Rabbit lymphocyte subpopulations. II. Separation and characterization of two Ig+ cell subpopulations.

Rabbit lymph node cells (Ig⁺Ig^?) were separated into Ig⁺ and Ig^? populations by rosette formation with anti-Ig antibody-coated erythrocytes and centrifugation on Ficoll-Hypaque. Subpopulations of Ig⁺ cells were obtained by treating rosetted cells with autologous serum which dissociated approximately half of the rosettes. The stable rosetted cells (Ig⁺ S) were separated from the labile unrosetted cells (Ig⁺L) by centrifugation on Ficoll-Hypaque. The Ig⁺S population contained most of the Ig-secreting cells and responded poorly to mitogens. The Ig⁺L population contained few Ig-secreting cells and responded well to mitogens. Approximately 50% of Ig⁺L cells became Ig⁺S when cultured with Ig^? cells but this transition did not occur if Ig⁺S cells were added to the culture at the start of the incubation period. Purified Ig⁺ L cells lost their ability to form rosettes when cultured by themselves but retained their ability to form rosettes when cultured wih Ig^? cells. The data indicate that the Ig⁺S and Ig⁺L populations are at different stages in the differentiation of Ig⁺ cells (B cells) and that the Ig⁺L cells are subject to the regulatory influences of both Ig^? and Ig⁺S cells.     

Characterization of T lymphocyte subpopulations responsible for deficient interleukin 2 activity in patients with systemic lupus erythematosus

Normal immunoregulation depends on a complex set of cellular interactions in which interleukin 2 (IL 2) appears to play an important role. We have examined the IL 2 activity in patients with systemic lupus erythematosus (SLE). IL 2 production by phytohemagglutinin (PHA)-stimulated T cells for 48 hr was measured by the ability of their culture fluid to induce proliferation of normal human T cells that had been activated for more than 20 days by PHA plus IL 2. To measure IL 2 responsiveness, T cells were blasted by preincubation with concanavalin A for 96 hr and stimulated for another 72 hr with lectin-free standard IL 2. SLE T cells failed to produce normal levels of IL 2 in vitro compared with normal control T cells. This failure resided in both OKT4+ and OKT8+ cells. Furthermore, the abnormality was due neither to soluble inhibitory factors produced by SLE T cells nor to active suppressor cells that might be induced by PHA-stimulation. Responsiveness to IL 2 of T cells from some, but not all, SLE patients was decreased significantly from that of normal controls. Absorption studies as well as studies with anti-Tac antibody demonstrated that the impaired responsiveness of T cells in the specific patients with SLE was due to inadequate expression of IL 2 receptors on the T cells upon activation. This defect was exclusively ascribed to the dysfunction of OKT4+, but not OKT8+, cells. The above defects in production of and responsiveness to IL 2 observed in patients with SLE were present at all times regardless of the disease activity or of corticosteroid therapy. Thus, the deficient IL 2 activity may be intrinsic to SLE lymphocytes and may contribute to impaired immunoregulation and to the development of SLE.     

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases. I. Human red blood cell subpopulations

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with 51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations. 51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained. It was further determined, by repartitioning 51Cr-labeled cells from the left or right ends of a CCD of human red blood cells admixed to unlabeled unfractionated erythrocytes, that a subpopulation with higher partition coefficient exists (probably constituting the old red cells). These experiments serve to illustrate (a) that human red blood cells, contrary to a previous report, can be subfractionated by partitioning and (b) the usefulness of this new method in detecting smaller surface differences between closely related cell populations than was heretofore possible by partitioning alone.     

Use of cell affinity chromatography for separation of lymphocyte subpopulations

We describe the use of affinity chromatography for separation of cell populations that do not differ significantly with respect to gross physical properties such as size, density, or charge. Cell affinity chromatography exploits differences in cell surface macromolecules by passage of mixtures of cell populations through a column containing beads to which are attached chemical ligands with specific binding affinity for particular cell surface receptors. In this article we focus on the application of this concept to separation of mature T lymphocytes from peripheral blood. This serves as a model for the separation of these cells from bone marrow in order to prevent graft-vs.-host disease in bone marrow transplantation. However, the concept of cell affinity chromatography should find more general widespread utility in a variety of biotechnological applications. Thus, we introduce a simple theoretical framework which is necessary in order to understand the results that might be expected in any given situation. Finally, we use this theory to provide a tentative explanation for experimental observation of the effects of temperature and flowrate on the degree of separation achieved for our current pplication.     

