Characteristics of BALB/C T cell lymphomas grown as continuous in vitro lines.

Transplanted lymphomas (Thy 1.2+, Ig-) of BALB/c mice, induced by the injection of 1-ethyl-1-nitrosourea, were adapted for growth as in vitro lines to provide potential tools for investigation of T lymphocyte differentiation and functions. All these tissue culture lines maintained the same pattern of surface differentiation antigens (Ly, TL, and Thy-1 antigens) as they had expressed during in vivo passages: BALENTL 13 was Thy 1.2+, TL.2-, and Ly 1+2-. BALENTL 3, 4, 5, 6, 7, 8, and 14 were Thy 1.2+, TL.2+, and Ly 1-2+. P1798 and BALENTL 9 were Thy 1.2+, TL.2+, and Ly 1-2+. There were various levels of terminal transferase activity present among these T cell tumor lines. The range of variation was from 4.6 units/10(8) cells to 29.3 units/10(8) cells (normal thymocytes, 5.0 units/10(8) cells). This 6-fold variation in TdT activity was present even among those cell lines which were Ly 1-2+, TL+. Most cultures lines had chromosome numbers near 40 and generation times of 11 to 22 hr. There were no significant morphologic changes after the adaptation of these tumors in culture except an increase in cytoplasmic C-type virus particles.     

In vitro targeting of erythrocytes to cytotoxic T-cells by coupling of Thy-1.2 monoclonal antibody.

A method was explored to develop a general means to target erythrocytes to T-cells in vitro. Mouse erythrocytes were coupled with an anti-Thy-1.2 monoclonal antibody by two methods. Chromic chloride coupling of antibody was preferred to biotinylation. The morphology, osmotic fragility, and the hematological values of treated cells were normal compared with those of control erythrocytes. Antibody-coupled erythrocytes were incubated with cytotoxic T-lymphocytes (CTLL) in vitro at a 20:1 ratio. Approximately 60-70% of the CTLL formed rosettes. The cell mixture was subjected to gradient centrifugation and separated into four fractions. THe rosettes were clearly identified only in the treated group containing anti-Thy-1.2-coupled erythrocytes. No rosettes were found when aspecific monoclonal antibody was coupled to erythrocytes. Examination by scanning and transmission electron microscopy revealed CTLL with 4-5 erythrocytes attached to them but did not show any evidence of membrane fusion. Rosettes of CTLL incubated in vitro proliferated as well as CTLL alone and maintained their dependency on interleukin-2. Targeting of erythrocyte carriers to lymphocytes offers the potential for delivery of molecules directly to the target cell.     

Immunoregulation by mouse T-cell clones. I. Suppression and amplification of cytotoxic responses by cloned H-Y-specific cytolytic T lymphocytes

H-Y-specific and H-2Db-restricted, Lyt-1-2+ T-cell clones ( CTLL ) with graded specific cytotoxic activities on male C57BL/6 (B6) target cells ( 1E3 , ; 2C5 , ++; 2A5 , +, 3E6 , +/-) were tested for their capacity to inhibit the generation of H-Y-specific cytotoxic T lymphocytes (CTL) in vitro. Addition of irradiated lymphocytes of CTLL 1E3 and CTLL 3E6 but not those of CTLL 2A5 or CTLL 2C5 abolished the generation of CTL from in vivo primed H-Y-specific precursor cells (CTLP) when added to fresh mixed-lymphocyte cultures (MLC). Exogenous sources of T-cell growth factors (TCGF) did not overcome suppression. Rather the presence of TCGF resulted in a further enhancement of suppressive activities in CTLL 1E3 and 3E6 and the induction of similar activities in cells from CTLL 2A5 and 2C5 , which by themselves were not inhibitory. Moreover when added to similar MLC on Day 1 instead of Day 0, only irradiated cells of CTLL 3E6 but not those of the other three CTLL were suppressive. Induction of suppressive activities in H-Y-specific CTLL was independent of the appropriate male stimulator cells since it was also observed in MLC induced by irrelevant antigens (H-2, trinitrophenol). Furthermore at low cell numbers, irradiated lymphocytes from any of the CTLL consistently enhanced CTL activities generated from H-Y-specific CTLP. This augmenting activity, which was not TCGF, could be transferred by soluble mediators present in antigen-sensitized CTLL cultures. Thus, these data indicate (i) that cytotoxic effector cells can function as suppressor cells in the generation of CTL, (ii) that the cytotoxic activity of cloned CTL does not correlate with their capacity to suppress CTL responses, (iii) that the inhibition of CTL responses by CTLL is not due to simple consumption of T-cell growth factors produced in MLC, and (iv) that different CTL clones may interfere with the generation of CTL at different stages of their maturation. Moreover, the experiments suggest an antigen-independent enhancement of suppression by the interaction of CTL with lymphokines. Together with the augmenting activity evoked by cloned CTL the data provide strong evidence for the expression of multiple immunological functions by one particular subset of T cells and suggest that cytotoxic effector cells can differentially regulate the maturation and/or clonal expression of their precursor cells.     

