CLINICAL AND DLAGNOSTIC LABORATORY IMMUNOLOGY, May 1994, p. 261-268 Vol. 1, No. 31071-412X/94/$04.00+0Copyright X 1994, American Society for Microbiology

In Vitro Cytokine Production by Normal Human Peripheral BloodMononuclear Cells as a Measure of Immunocompetence

or the State of ActivationDIANA FRIBERG,1 JOHN BRYANT,12 WILLIAM SHANNON,3 AND THERESA L. WHITESIDE'4*

Departments of Pathology,4 Mathematics,2 and Biostatistics,3 University of Pittsburgh, and Pittsburgh Cancer Institute,'Pittsburgh, Pennsylvania 15213

Received 23 November 1993/Accepted 2 February 1994

Measurements of cytokine levels in serum may not adequately reflect the cytokine-producing potential ofimmune cells because of the short half-lives of cytokines and the presence of various inhibitors in human sera.In vitro cytokine production by peripheral blood mononuclear cells (PBMCs) can be an important and reliablemeasure of immunocompetence. Also, spontaneous in vitro release of cytokines by PBMCs may serve as ameasure of their activation in vivo. In the present study, normal ranges for the in vitro production by PBMCsof interleukin-1l (IL-1i), tumor necrosis factor alpha (TNF-o), IL-2, and gamma interferon (IFN--y) wereestablished; the feasibility of using cryopreserved PBMCs for assays of in vitro cytokine production wasevaluated; and spontaneous (unstimulated) versus induced production of cytokines by fresh and cryopreservedPBMCs from healthy donors was compared. Supernatants obtained from paired fresh and frozen PBMCs werequantitated for IL-1", TNF-ot, IL-2, and IFN-,y by using enzyme-linked immunosorbent assay or a radioim-munoassay standardized against World Health Organization cytokine standards. Fresh or cryopreservedPBMCs activated with lipopolysaccharide produced comparable levels of IL-1p. However, the mean levels ofstimulated production of TNF-ot, IFN-,y, and IL-2 were significantly higher in cryopreserved versus freshPBMCs (P . 0.0004). Correlations between the level of production of each cytokine by fresh versuscryopreserved in vitro-stimulated PBMCs were statistically significant, although of moderate magnitude.Spontaneous cytokine release by fresh versus cryopreserved cells was not significantly different. No correlationwas observed between the TNF-oa and IL-1p or the IL-2 and IFN--y levels produced by PBMCs from healthydonors, indicating that in vitro production of each cytokine is independently regulated. Normal ranges forlevels of all four cytokines were established by using PBMCs from up to 60 healthy individuals. The use ofbatched cryopreserved rather than fresh PBMCs for the evaluation of spontaneous and induced cytokineproduction is more convenient, economical, and reliable for the serial monitoring of clinical trials. The biologicand clinical usefulness of serial cytokine production assays is illustrated in patients with advanced cancer whoreceived therapy with biologic response modifiers.

Cytokines are low-molecular-mass glycoproteins producedby immune cells and many other cell types in response tovarious activation stimuli (1, 3). Cytokines are not only partic-ipants in inflammation, immunity, and the host response toinfectious agents but are also involved in the regulation ofmany biologic systems, including the immune system. They areimplicated in normal homeostasis and defense against manydiseases and may contribute to the pathogenesis of a variety ofdisorders (10, 17). Abnormal cytokine production (i.e., either adeficiency or an excess) often accompanies and contributes topathologic conditions, including infectious diseases, cancer,autoimmunity, and allergy. Thus, when monitoring the immu-nocompetence of patients with immune-mediated disorders orpatients undergoing treatment with biologic response modifi-ers, it becomes particularly important to evaluate their abilityto produce appropriate levels of cytokines (4).Changes in the equilibrium of the immune system are likely

to be reflected by alterations in lymphokine and cytokineproduction and their levels in serum, plasma, or other bodyfluids. Hence, the usual approach to demonstrating thesechanges has been to measure cytokine levels in body fluids byimmunoassays or bioassays (5, 7, 9). However, cytokines are

* Corresponding author. Mailing address: Pittsburgh Cancer Insti-tute, W1041 Biomedical Science Tower, 200 Lothrop St., Pittsburgh,PA 15213. Phone: (412) 624-0096. Fax: (412) 624-0264.

