Peripheral Blood Mononuclear Cell Reconstitution Research Articles

Abstract 5200: Novel Immuno-Oncology drugs evaluation by humanised immune cells and cancer co-inoculated models

Abstract Currently more and more immunotherapeutic drugs and engineered cells have been developed to use human immune system against cancer. While humanized peripheral blood mononuclear cell reconstitution in immune deficient mice is a forward-straight model for evaluating therapeutic antibodies, this conventional model has several drawbacks to hinder its widespread use, including the graft-versus-host-disease happened after one month of reconstitution, insufficient immune cell infiltration from reconstituted circulatory system and hardly retention of functional innate immune cells, such as macrophages and nature killer cells. To overcome this, LIDE has developed a specific human immune cell and cancer cell co-inoculation model. Cancer-priming PBMC and/or its derivates were well mixed with the fresh cancer cells in MatriGel, co-transferring into NCG mice to form a relatively “hot tumor” tissue for immunotherapy, including drugs targeting T cells, dendritic cells and macrophages. Additionally, we also take advantage of human IL15 transgenic mice to study the function of ex vivo expanded NK cells. This novel method has successfully helped evaluate biological function of immune checkpoint blockers and immune agonists in multiple cancers, such as melanoma, breast cancer, lung cancer, hepatocellular carcinoma and ovarian cancer. Citation Format: Bin Xie, Xin Hou, Hanglu Chen, Pengfei Yang, Yanbin Zhou, Danyi Wen. Novel Immuno-Oncology drugs evaluation by humanised immune cells and cancer co-inoculated models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5200.

Phase 1 Study of Elotuzumab and Peripheral Blood Mononuclear Cell Reconstitution in Combination with Autologous Stem Cell Transplantation and Lenalidomide Maintenance for Multiple Myeloma

Phase 1 Study of Elotuzumab and Peripheral Blood Mononuclear Cell Reconstitution in Combination with Autologous Stem Cell Transplantation and Lenalidomide Maintenance for Multiple Myeloma

Correlation of CLAD in Lung Transplant Recipients with Transplant Arteriosclerosis in Humanized Mice: A 5-years Follow Up Analysis

Chronic lung allograft dysfunction (CLAD) is the leading cause of long-term mortality after LuTX. We studied correlations between CLAD five years after clinical LuTX and leukocyte-mediated development of transplant arteriosclerosis (TA) in a humanized mouse model. The pericardiophrenic artery was procured from surplus tissue of 25 donor lungs transplanted in our clinical program and each was implanted into the abdominal aorta of cohorts of at least four immune deficient mice. The mice from each experiment were then divided into four groups. Negative control mice received no human leukocyte reconstitution (neg. co). PBMC group mice received 5 × 106 allogeneic human peripheral blood mononuclear cells (PBMC) from the respective lung recipients. A further group of animals was reconstituted with the respective PBMC additionally enriched with autologous CD4+CD25high cells (putative regulatory T cells, Treg). Human leukocyte engraftment was monitored by FACS and development of TA was histologically assessed 28 days after PBMC reconstitution. The 25 lung recipients were divided into two groups at least 5 years post LTX according to their development of chronic rejection. Sixteen patients (64%) developed CLAD 74.5± 7.01 months after LuTX. The remaining nine patients (36%) did not develop CLAD within 67.81± 2.24 months after LuTX RESULTS: The neg. co group showed only mild thickening of the intima (7.78±4.60%). In the PBMC CLAD+ group, intimal thickening obliterating the vessel lumen was significantly more severe than in the PBMC CLAD- group (41.91 ± 6.12% vs. 15.45 ± 3.22%, p= 0.001). Then, intimal thickening was significantly inhibited in the PBMC+Treg CLAD+ group as compared to the PBMC CLAD+ group (0.29 ± 2.99% vs 41.91 ± 6.12%, p=0.012). In the experiments using PBMC from lung recipients without CLAD, enriching Treg also further suppressed the development of TA (12.77 ± 2.59% PBMC CLAD- vs. 0.39 ± 4.11% PBMC+Treg CLAD-, p=0.012). Lung transplant recipients, who later developed CLAD, had peripheral leukocytes already at the time of LTX that transferred pro-inflammatory properties leading to TA into a humanized mouse model. TA remained sensitive to inhibition by autologous regulatory T cells, suggesting a cell therapy-based approach for the prevention and treatment of CLAD after LuTX.

Phase 1 study of elotuzumab in combination with autologous stem cell transplantation and lenalidomide maintenance for multiple myeloma.