Epithelial lining fluid solute concentrations in chronic obstructive lung disease patients and normal subjects.

The exhaled breath condensate (EBC) method represents a new, noninvasive way to detect inflammatory and metabolic markers in the fluid that covers the airways [epithelial lining fluid (ELF)]. However, respiratory droplets represent only a very small and variable fraction of the EBC, most (approximately 99.99%) of which is water vapor. Our objective was to show that ELF concentrations could be calculated from EBC values by using any of three dilutional indicators (urea, total cations, and conductivity) in nine normal and nine chronic obstructive lung disease (COPD) subjects. EBC concentrations of Na(+), K(+), Ca(2+), Mg(2+), total cations, urea, and conductivity varied over a 10-fold range among individuals, but concentrations of these constituents (except Ca(2+)) remained well correlated (r(2) = 0.44-0.83, P < 0.001). Dilution (D) of respiratory droplets in water vapor was calculated by dividing plasma concentrations of the dilutional indicators by EBC concentrations. Estimates of D were not significantly different among these indicators, and urea D averaged 10,800 +/- 2,100 (SE) in normal and 12,600 +/- 3,300 in COPD subjects. Although calculated Na(+) concentrations in the ELF were less than one-half those in plasma, and concentrations of K(+), Ca(2+), and Mg(2+) exceeded those in plasma, total cation concentrations in ELF were not significantly different from those in plasma, indicating that ELF is isotonic in both normal and COPD subjects. EBC amylase concentrations (measured with an ultrasensitive procedure) indicated that saliva represented <10% of the respiratory (ELF) droplets in all but three samples. Dilutional and salivary markers are essential for interpretation of EBC studies.     

Characterization of lymphoid tumors induced by a recombinant murine retrovirus carrying the avian v-myc oncogene. Identification of novel (B-lymphoid) tumors in the thymus

Lymphoid tumors induced by a recombinant murine retrovirus carrying the v-myc oncogene of avian MC29 virus were characterized. The Moloney murine leukemia virus myc oncogene (M-MuLV (myc], carried by an amphotropic MuLV helper, induced tumors in NIH Swiss and NFS/N mice after a relatively long latency (8 to 24 wk). Tumor masses appeared in the thymus, spleen, and lymph nodes. Flow cytometry of the tumor cells indicated that approximately 50% were positive for Thy 1.2. Most of these tumors also expressed one or more other cell surface markers of thymocytes and mature T cells (CD4, CD8). Southern blot hybridization revealed genomic rearrangements for the TCR beta genes. The TCR beta analysis suggested that the M-MuLV(myc)-induced Thy 1.2+ tumors were derived from somewhat less mature cells than tumors induced by M-MuLV, which is a classical non-acute retrovirus lacking an oncogene. The remainder of the M-MuLV(myc)-induced tumors were Thy 1.2-, but they were positive for Ly-5 (B220) and also for MAC-2. The Thy 1.2- tumors were characteristically located in the thymus. However, they were negative for TCR beta gene rearrangements. Some, but not all, of the Thy 1.2- tumors contained rearrangements for Ig genes. Additionally, they typically expressed mRNA specific for B but not for T cells. Thus, these thymic tumors had characteristics of the B cell lineage. Tumor transplantation experiments demonstrated that the Thy 1.2- tumor cells could reestablish in the thymus and spleen of irradiated hosts, and low level expression of the Thy 1 molecule was observed in the thymus but not the spleen on the first passage. After serial passage, one Thy 1- tumor altered its cell surface phenotype to Thy 1low B220-.     

