Intrathymic immune modulation prevents acute rejection but not the development of graft arteriosclerosis (chronic rejection).

BACKGROUND: We showed previously that our intrathymic immune modulation protocol induces virtually permanent graft survival of simultaneously transplanted cardiac allografts in MHC-incompatible rat strain combinations. It is, however, unknown whether this procedure prevents the development of graft arterial disease (GAD). METHODS: Male AO recipient rats were intrathymically inoculated with 2.5x10(7) PVG splenocytes immediately followed by heterotopic transplantation of a PVG cardiac allograft (day 0). Immunosuppression consisted of 1 ml of antilymphocyte serum i.p. (day 0) and cyclosporine i.m. (15 mg/kg body weight) on days 1, 2, and 3 posttransplantation. Histological analysis, mixed lymphocyte reactions, and intragraft cytokine mRNA expression were performed at several time points after engraftment. RESULTS: Histological analysis revealed that GAD was already present 14 days after transplantation. At 200 days, virtually all vessels were affected and over 80% of the vessels showed severe intimal lesions. Infiltrate analysis displayed massive parenchymatous infiltrates (CD8+ cells and ED1+ macrophages) 2 weeks after transplantation. At later time points, infiltrates became epicardial and/or blood vessel associated and mainly consisted of CD4+, CD8+, and B cells. Mixed lymphocyte reactions showed nonspecifically decreased responses at 60 days but complete restoration of these responses at later time points (120 to 280 days). Intragraft cytokine mRNA expression showed decreased interleukin-2/interferon-gamma and sustained interleukin-10 expression 2 weeks after transplantation. Transforming growth factor-beta mRNA expression was increased >200 days after transplantation. CONCLUSIONS: Intrathymic immune modulation does not abolish alloreactivity, and despite induction of long-lasting graft survival, this procedure does not prevent and may even facilitate the development of GAD.

Liver cell proliferation after partial hepatectomy in rats with liver metastases.

OBJECTIVE: To validate proliferating cell nuclear antigen (PCNA) expression and flow cytometry as proliferation markers in regenerating rat liver containing metastases. STUDY DESIGN: Rats containing colorectal liver metastases were killed at various days after 70% partial hepatectomy or a sham operation. [3H]thymidine and 5-bromo-2'deoxyuridine (BrdU) incorporation, PCNA expression and flow cytometry were used to evaluate liver cell proliferation. RESULTS: The assessment of proliferating liver cells by PCNA expression and BrdU incorporation was more reliable than autoradiography. PCNA expression correlated well with BrdU incorporation (r = .68, P = .003) and autoradiography (r = .57, P = .02) in regenerating liver. BrdU incorporation and PCNA expression were higher in hepatectomized rats as compared to sham-operation rats at days 1-4 after hepatectomy. Flow cytometry of propidium-stained nuclei from livers of hepatectomized rats showed a higher proportion of S-phase nuclei as compared to S-phase nuclei in control rats. The correlation coefficients of the number of S-phase nuclei, BrdU-positive nuclei and PCNA-positive nuclei were .39 (P < .02) and .56 (P < .0005), respectively. CONCLUSION: Flow cytometry and PCNA expression are simple and reliable methods of studying proliferation in metastases containing rat liver after partial hepatectomy.

Reduction of EGP-2-positive pulmonary metastases by bispecific-antibody-redirected T cells in an immunocompetent rat model.

