Soe Than

Ontogeny of thymic B cells in normal mice

Ontogeny of thymic B cells and their surface characteristics were analyzed using monoclonal antibodies (mAbs) against B220 molecules (CD45, CD45R). A small number of B cells were detected in fetal thymus on Gestation Day 14 (approximately 3.5% of the low-density fraction). Similarly, the percentage of B cells in the low-density fraction was 3.2% on Gestation Day 18, and 3.5% on Day 1 after birth. These were the same level as that of adult mice. CD5+ B cells, which form the major population of thymic B cells, were also found in the fetal life (0.5% on Day 14 and 2.2% on Day 16 in the low-density cells). The percentage of CD5+ B cells in B cell-enriched fraction was about 65% on Day 1 after birth, which is the same level as that in adult mice. These results indicate that a small number of B cells or cells in the B-cell lineage are present in the fetal thymus and also suggest the importance of these thymic B cells in the negative selection of T cells during early developmental stages.

Characterization of B Cells in Human Thymus

The surface characteristics of B cells present in the human thymus were investigated. Cytofluorometrical and immunohistological studies, using anti-human IgM or anti-B cell monoclonal antibodies (mAbs; anti-Leu 12Ab, anti-Leu 16Ab, or L26), revealed that a small number of B cells are present in the human thymus. The thymic B cells were detected only in a low-density cell population, whereas in a high-density cell fraction, only T cells were found. In 15 cases, all of which the thymi were histologically normal, the percentages of B cells in the low-density fraction were 0.28% to 50% (6.8% in average), and Leu 1+ (CD5+) B cells in the low-density fraction were 0.1% to 26% (3.5% in average); approximately 50% of the thymic B cells were Leu 1+ B cells. These results indicate that B cells, especially Leu 1 (CD5)+ B cells, are also present in the human thymus, as suggested from our previous reports on mice.

Development of insulin-dependent diabetes mellitus in [(NOD + BALB/c) --> NOD] mixed allogeneic bone marrow chimeras.

To examine the possibility that the bone marrow cells of BALB/c genotype interfere with the development of insulitis and diabetes in NOD mice, we transplanted BALB/c bone marrow cells mixed with NOD bone marrow cells into NOD mice. The [(NOD + BALB/c) --> NOD] chimeras developed insulitis and diabetes, indicating that BALB/c bone marrow cells do not interfere with the development of the disease in NOD mice. Surprisingly, these mice have been reconstituted with only NOD hematolymphoid cells. When the pancreatic tissues from newborn NOD and BALB/c mice were grafted into [(NOD + BALB/c) --> NOD] chimeras, the BALB/c pancreatic tissues were rejected, whereas the NOD graft showed insulitis. Furthermore, the spleen cells of the chimeras showed responsiveness to BALB/c spleen cells in mixed lymphocyte reaction and generated cytotoxic T lymphocytes specific for the H-2d and third party targets. These findings indicate that the hematolymphoid cells (including hemopoietic stem cells) of NOD mice are more resilient than those of normal BALB/c mice, and that insulin-dependent diabetes mellitus will recur after bone marrow transplantation unless the hematolymphoid cells of NOD mice are completely destroyed by irradiation.

Analyses of Thymic Abnormalities in Autoimmune-Prone (NZW x BXSB) F1 Mice

Thymic abnormalities of autoimmune-prone (NZW x BXSB)F1 mice are investigated. After the onset of autoimmune diseases, severe thymic atrophy is observed. The atrophied thymus shows lower CD4+ CD8+ T cell and higher CD4+CD8- or CD4-CD8+ T cell counts than aged-matched normal strains or other autoimmune-prone (NZB x NZW)F1(B/WF1) mice; even at the age of 40 weeks, B/WF1 mice have a large number of double-positive cells and a small number of single-positive cells. Thymocytes in the atrophic thymus of (NZW x BXSB)F1 mice respond better than normal mice to T cell-mitogens (PHA and ConA). In addition, the single-positive T cells are J-11d-negative. These findings indicate that the atrophic thymus includes mature T cells and a large number of plasma cells and B cells; it therefore responds well to lipopolysaccharides. Experiments in reciprocal transplantation of the thymus and bone marrow between (NZW x BXSB)F1 and normal mice show that the thymic abnormalities are due to defects of the hemopoietic stem cells (HSCs) rather than either intrinsic abnormalities in the thymus or extrinsic abnormalities such as anti-thymus antibodies. It should be noted that the atrophic thymus recovers after transplantation of normal bone marrow cells; the atrophic thymus still has the capacity to induce the differentiation of normal T cells (including double-positive T cells) if normal HSCs are introduced.

Patterns of CD8 T cell clonal dominance in response to change in antiretroviral therapy in HIV-infected children.