Incorporation of Glycosylated β-Galactosidase into Bovine Brain Synaptosomes

Using a synthesized glycoprotein, beta-galactosidase modified with p-aminophenyl beta-D-galactopyranoside (beta-D-Gal beta-gal), the incorporation of the glycoprotein into bovine brain synaptosomes was studied. The uptake was mediated by a specific receptor to beta-D-galactoside, and was inhibited by GM1 ganglioside. The uptake was found to require energy and to be sensitive to metabolic inhibitors. Kinetic studies on beta-D-Gal beta-gal uptake indicated the presence of a saturable, carrier-mediated transport system in synaptosomes. By subcellular fractionation the beta-D-Gal beta-gal taken up was found in the fractions corresponding to the nucleus and membrane fragments, the soluble cytosomal fractions, and the mitochondria and lysosomes. The uptake was markedly increased by addition of Ca2+ to the incubation medium. The maximal uptake was obtained at pH 8.0 in the presence of 10 mM Ca2+ at 37 degrees C. By addition of a Ca2+ ionophore A23187, beta-D-Gal beta-gal uptake was increased in a dose-dependent way parallel to the increase in the intrasynaptosomal concentration of Ca2+. Preincubation of synaptosomes with calmodulin antagonists such as trifluoperazine and N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W-7) was found to inhibit the uptake markedly, and diazepam, an inhibitor of Ca2+/calmodulin-dependent protein kinase, also inhibited the uptake. At a concentration between 1 and 10 microM, 50% inhibition of the uptake was observed with either inhibitor. On the other hand, the addition of dibutyryl cyclic AMP did not affect the uptake of the glycoprotein into synaptosomes. These results suggest that the incorporation of this macromolecule is dependent on a Ca2+/calmodulin-dependent protein kinase.     

Efficient transduction and selection of human T-lymphocytes with bicistronic Thy1/HSV1-TK retroviral vector produced by a human packaging cell line

BACKGROUND: T-cells expressing the HSV1-TK suicide gene can be used for the control of graft-versus-host disease following allogeneic stem cell transplantation. To develop clinical trials based on such a strategy, we have generated under good manufacturing procedures a novel 'split genome' human packaging cell line (1704 cells). METHODS: To minimize the risk of generating replication-competent retroviruses, pol was truncated to remove sequences overlapping with env. To improve retroviral infection and selection of transduced T-cells, high titers of GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector coding for the CD90 GPI-anchored membrane molecule were produced by 1704 cells. RESULTS: Using 1704 cell supernatant and an optimized transduction protocol, approximately 50% of primary T-cells were transduced and could then be purified (approximately 95%) using clinical-grade immunomagnetic beads directed against CD90. Over 96% of these OKT3/IL-2-activated CD90(+)-selected T-cells were killed by ganciclovir. Cell proliferation and cytokine production of transduced T-cells and HLA-restricted cytotoxicity of transduced T-cell clones were identical to those of their non-transduced counterparts cultured under the same conditions. CONCLUSIONS: GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector allow efficient transduction and rapid selection of human T-cells under conditions applicable for clinical trials using the new human 1704 packaging cell line.     

Persistence of the irradiated host component in thymocyte populations from bone marrow radiation chimeras infected with lymphocytic choriomeningitis virus

The thymus of chimeras made using T cell-depleted donor bone marrow from Thy1.1+ mice and 950 rad Thy 1.2+ recipients is dominated initially by cells expressing the Thy 1.2+ phenotype of the irradiated host. The thymocyte population recovered at 2 weeks after reconstitution comprises 80% Thy 1.2+ cells (host), the remainder being Thy 1.1+ (donor). This situation is normally reversed within a further week, with the host Ty 1.2+ (donor). This situation is normally reversed within a further week, with the host Thy 1.2+ thymocytes being present at a frequency of less than 5% from Week 4. Infection with lymphocytic choriomeningitis virus (LCMV) at 1 week after reconstitution with bone marrow causes a profound and persistent drop in the total number of thymocytes. The decline is equivalent for all categories of donor-derived thymocytes defined by two-color flow microfluorometric analysis for CD4 and CD8. However, there is a partial compensation by the retention of cells originating from the Thy 1.2+ host, which constitute 30-40% of the total thymocyte pool as late as 8 weeks after administration of bone marrow in the LCMV-infected chimeras. These radiation-resistant precursors give rise to CD4-8-, CD4-8+, CD4+8-, and CD4+8+ thymocytes, with the latter category being present at increased frequency. The potential skewing of the mature T cell repertoire as a consequence of persistent virus infection is discussed.     

