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Effect of Contaminating Red Blood Cells on OKT3-Mediated Polyclonal Activation of Peripheral Blood Mononuclear Cells

Samuel Song,^1,2, Joseph Goodwin,^1, Jenny Zhang,^1, Bruce Babbitt,^1,|| and Janet L. Lathey^2*

Research Department, Cellcor Inc., Newton, Massachusetts 02139,¹ Zycos, Inc., Lexington, Massachusetts 02421²

Received 7 September 2001/ Returned for modification 23 October 2001/ Accepted 4 March 2002

	ABSTRACT

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Erythrocytes are typically present as impurities in the majority of peripheral blood mononuclear cell (PBMC) preparations. This study was undertaken to investigate the effects of contaminating red blood cells (RBC) on the ability of OKT3 to activate CD4⁺ and CD8⁺ T cells. Surprisingly, the levels of gamma interferon, tumor necrosis factor alpha, and interleukin-1ß (IL-1ß) produced by PBMC upon stimulation by OKT3 were increased (P < 0.05) in a dose-dependent manner when increasing amounts of autologous RBC (RBC-to-PBMC ratios of 2:1, 10:1, and 50:1) were spiked into PBMC preparations. The OKT3-driven induction of the IL-2 receptor (CD25) and the proliferation of T lymphocytes in response to phorbol myristate acetate were not affected by the addition of RBC.

	INTRODUCTION

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Lymphocytes are among the most extensively studied cells of the hematopoietic system because of their central role in the generation of immune responses. Information provided from the study of T lymphocytes is important not only in understanding the basic concepts of immune function but also in enabling the development of lymphocyte-based adoptive immune therapies. Lymphocytes can be collected from the peripheral blood, lymphoid tissues, and certain internal organs. In most cases, lymphocytes are initially isolated from the peripheral blood compartment and purified by Ficoll density gradient centrifugation. However, regardless of the method used to isolate T cells or peripheral blood mononuclear cells (PBMC), there always exists a low level of contaminating red blood cells (RBC). In addition, when PBMC are isolated on a large scale, as with most ex vivo adoptive immunotherapy approaches, the level of contaminating RBC increases even further. It has been shown previously that lymphocytes in whole blood stimulated with mitogen produce more interleukin-2 (IL-2) than Ficoll-Hypaque-purified lymphocytes in culture (5). What remains unknown is the effect various levels of contaminating RBC have on the ability of well-characterized T-cell stimulants to activate lymphocytes under normal cell culture conditions.

A unique form of outpatient adoptive immunotherapy referred to as autolymphocyte therapy (ALT) for the treatment of patients with metastatic renal cell carcinoma has been developed (6, 6a). Patients are infused monthly with 10⁹ T lymphocytes activated ex vivo in a conditioned medium containing a mixture of OKT3 (mouse monoclonal anti-CD3 antibody) and a broad panel of autologous cytokines. The cytokine mixture is generated by stimulation of patient PBMC ex vivo with 25 ng of OKT3/ml for 3 days during the first cycle of the therapy (8). During the secondary cycles, i.e., monthly, of therapy, patient PBMC are cultured with the autologous cytokine mixture from the first cycle of therapy for 5 days and then infused back into the patient.

For this procedure, lymphoapheresis is performed for each cycle to collect large numbers of PBMC. The resulting apheresis cell products (ACP) are highly enriched in white blood cells and contain various amounts of RBC, platelets, plasma, etc. The various ACP can vary greatly in their RBC content depending on the leukopheresis machine used, the skill of the apheresis technician, the clinical status of the patient, etc. In addition to the effects RBC could have on the preparation of cells on adoptive immunotherapy, RBC could also change immune parameters used in vitro to monitor immune responses in PBMC ex vivo during diseases or treatment trials.

Since it is difficult to generate large volume preparations of 100% pure PBMC, it would be desirable to know the potential effects of these contaminants on various critical parameters (cell phenotype, cell proliferation, and cytokine production) associated with the in vitro culture of human PBMCs. Herein, we report the results of a series of experiments in which the effect of increasing amounts of RBC on OKT3-mediated activation of PBMC was measured following the culture procedure used for outpatient adoptive immunotherapy, ALT.