8021 Background: Elotuzumab is a humanized monoclonal antibody directed against SLAMF7 that is approved for use in relapsed multiple myeloma. We initiated a single-center, open label, phase 1 trial based on the hypothesis that the addition of elotuzumab and autologous peripheral blood mononuclear cell (PBMC) reconstitution to standard-of-care autologous hematopoietic stem cell transplantation (auto-SCT) and lenalidomide maintenance for consolidation therapy in myeloma patients will be safe and feasible. Methods: This is a Phase 1b, open-label, trial investigating elotuzumab and autologous PBMC reconstitution with auto-SCT consolidation therapy and lenalidomide maintenance. The primary objective of this study is to assess the safety and tolerability of elotuzumab and autologous PBMC reconstitution in the setting of auto-SCT and lenalidomide maintenance. The secondary objectives are to assess myeloma disease status and progression-free survival (PFS) after one year of treatment. Subjects must be eligible for auto-SCT, and meet inclusion/exclusion criteria. Fifteen subjects participated in this study. The treatment plan is: In addition to PBSC harvest, subjects undergo steady-state leukopheresis for PBMC collection. Subjects receive standard melphalan (day -1) and autologous stem cell rescue (day 0). Autologous PBMC are reinfused on day +3 and cycle 1 of elotuzumab 20 mg/kg IV is given on day +4. Subjects receive elotuzumab every 28 days up to cycle 12. Lenalidomide maintenance at 10 mg orally daily begins with cycle 4 of elotuzumab. The evaluable population constitutes all subjects who received at least four of the first five planned doses of elotuzumab. Results: 15 subjects have been enrolled in the study. All of these subjects are included in the safety population, having received at least 1 dose of elotuzumab. Nine of 15 subjects have completed 4 of the first 5 planned elotuzumab infusions.. The majority of adverse events, including infusion reactions attributable to elotuzumab, have been grade 2 or lower. There were no delays in hematopoietic reconstitution. No AEs were attributed to PBMC reconstitution. Conclusions: The combination of elotuzumab and PBMC reconstitution with standard auto-SCT and lenalidomide maintenance for consolidation therapy appears to be safe and feasible. The trial is ongoing and has completed accrual. The clinical results will be updated for presentation.

Linking CLAD in Lung Transplant Recipients after 3 Years Follow Up with Transplant Arteriosclerosis in Humanized Mice

Purpose Chronic lung allograft dysfunction (CLAD) is a severe complication of lung transplantation (LTX) limiting long term survival. Here, we studied correlations between CLAD after clinical LTX and leukocyte-mediated development of transplant arteriosclerosis (TA) in a humanized mouse model. Methods The pericardiophrenic artery was procured from surplus tissue of 27 donor lungs transplanted in our clinical program and was implanted into the abdominal aorta of immune deficient mice. The experimental mice were then divided into four groups. Negative control mice received no human leukocyte reconstitution (neg. co). PBMC group mice received 5 × 106 allogeneic human peripheral blood mononuclear cells (PBMC) from the respective lung recipients (LR). A further group of animals was reconstituted with the respective PBMC additionally enriched with autologous CD4+CD25high cells (putative regulatory T cells, Treg). Human leukocyte engraftment was monitored by FACS and development of TA was histologically assessed 28 days after PBMC reconstitution. The 27 LR were divided into two groups at least 3 years post transplantation according to their development of chronic rejection. Nine patients (33.3%) developed CLAD 36.2± 4.16 month after LTX. The remaining eighteen patients (66,6%) did not develop CLAD within 35.9± 5.1 month after LTX. Results The neg. co group showed only mild thickening of the intima (6.38±5.70%). In the PBMC CLAD+ group, intimal thickening obliterating the vessel lumen was significantly more severe than in the PBMC CLAD- group (33.19 ± 7.02% vs. 13.37 ± 2.99%, p= 0.011). Then, intimal thickening was significantly inhibited in the PBMC+Treg CLAD+ group as compared to the PBMC CLAD+ group (0.39 ± 3.69% vs 33.19 ± 7.02%, p=0.012). In the experiments using PBMC from lung recipients without CLAD, enriching Treg also further suppressed the development of transplant arteriosclerosis (13.37 ± 2.99% PBMC CLAD- vs. 0.69 ± 5.88% PBMC+Treg CLAD-, p=0.007). Conclusion Lung transplant recipients, who later developed CLAD, had peripheral leukocytes already at the time of transplant that transfered pro-inflammatory properties leading to TA into a humanized mouse model. TA remained sensitive to inhibition by autologous regulatory T cells, suggesting a cell therapy-based approach for the prevention and treatment of CLAD after LTX.

