Combined negative and positive selection of mobilized CD34+ blood cells

We tested four negative and two positive selection methods for separation of CD34⁺ cells from mobilized blood cells, and analysed fold-enrichment, purity and recovery of CD34⁺ cells after selection procedures. The elimination of mature CD34⁻ cells was achieved by adhesion to nylon-wool fibre (5.9 ± 1.0 mean fold-enrichment and 65.2 ± 2.3 mean recovery of CD34⁺ cells). Standard or modified Ficoll-Hypaque and Percoll density gradients, as well as phagocytosis with magnetic beads, were less effective in eliminating CD34⁻ cells, both purity and fold-enrichment of CD34⁺ cells being lower than those obtained with separation by nylon-wool. Both positive selection methods tested, Ceprate and MiniMacs System, generated highly purified CD34⁺ cell populations ranging from 80% to 90%. The recovery of CD34⁺ cells was optimal with MiniMacs (77.9±3.6) and low with Ceprate (28.8±2.8). Based on these results, in two large-scale experiments we combined nylon-wool fibre and MiniMacs System in a two-step separation procedure obtaining a 36.9±2.6 mean fold-enrichment and a 50.5±0.3 mean recovery of CD34⁺ cells. In this way we achieved optimal enrichment and recovery of CD34⁺ cells, with a substantial saving of cost compared to either selection method alone.     

Increased number of peripheral blood CD34+ cells in lithium-treated patients

Eight adult patients with bipolar disorder were prospectively examined to find whether lithium carbonate increased their peripheral blood CD34⁺ haemopoietic stem cells. Following lithium therapy for 3–4 weeks their neutrophil counts increased by a mean of 88% (from 4625 ± 1350 × 10⁹/l, mean ± SD pretreatment, to a peak of 8300 ± 3910 × 10⁹/l). Concommitantly, there was a significant increment in their CD34⁺ cells (from 0.11 ± 0.01% to a peak of 0.18 ± 0.08%). There was a significant correlation between the rise in neutrophil count and that of the CD34⁺ cells ( r  = 0.795, P  = 0.019). Lithium therapy may be used to mobilize peripheral blood CD34⁺ cells for marrow transplantation.     

Erythropoietin receptor expression on human bone marrow erythroid precursor cells by a newly-devised quantitative flow-cytometric assay

In order to develop a non-isotopic quantitative assay of erythropoietin (Epo) receptor (EpoR) on human cells, we devised a flow-cytometric assay using cells stained with biotin-labelled and a streptavidine–RED670 conjugate. For quantification, we applied the Kolmogorov-Smirnov test and calculated the D value. The D value was evaluated from the degree of shift in two profiles according to the increase of fluorescence intensity due to the specific binding of biotin-labelled Epo to EpoR. A good correlation was observed between the number of EpoR calculated by ¹²⁵I-Epo binding assay and the D value. Then, EpoR expression on bone marrow cells from normal individuals was studied by three-colour flow cytometry. In normal bone marrow, the number of EpoR on cells was highest in CD34⁺CD38⁻ cells (approximately 1600 sites/cell), and decreased in the following order: CD34⁺CD38⁻ cells > CD34⁺CD38⁺ cells > CD34⁻CD38⁺ cells. Glycophorin A (GpA) positive erythroid cells also expressed EpoR, and their CD34⁺ fraction expressed more EpoR than their CD34⁻ fraction. However, the expression levels of EpoR of these fractions were lower than CD34⁺CD38⁻ cells. These results indicated that EpoR was highly expressed on CD34⁺ haemopoietic progenitors from very early stages of differentiation without expression of CD38 antigen, and that the level of expression decreased with erythroid differentiation as well as with various lineage commitment in human bone marrow cells.     

SLEx expression of normal CD 34 positive bone marrow haemopoietic progenitor cells

Summary. We investigated sialylated Lewis x (sLe^x) antigen expression on CD 34 positive (CD 34⁺) haemopoietic progenitors in the bone marrow of eight healthy volunteers using monoclonal antibodies. We found that in all the samples examined, CD 34⁺ bone marrow progenitors strongly expressed the sLe^x antigen. This contradicts previous publications which reported sLe^x expression on malignant blast cells but not on normal CD 34⁺ progenitor cells.     

