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Cellular immune response in coccidioidomycosis
CELLlII.AR
I?$MITNOLOGY
15,
Cellular ANT~NINO U~~kwsity
360-371 (1975)
Immune
CATANZARO,
Response in Coccidioidomycosisl LYNN
E.
~PITLER,’
AND
KENNETH
M.
MOSER
of C‘alifomiu, 5’1~~ IXcgo, Uniwrsif~~ Ilospital, Stm Diego, California 92103 ajzd Universit~l of California, San Frumisco, Mcdiral Ccntrr, .~clzool of Mrdicinc, San, Frarhsco, Califomitr 94122 Received May 14, 19i4
The CM1 response in vitro and in vim of 30 patients with a poor biologic response to infection with C. iwmitis was investigated. In patients with active pulmonary disease, skin reactivity to CDN was observed in 7/10. and to at least one of five other antigens in S/10. In patients with the most extensive infection, disseminated disease, skin reactivity to CDN and to at least one of five other antigens was observed in only 4/8. In patients with inactive disease, skin reactivity to CDN and to at least one of five other antigens was observed in 11/Q. Even when skin reactivity to CDN was present, MIF release and, more frequently, ‘H-thymidine incorporation were not consistently stimulated by CDN. Maximal 3H-thymidine incorporation in response to PHA and CDN is delayed in 50% of the patients studied. The defect also may be present in patients with inactive disease; however, in two patients followed serially, lymphocyte function very slowly returned to normal. Rosette-forming cells were normal in 18/30. The frequency with which patients with coccidioidal disease demonstrate a defect in cell-mediated immunity raises unanswered questions about the mechanisms responsible for the defect and the role it may play in the biologic defense against invasion by this fungus.
INTRODUCTION Coccidioides iunmitis is a fungus found in the soil in the semiarid southwestern United States, Mexico, and South America (1). Airborn spores enter the lungs and result in the infection of humans and many animal species (2). The rate of infection may reach 90% in areas of high endemicity (3). Within a few weeks following infection, cutaneous delayed hypersensitivity to coccidioidin (CDN)’ can be demonstrated. Extensive epidemiologic studies employing intradermal skin testing have established that the clinical response to the invading fungus is quite variable. Smith, who closely followed new arrivals to areas of high endemicity, reported that 50% of newly infected persons experienced no symptoms (4). 1 Supported in part by grants from the California Research and Education Fund and the National Institutes of Health (Pulmonary SCOR, HL-14169). 2 Recipient of a National Institutes of Health Research Career Development Award (,41-43012) and NIH Grant (Al-CA-10686). lymphocytes; CDN, coc3 Abbreviations used in this paper: B cells, bone marrow-derived minimal essential media; MIF, migration cidioidin ; LT, lymphocyte transformation ; MEM, inhibition factor ; PBS, phosphate-buffered saline ; PHA, phytohemagglutination : RFC, rosette-forming cell ; SI, stimulation index ; SK-SD, streptokinase-streptodonase ; T cells, thymus-derived lymphocytes.
Co>yr:ght 0 1975 by Academic Press, Inc. All rights of reproduction in any form rwzrved
CELLULAR
IMMUKE
RESPONSE
IN
COCCIDIOIDOMYCOSIS
361
In 4076, a flu-like syndrome was observed. The remainder developed an illness ranging from pulmonary disease of variable extent to widespread dissemination and death. The reasons for this variability in clinical course after infection have not been defined but may be related to the ability of the host to mount a cell-mediated response. It has been noted that half of the patients with disseminated coccidioidomycosis have a negative skin test to CDN (5). Furthermore, patients with a negative skin test have a less favorable clinical course than those with a positive skin test (4, 5). These observations suggest that patients with most severe disease may have the greatest impairment of T cell function. This report describes a study of T cell number and reactivity, as well as some of the factors which may affect T cell function, in normal subjects and patients with various types of coccidiodal infection. \Ve measured macrophage inhibition factor (MIF) production in response to CD?\ 1. \\“e examined the ability of lymphocytes to respond to CDN or phytohemagglutinin (PHA) with lymphocyte transformation (LT) (increase incorporation of 3H-tllymicline). The effect of patient plasma on 1-T was studied. M7e also examined the peripheral blood to determine the fraction of lymphocytes that form rosettes with sheep erythrocytes. The results indicate that patients with active coccidioidomycosis have decreased thymus-derived lymphocyte (T cell) reactivity which may affect their ability to mount a cell-mediated response. The impaired T cell reactivity as measured by in vitro tests is not related to a plasma factor nor to a decrease in the number of circulating T cells. MATERIALS
AND
METHODS
Study Subjects Thirty subjects who were free of disease upon clinical and radiologic examination, but who demonstrated greater than 10 mm of induration to intradermal injection of CDN after 48 hr, were designated as normal immune subjects. Eight normal subjects with negative intradermal skin tests to CDN were studied (nonimmune subjects). Thirty patients who had clinical symptoms and bacteriologic or histologic demonstration of C. iwwzitis, or a two-tube increase in titer by complement fixation to CDN, were diagnosed as having coccidioidomycosis. The thirty patients with coccidioidomycosis were subdivided into three groups. (1) Active pulmonary : 10 patients with infection confined to the lungs which was either increasing or decreasing in extent. Most of these patients had positive sputum cultures for C. iwnsitis. (2) Disseminated: 8 patients whose infection involved extrapulmonary sites (most commonly meninges, joints, and soft tissue). (3) Inactive : 12 patients who had well-documented active disease at some time in the past 2-8 years, but who had had ii0 evidence of activity in over 1 year. Skin Tests Antigens used were CDN 1 : 100 (Cutter Laboratories, Berkeley, CA), streptokinase-streptodonase (SK-SD) 5 units (Varidase, Lederle Laboratories, Pearl River, NY), Histoplasmin (Parke Davis and Company, Detroit, MI), Trichophyton 1 : 1000 ( Hollister-Steir, Spokane, WA), mumps, ( Letlerle TJaboratories, Pearl River, NY), PPD-S (United States Public Health Service, Atlanta,
362
CATAKZAKO,
SI’ITLER
AlGIl
MOSER
GA). Each antigen was injected intradermally in a volume of 0.1 ml. Induration was measured at 48 hr. A positive reaction is defined as over 5 mm induration. Antigen
and Mitogen
for in Vitro
Tests
Coccidiodin was prepared from a single strain, T. Silvera, by Dr. Demosthenes Pappagianas (TS-1-72). Previous studies have demonstrated that antigenicity of CDN in skin test does not correlate with either protein or carbohydrate concentration (6). Accordingly, the dose of CDN is expressed as a dilution of the crude extract. Each culture was stimulated with three doses of CDN, 0.01, 0.05, 0.1 ml of 1 : 10 dilution. Phytohemagglutinin was purchased from Burroughs Welcome (Research Triangle Park, NC). A dose range from 0.1 to 12 pg/ml was explored. Maximal stimulation was found at 3 pg/ml in both immune and nonimmune subjects. Patients with coccidioidomycosis also demonstrated maximal response at 3 pg/ml. Consequently, all studies reported here were performed at that dose. Lyophilized SK-SD was reconstituted in PBS, dialyzed exhaustively against PBS, and stored at a concentration of 1000 units/ml, 50 units/ml were used to stimulate cultures. Lymphocyte
Transformation
Peripheral venous blood was collected into a syringe containing 20 units of preservative-free heparin per milliliter of blood. The syringe was placed upright at 37°C to allow sedimentation of erythrocytes. The leukocyte-plasma layer was collected and centrifuged at 800 y for 10 min. The leukocytes were resuspended in F10 medium (Grand Island Biological Company, Grand Island, NY) and washed twice with media. The cells were suspendedin FlO medium supplemented with 20% plasma from a single AB+ donor and placed in culture at 750,000 lymphocytes/ml. Triplicate 2-ml cultures containing 1.25 X lo6 lymphocytes, were incubated at 37°C in 5% COz-enriched air atmosphere for l-9 days. Control unstimulated cultures, mitogen stimulated, and antigen stimulated were treated in the same manner. One hour before each culture was to be terminated, 1 &i “H-thymidine was added to each tube. Incubation was continued at 37°C for 1 hr. The culture was terminated with lOO-fold excess of unlabelled thymidine and centrifugation at 4°C. The cell mass was treated with 5 ml 6.7% TCA at 4°C. Precipitate was collected by centrifugation, redissolved in 1 ml 0.1 M NaOH. This procedure was repeated three times. Finally, the precipitate was dissolved in 2 ml Protosol (New England Nuclear, Boston, MA) and mixed with 10 ml of Aquasol (New England Nuclear, Boston, MA). 3H incorporation was measured as beta scintillation in a Packard liquid scintillation counter. Stimulation index (SI) was defined by cpm in culture with antigen or mitogen divided lay cpm in culture without antigen or mitogen. Mac-rophage
Inhibition
Factor
Macrophage inhibition factor (MIF) production was assayed by the technique of Rocklin (7). Briefly, peripheral blood lymphocytes are separated as described above. Eagle’s minimum essential medium (MEM), Spinner modification (Grand Island Biological Company, Grand Island, NY), was used to culture 3 X 10” lymphocytes in 1 ml. Triplicate cultures were stimulated with several dilutions of
CELLULAR
IMMUNE
RESPOKSE
IN
TABLE SKIN
Extent
REACTIONS
skin skin
reaction reaction
WITH
Positive CDNl‘
Active pulmonary disease Disseminated disease Inactive disease u Positive b Positive Trichophyton.
