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Precursor frequency of antigen-specific T cells: Effects of sensitization in vivo and in vitro
CELLULAR
IMMUNOLOGY
Precursor
79, 334-344 (1983)
Frequency of Antigen-Specific T Cells: Effects of Sensitization in Viva and in Vitro DENNISFORD’
ANDDENISBURGER'
Surgical Research Laboratory, Veterans Administration Medical Center, and Department of Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201 Received February 16. 1983; accepted April 8, I983 Limiting dilution analysis (LDA) of primary lymphocyte cultures was used to determine the frequency of keyhole limpet hemocyanin (KLH)-specific precursors in the peripheral blood of unimmunized individuals. The KLH-specific precursor frequencies ranged from 1: 150,000 to 1:340,000. In contrast, frequencies of KLH-specific cells in the blood from immune donors ranged from 1:25,000 to 1:42,000. LDA of KLH-stimulated primary cultures indicated that the frequency of KLH-specificcells increased with time in culture reaching a four- to fivefold expansion relative to the frequency obtained prior to culture. The data presented suggest that the enhanced kinetics of secondary T-cell responses observed after in vitro sensitization are due to a decrease in the proportion of lymphocytes which exhibit a suppressor phenotype.
INTRODUCTION Previously we have described the primary sensitization of human peripheral blood mononuclear cells (MNC) to hemocyanins in vitro (1,2). After a 12- to 14&y primary culture period in the presence of keyhole limpet hemocyanin (KLH), recovered cells proliferate in a secondary fashion (2-3 days) upon restimulation with KLH. This response is antigen specific (l), macrophage dependent (2), and HLA-DR restricted (2, 3). The present report determines the frequency of KLH-specific precursors before and during in vitro sensitization to this antigen. The frequency of T-cell precursors that respond to specific alloantigens in both cytolytic and proliferative systems has been documented in mice (4, 5) and man (6, 7) by limiting dilution analysis (LDA). The range of these estimated frequencies ( 1Oe2to 1Oe3) has exceeded the range reported for T-cell precursors specific for soluble antigens (5, 6, 8-12) by at least an order of magnitude. Van Oers et al. (6) reported the frequency of tetanus toxoid-reactive T cells in immunized and boosted donors to vary from 1:750 to 1:11,500. Sohnle and Collins-Lech (11) found the frequency of streptokinase/streptodornase (SK/SD)-reactive T cells to range from 1: 1000 to 1:25,000 in immune donors. Other studies have predicted similar frequencies for T cells reactive to antigens encountered previously. However, the frequency of antigenreactive precursors in nonimmune donors has only been recently addressed (13). ’ Research performed in partial fulfillment of the requirements for the Doctor of Philosophy degree at the Oregon Health Sciences University. 2 To whom correspondence should be addressed: VA Medical Center, Surgical Research Lab, Portland, Oreg. 9720 1. 334 OOO8-8749/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
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In this investigation we obtained estimated frequencies of KLH-specific cells ranging from 1: 150,000 to 1:340,000 in the peripheral blood of KLH-nonimmune individuals. We also observed that immunization of donors with KLH resulted in increases in the frequency of KLH-specific cells in the peripheral blood. Moreover, during sensitization to IUH in vitro, the frequency of IUH-specific cells gradually increased with the length of time in the culture. MATERIALS
AND
METHODS
Antigens. The preparation of keyhole limpet hemocyanin was previously described (14). As prepared, the KLH elicited dermal reactivity and induced lymphocyte proliferation in KLH-immunized individuals and exhibited negligible dermal reactivity in normal nonimmune individuals ( 15). Streptokinase/streptodornase (Varidase) was obtained from Lederle Laboratories Division, Pearl River, New York. PPD (tuberculin purified protein derivative) was obtained from Connaught Laboratories Limited, Ontario, Canada (Batch CT68). Isolation of blood mononuclear cells. Mononuclear cells were separated from heparinized (10 u/ml) peripheral blood from normal donors (ages 20-40) by FicollHypaque (Pharmacia Fine Chemicals, Piscataway, N.J.) density gradient centrifugation (16). Interface cells were subsequently washed two or three times with RPM1 1640 (Gibco, Grand Island, N.Y.). Macrophage isolation. Cells ofthe mononuclear phagocytic series (M4) were isolated from MNC by adherence to plastic. Tissue culture flasks (No. 30 13, Falcon, Oxnard, Calif.) were pretreated for 30-60 min at 4°C with 50% RPMI-50% AB serum (not heat inactivated) and rinsed. MNC (40-60 X lo6 cells) were added to the pretreated flasks in RPM1 with 10% AB serum (heat inactivated, 56°C 30 min). After l-2 hr incubation at 37°C and 5% COZ, the flasks were vigorously rinsed with medium to remove nonadherent cells. Subsequently, cold RPM1 with 5% AB serum and 0.2% EDTA was added and the flasks were held for 20-30 min at 4°C. The macrophages were removed by shaking and vigorous pipetting, washed twice, and counted. Limiting dilution assay. For limiting dilution analyses of primary cultures, MNC were suspended in RPM1 with 15% autologous plasma or heat-inactivated AB serum at 1 X lo6 cells/ml. This suspension of cells was serially diluted to achieve concentrations of 5 X 105, 2.5 X 105, lo5 , and 5 X 104/ml. From these cell suspensions, 100 ~1 was distributed to flat-bottom wells of 96-well microplates (Linbro, Flow Labs Ltd.). For each cell concentration 24 wells were plated (6 control, 18 with antigen). In order to keep the cell density constant in all wells, sufficient irradiated (1200 R, r3’Cs source) autologous MNC were added in 100~~1aliquots to achieve a concentration of lo5 cells in every well. Antigens were added in lo-p1 quantities. For LDA of primary cultures, microplates were incubated at 37°C and 5% CO2 for 1 l-l 2 days. For the last 24 hr of culture, 1.0 &i [3H]thymidine (Amersham, sp act 6.7 &i/mmol) was added. The cells were harvested, and 13H]thymidine uptake was measured by liquid scintillation spectrometry. LDA of secondary cultures was carried out after either sensitization in vitro or immunization in vivo. In experiments involving in vitro sensitization, MNC isolated as described above from nonimmune cell donors were cultured in tubes (Falcon No. 3033) at a cell density of 1 X 106/ml (2 X 106/tube) in RPM1 supplemented with antibiotics (PenStrep, Gibco), 15% heat-inactivated AB serum, and antigen (25-50 pg/ml KLH or HCH). After 12-14 days in a humidified 5% CO? (37’C) atmosphere,
