Jeng-Chang Chen

A kinetic study of the murine mixed lymphocyte reaction by 5,6-carboxyfluorescein diacetate succinimidyl ester labeling.

Alternatives to the use of radioisotopes to measure cell proliferation in mixed lymphocyte reactions (MLR) are desirable to avoid the hazards and costs associated with radioisotope use. The versatile fluorochrome 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) has been used to measure MLR and provides the opportunity to measure several different growth parameters. This study was aimed at determining which growth parameter is most practical and suitable for measuring murine MLR. The parameters measured were: the relative number of daughter T-cells, the relative number and frequency of reactive T-cell precursors and the relative number of mitotic events. Responder cells were CFSE-labeled unfractionated splenocytes from C57BL/6 mice. Stimulator cells included irradiated splenocytes from C57BL/6 (control), B6D2F(1) (haplo-allogeneic) or FVB/N (allogeneic) mice. Cultures were harvested daily for 1 week. Stimulator T-cells rapidly declined to less than 0.2-0.3% of the mixed population by day 2 of culture. Experimental groups had a significantly higher number of daughter T-cells and mitotic events after 2 days of culture with the number of daughter T-cells climbing exponentially after 5 days of culture. The number and frequency of reactive T-cell precursors were significantly higher in experimental groups on days 2-3, but this difference became insignificant by day 4. Among all the parameters, the relative number of daughter T-cells was the most practical for measuring MLR, after 5 days of culture, based upon the growth kinetics of responder T-cells and the survival of the stimulator cells.

Acute expression of hepatitis C core protein in adult mouse liver: Mitochondrial stress and apoptosis

Objective. In infection with hepatitis C virus (HCV), spontaneous clearance of the virus occurs in 30–40% of cases. By contrast, in chronic infection, this is rare. The basis for viral clearance in acute disease is unknown. Whereas cellular immune responses have been studied in detail, few data exist on the role of viral structural proteins, such as the core protein. The purpose of this study was to investigate the effects of core produced de novo within adult mouse hepatocytes by using a new transgenic mouse line in which expression of HCV core is regulated by tetracycline (tet-off). Material and methods. In this work, transgenic mice with conditional HCV core were created, to study the acute expression of HCV core protein in the context of the mature liver. The subcellular distribution of the core, hepatocellular oxidative stress and apoptosis were monitored. Results. Core protein is readily detectable and strongly associated with cytoplasmic lipid vesicles, endoplasmic reticulum and mitochondria. Mitochondrial oxidative stress was evidenced by a reduction in thioredoxin-2 (trx2). Concurrently, caspase-3 activity and TUNEL increased and, over time, the level of core protein in the liver declined. Conclusions. Mice that are conditionally transgenic for HCV core protein, which is readily detected and morphologically associated with steatosis in individual hepatocytes, were developed. Acute expression of core protein causes mitochondrial stress, as demonstrated by a reduction in trx2 and in the apoptosis of core-positive hepatocytes. We speculate that these events could be involved in the clearance of virus during acute hepatitis C, by both reducing the burden of virus in the liver and effectively priming the immune response.

Prenatal Tolerance Induction: Relationship Between Cell Dose, Marrow T-Cells, Chimerism, and Tolerance

It was reported that the dose of self-antigens can determine the consequence of deletional tolerance and donor T cells are critical for tolerance induction in mixed chimeras. This study aimed at assessing the effect of cell doses and marrow T cells on engraftment and tolerance induction after prenatal bone marrow transplantation. Intraperitoneal cell transplantation was performed in FVB/N (H-2Kq) mice at gestational day 14 with escalating doses of adult C57BL/6 (H-2Kb) marrows. Peripheral chimerism was examined postnatally by flow cytometry and tolerance was tested by skin transplantation. Transplantation of light-density marrow cells showed a dose response. High-level chimerism emerged with a threshold dose of 5.0 × 106 and host leukocytes could be nearly replaced at a dose of 7.5–10.0 × 106. High-dose transplants conferred a steady long-lasting donor-specific tolerance but were accompanied by >50% incidence of graft-versus-host disease. Depletion of marrow T cells lessened graft-versus-host disease to the detriment of engraftment. With low-level chimerism, tolerance was a graded phenomenon dependent upon the level of chimerism. Durable chimerism within 6 months required a threshold of = 2% chimerism at 1 month of age and predicted a 50% chance of long-term tolerance, whereas transient chimerism (<2%) only caused hyporesponsiveness to the donor. Tolerance induction did not succeed without peripheral chimerism even if a large amount of injected donor cells persisted in the peritoneum. Neither did an increase in cell doses or donor T-cell contents benefit skin graft survivals unless it had substantially improved peripheral chimerism. Thus, peripheral chimerism level can be a simple and straightforward test to predict the degree of prenatal immune tolerance.