Modulation of lymphocyte subsets in Peyer's patches of mice treated with monoclonal antibody against helper T-cells

Treatment of mice with anti-L3T4, a monoclonal antibody directed against helper T-cells, impairs clearance of intestinal Giardia muris infection. The present study examined the effect of anti-L3T4 treatment on mouse Peyer's patch cytoarchitecture and on the distribution of T-cell subsets within microenvironments of the follicle. Female BALB/c mice, aged 8 weeks, were given 4-7 weekly injections of either anti-L3T4 (1 mg/wk) or PBS (control group), and Peyer's patches were examined by immunohistochemistry or flow cytometry. In anti-L3T4-treated mice, Peyer's patch follicles (B-cell areas) were about two thirds the size of follicles in controls, and virtually all the size difference occurred in germinal centers. Peyer's patches were depleted of L3T4+ cells, yet the proportion of Thy-1.2+ (all T) cells was not decreased correspondingly, and the distribution of Thy-1.2+ cells in the patches was similar to that in control mice. In anti-L3T4-treated mice, Thy-1.2+ cells consisted of (a) Ly-2+ (cytotoxic/suppressor T) cells, and (b) a population of Thy-1.2+ cells that were neither L3T4+ nor Ly-2+. After treatment, Ly-2+ cells accounted for most of the T-cells in interfollicular areas and were also scattered in follicles, in germinal centers, and below the dome epithelium--in areas where L3T4+ cells predominated in control mice. Thy-1.2+ cells that were L3T4- and Ly-2- were mainly localized below the dome epithelium. These shifts indicate complex interrelationships among different lymphocyte subsets in Peyer's patches.     

Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.     

Ultrastructural and immunoelectron microscopic studies on infiltrating mononuclear cells in lymphocytic submandibulitis in NOD mice

The ultrastructural relations of the infiltrating mononuclear cells to the parenchymal tissues were studied in the submandibular gland of the female non-obese diabetic (NOD) mouse. In addition, the phenotype of mononuclear cells infiltrating the submandibular gland has been determined by light and electron microscopy by using monoclonal antibodies against T-cell subsets (Thy1.2, Lyt1, Lyt2). Ultrastructurally, lymphoid cells were frequently observed around and in the acini and ducts. Some of the lymphoid cells observed in the acini and ducts were irregular in shape and sometimes sent spike-like projections into acinar and ductal cells. Immunohistochemical study demonstrated that Thy1.2+ cells were predominant among the infiltrating cells, and the majority of these infiltrating T-cells were composed of Lyt1+ cells with a small proportion of Lyt2+ cells. By immunoelectron microscopy, lymphocytes carrying Thy1.2, Lyt1 or Lyt2 antigen were identified, as is demonstrated by an electron-dense reaction product on the entire cell surface, and these immunopositive cells were frequently observed around and in the acini and ducts. Some of the Thy1.2+, Lyt1+ or Lyt2+ cells observed in the acini and ducts demonstrated a close contact with acinar and ductal cells and both Lyt1+ and Lyt2+ cells sent spike-like projections into them. Occasionally, a partial degeneration of acinar cell adjacent to the invading lymphocytes was observed. These observations suggest that T-lymphocytes are involved in the direct destruction of acinar and ductal cells in the NOD mouse submandibular glands.     