Characterization of culture-induced cytotoxicity from human peripheral lymphocyte subpopulations

Cultured human lymphocyte subpopulations can generate cytotoxicity against K562 leukemia target cells under certain conditions. Such cytotoxicity arises during mixed leukocyte culture or in medium containing fetal calf serum (FCS), mitogenic factors, or interleukin 2. We cultured peripheral blood lymphocytes (PBL) in FCS-containing medium after fractionation of these cells on a Percoll discontinuous density gradient. Higher density cell fractions generated culture-induced spontaneous cytotoxicity (CIC) after 2-3 days in culture. CIC was not due to a loss of suppressor cells during fractionation since culture of PBL prior to fractionation yielded the same results. At least some CIC was associated with the differentiation of higher density cells to newly appearing lower density cells during culture. Most CIC required cells with the HNK-1- OKT3- OKM1+ phenotype. Culture-induced cytotoxicity has some similarities to the previously described lymphokine-activated killing but some important differences are also discussed.     

cAMP mediated cell growth regulation. II. Characterization of the biochemical change responsible for resistance to cAMP treatment

In this paper we characterize the biochemical defect of a mutant (10248) of CHO cells, resistant to the cAMP treatment. Cells cultured on MEM were collected each three days, homogenized and centrifuged. The cell extract was assayed for protein kinases activity and the binding of 8-N3-(32P)cAMP. The same extract was also applied on to a DEAE cellulose column, eluted with a linear gradient and the fractions tested for the phosphotransferase activity and 8-N3-(32P)cAMP binding. Mutant 10248 shows a different profile of protein kinases activity as compared to 10001 control. Protein kinases II is absent whereas a normal RII binding activity is present. RI shows altered affinity for cAMP.     

Selective proliferation of T cells in the mixed lymphocyte interaction

The contribution of avian thymus-derived (T) and bursa-derived (B₂) cells to the proliferating cell population in mixed lymphocyte cultures (MLC) was evaluated. When spleen cells of chickens containing chromosomally marked T and B₂ cells were stimulated in a one-way MLC by mitomycin C blocked allogeneic spleen cells, only T cells proliferated during a 4–9 day culture period. No evidence for significant recruitment of B₂ cells, expressed as proliferation of B₂ cells, was found. The initial viability and proliferative potential of B₂ cells was shown by a substantial and selective B₂ cell response to anti-immunoglobulin serum.     

T cell recognition in the mixed lymphocyte response. II. Ia-positive splenic adherent cells are required for non-I region-induced stimulation

Ia-positive splenic adherent cells (SAC) have been shown to be the predominant stimulators of a mixed lymphocyte response (MLR) to whole H-2 differences, in which most of the proliferative response is directed against I region-encoded determinants. The present studies were undertaken to examine the ability of several purified lymphoid subpopulations to activate T cells in response to the non-H-2-linked MIs products or to products of the K or D regions of H-2. The results demonstrated that adherent cell-depleted populations of T and B cells were nonstimulatory, whereas SAC were potent stimulators for responses involving each of these genetic differences. Treatment of these SAC with anti-Ia and C abrogated their MLR-stimulating ability. In contrast, whereas treatment of SAC with anti-Ia and C abrogated their ability to stimulate an MLR directed against K or D region-encoded determinants, this treatment had no effect on their ability to generate a cytotoxic T lymphocyte response against these same determinants. These findings suggest that in addition to presenting allogeneic I region-encoded determinants, Ia-positive SAC also play a unique role in the presentation of non-I region-encoded alloantigens to proliferating T cells.     

Peripheral blood lymphocyte subpopulations in sarcoidosis.