Prevention of Graft Rejection by Donor Type II CD8 + T Cells (Tc2 Cells) Is Not Sufficient to Improve Engraftment in Fetal Transplantation

Objectives: Tc2 cells, a subset of CD8+ T cells, are able to facilitate engraftment in a murine model of postnatal allogeneic bone marrow transplantation. The purpose of this study was to evaluate whether Tc2 cells could improve engraftment in fetal transplantation. Methods: Gestational day 13 C57BL/6 (H-2b) fetal mice were used as recipients, adult B6D2F1 mice (C57BL/6 × DBA/2, H-2b/d) as donors, and splenocytes from B6C3F1 (C57BL/6 × C3H/He, H-2b/k) mice were used as stimulators in cultures used to generate the Tc2 cells from B6D2F1 mice. Peripheral blood chimerism was examined monthly for 3 months. Thereafter, recipients were sacrificed to evaluate the levels of peritoneal, splenic and bone marrow chimerism. The T-cell responses of recipient splenocytes to cells of host origin were measured as a proliferative response in mixed lymphocyte cultures. Results: Low levels of peripheral blood cell chimerism (<0.3%) were observed at 1 month of age, which declined further by 3 months of age. The levels of donor cells in the spleen, bone marrow and peritoneal cavity were usually not more than 0.05%. The peritoneal cavity tended to have higher levels of donor cells with 1 recipient sustaining as high as 25.03% at the age of 3 months. Higher peritoneal chimerism correlated with a lower donor-specific T-cell response. Conclusions: Transplantation of Tc2 cells was insufficient to improve bone marrow engraftment in utero, suggesting that graft rejection is not the major barrier to successful in utero transplantation. Donor cells can persist in the peritoneal cavity and might play an important role in inducing immune tolerance in fetuses.

Characterization of Tolerance Induction through Prenatal Marrow Transplantation: The Requirement for a Threshold Level of Chimerism to Establish Rather than Maintain Postnatal Skin Tolerance:

Hematopoietic chimerism resulting from prenatal marrow transplantation does not consistently result in allotolerance for unidentified causes. In a C57BL/6-into-FVB/N murine model, we transplanted T-cell-depleted adult marrow on gestational day 14 to elucidate the immunological significance of chimerism towards postnatal tolerance. Postnatally, chimerism was examined by flow cytometry, and tolerance by skin transplantation and mixed lymphocyte reaction. Regulatory T cells were quantified by FoxP3 expression. Peripheral chimerism linearly related to thymic chimerism, and predicted the degree of graft acceptance with levels >3% at skin placement, yielding consistent skin tolerance. Low- and high-level chimeras had lower intrathymic CD3high expression than microchimeras or untransplanted mice. Regardless of the skin tolerance status in mixed chimeras, donor-specific alloreactivity by lymphocytes was suppressed but could be partially restored by exogenous interleukin-2. Recipients that lost peripheral chimerism did not accept donor skin unless prior donor skin had engrafted at sufficient chimerism levels, suggesting that complete tolerance can develop as a consequence of chimerism-related immunosuppression of host lymphocytes and the tolerogenic effects of donor skin. Thus, hematopoietic chimerism exerted immunomodulatory effects on the induction phase of allograft tolerance. Once established, skin tolerance did not fade away along with spontaneous regression of peripheral and tissue chimerism, as well as removal of engrafted donor skin. Neither did it break following in vivo depletion of increased regulatory T cells, and subcutaneous interleukin-2 injection beneath the engrafted donor skin. Those observations indicate that the maintenance of skin tolerance is multifaceted, neither solely dependent upon hematopoietic chimerism and engrafted donor skin nor on the effects of regulatory T cells or clonal anergy. We conclude that hematopoietic chimerism generated by in utero hematopoietic stem cell transplantation is critical to establish rather than maintain postnatal skin tolerance. Therefore, the diminution of hematopoietic chimerism below a threshold level does not nullify an existing tolerance state, but lessens the chance of enabling complete tolerance.

Cellular therapies supplement: the peritoneum as an ectopic site of hematopoiesis following in utero transplantation.

BACKGROUND: In utero transplantation (IUT) has the potential to treat birth defects early before full development of the immune system. Relatively small grafts, which are not matched for major histocompatibility antigens, can be delivered even before onset of disease symptoms. IUT of hematopoietic stem cells is usually performed via intraperitoneal injection, yet the fate of donor cells in the peritoneal cavity is not fully understood. We review our recent work and present new data demonstrating that the peritoneum can be a site of ectopic hematopoiesis with implications for IUT and immune tolerance induction.