Effectiveness of bispecific-monoclonal-antibody (BsMAb)-mediated cellular anti-tumour activity was evaluated in vitro and in vivo in relation to the additional need for T-cell activation in a new immunocompetent rat tumour model. L37 tumour cells, derived from a squamous-cell carcinoma of the lung of Wag/Rij rats, were transfected with the cDNA coding for the human 38-kDa transmembrane pan-carcinoma-associated antigen EGP-2. Intravenous inoculation of EGP-2-positive L37 cells resulted in a rapid outgrowth of EGP-2-positive tumour nodules in the lungs. A BsMAb BIS-19, recognizing EGP-2 on the transfected tumour cells and the T-cell receptor of the rat, was made and allowed specific lysis of EGP-2-transfected L37 tumour cells by activated rat T lymphocytes in vitro. In vivo T-cell activation, assessed by up-regulation of IL-2-receptor expression, could be induced by daily injection of rat rIL-2. Intravenous treatment of tumour-bearing EGP-2-positive L37 tumour with BIS-19 together with rat rIL-2 resulted in almost complete disappearance of established tumour. In contrast, animals treated with BIS-19 alone, IL-2 alone or a combination of anti-EGP-2, anti-TcR and IL-2 showed much less or no tumour reduction. These results show effectiveness of systemic treatment with BsMAbs to induce anti-tumour activity in established tumours. Immune activation prior to or during treatment with BsMAbs, as achieved with IL-2, appears to be a prerequisite for successful treatment.

High-frequency, but reduced absolute numbers of recent thymic migrants among peripheral blood T lymphocytes in diabetes-prone BB rats.

In rats, RT6 and CD45RC are expressed by mature peripheral T cells. The underrepresentation of T cells expressing these markers in the T lymphocytopenic BB rat might therefore be a reflection of a relatively immature T cell population. With the use of Thy-1 as a marker for recent thymic migrants, it was demonstrated that BB rats indeed have a phenotypically less mature T cell population than age-matched control rats. However, this could not account for the reduced percentages of RT6+ and CD45RC+ T cells, as these were also decreased among mature Thy-1- T cells of BB rats. Although relatively overrepresented, absolute numbers of Thy-1+ T cells were reduced in BB rats. Absolute numbers of mature Thy-1- T cells were also reduced in BB rats, but to a much larger degree than would proportionally be expected. Our findings taken together led us to conclude that both reduced thymic output and a defect in peripheral expansion are involved in the T lymphocytopenia of BB rats.

The extent of clonal structure in different lymphoid organs.

To gain insight into the clonal organization of lymphoid organs, we studied the distribution in situ of donor-derived cells in near-physiological chimeras. We introduced RT7b fetal liver cells into nonirradiated congenic RT7a neonatal rats. The chimerism 6-20 wk after injection ranged from 0.3 to 20%. The numbers of cell clones simultaneously contributing to cell generation in a particular histological feature were deduced from the variance in donor cell distribution. In bone marrow and thymus, donor-derived lymphoid cells were found scattered among host cells, indicating a high mobility of cells. In bone marrow, donor cells were evenly distributed over the entire marrow, even at low chimerism. This indicates that leukopoiesis is maintained by the proliferation of many clones. In the thymus, the various lobules showed different quantities of donor-derived lymphoid cells. Mathematical analysis of these differences indicated that 17-18 cell division cycles occur in the cortex. In spleen, the distribution of donor-derived cells over the germinal centers indicated that 5 d after antigenic stimulation, germinal centers develop oligoclonally. The main conclusions of this work are that (a) bone marrow and thymus are highly polyclonal; (b) 17-18 divisions occur between prothymocyte and mature T cell; and (c) lymphoid cells disperse rapidly while proliferating and differentiating.

Kinetics of thymocyte regeneration in adult adriamycin treated rats: evidence for an exclusive role of bone marrow derived cells.

This paper describes a new, less toxic and more selective approach to study the adult thymus. An adriamycin (ADR), sparing bone marrow (BM) stem cells and nontoxic to cells that are not in cycle during treatment, was used as a depleting agent in conjunction with vascular thymus transplantation. We were able to deplete the thymus of thymocytes without damaging its microenvironment as witnessed by intact antigen profiles of stromal cells. Two models were used in this study, (1) regeneration after ADR induced depletion with or without BM reconstitution either systemically or intrathymically and (2) thymocyte turnover or regeneration in vascularly transplanted thymi. In the latter model either normal thymus was grafted into ADR treated recipient or ADR depleted thymus was grafted into normal recipient. These experiments clearly show that intact BM function is a prerequisite for intact continued cellularity of the adult thymus. Although the resident thymocyte population possesses some limited proliferating potential, it clearly does not seem to have a permanent self-renewing capacity of intrathymic stem cells.