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the cells were washed twice and counted and the viability was assessed. The limiting dilution analysis of the secondary KLH response was then carried out as described above for primary cultures with fresh irradiated autologous MNC used as fillers. [3H]Thymidine incorporation was measured on Day 2 or 3 of secondary culture. In LDA of secondary responses after immunization in vivo, cell donors had either been exposed to the antigens environmentally (e.g., SK/SD or PPD) or actively immunized (e.g., intradermal injection of KLH). The protocol for these experiments was identical to that described above for primary cultures, except [3H]thymidine uptake was measured on Day 5 or 6 of incubation (17, 18). In limiting dilution experiments, the assumption was made that the constant cell density of lo5 cells/well provided for saturation of accessory cells (M6) thus making the presence of antigen-specific proliferating T cells the limiting factor in detecting a positive response. Upon application of Poisson statistics to the LDA, this assumption was borne out. That is, given a random and independent distribution of T cells among the wells, the number of antigen-specific T cells per well follows a Poisson distribution. By scoring cultures positive if the [3H]thymidine incorporation exceeded the mean of control cultures by two standard deviations, a frequency of positive wells was obtained at each cell concentration. Estimation of the frequency of specific T cells was then done with the use of the Poisson formula in which F, = (u’/Y!) X e-“, where F, is the probability of obtaining r specific T cells in a well when the average number of specific T cells per well is u at the cell concentration being considered. Assuming all wells with one or more antigen-specific T cells is scored positive, the fraction of negative wells is given by FO = CU. When u = 1, F,, = 0.37. Therefore, theoretically, when the average number of responding T cells per well is 1, 37% of the wells will be scored as negative. Extrapolation to this point in LDA gives a number of cells, the reciprocal of which represents the frequency of the antigen-specific T cells in question. The extrapolations in this report were done from lines fitted to data points by least squares analysis. Lines with an R value (correlation coefficient) greater than kO.80 and passing through or close to the origin were used, thus justifying the analysis by Poisson statistics. Figure 1 shows the counts per minute obtained in a representative LDA of a primary KLH culture. Indirect immunofluorescence. Either fresh MNC from peripheral blood or cells recovered from cultures after various time periods were suspended at 5 X lo6 cells/ ml in RPM1 with 5% fetal calf serum (Gibco). Monoclonal antibodies were added at optimal concentrations to 0.2-ml aliquots of the cell suspension. The antibodies employed were T28 (pan T, Peter Beverly), OKT4, OKT8, OKMl (Ortho Immunodiagnostics), 2.17 (anti-Ia), anti-K+ anti-L (Bethesda Research Labs), and MAC120 (Howard Ram. After a 30-min incubation on ice, during which the aliquots were vortexed twice, the cells were washed twice (at 4°C) with RPM1 (5% FCS) and then incubated for another 30 min with fluoresceinated goat anti-mouse Ig (Cappel). After two washes, cells were analyzed for specific cell surface fluorescence on a 5OH Cytofluorograf (Ortho). RESULTS Limiting Dilution Blood
Analysis
of the Frequency
of Antigen-Spebjic
T cells in Peripheral
The frequency of antigen-reactive cells estimated by LDA reflected the responsiveness of the donor to a particular antigen in all experiments. For example (Fig. 2 and Table
PRECURSOR
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CF.1"~
. . . .
Ag
2 . . . .
,I XI”.^C. CO"' Ag
m...
I
.I “I
OF KLH-SPECIFIC
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.
4
I 1 a
.
.
. .
.
6
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T CELLS
. IO
8
.
.
.
2
4
6
8
IO
2
4
6
8
IO
I xl04
5x103 I
I 0
‘,
COUNTS
PER
MINUTE
J
12
Cd)
FIG. 1. A representative limiting dilution analysis of KLH-specific precursor frequency in a nonimmune donor is shown. The dashed lines represent the division between negative and positive cultures (two standard deviations from the mean of control cultures).
1) a frequency of 3.3 X 10e5, or 1:30,000, was obtained to SK/SD and 183,000 to PPD, whereas the frequency of cells responding to KLH, an antigen not encountered previously, was 1:247,000 (average of four experiments). There was marked t3H]thymidine uptake in the standard 6-day proliferation assay to SK/SD (eightfold stimulation), marginal responses of PPD (twofold stimulation), and negative responses to KLH. The variability inherent in the frequency determinations was estimated at + 1:50,000 for KLH by evaluating the precursor frequency at different times. In testing different individuals who had not been exposed to KLH, precursor frequencies ranged from 1: 150,000 to 1:338,000 (Fig. 3 and Table 1, five representative experiments) with an average of 1:260,000. The frequency of KLH-specific cells in KLH-immunized donors was estimated to be 1:42,000 (A.V.) and 1:32,000 (D.B.). In donor D.B. the frequency of SK/SD-specific cells was 1:23,000 (Fig. 3). As an additional control the combined frequency of cells reactive to two hemocyanins (KLH and HCH) was determined by culturing cells to both antigens (donor A.W. unimmunized). A predicted combined frequency of 1:86,000 was expected on the basis of the individual experimental frequencies of 1:229,000 for KLH and 1: 139,000 for HCH (Fig. 4, Table 1). The experimentally determined combined frequency was 1:90,000. The Influence of Donor Immunization on the Frequency of KLH-Specific Precursors in Peripheral Blood
Active immunization with KLH (500 pg by a subcutaneous route) resulted in a twofold increase in the number of KLH-specific precursors after 1 week (from 1:252,000 to 1: 105,000 Table 1, Fig. 5) and remained constant over a 3-week period ( 1: 130,000 and 1: 106,000, respectively). LDA 2 days after a 10-pg intradermal boost, showed a decrease to 1:2 15,000, presumably due to recruitment of specific cells from the pe-
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FORD AND BURGER TABLE 1 Antigen-Specific Precursor Frequencies Calculated from Lines Fitted to Limiting Dilution Analysis Data from Figs. 2-6 Antigen Figure
Donor
KLH
SK/SD
PPD
2
D.F.
1:293,000 1:200,000 1:203,000 1:150,000
1:30,000
1:83,000
KLH
SK/SD
1:338,000 1:311,000 1:251,000 1:247,000 1:200,000 1:42,000 I :32,000
I :23,000
3
B.W. so. R.J. A.G. D.F. A.V. D.B.
KLH 4
A.W.
1:229,000
HCH
HCH + KLH
1:139,000
1:90,000
KLH 5
6
R.J.
D.F. S.O. D.F.
