Functional and phenotypic analysis of porcine peripheral blood CD4/CD8 double-positive T cells.

Functional and phenotypic properties of porcine peripheral blood CD4/CD8 double-positive (DP) lymphocytes were examined. In cross-sectional and longitudinal studies involving a total of 103 pigs, this lymphocyte population was found to increase gradually in proportion with age, comprising < 2% of the total peripheral blood lymphocyte pool in 1-week-old swine and reaching 30-55% by 3 years of age. CD4/CD8 DP lymphocytes were able to proliferate in response to stimulation with recall viral antigen. Furthermore, these cells mostly expressed high levels of the surface antigen recognized by monoclonal antibody (mAb) 4B4 (4B4hi), which is specific for the human beta 1 integrin. The CD4+4B4hi lymphocytes from pseudorabies virus-immune swine, proliferated in response to stimulation with the homologous virus, while CD4+4B4lo lymphocytes did not. Stimulation of CD4 single-positive (SP) cells with recall viral antigen, but not with mitogen, resulted in the generation of lymphoblasts which were predominantly of CD4/CD8 DP phenotype, suggesting a role for recall antigen in the generation of this lymphocyte subset. More than half of the CD4+ lymphocytes from palatine tonsils of 6-month-old swine were CD4/CD8 DP, while in the lymph nodes CD4/CD8 DP cells accounted for only one-third or less of CD4+ cells. In contrast, CD4/CD8 DP lymphocytes were absent from the palatine tonsils of 3-day-old swine, which only contained CD4 SP cells. Together, these results indicate that porcine CD4/CD8 DP lymphocytes, exhibit properties of mature antigen-experienced cells, and are inducible by stimulation with recall antigen. These data are consistent with the hypothesis that this population in swine includes memory/effector T cells.     

Distinct lymphokine production by CD4+ T cells isolated from mucosal and systemic lymphoid organs.

We have investigated the potential of CD4+ T cells isolated from Peyer's patches and mesenteric lymph nodes (mucosa-associated lymphoid tissues) and from spleens and peripheral lymph nodes (systemic lymphoid tissues) to secrete a variety of lymphokines. The results show several pronounced differences in the kinetics of lymphokine production and in the levels of lymphokines detected after primary and secondary in vitro stimulation with immobilized anti-CD3. The most striking difference was seen in the production of interleukin-4 (IL-4). Supernatants of splenic CD4-bearing cells collected at early time-points after primary stimulation contained high levels of IL-4. Supernatants of Peyer's patch and mesenteric lymph node CD4+ T cells contained considerably lower levels of IL-4 at all time-points analysed, while supernatants of peripheral lymph node CD4+ T cells contained high levels of IL-4, but only at late time-points. Neither cell concentration nor availability of accessory cells appeared to account for the differences observed in IL-4 production by the CD4+ T cells studied. Splenic CD4+ T cells most rapidly and effectively used the IL-4 they produced, as determined by analysing IL-4 production after restimulation. Therefore, we conclude that the spleen is a more potent source of IL-4 than are the mucosa-associated lymphoid tissues studied here. Amounts of IL-2, IL-3, IL-5, and IL-6 were similar in supernatants of all of the CD4+ T cells studied. Peyer's patch CD4+ T-cell supernatants contained the lowest levels of interferon-gamma and peripheral lymph node supernatants displayed the highest accumulation of this lymphokine.     

CD4+CD8+ murine intestinal intraepithelial lymphocytes

We have studied a population of CD4+ intestinal intraepithelial lymphocytes using two-color flow cytometric analyses, and in highly purified fluorescent-activated cell-sorted preparations. Although CD4+ T cells present within the epithelial immune compartment comprised only approximately 10-20% of the total intestinal epithelial lymphoid cells, an unusually high proportion of those CD4+ lymphocytes expressed a CD4+CD8+ phenotype which is rarely encountered in peripheral T cells. By comparison, CD4+ lymphocytes from spleen or lymph nodes existed exclusively as single-positive T cells. Analyses of CD4 and CD8 expression on lymphocytes from Peyer's patches, the lamina propria, and IEL indicated that CD4+CD8+ lymphocytes were unique to the IEL. Using CD4+CD8+ preparations obtained by fluorescent-activated cell sorting, CD4+CD8+ epithelial T cells were found also to express CD3 and Thy-1 surface markers. This heretofore undescribed extrathymic population of double-positive T cells constitutes a unique peripheral T cell subset which may be involved in intestinal T cell maturation and development, or could represent a highly specialized effector population.     

