Pig Blood Lymphocytes Research Articles

Flow cytometry complement-mediated cytotoxicity assay detects baboon xenoantibodies directed to porcine epitopes undetected by hemolytic assay

The pig-to-primate model is increasingly being utilized as the final preclinical means of assessing therapeutic strategies aimed at allowing discordant xenotransplantation. To obtain information about the nature of cytotoxic response in pig-to-baboon xenotransplants, we sought to determine if serum cytotoxicity in this model was assay dependent. Sera from nine kidney or heart xenotransplanted baboons were obtained before transplantation and at the time of acute humoral xenograft rejection (AHXR). Cytotoxicity was measured by an anti-pig haemolytic assay (APHA) and by a flow cytometry complement-dependent assay (FCCA), using pig blood lymphocytes (PBLs). Serum samples showing inter-assay differences were absorbed with pig erythrocytes and assayed by APHA and FCCA, as well as by measuring anti-αGal and total anti-pig xenoantibodies. The results showed that in four AHXR samples, FCCA cytotoxicity was higher than APHA cytotoxicity. Absorption with pig erythrocytes diminished FCCA and removed APHA cytotoxicity. Residual FCCA activity was due to total anti-pig and IgM anti-αGal and non-Gal antibodies. Our results indicate that some cytotoxic antibodies present in the sera of xenotransplanted baboons at time of AHXR are IgM antibodies directed against pig PBL antigens not detected by APHA.

Changes of lymphocyte subpopulations in pigs infected with porcine reproductive and respiratory syndrome (PRRS) virus

Abnormal changes of T-cell subpopulation were observed in the peripheral blood lymphocytes of pigs infected with porcine reproductive and respiratory syndrome (PRRS) virus. Pigs with naturally occurring PRRS revealed increases in CD2 + and CD8 + cells, and decreases in CD4 + cells and the ratios of CD4 + CD8 + cells. Specific-pathogen-free pigs inoculated with PRRS virus showed remarkable decreases in total lymphocytes, CD4 + and CD2 + cells on Postinoculation Day (PID) 3. The decline of CD4 + cells continued for at least 14 days, while CD2 + cells showed a tendency to increase thereafter. On the other hand, CD8 + cells slightly decreased in number on PID 3, and then increased remarkably; their number was significantly larger on PIDs 28 and 35 than on PID 0. The ratios of CD4 + CD8 + cells were significantly low between PIDs 3 and 28 as compared with PID 0. However, there were no differences in thymocyte subpopulations between infected and non-infected pigs, suggesting that the PRRS virus does not modulate intrathymic T-cell differentiation. In an experiment with peripheral blood mononuclear cell cultures, PRRS virus caused neither alteration of T-cell subpopulations nor cell proliferation, suggesting that the virus is not cytotoxic for CD4 + cells and not mitogenic for CD8 + cells.

Characterization of humoral human anti‐porcine xenoreactivity

The presence and specificity of natural xenoreactive human anti‐pig antibodies was studied. Titers of cytotoxic antibodies reactive with porcine blood lymphocytes were similar in sera from healthy controls, patients with high titers of HLA alloantibodies, uremic patients, diabetic uremic patients, kidney allografted patients on maintenance immunosuppressive therapy, patients who had been splenectomized and in bone marrow transplanted patients 6 months after transplantation. Similar titers were also recorded in patients with various autoimmune disorders, such as SLE and RA; there was no correlation to autoantibody titers. Furthermore, we found no correlation to natural isohemagglutinins; absorption with adequate human red cells did not reduce activity. However, absorption with pig red blood cells reduced the titer significantly and absorption with pig platelets completely abolished reactivity. Finally, antibodies were also present in cord blood samples indicating an endogenous ligand as an activating substance for antibody production. Most sera contained only IgM cytotoxic antibodies, but some also had a weak non‐IgM cytotoxic titer. Fluorescence studies with pig blood lymphocytes indicated that even sera that did not contain non‐IgM cytotoxic antibodies had IgG and IgA antibodies reactive with pig cells. Direct immunofluorescence experiments were also carried out to demonstrate the presence of target structures for naturally reactive antibodies on fetal pig islet cells. Pig blood lymphocytes were sensitive to ADCC‐mediated killing by IgG natural antibodies, whereas fetal pig islet cells were resistant. However, using adequate positive controls, such as anti‐pig MHC class I antibodies, it was shown that fetal pig islet cells can be sensitive to ADCC‐mediated killing. Finally, lysostrip and fluorostrip experiments were performed to test whether pig MHC antigens contain target determinants, possibly as oligosaccharide side chains, for natural antibodies. We found that pig MHC class I antigens do not contain such determinants.