1:251,000 1:105,000 (1 week) 1: 130,000 (2 weeks) 1:106,000 (3 weeks) I :2 15,000 (2 days postboost) 1:54,000 (2 weeks postboost) 1:26,000 (6 weeks postboost) KLH primary culture
KLH secondary culture
1:293,000 1:311,000 1:200,000
1:129,000 (8 days) 1:90,000 ( 10 days) 1:50,000 (12 days)
riphery in response to the challenge with antigen. Two and six weeks after the boost the IUH-specific precursor frequency had increased to 1:54,000 and 1:26,000, respectively (Fig. 5, Table 1). KLH-SpeciJic Precursor Frequency Increases during Primary Sensitization in Vitro
In comparison to active immunization, sensitization in vitro resulted in a steady increase of KLH-specific cells over the span of the primary culture period. After 12 days in primary culture an estimated frequency of 1:50,000 was obtained. This rep resented a 4-fold increase over the estimate (1:200,000) obtained from simultaneous primary cultures from the same donors without antigen (Fig. 6, Table I). The yield of cells harvested horn the primary culture was 62% of the original input. By harvesting cells at different time points during primary culture, changes in precursor frequency
PRECURSOR
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ADDED
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(x10-‘)
L. +...
\ \ \ 0
.
IO
FIG. 2. Limiting dilution analyses of KLH-specific precursors (four separate experiments) and a limiting dilution analysis of SK/SD- and PPD-specific precursors from a single donor (D.F.) are shown. Results are tabulated in Table 1. V V, KLH; we * *w, KLH; O---O, KLH; l - . -. 0, KLH; 0 - - - 0, PPD; m-.--m, SK/SD.
could be correlated to the culture interval. At 8 days a 2.3-fold increase had taken place (cell yield, 52%) and at 10 days a 3.5-fold increase was observed (cell yield, 72%, Fig. 6, Table 1). Accelerated Kinetics of Secondary Responsesto Antigen after Long-Term Culture is Due to a Loss of Nonspecific Suppression
In a previous communication (1) which described the methodology for in vitro primary sensitization to hemocyanins in detail, a distinct difference in kinetics was observed between KLH secondary responses after sensitization in vitro (2-3 days optimal) and after immunization in vivo (5-6 days optimal). From the data presented here it is evident that the frequency of KLH-specific cells at the beginning of both ADDED
5
I
MNC
IO
b.10”)
20
Y 3 3 Y F ii? Y ae
\
\. '\,
IO
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FIG. 3. Limiting dilution analysesofthe ICLH-specific precursor frequency from five nonimmune individuals (A**. A, B.W.; O-O, S.O.; o---O, A.G.; V---V, R.J.; O-.-.0, D.F.) and two KLH-immune individuals (A -. -. A, A.V.; w - * . - ‘I, D.B.) are shown. Additionally the SK-SD-specific precursor frequency for donor D.B. is shown (m --- n ). Results are tabulated in Table 1.
340
FORD AND BURGER ADDED
MNC
MO-‘)
FIG. 4. Limiting dilution analyses of IUH- (A A) and HCH-specific (0 - - - 0) precursor frequencies from donor A. W. are shown. Additionally the limiting dilution analysis of MNC that responds to a combination of KLH and HCH in cultures is shown (Cl- - -0). The results are tabulated in Table I.
types of secondary culture was essentially the same (1:40,000-l :50,000) and therefore could not account for the kinetic differences. The possibility that differences in regulatory elements in the secondary cultures could account for the altered kinetics was investigated. We found that cells recovered from 1Zday primary cultures showed a marked decrease in the T cells of the suppressor phenotype (OKT8+) as well as a decrease in macrophages and B cells. In the case of donor D.F. (Table 2), OKT8 cells constituted 10% of the T cells (OKT4 + OKT8) after 12 days in culture, compared to 40% of the T cells placed in culture. To evaluate this tentative explanation more directly, we attempted to alter the 6day optimal response of cells from immune donors by exposing them to a 12day culture period. If the hypothesis were correct, the la-day culture period would lead to a loss in regulatory control (loss of OKT8 and possibly other cells) and the response ADDED I
5
MNC IO
(~16~) 20
FIG. 5. The effects of immunization with KLH on the RLH-specific precursor frequency from donor R.J. are shown. Limiting dilution analyses were done prior to immunization (O-O), at weeks I (X---X),2(O-.-.),and3(U.--O)afterimmunization,andat2&ys(~---~),2weeks(A...A), and 6 weeks (A -. - - A) aRer booster immunization. The results are tabulated in Table 1.
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OF KLH-SPECIFIC
ADDED
MNC
341
T CELLS
d)
FIG. 6. The effects of in vitro culture on KLH-specific precursor frequencies are shown. MNC were analyzed for KLH-specific precursors after 8, 10, and 12 days of culture in the presence of KLH. These frequencies are compared to KLH-specific precursor frequencies obtained from LDA prior to in vitro culture. -0) and 10 days Donor D.F., 0 days (A. . . A) and 8 days (0 -e-e 0); donor S.O., 0 days (0 -.(X - X); donor D.F., 0 days (A---A) and 12 days (0 - - - 0).
to recall antigen would be optimal at an earlier time in secondary culture. When cells from an SK/SD-immune donor were primed to KLH for 12 days, accelerated secondary culture kinetics to both KLH and SK/SD were observed (Figs. 7a, b). The kinetics of the SK/SD response are accelerated relative to the SK/SD response of fresh cells from the same donor. These studies suggest that the early 2- to 3-day optimal responses after in vitro sensitization can be accounted for by a nonspecific loss of regulation during 12 days of cell culture. DISCUSSION In the present report we have demonstrated that the frequency of antigen-specific cells in peripheral blood of man reflects the immune status of the cell donor. Differences in estimates reported by other laboratories appear to be due primarily to differences TABLE 2 Phenotypic Profiles of Mononuclear Cells before and after Long-Term Culture Expt 1 (donor D.E.)
Expt 2 (donor M.H.)
Cell cultured marker”
Fresh cells
Culturedb cells
Fresh cells
Cultured cells
T28 T4 T8 2.17 Ml
64.9’ 31.6 21.5 11.3 18.2
39.3 38.4 4.1 20.0 4.3
67.0 44.3 22.0 6.3 6.0
32.9 35.1 10.0 8.5 3.0
a MNC were cultured for 12 days in the presence of KLH. b MNC were cultured for 12 days in the presence of SK/SD. ‘Percentage of cells expressing the phenotypic marker: T28, panT; T4, T helper; T8, T suppressor; 2.17, IA; M 1, monocyte.