Allogeneic lymphocytes exerted graft-versus-host rather than tolerogenic effects on preimmune fetuses

BACKGROUND Among cell suspensions from different origins, lymphocytes were reported to have the superiority of tolerance-conferring capacity in preimmune hosts. However, this belief was derived directly from murine combinations with fewer major histocompatibility complex (MHC) barriers that are exceptional in the clinical arena. Because of the potential for prenatal tolerance induction to facilitate postnatal therapies, it is important to examine the relative merits and hazards of fully MHC-mismatched naïve lymphocytes as the prenatal tolerogenic agent in the preimmune fetus to cross MHC barriers. MATERIALS AND METHODS In utero injection of C57BL/6 splenic lymphocytes was conducted in gestational day 14 FVB/N fetuses. Then, FVB/N recipients were subjected to the evaluation of hematopoietic chimerism, donor-specific tolerance, and graft-versus-host disease (GVHD). RESULTS With a dose of ≥ 5 × 10(5) C57BL/6 lymphocytes, the recipients born alive either died unexpectedly by maternal cannibalization or succumbed to GVHD within postnatal 1 mo. GVHD mice showed significant hematopoietic chimerism that was dominated by donor CD3 T cells. It was found that allogeneic lymphocytes could rapidly damage the fetal liver within 5 d after injection. Fetal recipients could survive a dose of ≤ 2 × 10(5) allogeneic lymphocytes beyond 1 mo of age, but at best showed microchimerism that was insufficient to confer donor-specific skin tolerance. CONCLUSIONS Fully MHC-mismatched lymphocytes injected in utero had lethal graft-versus-host effects, which might rapidly develop within 1 wk after injection in preimmune fetuses. They were incapable of conferring significant hematopoietic chimerism and graft tolerance even at bearable doses.

Persistence of allografts in the peritoneal cavity after prenatal transplantation in mice.

BACKGROUND: In utero transplantation (IUT) is usually performed by intraperitoneal injection, but there is little information regarding the fate of intraperitoneally transplanted cells in the first weeks after transplantation.

The various impacts of the hepatitis C virus core protein upon hepatic oxidative stress after common bile duct ligation and partial hepatectomy.

Abstract Introduction: Although it is uncertain how the hepatitis C virus (HCV) core protein influences hepatic oxidative stress after partial hepatectomy and common bile duct ligation (CBDL) this may be crucial for the prognosis of patients with HCV infection who have undergone hepatic resection, or who have complications due to a biliary tract obstruction. Materials and methods: A group of double transgenic mice (DTM) that express both the tetracycline transactivator (tTA) and the HCV core, with conditional, acute expression of the HCV core in the context of the mature liver were subjected to 43% partial hepatectomy and CBDL. The levels of thioredoxin-1, thiobarbituric acid reactive substances (TBARS), and 4-hydroxynonenal (4-HNE) were evaluated in liver samples taken 3 days after the operations. Results: The DTM had significantly higher TBARS levels than mice that were transgenic for only tTA (i.e. single transgenic mice; STM) and non-transgenic mice (NTM) after a sham laparotomy, CBDL and partial hepatectomy. Of the DTM, the TBARS levels were higher in female mice than in males after a sham laparotomy (P = 0.02) and CBDL (P = 0.0001). 4-HNE staining data were compatible with these results. Furthermore, male DTM exhibited higher levels of thioredoxin-1 than female DTM after sham laparotomy (P = 0.012) and CBDL (P = 0.008). Conclusions: The HCV core increases hepatic oxidative stress in vivo and female DTM are more vulnerable to the oxidative stress caused by acute core expression with, or without, CBDL. The fact that the female DTM had lower thioredoxin-1 levels may account for this observation.

In Utero Exposure to Exosomal and B-Cell Alloantigens Lessens Alloreactivity of Recipients’ Lymphocytes Rather than Confers Allograft Tolerance

According to actively acquired tolerance, antigen exposure before full immune development in fetal or early neonatal life will cause tolerance to this specific antigen. In this study, we aimed to examine whether allogeneic tolerance could be elicited by in utero exposure to surface MHC antigens of allogenic cells or soluble form of MHC exosomes. Gestational day 14 FVB/N fetuses were subjected to intraperitoneal injection of allogeneic major histocompatibility complex (MHC) exosomes or highly enriched B-cells. Postnatally, the recipients were examined for the immune responses to donor alloantigens by lymphocyte proliferative reactions and skin transplantation. In utero exposure to allogeneic MHC exosomes abolished the alloreactivity of recipients’ lymphocytes to the alloantigens, but could not confer skin allograft tolerance. In utero transplantation of highly enriched allogeneic B-cells generated low-level B-cell chimerism in the recipients. However, it only extended the survivals of skin allograft by a few days despite the lack of donor-specific alloreactivity of recipients’ lymphocyte. Thus, an early in utero contact with exosomal or B-cell alloantigens did not lead to full skin tolerance but rather, at best, only to delayed skin rejection in the presence of microchimerism made by B-cell inocula. These results argued against the theory of actively acquired tolerance, and implicated that in utero exposure to marrow cells in previous studies was a unique model of allo-tolerance induction that involved the establishment of significant hematopoietic chimerism. Taken together with the discovery of in utero sensitization to ovalbumin in our previous studies, the immunological consequences of fetal exposure to foreign antigens might vary according to the type or nature of antigens introduced.