1
OF PATIENTS
of disease
363
COCCIDIOIDOMYCOSIS
COCCIDIOIDOMYCOSIS
to
Positive to Histoplasmin(&
7/10 418 11/12
Positive to other antigens*
2/10 o/4 3/11
S/10
4/s 11/12
over 5 mm induration at 48 hr after intradermal injection. to at least one of the following: Tuberculin, Candida, mumps,
SK-SD,
CDN or SK-SD. Controls included media without cells or antigen, media with cells and no antigen, media with antigen and no cells. Tubes were incubated at 37°C in 55% COz-enriched air atmosphere. Supernatant was collected from each tube at 24, 48, and 72 hr. At 24 and 48 hr, 1 ml of fresh media containing the same concentration of antigen as originally used was added to the cells. Supernatant fluids from like cultures were pooled, dialyzed against distilled water, lyophilized, and reconstituted to 20% of their original volume. Peritoneal exudate cells were collected from Hartley strain guinea pigs. Intraperitoneal injection with 30 ml of mineral oil was made. Seventy-two hours later, the peritoneal cavity was drained and washed with MEM. The peritoneal exudate cells were collected and washed three times in MEM. Capillary tubes are loaded with a cell suspension and packed by centrifugation. Capillary tubes are cut at the cell interface and mounted in Mackaness-type chambers. Chambers were filled with processed supernatant fluid from the above-described cultures or controls. Migration of guinea pig peritoneal exudate cells was measured by planimetry at 24 and 48 hr. Results are expressed as percent migration inhibition compared with control unstimulated cultures. Inhibition of migration by 205% or more is indicative of MIF production. TABLE COKKELATION
OF M IF WTH
Extent
of disease
RELEASE SKIN
u Positive 6 Negative c Number
skin reaction skin reaction positive/number
SH-T~~~~~~~~
Positive
skin reaction” 3H-Thymidine incorporation
-
-
s/s< 6/7 3/4 9/11 18/22
27/30 J/7
over 5 mm induration over 5 mm induration tested.
INCOKPOKATION
TO CDN
REACTIVITY
nl IF release
Nonimmune subjects Normal immune subject Active pulmonary disease Disseminated disease Inactive disease All patients
2
AND
Negative MIF
skin reactionb
release
2/4 7/11 13/22 at 48 hr after at 48 hr after
intradermal intradermal
injection. injection.
3H-Thymidine incorporation
364
CATANZARO,
SPITLER
AiXD
MOSER
22 20 18
\
.
.G .
. i. \ 5 NORMAL IMMUNE SUBJECTS
7
5 ACTIVE PULMONARY DISEASE
DISSEMINATED DISEASE
7
INACTIVE DISEASE
FIG. 1. Kinetics of response to CDN stimulation.
Rosette-Forvning Cells Rosette-forming cells (RFC) were assayed by the technique of Wybran et al. (8). Briefly, lymphocytes are separated from peripheral blood by centrifugation in Ficoll-Hypaque density gradient. Lymphocytes were washed and resuspended in fresh media at a concentration of 1 x lo7 lymphocytes/ml. Plastic tubes (13 x 100 mm) were used; 0.05 ml of the lymphocyte suspensionwas added to 0.05 ml of fetal calf serum. Incubation was at 37°C for 1 hr in 5% COz atmosphere. were centrifuged at 200 g for 5 min at room temperature. After centrifugation as detailed above, the cells were allowed to incubate overnight. The next day the celIs were gently resuspendedand examined for rosette formation. Two hundred lymphocytes were counted in a hemacytometer and the percent RFC computed. Statistical Methods Standard deviation was calculated and is indicated after the +- sign. Statistical differences were tested by means of the Student t test. RESULTS Shin Tests Normal immune subjects were defined as having a positive skin reaction to CDN 1 : 100. Seven of ten patients with active pulmonary coccidioidomycosis had a positive skin response to CDN and 8/10 to one of the other antigens used (Table 1). Four of eight patients with disseminated coccidioidomycosis had
CELLULAR
IMMUNE
RESPONSE
IN
TABLE 3H-THYMIDINE
3
INCORPORATION
RESPONSE
TO PHA
Number
Subject
36.5
COCCIDIOIDOMYCOSlS
cpma
_____ Normal nonimmune Normal immune Active pulmonary disease Disseminated disease Inactive disease All patients with disease a cpm
= counts
per minute
30 30 10 8 12 30
in stimulated
tubes
after
46,531 28,618 7,326 15,703 10,443 10,863
z!z f f =t zt f
29,808 15,618 5,694 9,769 9,002 8,851
72 hr culture.
negative skin response to CDN and all antigens usecl. All but one patient with inactive diseasehad a positive skin test to CDN and one of the other antigens. Lymphocyte
Transformation
Response
to Coccidioidin
Lymphocytes from normal nonimmune subjects did not demonstrate nonspecific increased 3H-thymidine incorporation when cultured with CDN. Lymphocytes from 27/30 normal immune subjects when cultured in the presence of CDN had an SI greater than 2 (Table 2). Incorporation was maximal on day 5 in this group (Fig. 1). Patients with coccidioidomycosis were less reactive to CDN than were normal immune subjects. Lymphocytes from only 14/30 patients stimulated by 130 120 110 100
123456/12345/12345~12345
DAYS ACTIVE DISSEMINATED PULMONARY DISEASE I I DISEASE i I I
NORMAL IMMUNE SMJECTS
FIG. 2. Kinetics
of response
to PHA
INACTIVE DISEASE
stimulation.