261

short-range mediators, and their half-lives in the circulationare very short (1, 3). The presence of soluble receptors forcytokines, receptor antagonists, anti-cytokine antibodies, andother inhibitors further complicates cytokine detection or assayinterpretation (for a review, see reference 18). We thereforeevaluated an alternative approach to monitoring cytokines inhealthy individuals and patients with various disorders. Bymeasuring the production and release into supernatants ofcytokines by peripheral blood mononuclear cells (PBMCs) inthe presence or absence of exogenous stimulators, it is possibleto evaluate the immunocompetence or activation states ofthese cells, respectively. Since the cytokine production profilecan be established for PBMCs from healthy individuals, it isreasonable to expect that qualitative or quantitative changes inthis profile of cells obtained from the circulation (or thoseisolated from tissue sites or body fluids) of patients may beindicative of systemic (or local) dysregulation. Our hypothesisis that cytokine production by PBMCs is a more reliablemeasure of an individual's immunocompetence. To be able totest this hypothesis, we first evaluated cytokine production byPBMCs from healthy donors in order to establish the normalranges for spontaneous and induced cytokine release. We thendemonstrated that PBMCs obtained from cancer patientstreated with biological response modifiers have a distinctlydifferent cytokine production profile from that at the baselineand that the ability of PBMCs to produce cytokines in vitro

CLIN. DIAGN. LAB. IMMUNOL.

may reflect the altered functional states of these cells as a

result of immunotherapy.

MATERIALS AND METHODS

PBMC isolation. Heparinized venous blood was obtainedfrom healthy individuals or patients with cancer. PBMCs were

isolated on Ficoll-Hypaque gradients, washed twice with phos-phate-buffered saline, and resuspended in complete RPMImedium (CM) containing 20 mM L-glutamine, 100 U ofpenicillin per ml, 100 ,ug of streptomycin per ml, and 5%(vol/vol) fetal calf serum (FCS). All reagents were purchasedfrom GIBCO, Grand Island, N.Y., and were checked forendotoxin contamination by the Limulus amebocyte lysateassay (Whittaker Bioproducts, Walkersville, Md.). The PBMCswere either used immediately, i.e., they were tested as freshcells, or cryopreserved for future testing. For cryopreservation,the PBMCs were suspended (5 x 106 to 20 x 106/ml) inprecooled Dulbecco's minimal essential medium containing20% (vol/vol) FCS and 10% (vol/vol) dimethyl sulfoxide(DMSO). This cell suspension was divided into aliquots,placed into precooled freezing vials (Nunc), and immediatelycryopreserved by using a controlled-rate liquid N2 freezer.Vials containing 1-ml aliquots were stored in liquid N2 vapor.

Cells were thawed immediately before the assays by thefollowing procedure. Each vial was transferred from liquid N2to a 37°C water bath and was gently rotated for 1 or 2 min. Assoon as the cell suspension in the vial was thawed, it was

rapidly but gently pipetted into a 50-ml plastic conical tube.Next, 10 ml of medium containing 25% (vol/vol) FCS was

added dropwise with continuous gentle mixing to minimize thetoxic effects of DMSO. The cells were washed twice in mediumcontaining 10% (vol/vol) FCS, counted in the presence of a

trypan blue dye, and resuspended in CM for measurements ofcytokine production.

In vitro cytokine production. Cryopreserved and thawed orfreshly isolated PBMCs were adjusted to 106/ml in CM anddispensed at 100 ,u per well into wells of 96-well, round-bottom culture plates. To measure the production of interleu-kin-13 (IL-1>) and tumor necrosis factor alpha (TNF-a),aliquots of 100 ,ul (per well) of CM alone (for spontaneousrelease) or 100 RI (per well) of lipopolysaccharide (LPS;Escherichia coli 026:B6; Sigma, St. Louis, Mo.) at a concentra-tion of 10 ,ug/ml (for stimulated release) were added to wellscontaining PBMCs, and the plates were incubated for 24 h. Forthe detection of gamma interferon (IFN--y) and IL-2, aliquotsof 100 pAl of CM alone per well or 100 pI of phytohemagglu-tinin-P (PHA-P; Sigma) at a concentration of 20 ,ug/ml per wellwere added and were incubated with the PBMCs for 48 h. Thefinal concentrations of LPS (5 ,ug/ml) and PHA-P (10 ,ug/ml)used were previously shown to be optimal for proliferation ofhuman PBMCs in 3-day cultures. The optimal incubation timeswere determined in preliminary experiments in which LPS- or

PHA-P-induced cytokine production was measured after 24,48, and 72 h of incubation. The supernatants were harvestedfrom all wells and were stored at -70°C for future testing.Immunoassays. All cytokine measurements were done by

using commercially available reagent kits, as follows. TNF-awas measured by an enzyme-linked immunosorbent assay

(ELISA) purchased from T-Cell Sciences, Inc. (Cambridge,Mass.), and the sensitivity of the assay was .0.02 ng/ml. IFN--ywas measured by a radioimmunoassay (RIA) obtained fromCentocor (Malvern, Pa.); the sensitivity of the RIA was .1U/ml. IL-1lB levels were assayed by an ELISA purchased fromCistron Corp. (Pine Brook, N.J.). The sensitivity of this ELISAkit was .0.02 ng/ml. IL-2 levels were measured by an ELISA

purchased from Collaborative Research, Bedford, Mass., witha sensitivity of .3 U/ml.