We have determined the numbers of thymus-derived (T) and bone marrow-derived (B) lymphocytes in the peripheral blood of 20 patients with sarcoidosis and 15 healthy controls. T cells were estimated from the number of lymphocytes forming rosettes in vitro with unsensitized sheep red blood cells, and B cells were enumerated by immunofluorescent assesssment of membrane-bound immunoglobulins. The total lymphocyte count was lower in patients with sarcoidosis owing to a depletion of T lymphocytes from the blood. Nonetheless, the relative and absolute numbers of B lymphocytes were significantly increased. These alterations in lymphocyte subpopulations did not show any consistent correlation with the duration of the disease, clinical stage, activity, or treatment. Changes in the subpopulations may be related to both decreased cellular immunity and increased reactivity of the antibody-forming system as commonly seen in sarcoidosis.     

Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles

Poly(A)+RNA fractions prepared from free and loosely and tightly membrane-bound polysome populations (poly(A)+RNAfree, poly(A)+RNAloose, and poly(A)+RNAtight) were used to drive cDNA in homologous and heterologous hybridization reactions. A large fraction by mass of sequences was shared among the three poly(A)+RNA populations, but shared sequences exhibited distinct frequency distributions within the different populations. 13-15 in vitro translation products of poly(A)+RNAfree and poly(A)+RNAloose detected by gel electrophoresis were shared. Most of these were produced in different relative quantities by the two RNA populations. Five or six higher mol wt polypeptides were produced by poly(A)+RNAloose that were not detected as products of either poly(A)+free or poly(A)+RNAtight. We suggest that loosely bound polysomes may not be artifactually derived as reflected in their quantitatively distinct poly(A)+RNA population. Two tightly membrane-bound RNP fractions were prepared from rat liver on the basis of their release from or retention on purified rough microsomes or a crude membrane fraction after in vitro disaggregation of polysomes with high-salt and puromycin. Homologous and heterologous hybridizations involving their poly(A)+RNA fractions revealed that a large portion by mass of sequences was shared but that these sequences exhibited distinct frequency distributions in the two fractions. The RNA fractions produced exhibited distinct frequency distributions in the two fractions. The RNA fractions produced an identical set of in vitro translation products but individual polypeptides were produced in different relative quantities. This indicates that the two RNP fractions do not arise by any random artifactual process and suggests that they may represent functionally distinct populations.     

Characterization of lymphocyte populations in nonspecific interstitial pneumonia*

Study objectives

Nonspecific interstitial pneumonia (NSIP) has been identified as a distinct entity with a more favorable prognosis and better response to immunosuppressive therapies than usual interstitial pneumonia (UIP). However the inflammatory profile of NSIP has not been characterized.

Design

Using immunohistochemistry techniques on open lung biopsy specimens, the infiltrate in NSIP was characterized in terms of T and B cells, and macrophages, and the T cell population further identified as either CD4 (helper) or CD8 (suppressor-cytotoxic) T cells. The extent of Th1 and Th2 cytokine producing cells was determined and compared to specimens from patients with UIP.

Results

In ten NSIP tissue samples 41.4 ± 4% of mononuclear cells expressed CD3, 24.7 ± 1.8% CD4, 19.1 ± 2% CD8, 27.4 ± 3.9% CD20, and 14.3 ± 1.6% had CD68 expression. Mononuclear cells expressed INFγ 21.9 ± 1.9% of the time and IL-4 in 3.0 ± 1%. In contrast, biopsies from eight patients with UIP demonstrated substantially less cellular staining for either cytokine (INFγ; 4.6 ± 1.7% and IL-4; 0.6 ± 0.3%). Significant populations of CD20 positive B-cells were also identified.

Conclusion

The lymphocytic infiltrate in NSIP is characterized by an elevated CD4/CD8 T-cell ratio, and is predominantly of Th1 type, with additional populations rich in B-cells. Such features are consistent with the favorable clinical course observed in patients with NSIP compared to UIP.     

Glycolipid markers of murine lymphocyte subpopulations.

