofT4 + (helper) cells was markedly reduced below the lower limit of normal (3281cu mm)," ten patients had 2 to 25 T4 + cells per cubic millimeter; six had 3O/cu mm to 70/cu mm, and two had > l00/cu mm (llO/cu mm and 174/cu mm). The mean number ofT4 + cells per cubic millimeter for the 924
group with AIDS was 40 ± 11 vs 853 ± 53 for healthy individuals." In addition, in all 18 patients, the T4fr8 cell ratio was less than 0.55 (normal range, 0.84 to 3.4), with a mean of 0.15±0.04 (normal, 1.6±0.1).1l Although deficient numbers of T4 + cells probably explained the failure to respond to mycobacterial antigen,15,17 there was no apparent correlation between T4 + cell number and function. The two patients whose cells displayed either normal proliferative or 'V-interferon-generating activity had 35 and 18 T4 + cells per cubic millimeter, respectively; and, conversely, the two patients with more than 100 T4 + cells per cubic millimeter both showed less than 500 counts per minute and less than 5 units/ml in the proliferation and 'V-interferon assays, respectively. DISCUSSION
The failure of peripheral blood mononuclear cells to respond to stimulation with a previously encountered, specific microbial antigen is characteristic of patients with fully established AIDS and opportunistic infections. 10,11.18,19 In such patients, this defect has been demonstrated using a number of diverse microbial antigens (tetanus toxoid; cytomegalovirus; Herpes simplex virus; Toxoplasma gondii; Candida'?11.18,19) in assays which have measured T cell proliferation, interleukin 2 secretion, and 'V-interferon production. 1O,11.18,19 On the basis of the results of this study in which cells from 16 (89 percent) of18 patients with MAl bacillemia showed essentially no response, both particulate and soluble mycobacterial antigen can now be added to the list of specific microbial antigens to which T cells from infected patients with AIDS typically fail to respond; however, cells from two patients showed either proliferative or 'V-interferon-generating responses, which, although modest, were considered to be normal, given the broad range of control responses. While there is little doubt that successful host defense against mycobacteria is primarily dependent on T cells,1O-13 the results with these latter two patients indicate that an occasional patient with AIDS may nevertheless develop disseminated MAl infection despite antigen-reactive cells in vitro; however; in such patients the level of T cell reactivity as judged by 'Vinterferon secretion is low, and if the direct macrophage-activating effects of'V-interferonlO,12.i4 are important in defense against mycobacteria.f''" this level is still apparently inadequate to control or suppress systemic MAl infection. To test this possibility, we have proceeded to initiate a trial of recombinant 'Vinterferon therapy in patients with AIDS in whom MAl bacillemia persists despite a multi-drug chemotherapeutic regimen." Given that (1) AIDS mononuclear phagocytes including alveolar macrophages are fully responsive to recombinant 'V-interferon in vitro;lO,12.28 (2) the recombinant 'V-interferon-stimulated T Lymphocyte Responses to Mycobacterial Antigen In AIDS (Murray et aI)
monocyte can act against mycobacteria," (3) experimental M intraceUulare infection responds in vioo; to treatment with recombinant -y-interferon;15 (4) exogenous recombinant -y-interferon activates the human monocyte in vioo (patients with cancel;· lepromatous leprosy, i4 and AIDS),· including the induction of activity against mycobacteria (M lepr~); and, finally, (5) as demonstrated herein, that AIDS T cells fail to secrete mycobacterial antigen-induced -yinterferon, such a trial appears to be warranted. REFERENCES
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