All cytokine assays were performed at the Pittsburgh CancerInstitute's Immunologic Monitoring and Diagnostic Labora-tory, which maintains a rigorous quality assurance and controlprogram. As applied to cytokine testing, this program includedthe following procedures. All cytokine assays were calibratedagainst the World Health Organization's International Cyto-kine Standards (National Institute for Biologic Standards,London, United Kingdom, and the Biological Response Mod-ifier Program, Frederick, Md.). Interassay variability was mon-itored by the use of cytokine-rich supernatants, which wereprepared by the laboratory in bulk (by in vitro activation ofnormal PBMCs with PHA-P or LPS) every several months,stored (-80°C) in small aliquots, and included in every assay.Comparisons of the different lots of such supernatants weremade prior to their acceptance for such monitoring of inter-assay variability.

Intra-assay variability was monitored by incorporating blindduplicates of control samples in different positions each timethat the assay was performed.The laboratory has established its own criteria for accep-

tance and rejection of each assay on the basis of within-run andbetween-run precision measurements obtained for each assay.Precision was determined by assaying the same control speci-mens in each of at least 30 tests run over a period of time andby computing the means and standard deviations of test values.If the controls included with each experiment were outside ofthe established mean ± 2 standard deviations for a givencytokine, the assay was rejected.

Healthy individuals and patients. Healthy donors of PBMCs(ages, 25 to 50 years) were recruited from among laboratorypersonnel. Both males and females were represented equally.The PBMCs obtained from these donors were generally cryo-preserved for cytokine production assays except when freshversus cryopreserved cells were being compared. Patients withcancer (ages, 30 to 75 years) whose PBMCs were obtained forthe studies described here were participants in phase I clinicaltrials at the Pittsburgh Cancer Institute. The blood sampleswere obtained from patients with cancer at times specified inthe protocol schema. PBMCs were cryopreserved, and cellswere collected serially from each patient were thawed andevaluated together for cytokine production. The immunoas-says performed were a part of immunologic monitoring re-quired by the protocol and approved by the InstitutionalReview Board. All patients signed an informed consent forparticipation in the study.

Statistics. The levels of cytokines produced by fresh orcryopreserved PBMCs were compared as follows. For eachcytokine, the findings were confirmed in two separate experi-ments, in each of which multiple normal donors contributedblood samples that were split to obtain paired samples of freshand cryopreserved PBMCs. Stimulated production was statis-tically analyzed following logarithmic transformation to inducethe symmetry of distributions. Spontaneous production wasanalyzed by nonparametric methods, because the observedlevels were too close to the limits of assay sensitivity to bereliably transformed to normality.For in vitro-stimulated cytokine production, a preliminary

test of method (fresh versus cryopreserved)-by-experimentinteraction was carried out by parametric analysis of variance.Since no evidence of such an interaction was seen for any ofthe four cytokines, differences in the levels of fresh andcryopreserved PBMCs were tested by using the within-experi-ment donor-by-method interaction as the error term. Consis-tent results were also obtained by nonparametric methods.

262 FRIBERG ET AL.

CYTOKINE PRODUCTION BY HUMAN PBMCs 263

4---------'--------------------------E

Mean = 402 sd. - 5C.V. 6.68

30.

Days of Test

FIG. 1. Interassay variability of IFN-y immunoassay monitoredwith aliquots of the supernatant obtained in a large volume (200 ml)from PBMCs from healthy humans stimulated with PHA-P. Thesupernatant was divided into aliquots and placed into 1-ml vials, andthe vials were stored frozen at -80°C. Each time the assay was run, an

aliquot of the supernatant was included in the assay. s.d., standarddeviation; C.V., coefficient of variation.

For spontaneous production, method-by-experiment inter-action was tested by a Wilcoxon rank sum analysis of within-donor differences. Differences between fresh versus frozenPBMCs that were pooled over experiments were tested forsignificance by the signed rank test.

All correlations cited below are Spearman correlations andare corrected for possible between-experiment effects.

RESULTS

Quality control for cytokine assays. To measure the levels ofcytokines in cellular supernatants reliably, we not only stan-dardized the ELISA or RIA used against the World HealthOrganization's International Cytokine Standards but also mon-itored interassay and intra-assay variabilities. To determine theformer, we generated a cytokine-rich supernatant (100 ml) ofhealthy PBMCs (106/ml) stimulated with PHA-P (20 ,ug/ml for48 h), stored it frozen (-80°C), and assayed it each time thatan IFN--y or IL-2 immunoassay was performed. Similarly, wegenerated another cytokine-rich supernatant by stimulation ofnormal PBMCs with LPS (10 ,ug/ml for 24 h) and used it inTNF-a and IL-11 immunoassays. Figure 1 shows an exampleof the curve generated when the control supernatant was

repeatedly tested over a period of 3 months to monitor theinterassay variability of the RIA for IFN--y. This type of a

control curve is used to judge the performance of the RIA ona daily basis. On days when the control supernatant was notwithin the established mean ± 2 standard deviation range,results of the RIA were considered unacceptable and the assaywas rejected. The intra-assay variability was measured byincluding the control supernatant in two different positions inthe assay. The coefficient of variation for intra-assay variabilitywas <5%. Similar quality control measures were used for theother cytokine immunoassays: the interassay coefficient ofvariation for the IL-2 ELISA was 19%, that for the IL-11ELISA was 20%, and that for the TNF-a ELISA was 19%.These interassay coefficients of variation were determined in atleast 30 different independent assays performed by differenttechnologists over a period of more than 6 months.