Monitoring of CD95 and CD38 expression in peripheral blood T lymphocytes during active human cytomegalovirus infection after orthotopic liver transplantation

Aim:  The aim of the present study was to quantitatively monitor the response of CD95 molecules expressed on CD3⁺ T cells (CD95⁺CD3⁺ cells) and CD38 molecules expressed on CD8⁺ T cells (CD38⁺CD8⁺ cells) to ganciclovir treatment after orthotopic liver transplant (OLT) in recipients with active human cytomegalovirus (HCMV) infection.
Methods:  Blood samples were collected from 20 liver transplanted recipients with active HCMV infection and 24 recipients without HCMV infection. CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were quantitatively detected with QuantiBRITE bead methods by dual-color flow cytometry analysis during the post-transplantation period.
Results:  CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were not significantly different among different ages of healthy adults ( P  > 0.05). CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were drastically increased in the active HCMV infection group compared with that in the stable group or in the healthy group ( P  < 0.001), and then they were gradually decreased within the next several weeks after ganciclovir treatment when compared with active HCMV infection recipients ( P  < 0.001).
Conclusions:  The present study showed that CD38⁺CD8⁺ T cells can be an appropriate immunological marker for early detection and antiviral therapeutic monitoring of HCMV infection. The evaluation of CD95 molecule levels may be used routinely in clinical practice to assess the level of immunosuppression.     

Chronic myelogenous leukaemia CD34+ cells exit G0/G1 phases of cell cycle more rapidly than normal marrow CD34+ cells

To investigate the mechanisms behind the leukaemic expansion of chronic myelogenous leukaemia (CML), we examined the cell cycle status and activation kinetics of purified subpopulations of CD34⁺ cells from normal and CML bone marrow (BM). Propidium iodide staining was used to assess cell cycle status of fresh cells or those stimulated with cytokines. Although the cell cycle status of fresh low-density cells from CML and normal BM was similar, a larger percentage of CML CD34⁺ cells were cycling than those from normal BM. The HLA-DR⁻ compartment of CML CD34⁺ cells, a fraction enriched for normal, non-leukaemic progenitors, contained a higher percentage of quiescent cells than the CD34⁺ HLA-DR⁺ fraction. When the activation of CD34⁺ cells was examined in response to SCF or IL-3 alone, or SCF+IL-3+IL-6, CML CD34⁺ cells exited G₀/G₁ more rapidly than normal CD34⁺ cells. Interestingly, although normal BM CD34⁺ cells failed to cycle in response to IL-6 alone, or in the absence of exogenous cytokines, 30% of CML cells cycled under these conditions. No differences in the degree of apoptosis were documented among CML and normal CD34⁺ cells in these cultures. These data suggest that enhanced cell cycle activation of CML CD34⁺ cells, by either autocrine stimuli or via enhanced sensitivity to exogenous stimuli, may be partially responsible for the pronounced cellular expansion characteristic of CML.     

Decreased L-selectin expression in CD34-positive cells from patients with chronic myelocytic leukaemia

Abnormal adhesive interaction between bone marrow stroma and progenitors, one of the causes of unregulated proliferation in chronic myelocytic leukaemia (CML), may be caused by some alterations in adhesion molecules on CML progenitors. We investigated the expression of adhesion molecules (CD44, VLA-5, VLA-4, LFA-1, ICAM-1, L-selectin and c-kit) on bone marrow CD34⁺⁺ cells from 16 CML patients by three-colour flow cytometry. The mean percentage of cells expressing L-selectin in the CD34⁺⁺CD38^{+  ∼  ++} fraction from untreated CML patients was significantly lower, and that in the CD34⁺⁺CD38⁻ fraction tended to be lower than that from normal controls. Among 11 CML patients treated with interferon-α (IFN-α), the mean percentage of the cells expressing L-selectin in the CD34⁺⁺CD38⁻ fraction from three patients with a low percentage of Ph¹(+) cells in bone marrow was significantly higher than that from five patients with a high percentage of Ph¹(+) cells. In addition, L-selectin expression rate was inversely correlated to the percentage of Ph¹(+) cells. There was no significant difference between the untreated patients and normal controls with regard to the expression rates of the other adhesion molecules in each CD34⁺⁺ fraction except LFA-1. These data suggest that decreased L-selectin expression in CML CD34⁺⁺ cells reflects one of the features of malignant CML progenitors.     