Peripheral blood CD4+CD8+ lymphocytes in cynomolgus monkeys are of resting memory T lineage

In this study, we analyzed peripheral blood CD4+CD8+ double-positive (DP) lymphocytes in adult cynomolgus monkeys (Macaca fascicularis). Forty of 55 monkeys had > 5% of the peripheral blood DP subpopulation (9.3 +/- 5.9%; mean +/- SD) in peripheral blood lymphocytes (PBL) in contrast to a low percentage of peripheral blood DP cells in humans and mice. In a cross-sectional study, the peripheral blood DP cells were found to increase in proportion with age. To clarify whether peripheral blood DP lymphocytes were immature precursors released from thymus without prior differentiation, the expressions of CD8 chains and CD1b on peripheral blood DP lymphocytes were compared with those on thymocytes. The peripheral blood DP lymphocytes were CD8 alpha + beta- and CD1b-, while thymic DP lymphocytes were CD8 alpha + beta + and CD1b +, suggesting that the peripheral blood DP cells are extrathymic T lymphocytes. Furthermore, the peripheral blood DP lymphocytes exhibited a resting memory T cell phenotype with CD2hiCD3+CD28-CD29hiCD49dhiCD69- CD80lo. Taken together, adult cynomolgus monkeys possess a unique peripheral blood DP T cell subpopulation which expresses a resting memory T cell phenotype. In addition, similar phenotypic properties of DP lymphocytes were distributed in the spleen and lymph nodes, although the proportion was less in the spleen and much less in lymph nodes than in PBL.      

MHC class II-independent CD25+ CD4+ CD8alpha beta+ alpha beta T cells attenuate CD4+ T cell-induced transfer colitis

CD4+ alpha beta T cell populations that develop in mice deficient in MHC class II (through 'knockout' of either the Aalpha, or the Abeta chain of the I-A(b) molecule) comprise a major 'single-positive' (SP) CD4+ CD8- subset (60-90%) and a minor 'double-positive' (DP) CD4+ CD8alpha beta+ subset (10-40%). Many DP T cells found in spleen, mesenteric lymph nodes (MLN) and colonic lamina propria (cLP) express CD25, CD103 and Foxp3. Adoptive transfer of SP but not DP T cells from Aalpha(-/-) or Abeta(-/-) B6 mice into congenic RAG(-/-) hosts induces colitis. Transfer of SP T cells repopulates the host with only SP T cells; transfer of DP T cells repopulates the host with DP and SP T cells. Anti-CD25 antibody treatment of mice transplanted with DP T cells induces severe, lethal colitis; anti-CD25 antibody treatment of mice transplanted with SP T cells further aggravates the course of severe colitis. Hence, regulatory CD25+ T cells within (or developing from) the DP T cell population of MHC class II-deficient mice control the colitogenic potential of CD25- CD4+ T cells.     

Random entry of circulating lymphocyte subsets into peripheral lymph nodes and Peyer's patches: no evidence in vivo of a tissue-specific migration of B and T lymphocytes at the level of high endothelial venules.

