Peripheral Blood Mononuclear Cell Suspensions Research Articles

Induction of surface antigen CD69 expression in T-lymphocytes following exposure to actinomycin D

The expression of surface antigen CD69 in immune response cells is typically associated with the early stage(s) of cell activation, with maximal expression levels within 4 h of appropriate antigenic or mitogenic stimulation, and maintenance of these high expression levels for 18–24 h. The expression profiles of CD69 in human peripheral blood mononuclear cells (PBMC) cultured with actinomycin D prior to mitogenic stimulation were evaluated by direct immunofluorescence using flow cytometry. Pretreatment of PBMC suspensions with low, non-toxic levels of actinomycin D stimulated CD3 + T-lymphocytes to express CD69 in a concentration-dependent manner. Furthermore, CD4 + T-lymphocytes were the primary cells responding in this fashion. Secondary mitogenic stimulation following antibiotic treatment potentiated cellular CD69 expression in these assays. CD69 expression was profoundly suppressed with in vitro actinomycin D concentrations ≥1–2 μg/ml, presumably by interference with cellular transcription/translation mechanisms. Parallel thymidine incorporation assays indicated that actinomycin D effectively inhibited thymidine uptake in a concentration-dependent manner, with complete inhibition at ≥0.1 μg/ml. The evaluation of cell cycling dynamics following antibiotic treatment, with and without secondary mitogen stimulation, indicated no substantial changes in DNA synthesis over controls. The diversity of these responses suggests that expression of CD69 may not solely reflect mitogenic activation status but may, under some conditions, result from induced cellular stress.

Analysis of factors contributing to the formation of mononuclear cell aggregates (?escapees?) in flow cytometric immunophenotyping

During immunostaining, human lymphocytes may form aggregates with activated platelets and monocytes, resulting in increased forward (FSC) and sideward (SSC) light scatter signals. Consequently, aggregated cells "escape" from the standard FSC-SSC analysis gate, thereby producing erroneous results. We observed that the frequency of aggregate formation in peripheral blood mononuclear cell (PBMC) suspensions depended on cell donor, murine (m) monoclonal antibody (mAb) specificity and IgG subclass, type of fluorochrome conjugated to the mAb, amount of mAb used for immunostaining, time lapse between fixation of PBMC and flow cytometry, and other, as yet unidentified, factors. Platelets, monocytes, and granulocytes express the polymorphic class IIa IgG receptor (Fc gammaRIIa; CD32). A single amino acid difference, either arginine (R) or histidine (H) at amino acid position 131, underlies differential interaction with mIgG1. Because the Fc gammaRIIa-R131 allotypic form binds mIgG1 well in contrast to Fc gammaRIIa-H131, we studied the frequency of aggregate formation in PBMC suspensions from apparently healthy individuals allotyped for Fc gammaRIIa. The Fc gammaRIIa polymorphism contributed significantly to the frequency of mIgG1-induced cell aggregates, which was highest in Fc gammaRIIa-R/R131 individuals, intermediate in Fc gammaRIIa-R/H131 individuals, and lowest in Fc gammaRIIa-H/H131 individuals. The role of mIgG1-Fc gammaRIIa interactions in aggregate formation was confirmed by blocking Fc gammaRIIa by using F(ab')2 fragments of CD32 mAb. These data document the role of mIgG1 mAb binding by human class IIa IgG receptors in the formation of cell aggregates and show that inhibition of this interaction reduces this technical problem in flow cytometric immunophenotyping.

Rapid expression of the CD69 antigen on T cells and natural killer cells upon antigenic stimulation of peripheral blood mononuclear cell suspensions.

