Artificial Construction of Immune Tissues/Organoids and Their Application for Immunological Intervention.

Human-type lymphoid tissue organoids, which stably function in our body for a certain period of time or longer, may have a great potential as immune-stimulatory or immune-regulatory devices and could be utilized in the future for the treatment of various diseases such as cancer, severe infection, autoimmunity and congenital as well as acquired immunodeficiency resulting from severe infections or aging. In this review, we discuss about rationality and trials of the synthesis of immunologically functional lymphoid tissue organoids mainly in mouse. We have been recently trying to construct immunologically functioning human-type organoids, and the efforts are also briefly described.

Laparoscopic Free Omental Lymphatic Flap for the Treatment of Lymphedema.

UNLABELLED: Advances in microsurgery have displayed promising results for the treatment of lymphedema. The use of vascularized lymph node transfers has increased in popularity but incurs the potential risk for donor-site lymphedema. The omentum has been previously described for the treatment of lymphedema but has been overlooked because of presumed high morbidity, including the need for celiotomy and pedicled complications. The authors present a novel technique and early results of the laparoscopic free omental lymphatic flap for the management of lymphedema. The minimally invasive harvest successfully avoids both the previously associated morbidity of this flap and the risk of iatrogenic lymphedema to the donor site. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Human hemato-lymphoid system mice: current use and future potential for medicine.

To directly study complex human hemato-lymphoid system physiology and respective system-associated diseases in vivo, human-to-mouse xenotransplantation models for human blood and blood-forming cells and organs have been developed over the past three decades. We here review the fundamental requirements and the remarkable progress made over the past few years in improving these systems, the current major achievements reached by use of these models, and the future challenges to more closely model and study human health and disease and to achieve predictive preclinical testing of both prevention measures and potential new therapies.

Lymphatic tissue transplant in lymphedema--a minimally invasive, outpatient, surgical method: a 10-year follow-up pilot study.

Lymphedema is mainly characterized by swelling, fibrosis, and non-pitting edema. The aim of this study was evaluation of the long-term (10 years) effects of autologous lymphatic tissue implant in lymphedema. Lymphatic tissue from 9 patients (harvested form the same patient in areas not affected by lymphedema) was reimplanted into the affected limb, and these patients were followed for 10 years. Lymph nodes were harvested at the neck, axillary, or inguinal space (contralateral limb). Results showed that limb volume was decreased in the treatment group vs. controls. In ultrasound, black, low density, lymphatic spaces were visible in 100% of patients at inclusion but in only 23% of these subjects at 10 years. Thus, this early report proposes a new, minimally invasive method to improve lymphedema. Studies in progress will indicate the role of lymphatic transplant in the management of lymphedema and the best indications for this method.

Requirements for follicular exclusion and competitive elimination of autoantigen-binding B cells.

Results from several mouse tolerance models indicate that autoreactive B cells in peripheral lymphoid organs develop an anergic phenotype, migrate to the boundary between the T cell zone and the B cell follicle (T/B boundary), and undergo rapid cell death. We have used B cells from mice that are double-transgenic for soluble hen egg lysozyme (HEL) and an Ig that recognizes HEL with a high affinity to characterize the mechanisms underlying the migration and elimination of autoreactive B cells. In contrast to the situation for acutely activated B cells, we find that anergic B cells have reduced levels of CXCR5, the receptor for the follicular chemokine, CXCL13, and this contributes to their exclusion from follicles. CCR7 expression is required for follicular exclusion of anergic cells, although up-regulation of the receptor does not appear to be necessary. By TUNEL analysis, we observe that excluded anergic cells die in situ at the T/B boundary. We also show that this elimination occurs via a Fas-independent mechanism. Using CCR7(-/-)Ig(HEL)-transgenic B cells we find that localization to the T/B boundary is not a necessary event to achieve the competitive elimination of autoantigen-binding B cells. These findings characterize the mechanism for follicular exclusion of autoantigen-binding B cells and they indicate that B cells compete for survival by mechanisms that are separate from competition for the follicular niche.

[Microsurgical transplantation of "lymph flaps"]. / Mikrokhirurgicheskaia transplantatsiia "limfaticheskikh loskutov".