In vitro cytokine production by healthy human PBMCs. To

establish normal control ranges for spontaneous and inducedcytokine production, cryopreserved PBMCs obtained from

healthy volunteers were thawed and incubated in medium ormedium plus stimulators (PHA-P or LPS) for 24 or 48 h.Supernatants were harvested and tested for levels of IL-2,IFN--y, TNF-a, or IL-13 in the respective immunoassays. Asshown in Fig. 2A to D, the middle 80% range of spontaneousproduction of TNF-a by healthy PBMCs was 0 to 0.4 ng/ml,that of IL-1r3 was 0 to 0.09 ng/ml, that of IL-2 was 0 to 3 U/ml,and that of IFN--y was 0 to 15 U/ml. The middle 80% ranges ofinduced cytokine production for healthy PBMCs were muchhigher: TNF-a, 0.5 to 2.63 ng/ml; IL1-,, 0.9 to 9.91 ng/ml; IL-2,4 to 37 U/ml; and IFN--y, 31 to 442 U/ml (Fig. 2A to D).Comparison of cytokine production by fresh versus cryopre-

served PBMCs. To determine whether cryopreserved PBMCscould be reliably used for cytokine production assays, sponta-neous and induced cytokine production was measured inpaired (split) samples of fresh and cryopreserved PBMCsobtained from healthy individuals, and the data were analyzedas described in Materials and Methods. No significant differ-ence in LPS-induced production between fresh and cryopre-served PBMCs was observed for IL-113 (Table 1). On the otherhand, stimulated production of TNF-ot, IL-2, and IFN--y wassignificantly higher (P - 0.0004) for cryopreserved PBMCsthan freshly isolated PBMCs (Table 1). The spontaneousrelease of these four cytokines was not significantly different incryopreserved versus fresh PBMCs (Table 1).To further examine the relationship between IL-2 produc-

tion by fresh and cryopreserved PHA-P-stimulated PBMCs, wedetermined a degree of correlation between IL-2 levels pro-duced by paired fresh or frozen PBMCs (Fig. 3A). In 23 of 25donors, IL-2 production by cryopreserved PBMCs exceededthat by fresh PBMCs, as indicated by the fact that all but twopoints in Fig. 3A lie below and to the right of the line which isdrawn to show equal levels of IL-2 production by fresh orfrozen paired samples. On the other hand, we observed amoderate correlation (R = 0.70), which was statistically signif-icant at P = 0.0001, between IL-2 levels made by frozen versuscryopreserved PBMCs. Similar results (data not shown) wereobtained for the stimulated production of IFN-y (R = 0.42; P= 0.04) and TNF-ot (R = 0.40; P = 0.03). These data suggestthat although, as a group, cryopreserved cells tend to secretemore cytokines than fresh cells, high-level producers producedhigh levels of cytokines and low-level producers produced lowlevels of cytokines for both fresh and cryopreserved cells.Figure 3B shows that LPS-induced production of IL-1lB byboth fresh and cryopreserved PBMCs (R = 0.57; P = 0.001)showed no tendency for production by cryopreserved cells toexceed that by the paired fresh cells.To explain the observed differences in TNF-a, IL-2, or

IFN--y production between fresh and frozen PBMCs, weconsidered the possibility that during the freezing and thawingprocess the composition of the PBMCs was altered. To test thispossibility, we compared cell differentials of fresh versus frozenand thawed PBMCs by flow cytometry (Fig. 4A) and auto-mated differential cell counting (Fig. 4B). We found that thepercentages of lymphocytes, monocytes, and granulocytes werevirtually identical in fresh and paired frozen cell preparations.Our results (data not shown) indicated that there were no

differences in the proportions of CD3+, CD4+, CD8+, CD56+,CD16', CD19+, or CD25+ cells between the fresh and thecryopreserved paired samples. These results indicate that theproportions of various leukocytes or lymphocyte subsets are

not altered by cryopreservation of PBMCs, although the mean± standard deviation cell recovery after cryopreservation was

56% ± 15% for healthy PBMCs. The viability of cryopreservedcells was >95% by microscopy in the presence of trypan blue.A lack of concordance in production of various cytokines by

VOL. 1, 1994

264 FRIBERG ET AL.

TNF alpha Normal Controls - Spontaneous Production

N =49Mean ± s.d. ng/ml = 0.13 ± 0.2Middle 80% Range = 0 - 0.4

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FIG. 2. Normal values for spontaneous or stimulated cytokine production by cryopreserved PBMCs obtained from healthy volunteers agedbetween 25 and 50 years. (A) TNF-a; (B) IL-1a; (C) IL-2; (D) IFN--y. Cytokine levels were measured as described in the text; n = number ofindividuals; s.d., standard deviation.

