PLoS One. 2014 May 15;9(5):e97860. doi: 10.1371/journal.pone.0097860. eCollection 2014.
Immune humanization of immunodeficient mice using diagnostic bone marrow aspirates from carcinoma patients.
Werner-Klein M1, Proske J1, Werno C2, Schneider K3, Hofmann HS4, Rack B5, Buchholz S6, Ganzer R7, Blana A8, Seelbach-Göbel B9, Nitsche U10, Männel DN1, Klein CA11.
Institute of Immunology, University of Regensburg, Regensburg, Germany.
Project Group Personalized Tumor Therapy, Fraunhofer Institute of Toxicology and Experimental Medicine, Regensburg, Germany.
Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany.
Department of Thoracic Surgery, University Regensburg, Regensburg, Germany.
Department of Gynecology and Obstetrics, Ludwig-Maximilians-Universität München, Munich, Germany.
Department of Gynecology and Obstetrics, University Medical Center Regensburg, Regensburg, Germany.
Department of Urology, University of Leipzig, Leipzig, Germany.
Department of Urology, Fuerth Hospital, Fuerth, Germany.
Clinic of Gynecology and Obstetrics St. Hedwig, University of Regensburg, Regensburg, Germany.
Department of Surgery, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany.
Project Group Personalized Tumor Therapy, Fraunhofer Institute of Toxicology and Experimental Medicine, Regensburg, Germany; Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany.
Tumor xenografts in immunodeficient mice, while routinely used in cancer research, preclude studying interactions of immune and cancer cells or, if humanized by allogeneic immune cells, are of limited use for tumor-immunological questions. Here, we explore a novel way to generate cancer models with an autologous humanized immune system. We demonstrate that hematopoietic stem and progenitor cells (HSPCs) from bone marrow aspirates of non-metastasized carcinoma patients, which are taken at specialized centers for diagnostic purposes, can be used to generate a human immune system in NOD-scid IL2rγ(null) (NSG) and HLA-I expressing NSG mice (NSG-HLA-A2/HHD) comprising both, lymphoid and myeloid cell lineages. Using NSG-HLA-A2/HHD mice, we show that responsive and self-tolerant human T cells develop and human antigen presenting cells can activate human T cells. As critical factors we identified the low potential of bone marrow HSPCs to engraft, generally low HSPC numbers in patient-derived bone marrow samples, cryopreservation and routes of cell administration. We provide here an optimized protocol that uses a minimum number of HSPCs, preselects high-quality bone marrow samples defined by the number of initially isolated leukocytes and intra-femoral or intra-venous injection. In conclusion, the use of diagnostic bone marrow aspirates from non-metastasized carcinoma patients for the immunological humanization of immunodeficient mice is feasible and opens the chance for individualized analyses of anti-tumoral T cell responses.
HSPC engraftment in NSG-mice by intra-femoral or intra-hepatic injections.
Adult or neonatal NSG mice were sublethally irradiated and transplanted either intra-hepatically (neonatals: CB-HSPCs n = 8, 3 donors; BM-HSPCs n = 11 donors) or intra-femorally (adult: BM-HSPCs n = 14, 11 donors) with CD34+ HSPCs. Mice were analyzed at >20 weeks after transplantation by flow cytometry. (A) The number of injected CD34+ HSPCs for each group is given. (B) Engraftment was determined by human CD45 expression. The absolute number of hCD45+ cells in BM and spleen of reconstituted mice is shown. Indicated are values for significant differences between the groups. Bone marrow: CB i.h. vs. BM i.h., p = 0.003; CB i.h. vs. BM i.f., p = 0.004; Spleen: CB i.h. vs. BM i.h., p = 0.04; CB i.h. vs. BM i.f., p<0.0001; ANOVA, Kruskal-Wallis). Each symbol represents an individual animal. (C) The proportion of B cells and myeloid cells in BM and spleen is shown as percentage of human CD45+ cells. Each symbol represents an individual animal. (D) left: The percentage of mice with T cells in spleen at >20 weeks after transplantation is given. right: The absolute number of CD3+ T cells in the spleen of mice with T cells is shown. Due to the limited number of samples in two of the three groups no statistically analysis was performed.