Lymphocytes continuously migrate through the body and thus immune competent cells are constantly delivered to most tissues. They interact with high endothelial venules (HEV) via specific homing receptors and vascular addressins, and these molecules seem to be the reason for a preferential homing of B lymphocytes into Peyer's patches and of T lymphocytes into peripheral lymph nodes. When lymphocytes derived from lymph node cell suspensions were applied in the in vitro lymphocyte/endothelium binding assay, the well-known preference of mouse lymph node B lymphocytes for Peyer's patch HEV compared to peripheral lymph node HEV was confirmed in the rat (2.8 times). When in the same in vitro assay thoracic duct lymphocytes (TDL) were used this preference was far less obvious (1.4 times). However, by injecting rat TDL intravenously and by tracing them directly in HEV, B, T, CD4+ and CD8+ lymphocytes are seen to enter Peyer's patches and peripheral lymph nodes in vivo without preference. Thus, in contrast to lymphocytes from lymph node cell suspensions, no evidence was found of a tissue-specific migration of thoracic duct B, T, CD4+ and CD8+ lymphocytes at the HEV level. This finding demonstrates the importance of considering both experimental conditions and the cell source used when investigating lymphocyte traffic.     

Phenotypic discrimination between thymic and extrathymic CD4-CD8- and CD4+CD8+ porcine T lymphocytes

The composition of the T lymphocyte population in swine is special in that in addition to the CD4-CD8+ subpopulations, CD4-CD8- and CD4+CD8- and CD4+CD8+ subpopulations are prominent in the peripheral circulating as well as in the resident T lymphocyte pools. Since the same phenotypes are characteristic of thymic populations, it was asked whether the unusual distribution in swine may result from an emigration of thymic precursor phenotypes to the periphery. This explanation was refuted, as all thymic subpopulations were found to express CD1, albeit with differences in antigen density, whereas all extrathymic subpopulations lack CD1. The cellular distribution of CD2 in swine is without precedent among all species studied. Whereas in sheep and cattle the extrathymic CD4-CD8- subpopulation is known to entirely lack CD2 and to have a low propensity for homing to lymphoid tissues, the CD4-CD8- subpopulation in swine splits into CD2+ and CD2- subsets, both of which do reside in lymphoid tissues. While CD2+CD4-CD8- T lymphocytes are rare in the circulating pool, this subset accumulates in spleen and lymph nodes. This may indicate a role for CD2 in homing. Thus the species swine is immunologically unique, not only because of having CD1-CD2+CD4+CD8+ T lymphocytes in the periphery, but also with regard to subdivision and homing behavior of its CD4-CD8- T lymphocyte subpopulation.     

Proliferation of lymphocyte subsets in the adult rat: a comparison of different lymphoid organs

Adult, male Lewis rats received a single injection of 5-bromo-2'-deoxyuridine (BrdUrd) i.v. to label proliferating cells in the S phase of the cell cycle. After 1 and 24 h the thymus, bone marrow, blood, spleen, peripheral, cervical and mesenteric lymph nodes as well as Peyer's patches were removed. In cell suspensions surface staining was performed for B, T, T helper (Th) and cytotoxic/suppressor (Tc/s) T lymphocytes by identifying kappa light chain, CD5+, CD4+ and CD8+ cells, respectively. On the same slide the DNA label BrdUrd was demonstrated by a monoclonal antibody. B, T, Th and Tc/s lymphocytes proliferate locally both in central lymphoid organs such as the thymus and the bone marrow, and in peripheral lymphoid organs such as the spleen, lymph nodes and Peyer's patches. Within an organ the amount of proliferation among the lymphocyte subsets is similar, differing not more than threefold. Although concerning only a small fraction of cells within the organ, an unexpected finding is the high percentage of BrdUrd-labeled cells among B lymphocytes in the thymus (3%) and among T lymphocytes in the bone marrow (3%). One day after injection of BrdUrd the thymus contains 25% BrdUrd+ T lymphocytes, while the other organs investigated do not show more than about 2% BrdUrd+ B and T lymphocytes. Many of the newly formed lymphocyte subsets leave their organ of birth within 24 h. Thus the amount of proliferation in the lymphocyte subsets investigated is very similar and the differences between central (thymus and bone marrow) and peripheral lymphoid organs are much smaller than expected.     

Development of apoptosis and changes in lymphocyte subsets in thymus, mesenteric lymph nodes and Peyer's patches of mice orally inoculated with T-2 toxin.