The CD69 antigen has been identified as the earliest activation marker on the surface of cytokine- or mitogen-activated lymphocytes. The expression of this molecule may be a useful early marker of antigen- or allergen-specific activation of lymphocytes in vitro. We evaluated the expression of the CD69 and CD25 antigens on antigen- or allergen-stimulated lymphocytes and the proliferative responses as detected by thymidine incorporation. Peripheral blood mononuclear cells (PBMC) of allergic patients sensitized to Dermatophagoides pteronyssinus, bovine casein, or nickel sulfate were cultured in the absence or presence of clinically relevant allergens, tetanus toxoid, or recombinant interleukin (IL)-2. The respective binding of CD69 or CD25 antibodies to PBMC and thymidine incorporation were measured. An early expression of CD69, but not of CD25, antigen was detectable after 24-72 h of stimulation on up to 80% of natural killer (NK) cells and up to 10% of CD4+ T cells in PBMC cultures. Anti-IL-2 antibodies inhibited these increases of CD69 on NK cells and T cells by up to 60%. After 6 days of antigenic stimulation, the rates of both CD25+ and CD69+ lymphocytes were higher. Seventy-four percent of the CD25+ PBMC but only 55% of the CD69+ cells were CD3+ T lymphocytes at this time. No qualitative differences were detectable in allergen- or tetanus-toxoid-stimulated PBMC from allergic patients. The high expression of CD69 on NK cells in antigen-stimulated cultures suggests that these cells are easily activated by cytokines from antigen-stimulated T cells. CD69+ NK cells may serve as early-indicator cells in cultures with antigen- or allergen-stimulated mononuclear cells.

Transmembrane passage of cytokine-inducing bacterial products across new and reprocessed polysulfone dialyzers.

The widespread use of bicarbonate dialysate and reprocessed high-efficiency and "high-flux" dialyzers has raised concerns about the increased risk of reverse-transfer of dialysate contaminants into the blood compartment. To evaluate this concern, the reverse-transfer of bacterial products from contaminated bicarbonate dialysate into the blood compartment was compared during in vitro dialysis with new or reprocessed high-flux polysulfone dialyzers. In vitro dialysis was carried out at 37 degrees C by use of a counter-current recirculating loop dialysis circuit with either new high-flux polysulfone dialyzers or dialyzers reprocessed once or 20 times with formaldehyde (0.75%) and bleach (< 1%) with an automated system. Heparinized whole blood from healthy volunteers was circulated through the blood compartment, and bicarbonate dialysate was circulated in the dialysate compartment. The dialysate was challenged sequentially by 1:1000 and 1:100 dilutions of a sterile Pseudomonas aeruginosa culture supernatant (bacterial challenge). Samples were drawn from the blood and dialysate compartments 1 h after each challenge. Peripheral blood mononuclear cells (PBMC) were harvested by Ficoll-Hypaque separation from whole blood in the blood compartment and a 5 x 10(6) PBMC/mL cell suspension was prepared. Likewise, dialysate samples (0.5 mL) were added to 0.5 mL suspension of 5 x 10(6) PBMC/mL drawn at baseline. All PBMC suspensions were incubated upright in a humidified atmosphere at 37 degrees C with 5% CO2 for 24 h, and total interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF alpha) cytokine production (cell-associated and secreted) was measured by radioimmunoassay. Eight experiments were performed for each arm of the study with the same donor for each arm. One hour after contaminating the dialysate with a 1:1000 dilution of the bacterial challenge, IL-1 alpha production by PBMC harvested from the blood compartment was 160 +/- 0, 171 +/- 11, and 270 +/- 35 pg, respectively, for new dialyzers, dialyzers reprocessed once, and dialyzers reprocessed 20 times (P = 0.004). One hour after challenging the dialysate with 1:100 dilution, IL-1 alpha production by PBMC harvested from the blood compartment was 188 +/- 20, 228 +/- 35, and 427 +/- 67 pg, respectively, for new dialyzers, dialyzers reprocessed once, and dialyzers reprocessed 20 times (P = 0.006). IL-1 alpha production by PBMC from dialyzers reprocessed 20 times was significantly greater than both new and dialyzers reprocessed once. However, there were no significant differences between new dialyzers and dialyzers reprocessed once. Similarly, after the 1:1000 challenge, TNF alpha production by PBMC harvested from the blood compartment was 160 +/- 0, 160 +/- 0, and 213 +/- 22 pg, respectively, for new dialyzers, dialyzers reprocessed once, and dialyzers reprocessed 20 times (P = 0.008). After the 1:100 challenge, TNF alpha production was 168 +/- 8, 188 +/- 20, and 225 +/- 32 pg, respectively, for new dialyzers, dialyzers reprocessed once, and dialyzers reprocessed 20 times (P = 0.20). These results demonstrate that reprocessing of high-flux polysulfone dialyzers with bleach increases the risk of reverse-transfer of bacterial products from contaminated dialysate, and this risk appears to increase with the number of reuses. Consequently, units that reprocess membranes with bleach and have suboptimal water quality might subject their patients to a higher risk of cytokine-related morbidity.