G-CSF alone mobilizes sufficient peripheral blood CD34+ cells for positive selection in newly diagnosed patients with myeloma and lymphoma

We have evaluated CD34⁺ cell positive selection from granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) in 26 patients with either multiple myeloma (MM, n  = 18) or follicular non-Hodgkin's lymphoma (NHL, n  = 8). 26 PBPC were collected with two leukaphereses: 16 contained sufficient numbers of CD34⁺ cells and were selected. The absolute number of CD34⁺ cells in the leukapheresis products was found to be significantly related to the duration of underlying disease and exposure to prior treatment. CD34⁺ cell positive selection allowed recovery of a median of 35% of CD34⁺ cells, the selected fraction containing a median number of 1.43 × 10⁶/kg CD34⁺ cells/kg (range 0.48–41.5). 10 patients were transplanted and received a median dose of 1.51 × 10⁶ CD34⁺ cells (range 0.48–4.2). The median time to granulocyte (>0.5 × 10⁹/l) and platelet (>20 × 10⁹/l) engraftment was 12 and 13 d respectively (ranges 10–13 and 0–95). Lymphoma cells were found by a sensitive polymerase chain reaction technique in four out of five CD34⁺ cell fractions tested.     

Separation of G-CSF-mobilized PBSC transplants by counterflow centrifugal elutriation: modest enrichment of CD34+ cells but no loss of primitive haemopoietic progenitors

The suitability of counterflow centrifugal elutriation (CCE) for reduction of the number of non-stem cells in autologous G-CSF-mobilized peripheral blood stem cell (PBSC) transplants was investigated. By cell size-monitored CCE, small cells could be rapidly separated from the haemopoietic progenitor cells present in leukapheresis product (LP) samples. The large cell fraction contained an average 86 ± 25% of the CD34⁺ cells and 76 ± 20% of the granulocyte-macrophage progenitors (CFU-GM) loaded into the separation chamber, and was depleted of 75 ± 18% of the lymphocytes, 89 ± 7% of the erythrocytes and 98 ± 2% of the platelets ( n  = 21). Due to the presence of high numbers of large immature myeloid cells, which co-elutriated with progenitor cells, enrichment of CD34⁺ cells in the large cell fraction was only modest (average 1.8 times). No indication of preferential co-elutriation of primitive stem cells with the small cells was obtained. There was no difference in expression of CD38 or Thy-1 on CD34⁺ cells between the two elutriation fractions. Frequencies of cobblestone-area-forming cells (CAFC) week 6, which are considered to represent cells with long-term repopulating ability, were reduced in the small cell fractions as compared to those in the unseparated samples and the large cell fractions. On average, 100% of CAFC week 6 were recovered in the large cell fractions ( n  = 5). In conclusion erythrocytes, platelets and 40–50% of leucocytes can be depleted from G-CSF-mobilized PBSC samples by CCE with an almost complete recovery of both clonogenic and primitive stem cells.     

Thrombopoietin-independent effect of interferon-γ on the proliferation of human megakaryocyte progenitors

Flow cytometric study revealed that almost all CD34⁺ cells in human umbilical cord blood expressed interferon-γ receptor (IFN-γR). To clarify the precise functional roles of IFN-γR in human CD34⁺ cells, we examined the effect of IFN-γ alone and in combination with various cytokines on the growth of haemopoietic progenitor cells in CD34⁺ cells using a serum-free clonal culture. Surprisingly, IFN-γ alone supported only megakaryocyte (MK) colonies in a dose-dependent manner with a plateau level at 1000 U/ml of IFN-γ. IFN-γ at 1000 U/ml induced 10 ± 1.2 MK colonies from 1 × 10³ CD34⁺ cells, whereas thrombopoietin (TPO), interleukin (IL)-3, stem cell factor (SCF) or IL-6 alone induced 22 ± 4.0, 22 ± 4.2, 4 ± 0.6 and 0 MK colonies, respectively. The addition of anti-IFN-γ monoclonal antibody (mAb) to the IFN-γ culture completely abrogated MK colony formation, whereas the mAb had no effect on TPO-dependent production of MK colonies. In contrast, although anti-TPO polyclonal Ab almost completely blocked TPO-dependent MK colony formation, it failed to inhibit the generation of MK colonies induced by IFN-γ, suggesting that the observed effect of IFN-γ on the proliferation of human MK progenitor cells is independent of TPO. The addition of IFN-γ to culture with TPO or SCF significantly augmented the development of MK colonies, whereas it did not affect IL-3-dependent MK colony formation. Additionally, IFN-γ induced the increase of DNA content of cultured glycoprotein IIb/IIIa-positive megakaryocytes. These results suggest that IFN-γ may have regulatory roles in human megakaryocytopoiesis.     