Immune Humanization of Immunodeficient Mice Using Diagnostic Bone Marrow Aspirates from Carcinoma Patients
PLoS One. 2014;9(5):e97860.
Yield of CD34+ HSPCs isolated from cryoconserved and non-cryoconserved BM- MNCs.
(A) BM mononuclear cells from BM-aspirates were isolated by density centrifugation and frozen in liquid nitrogen. After maximally 6 months of storage cells were thawed and counted. (B) CD34+ HSPCs were isolated from BM-MNCs at the day of arrival (immediate), the next day after overnight storage of BM-MNCs at 4°C or from cryoconserved samples. The number of CD34+ cells per 10 million BM-MNCs is shown (p = 0.001 immediate vs. cryo and p = 0.0168 next day vs. cryo; ANOVA Kruskal-Wallis).
NSG mice engrafted with cryoconserved or non-cryoconserved BM-HSPCs by intra-femoral injections.
Adult NSG mice were sublethally irradiated and transplanted with cryoconserved (n = 14, 11 donors) or non-cryoconserved BM-HSPCs (n = 14, 13 donors). At >20 weeks after transplantation mice were analyzed by flow cytometry. (A) The number of injected CD34+ HSPCs for each group is given. As the yield of CD34+ cells is higher from non-cryoconserved BM samples, the respective group received significantly more HSPCs (p = 0.0045, student's t-test). (B) Engraftment in BM and spleen was determined by human CD45 expression. The absolute number of hCD45+ cells in BM and spleen of the reconstituted mice is shown, each symbol represents an individual animal. Indicated are values for significant differences between the groups (BM: p = 0.03, Spleen p = 0.0003, Mann-Whitney test) (C) Representative flow cytometric analysis of BM and spleen for CD19+ B cells, CD33+ myeloid cells and CD3+ or CD3+CD4+/CD8+ T cells. B cell developmental stages in BM and spleen are shown for CD24+CD38high/low cells. In BM pro/pre-B cells were defined as CD10+ IgM-, immature B cells as CD10+IgM+. In spleen mainly transitional (IgD+IgM+) B cells were detected. (D) Representative flow cytometric analysis of the spleen for plasmacytoid and myeloid dendritic cellls (pDC: Lin-CD123+CD11c-; mDC: Lin-CD123−CD11c+) and their expression of costimulatory molecules (CD40, CD86) (E) Representative flow cytometric analysis of CD3+ T cells in the thymus and their expression of CD4 and CD8 at >20 weeks after transplantation. The percentage of mice with CD3+ T cells and their absolute number in spleen at >20 weeks after transplantation is given (CB vs. non-cryo BM: p<0.01, Mann-Whitney test).
Reconstitution of a functional, HLA-restricted and self-tolerant T cell repertoire.
