Activated and memory T lymphocytes in human milk.

Since activated macrophages and cytokines are found in human milk (HM), a flow cytometry study was conducted to determine whether T cells in HM display phenotypic markers of recent or previous activation. HM was collected during the first 3 d of lactation. The Paint-a-Gate program was used to optimize gating on the lymphocyte population. A mean +/- 1 SD of 4 +/- 3% of total HM leukocytes were lymphocytes and 96 +/- 3% were macrophages and granulocytes (N = 33 subjects). HM lymphocyte populations were further analyzed in five subjects. T cells (CD3+) represented 83 +/- 11% and B cells (CD19+) were 6 +/- 4% of HM lymphocytes. The mean CD4/CD8 ratio of T cells in HM was 0.88 (range 0.40-1.25). This ratio was significantly decreased compared to the peripheral blood (PB) of control adults (P less than 0.02) and postpartum women (P less than 0.02), due mostly to a significant increase in CD8+ CD3+ cells in HM compared to the PB of control adults (P less than 0.002) and postpartum women (P less than 0.05). T cells bearing markers of recent activation were significantly increased in HM compared to the PB of control adults: 85 +/- 7% of CD3+ cells in HM were HLA-DR+ (controls, 10 +/- 4%; P less than 0.001), and 15 +/- 6% of CD3+ cells in HM were IL-2R+ (controls, 6 +/- 2%; P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Adriamycin extravasation.

Adriamycin extravasation creates a severe tissue necrosis which is unusual, because it may not appear until several weeks later, and may continue to worsen for several months. As soon as the progressive nature of the tissue necrosis is established, we recommend that an early wide excision be performed in an attempt to remove the necrotic area and the surrounding tissues containing the extravasated drugs--before it has had an opportunity to diffuse even further. Adequate debridement requires removal of any adjacent tissue that is indurated, reddened, edematous, or pale. Skin grafts take poorly if there are small amounts of Adriamycin left in the tissue of the recipient site. Synergistic effects with radiotherapy, and continued systemic Adriamycin therapy, can aggravate or recall necrosis. The administration of more dilute solutions of Adriamycin may decrease the hazard of extravasation necrosis.     

Activated T lymphocytes within human solid tumors

Summary The majority of lymphocytes separated from tumor cell suspensions were T cells. Conjugates of T lymphocytes and tumor cells were often seen. Variable numbers of T cells exhibited signs of activation such as the ability to form stable E rosettes and attachment to normal and malignant cells (a phenomenon designated natural attachment: NA). A proportion of T cells activated in vitro by allogeneic stimulation regularly exhibit these properties. The T cell-tumor conjugates in the suspensions may represent the NA phenomenon, but they could also be the product of T cells that adhere on the basis of specific recognition of cell surface antigens.Abbreviations BBS balanced salt solution - E rosettes rosettes formed with sheep erythrocytes - EA rosettes rosettes formed with ox erythrocytes coated with anti-ox IgG - FCS fetal calf serum - MLC mixed lymphocyte cultures - NA natural attachment - PBL peripheral blood lymphocytes - SRBC sheep erythrocytes - T lymphocytes thymusderived lymphocytes     

Activated lymphocytes during acute Epstein-Barr virus infection

Activated lymphocytes, as identified by HLA-DR expression, associated with acute Epstein-Barr virus (EBV)-induced infectious mononucleosis (IM) were shown to be a heterogeneous population containing significantly elevated cytotoxic/suppressor (CD8) T cells, natural killer (CD16) cells and helper (CD4) T cells. CD8 T cells were the primary activated population representing 24.5% of the total lymphocyte population. The activated CD4 T cells and natural killer cells accounted for 6.7% and 3.5% of the total lymphocyte population, respectively. Analysis of serum soluble interleukin 2 receptors (IL-2R) demonstrated significantly (p less than 0.001) elevated levels in the serum of acute IM patients compared with normal controls. Elevated levels of serum IL-2R were correlated (r = 0.67) with increased percentages of Leu 2a+/HLA-DR+T cells (i.e., activated CD8 T cells). Patients with X-linked lymphoproliferative syndrome and virus-associated hemophagocytic syndrome, two syndromes associated with severe acute EBV infections, demonstrated the most dramatic increase in serum IL-2R levels. These data demonstrate that EBV is associated with intense immune stimulation and that during acute IM activated lymphocytes, other than the CD8 T cells, may contribute to the immune response to EBV.     

Clonal evolution in human lymphoblast cultures.