342
FORD AND BURGER 5 a) 4 c-
I s 4
6
b)
5 4/
j
/
p’-- o-Q-,-
P
Q
(fl<\
day
day 2
day 3
day 4
FIG. 7. The effects of long-term culture (12 days) in the presence of KLH (0- - -0), SK/SD (O---O), or no antigen (X. * .X) on the kinetics of T-cell proliferative responses to (a) KLH and (b) SK/SD. The kinetics of SK/SD and KLH responses of fresh MNC are also shown (A-A). The donor (D.F.) is nonimmune to KLH and immune to SK/SD.
in the experimental protocols and the relative immune state of the cell donors. Geha estimated approximately 1: 12,000 T cells reactive to tetanus toxoid (10). van Oers et al. (6) using MNC obtained a range of 1:300 to 1:16,000 for PPD-specific cells and 1:750 to 1: 11,500 for tetanus toxoid-reactive cells. Macy and Stevens estimated the frequency of T helpers that would support a measurable tetanus-specific IgG response to be 1:23,250 (12). Sohnle and CollinsLech, by measuring the first generation lymphocyte response, obtained estimates ranging from 1: 1000 to 1:22,700 MNC reactive to SK/SD (11). Our estimates agree with these ranges. For example we found that the frequency of SK/SD-reactive cells ranged from 1:23,000 to 1:30,000, and KLH-reactive cells ranged between 1:26,000 and 1:42,000 when immune donors were used. In testing several unimmunized individuals to hemocyanins, we found an average frequency of 1:260,000 MNC responsive to IUH measured on Day 12 of primary culture (Fig. 3). Repeated analyses of the same donor (Fig. 2) confirmed that variation in the estimates obtained at different times was small. The estimated frequency was 7- to lo-fold less than the frequencies estimated for recall antigens, We tested the reliability of these estimates by comparing computed to experimentally determined combined frequencies for two noncross-reactive hemocyanins (Fig. 4). The calculated combined frequency was 1:86,000 (KLH + HCH) whereas the experimentally determined frequency was 1:90,000. It is interesting to note that Gebel et al. in a recent report ( 13) found a range of frequencies of 1:24,000 to 1:53,000 for IUH in unimmunized donors. This range appears closer to those observed for recall antigens. At this time it is not known whether these differences could be explained by differences in the donor population, the antigen, or experimental conditions. In any case, it is important to note that the increases in frequency during in vitro exposure to antigen
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KLH-SPECIFIC
T CELLS
343
reported by Gebel et al. (13) are consistent with what we report here, although the absolute values are not. The LDA estimates obtained should not be considered absolute values since a variation in sensitivity in the experimental system could modulate the observed responses. However, the linear nature of the semilogarithmic plot of the data minimizes that concern. Another concern is that the addition of “sensitized” fillers could lead to secretion of nonspecific factors that induce proliferation of bystander cells. This type of interference, should be minimal to LDA of primary responses, and is controlled in secondary responses by a constant density in all cultures. It should be noted that the estimated frequencies represent the contribution of the limiting cell type (antigenspecific T cell) to the original cell preparation cultured. In our experiments the cell preparation consisted of mononuclear cells from peripheral blood. Only about 4045% of these cells are of the T-helper phenotype which proliferates to soluble antigen ( 19). Therefore, estimates of the frequency of the antigen-reactive cells are at least off by a factor of about 2. Recently Eichmann and colleagues (20, 21) using a different technique for determining LDA frequencies have described three sets of T-cell precursors in frequencies of 1: 10 to 1:200, 1:200 to 1:3000, and less than 1:20,000. It appears that the T cells measured in our assay reflect the lower frequency set they describe. It will be of interest to see if the two sets with higher frequencies can be revealed when lower numbers of responding T cells are cultured in our system. Previously we reported accelerated kinetics of secondary responses to antigen after in vitro sensitization (2-3 days) relative to secondary responses of cells from individuals immunized in vivo (5-6 days). In the current investigations we asked if this could be explained by an increased frequency of antigen-reactive cells after in vitro primary sensitization. We found that although the frequency of KLH-reactive cells increased with time in primary culture (Fig. 6) about fourfold to 1:50,000, this was in the range of estimates from immune individuals ( 1:26,000-l :42,000). A loss of regulation was considered as a second alternative to explain the difference in kinetics. In initial experiments we observed a decrease in OKT8 cells during culture (Table 1) as has been reported previously (19). This appeared to account for the accelerated kinetics since immune cells cultured for 12 days without antigen subsequently responded in secondary culture with accelerated kinetics (3 days optimal, Fig. 7). We had the opportunity to compare the advent of antigen-specific cells in vitro (above) to their appearance in vivo during immunization (Fig. 4). The frequency of KLH-reactive cells increased from 1:25,000 to 1:54,000 during the observation period with a notable drop in frequency immediately after a booster dose of KLH. These results confirm those reported by Geha (10) who demonstrated a drop in frequency of tetanus toxoid-reactive cells after a booster dose followed by a increase to two and three times the prebooster level. In summary, we have demonstrated that the frequency of antigen-reactive cells in the peripheral blood is a reflection of the immune status of the cell donor. The measured frequency of reactive cells is increased by either immunization in vivo or sensitization in vitro. We have previously demonstrated (2) that the major histocompatibility complex-restricted nature of the secondary response to antigen is dictated by the HLA-DR phenotype of the antigen-presenting cells in primary culture. Utilizing LDA we are currently determining the frequency of KLHreactive cells that are able to respond to KLH presented by HLA-DR histoincompatible macrophages.
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FORD AND BURGER
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Ford, D. M., Hamblin, A., Dumonde, D., and Burger, D. R., Human Immunol. 4, 197, 1982. Ford, D. M., Hamblin, A., and Burger, D. R., J. Immunol. 128, 2 170, 1982. Rodey, G. E., Luehrman, L. K., and Thomas, D. W., J. Immunol. 123,2250, 1979. Ryser, J. E., and MacDonald, H. R., J. Immunol. 122, 1691, 1979. Swain, S. L., Panfili, P. R., Dutton, R. W., and Letkovits, I., J. Immunol. 123, 1062, 1979. van Oers, M. H. J., Pink&r, J., and Zeijlemaker, W. P., Eur. J. Immunol. 8, 477, 1978. Singal, D. P., Human Immunol. 1, 67, 1980. Waldmann, H., Letkovits, I., and Quintans, J., Immunology 28, 1135, 1975. Waldmann, H., and Pope, H., Immunology 31,343, 1976. Geha, R. S., Clin. Immunol. Immunopath. 19, 196, 1981. Sohnle, P. G., and Collins-Lech, C., J. Immunol. 127, 612, 198 1. Macy, E., and Stevens, R. H., J. Immunol. 124, 752, 1980. Gebel, H. M., Scott, J. R., Parvin, C. A., and Rodey, G. E., J. Immunol. 130, 29, 1983. Vandenbark, A. A., Yoshihara, P., Carveth, L., and Burger, D. R., Cell. Immunol. 60, 240, 1981. Burger, D. R., Vandenbark, A. A., Dunnick, W., Kraybill, W., Daves, G. D., and Vetto, R. M., J. Immunol. 122, 1091, 1979. Boyum, A., &and. J. Clin. Lab. Invest. 21, 3 1, 1968. Burger, D. R., Vandenbark, A. A., Finke, P., and Vetto, R. M., Cell. Immunol. 41, 62, 1977. Hensen, E. J., and Elferink, B. G., Nuture (London) 277, 223, 1979. Engleman, E. G., Benike, C. J., Grumet, F. C., and Evans, R. L., J. Immunol. 127, 2124, 1981. Melchets, I., Fey, K., and Eichmann, K., J. Exp. Med. 156, 1587, 1982. Hamann, V., Eichmann, K., and Krammer, P., J. Immunol. 130, 1983.