Adult sublethally irradiated NSG-HLA-A2/HHD mice were transplanted intra-venously with cryoconserved CD34+ HSPCs (n = 23 mice, 13 donors). At different time points after transplantation mice were analyzed by flow cytometry and final analysis of organs was performed at 18–20 weeks after reconstitution. (A) Flow cytometric analysis of peripheral blood of reconstituted mice (n = 12 mice) at 7–19 weeks after BM-HSPC transplantation. Shown is the % of hCD45+, hCD45+CD19+ (B cells), hCD45+CD33+ (myeloid cells) or hCD45+CD3+ (T cells) cells (median with interquartile range). (B) Engraftment in BM and spleen was determined by human CD45 expression. The absolute number of hCD45+ cells in BM and spleen of the reconstituted mice is shown and compared to NSG-mice reconstituted with non-cryoconserved BM-HSPCs (see Fig. 3B). Each symbol represents an individual animal. Indicated are values for significant differences between the groups (BM: p = 0.64; Spleen: p = 0.19, Mann-Whitney test). (C) In comparison to CB- (n = 8 mice) and BM-HSPC (n = 10 mice) transplanted NSG mice (i.h. and i.f. respectively), the absolute number of splenic CD3+ T cells is given for NSG-HLA-A2/HHD mice (n = 19 mice). Each symbol represents an individual animal. Indicated are values for significant differences between the groups (CB vs BM-NSG: p = 0.009; CB vs BM-HHD: p = 0.0027, Kruskal-Wallis test). (D) Representative flow cytometric analysis of splenic CD3+CD4+ and CD3+CD8+ T cells and their respective expression of CD27 and CD45RA. In addition, CD27 and CD45RA expression of peripheral blood T cells of a healthy volunteer is shown. (E) Mixed lymphocyte reaction of human T cells isolated from NSG-HLA-A2/HHD mice ( = chimeric T cells, cT) transplanted with CB- (n = 1 donor, 2 mice) or BM-HSPCs (n = 4 donors, each 2–3 mice). CFSE-labeled cT cells were stimulated with human T cell-depleted and irradiated peripheral blood mononuclear cells (hAPC, d5∶2×105 cells/well; d6∶2×105 cells/well). As control, human T cells from peripheral blood of the same (auto, n = 2) or different (alllo, n = 2) HLA-A2 negative donor were isolated. Self-tolerance was tested incubating cT cells or hT cells on autologous T cell depleted, irradiated cAPC or hAPC from the same humanized mouse or human volunteer. Proliferation of T cells was measured after 5 or 6 days based on the gradual loss of the CFSE-label.
Determination of the minimal number of BM-HSPCs, BM-MNCs and volume of BM-aspirate required for the reconstitution of NSG mice.
Six-to eight week-old sublethally irradiated NSG mice were transplanted intra-femorally with non-cryoconserved CD34+ HSPCs (n = 14, 11 donors). (A) At >20 weeks after transplantation mice were analyzed by flow cytometry and engraftment in BM and spleen was determined by human CD45 expression and plotted vs. the number of injected HSPCs. The number of BM-HSPCs is calculated, which is required to achieve reconstitution in BM and spleen comparable to the lowest number of transplanted CB-HSPCs (4.8×104 injected CD34+ HSPCs) that gives a reconstitution level matching with published data (BM: 6×106 hCD45+; spleen 1.7×106 hCD45+). Linear regression analysis revealed that at least 1.7–2.1×105 BM-HSPCs are needed. (B) The number of BM-MNCs at sample receipt is plotted vs. the number of isolated HSPCs. The number of BM-MNCs (108) is calculated that is required to isolate HSPCs sufficient for the reconstitution of six mice from one BM sample, given that each mouse receives 2.1×105 HSPCs. (C) The number of CD34+ HSPCs per 10 million BM-MNCs is plotted vs. the age of carcinoma (n = 77) or control-patients (n = 7). No correlation is observed. (D) Percentage of samples and the volume of samples at receipt, which contained ≥108 BM-MNCs allowing the isolation of HSPCs sufficient for the transplantation of six mice.
Antigens, CD34/metabolism
Bone Marrow/immunology
Bone Marrow/pathology
Breast Neoplasms/immunology
Breast Neoplasms/pathology
Disease Models, Animal
Esophageal Neoplasms/immunology
Esophageal Neoplasms/pathology
Hematopoietic Stem Cell Transplantation*
Hematopoietic Stem Cells/immunology
Hematopoietic Stem Cells/metabolism
Lung Neoplasms/immunology
Lung Neoplasms/pathology
Mice, Inbred NOD
Mice, SCID
Prostatic Neoplasms/immunology
Prostatic Neoplasms/pathology