366
CATANZARO,
SPITLER
AND
TABLE EFFECT
OF DILUTION
Day 3 base line
5 2.5 1 0.5
x 105 x 10’ x105 x 105
OF NORMAL
MOSER
4
CELLS
ON KINETIC
Number lymphocytes stimulated
SI
Day 5 base line
28,850 30,786 21,997 9,744
77 87 58 26
460 333 380 653
3.51 356 363 375
n Normal lymphocytes were cultured resulted in a reduction in the quantitative tion on day 5 than day 3 was not seen.
TO PHAfl
RESPONSE
Stimulated
SI
7,114 10,599 11,181 8,144
16 32 29 12
at low concentration. Reduction of cell concentration response to PHA stimulation. However, greater stimula-
CDN were able to incorporate more than twofold 3H-thynlidine over control on day 5. Furthermore, whereas lymphocytes from normal immune subjects showed a maximal response to CDN on day 5, 4/12 patients with coccidioidomycosis so studied showed a delay in maximal response to day 7 (Fig. 1) . MIF Release Coccidiodin stimulated the release of MIF in lymphocyte cultures in all normal immune subjects as shown in Table 2. Migration inhibition factor release was detected in lymphocyte cultures from 19/30 patients with coccidioidomycosis and generally correlated with skin test reactivity (see Table 2). Comparison
Between
in Viva and in Vitro
Tests of Cellular Immmit~
Table 2 shows the subgrouping of MIF release and stimulation of 3H-thymidine incorporation observed in each category of control subjects and patients. In vitro tests correlated best with skin test results in the control subjects. DisTABLE EFFECT
OF PKEINCUBATION
Patient
IN MEDIA
Preincubation of cells (hr)
WITH
NOKYAL
Base-line cpm
5 PLASMA
ON KINETIC
RESPONSE
TO PHA”
Day 3 stimulation
SI
H.O.
0 24 48
1,443 834 684
39,587 29,960 11,819
27 36 17
L.D.
0 24 48
622 903 952
21,559 53,930 32.148
35 60 34
W.A.
0 24 48
763 812 971
7,812 20,166 5,954
10 24 10
* H.O. had disseminated disease. L.D. and There was no consistent effect of preincubation
u’. A. had active on the reactivity
pulmonary PHA,
uf
coccidioidomycosis.
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367
3. Shaded area represents the fraction of total lymphocytes identified as RFC. Each point the fraction found in a patient with coccidioidomycosis.
FIG.
is
sociation or lack of correlation between skin test and in vitro results-that is, negative 3H-thymidine incorporation or MIF release when skin test is positive and the converse-is most frequent in patients with active disease. Migration inhibition factor release correlates more closely with skin test, being detectable in 18/22 patients with positive skin test and also detected in l/8 with negative skin test. Lymfihocyte Transformation: Responseto PHA 3H-Thymidine incorporation in response to PHA stimulation, assayed on the third day, is shown in Table 3. The difference between the PHA response of normal immune subjects and normal nonimmune subjects was not statistically significant (P < 0.01). However, the PHA response of lymphocytes from 30 patients with coccidioidomycosis was significantly lower than the response in normal immune subjects (P < 0.01). Depressed response to PHA was observed in each subgroup of patients with coccidioidomycosis. Depression was most marked in patients with active pulmonary disease, but was not significantly different from that in patients with inactive disease (P > 0.01). We also explored the time/dose-response curve to PHA in patients with coccidioidomycosis and in normal immune subjects. Both groups responded maximally to 3 pg/ml of culture medium. Normal immune subjects demonstrated maximal incorporation on day 3 ; couiits on da! 5 were never as high as on day 3. In contrast, five patients with coccidioidomycosis had a greater response to PHA on day 5. Three of these fire had active pulmonary disease (Fig. 2). In order to explore whether reduced reactivity to PHA might be due to a reduction in the number of T cells available in culture, the effect of culturing normal cells at reduced concentration was studied. \Vhen normal cells were cultured at reduced concentration, the response to PHA was diminished. However, an abnormal kinetic pattern was not observed (Table 4). In order to determine whether plasma factors played a role in the reduced reacti+ observed in patients with coccidioidomycosis, an attempt was made to elute depressor substance from patient cells. Lymphocytes from patients with coccidioidomycosis were washed and resuspended in fresh media and normal
plasma for 24 hr, Cells were incubated in media and normal plasma for two
368
CATANZARO,
SPITLER
AND
MOSER
MONTHS FIG. 4. Serial
observations
consecutive 24-hr periods. did not return to normal reduced reactivity to PHA, of these cells to PHA. Enumeration
over
13 months
in case 1 are illustrated.
Phytohemagglutination reactivity of cells so treated (Table 5). Nor did the plasma from patients with when incubated with normal cells, inhibit the response
of T LyPYzphocytes
The reduced T cell reactivity described above could a decrease in the percentage of circulating lymphocytes formation of sheep erythrocytes around lymphocytes was As shown in Fig. 3, patients with coccidioidomycosis controls.
possibly be the result of that are T cells. Rosette used to assay for T cells. were not different from
Serial Studies We had an opportunity to perform serial studies on two patients with active coccidioidomycosis. The first case was a 37-year-old Negro man with acute pulmonary coccidioidomycosis. He improved clinically durmg a 6-week course and he remained well throughout the subsequent of intravenous Amphotericin, 60 50 40
-
z o “0 ;
3020
-
IO 0 2-
9
I
3
5
7
9
MONTHS
FIG. 5. Serial
observations
over
10 months
in case 2 are illustrated.