Development of apoptosis and changes in lymphocyte subsets were examined mainly by flow cytometer in thymus, mesenteric lymph nodes and Peyer's patches of mice up to 24 hours after oral inoculation with T-2 toxin (10 mg/kg). T-2 toxin attacked Peyer's patches first, then mesenteric lymph nodes, and finally thymus in relation to the course of enteric absorption of orally inoculated T-2 toxin. The degree of lymphocyte apoptosis was prominent in the thymus, moderate in the Peyer's patches, and somewhat mild in the mesenteric lymph nodes, suggesting the difference in lymphocyte population susceptible to T-2 toxin. As to the changes in lymphocyte subsets, CD4+ CD8+ T cells were most sensitive to T-2 toxin, and CD4+ CD8- T cells were more severely depressed than CD4- CD8+ T cells in the thymus. In the mesenteric lymph nodes, CD3+ cells was more clearly affected than CD19+ cells, and the numbers of CD4+ and CD8+ cells were similarly decreased. In the Peyer's patches, the numbers of CD3+, CD 19+, CD4+ and CD8+ cells were unexceptionally decreased. In addition, among IgM+, IgG+ and IgA+ B cells, the number of IA+ B cells which are more important in the mucosal immunity was most severely affected.     

Restricted expression of CD2 among subsets of sheep thymocytes and T lymphocytes.

A monoclonal antibody (mAb) generated against sheep T-cell blasts, called I/35 A, blocks sheep autologous E rosetting and competes with purified T11 target structure (TS), the sheep form of LFA3, for binding sites on the sheep T-cell surface. Immunoprecipitation from lysates of surface iodinated sheep T cells identifies the cell surface molecule recognized by mAb I/35 A as a single chain polypeptide migrating as a diffuse band of MW 55,000. From its binding properties and the biochemical nature of the target antigen, we conclude that mAb I/35 A is directed at sheep CD2. This finding makes sheep the first animal model in which the CD2-LFA3 (T11TS) system is defined by mAbs to both receptor and ligand. When analysed by two-colour flow cytometry and by immunohistochemistry, the cellular expression of CD2 in sheep differs significantly to that reported in humans. In peripheral blood, CD2 is found exclusively on CD4+8- and CD4-8+ T cells, while the third, CD4-8- (predominantly SBU-T19+) subset of sheep T cells (around 20% in peripheral blood) is CD2-. In thymus, only low to moderate levels of CD2 expression occurs on 80% of cells. Among these, medullary 'single positive' thymocytes express the highest level of CD2, whereas the CD4-8- 'double negative' population (which in contrast to peripheral CD4-8- T cells contains only very few SBU-T19+ cells) consists of CD2- and weakly positive cells. In peripheral lymphoid organs, CD2+ lymphocytes occur in the T-cell regions of spleen, lymph nodes and jejunal Peyer's patches (JPP). Tissue macrophages found in B-cell follicles of lymph nodes and JPP are also CD2+. The implications of these findings are discussed in terms of the role CD2 plays in the proliferation of immature thymocytes and of the possible importance of CD2/LFA3 interactions in lymphocyte recirculation.     

FTY720, a novel immunosuppressant, induces sequestration of circulating mature lymphocytes by acceleration of lymphocyte homing in rats, III. Increase in frequency of CD62L-positive T cells in Peyer's patches by FTY720-induced lymphocyte homing.