Staining of pig lymphocytes subpopulations with acid α-naphthyl acetate esterase

The use of α-naphthyl acetate esterase (ANAE) as a T cell marker in some other species and the broad correlation of incidence of ANAE-positivity and E rosette-formation in the pig suggest that ANAE-staining may be a T-cell marker in the pig. However, by studying the staining of lymphocytes within a variety of rosettes in fixed preparations a similar incidence of pig blood lymphocytes were found to be ANAE + among T cells (E rosettes formed in dextran), B cells antiglobulin rosettes) and Fc-γ receptor-bearing B and T cells (EA rosettes in saline and dextran): complement (C′) receptor-bearing cells showed a higher incidence of staining than other lymphocytes. Analysis of staining morphology suggested that certain morphologies within the B and T lineages may be confined to subpopulations. Thus ANAE positivity is certainly not a marker identifying blood T lymphocytes but could be of some value indicating subpopulations of B and T lymphocytes.

Migration of fluoresceinated pig lymphocytes in vivo: technical aspects and use in studies of autologous and homologous cell survival for up to three weeks.

Optimum conditions were determined for FITC and RITC labelling of pig blood lymphocytes with bright fluorescence and high viability and for their enumeration during their long survival after infusion in vivo. For at least 9 days, FITZ-labelled autologous lymphocytes and lymphocytes from MHC-identical donors showed no detectable difference in patterns of survival in the blood. After initial equilibration with extra-vascular lymphoid tissues, the fall-off suggested the expected presence of populations with a variety of life-spans, with some autologous labelled lymphocytes surviving beyond 3 weeks. A trace of cytotoxic antibody specific for labelled cells developed in these pigs later and could be boosted by a further FITC-labelled cell infusion.

Immunoglobulin biosynthesis in vitro after stimulation of pig blood lymphocytes with mitogen.

The biosynthesis of immunoglobulin (Ig) by pig blood lymphocytes after mitogen stimulation with lipopolysaccharide (LPS) and anti-Ig serum has been assessed by incorporation of 3H--leucine into secreted Ig. Lymphocytes prepared from defibrinated and heparinised blood responded to LPS with mitosis, but Ig biosynthesis (predominantly IgM) only occurred with heparinised blood lymphocytes. Anti-Ig treatment produced pronounced mitosis in both preparations but no significant Ig biosynthesis in either. Mitosis was induced by anti-IgM (mu- and L chains), anti-mu and anti-IgG (gamma- and L chains) but only weakly by anti-gamma in both types of preparation. Lymphocytes from heparinised blood gave preparations with higher percentages of surface-Ig-bearing cells by direct immunofluorescence (defibrinated 14.0%, heparinised 25.2%).

Stimulation of Pig Lymphocytes with Anti-Immunoglobulin Serum and Mitogens

Mitogenic stimulation of pig lymphocytes by anti-immunoglobulin serum and by Con A, PHA, PWM and LPS has been measured by 3H-thymidine incorporation. Lymphocytes from blood, spleen and lymph node were readily stimulated by antiglobulin. Specific anti-micron serum was mitogenic for blood lymphocytes, indicating that those B cells with surface IgM can be stimulated by complexing of this surface component with antibody. An initial 2-hour incubation with anti-Ig was as effective as continuous incubation. Differentiation of stimulated cells into high rate synthesising plasmablasts was not observed. Pig blood lymphocytes were reactive to Con A, PHA, PWM and LPS. LPS was also tested against spleen and lymph node cells, which were responsive to this mitogen.

Isolation and characterization of pokeweed mitogen-like phytomitogens from shoriku, Phytolacca esculenta