period of observation. When first tested 5 weeks after the onset of clinical symptoms, he had no response to intradermal CDN, and 3H-tllyniidine incorporation was at a low level in both base-line and Of\‘-stimulated cultures (Fig. 4). There was no response to PHA initially or over the next 8 months. Ten weeks after symptom onset, responsiveness to intradermal CDN was present and was maintained. At S months, the kinetic response to PHA was found to be abnormal, with greater incorporation on day 5 than on day 3. At the tenth month SI response to PHA was in the normal range, but the kinetics were still abnormal. In the 13th month, the response to PHA was normal in both quantitative and kinetic aspects. The second case was a 38-year-old Caucasian diabetic woman, first seen during a recurrence of pulmonary cavitary coccidioidomyocsis. Her disease smoulderecl during the period of observation, improving slowly with intravenous Amphotericin treatment. Her response to PHA was less than normal initially and remained below normal during the first and third months (Fig. 5). In the seventh month, the time-response curve to PHA showed maximal stimulation on day 5. In the tenth month, a more normal quantitative and kinetic response to PHA was observed. There was no response to CDN at 1 or 3 months. At 6 months and thereafter, the SI to CDN was often greater than 2.5. DISCUSSION The data presented indicate that patients with coccidioidal disease consistently have evidence of impaired cell-mediated immunity, although the pattern of abnormality is not homogeneous. There is no data to determine whether the defect is the cause or effect of coccidioidal infection. Skin reactivity to CDN is nearly always (11/12) present in patients with inactive disease, usually (7/10) present in patients with active pulmonary disease, and present only half of the time (4/8) in patients with disseminated disease. An equal number in each subgroup of patients with disease failed to react to all five other antigens tested. Patients with disseminated disease and two of the patients with active pulmonary disease who failed to react to CDK also failed to react to any skin test antigen, leaving open the question as to whether there is a specific lack of lymphocyte function in addition to a nonspecific defect in augmentation of inflammatory mechanisms. The remaining nonresponders to CDN did respond to at least one of the other antigens tested. In the latter group, specific loss of lymphocyte reactivity to CDN appears to be present. Lawrence (10) had demonstrated that, when lymphocytes from a subject sensitive to a specific antigen are incubated with that antigen, they lose their ability to transfer reactivity to that antigen. When the antigen is an infectious agent, proliferation of that agent and overwhelming exposure of sensitized lymphocytes to antigen in viva may result in loss of lymphocyte reactivity to further stimulation in viva or in vitro. This mechanism has been suggested to be operative in other infectious agents (lo), Patients who lack skin reactivity to CDN usually also lack in vititvo reactivity as measured by 3H-thymidine incorporation and MIF release. This suggests that lymphocyte sensitization to this antigen either has not occurred or cannot be expressed in the assays used in this study. Problems may exist at the level of antigen processing, the response to antigen by the effector cells, or the function of a nonspecific cell type such as the macrophage which is important in in V~VO and in vitro
370
CATANZARO,
SPITLER
AND
MOSER
response. Failure to detect reactivity in these studies may rest in part with the antigen utilized. but this is unlikely to be the complete explanation. The CDN utilized for in z&o studies was prepared from a single strain of C‘. i~z&ir, T. SilL4era. T. Silvera is a strain that has an abundance of morphologic, biochemical, and immunologic features used to identify C. i~z~~zifis. Many preparations of CDN used by others incorporate several strains to increase the number of antigenic determinants in an effort to increase sensitivity (11). On the other hand, all normal subjects with skin reactivity on CDN also had in vifro reactivity to CDN derived from T. Silvera. Lymphocytes from skin nonreactors to CDN were not stimulated in vitro in a nonspecific manner by CDS. All patients during the active phase of their diseasehad circulating antibody to this antigen. In normal subjects, immune and nonimmune, the skin reactivity, MIF release, and stimulation of 3H-thymidine incorporation parallel one another closely. Dissociation has been reported to occur in patients with the Wiskott-Alrich syndrome. These patients also may show skin reactivity and MIF production in response to specific antigen, but lack lymphocyte stimulation with the same antigen (12). Animals immunized with tobacco mosaic virus protein show skin reactivity and MIF production to a pentapeptide, which represents the major antigenic determinant, but they do not show lymphocyte stimulation to this antigen (13). This led to the postulate that there may be subpopulations of T lymphocytes. Subsequent work by others has given more direct evidence of T cell heterogeneity ( 14). We also found that the kinetics of lymphocyte stimulation in response to both CDN and to PHA was abnormal in patients with coccidioidomycosis. The delay in peak reactivity to both stimuli observed may be due to one of several mechanisms. Patient lymphocyte response to stimuli may simply be slower because of reduction of an enzyme or cofactor in the cell, or it may indicate a basic difference in the mode of antigen recognition. That the abnormal in vitro response to mitogen and specific antigen observed in patients was not due to decrease in the number of T cells available in peripheral blood at the time of study was shown by two techniques. T cells, identified by their ability to form rosettes with sheep erythrocytes, were not decreased. Furthermore, when we reduced the number of lymphocytes in culture, including the number of T cells, we were unable to produce the abnormal kinetic pattern of response to PHA. It is of interest that the percentages of rosette-forming cells were normal despite the demonstration of impaired cellular immunity to specific antigen in viva and to specific antigen and mitogen in vitro. Rosette-forming cells have been shown to represent T cells (8). The percentage of RFC is known to be diminished in patients with immunologic deficiency disease associated with defects in cellular immunity ( 15), and lymphocytes from patients with these diseasesusually show a diminished responseto PHA. It thus appears that the defect in cellular immunity observed in patients with coccidioidomycosis does not result from diminished numbers of T cells but rather from actual impairment of the function of these cells. A particularly interesting group of patients included in this study was that with inactive disease.This group consisted of subjects who had well-documented active coccidioidomycosis in the past, but who had had no evidence of activity of the disease in over 1 year and were in apparent good health at the time of the
CELLITLAR
IMMUKE
RESPONSE
IN
COCCIDIOIDOhlYCOSIS
351
study. The finding that such patients had reduced T cell reactivity to CDN and as well as an al)normal kinetic response Ion,v after their active infection may be of some importance. It raises the question of whether the defect observed in patients with active disease may not be simply the result of overwhelming infection but rather an underlying defect casually related to the infection. Alternatively, the encounter with C. i~ktis may have caused profound effects which persist for many months or years after apparent cure. Our data also raise questions about the relationship between response to an invading fungus and the events measured by antigen or mitogen stimulation of 3H-thymidine incorporation. Our serial studies on two patients demonstrate that impaired T cell function can be reversible. With clinical recovery, in z4ro tests became normal, albeit very slowly. In these patients the data suggest that impairment of CnlI may be associated with active disease and that, if the mechanism for such impairment were understood, it might be reversible. With regard to the pathogenesis of this unusual infection, it would be important to determine whether the defect in lymphocyte function preceded infection and allowed its progression or whether defect is induced proliferation of c‘. ilrnnifis in the host and contributes to dissemination.
PHA
ACKNO\\‘LEI)GMENTS The authors wish to extend their appreciation to Sharon Batcher, Patricia Flanagan, Hazel Tally, James Marsh, Mae Hsu, and Christine von Muller for technical assistance, and to Mary Gortmaker for preparation of the manuscript.
REFERENCES 1. Swatek, F. E., Myropat/zol. Mycol. AppI. 40, 1, 1970. 2. Ajello, L., Maddy, K. T., and Crecilius, G., Snzrbnzlmzrdia 4, 92, 1965. 3. Smith, C. E., Beard, R. R., Rosenberger, H. G.. and Whiting, E. C., .41zzcr. J. Pzrhlir Health 36, 1394, 1946. 4. Smith, C. A., Pappagianas, D., Levine, H. B., and Saito, M., Bnctcriol. Rsv. 25, 310, 1961. 5. Fiese, M. J., ilz “Coccidioidomycosis” (S. Ajello, Ed.), p. 96. Thomas, Springfield, IL, 1965. 6. Pappagianas, D., Putnam, E. W., and Kabayashi, G. S., J. Bacterial. 82, 714, 1961. 7. Rocklin, R. E., Myers, 0. L., and David, J. R., J. Ilzznzrf>tol. 104, 95, 1970. 8. Wybran, J., Carr, M. C., and Fudenberg, H. H., J. Clilz. Itzzvst. 51, 2537, 1972. 9. Shortman, K., Byrd, W. J., Cerottini, J. C., and Brunner, K. T., Cell. Iwzz~zzozol. 6, 25, 1973. 10. Lawrence, H. S., and Pappenheimer, A. M., J. Exp. A4cd. 104, 321, 1956. 11. Kobayashi, G. S., and Pappagianas, D., Mycopathol. Mycol. Appl. 41, 139, 1970. 12. Spitler, L. E., Levin, A. S., Stites, D. P., Fudenberg, H. H., Pirofsky, B., August, C. S., Stiem, E. R., Hitzig, W. H., and Gatti, R. A., J. C/it?. I~IZVS~. 51, 3216, 1972. 13. Rocklin, R. E., J. Znzn2zznol. 110, 674, 1973. 14. Wybran, J., Levin, A. S., Spitler, L. E., and Fudenberg, H. H., Ne~w Eugf. J. Med. 228, 710, 1973. 15. Spitler, L. E., Benjamini, E., Young, J. D., Kaplan, H., and Fudenberg, H. H., J. l&p. Med. 131, 133, 1970.