The review is concerned with the main historical stages in the development of "lymph flaps" transplantation. The author describes their blood supply, the topography of the constituent lymph structures, the feeding vascular pedicle, techniques of the harvesting, revascularization and restoration of lymph drainage. The review is intended for familiarization of a wide circle of physicians with the current strategy of microsurgical treatment of extremity lymphedema. This will allow to widen the outlook of practicing physicians and will give a possibility of further perfection of this trend.

Distribution of cycling T lymphocytes in blood and lymphoid organs during immune responses.

Proliferation of murine T lymphocytes in blood, lymph nodes, and spleen was studied in four in vivo stimulation systems, using BrdU pulse-labeling of DNA-synthesizing cells. The T cell response to the superantigen Staphylococcus enterotoxin B (SEB) was studied in detail. Vbeta8+ T cells showed a peak of DNA synthesis 16-24 h after SEB injection, and the percentage of BrdU+ CD4 and CD8 T cells was higher in blood than in lymph nodes and spleen. DNA synthesis was preceded by massive migration of Vbeta8+ cells from blood to lymphoid organs, in which the early activation marker CD69 was first up-regulated. SEB-nonspecific Vbeta6+ cells showed minimal stimulation but, when cycling, also expressed a high level of CD69. The other systems studied were injection of the IFN-gamma inducer polyinosinic:polycytidylic acid, infection by the BM5 variants of murine leukemia virus (the causative agent of murine AIDS), and T cell expansion after transfer of normal bone marrow and lymph node cells into recombinase-activating gene-2-deficient mice. In each case, a peak of T cell proliferation was observed in blood. These data demonstrate the extensive redistribution of cycling T cells in the first few hours after activation. Kinetic studies of blood lymphocyte status appear crucial for understanding primary immune responses because cycling and redistributing T lymphocytes are enriched in the circulating compartment.

Follicular dendritic cell (FDC) precursors in primary lymphoid tissues.

The origin of follicular dendritic cells (FDC) is unresolved, and as such, remains controversial. Based on the migration of Ag-transporting cells (ATC) into lymphoid follicles and the phenotypic similarity between FDC and ATC, one hypothesis is that ATC may represent emigrating FDC precursors. This contrasts with the view that FDC originate from local stromal cells in the secondary lymphoid tissues. Mice homozygous for the severe combined immunodeficiency (prkdc(scid)) mutation (scid) lack FDC. Thus, they provide a powerful tool for assessing de novo generation of FDC. To test whether FDC precursors could be found in bone marrow or fetal liver, scid/scid mice were reconstituted with either: 1) bone marrow cells from (BALB/c x C57BL/6)F1 donors, 2) bone marrow cells from ROSA BL/6 F1 (lacZ-transfected) mice, 3) rat bone marrow cells, or 4) rat fetal liver cells. Six to eight weeks after reconstitution with F1 bone marrow, cells reactive with the FDC-labeling mAb, FDC-M1, also expressed donor class I molecules on their surfaces. Similarly in mice reconstituted with lacZ-transfected bone marrow cells, these cells were also positive for the lacZ gene product. Furthermore, in spleens of animals reconstituted with either rat bone marrow or rat fetal liver, rat FDC were identified using the specifically labeling mAb, ED5. In all cases, host FDC were also present, indicating that scid/scid mice have FDC precursors that will mature in the presence of allogeneic or xenogeneic lymphoid cells. In summary, FDC can be derived from progenitor cells present in primary lymphoid tissues.

Both resting and activated B lymphocytes expressing engineered peptide-Ig molecules serve as highly efficient tolerogenic vehicles in immunocompetent adult recipients.