IMMUNOLOGY
Precursor
79, 334-344 (1983)
Frequency of Antigen-Specific T Cells: Effects of Sensitization in Viva and in Vitro DENNISFORD’
ANDDENISBURGER'
Surgical Research Laboratory, Veterans Administration Medical Center, and Department of Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201 Received February 16. 1983; accepted April 8, I983 Limiting dilution analysis (LDA) of primary lymphocyte cultures was used to determine the frequency of keyhole limpet hemocyanin (KLH)-specific precursors in the peripheral blood of unimmunized individuals. The KLH-specific precursor frequencies ranged from 1: 150,000 to 1:340,000. In contrast, frequencies of KLH-specific cells in the blood from immune donors ranged from 1:25,000 to 1:42,000. LDA of KLH-stimulated primary cultures indicated that the frequency of KLH-specificcells increased with time in culture reaching a four- to fivefold expansion relative to the frequency obtained prior to culture. The data presented suggest that the enhanced kinetics of secondary T-cell responses observed after in vitro sensitization are due to a decrease in the proportion of lymphocytes which exhibit a suppressor phenotype.
INTRODUCTION Previously we have described the primary sensitization of human peripheral blood mononuclear cells (MNC) to hemocyanins in vitro (1,2). After a 12- to 14&y primary culture period in the presence of keyhole limpet hemocyanin (KLH), recovered cells proliferate in a secondary fashion (2-3 days) upon restimulation with KLH. This response is antigen specific (l), macrophage dependent (2), and HLA-DR restricted (2, 3). The present report determines the frequency of KLH-specific precursors before and during in vitro sensitization to this antigen. The frequency of T-cell precursors that respond to specific alloantigens in both cytolytic and proliferative systems has been documented in mice (4, 5) and man (6, 7) by limiting dilution analysis (LDA). The range of these estimated frequencies ( 1Oe2to 1Oe3) has exceeded the range reported for T-cell precursors specific for soluble antigens (5, 6, 8-12) by at least an order of magnitude. Van Oers et al. (6) reported the frequency of tetanus toxoid-reactive T cells in immunized and boosted donors to vary from 1:750 to 1:11,500. Sohnle and Collins-Lech (11) found the frequency of streptokinase/streptodornase (SK/SD)-reactive T cells to range from 1: 1000 to 1:25,000 in immune donors. Other studies have predicted similar frequencies for T cells reactive to antigens encountered previously. However, the frequency of antigenreactive precursors in nonimmune donors has only been recently addressed (13). ’ Research performed in partial fulfillment of the requirements for the Doctor of Philosophy degree at the Oregon Health Sciences University. 2 To whom correspondence should be addressed: VA Medical Center, Surgical Research Lab, Portland, Oreg. 9720 1. 334 OOO8-8749/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
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In this investigation we obtained estimated frequencies of KLH-specific cells ranging from 1: 150,000 to 1:340,000 in the peripheral blood of KLH-nonimmune individuals. We also observed that immunization of donors with KLH resulted in increases in the frequency of KLH-specific cells in the peripheral blood. Moreover, during sensitization to IUH in vitro, the frequency of IUH-specific cells gradually increased with the length of time in the culture. MATERIALS
AND
METHODS
Antigens. The preparation of keyhole limpet hemocyanin was previously described (14). As prepared, the KLH elicited dermal reactivity and induced lymphocyte proliferation in KLH-immunized individuals and exhibited negligible dermal reactivity in normal nonimmune individuals ( 15). Streptokinase/streptodornase (Varidase) was obtained from Lederle Laboratories Division, Pearl River, New York. PPD (tuberculin purified protein derivative) was obtained from Connaught Laboratories Limited, Ontario, Canada (Batch CT68). Isolation of blood mononuclear cells. Mononuclear cells were separated from heparinized (10 u/ml) peripheral blood from normal donors (ages 20-40) by FicollHypaque (Pharmacia Fine Chemicals, Piscataway, N.J.) density gradient centrifugation (16). Interface cells were subsequently washed two or three times with RPM1 1640 (Gibco, Grand Island, N.Y.). Macrophage isolation. Cells ofthe mononuclear phagocytic series (M4) were isolated from MNC by adherence to plastic. Tissue culture flasks (No. 30 13, Falcon, Oxnard, Calif.) were pretreated for 30-60 min at 4°C with 50% RPMI-50% AB serum (not heat inactivated) and rinsed. MNC (40-60 X lo6 cells) were added to the pretreated flasks in RPM1 with 10% AB serum (heat inactivated, 56°C 30 min). After l-2 hr incubation at 37°C and 5% COZ, the flasks were vigorously rinsed with medium to remove nonadherent cells. Subsequently, cold RPM1 with 5% AB serum and 0.2% EDTA was added and the flasks were held for 20-30 min at 4°C. The macrophages were removed by shaking and vigorous pipetting, washed twice, and counted. Limiting dilution assay. For limiting dilution analyses of primary cultures, MNC were suspended in RPM1 with 15% autologous plasma or heat-inactivated AB serum at 1 X lo6 cells/ml. This suspension of cells was serially diluted to achieve concentrations of 5 X 105, 2.5 X 105, lo5 , and 5 X 104/ml. From these cell suspensions, 100 ~1 was distributed to flat-bottom wells of 96-well microplates (Linbro, Flow Labs Ltd.). For each cell concentration 24 wells were plated (6 control, 18 with antigen). In order to keep the cell density constant in all wells, sufficient irradiated (1200 R, r3’Cs source) autologous MNC were added in 100~~1aliquots to achieve a concentration of lo5 cells in every well. Antigens were added in lo-p1 quantities. For LDA of primary cultures, microplates were incubated at 37°C and 5% CO2 for 1 l-l 2 days. For the last 24 hr of culture, 1.0 &i [3H]thymidine (Amersham, sp act 6.7 &i/mmol) was added. The cells were harvested, and 13H]thymidine uptake was measured by liquid scintillation spectrometry. LDA of secondary cultures was carried out after either sensitization in vitro or immunization in vivo. In experiments involving in vitro sensitization, MNC isolated as described above from nonimmune cell donors were cultured in tubes (Falcon No. 3033) at a cell density of 1 X 106/ml (2 X 106/tube) in RPM1 supplemented with antibiotics (PenStrep, Gibco), 15% heat-inactivated AB serum, and antigen (25-50 pg/ml KLH or HCH). After 12-14 days in a humidified 5% CO? (37’C) atmosphere,