	MATERIALS AND METHODS

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Cell sources. ACP from nine normal donors were used as sources of PBMC and RBC in this study, and they were collected using three different apheresis machines (three each from Haemonetics V-50, Fenwal CS-3000, and Cobe Spectra apheresis machines). The ACP were shipped overnight from multiple collection sites to the cell processing laboratory in thermally insulated boxes at room temperature. Previous studies had shown that viability (>70%) and CD3/CD25 expression (>50% of preculture values) were acceptable after 3 days of culture when cells were processed within 24 to 48 h. Cells held for 72 h before processing did not meet these specifications.

Cell separation. ACP were divided into two equal volumes, one aliquot to be used for isolation of PBMC and another for isolation of RBC. To isolate PBMC, 15-ml aliquots of ACP were diluted to 50 ml with saline. To remove platelets, the diluted ACP were centrifuged at 200 x g in a Sorvall RT 6000B centrifuge for 15 min. The supernatants, which contained platelets, were discarded. The cell pellets were resuspended with 35 ml of 0.9% saline (Baxter I.V. System, catalog no. 2B1323Q). The cell suspensions were underlaid with 14 ml of Lymphoprep (Nycomed Pharma AS, Oslo, Norway) and centrifuged at 400 x g for 30 min without brake. The PBMC layer was collected and washed twice with saline by centrifuging at 350 x g for 10 min. The cells were resuspended with 10 ml of complete AIM-V medium to which was added 50 µM cimetidine (Tagamet; Smith Kline Beecham Pharmaceutical, Cidra, Pa.) and 10 nM indomethicin (Indocin; Merck Sharp & Dohme, West Point, Pa.), 1 mM sodium pyruvate (Gibco-BRL, Grandview, N.Y.), and 100 µg of gentamicin (Gibco-BRL)/ml. Indomethacin and cimetidine were added to reduce tumor-related suppression (1, 2), keeping this protocol in line with the clinical protocol. Cell count was determined with a Coulter Counter, and cell viability was determined by trypan blue exclusion.

RBC were isolated from the ACP following the removal of platelets, as described above, using high-speed centrifugation (950 x g, 20 min). Following the centrifugation, the buffy coat and 2 cm of RBC right underneath the buffy coat were removed and discarded. The pelleted RBC were washed twice with 0.9% saline and then resuspended in 10 ml of complete AIM V medium. A cell count was determined with a hemacytometer. In certain cases where the ACP RBC-to-PBMC ratio was very low, Histopaque 1119 (Sigma, St. Louis, Mo.) instead of Ficoll was used to isolate the RBC. Briefly, the ACP collected following the removal of platelets was layered on top of Histopaque 1119 and centrifuged for 30 min at 550 x g. Following centrifugation, the pelleted RBC were collected from the bottom of the centrifuge tube, washed in saline, resuspended, and counted as outlined above.

Activation cultures. Two different types of activation cultures were evaluated in this study to mimic the therapeutic cell generation. The primary cell activation culture (first clinical cycle) generated conditioned medium (CM) containing autologous cytokines for the secondary cell activation culture (secondary clinical cycles). The secondary cell activation culture generated ex vivo-activated T cells from the PBMC in the presence of CM from the primary culture. An autologous cytokine-enriched CM was generated by the stimulation of PBMC for 3 days with 25 ng of OKT3/ml in the primary culture. The ex vivo-activated T cells for the secondary culture were generated from PBMC with stimulation for 5 days by using 25% of the volume of the previously generated CM. The CM also contained residual OKT3 at concentrations of <10 ng/ml.

Primary cultures were generated on a 40-ml scale using PBMC alone or various ratios (2:1, 10:1, and 50:1) of RBC to PBMC. The starting PBMC concentration was 10⁶/ml. The PBMC or PBMC-RBC mixtures were incubated in Stericell NC920 culture bags at 37°C for 3 days in the complete AIM-V medium described above with addition of 25-ng/ml OKT3 (Orthoclone OKT3; Ortho Pharmaceutical Corp., Raritan, N.J.). After 3 days, the supernatants were harvested for cytokine analysis, and the cells were harvested for the measurement of CD3/CD25 levels.