ObjectiveTo examine the influence of change in antiretroviral therapy (ART) on patterns of CD8 T cell clonal dominance in HIV-infected children. DesignSeventeen HIV-infected children with plasma virus loads between 3.1 and 5.7 log10 were investigated before and after changes in ART. MethodsCDR3 spectratyping was performed in 22 T cell receptor (TCR) Vβ subfamilies by multiplex polymerase chain reaction (PCR) in purified peripheral blood CD8 T cells in conjunction with CD4 cell counts, plasma HIV-RNA copies and lymphoproliferative assays (LPA). ResultsCD8 T cell clonal dominance in two or more Vβ families was present in eight out of 17 children. After a change in therapy, 13 patients (76%) acquired new clones whereas three patients (17.6%) showed a loss in CD8 cell clones. An increase in the numbers of dominant clones correlated with an increase in percentage CD4 cell counts (P < 0.001) and with improved LPA responses to tetanus (P < 0.05) and alloantigens (P < 0.01). CD4 cell increase was associated with an initial mean gain of 3.1 ± 2.1 CD8 cell clones, independent of a virological response. A loss of CD8 cell clones or failure to achieve CD4 T cell increase was associated with failure to achieve virological suppression. ConclusionChildren with chronic HIV infection manifest CD8 T cell clonal dominance, which appears to be dependent upon the adequacy of the CD4 cells. With optimization of therapy, a gain in clonal dominance is the predominant response, except in situations of failure to contain viral replication.

Repair Mechanism of Lupus Nephritis in (NZB × NZW) F1 Mice by Allogeneic Bone Marrow Transplantation

We have recently found that allogeneic bone marrow transplantation (BMT) can be used to treat lupus nephritis in (NZB x NZW)F1(B/WF1), BXSB, MRL/lpr and (NZW x BXSB)F1 mice. To elucidate why and how glomerular damage is repaired by BMT, serial renal biopsies were carried out using B/WF1 mice before and after BMT. Donor-derived B cells and macrophages with normal functions developed two weeks (wks) after BMT. At this stage, the macrophages did not show immune complex (IC) clearance activity. Donor-derived T cells with normal functions were generated six wks after BMT. At this stage, visceral epithelial cells macrophages and mesangial cells in the glomeruli were activated by T cells and showed marked phagocytic activity; macrophages and mesangial cells were found to be responsible for the clearance of ICs, whereas, to our surprise, epithelial cells were found to be responsible for the repair of injured basement membranes. These findings suggest that T cells with normal functions, which have the capacity to activate macrophages, mesangial cells and epithelial cells, play a crucial role in repairing IC-mediated glomerular damage.

RANTES and MIP-1β mRNA expression in human peripheral blood mononuclear cells: transcript quantification using NASBA technology

The importance of chemokines in the immune response, as well as in a range of specific disease states, is becoming increasingly apparent. The role of CC- (or beta-) chemokines and their receptors in the pathology and mechanisms of HIV-1 infection has served to intensify interest in these factors. Although the functionality of these factors resides in their protein forms, assays for the detection and quantification of these protein factors in clinical samples are not readily available. Consequently, we designed NASBA-based assays for the quantification of the mRNA encoding two members of the CC-chemokine family: RANTES and MIP-1beta. The NASBA-based assays are extremely sensitive, accurate, and reproducible across a dynamic range of at least four orders of magnitude. Inter-assay performance is comparable to intra-assay performance. We applied these methods to the analysis of normal human PBMC and PBMC from HIV-1 infected individuals. Although MIP-1beta mRNA levels are higher than RANTES levels in both populations, RANTES levels in HIV-1+ patients are higher than in normal individuals. The utility of these assays in longitudinal studies of specific subpopulations of cells, as well as their potential use in clinical diagnostics, is discussed.

Analyses of development of insulitogenic T lymphocytes in NOD mice by transplantation of bone marrow, thymus, and pancreas.

To estimate the functional participation of the insulitogenic genes in the development of insulitogenic lymphocytes, we attempted to induce insulitis in normal allogeneic BALB/c hosts by bone marrow transplantation from NOD mice. The development of the insulitogenic lymphocytes was histologically examined using the hosts pancreas and pancreatic tissue from NOD mice which had been grafted under the renal capsules 2 weeks before sacrifice. Adult-thymectomized NOD mice that had been reconstituted with the thymus and bone marrow from NOD mice showed insulitis in both the hosts pancreas and grafted pancreatic tissue from newborn NOD mice. When BALB/c mice were lethally irradiated and then reconstituted with NOD bone marrow cells, no insulitis developed in the pancreatic grafts from NOD and BALB/c mice or in the hosts pancreas. Since the specificities and functions of T lymphocytes are controlled by the thymic microenvironment during the differentiation within the thymus, the thymus of BALB/c genotype may be responsible for the failure to induce the insulitogenic lymphocytes. Therefore, athymic BALB/c mice were reconstructed with bone marrow cells and thymus of NOD genotype. No insulitis developed, however, in either the hosts pancreas or grafted pancreatic tissue. These results suggest that the bone marrow cells and thymus of NOD genotype are not sufficient to induce insulitogenic lymphocytes in the allogeneic BALB/c environment.