Engraftment Of Lymphoid Cells Research Articles

Ebola Virus Replication and Disease Without Immunopathology in Mice Expressing Transgenes to Support Human Myeloid and Lymphoid Cell Engraftment.

The study of Ebola virus (EBOV) pathogenesis in vivo has been limited to nonhuman primate models or use of an adapted virus to cause disease in rodent models. Herein we describe wild-type EBOV (Makona variant) infection of mice engrafted with human hematopoietic CD34+ stem cells (Hu-NSG™-SGM3 mice; hereafter referred to as SGM3 HuMice). SGM3 HuMice support increased development of myeloid immune cells, which are primary EBOV targets. In SGM3 HuMice, EBOV replicated to high levels, and disease was observed following either intraperitoneal or intramuscular inoculation. Despite the high levels of viral antigen and inflammatory cell infiltration in the liver, the characteristic histopathology of Ebola virus disease was not observed, and this absence of severe immunopathology may have contributed to the recovery and survival of some of the animals. Future investigations into the underlying mechanisms of the atypical disease presentation in SGM3 HuMice will provide additional insights into the immunopathogenesis of severe EBOV disease.

A Radio-Resistant Perforin-Expressing Lymphoid Population Controls Allogeneic T Cell Engraftment, Activation and Onset of Gvhd

IntroductionImmunosuppressive pre-transplantation conditioning is essential for donor cell engraftment in allogeneic bone marrow transplant (BMT). The role of residual post-conditioning recipient immunity in determining engraftment is poorly understood. Although recipient perforin has previously been shown to modulate myeloid engraftment from mouse bone marrow, and adoptive transfer of natural killer (NK) cells can ameliorate the onset of graft-versus host disease (GVHD), the role of perforin-expressing endogenous recipient NK and NKT cells in modulating donor T cell engraftment has not been described. Using an MHC-mismatched mouse model, we examined the role of perforin-expressing NK cells in regulating both the rate and activation status of donor lymphocyte engraftment after allogeneic transplantation.MethodsAn MHC-disparate model of BMT was established transplanting BALB/c donor BM (H-2Kd), into female 6-14 week old C57BL/6 WT and C57BL/6.perforin KO (H-2Kb) recipients. On day 0, recipient mice were administered a split dose of lethal radiation using a caesium source (2 x 6 gray), and injected i.v. with 5e6 T cell depleted BM (TCD-BM) cells from MHC-mismatched or syngeneic donors. On day 2, recipient mice were injected i.v. with 1e6 BALB/c purified splenic T cells at a 2:1 CD4+:CD8+ T cell ratio. Mice were monitored daily, and killed at selected time points post-transplant (typically day 5-7 for short term engraftment, or day 20 for long term engraftment) to examine donor lymphoid cell engraftment.ResultsAn HLA-mismatched BMT mouse model demonstrated that both the rate and proportion of donor lymphoid cell engraftment, and expansion of effector memory donor T cells in both spleen and BM were significantly increased within 5-7 days post BMT in perforin-deficient (pfn-/-) recipients, compared with wild-type (WT). Critically, we found that the absence of perforin resulted in the increased production of pro-inflammatory cytokines, in particular IL-6, from engrafting donor T cells. IL-6 has recently been identified as a primary driver of both mouse model and clinical GVHD. Correlating with pro-inflammatory cytokine secretion, effector memory donor T lymphocytes expanded more rapidly in pfn-/- recipients than in WT mice, possibly arising from more rapid proliferation or selective survival of these donor cells. In WT recipients, donor lymphoid cell engraftment was enhanced to that seen in pfn-/- recipients, by depleting NK cells prior to BMT, demonstrating that a perforin-dependent, NK-mediated host-versus graft effect limits the rate of donor cell engraftment and T cell activation. We found that radiation-resistant NKT cells survived in the BM of lethally irradiated mice and may drive NK cell activation, resulting in the host-versus graft effect. Furthermore, reduced pre-transplant irradiation doses in pfn-/- recipients permitted long-term donor lymphoid cell engraftment and improved survival (Figure 1).Figure 1: Reduced total body irradiation in perforin-deficient mice permits rapid donor lymphoid cell engraftment. On day 0, WT or pfn-/- (KO) mice were irradiated with variable doses (12-6 gray), and injected i.v. with 5e6 TCD-BM cells from BALB/c donors. On day 2, recipient mice were injected i.v. with 1e6 splenic BALB/c T cells. On day 7 (A) and day 20 (B) after BMT, the BM cells of WT or KO mice were stained for H-2Kd (donor cells) and engraftment determined as a percentage of donor lymphocytes. (C) Survival of KO mice administered 9 gray (closed circles), 7.5 gray (triangles) and 6 gray (lines) after BMT described above. [Display omitted] [Display omitted] [Display omitted] ConclusionsThe challenge in clinical HSCT is to promote reliable donor engraftment and full immunological reconstitution whilst minimizing pre-transplant conditioning and associated toxicity. Our findings suggest that suppression of perforin activity or selective depletion of recipient NK cells prior to BMT may allow the combined advantage of reduced transplant toxicity whilst maintaining donor engraftment and promoting the graft versus tumour effect. DisclosuresNo relevant conflicts of interest to declare.