The secondary ex vivo activation culture was also set up by using PBMC alone or various ratios (2:1, 10:1, and 50:1) of RBC to PBMC. The 2 x 10⁶ cells of PBMC/ml were cultured in the complete AIM-V culture medium mentioned above but with no OKT3 addition; instead, 25% of the volume of CM previously generated from primary PBMC-only cultures (no RBC had been present in the CM generating culture [see above]) was added. The cells were cultured for 5 days. At the end of the culture, a panel of cell surface phenotypes was measured and the activated T cells were further stimulated with a low dose of phorbol myristate acetate (PMA) to measure their proliferative capacity.

Cytokine detection. The CM generated in the 3-day cultures were stored at -80°C. IL-1ß, gamma interferon (IFN-), and tumor necrosis factor alpha (TNF-) levels were determined utilizing enzyme-linked immunosorbent assay (ELISA) kits obtained from R&D systems (Minneapolis, Minn.).

Phenotyping. Activated PBMC collected after the 5 days of secondary culture were rested overnight and analyzed for the expression of a panel (CD4, CD8, CD25 [IL-2R], major histocompatibility complex class II [MHC-II], and CD45RO) of T-cell activation and differentiation markers. Activated PBMC derived from the third day of primary cultures were analyzed for CD3/CD25 expression only.

The cells were stained with specific monoclonal antibody conjugates at 4°C for 30 min by following the manufacturer's recommendations. Residual RBC were lysed using Opilyse C (Immunotech, Marseille, France). The stained cell samples were treated with 0.5 ml of Optilyse, vortexed, and incubated at room temperature for 15 min. Then, stained cells were washed and resuspended in phosphate-buffered saline (PBS) for analysis. Monoclonal antibodies used for phenotypic determinations were as follows: CD3 phycoerythrin, CD25 fluorescein isothiocyanate (FITC), CD4 phycoerythrin, CD8 FITC, and I3 FITC (MHC-II) and Mo2-RD1/KC56-FITC (CD14 and CD45), all purchased from Coulter Immunology (Hialeah, Fla.), and CD45RO FITC obtained from Dako (Carpinteria, Ca.).

Cell surface phenotypes were measured utilizing a Profile II flow cytometer (Coulter Corp.) equipped with an air-cooled argon laser (488 nm). Lymphocytes were acquired using forward and side scatter and identified using CD14 and CD45 as markers. CD3 dual-phenotype cells were identified as CD3-positive cells that also expressed a second phenotype, i.e., CD25, MHC-II, or CD45RO.

T-cell proliferation assay. PMA (Sigma Chemical Co.) was used to evaluate the degree of activation of T cells within the 5-day activation culture with addition of RBC. Following the 5 days of culture and overnight storage at 4°C, the T cells were further cultured in triplicate for 2 days at 2 x 10⁶/ml with 1 ng of PMA/ml. During the final 6 h of culture, the cells were pulsed with [³H]thymidine. Cell proliferation was determined by cellular incorporation of [³H]thymidine measured with scintillation counting using an LKB 1205 Betaplate Liquid Scintillation Counter (Wallac Oy, Turku, Finland).

Statistical analysis. Statistical differences in parameters measured on PBMC alone versus PBMC spiked with RBC were determined using a paired two-tailed Student test with Excel statistical function. A P value of <0.05 was considered significant.

	RESULTS

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Cell yield. Cell yield was determined using a Coulter ZM counter. Cell counts were taken immediately before adding the cells into the culture bags and immediately after harvesting the cells. Cell yields in the 3-day cultures varied from 83 to 98% of the starting cell population and in the 5-day cultures from 124 to 134% of the starting cell population. There was no statistically significant difference in cell yield when RBC were added to the PBMC.