Germinal centers develop oligoclonally.

As part of our studies into the role of germinal centers, we investigated whether each de novo generated germinal center (GC) develops from one single GC precursor cell (GCPC, monoclonal development), a few GCPC (oligoclonal development) or from many GCPC (polyclonal development). Thus, lethally (9 Gy) X-irradiated AO (RT1u) rats were reconstituted with 10(8) thoracic duct lymphocytes (TDL) containing mixtures of AO and AO X BN cells in various ratios. The AO TDL were tolerant for AO X BN cells by using TDL from AO----(AO X BN)F1 (RT1u/n) X-irradiation bone marrow chimeras. To induce GC formation in the spleen of TDL-reconstituted rats, animals were i.v. injected with 10(9) sheep red blood cells. Five days after reconstitution and antigenic challenge spleens were taken for analysis of cellular make up of de novo generated GC. Spleen sections were immunohistochemically stained with monoclonal antibody F17-23-2, recognizing major histocompatibility complex class II antigens of the RT1n haplotype but not the RT1u haplotype, to discriminate between B cells of AO and AO X BN origin. Analysis of the GC in spleens of rats reconstituted with a mixture of AO and AO X BN TDL revealed three types of GC: GC entirely composed of AO cells, GC entirely composed of AO X BN cells and GC containing a mixture of both. The relative frequencies of these three types of GC indicated that in our experimental system, de novo GC developed oligoclonally from one to three GCPC. These data strongly suggest that GC are sites of antigen-driven expansion of peripheral B cells to very large clones.

B lymphocyte differentiation in the rat: production and characterization of monoclonal antibodies to B lineage-associated antigens.

Three mouse monoclonal antibodies (mAb) directed against rat B lineage antigens were produced. The mAb, designated HIS14 (IgG1), HIS22 (IgM) and HIS24 (IgG2b), were characterized for binding to lymphoid and nonlymphoid tissues by immunoperoxidase staining of frozen sections and by (double-) immunofluorescence staining of single cell suspensions from lymphoid organs. HIS14 recognized a pan B cell determinant: it reacted with virtually all cells of each anatomic B cell compartment and with about 95% of surface (s)Ig+ cells in thoracic duct lymph and in suspensions of spleen and lymph nodes. HIS22 and HIS24 detected B lineage-associated antigens expressed by major subpopulations of B cells. HIS22 predominantly stained the lymphocyte corona, but not (or weakly) the germinal centers and splenic marginal zones, whereas HIS24 reacted with both corona and germinal center and not (or weakly) with marginal zone. In accordance with this, substantial proportions of sIg+ cells in spleen cell suspensions did not express HIS22 or HIS24 determinants (20% and 27%, respectively). In bone marrow the vast majority of cytomplasmic mu+ pre-B cells were HIS14+ and HIS24+, and up to one third also HIS22+, indicating an appearance of the determinants early in B lymphocytopoiesis. The antigens recognized by HIS14, HIS22 and HIS24 are lost during the final stage of B cell differentiation: none of the mAb bound to plasma cells. As far as detectable, neither cells of myeloid and erythroid lineages in bone marrow nor thymocytes were stained by HIS14, HIS22, or HIS24. In suspensions of peripheral lymphoid organs (spleen and lymph nodes) but not in thoracic duct lymph, HIS14 and HIS24 labeled a small proportion (12% and 14%, respectively) of Ig- cells. HIS22 did not bind to Ig- peripheral lymphocytes. Reactivity of HIS14, HIS22 and HIS24 with nonlymphoid tissues was virtually absent; HIS22 stained the high endothelial venules in lymph nodes and Peyer's patches. As determined by immunoblotting, the antigenic determinants on lymph node cells recognized by HIS14, HIS22 and HIS24 were present on molecules with an apparent molecular mass of 205 kDa, 210 (and 175) kDa and 205 kDa, respectively, which is similar to the molecular mass of the B cell form of the rat leukocyte common antigen. In addition, the antigens recognized by HIS14, HIS22 and HIS24 co-capped with the leukocyte common antigen. This suggests that each of the three mAb recognize determinants present on the B cell form of the leukocyte common antigen.

