Severe Combined Immune Deficiency Recipients Research Articles

Engraftment of Severe Combined Immune Deficient Mice Receiving Allogeneic Bone Marrow Via In Utero or Postnatal Transfer

Although in utero transplantation (IUT) has been shown to be effective in treating human severe combined immune deficiency (SCID), the relative merit of IUT as compared with postnatal bone marrow transplantation (BMT) for SCID is unknown. Therefore, comparative studies were undertaken in mice to determine the engraftment outcome in these two settings. Because T-cell depletion (TCD) reduces graft-versus-host disease (GVHD) severity but compromises alloengraftment, studies were performed with TCD or non-TCD BM and GVHD risk was assessed using a tissue scoring system and by the adoptive transfer of splenocytes from engrafted mice into secondary recipients. Non-SCID recipients received pre-BMT irradiation to simulate those circumstances in which conditioning is required for alloengraftment. IUT recipients of non-TCD and especially TCD BM cells in general had higher levels of donor T-cell and myeloid peripheral blood (PB) engraftment than nonconditioned SCID recipients. Increased TCD or non-TCD BM cell numbers in adult SCID recipients resulted in similar levels of PB engraftment as IUT recipients. However, under these conditions, mean GVHD scores were higher than in IUT recipients. The majority of adoptive transfer recipients of splenocytes from IUT recipients were GVHD-free, consistent with the in vitro evidence of tolerance to host alloantigens. Total body irradiation (TBI)-treated mice that had the highest engraftment had evidence of thymic damage as denoted by a higher proportion of thymic and splenic T cells with a memory phenotype as compared with IUT recipients. IUT mice had vigorous thymic reconstitution by 3 weeks of age. Our data indicate that IUT has a number of advantages as compared with postnatal BMT. Future studies examining the fine specificity of immunoreconstitution in IUT versus postnatal BMT are indicated.

Engraftment of Severe Combined Immune Deficient Mice Receiving Allogeneic Bone Marrow Via In Utero or Postnatal Transfer

Although in utero transplantation (IUT) has been shown to be effective in treating human severe combined immune deficiency (SCID), the relative merit of IUT as compared with postnatal bone marrow transplantation (BMT) for SCID is unknown. Therefore, comparative studies were undertaken in mice to determine the engraftment outcome in these two settings. Because T-cell depletion (TCD) reduces graft-versus-host disease (GVHD) severity but compromises alloengraftment, studies were performed with TCD or non-TCD BM and GVHD risk was assessed using a tissue scoring system and by the adoptive transfer of splenocytes from engrafted mice into secondary recipients. Non-SCID recipients received pre-BMT irradiation to simulate those circumstances in which conditioning is required for alloengraftment. IUT recipients of non-TCD and especially TCD BM cells in general had higher levels of donor T-cell and myeloid peripheral blood (PB) engraftment than nonconditioned SCID recipients. Increased TCD or non-TCD BM cell numbers in adult SCID recipients resulted in similar levels of PB engraftment as IUT recipients. However, under these conditions, mean GVHD scores were higher than in IUT recipients. The majority of adoptive transfer recipients of splenocytes from IUT recipients were GVHD-free, consistent with the in vitro evidence of tolerance to host alloantigens. Total body irradiation (TBI)-treated mice that had the highest engraftment had evidence of thymic damage as denoted by a higher proportion of thymic and splenic T cells with a memory phenotype as compared with IUT recipients. IUT mice had vigorous thymic reconstitution by 3 weeks of age. Our data indicate that IUT has a number of advantages as compared with postnatal BMT. Future studies examining the fine specificity of immunoreconstitution in IUT versus postnatal BMT are indicated.

Donor‐specific growth factors promote swine hematopoiesis in severe combined immune deficient mice