Primary culture—cytokine production. To determine the effect of RBC on PBMC cytokine production, primary cultures were stimulated with OKT3 for 3 days. Cytokine production from PBMC increased with the addition of increasing amounts of RBC (Fig. 1A, B, and C). The levels of cytokine production from unstimulated cells were 17.7 ± 9.4 pg/ml for IL-ß, 37.9 ± 6.5 pg/ml for TNF-, and 74.8 ± 19.2 pg/ml for IFN-. IL-1ß (Fig. 1A) production increased from 328 ± 109 pg/ml in the PBMC-alone group to 462 ± 154 pg/ml in the 10:1 mixtures of RBC and PBMC (P = 0.02) and to 624 ± 171 pg/ml in the 50:1 mixtures (P = 0.003). The IL-1ß production in the 2:1 group was increased compared to that in the PBMC-alone group, but the difference was not significant statistically (P = 0.07). TNF- (Fig. 1B) production increased from 1,174 ± 391 pg/ml in the PBMC-alone group to 1,377 ± 459 pg/ml in the 2:1 mixture (P = 0.02), to 1,624 ± 541 pg/ml in the 10:1 mixture (P = 0.02), and to 1,946 ± 266 pg/ml in the 50:1 mixture (P = 0.02). IFN- (Fig. 1C) production increased from 2,925 ± 975 pg/ml in the PBMC-alone group to 4,181 ± 1,394 pg/ml in the 2:1 mixture (P = 0.03), to 6,850 ± 2,283 pg/ml in the 10:1 mixture of RBC and PBMC (P = 0.01), and to 8,733 ± 1,309 pg/ml in the 50:1 mixtures of RBC and PBMC (P = 0.0003). The increases in cytokine production at higher RBC-to-PBMC ratios varied for the different cytokines. IFN- production showed the largest increase, threefold in the 50:1 mixture of RBC and PBMC relative to the PBMC-alone group. TNF- production had a 1.7-fold increase, followed by IL-1ß, which had a 1.9-fold increase.

View larger version (24K): [in this window] [in a new window]   FIG. 1. Cytokine (IL-1ß [A], TNF- [B], and IFN- [C]) production from PBMC in the primary activation culture with OKT3 plus various amounts of RBC. The primary activation culture was set up with PBMC (106/ml) stimulated with OKT3 (25 ng/ml) in the presence of indomethacin and cimetidine. RBC were added to the culture at RBC-to-PBMC ratios of 0:1 (no RBC addition), 2:1, 10:1, and 50:1. The culture supernatants were harvested for cytokine detection on day 3. These data are experimental results from nine healthy donors. *, significantly different from the 0:1 group; #, significantly different from the 2:1 group; , significantly different from the 10:1 group; P < 0.05.

Primary culture—phenotype. To evaluate the effect of RBC on the activation level of PBMC by OKT3 in primary cultures, the percentage of IL-2 receptor (CD25)-expressing T cells was measured. The mean (±standard deviation) of CD3/CD25-positive T cells was 61.1% ± 3.8% in the PBMC-alone group, 62.8% ± 3.8% in the 2:1 RBC/PBMC mixture, 62.0% ± 3.6% in the 10:1 RBC/PBMC mixture, and 58.9% ± 3.3% in the 50:1 RBC/PBMC mixture. These differences were not statistically significant (n = 9, P > 0.05).

Secondary culture—phenotype. Autologous PBMC were cultured with or without RBC in CM containing OKT3 for 5 days. Cells were then analyzed for the expression of a panel of differentiation and activation markers. The level of CD3⁺ T cells was 83.9% ± 4.9% when PBMC were cultured alone (Table 1). The addition of RBC to the PBMC population had very little effect on the level of CD3⁺ T cells present at the end of the culture. In addition, there was little effect on the levels of CD4⁺ and CD8⁺ T cells. The ratio of CD4⁺ T cells to CD8⁺ T cells was approximately 1:1 whether or not RBC were added to the PBMC at the beginning of the culture. The percentage of CD3/CD25-positive T cells generated when PBMC were cultured alone was 17.3 ± 2.5. Although there was an increase in CD3/CD25⁺ T cells at higher RBC-to-PBMC ratios, the difference was not statistically significant (Table 1). When PBMC were cultured alone, 62% ± 4.2% of the CD3⁺ T cells expressed the CD45RO marker; once again, the addition of RBC had very little effect. Unlike CD45RO and CD25, the percentage of CD3/MHC-II-positive T cells decreased at higher RBC-to-PBMC ratios (P < 0.05) (Table 1). Thus, the only marker measured that was significantly affected by the addition of RBC was MHC-II.

	DISCUSSION

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Erythrocytes are a major cellular component of blood, and the normal RBC-to-PBMC ratio is approximately 600:1. RBC are also the major cellular impurity in ACP, which was used to isolate PBMC and generate therapeutic T cells. The range of the RBC-to-PBMC ratio in the apheresis products before PBMC isolation was from 1.4:1 to 105.0:1, with a large variation dependent upon which commercially available aphaeresis machine (Cobe, Fenwall, Haemonetics) was used. Here, RBC at a similar ratio range were added back to purified PBMC and the effects on T-cell activation were evaluated.