Quantification of fluorescence properties of lymphocytes in peripheral blood mononuclear cell suspensions using a latent class model

Lymphocytes, monocytes, granulocytes, and other blood cells can be distinguished on the basis of their forward (FSC) and sideward (SSC) light scatter properties and their expression of CD45 and CD14. A FSC,SSC gate can be set to include > 95% of the lymphocytes using a "back gating" procedure on the CD45+, CD14- cells. However, nonlymphoid cells such as monocytes have light scattering properties similar to lymphocytes. This problem occurs particularly in patient populations where the light scattering properties of lymphocyte subsets have changed (e.g., due to activation) and are similar to those of the monocytes. Thus, immunophenotyping using antibodies specific for other markers than CD45 and CD14 does not allow a direct assessment of the percentage of all lymphocytes positive for those markers. In order to optimize immunophenotyping we have developed analytic model in which the FSC,SSC dot plot is partitioned into six nonoverlapping light scatter regions. Each light scatter region contains a mixture population of different cell types, i.e., lymphocytes, monocytes, granulocytes, and other cells. The proportions of each cell type are known from the CD45,CD14 expression within each light scatter region. Under the assumption of independence of fluorescence and scatter properties conditional on cell type, the expression of markers other than CD45 or CD14 are derived from the cell type composition and the fluorescence properties on the other markers of each light scatter region. The underlying statistical model is a latent class model, and maximum likelihood estimates are computed using the expectation-maximization (EM) algorithm. The application of the model for immunophenotyping of lymphocytes of healthy individuals and cancer patients receiving immunotherapy is shown.

Positive Selection of Human Blood Cells Using Improved High Gradient Magnetic Separation Filters

High gradient magnetic separators (HGMS) create magnetic field gradients that can be used to attract much smaller and less magnetic particles than those required for conventional magnetic separation techniques. As a result cells can be labeled with submicron magnetic particles and still be separated using an HGMS filter. Typically, HGMS filters consist of random arrays of wire such as stainless steel wool. Wire elements arranged regularly in a filter should allow more efficient separation of cells. Filters were constructed containing ordered wire arrays composed of 430 series stainless steel wire mesh with wire diameters of 50, 100, or 150 microns. The ability of these filters to separate T cells from peripheral blood mononuclear cell suspensions was tested and found superior to random arrays of 302 series stainless steel wire (Thomas et al, 1992). Target cells recognized by OKT5 monoclonal antibody were cross-linked to dextran-iron particles of approximately 20 nm in diameter. Separation conditions were optimized and after one passage through the filter 88% of the OKT5+ cells were recovered in the enriched fraction with 85% purity (%OKT5+). Multiple passages (3 times) could achieve 99% purity with 68% recovery. Variations in separation flow rate had a large effect on the balance between purity and recovery. Optimum separation efficiencies were achieved only when > 10(8) cells were processed. The primarily cause of nonspecific entrapment of CD8- cells was not nonspecific magnetic labeling of cells but the physical (nonmagnetic) characteristics of the filter/filter chamber.

The effect of T-cell depletion on enhanced basophil histamine release after in vitro incubation with live influenza A virus.