Abstract: Induction of a state of mixed hematopoietic chimerism following bone marrow transplantation is associated with donor‐specific tolerance in the concordant xenogeneic rat‐to‐mouse species combination. We are now attempting to induce such tolerance in a discordant species combination, pig to mouse. Our initial studies showed that non‐immune physiologic factors limited the level of swine hematopoietic reconstitution in severe combined immune deficient (SCID) mice. We have now examined the ability of swine‐specific growth factors, interleukin‐3 (IL‐3), granulocyte macrophage‐colony stimulating factor (GM‐CSF), and stem cell factor (SCF) to enhance repopulation by swine bone marrow cells in SCID recipients. The results indicate that swine IL‐3 promotes pig hematopoiesis in SCID mouse recipients. The percentage of swine class I+ cells in bone marrow, spleen, and peripheral blood was markedly increased by a 3‐week treatment course with porcine IL‐3. In longer‐term studies, the effect of IL‐3 was further enhanced by combining it with porcine GM‐CSF. Almost all repopulating porcine cells expressed a swine myeloid marker. Colony‐forming assays showed a correlation of the number of pig‐specific colonies with the number of swine cells detected by flow cytometry in transplanted‐SCID bone marrow recipients. Porcine CD2+ cells which did not express CD4 or CDS coreceptors were also detected in SCID mouse recipients of pig bone marrow, and their numbers were also increased by swine cytokine treatment. Swine IgG, but not B cells were detected in SCID recipients at 3 and 6 weeks following bone marrow transplantation, and declined over time, suggesting that mature B cells engrafted, but that de novo B lymphopoiesis did not occur in these mice. Thus, our study demonstrates that donor‐specific growth factors can help to overcome the physiologic barrier to xenogeneic hematopoiesis in the discordant pig to mouse species combination.

T helper cell-dependent, microbial superantigen-mediated B cell activation in vivo.

We have utilized a severe combined immune-deficient (SCID) mouse adoptive transfer model to explore the in vivo immunostimulatory effects of bacterial superantigens (SAg). B cell reconstituted SCID recipients were treated with the Staphylococcus aureus-derived toxic shock syndrome toxin (TSST-1) alone or in conjunction with syngeneic L3T4+ TSST-1-reactive Th cells. Over several months of study, the repetitive administration of TSST-1 resulted in a prompt, transient increase in serum IgG levels. This response required both biologically active TSST-1 and Th cells. These findings demonstrate that certain bacterial SAgs can promote Th cell-dependent B cell activation and differentiation in vivo. These studies strengthen the analogy between SAg-mediated and allospecific Th-B cell interactions responsible for the autoimmune sequelae of graft-versus-host disease.

Antibodies to CD40 Prevent Epstein-Barr Virus-Mediated Human B-Cell Lymphomagenesis in Severe Combined Immune Deficient Mice Given Human Peripheral Blood Lymphocytes

CD40 is expressed on both normal and neoplastic B lymphocytes. Signal transduction through CD40 in vitro has been shown to exert stimulatory effects on normal B cells and inhibitory effects on Epstein-Barr virus (EBV)-induced B-cell lymphoma lines and some other cell lines derived from patients with aggressive histology lymphoma. The transfer of normal human peripheral blood lymphocytes (huPBL) from EBV-seropositive donors into severe combined immune deficient (SCID) mice has been previously shown to result in the generation of human B-cell lymphomas. These tumors are similar to the highly aggressive EBV-induced lymphomas that can arise clinically after transplantation or in the setting of immunodeficiency. Treatment of huPBL-SCID chimeric mice with anti-CD40 or anti-CD20 monoclonal antibodies (MoAb) significantly delayed the development of EBV-induced B-cell lymphoma. However, the effects of the two MoAb were mechanistically distinct. Anti-CD40 treatment prevented lymphoma generation, while still allowing for functional human B-cell engraftment in the huPBL-SCID mice compared with mice receiving no treatment, all of which succumbed to lymphoma. By contrast, treatment with anti-CD20 significantly inhibited total human B-cell engraftment in the SCID recipients, which accounted for the absence of lymphomas. In vitro assays examining the transformation of human B cells by EBV also indicated that anti-CD40 could directly inhibit EBV-transformation, whereas anti-CD20 antibodies had no effect. Thus, anti-CD40 exerts selective effects to allow for the engraftment of normal human B cells and prevent the emergence of EBV lymphomas. Stimulation of CD40 by antibodies or its physiologic ligand may, therefore, be of significant clinical use in the prevention of EBV-induced B lymphomas that may arise when EBV-seropositive individuals receive immunosuppressive regimens after transplantation or in immune deficiency states, such as acquired immune deficiency syndrome.

Human/Mouse Radiation Chimera Are Capable of Mounting a Human Primary Humoral Response