Stromal Cells Act as Guardians for Endothelial Progenitors by Reducing Their Immunogenicity After Co-Transplantation.

Regeneration of injured tissues requires effective therapeutic strategies supporting vasculogenesis. The lack of instantly available autologous cell sources and immunogenicity of allogeneic endothelial (progenitor) cells limits clinical progress. Based on the immunosuppressive potency of mesenchymal stem/progenitor cells (MSCs), we investigated whether crosstalk between endothelial colony-forming progenitor cells (ECFCs) and MSCs during vasculogenesis could lower allogeneic T cell responses against ECFCs allowing long-term engraftment in vivo. Immunodeficient mice received subcutaneous grafts containing human ECFCs alone, or pairs of human ECFCs/MSCs from the same umbilical cord (UC) to study vasculogenesis in the presence of human leukocyte antigen (HLA)-mismatched human peripheral blood mononuclear cells (PBMCs). In vitro, cell surface marker changes due to interferon gamma (IFNγ) stimulation during ECFC/MSC coculture were determined and further effects on allostimulated T cell proliferation and cytotoxic lysis were measured. IFNγ-induced HLA-DR expression on ECFCs and MSCs, but both cell types had significantly less HLA-DR in cocultures. ECFC-induced T cell proliferation was abolished after MSC coculture as a result of HLA-DR downregulation and indolamin-2,3-dioxygenase activation. Additionally, allospecific CD8+ T cell-mediated lysis of ECFCs was reduced in cocultures. ECFC/MSC coapplication in immunodeficient mice not only promoted the generation of improved blood vessel architecture after 6 weeks, but also reduced intragraft immune cell infiltration and endothelial HLA-DR expression following PBMC reconstitution. Crosstalk between UC-derived ECFCs and MSCs after combined transplantation can lower the risk of ECFC rejection, thus enabling their coapplication for therapeutic vasculogenesis. Stem Cells 2017;35:1233-1245.

Phase 1 Study of Elotuzumab in Combination with Autologous Stem Cell Transplantation and Lenalidomide Maintenance for Multiple Myeloma

Introduction: Elotuzumab is a humanized monoclonal antibody directed against SLAMF7 that is approved for use in relapsed multiple myeloma patients in combination with lenalidomide and dexamethasone. This agent appears to have several modes of action, including facilitation of antibody-dependent, cell-mediated cytotoxicity (ADCC) through binding to SLAMF7 on myeloma cells and activation of natural killer (NK) cells to kill tumor cells through ligation of the target. We initiated a single-center, open label, phase 1 trial based on the hypothesis that the addition of elotuzumab and autologous peripheral blood mononuclear cell (PBMC) reconstitution to standard-of-care autologous hematopoietic stem cell transplantation (auto-SCT) and lenalidomide maintenance for consolidation therapy in myeloma patients after induction therapy will be safe and feasible. We hypothesize that early PBMC reconstitution post-auto-SCT will restore a viable NK cell population for activation by elotuzumab, which may target residual myeloma cells and promote tumor-specific humoral and cellular immune responses against myeloma cells. Subsequent maintenance therapy with elotuzumab and lenalidomide may amplify this response, resulting in long-term maintenance of the minimal residual disease state.Methods. This is a Phase 1b, open-label, trial investigating elotuzumab and autologous PBMC reconstitution with auto-SCT consolidation therapy and lenalidomide maintenance. The primary objective of this study is to assess the safety and tolerability of elotuzumab and autologous PBMC reconstitution in the setting of auto-SCT and lenalidomide maintenance in multiple myeloma patients. The secondary objectives are to assess myeloma disease status and progression-free survival (PFS) after one year of treatment. Subjects must achieve partial response or better by IMWG criteria with induction chemotherapy, be eligible for auto-SCT by institutional standards, and meet inclusion/exclusion criteria. Fifteen subjects are planned in this pilot study. The treatment plan is as follows: In addition to standard peripheral blood stem cell mobilization and harvest, subjects undergo steady-state leukopheresis for PBMC collection. Subjects receive standard melphalan conditioning (day -1) and autologous stem cell rescue (day 0). Autologous PBMC are reinfused on day +3 post-stem cell infusion and cycle 1 of elotuzumab 20 mg/kg IV is given on day +4. Subjects receive subsequent cycles of elotuzumab every 28 days up to cycle 12. Lenalidomide maintenance at 10 mg orally daily days 1-21 of every 28-day cycle begins with cycle 4 of elotuzumab, and may continue off study beyond cycle 12 at the investigator's discretion. Bone marrow aspirates and peripheral blood are collected for correlative studies at screening, cycle 2, cycle 4, and at the end of study after cycle 12. For the primary endpoint analysis, the safety population includes all subjects who received at least one dose of study treatment. The evaluable population constitutes all subjects who received at least four of the first five planned doses of elotuzumab.Results: Fourteen of the planned 15 subjects have been enrolled in the study. Demographic and staging data reflect the general transplant-eligible myeloma patient population at our institution. All 14 of these subjects are included in the safety population, having received at least 1 dose of elotuzumab. Nine of 14 subjects have completed at least 4 of the first 5 planned elotuzumab infusions and are evaluable. The majority of adverse events, including infusion reactions attributable to elotuzumab, have been grade 2 or lower. Grade 3 or higher hematologic AEs, including anemia, neutropenia, lymphopenia, thrombocytopenia, and non-hematologic AEs including nausea, vomiting, and dehydration, were attributable to the auto-SCT procedure. There were no delays in hematopoietic reconstitution observed. One episode of grade 3 hypertension was attributed to elotuzumab infusion and resolved with supportive care. No AEs were attributed to PBMC reconstitution.Conclusions: The combination of elotuzumab and PBMC reconstitution with standard auto-SCT and lenalidomide maintenance for consolidation therapy of multiple myeloma appears to be safe and feasible. One subject withdrew for personal reasons. The trial is ongoing and is expected to complete accrual and the clinical results will be updated for presentation. DisclosuresChari:Celgene: Consultancy, Research Funding; Array Biopharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Research Funding; Amgen Inc.: Honoraria, Research Funding; Takeda: Consultancy, Research Funding. Geerlof:Bristol-Myers Squibb: Employment. Jagannath:Novartis: Consultancy; Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Merck: Consultancy. Cho:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agenus, Inc.: Research Funding; Genentech Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Research Funding; Ludwig Institute for Cancer Research: Membership on an entity's Board of Directors or advisory committees.