PBMCs. Our results (Fig. 2) indicated that PBMCs obtainedfrom healthy individuals differ considerably in their ability toproduce various cytokines in response to in vitro stimulationwith PHA-P or LPS. To further investigate whether productionof various cytokines by PBMCs from healthy individuals was

concordant, we compared the levels of IL-2 and IFN--y as wellas those of IL-l1 and TNF-ot produced by in vitro-activated

PBMCs. No concordance between TNF-ot and IL-13 levelsproduced by PBMCs from the same individuals could bedemonstrated (for fresh PBMC, R = 0.13 and P = 0.48; forfrozen PBMCs, R = 0.21 and P = 0.27). These data indicatethat, as expected, the production of each cytokine is indepen-dently regulated at the cellular level in healthy PBMCs. Thus,individuals whose cells can be induced to produce high levels

TABLE 1. Cytokine production by fresh or cryopreserved PBMCs from healthy human volunteersg

PBMC IL-1f concn (ng/ml) TNF-a concn (ng/ml) IL-2 concn (U/ml) IFN-y concn (U/ml)(n = 30) (n = 30) (n = 25) (n = 25)

SpontaneousFresh 0.03 + 0.006 0.13 ± 0.023 0.4 + 0.3 1.0 ± 0.5Cryopreserved 0.065 ± 0.019 0.17 + 0.038 0.4 ± 0.4 0.4 ± 0.3

Difference -0.035 + 0.02 -0.044 ± 0.034 0 ± 0.5 0.6 ± 0.6P value 0.07 0.19 1.00 0.25

StimulatedFresh 4.1 ± 0.2 1.7 ± 0.2 14 ± 3 133 ± 24Cryopreserved 4.0 ± 0.2 2.1 ± 0.3 27 + 4 291 ± 34

Difference 0.15 ± 0.23 -0.42 ± 0.14 -13 ± 3 -158 ± 35P value 0.47 0.0004 0.0001 0.0001

a Data are means + standard errors of the mean. P values were calculated by the sign rank test.

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CYTOKINE PRODUCTION BY HUMAN PBMCs 265

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FIG. 3. Correlations between the level of cytokine production byfresh (Frsh. Stim.) versus cryopreserved stimulated (Froz. Stim.)PBMCs. (A) Comparison of levels of IL-2 production (log scale) forfresh versus frozen cells from 26 normal individuals. White circles,results of the first experiment; black circles, results of the secondexperiment. A solid line is drawn at equal levels of IL-2 production.For 23 of 25 donors, production of IL-2 by cryopreserved cellsexceeded that by fresh cells (fresh versus cryopreserved, R = 0.69; P =

0.0001). (B) LPS-induced production of IL-lp for fresh (Frsh. Stim.)versus cryopreserved stimulated (Froz. Stim.) PBMCs from 30 donorswas compared (R = 0.57; P = 0.001). Symbols are as described abovefor panel A.

of TNF-a in vitro do not generally produce high levels ofIL-1a. Similar results were obtained for PHA-P-induced IL-2and IFN-y production, with R = 0.28 for cryopreserved cells(not significant) and R = 0.48 and P = 0.02 (marginallysignificant) for fresh cells.

Cytokine production by PBMCs of patients treated withbiologic response modifiers. To validate in vitro production ofcytokines by PBMCs as a reliable and biologically importantassay, we obtained PBMCs from cancer patients treated withbiological response modifiers in three different phase I proto-cols. In the first instance, 14 male and 8 female patients (ages,31 to 75 years) with stage III/IV recurrent or metastatic diseasewere treated with picibanil (OK-432). The drug was adminis-tered in escalating doses (0.1, 0.5, 1, 2, and 3 mg) to groups offour to five patients each (11). Cytokine production (IL-1p,TNF-a, and IL-2) by PBMCs collected pretreatment and

posttreatment at weeks 1, 2, 4, and 5 as well as at months 2 and3 was measured. On the basis of the Mack-Skillings nonpara-metric repeated measures test, statistically significant overalltreatment effects of picibanil were demonstrated (P < 0.01)only for stimulated IL-2 production by PBMCs (Fig. 5; the Pvalues in Fig. 5 were not adjusted for multiple end points).Increased production over baseline levels was accompanied byother drug-induced immunomodulatory changes, includingincreases in CD56+ CD16+ natural killer (NK) cells and inlectin- or alloantigen-dependent proliferation (11). Thus, sta-tistically significant changes from the baseline in PBMC IL-2production were induced by picibanil in patients with advancedcancer, and they were accompanied by other immunostimula-tory effects of picibanil in this phase IA study.

