Target Cell Formation Research Articles

LPA suppresses T cell function by altering the cytoskeleton and disrupting immune synapse formation

Cancer and chronic infections often increase levels of the bioactive lipid, lysophosphatidic acid (LPA), that we have demonstrated acts as an inhibitory ligand upon binding LPAR5 on CD8 T cells, suppressing cytotoxic activity and tumor control. This study, using human and mouse primary T lymphocytes, reveals how LPA disrupts antigen-specific CD8 T cell:target cell immune synapse (IS) formation and T cell function via competing for cytoskeletal regulation. Specifically, we find upon antigen-specific T cell:target cell formation, IP3R1 localizes to the IS by a process dependent on mDia1 and actin and microtubule polymerization. LPA not only inhibited IP3R1 from reaching the IS but also altered T cell receptor (TCR)–induced localization of RhoA and mDia1 impairing F-actin accumulation and altering the tubulin code. Consequently, LPA impeded calcium store release and IS-directed cytokine secretion. Thus, targeting LPA signaling in chronic inflammatory conditions may rescue T cell function and promote antiviral and antitumor immunity.

A hypothesis of target cell formation in sickle cell disease

A fraction of erythrocytes appear as target cells in stained blood smears in sickle cell disease, due to a inheritance of the hemoglobin variant Hb S, polymerizing upon deoxygenation. These cells appear in a three dimension as thin cups. A process of their formation in this disease is proposed based on a band 3-based mechanism of the erythrocyte shape control, able to explain the erythrocyte echinocytosis by glucose depletion. It indicates that their formation is due to a stomatocytogenic slow outward transport of the dibasic form of endogenous Pi with an H+ by band 3, promoted by the decrease of the Donnan ratio, which decreases cell pH and volume, attributed by a decrease of cell KCl concentration by the higher efflux of K+Cl− cotransport and Ca2+ activation of the Gardos channel. Its implications are briefly discussed with respect to target cells per se, target cell formation in other hemoglobinopathies, acquired and inherited disorders of the lipid metabolism and dehydrated hereditary stomatocytosis as well as a stomatocyte presence in a double heterozygote of Hb S and Hb C and of an involvement of the process of target cell formation in acanthocytosis in acquired and inherited disorders.

Target cell formation leading to pseudoleukocytosis in patients with malignant disorders

Article Target cell formation leading to pseudoleukocytosis in patients with malignant disorders was published on August 1, 2010 in the journal Clinical Chemistry and Laboratory Medicine (CCLM) (volume 48, issue 8).

Danazol therapy renders red cells resistant to osmotic lysis 1

Danazol, an attenuated androgen, is useful in endometriosis, idiopathic thrombocytopenic purpura (ITP), and autoimmune hemolytic anemia (AIHA). However, its mechanism of action is unknown. We investigated the possibility that danazol affects cell membranes directly. Red cell osmotic fragility was studied in patients receiving danazol. A significant decrease in osmotic fragility was observed. Accompanying the change, peripheral blood smears showed many target cells and electron microscopy revealed extra folds in erythrocyte membranes. Twenty-two patients were studied prospectively before and after danazol. Osmotic fragility decreased significantly (P less than 0.001) in 1 month of therapy and progressed with further treatment. A rebound increase (P less than 0.01) was observed in 1 month after discontinuation of danazol among 16 patients. Incubation experiments showed that danazol-induced changes are not reversed with normal sera. Patient sera did not induce the changes in normal red cells. Danazol in vitro protected red cells from osmotic lysis at low concentrations but enhanced lysis at high concentrations. We suggest that danazol alters red cell membranes directly to increase their surface area, inducing target cell formation and increasing their resistance to osmotic lysis.

Effects of chloramphenicol on the development of immune responses to canine distemper virus in beagle pups.

The effect of oral chloramphenicol (CHPC) on the development of immune responses to canine distemper virus (CDV) in Beagle pups was studied. Dogs were treated with CHPC for 14 days at a dose of 50 mg/kg, three times a day. Hematologic changes in CHPC-treated dogs included: polychromasia, anisocytosis, and target cell formation of red blood cells concurrent with vacuolation of lymphocytes and basophilic granule formation in neutrophils. Dogs given this therapy showed normal in vivo and in vitro immune responses after CDV vaccination and survived a virulent CDV challenge, whereas untreated, unvaccinated dogs became ill or died after challenge exposure. The results of this study indicate that CHPC therapy does not interfere with either the prechallenge immune response to attenuated viral antigen or the efficient immune mechanisms invoked during virulent virus challenge.