FTY720, a novel immunosuppressant, sequesters circulating mature lymphocytes, especially T cells, within lymph nodes and Peyer's patches by accelerating lymphocyte homing, and thereby causes lymphocyte depletion in the blood. The FTY720-induced acceleration of lymphocyte homing appears to be mediated by lymphocyte homing receptors including CD62L, CD49d/beta7, and CD11a/CD18. In this study, expressions of CD62L, CD49d and CD11a on T cells in the peripheral blood, lymph nodes and Peyer's patches were analysed by flow cytometry in rats given FTY720 (1 mg/kg) orally. FTY720 markedly decreased the number of peripheral blood T cells, while not affecting CD62L, CD49d and CD11a expressions at 1-3 hr after administration. In contrast, both the frequency of CD62L-positive T cells and intensity of CD62L expression on T cells were increased in Peyer's patches but not lymph nodes at 3 hr after administration of FTY720. CD49d and CD11a expressions on T cells were unaffected by FTY720 in both Peyer's patches and lymph nodes at the same point in time. On the other hand, analysis of lymphocyte homing with calcein-labelled lymphocytes and anti-CD62L monoclonal antibody (mAb) confirmed that FTY720 predominantly increased CD62L-dependent lymphocyte homing to Peyer's patches. These findings indicate that FTY720 increases the frequency of CD62L-positive T cells by accelerating CD62L-predominant homing in Peyer's patches.     

Analysis of cell division among subpopulations of lymphoid cells in sheep. II. Peripheral lymphocytes

The number, distribution and surface phenotype of dividing cells in lymph nodes and blood and differences between the cell-cycle status of lymphocyte subpopulations were studied in lambs using double-labelling techniques. Dividing cells were labelled in vivo for various time periods with 5-bromo-2-deoxyuridine (BrdU). After removal of lymphoid tissues, the proportions of constituent lymphocyte subpopulations which had synthesized DNA during the labelling period were measured by flow cytometry or immunohistochemistry using a panel of monoclonal antibodies (mAbs) specific for sheep lymphocyte differentiation antigens and MHC class I and class II antigens in conjunction with an anti-BrdU mAb. There was a higher overall level of cell division in the ileocaecal lymph node than in either the prescapular or parathymic lymph nodes. In all three lymph nodes, the majority of lymphocytes which incorporated BrdU occurred in B-cell follicles or germinal centers. CD4+ and CD8+ T cells had a higher level of cell division (LI 5-14%) than those recognized by mAb 197 (CD4- CD8- subset) (LI less than or equal to 3%).     

Presence of a distinct CD8+ and CD5- leucocyte subpopulation in the sheep liver.

The relative proportions of CD5, CD4, CD8, SBU-T19 and surface immunoglobulin (sIg)-positive lymphocytes in normal sheep livers were determined by flow cytometry analysis of isolated liver leucocytes and compared to lymphocyte subpopulations found in peripheral blood and hepatic lymph nodes. In contrast to the blood, very low numbers of SBU-T19+ cells were found in both the liver and hepatic lymph nodes. The ratio of helper versus suppressor/cytotoxic T cells in the liver was less than 1, which was much lower than in blood or hepatic lymph nodes. A large proportion of liver lymphocytes were characterized by a low intensity staining for the CD8 antigen (CD8L). Two-colour flow cytometry analysis revealed that the CD8L cells were negative for the CD5 pan T-cell marker. It is suggested that these CD8LCD5- cells are natural killer (NK) cells similar to those found in the gut. An influx of CD8LCD5- cells was observed in leucocyte-infiltrated liver lesions at later stages of a rejection response to the cestode Taenia hydatigena.     

Effects of HIV-1 infection on lymphocyte phenotypes in blood versus lymph nodes

Most immunopathogenesis studies of HIV-1 use peripheral blood. Most lymphocytes reside in lymphoid tissues, however, and the extent to which blood mirrors tissues is unclear. Here, we analyze lymphocytes in blood and lymph nodes of HIV-1-uninfected and -infected persons. Baseline comparison of node and blood lymphocytes in seronegative persons demonstrates a lower ratio of CD8+ versus CD4+ T lymphocytes, a lower number of effector cells (CD28-) within the CD8+ compartment, and greater activation (D-receptor [DR+]) within the CD4+ compartment. In infected versus uninfected persons, nodes exhibit elevated CD8+ T lymphocytes with an increased memory-effector phenotype (CD62L-/CD45RA-) and activation (CD38+ and DR+) but minimal differences in the CD4+ compartment. Changes attributable to HIV-1 infection are markedly greater in node lymphocytes than in blood. Comparisons of CD8+ T-lymphocyte parameters and viremia in infected persons reveal positive correlations of CD38+ expression on cells in blood and nodes and a negative correlation of terminal effector cells (CD62L-/CD45RA+) in the nodes to viremia. Multiple linear regression analysis indicates that CD38 expression on node (not blood) CD8+ T lymphocytes is the sole independent predictor for viremia. Thus, blood indirectly reflects processes in lymphoid tissues, and caution should be applied when interpreting immunopathogenesis studies of blood.     