Development of a diphtheria toxin based antiporcine CD3 recombinant immunotoxin

Anti-CD3 immunotoxins, which induce profound but transient T-cell depletion in vivo by inhibiting eukaryotic protein synthesis in CD3+ cells, are effective reagents in large animal models of transplantation tolerance and autoimmune disease therapy. A diphtheria toxin based antiporcine CD3 recombinant immunotoxin was constructed by fusing the truncated diphtheria toxin DT390 with two identical tandem single chain variable fragments (scFv) derived from the antiporcine CD3 monoclonal antibody 898H2-6-15. The recombinant immunotoxin was expressed in a diphtheria-toxin resistant yeast Pichia pastoris strain under the control of the alcohol oxidase promoter. The secreted recombinant immunotoxin was purified sequentially with hydrophobic interaction chromatography (Butyl 650 M) followed by strong anion exchange (Poros 50 HQ). The purified antiporcine CD3 immunotoxin was tested in vivo in four animals; peripheral blood CD3+ T-cell numbers were reduced by 80% and lymph node T-cells decreased from 74% CD3+ cells pretreatment to 24% CD3+ cells remaining in the lymph node following 4 days of immunotoxin treatment. No clinical toxicity was observed in any of the experimental swine. We anticipate that this conjugate will provide an important tool for in vivo depletion of T-cells in swine transplantation models.     

Clinical,Immunological and Treatment-Related Factors Associated with Normalised CD4+/CD8+ T-Cell Ratio: Effect of Na?ve and Memory T-Cell Subsets

Background

Although effective antiretroviral therapy(ART) increases CD4+ T-cell count, responses to ART vary considerably and only a minority of patients normalise their CD4+/CD8+ ratio. Although retention of naïve CD4+ T-cells is thought to predict better immune responses, relationships between CD4+ and CD8+ T-cell subsets and CD4+/CD8+ ratio have not been well described.

Methods

A cross-sectional study in a cohort of ambulatory HIV+ patients. We used flow cytometry on fresh blood to determine expanded CD4+ and CD8+ T-cell subsets; CD45RO+CD62L+(central memory), CD45RO+CD62L-(effector memory) and CD45RO-CD62L+(naïve) alongside routine T-cell subsets(absolute, percentage CD4+ and CD8+ counts), HIVRNA and collected demographic and treatment data. Relationship between CD4+/CD8+ T-cell ratio and expanded T-cell subsets was determined using linear regression analysis. Results are median[IQR] and regression coefficients unless stated.

Results

We recruited 190 subjects, age 42(36–48) years, 65% male, 65.3% Caucasian, 91% on ART(52.6% on protease inhibitors), 78.4% with HIVRNA<40cps/ml and median ART duration 6.8(2.6–10.2) years. Nadir and current CD4+ counts were 200(112–309) and 465(335–607) cells/mm³ respectively. Median CD4+/CD8+ ratio was 0.6(0.4–1.0), with 26.3% of subjects achieving CD4+/CD8+ ratio>1. Of the expanded CD4+ T-cell subsets, 27.3(18.0–38.3)% were naïve, 36.8(29.0–40.0)% central memory and 27.4(20.0–38.5)% effector memory. Of the CD8+ T-cells subsets, 16.5(10.2–25.5)% were naïve, 19.9(12.7–26.6)% central memory and 41.0(31.8–52.5)% effector memory. In the multivariable adjusted analysis, total cumulative-ART exposure(+0.15,p = 0.007), higher nadir CD4+ count(+0.011,p<0.001) and higher %CD8+ naive T-cells(+0.0085,p<0.001) were associated with higher CD4+/CD8+ ratio, higher absolute CD8+ T-cell(-0.0044,p<0.001) and higher %CD4+ effector memory T-cells(-0.004,p = 0.0036) were associated with lower CD4+/CD8+ ratio. Those with CD4+/CD8+ ratio>1 had significantly higher median %CD8+ naive T-cells; 25.4(14.0–36.0)% versus 14.4(9.4–21.6)%, p<0.0001, but significantly lower absolute CD8+ count; 464(384.5–567) versus 765(603–1084) cells/mm³, p<0.001.

Conclusions

Study suggests important role for naïve CD8+ T-cell populations in normalisation of the immune response to HIV-infection. How these findings relate to persistent immune activation on ART requires further study.     