There were no significant differences in total cell yields whether or not RBC were added. CD3⁺ T cells expanded preferentially in both primary (data not shown) and secondary activation culture, composing about 80% of the cultured cells. CD4⁺ and CD8⁺ cells each made up about 50% of the T cells. Compositions of CD3⁺, CD4⁺, and CD8⁺ T cells were not changed with addition of RBC to the culture.

In the primary culture, 66.8% of CD3⁺ T cells expressed CD25 (IL-2 receptor) on their cell surfaces, while only 21.4% of CD3⁺ T cells in the secondary culture expressed CD25. Although CD25 expression on the T cells in the secondary culture was lower than that in the primary culture, RBC did not influence CD25 expression on T cells in either culture. In secondary cultures, CD3⁺ CD45RO⁺ T cells were 62% of the T cells in the PBMC-alone group; 35% would be observed for resting T cells (1, 2), indicating that the CM could stimulate the T cells. However, RBCs had no effect on the percentages of cells expressing CD45RO in the culture. Unlike CD45RO and IL-2R expression, the percentages of MHC-II-positive cells in the RBC groups were lower than in the PBMC-alone group. Although this could interfere with antigen presentation/stimulation through MHC-II in the secondary culture being prepared for adoptive transfer, the decreases were small and cells could be induced to proliferate.

The PMA-induced proliferation assay used here was the Cellcor-patented technique, and it is routinely used to measure the activation level of ex vivo-activated T cells in the adoptive immunotherapy. The assay is based on the ability of activated, not resting, T cells to proliferate in response to the low dose (1 ng/ml) of PMA, the protein kinase C activator (3, 4). When this assay was used to evaluate the effect of RBC on the levels of proliferative response of the PBMC in the secondary culture, there were no statistical differences of proliferation of the ex vivo-activated T cells between PBMC with and without RBC addition.

Cytokine production increased with the addition of RBC to the OKT3 activation cultures. The underlying reason for the enhancement in PBMC cytokine production when RBC were added to the OKT3 activation culture is unclear. One could hypothesize that the RBC membranes could activate the macrophages through membrane interactions. These interactions could occur as the two cell types come in contact at the beginning of macrophage phagocytosis of RBC. Whatever the mechanism, this increase is supported by previous reports using whole blood (5, 7; J. L. Lathey, R. Trout, B. Roberts, and S. A. Spector, 12th World AIDS Conf., abstr. 21184, p. 275, 1998). Suni et al. (7) showed that intracellular cytokine production by antigen-specific memory T cells stimulated in vitro with soluble antigen was enhanced when whole-blood cultures were utilized. We have also observed that whole-blood culture stimulated with lipopolysaccharide (LPS) or phytohemagglutinin (PHA) produced higher levels of TNF- and IFN-, respectively, than their paired PBMC cultures (Lathey et al., 12th World AIDS Conf.). In their systems and ours, it is possible that the presence of RBC might provide a more favorable physiological environment for PBMC or activated T cells to respond to stimulants.

In summary, the presence of contaminating RBC in a population of PBMC had minimal influence on the in vitro activation of T lymphocytes by a mixture of OKT3 and autologous cytokines if the RBC-to-PBMC ratio was less than 50:1. However, the presence of RBC in a culture of PBMC appeared to enhance the production of TH1/proinflammatory type of cytokines by mononuclear cells. Thus, extra processing of PBMC samples to remove low levels of contaminating RBC may not be necessary when inducing T-cell activation via cytokines. However, RBC could have a confounding effect on immunological assays based on serial or prognostic cytokine measurements. For serial measurements of cytokine from PBMC, RBC contamination should be kept to a minimum.

	ACKNOWLEDGMENTS

 
We thank Mary Lynne Headley and Robert Urban for their critical evaluation and support of the manuscript.

	FOOTNOTES

 
* Corresponding author. Present address: 44 Hartwell Ave., Lexington, MA 02421. Phone: (781) 274-6500, ext. 234. Fax: (781) 274-0839. E-mail: jlathey{at}hotmail.com.

Present address: Biotransplant, Medford, Mass.

Present address: BD Biosciences, Bedford, Mass.

Present address: Purdue Pharma L.P., Princeton, N.J.

^|| Present address: Parexel, Waltham, Mass.

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