We established lymphoblast cultures from normal females heterozygous for electrophoretic variants of glucose-6-phosphate dehydrogenase (G6PD), and the X-linked markers have permitted us to look at evolution of these cell populations in culture. The established cultures were phenotypically heterozygous at onset, having both of the mosaic cell populations resulting from X chromosome inactivation. However, by the tenth subculture, the population of cells no longer reflected the heterozygous genotype in 50% of the cultures, as only a single G6PD isozyme was expressed. The ultimate cell composition seems to be influenced by the initial composition, by the nature of alleles at heterozygous X-linked loci that may provide a growth advantage (or disadvantage), as well as by stochastic events. Our results show that lymphoblast cultures may not reflect the X-linked phenotype of the cells from which they were derived. The fate of such cultures seems to be evolution toward clonal cell populations.     

Activated v-myc and v-ras oncogenes do not transform normal human lymphocytes.

Activated v-myc (pSV v-myc) and v-Ha-ras (GT10) oncogenes were introduced into normal human lymphocytes, NIH 3T3 fibroblasts, B-lymphoblastoid cells, and human epithelial cells, using a reconstituted Sendai virus envelope-mediated gene transfer technique. Efficient transfer of the plasmid in each cell type was demonstrable within 1.5 h of transfection by Southern blotting of extrachromosomal DNA extracts, which unexpectedly revealed that v-myc plasmid DNA was unstable in normal lymphocytes but not in the other cell types. The v-myc plasmid was stabilized when cotransfected into lymphocytes together with v-Ha-ras. The transfected v-Ha-ras plasmid was stable in all the cell types tested. v-myc plasmid expression was clearly detectable by 5 h in all cell types except human lymphocytes. Lymphocytes expressed v-myc when transfected together with v-Ha-ras. Transfected ras oncogene was efficiently expressed in all the cell types tested. Expression of the transfected genes increased at 24 and 48 h after transfection. Even though plasmid stability and expression were achieved in myc-ras-cotransfected lymphocytes, no effects on cellular DNA synthesis or immortalization were observed, in contrast to efficient transformation of NIH 3T3 fibroblasts by the same procedure. Our data suggest that efficient expression of transfected myc and ras oncogenes in normal quiescent human lymphocytes is not sufficient for the induction of cell growth and immortalization.     

Spontaneous cytotoxicity of human lymphoblast cell lines mediated by normal peripheral blood lymphocytes. I. Differential susceptibility of T-versus B-cell lines.

Human lymphoblast cell lines of B- and T-cell origin have been tested for their ability to serve as targets in a 4-hr ⁵¹Cr release microcytotoxicity assay using normal human peripheral blood lymphocytes as effector cells. Cell lines of T-cell origin were susceptible to lysis in this assay by effector lymphocytes from all normal donors tested. Cell lines of B-cell origin were repeatably lysed by normal lymphocytes from some, but not all donors. Spontaneous cytotoxicity of B-cell lines, when observed, was also quantitatively less than was obtained using T-cell lines as targets. One cell line (RPMI-7666), of B-cell origin, was not susceptible to spontaneous cytotoxicity by almost all of the normal lymphocyte effectors tested. Lymphocytes from patients with acute lymphoblastic leukemia in remission were less capable of effecting lysis in this assay.     

Immortalized myeloid suppressor cells trigger apoptosis in antigen-activated T lymphocytes

We described a generalized suppression of CTL anamnestic responses that occurred in mice bearing large tumor nodules or immunized with powerful recombinant viral immunogens. Immune suppression entirely depended on GM-CSF-driven accumulation of CD11b(+)/Gr-1(+) myeloid suppressor cells (MSC) in secondary lymphoid organs. To further investigate the nature and properties of MSC, we immortalized CD11b(+)/Gr-1(+) cells isolated from the spleens of immunosuppressed mice, using a retrovirus encoding the v-myc and v-raf oncogenes. Immortalized cells expressed monocyte/macrophage markers (CD11b, F4/80, CD86, CD11c), but they differed from previously characterized macrophage lines in their capacities to inhibit T lymphocyte activation. Two MSC lines, MSC-1 and MSC-2, were selected based upon their abilities to inhibit Ag-specific proliferative and functional CTL responses. MSC-1 line was constitutively inhibitory, while suppressive functions of MSC-2 line were stimulated by exposure to the cytokine IL-4. Both MSC lines triggered the apoptotic cascade in Ag-activated T lymphocytes by a mechanism requiring cell-cell contact. Some well-known membrane molecules involved in the activation of apoptotic pathways (e.g., TNF-related apoptosis-inducing ligand, Fas ligand, TNF-alpha) were ruled out as candidate effectors for the suppression mechanism. The immortalized myeloid lines represent a novel, useful tool to shed light on the molecules involved in the differentiation of myeloid-related suppressors as well as in the inhibitory pathway they use to control T lymphocyte activation.     

Activated human T lymphocytes express albumin binding proteins which cross-react with alpha-fetoprotein.