A number of mechanisms participate in virus-induced asthma. Previously, we described enhanced basophil histamine release (HR) during an experimentally induced rhinovirus infection and after in vitro incubation of peripheral blood mononuclear cells (PBMC) with influenza virus. This study extends our previous observations and examines the effect of influenza A virus on basophil leukotriene C4 (LTC4) release as well as the effect of T-cell depletion on virus-enhanced basophil HR. PBMC were isolated from ragweed-allergic subjects and incubated with live influenza A virus or control medium (allantoic fluid). After incubation with influenza A, ragweed antigen (AgE) stimulated LTC4 and HR were enhanced (P less than 0.05). To further define the role of T cells in virus-enhanced basophil secretion, PBMC were isolated and divided into two aliquots. In one aliquot, T cells were removed by magnetic bead separation of mouse monoclonal anti-CD3-coated lymphocytes. T-cell-depleted and nontreated PBMC suspensions were incubated with influenza A or control medium, collected, and challenged with AgE to release histamine. Basophil HR was enhanced in the virus-treated group of PBMC that had not undergone T-cell depletion. In contrast, virus incubation did not enhance HR in the T-cell-depleted fraction. Finally, preliminary analysis of the supernate from virus-treated leukocytes indicates the presence of interferon-gamma. These findings suggest that T cells, and their cytokine products, play an integral role in the process by which viruses enhance basophil HR.

Antigen presentation in patients with recrudescent orofacial herpes simplex virus infections

Recovery from epidermal herpes simplex virus (HSV) infection depends primarily on development of an effective cell-mediated immune response, possibly generated following antigen (Ag) presentation by epidermal cells (EC). The ability of EC to present HSV Ag was investigated in 12 subjects with occasional recrudescent facial HSV infections. All had circulating HSV specific antibodies and cell-mediated immunity to the virus. Peripheral blood mononuclear cell suspensions, depleted of antigen presenting cells (APC) by glass adherence and then enriched for T cells by adsorption on nylon wool columns, did not proliferate in response to HSV Ag. Both EC suspensions, prepared from suction blister roofs, and glass-adherent peripheral blood mononuclear cells (AC) preincubated with ultraviolet-inactivated HSV, reconstituted the T-cell proliferative response to HSV. EC were more efficient than AC at presenting HSV Ag to T cells. Depletion of CD1+ cells from EC suspensions by cell sorting reduced their ability to present HSV Ag and augmentation of CD1+ cell numbers supplemented it. Preincubation of EC or AC with monoclonal antibodies to major histocompatibility complex class II antigens DP, DQ or DR, blocked the lymphoproliferative response to HSV Ag. Evidence was obtained that cells co-ordinately expressing products of the DP, DQ and DR loci are involved in presentation of HSV Ag by both EC and AC.

Immunoadsorption of T cells onto glass beads using tetramolecular complexes of monoclonal antibodies

Tetramolecular monoclonal antibody complexes were used to selectively cross-link a subset of human peripheral blood T cells (CD8 positive) to glass beads coated with hapten (fluorescein) modified bovine serum albumin. Tetramolecular antibody complexes were prepared with anti-CD8 (Leu 2a), anti-FITC mouse IgG1 monoclonal antibodies and monoclonal rat anti-mouse IgG1. Optimum conditions for depletion of CD8 positive cells from peripheral blood mononuclear cell suspensions were determined with 1 ml columns. 90–99% of the CD8 positive cells could be removed with 0–17% non-specific adsorption of CD8 negative cells. The weakest link in this system was the bond between hapten-modified albumin and the glass beads. These results indicate that tetramolecular antibody complexes are useful for the specific immunoadsorption of cells to a defined affinity matrix.

Expression of receptors for IgA on mitogen-stimulated human T lymphocytes.