Phenotypic and functional characterization of lymphocytes derived from normal and HIV-1-infected human lymph nodes.

Lymph nodes are the major site of cell-to-cell transmission and replication of HIV-1. Trafficking of CD4+ T lymphocytes into lymph nodes provides a continual supply of susceptible target lymphocytes, and conversely, recruitment of CD8+ T lymphocytes may be critical for the host response that attempts to control HIV-1 replication. The present study was undertaken as no detailed assessment of lymphocyte subpopulations in HIV-1-infected lymph nodes has previously been reported. Peripheral blood and single-cell suspensions prepared from lymph nodes of patients with HIV-1 and control subjects were analysed using three-colour flow cytometry. Approximately 80% of the lymphocytes in control lymph nodes were CD3+ T lymphocytes, of which over 65% were CD4+. The majority of the CD4+ and CD8+ T lymphocytes obtained from both lymph nodes and blood of control subjects were immunologically naive (CD45RA+). By contrast, in HIV-1-infected patients there was a significant reduction in the proportion of CD4+ T lymphocytes and an expansion of the CD8+ T lymphocyte subset in both lymph nodes and peripheral blood. Furthermore, a high proportion of these T lymphocytes displayed a marker for immunological memory (CD45RO+). T lymphocytes derived from HIV-1-infected lymph nodes also showed altered expression of the adhesion molecules, L-selectin and very late antigen-4 (VLA-4), but not leucocyte function-associated antigen-1 (LFA-1). In an in vitro adhesion assay, lymphocytes from HIV-1-infected nodes were significantly more adhesive than control lymphocytes on fibronectin, as well as recombinant human intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) substrates. This combination of altered lymphocyte subpopulations in the HIV-1-infected lymph nodes, as well as enhanced adhesion phenotype and function, suggests that T lymphocyte traffic to lymph nodes in HIV disease may be an important determinant of pathogenesis.     

Transient T cell accumulation in lymph nodes and sustained lymphopenia in mice treated with FTY720

FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride) is an orally available immunomodulatory agent that induces severe peripheral blood lymphopenia. Most studies of these lymphopenic effects have been limited to short-term exposure to FTY720. FTY720 alters the ability of lymphocytes to respond to sphingosine-1-phosphate (S1P) through S1P receptors, particularly S1P1. FTY720 affects different leukocyte populations and their trafficking through major lymphoid organs. We show the dynamics of CD4 T, CD8 T, and B lymphocyte recirculation in all major lymphoid compartments during 21-day FTY720 treatment of normal C57BL/6 mice. Following a transient increase in peripheral lymph nodes and Peyer's patches, lymphocyte recirculation reaches a new steady state. Other lymphoid organs show transient changes in lymphocyte composition with various patterns. At 21 days of FTY720 treatment, total body lymphocyte content is reduced by 20% and blood lymphocytes by 80%. Modeling suggests that the new steady state is due to a combination of reduced naive lymphocyte release from the thymus and a transient reduction of lymphocyte egress from lymph nodes. Our data indicate that the commonly held belief that FTY720 blocks lymphocyte egress from lymph nodes cannot fully explain the lymphocyte dynamics observed with prolonged treatment.     