To test the potential for genetically transferring foreign sequences into autologous cells for specific modulation of immunity, we have generated transgenic mice that express an engineered peptide-IgG construct in the peripheral B cell compartment. B cells from these mice express and can be stimulated to secrete a murine IgG1 chain grafted with residues 12-26 from bacteriophage A cI repressor protein in-frame at the heavy chain N terminus. As expected, 12-26-IgG transgenic mice are profoundly tolerant to the peptide at both the T and B cell levels. Importantly, the injection of transgenic whole spleen, purified B cells, or even bone marrow cells into normal, immunocompetent adults results in profound peptide-specific T cell tolerance, as well as partial B cell tolerance. Injection of LPS-activated peptide-Ig-expressing B cells was uniquely effective at diminishing an ongoing humoral immune response typical of both Th1 and Th2 help. Since fixed transgenic B cells were tolerogenic, this suggests that secretion of the fusion protein is not required for tolerogenicity. These results show that an engineered self Ig, as well as B lymphocytes expressing epitopes from such a fusion protein, can regulate both cellular and humoral immune responses. Moreover, these studies provide the basis for expressing foreign epitopes on engineered IgG for the induction of gene-transferred tolerogenesis in autoimmune states.

Reduced provirus burden and enhanced humoral immune function in AZT-treated SCID-feline mice inoculated with feline immunodeficiency virus.

The lack of a safe, economical murine lentivirus model for human immunodeficiency virus type 1 (HIV-1) infection of humans has hampered the preclinical evaluation of potential antiviral compounds, vaccines, and biological response modifiers. A small animal model that does not employ HIV-1 is needed to minimize risk of accidental human exposure, enhance efficient use of scarce experimental compounds, and reduce laboratory space necessary to conduct statistically significant in vivo trials. Feline immunodeficiency virus (FIV), an immunosuppressive lentivirus of domestic cats, has been used extensively as an animal model for the pathogenesis and therapy of human HIV-1 infection. Cats, however, are not amenable to large-scale efficacy trials because of their relatively large size, high cost, and limited degree of physiologic characterization, particularly with regard to drug metabolism. To adapt the feline immune system to a small laboratory animal host, severe combined immunodeficient mice (SCID mice) were engrafted with feline lymphoid tissues (forming the SCID-fe mouse) and inoculated with FIV. Two quantitative parameters, the incidence of provirus detection in feline tissue grafts and the level of feline IgG in plasma, were used to demonstrate the antiviral efficacy of 3'-azido-3'-deoxythymidine (AZT, azidothymidine, Retrovir, zidovudine) in the SCID-fe system. Of 17 SCID-fe mice inoculated with 7 x 10(6) peripheral blood mononuclear cells (PBMC) from an FIV-infected cat, eight had detectable FIV provirus in both the feline thymus and feline lymph node implants, as measured by polymerase chain reaction (PCR)/Southern blot analysis. Treatment of these mice with AZT at a dose of 125 mg kg-1 day-1 in drinking water beginning 1 day prior to FIV inoculation and continuing throughout the study interval prevented the dual detection of provirus in feline lymph node and thymus grafts of all mice tested. In a separate experiment, the level of spontaneous feline IgG production was quantified by ELISA 2 weeks after FIV inoculation with and without AZT treatment. Mean plasma feline IgG level of five SCID-fe mice inoculated with 10(3) TCID50 cell-free FIV was 2.23 mg ml-1. Mean feline IgG level of five mice inoculated with the same quantity of FIV and treated with AZT beginning 1 day prior to virus inoculation and continuing for 2 weeks thereafter was 14.98 mg ml-1. AZT significantly (P < 0.05) enhanced feline humoral immune function at a virus inoculum titer of 10(3) TCID50.(ABSTRACT TRUNCATED AT 400 WORDS)

Feline lymphoid tissues engrafted into scid mice maintain morphologic structure and produce feline immunoglobulin.

To adapt the feline immune system to a small laboratory animal host, severe combined immunodeficient (scid) mice were engrafted with neonatal feline lymphoid tissues, including lymph node, thymus, spleen, and bone marrow. Lymph node and thymus tissue were implanted subcutaneously within the mammary fat pad, and a single-cell suspension of spleen, thymus, and bone marrow was inoculated intraperitoneally (IP). Seven groups of mice (three mice per group) were engrafted on day 0, and members of one group were euthanatized weekly from 2 to 8 weeks after engraftment. For each mouse, graft morphology was evaluated by light microscopy, feline DNA was detected in peripheral blood by polymerase chain reaction (PCR) amplification of feline-specific DNA sequences, and serum IgG concentration was measured by ELISA. Ten of 13 feline grafts evaluated histologically between 3 and 8 weeks after engraftment contained large focal aggregates of lymphocytes bordered by plasma cells. Of 14 thymus grafts evaluated histologically during the same period, 5 were characterized by dense accumulations of small lymphocytes surrounding thymic epithelial cells. Two of these thymus grafts were indistinguishable from age-matched feline thymus. At 2 weeks after engraftment, feline lymph node and thymus contained extensive central necrosis bordered by a narrow zone of lymphocytes and small-caliber blood vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplantation of T cell-mediated, lymphoreticular disease from the scurfy (sf) mouse.