I?$MITNOLOGY
15,
Cellular ANT~NINO U~~kwsity
360-371 (1975)
Immune
CATANZARO,
Response in Coccidioidomycosisl LYNN
E.
~PITLER,’
AND
KENNETH
M.
MOSER
of C‘alifomiu, 5’1~~ IXcgo, Uniwrsif~~ Ilospital, Stm Diego, California 92103 ajzd Universit~l of California, San Frumisco, Mcdiral Ccntrr, .~clzool of Mrdicinc, San, Frarhsco, Califomitr 94122 Received May 14, 19i4
The CM1 response in vitro and in vim of 30 patients with a poor biologic response to infection with C. iwmitis was investigated. In patients with active pulmonary disease, skin reactivity to CDN was observed in 7/10. and to at least one of five other antigens in S/10. In patients with the most extensive infection, disseminated disease, skin reactivity to CDN and to at least one of five other antigens was observed in only 4/8. In patients with inactive disease, skin reactivity to CDN and to at least one of five other antigens was observed in 11/Q. Even when skin reactivity to CDN was present, MIF release and, more frequently, ‘H-thymidine incorporation were not consistently stimulated by CDN. Maximal 3H-thymidine incorporation in response to PHA and CDN is delayed in 50% of the patients studied. The defect also may be present in patients with inactive disease; however, in two patients followed serially, lymphocyte function very slowly returned to normal. Rosette-forming cells were normal in 18/30. The frequency with which patients with coccidioidal disease demonstrate a defect in cell-mediated immunity raises unanswered questions about the mechanisms responsible for the defect and the role it may play in the biologic defense against invasion by this fungus.
INTRODUCTION Coccidioides iunmitis is a fungus found in the soil in the semiarid southwestern United States, Mexico, and South America (1). Airborn spores enter the lungs and result in the infection of humans and many animal species (2). The rate of infection may reach 90% in areas of high endemicity (3). Within a few weeks following infection, cutaneous delayed hypersensitivity to coccidioidin (CDN)’ can be demonstrated. Extensive epidemiologic studies employing intradermal skin testing have established that the clinical response to the invading fungus is quite variable. Smith, who closely followed new arrivals to areas of high endemicity, reported that 50% of newly infected persons experienced no symptoms (4). 1 Supported in part by grants from the California Research and Education Fund and the National Institutes of Health (Pulmonary SCOR, HL-14169). 2 Recipient of a National Institutes of Health Research Career Development Award (,41-43012) and NIH Grant (Al-CA-10686). lymphocytes; CDN, coc3 Abbreviations used in this paper: B cells, bone marrow-derived minimal essential media; MIF, migration cidioidin ; LT, lymphocyte transformation ; MEM, inhibition factor ; PBS, phosphate-buffered saline ; PHA, phytohemagglutination : RFC, rosette-forming cell ; SI, stimulation index ; SK-SD, streptokinase-streptodonase ; T cells, thymus-derived lymphocytes.
Co>yr:ght 0 1975 by Academic Press, Inc. All rights of reproduction in any form rwzrved
CELLULAR
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361
In 4076, a flu-like syndrome was observed. The remainder developed an illness ranging from pulmonary disease of variable extent to widespread dissemination and death. The reasons for this variability in clinical course after infection have not been defined but may be related to the ability of the host to mount a cell-mediated response. It has been noted that half of the patients with disseminated coccidioidomycosis have a negative skin test to CDN (5). Furthermore, patients with a negative skin test have a less favorable clinical course than those with a positive skin test (4, 5). These observations suggest that patients with most severe disease may have the greatest impairment of T cell function. This report describes a study of T cell number and reactivity, as well as some of the factors which may affect T cell function, in normal subjects and patients with various types of coccidiodal infection. \Ve measured macrophage inhibition factor (MIF) production in response to CD?\ 1. \\“e examined the ability of lymphocytes to respond to CDN or phytohemagglutinin (PHA) with lymphocyte transformation (LT) (increase incorporation of 3H-tllymicline). The effect of patient plasma on 1-T was studied. M7e also examined the peripheral blood to determine the fraction of lymphocytes that form rosettes with sheep erythrocytes. The results indicate that patients with active coccidioidomycosis have decreased thymus-derived lymphocyte (T cell) reactivity which may affect their ability to mount a cell-mediated response. The impaired T cell reactivity as measured by in vitro tests is not related to a plasma factor nor to a decrease in the number of circulating T cells. MATERIALS
AND
METHODS
Study Subjects Thirty subjects who were free of disease upon clinical and radiologic examination, but who demonstrated greater than 10 mm of induration to intradermal injection of CDN after 48 hr, were designated as normal immune subjects. Eight normal subjects with negative intradermal skin tests to CDN were studied (nonimmune subjects). Thirty patients who had clinical symptoms and bacteriologic or histologic demonstration of C. iwwzitis, or a two-tube increase in titer by complement fixation to CDN, were diagnosed as having coccidioidomycosis. The thirty patients with coccidioidomycosis were subdivided into three groups. (1) Active pulmonary : 10 patients with infection confined to the lungs which was either increasing or decreasing in extent. Most of these patients had positive sputum cultures for C. iwnsitis. (2) Disseminated: 8 patients whose infection involved extrapulmonary sites (most commonly meninges, joints, and soft tissue). (3) Inactive : 12 patients who had well-documented active disease at some time in the past 2-8 years, but who had had ii0 evidence of activity in over 1 year. Skin Tests Antigens used were CDN 1 : 100 (Cutter Laboratories, Berkeley, CA), streptokinase-streptodonase (SK-SD) 5 units (Varidase, Lederle Laboratories, Pearl River, NY), Histoplasmin (Parke Davis and Company, Detroit, MI), Trichophyton 1 : 1000 ( Hollister-Steir, Spokane, WA), mumps, ( Letlerle TJaboratories, Pearl River, NY), PPD-S (United States Public Health Service, Atlanta,
362
CATAKZAKO,
SI’ITLER
AlGIl
MOSER
GA). Each antigen was injected intradermally in a volume of 0.1 ml. Induration was measured at 48 hr. A positive reaction is defined as over 5 mm induration. Antigen
and Mitogen
for in Vitro
Tests
Coccidiodin was prepared from a single strain, T. Silvera, by Dr. Demosthenes Pappagianas (TS-1-72). Previous studies have demonstrated that antigenicity of CDN in skin test does not correlate with either protein or carbohydrate concentration (6). Accordingly, the dose of CDN is expressed as a dilution of the crude extract. Each culture was stimulated with three doses of CDN, 0.01, 0.05, 0.1 ml of 1 : 10 dilution. Phytohemagglutinin was purchased from Burroughs Welcome (Research Triangle Park, NC). A dose range from 0.1 to 12 pg/ml was explored. Maximal stimulation was found at 3 pg/ml in both immune and nonimmune subjects. Patients with coccidioidomycosis also demonstrated maximal response at 3 pg/ml. Consequently, all studies reported here were performed at that dose. Lyophilized SK-SD was reconstituted in PBS, dialyzed exhaustively against PBS, and stored at a concentration of 1000 units/ml, 50 units/ml were used to stimulate cultures. Lymphocyte
Transformation
Peripheral venous blood was collected into a syringe containing 20 units of preservative-free heparin per milliliter of blood. The syringe was placed upright at 37°C to allow sedimentation of erythrocytes. The leukocyte-plasma layer was collected and centrifuged at 800 y for 10 min. The leukocytes were resuspended in F10 medium (Grand Island Biological Company, Grand Island, NY) and washed twice with media. The cells were suspendedin FlO medium supplemented with 20% plasma from a single AB+ donor and placed in culture at 750,000 lymphocytes/ml. Triplicate 2-ml cultures containing 1.25 X lo6 lymphocytes, were incubated at 37°C in 5% COz-enriched air atmosphere for l-9 days. Control unstimulated cultures, mitogen stimulated, and antigen stimulated were treated in the same manner. One hour before each culture was to be terminated, 1 &i “H-thymidine was added to each tube. Incubation was continued at 37°C for 1 hr. The culture was terminated with lOO-fold excess of unlabelled thymidine and centrifugation at 4°C. The cell mass was treated with 5 ml 6.7% TCA at 4°C. Precipitate was collected by centrifugation, redissolved in 1 ml 0.1 M NaOH. This procedure was repeated three times. Finally, the precipitate was dissolved in 2 ml Protosol (New England Nuclear, Boston, MA) and mixed with 10 ml of Aquasol (New England Nuclear, Boston, MA). 3H incorporation was measured as beta scintillation in a Packard liquid scintillation counter. Stimulation index (SI) was defined by cpm in culture with antigen or mitogen divided lay cpm in culture without antigen or mitogen. Mac-rophage
Inhibition
Factor
Macrophage inhibition factor (MIF) production was assayed by the technique of Rocklin (7). Briefly, peripheral blood lymphocytes are separated as described above. Eagle’s minimum essential medium (MEM), Spinner modification (Grand Island Biological Company, Grand Island, NY), was used to culture 3 X 10” lymphocytes in 1 ml. Triplicate cultures were stimulated with several dilutions of
CELLULAR
IMMUNE
RESPOKSE
IN
TABLE SKIN
Extent
REACTIONS
skin skin
reaction reaction
WITH
Positive CDNl‘
Active pulmonary disease Disseminated disease Inactive disease u Positive b Positive Trichophyton.