This study demonstrates that activation of human peripheral blood mononuclear cells (PBMC) by the T cell mitogens phytohemagglutinin (PHA) and concanavalin A (Con A) induces the expression of receptors for IgA without addition of IgA to the culture medium. Cells bearing receptors for IgA were determined by indirect immunofluorescence using human secretory IgA and fluoresceinated goat anti-human IgA or goat anti-secretory component antibodies. Among freshly isolated PBMC, 4.7 +/- 1.7% of T cells, 12.7 +/- 12.5% of B cells and 14.4 +/- 7.6% of monocytes were found to be IgA receptor positive. In unstimulated PBMC cultures the percentage of IgA receptor-positive cells slightly increased at 48 h and was more elevated after 7 days. In Con A-stimulated cultures 24.3 +/- 18.5% of the cells expressed receptors for IgA after 48 h. Then, the number decreased and rose again thereafter. PHA stimulation induced an increase of smaller magnitude with similar kinetics. Induction of receptor for IgA on activated T cells was demonstrated by double-labelling experiments showing more CD8+ than CD4+ cells with receptors for IgA among Con A-activated PBMC. Furthermore, PHA or Con A stimulation of B cell-depleted PBMC suspensions resulted in a marked increase of cells bearing receptors for IgA. Expression of these receptors was down-regulated by recombinant interferon-gamma (250 units/ml) and by prostaglandin PGE2 (100 nM) both on unstimulated and mitogen-activated PBMC. The receptor for IgA was distinct from the asialoglycoprotein receptor and did not cross-react with the poly-Ig receptor of epithelial cells. It was concluded that, in the absence of inducing exogenous IgA, T cell mitogens trigger the expression of receptors for IgA. Therefore, T cell activation is associated with the down-regulation of receptors for IgM and the increased expression of receptors for IgG, IgA and IgE.

Suppression of human IgA-B-cell maturation by IgA-binding factors requires T cells.

IgA-binding factors (IgA-BFs) prepared from human peripheral blood mononuclear cells selectively decrease the generation of cytoplasmic IgA-positive cells in peripheral blood mononuclear cell cultures stimulated by pokeweed mitogen or by Nocardia opaca delipidated cell mitogen. In N. opaca delipidated cell mitogen stimulated cultures, the suppressive effect of IgA-BFs was no longer demonstrable after removal of sheep erythrocyte rosetting T cells or after depletion of CD8+ cells, but it was not altered by depletion of CD4+ cells. In pokeweed mitogen stimulated cultures of T-depleted peripheral blood mononuclear cell suspensions, IgA-BFs were ineffective when irradiated (12 Gy) instead of control T cells were used for reconstitution. The data indicate that radiosensitive and CD8+ T cell subsets may be required for IgA-BFs to suppress the generation of IgA-containing B cells after polyclonal activation of human peripheral blood mononuclear cells.

Isolation of pure functionally active CD8 + T cells positive selection with monoclonal antibodies directly conjugated to monosized magnetic microspheres

A monoclonal antibody of the IgM isotype, ITI-5C2, which binds with high affinity to CD8 molecules, was directly conjugated to the monosized magnetic microspheres M-450. This permits selective removal of the CD8 + T cell subset (T8) from peripheral blood mononuclear cell suspensions in a rapid one-step procedure. With a low ratio of microspheres to cells (2:1), functionally active T8 cells can be recovered. In vitro experiments involving such positively selected T8 cells or recombinations of isolated T8 and T4 subsets, demonstrate that the presence of M-450 microspheres coated with ITI-5C2 do not interfere with the immunological functions of the positively selected cells. The method has possible application in the isolation of all cell populations where high avidity mAbs of appropriate specificity are available.

The Mixed Epidermal Cell-Lymphocyte Reaction. II. Epidermal Langerhans Cells Are Responsible for the Enhanced Allogeneic Lymphocyte-Stimulating Capacity of Normal Human Epidermal Cell Suspensions