Effects of SIVmac Infection on Peripheral Blood CD4CD8T Lymphocytes in Cynomolgus Macaques

We have previously reported that CD4+CD8+ double-positive (DP) T cells with a resting memory phenotype exist in a substantial proportion of peripheral blood lymphocytes of adult cynomolgus macaques. In this study, we examined the effects of simian immunodeficiency virus of macaque (SIVmac) infection on DP T cells. In vitro, SIVmac239 nef-open (239) and its nef-deletion mutant replicated well in both CD4+CD8- and DP T cells. However, when the macaques were infected with 239, DP, but not CD4+CD8-, T cells were transiently increased in parallel with cell activation and viral replication, followed by depletion within 1 month postinfection. Interestingly, the nef gene was required for depletion but not for the increase and activation of DP T cells. These data suggest that the pathogenic SIV infection may downmodulate production and/or blood circulation of DP T cells by a Nef function-related mechanism(s) different from that for the depletion of CD4+CD8- T cells.     

Changes in CD4(+), CD8(+), CD4(+) CD8(+), and immunoglobulin M-positive peripheral blood mononuclear cells of postweaning multisystemic wasting syndrome-affected pigs and age-matched uninfected wasted and healthy pigs correlate with lesions and porcine circovirus type 2 load in lymphoid tissues

Forty-one 8- to 12-week-old wasted pigs were selected from several conventional farms with histories of postweaning multisystemic wasting syndrome (PMWS) and classified into two groups according to their porcine circovirus type 2 (PCV2) infection status, as determined by in situ hybridization (ISH). Twenty-four pigs tested positive for PCV2 (PCV2-positive group), while 17 pigs tested negative for PCV2 (PCV2-negative group). In addition, eight uninfected healthy pigs from an experimental farm were used as controls. Heparinized blood samples were taken to obtain peripheral blood mononuclear cells. The CD4(+), CD8(+), CD4(+) CD8(+) (double-positive [DP]), and immunoglobulin M-positive (IgM(+)) cell subsets were analyzed by flow cytometry with appropriate monoclonal antibodies. Histopathological studies were done to evaluate the apparent degrees of lymphocyte depletion in different lymphoid organs (superficial inguinal and mesenteric lymph nodes, Peyer's patches, tonsils, and spleen) and to determine the viral load of the PCV2 genome by using an ISH technique. Animals of the PCV2-positive group showed a significant downshift of the CD8(+) and DP cell subsets compared to the other groups (P < 0.05). Moreover, in PCV2-positive pigs, the amount of PCV2 genome in lymphoid tissues was related to the degree of cell depletion in those tissues (P < 0.05) as well as to the relative decrease in IgM(+) and CD8(+) cells in peripheral blood. These data support the notion that PCV2-positive pigs might have an impaired immune response.     

Size, CD4 and CD8 marker profiles and functions of lymphocyte subpopulations in mucosal-associated lymph nodes.

We analyzed phenotypic and functional characteristics of T cell populations in mucosal-associated supramammary and mesenteric lymph nodes in goats. Here we demonstrate, by flow cytometry, quantitative differences in CD4 and CD8 T cell subsets among large and small mucosal-associated lymphocyte populations and their differential regulatory activities on resident lymph node B cells stimulated with Staphylococcus aureus Cowan I or pokeweed mitogen. The CD4/CD8 T cell ratio was lower in mesenteric lymph nodes (1.46) when compared to that of supramammary lymph nodes (2.18). Analysis of large and small lymphocyte subpopulations from lymph nodes showed nearly 62% of the lymphocytes from mesenteric lymph nodes being of large cell phenotype with CD4/CD8 ratios of 1.34. In contrast, large cell subpopulations in supramammary lymph nodes showed a significantly lower number (50%) with a higher CD4/CD8 ratio of 2.05. Functionally, mesenteric lymph node T cells, isolated by nylon wool, showed heightened suppressive activity in mitogen-driven B cell proliferation responses, whereas T cells from supramammary lymph nodes were stimulatory. These findings clearly demonstrate distinctive functional properties between resident T cell populations of supramammary and mesenteric lymph nodes, suggesting that different proportions of T cell subsets in these nodes are activated and thus regulate regional immune responses via different pathways.