In another clinical trial, patients with metastatic melanomaor renal cell carcinoma were given adoptive immunotherapywith autologous activated NK cells (A-NK cells) and IL-2 (19).Cytokine levels in serum were serially measured, as was theability of the patients' PBMCs to produce cytokines. For onepatient, these cells obtained prior to and at various time pointsduring the course of therapy did not spontaneously produceIFN--y (Fig. 6), even though the patient was receiving treat-ments with multiple cycles of IL-2 at a dose of 18 x 106IU/m2/day x 5. On the other hand, PHA-P-stimulated produc-tion of IFN--y by the patient's PBMCs was detectable betweenthe 5-day courses of IL-2 and rapidly declined at the time ofIL-2 administration. No IFN--y was detected in the serum ofthis patient at any time during therapy.

Finally, patients with advanced cancer were treated withrecombinant TNF, which was infused systemically for 5 daysfor four courses given 2 weeks apart at doses of 80, 160, 200,and 240 p,g/m2, and the PBMCs were harvested from thosepatients and assayed serially for IL-2 production after PHA-Pstimulation in vitro. As shown in Fig. 7, the mean level of IL-2production by the PBMCs from these patients was statisticallysignificantly elevated (P < 0.01) immediately after TNF infu-sions, but decreased to near baseline levels between theinfusions.

DISCUSSION

Cytokines are involved in multiple aspects of physiology andpathology and are being used clinically (4). In view of theincreasing awareness of the key role of cytokines in a variety ofhuman diseases, assays for cytokine quantification have be-come a desirable and rapidly expanding part of the laboratoryrepertoire. The choice of an appropriate cytokine assay amongthe bioassays and immunoassays available depends, to a largeextent, on the clinical or research objective to be achieved.Thus, it may be important to measure cytokine pharmacoki-netics in the sera of patients receiving cytokine therapy. On theother hand, the assessment of patients with various immuno-deficiencies is likely to require information about their abilityto produce cytokines. In vitro cytokine production by PBMCscan be used as an assay of immunocompetence, because itmeasures the ability of an individual's cells to produce cyto-kines in response to an exogenous activation signal. In evalu-ating the immunocompetence of patients with various diseases,it is important to determine whether their PBMCs are capableof making and releasing cytokines comparably to normalPBMCs in response to the same in vitro stimulators. The assayis applicable to cytokine measurements with whole blood,separated PBMCs, or isolated and purified subsets of mono-

nuclear cells.When used to monitor immunologic responses following

treatment or during therapy with immunomodulating agents,

VOL. 1, 1994

CLIN. DIAGN. LAB. IMMUNOL.

A FRESH CRYOPRESERVED

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FIG. 4. Flow cytometry of fresh versus cryopreserved and thawed healthy PBMCs to show an almost identical gating profile of both populations.(A) Percentages of different leukocyte populations determined by automated cell counting. Among the fresh cells, there were 79% lymphocytes,17% monocytes, and 2% granulocytes. Among the cryopreserved cells, there were 80% lymphocytes, 17% monocytes, and no granulocytes. (B)Percentages of lymphocytes (Lymphs) and monocytes (Monos) in fresh PBMCs compared with those of cryopreserved PBMCs for 15 differenthealthy volunteers. SSC, side scatter; FSC, forward scatter.

the assay for in vitro cytokine production may be moreinformative than the widely used measurements of cytokinelevels in serum. Cytokine production in vitro does not seem tobe influenced by cytokine inhibitors (12), which are commonlypresent in patients' sera in the form of soluble cytokinereceptors (14, 15), receptor antagonists (2, 6), or anticytokineantibodies. In addition, the assay can also provide informationabout the degree of in vivo activation of PBMCs. Properlyhandled, these cells should be capable of spontaneous cytokineproduction in vitro only as a consequence of their previous invivo activation. In patients undergoing treatment with biolog-ical response modifiers, in which the in vivo activation ofimmune cells is one of the major goals, spontaneous in vitrorelease of cytokines by serially tested PBMCs is likely toprovide evidence for the in vivo immunostimulatory effects of

the drug or for its ability to down-regulate selected functions,much as shown by us for spontaneous IFN-,y production (Fig.6) by PBMCs from patients with cancer treated systemicallywith autologous A-NK cells plus IL-2 (19). We have selectedand presented examples of the clinical use of the cytokineproduction assay performed with cells from patients withcancer receiving various immunotherapies (11, 13, 19). Theassay appears to be at least as sensitive a marker of the changesinduced in circulating mononuclear cells by immunotherapy asother more conventional immunoassays are. In fact, the assaymay turn out to be more selective, in that production of onlyone of several cytokines was significantly altered by a givendrug; i.e., only IL-2 production was induced by picibanil (Fig.5); only IFN--y production was induced by systemic IL-2 plusA-NK cell therapy (Fig. 6).