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the cells were washed twice and counted and the viability was assessed. The limiting dilution analysis of the secondary KLH response was then carried out as described above for primary cultures with fresh irradiated autologous MNC used as fillers. [3H]Thymidine incorporation was measured on Day 2 or 3 of secondary culture. In LDA of secondary responses after immunization in vivo, cell donors had either been exposed to the antigens environmentally (e.g., SK/SD or PPD) or actively immunized (e.g., intradermal injection of KLH). The protocol for these experiments was identical to that described above for primary cultures, except [3H]thymidine uptake was measured on Day 5 or 6 of incubation (17, 18). In limiting dilution experiments, the assumption was made that the constant cell density of lo5 cells/well provided for saturation of accessory cells (M6) thus making the presence of antigen-specific proliferating T cells the limiting factor in detecting a positive response. Upon application of Poisson statistics to the LDA, this assumption was borne out. That is, given a random and independent distribution of T cells among the wells, the number of antigen-specific T cells per well follows a Poisson distribution. By scoring cultures positive if the [3H]thymidine incorporation exceeded the mean of control cultures by two standard deviations, a frequency of positive wells was obtained at each cell concentration. Estimation of the frequency of specific T cells was then done with the use of the Poisson formula in which F, = (u’/Y!) X e-“, where F, is the probability of obtaining r specific T cells in a well when the average number of specific T cells per well is u at the cell concentration being considered. Assuming all wells with one or more antigen-specific T cells is scored positive, the fraction of negative wells is given by FO = CU. When u = 1, F,, = 0.37. Therefore, theoretically, when the average number of responding T cells per well is 1, 37% of the wells will be scored as negative. Extrapolation to this point in LDA gives a number of cells, the reciprocal of which represents the frequency of the antigen-specific T cells in question. The extrapolations in this report were done from lines fitted to data points by least squares analysis. Lines with an R value (correlation coefficient) greater than kO.80 and passing through or close to the origin were used, thus justifying the analysis by Poisson statistics. Figure 1 shows the counts per minute obtained in a representative LDA of a primary KLH culture. Indirect immunofluorescence. Either fresh MNC from peripheral blood or cells recovered from cultures after various time periods were suspended at 5 X lo6 cells/ ml in RPM1 with 5% fetal calf serum (Gibco). Monoclonal antibodies were added at optimal concentrations to 0.2-ml aliquots of the cell suspension. The antibodies employed were T28 (pan T, Peter Beverly), OKT4, OKT8, OKMl (Ortho Immunodiagnostics), 2.17 (anti-Ia), anti-K+ anti-L (Bethesda Research Labs), and MAC120 (Howard Ram. After a 30-min incubation on ice, during which the aliquots were vortexed twice, the cells were washed twice (at 4°C) with RPM1 (5% FCS) and then incubated for another 30 min with fluoresceinated goat anti-mouse Ig (Cappel). After two washes, cells were analyzed for specific cell surface fluorescence on a 5OH Cytofluorograf (Ortho). RESULTS Limiting Dilution Blood
Analysis
of the Frequency
of Antigen-Spebjic
T cells in Peripheral
The frequency of antigen-reactive cells estimated by LDA reflected the responsiveness of the donor to a particular antigen in all experiments. For example (Fig. 2 and Table
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.
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8
.
.
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I 0
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FIG. 1. A representative limiting dilution analysis of KLH-specific precursor frequency in a nonimmune donor is shown. The dashed lines represent the division between negative and positive cultures (two standard deviations from the mean of control cultures).
1) a frequency of 3.3 X 10e5, or 1:30,000, was obtained to SK/SD and 183,000 to PPD, whereas the frequency of cells responding to KLH, an antigen not encountered previously, was 1:247,000 (average of four experiments). There was marked t3H]thymidine uptake in the standard 6-day proliferation assay to SK/SD (eightfold stimulation), marginal responses of PPD (twofold stimulation), and negative responses to KLH. The variability inherent in the frequency determinations was estimated at + 1:50,000 for KLH by evaluating the precursor frequency at different times. In testing different individuals who had not been exposed to KLH, precursor frequencies ranged from 1: 150,000 to 1:338,000 (Fig. 3 and Table 1, five representative experiments) with an average of 1:260,000. The frequency of KLH-specific cells in KLH-immunized donors was estimated to be 1:42,000 (A.V.) and 1:32,000 (D.B.). In donor D.B. the frequency of SK/SD-specific cells was 1:23,000 (Fig. 3). As an additional control the combined frequency of cells reactive to two hemocyanins (KLH and HCH) was determined by culturing cells to both antigens (donor A.W. unimmunized). A predicted combined frequency of 1:86,000 was expected on the basis of the individual experimental frequencies of 1:229,000 for KLH and 1: 139,000 for HCH (Fig. 4, Table 1). The experimentally determined combined frequency was 1:90,000. The Influence of Donor Immunization on the Frequency of KLH-Specific Precursors in Peripheral Blood
Active immunization with KLH (500 pg by a subcutaneous route) resulted in a twofold increase in the number of KLH-specific precursors after 1 week (from 1:252,000 to 1: 105,000 Table 1, Fig. 5) and remained constant over a 3-week period ( 1: 130,000 and 1: 106,000, respectively). LDA 2 days after a 10-pg intradermal boost, showed a decrease to 1:2 15,000, presumably due to recruitment of specific cells from the pe-
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FORD AND BURGER TABLE 1 Antigen-Specific Precursor Frequencies Calculated from Lines Fitted to Limiting Dilution Analysis Data from Figs. 2-6 Antigen Figure
Donor
KLH
SK/SD
PPD
2
D.F.
1:293,000 1:200,000 1:203,000 1:150,000
1:30,000
1:83,000
KLH
SK/SD
1:338,000 1:311,000 1:251,000 1:247,000 1:200,000 1:42,000 I :32,000
I :23,000
3
B.W. so. R.J. A.G. D.F. A.V. D.B.
KLH 4
A.W.
1:229,000
HCH
HCH + KLH
1:139,000
1:90,000
KLH 5
6
R.J.
D.F. S.O. D.F.
1:251,000 1:105,000 (1 week) 1: 130,000 (2 weeks) 1:106,000 (3 weeks) I :2 15,000 (2 days postboost) 1:54,000 (2 weeks postboost) 1:26,000 (6 weeks postboost) KLH primary culture
KLH secondary culture
1:293,000 1:311,000 1:200,000
1:129,000 (8 days) 1:90,000 ( 10 days) 1:50,000 (12 days)
riphery in response to the challenge with antigen. Two and six weeks after the boost the IUH-specific precursor frequency had increased to 1:54,000 and 1:26,000, respectively (Fig. 5, Table 1). KLH-SpeciJic Precursor Frequency Increases during Primary Sensitization in Vitro
In comparison to active immunization, sensitization in vitro resulted in a steady increase of KLH-specific cells over the span of the primary culture period. After 12 days in primary culture an estimated frequency of 1:50,000 was obtained. This rep resented a 4-fold increase over the estimate (1:200,000) obtained from simultaneous primary cultures from the same donors without antigen (Fig. 6, Table I). The yield of cells harvested horn the primary culture was 62% of the original input. By harvesting cells at different time points during primary culture, changes in precursor frequency
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(x10-‘)
L. +...
\ \ \ 0
.