The kinetics of iodinated human serum albumin ([125I]Hu-SA) and alpha-fetoprotein ([125I]Hu-AFP) binding and endocytosis by resting and phytohemagglutinin (PHA)-activated human T lymphocytes were studied comparatively. The binding of both SA and AFP appeared considerably increased upon blastic transformation. SA, like AFP, binds in a saturable way to the surface of PHA-stimulated human T lymphocytes at 4 degrees C and is endocytosed at 37 degrees C. Two saturation plateaus were observed by incubating at 4 degrees C activated T lymphocytes with [125I]Hu-AFP at different concentrations (10 ng-250 micrograms/ml), while only one saturation plateau was obtained by incubating cells with [125I]Hu-SA in the same conditions. Scatchard analysis of binding data revealed two types of binding sites for Hu-AFP and one for Hu-SA. Competition experiments using proteins of human and bovine origin are in favor of the presence on the surface of these cells of a common binding site for AFP and SA. Pulse-chase experiments showed that internalized [125I]SA was released mainly in a degraded form from the cells, in agreement with detection by ultrastructural cytochemistry of peroxidase-conjugated SA in lysosome-like bodies by ultrastructural cytochemistry. This contrasts with the intracellular pathway of AFP, which as previously described (Geuskens, M., et al., Eur. J. Cell Biol. 50, 418-427 (1989)), moves to tubular-vesicular structures in the Golgi region and is recycled for the most part undegraded.     

Activated T lymphocytes and macrophages secrete fibronectin which strongly supports cell adhesion.

Matrix-bound fibronectin (FN) appears to be involved in cell adhesion and motility mediated by integrin receptors. Although lymphoid cells and other cell types are capable of producing and secreting FN, the precise role of this secreted FN-like factor in regulating immune reactions is unclear. In the present study we analyzed the adhesive properties of FN secreted by rat CD4+ T cells and clone cells activated by the T cell mitogen concanavalin A (Con A), antigen, or via the CD2 pathways, or by macrophages (M phi) activated by lipopolysaccharide (LPS). Immobilized culture supernatant (CS) from the activated T cells or M phi supports the adhesion of activated rat or human CD4+ T cell or murine tumor cell. These CS contained FN and were more potent at facilitating cell adhesion then plasma FN. The adhesion activity of CS was attributed to FN because (a) gelatin columns depleted the FN present in the CS and (b) pretreating the cells with peptides of the cell-binding domain of FN abrogated their ability to bind CS. CS-mediated adhesion appears to occur primarily via the recognition of the Arg-Gly-Asp (RGD) by the beta 1-integrin-specific receptors of the adhesive cells. Thus, we postulate that FN secreted by various types of leukocytes is involved in promoting essential cell-matrix interactions, possibly affecting cell-adhesive and migratory processes at inflammatory or extravasation sites.     

Analysis of HGPRT- CRM+ human lymphoblast mutants.

Three 6-thioguanine-resistant mutants of the human diploid lymphoblast line MGL-8 were studied. The inactivation by heat of both HGPRT activity and antigenicity of the HGPRT immunologically cross-reacting material of the A30 mutant cells were not protected by PRPP, indicating that the HGPRT in A30 cells has an altered PRPP binding site, leading to lack of stabilization and rapid degradation of the enzyme. Two dimensional separations of the immunoprecipitates from extracts of the parental and mutant cell lines showed that the A35 mutant CRM has a more acidic isoelectric pH, while the A30 CRM has a more basic isoelectric pH and that the A30 protein has a faster rate of degradation than the wild-type HGPRT. The A30 CRM also has a smaller molecular size than the wild-type enzyme.     

Invariant natural killer T cells trigger adaptive lymphocytes to churn up bile

How innate immune response causes autoimmunity has remained an enigma. In this issue of Cell Host & Microbe, Mattner et al. demonstrate that invariant natural killer T cells activated by the mucosal commensal Novosphingobium aromaticivorans precipitate chronic T cell-mediated autoimmunity against small bile ducts that mirrors human primary biliary cirrhosis. These findings provide a mechanistic understanding of the role of innate immunity toward a microbe in the development of autoimmunity.     