Progenitor cell subsets and engraftment kinetics in children undergoing autologous peripheral blood stem cell transplantation

The main objective of the present study was to determine the role of CD34⁺ cell subsets in the haemopoietic recovery of children undergoing peripheral blood stem cell transplantation. For this purpose, 38 leukaphereses from 33 children with malignancies mobilized with G-CSF were analysed. Using dual-colour flow cytometry, different subpopulations of CD34⁺ cells were quantified and the number of each reinfused subsets correlated with haemopoietic resurgence. Multivariate analysis showed that the number of CD34⁺CD38⁻ cells and CD34⁺CD38⁺ cells correlated better with time to neutrophil and platelet recovery, respectively, than the total number of CD34⁺ cells. Threshold values for rapid haemopoietic recovery, determined by the receiver operating characteristic analysis, were found to be 0.5 × 10⁶ CD34⁺CD38⁻ cells for neutrophil engraftment, and 2.0 × 10⁶ CD34⁺CD38⁺ cells for platelet recovery. It is suggested that the analysis of CD34⁺ cell subsets could increase understanding of the repopulation capacity of a given leukapheresis product in peripheral blood stem cell transplantation procedures in children. In particular, this procedure could be extremely useful when low numbers of CD34⁺ cells are collected.     

Cytokine and chemokine production by CD34+ haemopoietic progenitor cells: detection in single cells

In vitro expansion of haemopoietic progenitor cells (HPC), lineage-specific differentiation, and gene transfer are all based on in vitro culture systems using haemopoietic growth factors (HGF). A close control of the actual culture conditions, however, is difficult due to secondary mediators secreted by the heterogenous population of mature and immature cells in culture. Although monocytes and granulocytes have already been identified as active producers, this study specifically addressed the role of CD34⁺ progenitor cells in this respect. Using an immunostaining method that enables simultaneous detection of cytokines and phenotype, 56±6% CD34⁺ peripheral blood progenitor cells (PBPC) were found to contain cytoplasmic IL-8 after stimulation with phorbol myristate acetate+ionomycin for 90 min, 19±4% stained positive after TNF-α induction (20 h), and 7±1% expressed IL-8 in the presence of culture medium alone. Intra-cytoplasmic TNF-α and IL-1β were detected at lower frequency, and <1% of CD34⁺ cells expressed IL-1ra or IL-6, whereas IL-1α, IL-10 and G-CSF were not detected. Thus, CD34⁺ HPC are able to synthesize chemo- and cytokines that may operate in an auto- or paracrine manner to modulate in vivo as well as in vitro growth and differentation of haemopoietic cells.     

The effect of interleukin-12 in ex-vivo expansion of human haemopoietic progenitors

Summary. We evaluated progenitor cell proliferation in cultures supplemented by different cytokine combinations in the presence or absence of IL-12. In cultures of low density cells, cytokine combinations including IL-12 were associated to a greater proliferation (up to 6.7 ± 2.5 CFU-GM fold expansion). However, in cultures of purified CD34⁺ cells the more efficient cytokine combination (147 ± 49 CFU-GM fold expansion) was SCF, IL-3, IL-11 and MlP-la, and the addition of IL-12 did not further enhance expansion of progenitors.
These results indicate that accessory cells, lost in CD34⁺ cell purification, could be in part responsible for IL-12 effect on progenitor cell proliferation. In CD34⁺ cell cultures the addition of IL-12 led to CD19 mRNA generation, suggesting that IL-12 acts on haemopoietic cells with both myeloid and lymphoid potential.     