1
OF PATIENTS
of disease
363
COCCIDIOIDOMYCOSIS
COCCIDIOIDOMYCOSIS
to
Positive to Histoplasmin(&
7/10 418 11/12
Positive to other antigens*
2/10 o/4 3/11
S/10
4/s 11/12
over 5 mm induration at 48 hr after intradermal injection. to at least one of the following: Tuberculin, Candida, mumps,
SK-SD,
CDN or SK-SD. Controls included media without cells or antigen, media with cells and no antigen, media with antigen and no cells. Tubes were incubated at 37°C in 55% COz-enriched air atmosphere. Supernatant was collected from each tube at 24, 48, and 72 hr. At 24 and 48 hr, 1 ml of fresh media containing the same concentration of antigen as originally used was added to the cells. Supernatant fluids from like cultures were pooled, dialyzed against distilled water, lyophilized, and reconstituted to 20% of their original volume. Peritoneal exudate cells were collected from Hartley strain guinea pigs. Intraperitoneal injection with 30 ml of mineral oil was made. Seventy-two hours later, the peritoneal cavity was drained and washed with MEM. The peritoneal exudate cells were collected and washed three times in MEM. Capillary tubes are loaded with a cell suspension and packed by centrifugation. Capillary tubes are cut at the cell interface and mounted in Mackaness-type chambers. Chambers were filled with processed supernatant fluid from the above-described cultures or controls. Migration of guinea pig peritoneal exudate cells was measured by planimetry at 24 and 48 hr. Results are expressed as percent migration inhibition compared with control unstimulated cultures. Inhibition of migration by 205% or more is indicative of MIF production. TABLE COKKELATION
OF M IF WTH
Extent
of disease
RELEASE SKIN
u Positive 6 Negative c Number
skin reaction skin reaction positive/number
SH-T~~~~~~~~
Positive
skin reaction” 3H-Thymidine incorporation
-
-
s/s< 6/7 3/4 9/11 18/22
27/30 J/7
over 5 mm induration over 5 mm induration tested.
INCOKPOKATION
TO CDN
REACTIVITY
nl IF release
Nonimmune subjects Normal immune subject Active pulmonary disease Disseminated disease Inactive disease All patients
2
AND
Negative MIF
skin reactionb
release
2/4 7/11 13/22 at 48 hr after at 48 hr after
intradermal intradermal
injection. injection.
3H-Thymidine incorporation
364
CATANZARO,
SPITLER
AiXD
MOSER
22 20 18
\
.
.G .
. i. \ 5 NORMAL IMMUNE SUBJECTS
7
5 ACTIVE PULMONARY DISEASE
DISSEMINATED DISEASE
7
INACTIVE DISEASE
FIG. 1. Kinetics of response to CDN stimulation.
Rosette-Forvning Cells Rosette-forming cells (RFC) were assayed by the technique of Wybran et al. (8). Briefly, lymphocytes are separated from peripheral blood by centrifugation in Ficoll-Hypaque density gradient. Lymphocytes were washed and resuspended in fresh media at a concentration of 1 x lo7 lymphocytes/ml. Plastic tubes (13 x 100 mm) were used; 0.05 ml of the lymphocyte suspensionwas added to 0.05 ml of fetal calf serum. Incubation was at 37°C for 1 hr in 5% COz atmosphere. were centrifuged at 200 g for 5 min at room temperature. After centrifugation as detailed above, the cells were allowed to incubate overnight. The next day the celIs were gently resuspendedand examined for rosette formation. Two hundred lymphocytes were counted in a hemacytometer and the percent RFC computed. Statistical Methods Standard deviation was calculated and is indicated after the +- sign. Statistical differences were tested by means of the Student t test. RESULTS Shin Tests Normal immune subjects were defined as having a positive skin reaction to CDN 1 : 100. Seven of ten patients with active pulmonary coccidioidomycosis had a positive skin response to CDN and 8/10 to one of the other antigens used (Table 1). Four of eight patients with disseminated coccidioidomycosis had
CELLULAR
IMMUNE
RESPONSE
IN
TABLE 3H-THYMIDINE
3
INCORPORATION
RESPONSE
TO PHA
Number
Subject
36.5
COCCIDIOIDOMYCOSlS
cpma
_____ Normal nonimmune Normal immune Active pulmonary disease Disseminated disease Inactive disease All patients with disease a cpm
= counts
per minute
30 30 10 8 12 30
in stimulated
tubes
after
46,531 28,618 7,326 15,703 10,443 10,863
z!z f f =t zt f
29,808 15,618 5,694 9,769 9,002 8,851
72 hr culture.
negative skin response to CDN and all antigens usecl. All but one patient with inactive diseasehad a positive skin test to CDN and one of the other antigens. Lymphocyte
Transformation
Response
to Coccidioidin
Lymphocytes from normal nonimmune subjects did not demonstrate nonspecific increased 3H-thymidine incorporation when cultured with CDN. Lymphocytes from 27/30 normal immune subjects when cultured in the presence of CDN had an SI greater than 2 (Table 2). Incorporation was maximal on day 5 in this group (Fig. 1). Patients with coccidioidomycosis were less reactive to CDN than were normal immune subjects. Lymphocytes from only 14/30 patients stimulated by 130 120 110 100
123456/12345/12345~12345
DAYS ACTIVE DISSEMINATED PULMONARY DISEASE I I DISEASE i I I
NORMAL IMMUNE SMJECTS
FIG. 2. Kinetics
of response
to PHA
INACTIVE DISEASE
stimulation.