Target Antigen of Vaccinia‐Infected Cells Recognized by Virus‐Specific Cytotoxic T Lymphocytes

A vaccinia-specific target antigen for recognition of anti-vaccinia cytotoxic T lymphocytes (CTL) was found to be formed on the surface of infected cells through two distinct processes. In the first phase, the expression of the target antigen was dependent on the dose of inoculated virus, without specific protein synthesis. The target antigen seems to be produced by virus-cell fusion. In the second phase, the expression of the target antigen was accompanied by synthesis of an early protein. In spite of the difference in their mode of expression, the first-phase and the second-phase target antigens were cross-reactive in cytotoxicity inhibition assays. Cowpox virus, CPR Cl strain, brought about a lower response than vaccinia virus, IHD-J strain, in both sensitization of CTL and formation of CTL-susceptible cells at both the first and the second phase. The cross-reactive, but inefficient, recognition of anti-vaccinia CTL for cowpox-infected cells suggested a slight difference in the target antigens of the two viruses. Attempts to identify the target antigen were then made by comparing the polypeptide composition of vaccinia virus, cowpox virus, and their recombinants. SDS-PAGE analysis of trypsin-activated viruses revealed 44K (cowpox)/45K (vaccinia) polypeptides which corresponded to the difference in target cell formation. Trypsinization of the viruses also increased the ability of the virus to induce the production of CTL-susceptible target cells.

Recognition of viral glycoproteins by influenza A-specific cross-reactive cytolytic T lymphocytes.

Two populations of cytolytic T lymphocytes (CTL) generated after influenza A virus infection can be distinguished into one with specificity for the sensitizing hemagglutinin type and a second with cross-reactivity for antigens induced by other type-A influenza viruses. The molecules carrying the antigenic determinants recognized by the cross-reactive CTL were studied. In L-929 cells abortively infected with fowl plague virus, matrix (M) protein synthesis is specifically inhibited, whereas the envelope glycoproteins, hemagglutinin and neuraminidase, are synthesized and incorporated into the plasma membrane. These target cells were lysed by cross-reactive CTL. The envelope proteins of type A/Victoria virus were separated from the other virion components and reconstituted into lipid vesicles that lacked M protein that subsequently were used to prepare artificial target cells. Target-cell formation with vesicles was achieved by addition of fusion-active Sendai virus. These artificial target cells were also susceptible to lysis by cross-reactive CTL. In contrast to previous observations that suggested that the M protein of influenza viruses is recognized by these effector cells, we present evidence that the antigencic determinants induced by the viral glycoproteins are recognized.

Difference in capacity of Sendai virus envelope components to induce cytotoxic T lymphocytes in primary and secondary immune responses

Studies were made on the abilities of Sendai virus envelope components to induce primary and secondary generations of virus-specific cytotoxic mouse T lymphocytes (CTL). The primary CTL response in BALB/c mice was induced by reassembled envelope particles that had fusion activity but not by envelope glycoproteins without fusion activity, although both preparations induced a humoral immune response. Reconstitution of membrane-bound envelope proteins from envelope glycoproteins with lipids restored the fusion activity and the capacity to induce CTL. Target cells susceptible to virus-specific CTL could be induced by reassembled envelope particles, but not by envelope glycoproteins or LLC-MK2 cell-grown Sendai virus, neither of which had fusion activity. On the other hand, all the viruses and envelope components tested were found to stimulate a virus-specific CTL response in the in vitro secondary generation of CTL from virus-primed spleen cells. These results suggest that Senaei virus fusion activity is involved in primary induction of the CTL response as well as in target cell formation, but that it is not essential for secondary stimulation of the CTL response.

Influenza virus‐specific T cell‐mediated cytotoxicity: integration of the virus antigen into the target cell membrane is essential for target cell formation

This study deals with the requirements for target cell recognition by influenza A virus-specific cytotoxic T lymphocytes (CTL). H-2-identical cells were incubated with infectious or UV light-inactivated influenza A virus expressing either cleaved or uncleaved hemagglutinin (HA). Thereafter, the treated cells were tested in a 4-h 51Cr assay for susceptibility to CTL-mediated cytolysis. Regardless whether the influenza virus was infectious, virions expressing cleaved HA were efficient in target cell formation. In contrast, cells incubated with either active or UV-inactivated virions expressing uncleaved HA were not lysed by virus-specific CTL. Yet, after mere trypsin-mediated cleavage of the HA of cell-absorbed viroins, strong cytolysis could be observed. On the other hand, solubilization of the envelope lipid bilayer by ethylether abolished the capacity of the remaining HA to induce target cell formation. The results clearly suggest that mere absorption of virions to the membrane of cells, which is performed by virus with uncleaved HA, is insufficient for target cell formation. For this, both cleaved HA and an intact envelope appear to be crucial. We conclude that fusion of the virion into the cell membrane is essential for target cell formation.