A Radio-Resistant Perforin-Expressing Lymphoid Population Controls Allogeneic T Cell Engraftment, Activation, and Onset of Graft-versus-Host Disease in Mice

Immunosuppressive pretransplantation conditioning is essential for donor cell engraftment in allogeneic bone marrow transplantation (BMT). The role of residual postconditioning recipient immunity in determining engraftment is poorly understood. We examined the role of recipient perforin in the kinetics of donor cell engraftment. MHC-mismatched BMT mouse models demonstrated that both the rate and proportion of donor lymphoid cell engraftment and expansion of effector memory donor T cells in both spleen and BM were significantly increased within 5 to 7 days post-BMT in perforin-deficient (pfn−/−) recipients, compared with wild-type. In wild-type recipients, depletion of natural killer (NK) cells before BMT enhanced donor lymphoid cell engraftment to that seen in pfn−/− recipients. This demonstrated that a perforin-dependent, NK-mediated, host-versus-graft (HVG) effect limits the rate of donor engraftment and T cell activation. Radiation-resistant natural killer T (NKT) cells survived in the BM of lethally irradiated mice and may drive NK cell activation, resulting in the HVG effect. Furthermore, reduced pretransplant irradiation doses in pfn−/− recipients permitted long-term donor lymphoid cell engraftment. These findings suggest that suppression of perforin activity or selective depletion of recipient NK cells before BMT could be used to improve donor stem cell engraftment, in turn allowing for the reduction of pretransplant conditioning.

High level functional engraftment of severe combined immunodeficient mice with human peripheral blood lymphocytes following pretreatment with radiation and anti-asialo G M1

The severe combined immunodeficient (SCID) mouse engrafted with human peripheral blood lymphocytes (PBLs) is a potentially useful model for the study of cancer immunotherapy. For this application, rapid, consistent, and high level engraftment of SCID mice with functional human cytotoxic effector cells is necessary. To date, short term human lymphoid cell engraftment in SCID mice has generally been low and variable. Further, most of the human cells detected within the first 30 days are found in the peritoneal cavity. The purpose of the present study was to improve short term reconstitution of human PBLs in the SCID mouse. When untreated SCID mice were injected with human PBLs, the mean level of CD3 + cells in the spleens was < 5% on days 6–32 after injection, as determined by flow cytometry (FCM). Depletion of SCID mouse natural killer (NK) cells with anti-asialo G M1 only marginally improved short term reconstitution with human CD3 + cells. Preirradiation of SCID mice with 3 Gy improved reconstitution to over 16% CD3 + cells on days 12–14 following engraftment. However, the combination of pretreatment with anti-asialo G M1 plus radiation, significantly increased the mean percentage of human CD3 + cells in the spleen to 40% within 2 weeks following injection of PBLs. Human T cells positive for CD4, CD8, TcRαβ, and TcRγδ, and human NK and B cells were detected in the spleens of irradiated plus anti-asialo G M1 pretreated SCID mice. The presence of human lymphoid cells was confirmed by immunohistologic staining. The human immune cells in these mice were shown to be functional by the in vivo demonstration of an appropriate secondary immune response to the injection of tetannus toxoid and by an in vivo proliferative response to phytohemagglutinin. Human NK cells could be found in the spleens and peripheral blood of irradiated plus anti-asialo G M1 pretreated mice. These cells were also shown to be competent by their ability to lyse the human NK sensitive tumor targets K562 and MOLT-4 in 51Cr release assays. Thus, pretreatment of SCID mice with radiation plus anti-asialo G M1 significantly improves short term human PBL engraftment and provides a potentially useful model for the study of cancer immunotherapy.

Engraftment of human lymphoid cells into newborn SCID mice leads to graft-versus-host disease.