Lubin et al recently described a new approach that enables the generation of human/mouse chimera by adoptive transfer of human peripheral blood mononuclear cells (PBMC) into lethally irradiated normal strains of mice, radioprotected with bone marrow (BM) from donors with severe combined immune deficiency (SCID). In the present study, we demonstrate in such human/mouse chimera a marked humoral response to recall antigen, such as tetanus toxoid (TT) or hepatitis B surface antigen (HBsAg), as well as a significant primary response to keyhole limpet hemocyanin (KLH). Maximal anti-KLH response in human/Balb chimera was attained 2 to 4 weeks after the immunization and declined thereafter. One week after transplantation, the predominant anti-KLH subtype was IgM, while after 2 weeks, the dominance had shifted to IgG. Similar primary antibody response was also demonstrated against the human immunodeficiency virus (HIV) Nef protein. Comparison between human/Balb and human/SCID chimera showed a major difference in their ability to mount a primary response against KLH. In Balb/c recipients, more than half of the mice exhibited marked IgM titers against KLH, while there was hardly any anti-KLH IgM response in the SCID recipients. From the earliest time point onwards, when anti-KLH antibodies were found in the latter chimera, they were predominantly of the IgG type. We have previously shown that in human/Balb chimera, unlike in SCID recipients, dissemination of transplanted PBMC into the spleen and other internal organs occurs within 24 hours. Therefore, it is likely that the early seeding in the appropriate microenvironment of the lymphoid tissues, is crucial for the maintenance of virgin human B cells.

Engraftment and activity of anti-CD3-activated human peripheral blood lymphocytes transferred into mice with severe combined immune deficiency.

Human PBL (huPBL) were activated with anti-CD3 mAb in vitro and then were transferred into mice with severe combined immune deficiency (SCID) to determine the effect of activation on engraftment and to determine if the engrafted human cells could provide antitumor effects in mice. Some mice were also treated with human rIL-2 after huPBL transfer. Mice were analyzed 6 to 8 wk after cell transfer and the number of human cells in the peripheral lymphoid organs was determined. Mice receiving anti-CD3-activated huPBL demonstrated a significant increase in the incidence of human T cell engraftment in the periphery as assessed by flow cytometry. Human cells were also detected in the murine thymus after anti-CD3 stimulation indicating that human T cells can migrate to the murine thymus provided that they are activated before the transfer. The transfer of anti-CD3-activated huPBL also resulted in an xenogeneic graft-vs-host reaction manifested primarily by proliferation of murine splenic hemopoietic cells. When SCID mice received the human colon carcinoma HT29, the concurrent transfer of anti-CD3-activated human cells resulted in a significant increase in survival. However, the human cells displayed low cytolytic activity toward human tumor targets when they were recovered from the lymphoid organs of the SCID recipients. Supernatants from the anti-CD3-activated cells were able to inhibit the growth of HT29 in vitro, partly because of the presence of IFN-gamma, suggesting that the human T cells are producing cytokines in vivo that have antitumor effects. Thus, the use of anti-CD3-activated huPBL in SCID mice may be of value for optimizing human cell engraftment in the human/mouse lymphoid chimeras and may be used to evaluate potential anti-neoplastic therapies that employ human effector cells.

Human-mouse lymphoid chimeras: host-vs.-graft and graft-vs.-host reactions.

Human peripheral blood lymphocytes (huPBL) were injected into mice with severe combined immune deficiency (SCID). It was ascertained that murine natural killer (NK) cells were capable of affecting engraftment of human lymphocytes in SCID mice. The presence of host NK cells resulted in the clearance of the human lymphocytes. Human T lymphocytes were the primary cell to engraft in the SCID recipients, with human T cells being detected in the spleen, lymph nodes, bone marrow and peritoneal cavity of the mice up to several months after transfer. No human T cells were detected in the murine thymus and the level of engraftment in the periphery could be highly variable. Additionally, there appeared to be significant reactions between the human lymphocytes and the murine host resulting in a xenogeneic graft-vs.-host reaction (XGVHR). The predominant manifestation involved splenomegaly resulting from an expansion of murine hematopoietic cells in the spleens of these xenogeneic chimeras. The severity of the XGVHR could be correlated with the extent of human T cell engraftment and the recovered human T cells were found to be in a proliferative state. Thus, there appear to be significant host-vs.-graft and graft-vs.-host interactions occurring in human/mouse lymphocyte chimeras.

Human growth hormone promotes engraftment of murine or human T cells in severe combined immunodeficient mice.

Growth hormone (GH) has previously been implicated in T-cell development, and here we test its efficacy in promoting T-lymphocyte engraftment in mice with severe combined immune deficiency (SCID). SCID mice receiving syngeneic thymocytes and treated with recombinant human GH (rhGH) had significantly better T-cell engraftment in their lymph nodes. Human T-cell engraftment was also strongly promoted by rhGH in SCID recipients receiving human peripheral blood lymphocytes. Additionally, although mature human cells have not been thought to enter the murine thymus, human T cells were detected in the SCID thymus after rhGH treatment. Thus, rhGH can be used to optimize long-term peripheral T-cell engraftment in these human-mouse chimeras and may also be useful clinically in treating T-cell deficiencies.