IO
FIG. 2. Limiting dilution analyses of KLH-specific precursors (four separate experiments) and a limiting dilution analysis of SK/SD- and PPD-specific precursors from a single donor (D.F.) are shown. Results are tabulated in Table 1. V V, KLH; we * *w, KLH; O---O, KLH; l - . -. 0, KLH; 0 - - - 0, PPD; m-.--m, SK/SD.
could be correlated to the culture interval. At 8 days a 2.3-fold increase had taken place (cell yield, 52%) and at 10 days a 3.5-fold increase was observed (cell yield, 72%, Fig. 6, Table 1). Accelerated Kinetics of Secondary Responsesto Antigen after Long-Term Culture is Due to a Loss of Nonspecific Suppression
In a previous communication (1) which described the methodology for in vitro primary sensitization to hemocyanins in detail, a distinct difference in kinetics was observed between KLH secondary responses after sensitization in vitro (2-3 days optimal) and after immunization in vivo (5-6 days optimal). From the data presented here it is evident that the frequency of KLH-specific cells at the beginning of both ADDED
5
I
MNC
IO
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20
Y 3 3 Y F ii? Y ae
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FIG. 3. Limiting dilution analysesofthe ICLH-specific precursor frequency from five nonimmune individuals (A**. A, B.W.; O-O, S.O.; o---O, A.G.; V---V, R.J.; O-.-.0, D.F.) and two KLH-immune individuals (A -. -. A, A.V.; w - * . - ‘I, D.B.) are shown. Additionally the SK-SD-specific precursor frequency for donor D.B. is shown (m --- n ). Results are tabulated in Table 1.
340
FORD AND BURGER ADDED
MNC
MO-‘)
FIG. 4. Limiting dilution analyses of IUH- (A A) and HCH-specific (0 - - - 0) precursor frequencies from donor A. W. are shown. Additionally the limiting dilution analysis of MNC that responds to a combination of KLH and HCH in cultures is shown (Cl- - -0). The results are tabulated in Table I.
types of secondary culture was essentially the same (1:40,000-l :50,000) and therefore could not account for the kinetic differences. The possibility that differences in regulatory elements in the secondary cultures could account for the altered kinetics was investigated. We found that cells recovered from 1Zday primary cultures showed a marked decrease in the T cells of the suppressor phenotype (OKT8+) as well as a decrease in macrophages and B cells. In the case of donor D.F. (Table 2), OKT8 cells constituted 10% of the T cells (OKT4 + OKT8) after 12 days in culture, compared to 40% of the T cells placed in culture. To evaluate this tentative explanation more directly, we attempted to alter the 6day optimal response of cells from immune donors by exposing them to a 12day culture period. If the hypothesis were correct, the la-day culture period would lead to a loss in regulatory control (loss of OKT8 and possibly other cells) and the response ADDED I
5
MNC IO
(~16~) 20
FIG. 5. The effects of immunization with KLH on the RLH-specific precursor frequency from donor R.J. are shown. Limiting dilution analyses were done prior to immunization (O-O), at weeks I (X---X),2(O-.-.),and3(U.--O)afterimmunization,andat2&ys(~---~),2weeks(A...A), and 6 weeks (A -. - - A) aRer booster immunization. The results are tabulated in Table 1.
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T CELLS
d)
FIG. 6. The effects of in vitro culture on KLH-specific precursor frequencies are shown. MNC were analyzed for KLH-specific precursors after 8, 10, and 12 days of culture in the presence of KLH. These frequencies are compared to KLH-specific precursor frequencies obtained from LDA prior to in vitro culture. -0) and 10 days Donor D.F., 0 days (A. . . A) and 8 days (0 -e-e 0); donor S.O., 0 days (0 -.(X - X); donor D.F., 0 days (A---A) and 12 days (0 - - - 0).
to recall antigen would be optimal at an earlier time in secondary culture. When cells from an SK/SD-immune donor were primed to KLH for 12 days, accelerated secondary culture kinetics to both KLH and SK/SD were observed (Figs. 7a, b). The kinetics of the SK/SD response are accelerated relative to the SK/SD response of fresh cells from the same donor. These studies suggest that the early 2- to 3-day optimal responses after in vitro sensitization can be accounted for by a nonspecific loss of regulation during 12 days of cell culture. DISCUSSION In the present report we have demonstrated that the frequency of antigen-specific cells in peripheral blood of man reflects the immune status of the cell donor. Differences in estimates reported by other laboratories appear to be due primarily to differences TABLE 2 Phenotypic Profiles of Mononuclear Cells before and after Long-Term Culture Expt 1 (donor D.E.)
Expt 2 (donor M.H.)
Cell cultured marker”
Fresh cells
Culturedb cells
Fresh cells
Cultured cells
T28 T4 T8 2.17 Ml
64.9’ 31.6 21.5 11.3 18.2
39.3 38.4 4.1 20.0 4.3
67.0 44.3 22.0 6.3 6.0
32.9 35.1 10.0 8.5 3.0
a MNC were cultured for 12 days in the presence of KLH. b MNC were cultured for 12 days in the presence of SK/SD. ‘Percentage of cells expressing the phenotypic marker: T28, panT; T4, T helper; T8, T suppressor; 2.17, IA; M 1, monocyte.