The ontogeny of germinal centre forming capacity of neonatal rat spleen.

In non-specifically immunized rats, bred under conventional conditions, the first 'spontaneous' germinal centres were observed by 21 days after birth. Deliberate antigenic stimulation led to an earlier appearance of germinal centres in neonatal spleen: immunization with sheep red blood cells as early as 7 days after birth resulted in germinal centre formation in the spleen as observed 7 days later. By that time the first primary follicles could also be observed, in both immunized and non-immunized rats. Although 3-day-old rats upon antigenic stimulation failed to generate germinal centres in their spleen, transfer experiments of 3-day-old spleen cells to lethally X-irradiated syngeneic adult recipients indicated that 3-day-old spleens at least contained all the essential lymphoid elements (B and T cells) needed for germinal centre formation. These results strongly suggest that the failure to induce germinal centres in 3-day-old rats is most probably due to an immaturity of their splenic microenvironment. Immunohistochemical staining of frozen sections of neonatal rat spleen using mAb ED 5 and MRC OX-2 showed that follicular dendritic cells (FDC) were found as soon as primary follicles were found (i.e. by 14 days after birth). The appearance of FDC in neonatal spleens was not influenced by deliberate antigenic stimulation nor by the administration of adult spleen cells. We postulate that, during the development of FDC, a splenic microenvironment is created that allows primary follicle formation and the generation of germinal centres.

Germinal centre formation and follicular antigen trapping in the spleen of lethally X-irradiated and reconstituted rats.

In this study, the relationship between germinal centre formation and the follicular trapping of immune complexes in the rat spleen was investigated. Lethally (9 Gy) X-irradiated rats were reconstituted with thoracic duct lymphocytes and subsequently challenged with sheep red blood cells to induce germinal centre formation. Rats were killed at daily intervals from 1 to 8 days after reconstitution and antigenic stimulation. Antigen trapping capacity during this interval was assessed by intravenous injection of HRP-anti-HRP immune complexes, 24 hr before killing of the animals. Germinal centre formation could be observed from Day 4 onwards. The follicular trapping capacity, which had been abolished by the X-irradiation, however, returned 2 days later (i.e. by Day 6). Apparently, in these experiments, early germinal centre formation could occur without an intact follicular trapping mechanism. It was, therefore, concluded that in this transfer system follicular immune complex trapping is not a prerequisite for the induction of de novo germinal centre formation. Previous studies have shown that both follicular dendritic cells and marginal zone B cells may play a role in the follicular antigen trapping mechanism. FDCs, as detectable with mAB MRC OX 2 in immunoperoxidase-stained frozen sections, were present in spleens at any time after irradiation and reconstitution; the impaired follicular trapping of HRP-anti-HRP immune complexes was therefore not due to an absence of FDCs. Marginal zone B cells, however, were almost absent until 5 days after reconstitution, as observed by immunohistochemical staining with anti-B cell mABs (HIS 14 and HIS 22) and a polyclonal anti-IgD antiserum. Our data suggest that regeneration of follicular antigen trapping capacity is dependent on the presence of marginal zone B cells.

Germinal centres and the B-cell system. V. Presence of germinal centre-precursor cells among lymphocytes of the thoracic duct in the rat.

Thoracic duct lymphocytes (TDL) were studied with respect to their capacity to give rise to germinal centres (GC) and to form primary antibody in an adoptive transfer system of the rat. Challenge with sheep erythrocytes (SRBC) 24h after lethal irradiation (900 rads) and syngeneic TDL reconstitution (10(8)) lead to conspicuous GC activity already 7 days after transfer. In contrast, using syngeneic bone marrow (BM) in the adoptive transfer system, no GC formation was observed over the period studied (14 days after reconstitution). Reconstitution experiments using in vivo-separated T-TDL (1-5% s-Ig+) and B-TDL (greater than 90% s-Ig+) subpopulations, either separately or in combination, indicated that GC originate from B-TDL but require T-TDL for induction.