Earlier studies carried out in our laboratory which demonstrated that disaggregated human epidermal cells isolated from normal flexor forearm skin produced a greater degree of primary allogeneic lymphocyte blastogenic response than did autologous peripheral blood mononuclear cells have since been confirmed by others. We have now completed a series of additional studies designed to determine the basis for this difference. Blocking studies with anti-HLA-DR antibodies revealed that the allogeneic response triggered by epidermal cells was completely dependent on the presence of unbound HLA-DR molecules eliminating the possibility that nonspecific mitogenic effects produced by the epidermal cell suspension might be responsible for the difference. In addition we were unable to demonstrate that epidermal keratinocytes were supplying a nonspecific helper or growth-promoting effect to the interaction between stimulating HLA-DR bearing Langerhans cells and responding T lymphocytes. Since it has recently been suggested that the entire alloantigen-presenting capacity of unfractionated peripheral blood mononuclear cells can be attributed to a small population of HLA-DR antigen-bearing dendritic cells, we have considered the possibility that the greater degree of allogeneic lymphocyte response produced by epidermal cells could be due to the presence of a greater percentage of HLA-DR positive dendritic cells present in epidermal cell suspensions (i.e., Langerhans cells). Peripheral blood dendritic cell-enriched fractions and epidermal cell suspensions that contained similar percentages of dendritic cells produced equivalent amounts of allogeneic lymphocyte stimulation, whereas peripheral blood dendritic cell-enriched fractions that contained greater percentages of dendritic cells than were present in epidermal cell suspensions produced greater amounts of allogeneic stimulation. We therefore conclude that the enhanced mixed lymphocyte reaction stimulating capacity of human epidermal cell suspensions could be explained by the fact that epidermal cell suspensions contain a greater percentage of HLA-DR bearing alloantigen-presenting dendritic cells than do unfractionated peripheral blood mononuclear cell suspensions.

The effect of angiotensin II on human mononuclear cell reactivity: suppression of PHA-P-induced thymidine incorporation.

Recent evidence suggests that angiotensin II may participate in the regulation of inflammation. We therefore studied the effects of angiotension II on human peripheral blood mononuclear cell reactivity. Peripheral blood mononuclear cell suspensions stimulated with PHA-P revealed decreased thymidine incorporation when simultaneously cultured for 48 or 72 hours with angiotensin II. Experiments were next conducted to determine whether angiotensin II acted directly on lymphocytes. Complete monocyte depletion (greater than 99.5%) resulted in low PHA-P-induced reactivity which was still inhibited by the addition of angiotensin II. Purified (greater than 99.5%) lymphocytes incubated with angiotensin II prior to reconstitution of mixed mononuclear cell suspensions led to reduced PHA-P-stimulated thymidine incorporation. These findings suggest that angiotensin II can effect mononuclear cell uptake possibly through actions on the lymphocyte population.

Modulation of Pokeweed Mitogen‐Induced Human B‐Cell Differentiation by Aggregated IgG

The modulatory effect of human heat-aggregated IgG on human B-cell differentiation induced by pokeweed mitogen was investigated with three experimental protocols. Pulse exposure to aggregated IgG, followed by extensive washings before culture, of peripheral blood mononuclear cell suspensions rigorously depleted of platelets and containing less than 4% monocytes resulted in a selective decrease of the numbers of IgG-containing cells and IgG-secreting cells, whereas a simultaneous decrease of the numbers of cells producing IgG and, to a lesser extent, of those producing IgM or IgA was observed when the pulsed suspensions contained platelets and more than 4% monocytes. This non-isotype-specific suppression was shown to be more pronounced when aggregated IgG and platelets were present in the cell suspensions throughout the cultures. The results suggest that two distinct suppressor pathways can be triggered by aggregated IgG. The first one is restricted to cells producing the matching isotype, in the absence of platelets, with few monocytes in the cell suspensions. The second one leads to a nonspecific suppression of the three major Ig classes. It requires the presence of platelets and/or a high percentage of monocytes and, although it remains to be demonstrated, is probably mediated by prostaglandin E2.

Leu 7+ (HNK-1+) cells. I. Selective compartmentalization of Leu 7+ cells with different immunophenotypes in lymphatic tissues and blood.