Activated human lymphocytes secrete a soluble factor that aggregates leukocytes

Human polymorphonuclear leukocytes can be activated by various inflammatory stimuli to display increased cell aggregation which is potentially an important pathogenetic mechanism. This study describes a soluble factor produced by concanavalian A-stimulated lymphocytes that causes human leukocytes to aggregate. This factor could be assayed quantitatively by measuring the light absorbance of polynuclear leukocyte suspension using a spectrophotometer. The lymphokine involved, namely the leukocyte aggregating factor (LAgF) was released by non pulse exposure to the mitogen for up to 72 hr with a maximum at 48 hr. LAgF was characterized by Sephadex gel filtration, chromatofocusing, enzymatic and chemical treatment. Sephadex G 100 gel filtration showed LAgF activity in a molecular range of 40,000-65,000. Chromatofocusing of culture supernatant showed LAgF in a single broad peak (4.8-5.4) with a maximum activity at pI 5.2. Human LAgF was heat sensitive, inactivated by treatment with chymotrypsin, and not affected by neuraminidase. Activity was partially recovered from the supernatant after protein precipitation with 1 M perchloric acid and not destroyed by 0.02 M sodium periodate. These findings characterize LAgF as a protein. These data suggest that LAgF is not different from leukocyte inhibiting factor by virtue of its size and physiological properties.     

Activated lymphocytes depress phagocytosis of latex particles by human monocyte-macrophages

In this study, monocyte-macrophages of normal human donors were cultured with and without lymphocytes and antigen in order to define the effect of antigen-stimulated lymphocytes on phagocytosis by macrophages. Phagocytosis was assessed by uptake of radio-labeled latex particles by macrophage monolayers. Although no effect was noted when purified macrophage monolayers were cultured in the presence of antigen, marked inhibition of phagocytosis was observed when macrophages were cultured in the presence of autochthonous antigen-stimulated lymphocytes. The degree of phagocytic depression correlated with the delayed cutaneous hypersensitivity response of the donor and with the concentration of antigen present in the system.     

Uptake of alpha-ketoisocaproic acid in lymphoblast line WI-L2.

The uptake of α-ketoisocaproate by the cultured human lymphoblast line WI-L2 appears to be mediated by a transport system which has an apparent Km of 125 μM. The rate of uptake of α-ketoisocaproate decreases with increasing pH values, i.e., pH 6 > 7 > 8 and is stimulated by sodium at all pH values. Closely related branched chain α-ketoacids, α-keto-β-methylvaleric and α-ketoisovaleric exhibited the greatest inhibition of α-ketoisocaproate transport. Straight chain α-keto acids inhibited α-ketoisocaproic acid uptake to a lesser degree as did the α-hydroxy analogs of the branched chain α-keto acids. Inhibitors of the general anion transport system of erythrocytes, 1-anilino-8-napthalene sulfonic acid and 4-acetamido-4-isothiocyanostilbene-2-1′-disulfonic acid did not affect α-ketoisocaproate transport. A reduced sulfhydryl group is critical for α-ketoisocaproate acid uptake; transport is partially or completely inhibited by sulfhydryl reagents such as dithio-bis-nitrobenzoate, iodoacetamide, and p-chloromercuribenzoate. Inhibition by the sulfhydryl reagents is reversed with β-mercaptoethanol or partially with dithiothreitol.     

Purification and characterization of human lymphoblast N-acetylglucosamine-1-phosphotransferase.

N-Acetylglucosamine-1-phosphotransferase (GlcNAcPTase) was solubilized with 2% Tergitol NP-10 from cultured human lymphoblast cells and purified 3840-fold with 14% recovery using lentil lectin-Sepharose 4B, DEAE-Sephacel and Sephacryl S-400 chromatographies. The partially purified enzyme requires the non-ionic detergent Tergitol NP-10 and a divalent cation, Mn2+ or Mg2+, for its activity and exhibits an optimal pH at 7.2-7.5 in Tris-maleate buffer. Kinetic studies demonstrated an apparent Km of 24 microM for the donor UDP-N-acetylglucosamine and of 117 mM for the artificial acceptor alpha-methylmannoside. The GlcNAcPTase is inhibited by UDP and UDP-glucose, and by negatively charged phospholipids including phosphatidylserine, phosphatidylglycerol and phosphatidic acid. The apparent mol. wt of the human lymphoblast GlcNAcPTase is approximately 1000 kDa, which is analogous to that reported for the partially purified enzyme from rat liver (Waheed et al., 1982).     

Mitogenic and non-mitogenic ligands trigger a calcium-dependent cytosolic acidification in human T lymphocytes

We have investigated the patterns of cytosolic pH and Ca2+ ([Ca2+]i) changes after exposure of human peripheral blood T cells to different mitogenic and non-mitogenic ligands. Using ligands that have different accessory cell requirements and varying effect on [Ca2+]i or cell proliferation, we observed that intracellular acidification occurred only with agents that increased [Ca2+]i. However, treatment of the cells with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, results in significant cytosolic alkalinization without detectable acidification, but did not affect the proliferative responses to mitogenic ligands and was a potent co-mitogen with non-mitogenic ligands. These data indicate that initial acidification or alkalinization responses are not essential for early activation or triggering of DNA synthesis.