The X-linked mutation, scurfy (sf), causes a fatal lymphoreticular disease characterized by runting, lymphadenopathy, splenomegaly, hypergammaglobulinemia, exfoliative dermatitis, Coombs'-positive anemia, and death by 24 days of age. T lymphocytes are required to mediate this syndrome as shown by a total absence of disease in mice bred to be scurfy and nude (sf/Y; nu/nu). The scurfy phenotype is not transmitted by sf/Y bone marrow transplants, though cells of scurfy origin do reconstitute all lymphoid organs in the recipient mouse. These data suggest that scurfy disease results from an abnormal T cell development process and not from an intrinsic stem cell defect. We therefore tested the ability of transplanted scurfy thymuses to transmit scurfy disease to congenic euthymic mice, to athymic (nude) mice, and to severe combined immunodeficiency (SCID) mice. Euthymic recipients of sf/Y thymic grafts remained clinically normal as did all SCID and nude recipients of normal thymus transplants. Morphological lesions similar to those found in scurfy mice occurred in all H-2-compatible nude and SCID recipients of sf/Y thymic grafts. Intraperitoneal injections of scurfy thymocytes, splenocytes, and lymph node cells also transmitted the scurfy phenotype to H-2-compatible nude mice and SCID mice. Our findings indicate that scurfy disease can be transmitted to T cell-deficient mice by engraftment of scurfy T cells, but that pathogenic scurfy T cell activities can be inhibited (or prevented) in immunocompetent recipient mice.

Human hematolymphoid cells in SCID mice.

The severe combined immunodeficient C.B.-17 scid/scid (SCID) mouse has been widely used to study the normal processes of murine lymphoid differentiation. To create an in vivo model of the human hematolymphoid system, this mouse strain has been engrafted with human organ systems (the SCID-hu mouse) or with human peripheral blood mononuclear cells (the hu-PBL-SCID mouse). These mouse models have now been characterized and used to analyze human infectious diseases, hematopoiesis, malignancies and vaccines.

Phenotypic progression of a rat lymphoid cell line immortalized by human T-lymphotropic virus type I to induce lymphoma/leukemia-like disease in rats.

Rat lymphoid cells, TARS-1, immortalized by coculture with adult T-cell leukemia cells, were intraperitoneally injected into 65 newborn, inbred WKAH/Hkm rats. In most of the rats, tumor nodules were discernible 7 to 15 days after transplantation but were completely rejected within 5 to 6 weeks. Two rats with no tumor nodules exhibited gait disturbances and paralysis of the hind legs 3 to 4 weeks after transplantation. Histological and hematological examinations revealed that a lymphoma/leukemia-like disease had developed in one of the two rats, and the T-lymphoid cell line WLeuk-1 was established from peripheral blood mononuclear cells from this rat. When the WLeuk-1 cells were transplanted into newborn WKAH/Hkm rats, the animals died of a lymphoma/leukemia-like disease within several weeks after transplantation, in contrast to their rejection of the TARS-1 cells. Southern blot and karyotype analyses revealed that WLeuk-1 cells had retained the marker chromosomes and human T-lymphotropic virus type I (HTLV-I) integration patterns of the parent cell line, TARS-1. The additional specific chromosome abnormalities 3p+,t (12;13), and Xq+ were found in the WLeuk-1 cells. Moreover, the expression of HTLV-I structural proteins was slightly depressed in WLeuk-1 cells, while that of the transacting factors p40tax and p21x, but not that of p27rex, was enhanced about fivefold compared with that in TARS-1. The transactivating function of p40tax was intact in WLeuk-1, as evidenced by enhanced interleukin-2 receptor alpha chain expression. These results suggest that aberrant expression of HTLV-I regulatory genes and alteration of cellular genes were associated with the phenotypic progression of the WLeuk-1 cell line.