Fusion of Sendai virus with the target cell membrane is required for T cell cytotoxicity

INFECTION of mice with viruses can generate cytotoxic T lymphocytes (CTL) which show restricted specificity for target cell lysis. Specific lysis requires that the virus used to prime the target cells must be of the same type as that used to sensitise the CTL, and that both target and CTL cells must express the same major histocompatability complex (MHC) gene product(s). The nature of the viral gene product(s) and their interaction with the MHC gene product(s) have been the subject of recent stud1–5. Previously we used Sendai virus to show that lysable target cells can be obtained using membrane vesicles which contain only the viral glycoproteins, indicating that these may be the specific viral gene products involved in target formation5. Sendai virus contains two glycoproteins—the haemagglutinin-neuraminidase (HANA) which promotes attachment of virus to cells and the fusion protein (F) which is involved in subsequent virus cell fusion7–9. Both activities are necessary for insertion of these viral glycoproteins into the plasma membrane of the cell10. In this letter we suggest that the insertion of the viral glycoproteins into the cell membrane is an essential step in target cell formation since we can show that virus containing an inactive fusion protein precursor (F0) cannot elicit T cell cytotoxicity unless the fusion activity is generated by proteolytic cleavage of the precursor. Sugamura et al.6 have suggested that it is primarily the F glycoprotein of the Sendai virus envelope which is essential for the formation of the target antigen, as virus lacking the functional activities of F following trypsin digestion was inactive in priming target cells for T cell killing. However, we show that proteolytic inactivation of either of the two glycoproteins (F or HANA) of virus used to prime target cells will abolish the cytotoxic response.

Mild "unstable haemoglobin haemolytic anaemia" caused by haemoglobin Shepherds Bush(B74(E18) gly--asp).

A 30 YEAR old South African woman of British extraction was investigated because of a persistent reticulocytosis. As a child she had been chronically anaemic, and at the age of six her spleen had been removed. After that time she had been well, but on one occasion she became “ill” after a course of sulphonamide. She was found to have a well compensated haemolytic anaemia; the haemoglobin level was 13 g/100 ml. of blood, and 5–8 per cent of her red cells were reticulocytes. Though of normal size the red cells showed target cell formation, basophilic stippling, and 75–80 per cent contained Heinz bodies. The test for abnormally heat labile haemoglobin1 was positive. The patient's mother was found to carry the same haemoglobin abnormality but was not thought to suffer from severe haemolysis. She did, however, experience an episode of severe haemoglobinuria following sulphonamides.

X-linked hypochromic anaemia of mice.

SUMMARYAn X‐linked recessive mutant gene (symbol sla) in the mouse causes hypochromic anaemia in hemizygous males and homozygous females; heterozygous carrier females show only slight changes. A detailed assessment of the anaemia is presented here. The red cells show a wide variety of morphological changes, including target cell formation, and the MCV is strikingly reduced. The anaemia and morphological changes tend to regress with increasing age. The anaemia also responds to parenteral iron‐dextran treatment, but not to pyridoxine and other agents.These findings and preliminary results of other investigations suggest that the underlying metabolic disturbance is likely to involve some step in the supply of iron to the erythron. It thus appears that in the mouse an X‐linked locus plays a role in iron metabolism, and this mutant provides an opportunity further to elucidate mechanisms of iron transfer.

Clinical Manifestations of Inherited Abnormal Hemoglobins

Abstract A clinical and hematologic description is presented of the family demonstrating the existence of the genetically determined hemoglobin-D originally described by Itano. The interaction of hemoglobin-D with hemoglobin-S in two members of this family has resulted in a hemolytic process and a mild anemia. Clinically, one of the patients is asymptomatic. The other has repeated painful episodes characteristic of those seen in sickle cell crises. Clinical and hematologic evaluations of two members of the family inheriting normal hemoglobin and hemoglobin-D revealed no abnormalities. The only feature of hemoglobin-D which distinguishes it from normal hemoglobin is its electrophoretic mobility which is similar to that of hemoglobin-S. Hemoglobin-D is distinguished from hemoglobin-S by a higher solubility and by a failure to sickle and from hemoglobin-C by a lack of target cell formation and a different electrophoretic mobility. Clinical, hematologic and special hemoglobin studies are presented of a family exhibiting a new abnormal hemoglobin (hemoglobin-E). Its interaction with thalassemia trait in one of its members also is described. Hemoglobin-E does not sickle; under alkaline conditions it migrates slowly at a rate comparable to hemoglobin-C, while at an acid pH it migrates at a rate similar to hemoglobin-S. The result of its interaction with thalassemia is a microcytic, hypochromic anemia clinically resembling thalassemia major of an intermediate degree of severity. The major defect appears to be in hemoglobin synthesis, although the slight persistent reticulocytosis suggests a mild hemolytic process.