(107) - Leukocytes Transfer More Severe Rejection from Lung Transplant Recipients with Severe PGD into Humanized Mice and Are Sensitive to T Cell Regulation

Primary graft dysfunction (PGD) is the main cause of early morbidity and mortality after lung transplantation. Here, we studied correlations between PGD after clinical lung transplantation and leukocyte-mediated development of transplant arteriosclerosis in a humanized mouse model. The pericardiophrenic artery was procured from donor lungs transplanted in our clinical program and was implanted into the abdominal aorta of immune deficient mice. Of 17 lung recipients, 10 (43.47%) developed severe PGD (grade 2-3) and 13 (56.52%) did not develop PGD (grade 0-1) at 24 h. Negative control mice received no human leukocyte reconstitution (neg. co). PBMC PGD+ group mice received 5x106 allogeneic human peripheral blood mononuclear cells (PBMC) from recipients with severe PGD, whereas PBMC PGD- group mice received PBMC from patients without PGD. Two groups of animals were reconstituted with the respective PBMC additionally enriched with autologous CD4+CD25 high cells (putative regulatory T cells, Treg) from recipients with severe PGD (PBMC+Treg PGD+ group) or without PGD (PBMC+Treg PGD- group). Transplant arteriosclerosis was histologically assessed 28 days after PBMC reconstitution. The neg. co group showed only mild thickening of the intima (13.86±14.22%). In the PBMC PGD+ group, intimal thickening obliterating the vessel lumen was significantly more severe than in the PBMC PGD- group (41.71 ± 15.01% vs. 10.52 ± 10.22%, p= 0.001). Then, intimal thickening was significantly inhibited in the PBMC+Treg PGD+ group as compared to the PBMC PGD+ group (3,42 ± 5,41% vs. 41.71 ± 15.01%, p=0.016). In the experiments using PBMC from lung recipients without PGD, enriching Treg also further suppressed the development of transplant arteriosclerosis (3.88 ± 5.54% PBMC PGD- vs. 10.52 ± 10.22%, PBMC+Treg PGD-, p=0.010). Leukocytes from lung transplant recipients with PGD transfer pro-inflammatory properties eliciting transplant arteriosclerosis into a humanized mouse model. This indicates a link between early graft dysfunction and the later development of chronic rejection. Importantly, however, transplant arteriosclerosis remains sensitive to inhibition by autologous Treg, suggesting a novel cell therapy-based approach for the treatment of patients with severe PGD.