342
FORD AND BURGER 5 a) 4 c-
I s 4
6
b)
5 4/
j
/
p’-- o-Q-,-
P
Q
(fl<\
day
day 2
day 3
day 4
FIG. 7. The effects of long-term culture (12 days) in the presence of KLH (0- - -0), SK/SD (O---O), or no antigen (X. * .X) on the kinetics of T-cell proliferative responses to (a) KLH and (b) SK/SD. The kinetics of SK/SD and KLH responses of fresh MNC are also shown (A-A). The donor (D.F.) is nonimmune to KLH and immune to SK/SD.
in the experimental protocols and the relative immune state of the cell donors. Geha estimated approximately 1: 12,000 T cells reactive to tetanus toxoid (10). van Oers et al. (6) using MNC obtained a range of 1:300 to 1:16,000 for PPD-specific cells and 1:750 to 1: 11,500 for tetanus toxoid-reactive cells. Macy and Stevens estimated the frequency of T helpers that would support a measurable tetanus-specific IgG response to be 1:23,250 (12). Sohnle and CollinsLech, by measuring the first generation lymphocyte response, obtained estimates ranging from 1: 1000 to 1:22,700 MNC reactive to SK/SD (11). Our estimates agree with these ranges. For example we found that the frequency of SK/SD-reactive cells ranged from 1:23,000 to 1:30,000, and KLH-reactive cells ranged between 1:26,000 and 1:42,000 when immune donors were used. In testing several unimmunized individuals to hemocyanins, we found an average frequency of 1:260,000 MNC responsive to IUH measured on Day 12 of primary culture (Fig. 3). Repeated analyses of the same donor (Fig. 2) confirmed that variation in the estimates obtained at different times was small. The estimated frequency was 7- to lo-fold less than the frequencies estimated for recall antigens, We tested the reliability of these estimates by comparing computed to experimentally determined combined frequencies for two noncross-reactive hemocyanins (Fig. 4). The calculated combined frequency was 1:86,000 (KLH + HCH) whereas the experimentally determined frequency was 1:90,000. It is interesting to note that Gebel et al. in a recent report ( 13) found a range of frequencies of 1:24,000 to 1:53,000 for IUH in unimmunized donors. This range appears closer to those observed for recall antigens. At this time it is not known whether these differences could be explained by differences in the donor population, the antigen, or experimental conditions. In any case, it is important to note that the increases in frequency during in vitro exposure to antigen
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reported by Gebel et al. (13) are consistent with what we report here, although the absolute values are not. The LDA estimates obtained should not be considered absolute values since a variation in sensitivity in the experimental system could modulate the observed responses. However, the linear nature of the semilogarithmic plot of the data minimizes that concern. Another concern is that the addition of “sensitized” fillers could lead to secretion of nonspecific factors that induce proliferation of bystander cells. This type of interference, should be minimal to LDA of primary responses, and is controlled in secondary responses by a constant density in all cultures. It should be noted that the estimated frequencies represent the contribution of the limiting cell type (antigenspecific T cell) to the original cell preparation cultured. In our experiments the cell preparation consisted of mononuclear cells from peripheral blood. Only about 4045% of these cells are of the T-helper phenotype which proliferates to soluble antigen ( 19). Therefore, estimates of the frequency of the antigen-reactive cells are at least off by a factor of about 2. Recently Eichmann and colleagues (20, 21) using a different technique for determining LDA frequencies have described three sets of T-cell precursors in frequencies of 1: 10 to 1:200, 1:200 to 1:3000, and less than 1:20,000. It appears that the T cells measured in our assay reflect the lower frequency set they describe. It will be of interest to see if the two sets with higher frequencies can be revealed when lower numbers of responding T cells are cultured in our system. Previously we reported accelerated kinetics of secondary responses to antigen after in vitro sensitization (2-3 days) relative to secondary responses of cells from individuals immunized in vivo (5-6 days). In the current investigations we asked if this could be explained by an increased frequency of antigen-reactive cells after in vitro primary sensitization. We found that although the frequency of KLH-reactive cells increased with time in primary culture (Fig. 6) about fourfold to 1:50,000, this was in the range of estimates from immune individuals ( 1:26,000-l :42,000). A loss of regulation was considered as a second alternative to explain the difference in kinetics. In initial experiments we observed a decrease in OKT8 cells during culture (Table 1) as has been reported previously (19). This appeared to account for the accelerated kinetics since immune cells cultured for 12 days without antigen subsequently responded in secondary culture with accelerated kinetics (3 days optimal, Fig. 7). We had the opportunity to compare the advent of antigen-specific cells in vitro (above) to their appearance in vivo during immunization (Fig. 4). The frequency of KLH-reactive cells increased from 1:25,000 to 1:54,000 during the observation period with a notable drop in frequency immediately after a booster dose of KLH. These results confirm those reported by Geha (10) who demonstrated a drop in frequency of tetanus toxoid-reactive cells after a booster dose followed by a increase to two and three times the prebooster level. In summary, we have demonstrated that the frequency of antigen-reactive cells in the peripheral blood is a reflection of the immune status of the cell donor. The measured frequency of reactive cells is increased by either immunization in vivo or sensitization in vitro. We have previously demonstrated (2) that the major histocompatibility complex-restricted nature of the secondary response to antigen is dictated by the HLA-DR phenotype of the antigen-presenting cells in primary culture. Utilizing LDA we are currently determining the frequency of KLHreactive cells that are able to respond to KLH presented by HLA-DR histoincompatible macrophages.
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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Ford, D. M., Hamblin, A., Dumonde, D., and Burger, D. R., Human Immunol. 4, 197, 1982. Ford, D. M., Hamblin, A., and Burger, D. R., J. Immunol. 128, 2 170, 1982. Rodey, G. E., Luehrman, L. K., and Thomas, D. W., J. Immunol. 123,2250, 1979. Ryser, J. E., and MacDonald, H. R., J. Immunol. 122, 1691, 1979. Swain, S. L., Panfili, P. R., Dutton, R. W., and Letkovits, I., J. Immunol. 123, 1062, 1979. van Oers, M. H. J., Pink&r, J., and Zeijlemaker, W. P., Eur. J. Immunol. 8, 477, 1978. Singal, D. P., Human Immunol. 1, 67, 1980. Waldmann, H., Letkovits, I., and Quintans, J., Immunology 28, 1135, 1975. Waldmann, H., and Pope, H., Immunology 31,343, 1976. Geha, R. S., Clin. Immunol. Immunopath. 19, 196, 1981. Sohnle, P. G., and Collins-Lech, C., J. Immunol. 127, 612, 198 1. Macy, E., and Stevens, R. H., J. Immunol. 124, 752, 1980. Gebel, H. M., Scott, J. R., Parvin, C. A., and Rodey, G. E., J. Immunol. 130, 29, 1983. Vandenbark, A. A., Yoshihara, P., Carveth, L., and Burger, D. R., Cell. Immunol. 60, 240, 1981. Burger, D. R., Vandenbark, A. A., Dunnick, W., Kraybill, W., Daves, G. D., and Vetto, R. M., J. Immunol. 122, 1091, 1979. Boyum, A., &and. J. Clin. Lab. Invest. 21, 3 1, 1968. Burger, D. R., Vandenbark, A. A., Finke, P., and Vetto, R. M., Cell. Immunol. 41, 62, 1977. Hensen, E. J., and Elferink, B. G., Nuture (London) 277, 223, 1979. Engleman, E. G., Benike, C. J., Grumet, F. C., and Evans, R. L., J. Immunol. 127, 2124, 1981. Melchets, I., Fey, K., and Eichmann, K., J. Exp. Med. 156, 1587, 1982. Hamann, V., Eichmann, K., and Krammer, P., J. Immunol. 130, 1983.