In the present immunohistochemical studies, Leu 7+ (HNK-1+, human natural killer and killer) cells were found to occupy preferentially germinal centres of follicles in lymph nodes and tonsils. Leu 7+ cells were also present in germinal-like zones of spleen follicles and in mantle zones of hyperplastic thymus follicles and varied in localization in lymph nodes involved in different types of follicular centre cell-derived malignant lymphomas. Most of the Leu 7+ cells in the follicles expressed the Leu 3 (helper/inducer) marker. Double staining studies of tonsil sheep erythrocyte-rosetting and peripheral blood mononuclear cell suspensions showed that two main, mutually complementary, subpopulations of Leu 7+ cells could be distinguished in both cases, namely Leu 7+/Leu 4+ (subdivided into Leu 2+ (suppressor/cytotoxic) and Leu 3+) and Leu 7+/Leu 4-, including mostly cells with OKM 1 (myelomonocytic) characteristics. Thus, in the tonsil cell suspension the cells with Leu 7+ Leu 3+/OKM 1- immunophenotype strongly predominated, whereas among peripheral blood mononuclear cells Leu 7+Leu 2+/OKM 1- and Leu 7+/OKM 1+ immunophenotypes were mostly observed.

Iron Binding Proteins in Selected Human Peripheral Blood Cell Sets: Immunofluorescence

Selected populations of human peripheral blood T lymphocytes, B lymphocytes, monocytes and polymorphonuclear cells were examined for their intracellular content of lactoferrin transferrin and ferritin by an indirect immunofluorescence technique. Lactoferrin was found in polymorphs but not in lymphoid cells. Two different lactoferrin staining patterns were observed which we designated 'perinuclear', characterized by a ring of positive material round the nucleus, and cytoplasmic, in which most positive material was distributed in the cytoplasm. The former staining pattern was found in high density polymorphs the latter was associated with low density polymorphs occasionally found contaminating the peripheral blood mononuclear cell suspension. After incubation in vitro, the perinuclear pattern changed to cytoplasmic staining. Transferrin was found in T cells and polymorphs. In contrast, only a few transferrin containing cells were detected in the B cell and monocyte fractions. Following overnight incubation, a halo of positive material was found surrounding T cells stained for transferrin, suggesting that T cells released transferrin during incubation. Ferritin was also found in T cells and adherent cells. Following latex particle ingestion, the intensity of ferritin staining was markedly increased. Only a small proportion of lymphoid cells and monocytes stained for transferrin or ferritin in the total peripheral blood mononuclear cell suspension. The results indicate, therefore, that in response to manipulation procedures used routinely for the selection of human peripheral blood lymphoid cells, detectable amounts of transferrin and ferritin are present in T but not B cells. The fact that T cells are equipped with proteins known to participate in binding and storage of iron may constitute, at least in part, the basis for the contribution of these cells to the regulation of other major biological systems.

Importance of HLA-D antigens for the cooperation between human monocytes and T lymphocytes.

AbstractThe ability of autologous and allogeneic monocytes to restore the responsiveness of purified T lymphocytes to tuberculin (PPD) was studied in twelve different experiments involving eleven unrelated T lymphocyte donors and a large number of related and unrelated monocyte donors. T lymphocyte suspensions were prepared by passage of peripheral blood mononuclear cell suspensions through Ig‐anti‐Ig‐coated columns. Monocyte‐enriched suspensions were prepared from mononuclear suspensions by density gradient centrifugation on albumin solution and added to the T cell suspension in a final concentration of 3%. Autologous monocytes and monocytes from related donors sharing one HLA haplotype with the T cell donor partly restored the responsiveness to PPD, and so did monocytes from unrelated donors who shared HLA‐D alleles with the T cells. None of eight monocyte suspensions from eight unrelated donors sharing no HLA‐D antigens with the T cell donor were able to restore the responsiveness to PPD. The difference between HLA‐D‐sharing and nonsharing monocytes could not be explained by a simultaneous allogeneic reaction, as the addition of monocytes autologous with the T cells to mixtures of non‐HLA‐D‐sharing T cells and monocytes partly restored the response. Moreover, experiments with the addition of PPD to ordinary mixed leukocyte cultures revealed only a moderate depression on the PPD response. It is concluded that in man – as in mice and guinea pigs – some HLA‐D identity between monocytes and T lymphocytes is necessary for a cooperation to take place in the secondary immune response. This observation emphasizes the homology between HLA‐D antigens in man and Ia antigens in animals.