Presence of transplantable T-lymphoid cells in C57BL/6 mice infected with murine AIDS virus.

The Duplan strain of murine leukemia virus induces murine AIDS in C57BL/6 mice. When spleen cells from C57BL/6 mice infected with the virus were transplanted into nude mice, subcutaneous solid tumors at the transplanted sites were formed and splenomegaly and lymphadenopathy were induced. These transplantable cells were Thy-1- CD4+ alpha-beta T-cell receptor-positive T cells and integrated with the pathogenic defective viral genome. These results indicate that neoplastic cells of T-cell lineage were induced by infecting C57BL/6 mice with murine AIDS virus.

Permanent acceptance of liver allografts by intraportal injection of donor spleen cells in rats.

Antigen pretreatment through the oral or intraportal route has been reported to suppress antibody formation or delayed-type hypersensitivity (DTH) response to the same antigens. However, this effect on allografted organs has not been well studied. In this study we evaluated the efficacy for suppression of antibody formation and DTH response to the allogeneic antigens and tried to prolong liver allograft survival in rats. Male ACI (RT1a) rats were used as donors and male BUF (RT1b) rats as recipients. Intraportal or intravenous injection of spleen cells (SPCs) (5 x 10(7)) was performed through the mesenteric vein or the tail vein, respectively. The anti-ACI DTH response was tested by ear challenge of ACI SPCs. Cytotoxic antibody was assessed by complement-dependent cytotoxicity assay. ACI rat liver was transplanted orthotopically to BUF rat by the cuff technique 10 days after SPC injection. Cytotoxic antibody titer rose to X2(6) to X2(8) at 7 to 10 days after intravenous injection of SPCs. However, intraportal injection of the cells rarely caused a rise in antibody titer and even strongly suppressed subsequent antibody formation induced by intravenous injection when given 10 days before. The DTH response was also suppressed by intraportal injection of SPCs, with a mean value of 0.18 +/- 0.13 mm versus 0.67 +/- 0.19 mm for controls or 0.46 +/- 0.04 mm with intravenous injection. Liver-allografted rats died between 9 and 11 days, averaging 10.1 +/- 0.7 days in the control group. All seven transplants injected intraportally with donor SPCs survived more than 100 days, whereas six of eight rats injected intravenously with donor SPCs died of bleeding from the surface of the liver grafts within 12 hours after grafting, with signs similar to those of hyperacute rejection. Four of five rats injected intraportally with F344 (third-party) SPCs died of acute rejection in the same way the control rats died. The liver allograft-bearing rats had permanently accepted ACI skin grafts when tested 60 days after liver transplantation but rejected F344 skin grafts in the normal fashion. Intraportal injection of donor SPCs markedly suppressed the antibody formation, as well as the DTH response, and completely blocked liver allograft rejection. Moreover, the liver allograft-bearing rats proved to be fully tolerant of the donor antigen. This method might be a promising modality for inducing donor-specific tolerance in liver transplantation.

Replacement of donor lymphoid tissue in small-bowel transplants.

The presence of recipient lymphocytes in grafts is thought to equate with rejection. Thus, we wished to follow the fate of lymphocytes after transplant of the small bowel. Three complete small-bowel transplants, two with the liver from the same donor also transplanted, were done successfully. Patients were immunosuppressed with FK 506. 5 to 11% of lymphocytes in the recipients' peripheral blood were of donor origin during the early postoperative period when there were no clinical signs of graft-versus-host disease. However, donor cells were no longer detectable after 12 to 54 days. Serial biopsy specimens of the grafted small bowel showed progressive replacement of lymphocytes in the lamina propria by those of the recipient's HLA phenotype. Lymphoid repopulation was complete after 10 to 12 weeks but the epithelial cells of the intestine remained those of the donor. The patients are on enteral alimentation after 5, 6, and 8 months with histopathologically normal or nearly normal intestines. Re-examination of assumptions about the rejection of intestinal grafts and strategies for its prevention are required following these observations.