Characterization of cytotoxic cells generated from in vitro cultures of murine bone marrow cells

Bone marrow cells cultured for 5-6 days generate cytotoxic activity against a number of natural killer (NK)-susceptible tumor cells. In this study, these bone marrow cytotoxic cells were compared to cells with NK activity obtained either from spleen cells activated in vitro with interferon (IFN-alpha/beta) or mitogen or from peritoneal exudate cells (PEC) obtained 4 days after bacillus Calmette-Guerin (BCG) infection. Splenic and PEC cytotoxic cells were shown to be Thy 1.2+, NK 1.1+, Asialo GM+1, Lyt 1.2-, Lyt 2.2-. In contrast, bone marrow cytotoxic cells were Thy 1.2+, NK 1.1-, Lyt 1.2-, Lyt 2.2- and expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Precursor cells for bone marrow cytotoxic activity were shown to be Thy 1.2-, NK 1.1-, Lyt 1.2-, Lyt 2.2- but also expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Cytotoxic activity for both bone marrow and spleen cells peaked in the low-density fractions of discontinuous Percoll density gradients. The cytotoxic activity of these bone marrow cells was augmented by pretreatment with IFN (-alpha/beta, -gamma) or soluble factors (IFN free) from activated EL-4 thymoma cells. Surprisingly, the ability of bone marrow cells to generate high levels of cytotoxic activity following in vitro culture appeared to be associated primarily with mice which were of the H-2b haplotype.     

T hybridoma alpha/beta gene transfected in a murine T cell hybridoma: role of CD4 molecule in vitro and in vivo--engraftment in SCID mice induces T cell maturation.

In the present work, we tested in SCID and Balb/c mice the activity of T hybridoma transfected with T cell receptor (TCR) alpha/beta chain genes. A T cell hybridoma denoted D011107 was used as recipient for transfection of cytotoxic KB5C20 TCR alpha/beta heterodimer genes by protoplast fusion or electroporation. After transfection, the parental D011107 T cell line reexpressed CD5 and CD4 surface molecules. In vitro, we noted strong proliferation and unusual cytotoxic reactivities against H-2k target cells although the transfected cell line does not express the CD8 molecule. The fate of parental and transfected cells was examined in severe combined immunodeficient (SCID) and Balb/c mice at Day 16 after intravenous injection. Cells from bone marrow, thymus, and spleen tissues were analyzed by immunofluorescence. The transfected T cell hybridoma was CD3+ Desire 1+ CD4+ Thy1.2. The SCID mice grafted with the transfected T cell hybridoma presented a high percentage of CD3+ (15%), CD4+ (27%), Thy1.2+ (27.52%), and Desire 1+ (8.74%) cells in the spleen. The percentages of CD3+ (6.2%) and Thy1.2+ (5.06%) cells in the spleen from SCID mice grafted with parental T cell D011107 and from untreated SCID were similar and lower (CD3+, 3.52%; Thy1.2+, 4.34%). It seems that transfected T cells hybridoma grafted in the SCID mice induce significant expression of CD4+ Thy1.2+ Desire 1- cells (17%) in the spleen. These results indicate that transfected T cells graft may allow T cell differentiation. In Balb/c mice, the percentage of different T cell subsets in bone marrow, thymus, or spleen cells in mice injected with transfected T cells was similar to that in untreated mice. We did not observe any cytotoxic or significant allogeneic proliferation in vitro.     

Human placental alkaline phosphatase. An improved purification procedure and kinetic studies.

An improved method for the purification of human placental alkaline phosphatase is described. The partially purified enzyme from Sigma was further purified by successive Concanavalin A-Sepharose and Q-Sepharose chromatography. The whole procedure may be completed in one working day. Highly purified enzyme was obtained with a 39% yield. The intrinsic fluorescence of the enzyme decreased at elevated temperature. The conformation of the enzyme molecule was studied by the fluorescence quenching technique. Upward Stern-Volmer plots were obtained for the quenching data which suggested that, in addition to collisional quenching, static quenching was involved in the quenching mechanism. The dynamic and static quenching constants were found to be 0.7 +/- 0.16 M-1 and 0.44 +/- 0.1 M-1, respectively, using acrylamide as the quenching agent. The corresponding values were 0.43 +/- 0.23 M-1 and 0.84 +/- 0.18 M-1, respectively, with KI as the quenching agent. Mg2+ and PO4(3-) induced protein conformational changes which altered both the dynamic and static quenching constants. Mg2+ was found to be a non-essential activator for the placental alkaline-phosphatase-catalyzed hydrolysis of 4-nitrophenyl phosphate. At pH 9.8, Mg2+ increased Vmax by 1.2-fold without affecting the Kd of the substrate. The tetranitromethane-modified enzyme showed slower migration toward the anode on electrophoresis and increased Kd for Mg2+.