A flow cytometric assay of CD34-positive cell populations in the bone marrow

Summary CD34⁺ BM cells form a heterogenous population of primitive stem cells and more mature progenitors committed to different lineages of differentiation. By combining CD45 expression with SSC, it is possible to separate immature cells from more diferentiated BM cells, and, by three-colour flow cytometry, analyse the antigens expressed in various subsets of cells. In this paper we show that in the normal BM at least four distinct CD34⁺ cell populations can be identified by their different patterns of CD45 expression and SSC. The most immature CD34⁺ cell population (0.1% of the BM cellularity) lacked all signs of lineage commitment and was CD45RA negative and only weakly CD45 positive. With increasing expression of the CD45 antigens, a second CD34⁺ population (0.2% of the BM cellularity) was formed expressing mainly primitive lymphatic antigens. However. 30% of the cells co-expressed B-cell line antigens and myeloid antigens. Cells committed to the myeloid cell line lost B-cell line antigens, gained CD45 antigen expression and SSC and formed two CD34⁺ cell population (0.2% and 0.1% of the BM cellularity. respectively) differing only with respect to the pattern of myeloid antigen expression and SSC characteristics. Similarly, differentiation along the lymphatic pathway implicated down-regulation of myeloid antigens, loss of the stem cell antigen and immature lymphatic antigens and gain of CD45 expression and mature lymphatic antigens.     

Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anaemia

Summary Fas antigen, a receptor molecule that mediates signals for programmed cell death, is involved in T-cell-mediated killing of malignant, virus-infected or allogeneic target cells. Interferon- γ (IFN- γ ) and tumour necrosis factored (TNF- α ), potent inhibitors of haemopoiesis, enhance Fas receptor expression on bone marrow (BM) CD34⁺ cells, and both cytokines render haemopoietic progenitor cells susceptible to Fas-mediated inhibition of colony formation due to the induction of apoptosis. Haemopoietic suppression in aplastic anaemia (AA) has been associated with aberrant IFN- γ , increased TNF- β expression, and elevated numbers of activated cytotoxic T-cells in marrow. We have now examined Fas antigen expression in fresh AA BM samples. In normal individuals few CD34⁺ cells expressed Fas antigen and normal marrow cells had low sensitivity to Fas-mediated inhibition of colony formation. In contrast, in early AA, BM CD34⁺ cells showed markedly increased percentages of Fas receptor-expressing CD34⁺ cells, which correlated with increased sensitivity of AA marrow cells to anti-Fas antibody-mediated inhibition of colony formation. The proportion of Fas antigen-bearing cells was lower in recovered patients'BM. Fas antigen was also detected in the marrow of some patients with myelodysplasia, especially the hypocellular variant. These results are consistent with the hypothesis that AA CD34⁺ cells, probably including haemopoietic progenitor cells, express high levels of Fas receptor due to in vivo exposure to IFN- γ and/or TNF-α and are suitable targets for T-cell-mediated killing. Our results suggest that the Fas receptor/Fas ligand system are involved in the pathophysiology of BM failure.     

Direct and reversible inhibition of platelet factor 4 on megakaryocyte development from CD34+ cord blood cells: comparative studies with transforming growth factor β1

Mechanisms of the action of platelet factor 4 (PF4) on the growth of megakaryocyte (MK) progenitor cells in CD34⁺ cord blood (CB) cells were studied in comparison with transforming growth factor β1 (TGFβ1). Development of MK from CD34⁺ CB cells in both plasma clot culture and liquid culture was significantly inhibited by either purified human PF4 and by recombinant human TGFβ1. Inhibition of MK colony formation by PF4 was reversible, because CD34⁺ cells preincubated with PF4 could regenerate colonies after washing and replating into secondary cultures. In contrast, TGFβ1-preincubated CD34⁺ cells gave rise to few colonies following replating. Moreover, incubation of CD34⁺ cells with PF4 in liquid culture caused the increased number of both stem cell factor (SCF)-binding cells and CD34 antigen-bearing cells. In addition, PF4-preincubated CD34⁺ cells exibited a higher potential in MK colony formation in the presence of 5-fluorouracil (5FU). These results demonstrate that both PF4 and TGFβ1 inhibit MK development from CD34⁺ CB cells by different mechanisms, and suggest that PF4, unlike TGFβ1, exerts its inhibitory effect on the growth of the target cells in a reversible manner which results in a preservation of a more immature and 5FU-resistant cell population.     