1. 1.|From saline extracts of Phytolacca esculenta (shoriku) roots, two phytomitogenes were isolated by salting out with (NH 4) 2SO 4 and chromatography on DEAE-cellulose and Sephadex G-100 columns. Both fractions were homogeneous on disc electrophoresis and on immunoelectrophoresis. One of these (Fraction E-2) was shown to be similar to pokeweed mitogen in respect to mol. wt (32 000) and amino acid composition. The other (Fraction E-3) was a protein of 18 000 mol. wt. Both fractions had similar biological activities to pokeweed mitogen in their ability to stimulate pig blood lymphocytes in vitro to incorporate tritiated thymidine, and to induce blastoid transformation. 2. 2.|Both fractions contained an unusually large amount of cystine, i.e., 18 half-cystine residues % for Fraction E-2 and 22 residues % for Fraction E-3. 3. 3.|Although these mitogens were resistant to deproteinizing procedures such as perchloric acid treatment and Sevag's procedure, the DNA synthesis-stimulating activity was inactivated by digestion with Pronase E and Nagarse, but resistant to trypsin, chymotrypsin, deoxyribonuclease, ribonuclease, lysozyme and neuraminidase. 4. 4.|The activity was stable at acidic and neutral pH (4–7) but unstable at alkaline pH. 5. 5.|The activity at pH 7.3 was stabilized by the addition of Ca 2+ or Mg 2+. On the addition of more than 2 mM of Ca 2+, precipitation of mitogen occurred. 6. 6.|From the above results the molecular basis of the mitogenic activity of shoriku mitogen is discussed.

Characteristics of the in Vitro Response of Guinea Pig Blood Lymphocytes to PHA and Antigen

Abstract The in vitro response of guinea pig lymphocytes to phytohemagglutinin and tuberculin in tuberculin-sensitive and non-sensitive animals was studied. Measurements of tritiated thymidine incorporation, mitotic activity, and blast cell transformation were used to establish optimal conditions of culture and to investigate the mechanisms of in vitro interactions of antigen and cells. The pattern of response to phytohemagglutin (PHA) was found to be similar to that reported with human lymphocytes. Tuberculin-induced proliferative responses were limited to cells from sensitized animals, with a fairly consistent antigen dose-response and period of optimal incubation. Evidence was found to suggest a considerable dependence of the degree of in vitro responses to PHA or antigen upon the source of serum used in the culture medium. A high degree of correlation was found between isotope uptake and mitotic division in both PHA- and tuberculin-induced proliferative responses, with a less impressive correlation of these findings with the degree of blast cell transformation. When high concentrations of antigen were employed, there was an apparent dissociation of the blast cell transformation from DNA synthesis and mitotic activity in the lymphocytes of sensitized animals.

Incorporation of [5-3H]uridine and attachment of cells to glass during activation of lymphocytes induced by phytohaemagglutinin.

1. Shortly after the addition of phytohaemagglutinin to cultures, partially purified pig blood lymphocytes showed increased labelling of RNA by [5-(3)H]uridine and began to attach to the glass of the culture tubes. 2. In the presence of phytohaemagglutinin most cells became attached to glass during the first 5hr. of culture; those first attaching had a low mean RNA/DNA ratio. In the absence of phytohaemagglutinin cell attachment to glass was diminished. 3. During the first 5hr. of culture the rates of increase of radioactivity/unit of DNA of the attached and unattached populations in the presence of phytohaemagglutinin were of the same order and exceeded that of cultures without phytohaemagglutinin. 4. The increase of labelling in cultures to which phytohaemagglutinin was added after cells had sedimented to glass was greater than that occurring in cultures in which cells were resuspended before phytohaemagglutinin was added.

Studies on the incorporation of [5-3H] uridine during activation and transformation of lymphocytes induced by phytohaemagglutinin. Dependence on the incorporation rate on uridine concentration at certain critical concentrations.

1. Partially purified pig blood lymphocytes were stimulated to transform in culture with phytohaemagglutinin. Initial cell activation was assessed by measuring the increase of uridine incorporation into RNA induced by phytohaemagglutinin. The phytohaemagglutinin/serum ratio for this effect was similar to that required for transformation; however, no inhibition at high phytohaemagglutinin/serum ratios was found during cell activation. 2. Without replenishment of medium the pool of competitors with added uridine for incorporation fell to zero during 2 days of culture. At certain critical pool concentrations uridine itself could stimulate the rate of uridine incorporation. 3. Most of the tritium from [5-(3)H]uridine added at the initiation of culture had been incorporated into RNA by the end of the second day of culture; the subsequent loss of radioactivity preceded a fall in the total RNA content of cultures. 4. RNA was qualitatively examined on sucrose density gradients. In the first 30min. after the addition of phytohaemagglutinin, increased labelling occurred predominantly between the 28s and 4s regions of the gradients. On the second day of culture with phytohaemagglutinin mainly RNA sedimenting beyond the 28s region of gradients was labelled in 30min.