IWN AM 03 VWL E RM *G

266 FRIBERG ET AL.

CYTOKINE PRODUCTION BY HUMAN PBMCs 267

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FIG. 5. Changes in PHA-P-stimulated production of IL-2 byPBMCs obtained from patients with cancer (n = 22) during the courseof therapy with picibanil. The data are shown as the log10 ratio ofstimulated IL-2 production at various time points during therapy tobaseline production. Dots represent individual patients. Results frommultiple blood samples for each patient within a time interval wereaveraged. The boxes drawn for each time interval enclose the middle50% of the ratios. Dotted reference lines correspond to doubled orhalved production levels relative to the baseline production. Resultswere pooled over all dose tiers, since no dose-response relationshipcould be demonstrated statistically. The increase in IL-2 productionwas significant at P < 0.01.

Before the cytokine production assay could be applied tomonitoring of patients' cells, it had to be extensively evaluatedfor its sensitivity and reproducibility by using PBMCs obtainedfrom healthy volunteers. The normal range of cytokine pro-duction by PBMCs was defined and was found to be ratherbroad for each of the four cytokines tested. While spontaneouscytokine production was essentially not detected with healthyPBMCs, both high and low responders for induced cytokinerelease were identified among healthy individuals. It is notclear at this time whether the high versus low responder statusamong healthy individuals has any importance for health, but itis possible that the assay discriminates between immunologi-cally more competent versus less immunologically competentindividuals. On the other hand, individuals whose cells pro-duced a high level of one cytokine were not necessarily goodproducers of all cytokines. We found that PBMCs from healthyvolunteers were not concordant for cytokine production, atleast for TNF-aL and IL-1r, because no significant correlationcould be established for this pair of cytokines.

Cellular production of cytokines can be standardized in aclinical laboratory, much like lymphocyte proliferation assays(16, 18). Control supernatants generated in large quantitiesand standardized against the World Health Organization'sInternational Cytokine Standard were prepared for each cyto-kine assay, divided into aliquots and placed in small vials, andstored at -80°C. These supernatants can be used to measureinter- and intra-assay variabilities. We determined that theinterassay coefficients of variation were <10% for RIA and<20% for ELISA in our laboratory. In many situations, it maybe advantageous to use cryopreserved PBMCs rather thanfresh PBMCs for cytokine production to avoid the variabilityassociated with cytokine generation on different days. Theability to use cryopreserved PBMCs facilitates serial studies, inwhich PBMCs collected over time can be batched and testedtogether in order to diminish interassay variability. Whilespontaneous cytokine production was unaffected by cryo-

A.NK A.NK AJJKRIA.SPONTANEOUS

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FIG. 6. IFN--y production by PBMCs obtained from one patientwith melanoma over the course of therapy with A-NK cells and IL-2.No spontaneous IFN-y production was detected (A). Stimulatedproduction of IFN-y increased after each 5-day course of IL-2 therapybut then decreased dramatically to very low levels before the start ofeach IL-2 infusion (B). The shaded boxes indicate the times of IL-2administration.

preservation of PBMCs, stimulated production of TNF-ct,IFN-y, and IL-2, but not of IL-1,, was higher with cryopre-served than fresh PBMCs in paired assays. This unexpected,yet reproducible, observation could not be explained by a lossof mononuclear cell subsets because of cryopreservation, be-cause no differences in the cellular phenotype would bedetected by flow cytometry when cryopreserved PBMCs werecompared with their fresh counterparts. The higher functionalresponses of cryopreserved versus fresh PBMCs are not re-stricted to TNF-ax, IL-2, or IFN--y production, because weobserved that they also have higher proliferative responses tomitogens. On the other hand, considerable losses in thecytolytic functions of cryopreserved PBMCs have been notedin our laboratory and in other laboratories (20). Thus, the factthat cryopreserved PBMCs tend to produce more and notfewer cytokines upon in vitro stimulation is reassuring forefforts aimed at the use of cryopreserved PBMCs instead offresh PBMCs for cytokine production studies.The issue of the biologic significance of cytokine production

as opposed to cytokine levels in serum is not easy to address.Determinations of cytokine levels in serum are not informativefor many of the reasons discussed above (2, 6, 14, 15, 18)

VOL. 1, 1994

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CLIN. DIAGN. LAB. IMMUNOL.