Although there has been considerable progress in transplanting normal human hematopoietic cells into immune-deficient mice, the establishment of a functional human immune system has proved to be difficult. Current methods of introducing mature human lymphoid cells into adult SCID mice lead to oligoclonal engraftment with restricted dissemination to various organs. We have attempted to improve human lymphoid cell engraftment in mice, both qualitatively and quantitatively, by injecting human bone marrow cells and peripheral blood leukocytes intraperitoneally into newborn SCID mice. Newborn mice were used as recipients because certain immune functions such as natural killer cell activity do not develop until several weeks after birth and the numerous growth factors secreted in young mice may facilitate the engraftment and proliferation of transplanted human cells. At various times after transplantation, the presence of human cells in different organs was determined by Southern blot analysis using a human specific probe. Within 4 weeks, 70% of the mice were engrafted with human cells. Human cell engraftment of the bone marrow, spleen, lungs, kidneys, liver, and thymus exceeded 10% in at least 40% of the transplanted mice; most of these highly engrafted mice were sick. Flow cytometry and immunocytochemistry indicated these organs were heavily infiltrated with mature T and B lymphocytes. Histologic and molecular analysis showed massive human cell infiltrates within the liver, lung and spleen. The presence of human IgG and IgM antibodies against mouse red blood cells provided evidence that the engrafted human cells retained some immune function. Mice transplanted with peripheral blood leukocytes from donors that were allergic to mouse antigens engrafted to the same extent as normal cells but in addition developed the classical symptoms of acute allogeneic graft-versus-host disease (GVHD) including infiltrates of the skin, gut, and liver. The newborn SCID system provides a new in vivo model to study human xenoreactivity and GVHD.

Lymphoid chimerism after allogeneic bone marrow transplantation. Y-chromatin staining of peripheral T and B lymphocytes and allotyping of serum immunoglobulins.

Lymphoid cell engraftment was monitored for several years after bone marrow transplantation by Y-chromatin staining of T and B lymphocytes in the peripheral blood and/or by immunoglobulin allotyping in the serum of 20 of 52 pediatric patients grafted successively between October 1973 and October 1983. Data on 2 patients with severe combined immunodeficiency, grafted earlier in December 1968 and April 1971, are also included. These children received an allogeneic bone marrow graft for leukemia (n = 7), severe aplastic anemia (n = 11), or severe combined immunodeficiency (n = 4) and were informative for this study, because they differed from their donor by sex (n = 16) and/or by immunoglobulin phenotype (n = 13). Of 16 pairs in which the donor was of the opposite sex, 11 patients ultimately showed circulating T and B lymphocytes of donor origin after bone marrow transplantation; in the remaining 5, there was an incomplete chimerism of the circulating lymphoid cells. Of 13 pairs with a difference in immunoglobulin phenotype between donor and recipient, 8 patients exhibited donor allotypes 3 months or later after transplantation, in 3 of them together with recipient allotypes. In the remaining 5 patients, recipient allotypes were detected after transplantation, but the simultaneous presence of donor-type immunoglobulin production could not be excluded in 4. The persistence of either a split (T lineage of donor origin and B lineage of recipient origin) or mixed (T and/or B lineage of donor and recipient origin) chimerism was related to the type of disease. In 3 children circulating B cells of donor-origin did not fit with the recipient origin of the sessile immunoglobulin-secreting plasma cells. This implies that different immune compartments--e.g., bone marrow and peripheral lymphoid tissues--should be investigated following allogeneic bone marrow transplantation. A prolonged presence of recipient-type lymphoid cells increased the risk of leukemic relapse in the patients investigated.

Bone marrow transplantation only partially restores purine metabolites to normal in adenosine deaminase-deficient patients.

To delineate the extent to which bone marrow transplantation provides "enzyme replacement therapy", we have determined metabolite concentrations in two patients with adenosine deaminase (ADA) deficiency treated with bone marrow transplants and rendered immunologically normal. 10 yr after engraftment of lymphoid cells, erythrocyte deoxy ATP was markedly decreased compared to the marked elevations of deoxy ATP observed in untreated patients, but was still significantly elevated (62 and 90 vs. normal of 6.0 +/- 6.0 nmol/ml packed erythrocytes). Similarly, deoxyadenosine and adenosine excretion were both markedly diminished compared to that of untreated patients but deoxyadenosine excretion was still clearly increased (20.1 and 38.6 vs. normal of less than 0.2 nmol/mg creatinine) while adenosine excretion was in the upper range of normal (7.0 and 8.1 vs. normal of 5.6 +/- 3.6 nmol/mg creatinine). Mononuclear cell deoxy ATP content was also elevated compared to normal (5.25 and 14.4 vs. 1.2 +/- 0.3). Separated mononuclear cells of bone marrow transplanted patients contain both donor lymphocytes and recipient monocytes. When mononuclear cells were depleted of the cells enriched for donor lymphocytes (i.e. monocyte depleted) was lower than that of the mixed mononuclear cells (2.2 vs. 5.26). Surprisingly, plasma adenosine was as high as in untreated ADA-deficient patients (3.2 and 1.5 vs. untreated of 0.3-3 microM). Consistent with the elevated plasma adenosine and urinary deoxyadenosine, erythrocyte S-adenosyl homocysteine hydrolase activity was diminished (0.88 and 1.02 vs. normal of 5.64 +/- 0.25). Thus, bone marrow transplantation of ADA-deficient patients not only provides lymphoid stem cells, but also partially, albeit incompletely, clears abnormally increased metabolites from nonlymphoid body compartments.