366
CATANZARO,
SPITLER
AND
TABLE EFFECT
OF DILUTION
Day 3 base line
5 2.5 1 0.5
x 105 x 10’ x105 x 105
OF NORMAL
MOSER
4
CELLS
ON KINETIC
Number lymphocytes stimulated
SI
Day 5 base line
28,850 30,786 21,997 9,744
77 87 58 26
460 333 380 653
3.51 356 363 375
n Normal lymphocytes were cultured resulted in a reduction in the quantitative tion on day 5 than day 3 was not seen.
TO PHAfl
RESPONSE
Stimulated
SI
7,114 10,599 11,181 8,144
16 32 29 12
at low concentration. Reduction of cell concentration response to PHA stimulation. However, greater stimula-
CDN were able to incorporate more than twofold 3H-thynlidine over control on day 5. Furthermore, whereas lymphocytes from normal immune subjects showed a maximal response to CDN on day 5, 4/12 patients with coccidioidomycosis so studied showed a delay in maximal response to day 7 (Fig. 1) . MIF Release Coccidiodin stimulated the release of MIF in lymphocyte cultures in all normal immune subjects as shown in Table 2. Migration inhibition factor release was detected in lymphocyte cultures from 19/30 patients with coccidioidomycosis and generally correlated with skin test reactivity (see Table 2). Comparison
Between
in Viva and in Vitro
Tests of Cellular Immmit~
Table 2 shows the subgrouping of MIF release and stimulation of 3H-thymidine incorporation observed in each category of control subjects and patients. In vitro tests correlated best with skin test results in the control subjects. DisTABLE EFFECT
OF PKEINCUBATION
Patient
IN MEDIA
Preincubation of cells (hr)
WITH
NOKYAL
Base-line cpm
5 PLASMA
ON KINETIC
RESPONSE
TO PHA”
Day 3 stimulation
SI
H.O.
0 24 48
1,443 834 684
39,587 29,960 11,819
27 36 17
L.D.
0 24 48
622 903 952
21,559 53,930 32.148
35 60 34
W.A.
0 24 48
763 812 971
7,812 20,166 5,954
10 24 10
* H.O. had disseminated disease. L.D. and There was no consistent effect of preincubation
u’. A. had active on the reactivity
pulmonary PHA,
uf
coccidioidomycosis.
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367
3. Shaded area represents the fraction of total lymphocytes identified as RFC. Each point the fraction found in a patient with coccidioidomycosis.
FIG.
is
sociation or lack of correlation between skin test and in vitro results-that is, negative 3H-thymidine incorporation or MIF release when skin test is positive and the converse-is most frequent in patients with active disease. Migration inhibition factor release correlates more closely with skin test, being detectable in 18/22 patients with positive skin test and also detected in l/8 with negative skin test. Lymfihocyte Transformation: Responseto PHA 3H-Thymidine incorporation in response to PHA stimulation, assayed on the third day, is shown in Table 3. The difference between the PHA response of normal immune subjects and normal nonimmune subjects was not statistically significant (P < 0.01). However, the PHA response of lymphocytes from 30 patients with coccidioidomycosis was significantly lower than the response in normal immune subjects (P < 0.01). Depressed response to PHA was observed in each subgroup of patients with coccidioidomycosis. Depression was most marked in patients with active pulmonary disease, but was not significantly different from that in patients with inactive disease (P > 0.01). We also explored the time/dose-response curve to PHA in patients with coccidioidomycosis and in normal immune subjects. Both groups responded maximally to 3 pg/ml of culture medium. Normal immune subjects demonstrated maximal incorporation on day 3 ; couiits on da! 5 were never as high as on day 3. In contrast, five patients with coccidioidomycosis had a greater response to PHA on day 5. Three of these fire had active pulmonary disease (Fig. 2). In order to explore whether reduced reactivity to PHA might be due to a reduction in the number of T cells available in culture, the effect of culturing normal cells at reduced concentration was studied. \Vhen normal cells were cultured at reduced concentration, the response to PHA was diminished. However, an abnormal kinetic pattern was not observed (Table 4). In order to determine whether plasma factors played a role in the reduced reacti+ observed in patients with coccidioidomycosis, an attempt was made to elute depressor substance from patient cells. Lymphocytes from patients with coccidioidomycosis were washed and resuspended in fresh media and normal
plasma for 24 hr, Cells were incubated in media and normal plasma for two
368
CATANZARO,
SPITLER
AND
MOSER
MONTHS FIG. 4. Serial
observations
consecutive 24-hr periods. did not return to normal reduced reactivity to PHA, of these cells to PHA. Enumeration
over
13 months
in case 1 are illustrated.
Phytohemagglutination reactivity of cells so treated (Table 5). Nor did the plasma from patients with when incubated with normal cells, inhibit the response
of T LyPYzphocytes
The reduced T cell reactivity described above could a decrease in the percentage of circulating lymphocytes formation of sheep erythrocytes around lymphocytes was As shown in Fig. 3, patients with coccidioidomycosis controls.
possibly be the result of that are T cells. Rosette used to assay for T cells. were not different from
Serial Studies We had an opportunity to perform serial studies on two patients with active coccidioidomycosis. The first case was a 37-year-old Negro man with acute pulmonary coccidioidomycosis. He improved clinically durmg a 6-week course and he remained well throughout the subsequent of intravenous Amphotericin, 60 50 40
-
z o “0 ;
3020
-
IO 0 2-
9
I
3
5
7
9
MONTHS
FIG. 5. Serial
observations
over
10 months
in case 2 are illustrated.