The development of human megakaryocytes. II. CD4 expression occurs during haemopoietic differentiation and is an early step in megakaryocyte maturation

CD4 expression is not limited to T cells and monocytes. In both mouse and man the antigen has been detected on some early haemopoietic progenitors and we have shown that some mature megakaryocytes (MK) express CD4, the surface molecule that serves as the high-affinity receptor for human immunodeficiency virus, type-1 (HIV-1). Using a serum-free culture system in which sorted CD34⁺ haemopoietic progenitors are cultured with thrombopoietin (TPO), IL-3, IL-6 and SCF, we now show that CD4 expression is induced in virtually all developing haemopoietic cells. This phenomenon was particularly striking in the MK lineage, where CD4 expression began whilst the cells were still CD34⁺ but after they expressed CD41 (GPIIb/IIIa). CD4 expression and endomitotic polyploidization occur at the same time in MK development. In culture, maximum CD4 expression occurred 4–6 d after CD41 expression and lasted for a few days. Expression of CD4 declined gradually thereafter and most MK were CD4⁻ by the end of the culture period. The amount of CD4 on the surface of some MK, as measured by intensity of fluorescence staining, exceeded that of normal monocytes and approached the brightness of T cells. Appearance of the surface antigen correlated with the presence of mRNA for CD4, as measured by RT-PCR.     

Immunomagnetic selection of CD34+peripheral blood stem cells for autografting in patients with breast cancer

Contamination of transplants with tumour cells may contribute to relapse after peripheral blood stem cell transplantation (PBSCT). We studied the feasibility of CD34⁺ cell selection from blood-derived autografts obtained following G-CSF-supported cytotoxic chemotherapy in a group of 25 patients with breast cancer (10 with high-risk stage II/III and 15 with stage IV without bone or bone marrow involvement).
Using immunomagnetic beads (Isolex 300 SA, Baxter) CD34⁺ cells were enriched and released by chymopapain resulting in a median purity of 95% (range 82–99%) and a median recovery of 80% (range 27–132%). The enrichment procedure did not change the proportion of CD34⁺ subsets coexpressing HLA-DR, CD38 and Thy-1, while L-selectin was removed from the cell surface following selection. Using a sensitive immunocytological technique with a cocktail of epithelial-specific antibodies (anti-cytokeratin 8, 18 and 19; HEA125; BM7 and BM8), five leukaphereses products contained epithelial cells, whereas the selected CD34⁺ cell fraction was free of tumour cells. A neutrophil count of 0.5×10⁹/l and a platelet count of 20×10⁹/l was reached after a median time of 14 and 10 d following 40 high-dose chemotherapy (HDC) cycles. Our results indicate that immunomagnetic selection of CD34⁺ cells yields highly purified autografts devoid of tumour cells whereas the engraftment ability of the progenitor and stem cells is fully retained.     

Blastogenic responses, interleukin-2 production and interleukin-2 receptor expression on CD4+ and CD8+ lymphocytes in rhesus monkeys experimentally inoculated with Loa loa

To better understand cellular responses in loiasis infection, in vitro blastogenesis of peripheral blood mononuclear cells (PBMC) to filarial antigen was assessed in 12 Loa loa -inoculated rhesus monkeys over a two-year period. Cellular reactivity to antigen was observed between 10–35 weeks postinoculation (WPI), but had declined by week 50. The roles of interleukin-2 (IL-2) and IL-2 receptor (IL-2R) expression on CD4⁺ and CD8⁺ T cells in regulating the response to antigen were examined during the initial (57 WPI) and late (92 WPI) time points of the observed diminished reactivity to antigen. The levels of IL-2 in antigen cultures at both time points were not significantly different from those in unstimulated cultures. Also, exogenous IL-2 partially reversed the PBMC response to antigen. The percentages of CD4⁺ and CD8⁺ T cells expressing IL-2R in antigen cultures at 57 WPI were not different from those of control animals. Likewise at 92 WPI, the percentage of CD4⁺ T cells expressing IL-2R in antigen cultures, were not increased above those of control animals. In contrast, the percentage of CD8⁺ T cells expressing IL-2R in antigen cultures were significantly increased above those of control animals ( P  < 0.0001), coinciding with an increase in CD8⁺ T cell numbers in these cultures. The data show that factors besides IL-2, and probably an imbalance in the percentages of CD4⁺ and CD8⁺ T cells bearing IL-2R in antigen cultures, may contribute to the diminished reactivity to antigen in L. loa -inoculated rhesus monkeys .