' . . .Pre 1 2 3 4 5 6 7Tx Weeks on Study

A 1%i-

I I I 18 9 10 11 12 Post

Tx

FIG. 7. Stimulated mean IL-2 production (±2 standard errors) byPBMCs obtained from the peripheral blood of patients with cancer

treated with TNF-ot as described in the text. The changes from thebaseline after each course of TNF-ot for 13 patients were significant atP < 0.01. The solid boxes indicate the times of TNF-ot infusion. Tx,therapy.

except for studies of cytokine kinetics following therapeuticadministration of cytokines (8). The ability of PBMCs tosecrete cytokines in vitro can be modulated by immunothera-pies administered in vivo, as shown in the three examples thatwe provided. Whether monitoring of this ability will be a moreuseful correlate of response to therapy than determination ofcytokine levels in serum remains to be determined.

ACKNOWLEDGMENT

This study was supported in part by the Pathology Education andResearch Foundation.

REFERENCES1. Akira, S., T. Hirano, T. Taga, and T. Kishimoto. 1990. Biology of

the multifunctional cytokines: IL6 and related molecules (ILl andTNF). FASEB J. 4:2860-2867.

2. Arend, W. P. 1991. Interleukin 1 receptor antagonist. A newmember of the interleukin 1 family. J. Clin. Invest. 88:1445-1451.

3. Balkwill, F. R., and F. Burke. 1989. The cytokine network.Immunol. Today 10:299-304.

4. Borden, E. C., and P. M. Sondell. 1990. Lymphokines and cyto-kines in cancer treatment. Immunotherapy realized. Cancer 65:800-814.

5. Conlon, P. J. 1993. A rapid biologic assay for the detection of

interleukin 1. J. Immunol. 131:1280-1287.6. Dinarello, C. A. 1991. Interleukin 1 and interleukin 1 antagonism.

Blood 72:1627-1652.7. Gaffney, E. V. 1989. Interleukin 1: clinical significance and current

assay protocols. Clin. Biotechnol. 1:5-11.8. Gemlo, B. T., Jr., M. A. Palladino, H. S. Jaffe, T. P. Espevik, and

A. A. Rayner. 1988. Circulating cytokines in patients with meta-static cancer treated with recombinant interleukin 2 and lympho-kine-activated killer cells. Cancer Res. 48:5864-5867.

9. Gillis, S., M. M. Ferm, W. Ou, and K. A. Smith. 1978. T cell growthfactor: parameters of production and quantitative microassay foractivity. J. Immunol. 120:2027-2032.

10. Kantor, T. V., D. F. Friberg, Jr., T. A. Medsger, R. B. Buckingham,and T. L. Whiteside. 1992. Cytokine production and serum levelsin systemic sclerosis. Clin. Immunol. Immunopathol. 65:278-285.

11. Kirkwood, J. M., J. Wilson, T. L. Whiteside, J. Bryant, M. S.Ernstoff, and R. B. Herberman. 1993. Phase IA evaluation of theimmunologic effects of immunostimulant picibanil (OK-432) inpatients with cancer. Unpublished data.

12. Larrick, J. W. 1989. Native interleukin 1 inhibitors. Immunol.Today 10:61-66.

13. Logan, T. F., W. F. Gooding, M. S. Ernstoff, T. L. Whiteside, S. S.Kaplan, and J. M. Kirkwood. Submitted for publication.

14. Lydyard, P. M., and M. W. Fanger. 1982. Characteristics andfunction of Fc receptors on human lymphocytes. Immunology47:1-17.

15. Pizzolo, G. 1988. The soluble interleukin 2 receptor as a newbiological marker in diseases. Immunol. Clin. 7:13-21.

16. Rossio, J. L., H. C. Rager, C. S. Goundry, and E. A. Crisp. 1992.Cytokine testing in clinical trial monitoring, p. 942-947. In N. R.Rose, E. C. DeMarcario, J. L. Fahey, H. Friedman, and G. M.Penn (ed.), Manual of clinical laboratory immunology, 4th ed.American Society for Microbiology, Washington, D.C.

17. Waage, A., P. Brandtzaeg, A. Halsteusen, P. Kierulf, and T.Esperile. 1989. The complex pattern of cytokines in serum frompatients with meningococcal septic shock. J. Exp. Med. 169:333-338.

18. Whiteside, T. L. 1994. Cytokines and cytokine measurements in aclinical laboratory. Clin. Diagn. Lab. Immunol. 1:257-260.

19. Whiteside, T. L., and R. B. Herberman. 1990. Characteristics ofnatural killer cells and lymphokine-activated killer cells. Their rolein the biology and treatment of human cancer. Immunol. AllergyClin. N. Am. 10:663-704.

20. Whiteside, T. L., Jr., C. R. Rinaldo, and R. B. Herberman. 1992.Cytolytic cell functions, p. 220-230. In N. R. Rose, E. C. DeMar-cario, J. L. Fahey, H. Friedman, and G. M. Penn (ed.), Manual ofclinical laboratory immunology, 4th ed. American Society forMicrobiology, Washington, D.C.

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