The SCID-hu mouse: a small animal model for HIV infection and pathogenesis.

The SCID-hu mouse is a heterochimeric small animal model designed to support hematopoietic differentiation and function in vivo. Multiple organs of the human hematolymphoid system have been successfully engrafted into the immunodeficient C.B-17 scid scid mouse, including fetal liver, thymus, lymph node, and skin. Co-implantation of human fetal liver and human fetal thymus results in long-term, multilineage human hematopoiesis in vivo. Mature human lymphocytes within the SCID-hu mouse are phenotypically and functionally normal. HIV infection of the SCID-hu mouse reflects a tropism similar to that found in humans: only human organs with CD4+ cells are infected. Viral replication can thereafter be monitored with assays that are safe, reproducible, and quantitative. Given this small animal model, it is now possible to study systematically the infective process of HIV and to address questions about the efficacy of novel antiviral compounds or vaccines in vivo.

Location of hemopoietic stem cells influences frequency of lymphoid engraftment in Xenopus embryos.

The first hemopoietic stem cells to differentiate in Xenopus embryos arise from ventral blood island (VBI) mesoderm. Progeny of these stem cells contribute to larval E, macrophage, thymocyte, and B lymphocyte populations. When small pieces of mesoderm are transplanted to a central location within the VBI, the contribution of this mesoderm is predominantly to erythropoiesis and engraftment of lymphoid populations is minimal. The present experiments examined the influence of position within the VBI on the contribution of single stem cells to lymphoid populations. Pieces of diploid VBI mesoderm, containing an average of one hemopoietic stem cell, were transplanted to either a central or a peripheral location within the defined boundaries of the VBI of triploid, stage matched embryos. The number of animals with donor-derived cells in lymphoid populations was markedly increased when stem cells were grafted to a peripheral position. In three cases, stem cells contributed to lymphoid populations at the exclusion of erythroid populations. These data were consistent with the notion of either a lymphoid stem cell or restricted B and T lymphocyte precursors. These data also suggested that during embryogenesis, stochastic differentiation of hemopoietic stem cells was influenced by regional differences in the VBI microenvironment.

An analysis of pulmonary natural killer cell activity in F1-hybrid mice with acute graft-versus-host reactions.

The kinetics of activation, cell surface phenotype, target cell specificity and anatomic localization of pulmonary natural killer cells was examined in (C57BL/6 X A/J)F1-hybrid mice with acute graft-versus-host reactions induced by intravenous injection of 50 x 10(6) parental strain lymph node and spleen cells. Results showed that there was a marked increase in NK cell activity directed against YAC-1 tumor cells. This activity remained elevated in the lung over almost the entire course of the reaction, whereas it was only transiently increased in the spleen during the early stages of the reaction and then fell to control values. During the reaction, NK cells from both organs acquired the ability to kill P815 targets, cells that are normally insensitive to NK cell lysis. The level of P815 killing never reached that achieved against YAC-1 cells, but was significantly higher in the lung than in the spleen. Antibody and complement depletion experiments showed that both anti-YAC-1 and anti-P815 activity could be depleted with antiserum to the asialo-GM1 cell surface marker, but was unaffected by anti-Lyt-1.2 and anti-Lyt-2.2 treatment. Anti-YAC-1 activity was partly sensitive to depletion with anti-Thy-1.2. Cytotoxicity to P815 target cells acquired during the reaction was completely abrogated by anti-Thy-1.2. These findings suggest that during the reaction two phenotypically distinct types of NK cells are activated: a conventional, Thy-1-negative cell that kills only YAC-1 targets, and a Thy-1-positive cell with a broadened spectrum of lytic activity. We suggest that the latter may be generated in response to interleukin-2 released during the lymphoproliferative phase of the reaction and may represent a type of lymphokine-activated killer cell. Our results revealed that virtually all of the NK cell activity in the lung could be attributed to cells residing in the interstitium, or to cells tightly adherent to endothelium or epithelium. There was no correlation between augmented NK cell activity in the lung and the presence of peribronchial and perivascular mononuclear cell infiltrates seen in histological sections of the lung. These findings do not appear to support the idea that NK cells are by themselves directly responsible for the pathological changes produced by the reaction.