period of observation. When first tested 5 weeks after the onset of clinical symptoms, he had no response to intradermal CDN, and 3H-tllyniidine incorporation was at a low level in both base-line and Of\‘-stimulated cultures (Fig. 4). There was no response to PHA initially or over the next 8 months. Ten weeks after symptom onset, responsiveness to intradermal CDN was present and was maintained. At S months, the kinetic response to PHA was found to be abnormal, with greater incorporation on day 5 than on day 3. At the tenth month SI response to PHA was in the normal range, but the kinetics were still abnormal. In the 13th month, the response to PHA was normal in both quantitative and kinetic aspects. The second case was a 38-year-old Caucasian diabetic woman, first seen during a recurrence of pulmonary cavitary coccidioidomyocsis. Her disease smoulderecl during the period of observation, improving slowly with intravenous Amphotericin treatment. Her response to PHA was less than normal initially and remained below normal during the first and third months (Fig. 5). In the seventh month, the time-response curve to PHA showed maximal stimulation on day 5. In the tenth month, a more normal quantitative and kinetic response to PHA was observed. There was no response to CDN at 1 or 3 months. At 6 months and thereafter, the SI to CDN was often greater than 2.5. DISCUSSION The data presented indicate that patients with coccidioidal disease consistently have evidence of impaired cell-mediated immunity, although the pattern of abnormality is not homogeneous. There is no data to determine whether the defect is the cause or effect of coccidioidal infection. Skin reactivity to CDN is nearly always (11/12) present in patients with inactive disease, usually (7/10) present in patients with active pulmonary disease, and present only half of the time (4/8) in patients with disseminated disease. An equal number in each subgroup of patients with disease failed to react to all five other antigens tested. Patients with disseminated disease and two of the patients with active pulmonary disease who failed to react to CDK also failed to react to any skin test antigen, leaving open the question as to whether there is a specific lack of lymphocyte function in addition to a nonspecific defect in augmentation of inflammatory mechanisms. The remaining nonresponders to CDN did respond to at least one of the other antigens tested. In the latter group, specific loss of lymphocyte reactivity to CDN appears to be present. Lawrence (10) had demonstrated that, when lymphocytes from a subject sensitive to a specific antigen are incubated with that antigen, they lose their ability to transfer reactivity to that antigen. When the antigen is an infectious agent, proliferation of that agent and overwhelming exposure of sensitized lymphocytes to antigen in viva may result in loss of lymphocyte reactivity to further stimulation in viva or in vitro. This mechanism has been suggested to be operative in other infectious agents (lo), Patients who lack skin reactivity to CDN usually also lack in vititvo reactivity as measured by 3H-thymidine incorporation and MIF release. This suggests that lymphocyte sensitization to this antigen either has not occurred or cannot be expressed in the assays used in this study. Problems may exist at the level of antigen processing, the response to antigen by the effector cells, or the function of a nonspecific cell type such as the macrophage which is important in in V~VO and in vitro
370
CATANZARO,
SPITLER
AND
MOSER
response. Failure to detect reactivity in these studies may rest in part with the antigen utilized. but this is unlikely to be the complete explanation. The CDN utilized for in z&o studies was prepared from a single strain of C‘. i~z&ir, T. SilL4era. T. Silvera is a strain that has an abundance of morphologic, biochemical, and immunologic features used to identify C. i~z~~zifis. Many preparations of CDN used by others incorporate several strains to increase the number of antigenic determinants in an effort to increase sensitivity (11). On the other hand, all normal subjects with skin reactivity on CDN also had in vifro reactivity to CDN derived from T. Silvera. Lymphocytes from skin nonreactors to CDN were not stimulated in vitro in a nonspecific manner by CDS. All patients during the active phase of their diseasehad circulating antibody to this antigen. In normal subjects, immune and nonimmune, the skin reactivity, MIF release, and stimulation of 3H-thymidine incorporation parallel one another closely. Dissociation has been reported to occur in patients with the Wiskott-Alrich syndrome. These patients also may show skin reactivity and MIF production in response to specific antigen, but lack lymphocyte stimulation with the same antigen (12). Animals immunized with tobacco mosaic virus protein show skin reactivity and MIF production to a pentapeptide, which represents the major antigenic determinant, but they do not show lymphocyte stimulation to this antigen (13). This led to the postulate that there may be subpopulations of T lymphocytes. Subsequent work by others has given more direct evidence of T cell heterogeneity ( 14). We also found that the kinetics of lymphocyte stimulation in response to both CDN and to PHA was abnormal in patients with coccidioidomycosis. The delay in peak reactivity to both stimuli observed may be due to one of several mechanisms. Patient lymphocyte response to stimuli may simply be slower because of reduction of an enzyme or cofactor in the cell, or it may indicate a basic difference in the mode of antigen recognition. That the abnormal in vitro response to mitogen and specific antigen observed in patients was not due to decrease in the number of T cells available in peripheral blood at the time of study was shown by two techniques. T cells, identified by their ability to form rosettes with sheep erythrocytes, were not decreased. Furthermore, when we reduced the number of lymphocytes in culture, including the number of T cells, we were unable to produce the abnormal kinetic pattern of response to PHA. It is of interest that the percentages of rosette-forming cells were normal despite the demonstration of impaired cellular immunity to specific antigen in viva and to specific antigen and mitogen in vitro. Rosette-forming cells have been shown to represent T cells (8). The percentage of RFC is known to be diminished in patients with immunologic deficiency disease associated with defects in cellular immunity ( 15), and lymphocytes from patients with these diseasesusually show a diminished responseto PHA. It thus appears that the defect in cellular immunity observed in patients with coccidioidomycosis does not result from diminished numbers of T cells but rather from actual impairment of the function of these cells. A particularly interesting group of patients included in this study was that with inactive disease.This group consisted of subjects who had well-documented active coccidioidomycosis in the past, but who had had no evidence of activity of the disease in over 1 year and were in apparent good health at the time of the
CELLITLAR
IMMUKE
RESPONSE
IN
COCCIDIOIDOhlYCOSIS
351
study. The finding that such patients had reduced T cell reactivity to CDN and as well as an al)normal kinetic response Ion,v after their active infection may be of some importance. It raises the question of whether the defect observed in patients with active disease may not be simply the result of overwhelming infection but rather an underlying defect casually related to the infection. Alternatively, the encounter with C. i~ktis may have caused profound effects which persist for many months or years after apparent cure. Our data also raise questions about the relationship between response to an invading fungus and the events measured by antigen or mitogen stimulation of 3H-thymidine incorporation. Our serial studies on two patients demonstrate that impaired T cell function can be reversible. With clinical recovery, in z4ro tests became normal, albeit very slowly. In these patients the data suggest that impairment of CnlI may be associated with active disease and that, if the mechanism for such impairment were understood, it might be reversible. With regard to the pathogenesis of this unusual infection, it would be important to determine whether the defect in lymphocyte function preceded infection and allowed its progression or whether defect is induced proliferation of c‘. ilrnnifis in the host and contributes to dissemination.
PHA
ACKNO\\‘LEI)GMENTS The authors wish to extend their appreciation to Sharon Batcher, Patricia Flanagan, Hazel Tally, James Marsh, Mae Hsu, and Christine von Muller for technical assistance, and to Mary Gortmaker for preparation of the manuscript.
REFERENCES 1. Swatek, F. E., Myropat/zol. Mycol. AppI. 40, 1, 1970. 2. Ajello, L., Maddy, K. T., and Crecilius, G., Snzrbnzlmzrdia 4, 92, 1965. 3. Smith, C. E., Beard, R. R., Rosenberger, H. G.. and Whiting, E. C., .41zzcr. J. Pzrhlir Health 36, 1394, 1946. 4. Smith, C. A., Pappagianas, D., Levine, H. B., and Saito, M., Bnctcriol. Rsv. 25, 310, 1961. 5. Fiese, M. J., ilz “Coccidioidomycosis” (S. Ajello, Ed.), p. 96. Thomas, Springfield, IL, 1965. 6. Pappagianas, D., Putnam, E. W., and Kabayashi, G. S., J. Bacterial. 82, 714, 1961. 7. Rocklin, R. E., Myers, 0. L., and David, J. R., J. Ilzznzrf>tol. 104, 95, 1970. 8. Wybran, J., Carr, M. C., and Fudenberg, H. H., J. Clilz. Itzzvst. 51, 2537, 1972. 9. Shortman, K., Byrd, W. J., Cerottini, J. C., and Brunner, K. T., Cell. Iwzz~zzozol. 6, 25, 1973. 10. Lawrence, H. S., and Pappenheimer, A. M., J. Exp. A4cd. 104, 321, 1956. 11. Kobayashi, G. S., and Pappagianas, D., Mycopathol. Mycol. Appl. 41, 139, 1970. 12. Spitler, L. E., Levin, A. S., Stites, D. P., Fudenberg, H. H., Pirofsky, B., August, C. S., Stiem, E. R., Hitzig, W. H., and Gatti, R. A., J. C/it?. I~IZVS~. 51, 3216, 1972. 13. Rocklin, R. E., J. Znzn2zznol. 110, 674, 1973. 14. Wybran, J., Levin, A. S., Spitler, L. E., and Fudenberg, H. H., Ne~w Eugf. J. Med. 228, 710, 1973. 15. Spitler, L. E., Benjamini, E., Young, J. D., Kaplan, H., and Fudenberg, H. H., J. l&p. Med. 131, 133, 1970.