We have shown previously that purified antibodies to ganglioside GM1 react with peripheral T cells and most thymocytes in several strains of mice, independent of Thy-1 phenotype. GM1 and the Thy-1.2 antigen cap independently on C3H thymocytes, which provides additional evidence that GM1 is not the Thy-1.2 antigen. In C3H and nude mice antibodies to GM1 also react with a population of cells, comprising about 25% of lymphocytes from lymph nodes or spleen, that bear surface immunoglobulin. After removal of immunoglobulin from these cells by digestion with proteolytic enzyme, the GM1+ cells regenerate their surface immunoglobulin during 18 hr in culture, which indicates that these double-labeled cells synthesize their surface immunoglobulin. Protease treatment of lymphocytes reveals receptors for antibodies to GM1 on most cells. These data indicate that T and B cells differ in the accessibility of GM1 to antibody, and not necessarily in their content of GM1. Purified antibodies to asialo GM1 react with mature T cells in all strains of mice tested. In contrast to anti-GM1, these antibodies do not react with most thymocytes, with immunoglobulin-bearing lymphocytes of C3H or nude mice, nor with pronase-treated B cells.     

Functional properties of lymphocyte subpopulations in hepatitis B virus infection. I. Suppressor cell control of T lymphocyte responsiveness

Hepatocellular injury in hepatitis B virus infection may be produced by an autoaggressive hepatocytotoxic immune response. To test the hypothesis that acquired suppressor cell defects may participate in such a response, we assessed the functional integrity of 2 suppressor cell populations in patients with type B viral hepatitis. Spontaneous suppression of the 1-way mixed lymphocyte response by radiation-resistant, adherent peripheral blood mononuclear cells decreases during the acute phase of disease, returns towards normal with clinical recovery, but remains depressed in patients with chronic hepatitis. The degree of spontaneous suppressor cell dysfunction correlates inversely with at least 1 biochemical parameter of hepatocellular injury (SGPT). The functional integrity of this suppressor cell fluctuates during chronic hepatitis and may reflect currently undefined biologic variables in this disease. Mitogen-induced suppression on lymphocyte activation by radiation resistant, nonadherent suppressor cells is also depressed in acute and chronic hepatitis, but it does not correlate with biochemical evidence of hepatocellular injury on an individual-patient basis. Documentation of these generalized defects of nonspecific suppressor cell function establishes a basis for the possible existence of specific anomalies of immuno-regulation that may permit the expression of normally suppressed auoaggressive hepatocytotoxic immune mechanisms in viral hepatitis.     

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases. II. A correction

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with 51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations. 51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between lymphocyte membrane molecules. II. Characterization of an interaction between B lymphocyte surface IgD and Fc IgG receptors that differs from the surface IgM-Fc IgG receptor interaction

The B lymphocyte surface membrane receptors IgD (sigD) and Fc IgG receptors (Fc gamma R) were evaluated for interactions by means of immunofluorescence. Ligand-(F(ab')2 anti-delta) induced capping of sIgD resulted in co-capping of Fc gamma R if the latter were occupied during the capping process by soluble antigen-antibody complexes (which themselves provided insufficient cross-linking to result in capping), but not if the Fc gamma R were occupied by monomeric IgG or unoccupied. Capping of Fc gamma R by highly cross-linked complexes did not cause co-capping of sIgD occupied by monomeric F(ab') anti-delta. The interaction between sIgD and Fc gamma R was specific in that cross-reactions between ligands were excluded and ligand-induced capping of sIgD did not cause co-capping of ligand-occupied sIgM or I-A antigens. The sIgD-Fc gamma R interaction occurred on only approximately 60% of B lymphocytes, and this B cell subpopulation did not correlate with other B cell subpopulations (CBA/N strain B cells and B cells bearing either large or small amounts of sIgD). The sIgD-Fc gamma R interaction differed from the sIgM-Fc gamma R interaction in that co-redistribution of the Fc gamma R was occupied by monomeric IgG and involved nearly all B lymphocytes. The qualitative and quantitative differences between the sIgD-Fc gamma R and sIgM-Fc gamma R interactions suggest a mechanism whereby the two antigen receptors could provide different signals to the B lymphocyte.