(105) - The Real Time Visualization of Neutrophil Extracellular Trap Formation in Lung Transplants Reveals a Critical Role in Tolerance

Methods: The pericardiophrenic artery was procured from donor lungs transplanted in our clinical program and was implanted into the abdominal aorta of immune deficient mice. Of 17 lung recipients, 10 (43.47%) developed severe PGD (grade 2-3) and 13 (56.52%) did not develop PGD (grade 0-1) at 24 h. Negative control mice received no human leukocyte reconstitution (neg. co). PBMC PGD+ group mice received 5x106 allogeneic human peripheral blood mononuclear cells (PBMC) from recipients with severe PGD, whereas PBMC PGDgroup mice received PBMC from patients without PGD. Two groups of animals were reconstituted with the respective PBMC additionally enriched with autologous CD4+CD25 high cells (putative regulatory T cells, Treg) from recipients with severe PGD (PBMC+Treg PGD+ group) or without PGD (PBMC+Treg PGDgroup). Transplant arteriosclerosis was histologically assessed 28 days after PBMC reconstitution. Results: The neg. co group showed only mild thickening of the intima (13.86±14.22%). In the PBMC PGD+ group, intimal thickening obliterating the vessel lumen was significantly more severe than in the PBMC PGDgroup (41.71 ± 15.01% vs. 10.52 ± 10.22%, p= 0.001). Then, intimal thickening was significantly inhibited in the PBMC+Treg PGD+ group as compared to the PBMC PGD+ group (3,42 ± 5,41% vs. 41.71 ± 15.01%, p= 0.016). In the experiments using PBMC from lung recipients without PGD, enriching Treg also further suppressed the development of transplant arteriosclerosis (3.88 ± 5.54% PBMC PGDvs. 10.52 ± 10.22%, PBMC+Treg PGD-, p= 0.010). Conclusion: Leukocytes from lung transplant recipients with PGD transfer proinflammatory properties eliciting transplant arteriosclerosis into a humanized mouse model. This indicates a link between early graft dysfunction and the later development of chronic rejection. Importantly, however, transplant arteriosclerosis remains sensitive to inhibition by autologous Treg, suggesting a novel cell therapy-based approach for the treatment of patients with severe PGD.

ESDN Is a Marker of Vascular Remodeling and Regulator of Cell Proliferation in Graft Arteriosclerosis

Vascular remodeling is a common feature of many vasculopathies, including graft arteriosclerosis (GA). We investigated whether endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is a marker of vascular remodeling in GA. Immunostaining of human coronary arteries demonstrated high levels of ESDN in GA, but not in normal arteries. In a model of GA, where a segment of human coronary is transplanted into a severe combined immunodeficient mouse, followed by allogeneic human peripheral blood mononuclear cell (PBMC) reconstitution, ESDN was minimally expressed in transplanted human arteries in the absence of reconstitution. By 2 weeks following PBMC reconstitution, at a time corresponding to maximal vascular cell proliferation, high levels of ESDN were detected in the transplanted arteries. Similarly, injury-induced vascular remodeling in apoE(-/-) mice was associated with early and transient ESDN upregulation, in parallel with cell proliferation. In vascular smooth muscle cell (VSMC) cultures, ESDN expression was significantly higher in proliferating, as compared to growth-arrested cells. ESDN overexpression in VSMC led to a decline in growth curves, while ESDN knock down had the opposite effect. We conclude that ESDN is a marker of vascular remodeling and regulator of VSMC proliferation. ESDN may serve as a therapeutic or diagnostic target for GA.

αvβ3‐Targeted detection of arteriopathy in transplanted human coronary arteries: an autoradiographic study

Graft arteriopathy (GA), characterized by diffuse concentric narrowing of coronary arteries, is the major cause of late graft failure in cardiac transplantation. alphavbeta3 Integrin is up-regulated in proliferating vascular cells and may constitute an appropriate target for imaging GA. We used a human/mouse chimeric model of GA, in which segments of human coronary artery were transplanted to severe combined immunodeficiency mice, followed by reconstitution with allogeneic human peripheral blood mononuclear cells (PBMC). This led to vascular remodeling characterized by neointima formation over a period of 4 wk. alphavbeta3 expression in the graft was minimal in animals without PBMC, considerably increased by 2 wk, and decreased toward baseline by 4 wk after PBMC reconstitution. Cell proliferation was maximal at 2 wk, correlating with peak alphavbeta3 expression. RP748, an 111In-labeled alphavbeta3 (active conformation)-targeted radiotracer was injected into groups of 5 recipients at 0, 2, and 4 wk after PBMC reconstitution. Relative uptakes, defined as autoradiographic intensity in the graft/native aortas closely tracked the proliferative process. Specificity of uptake was demonstrated using excess nonlabeled tracer. In conclusion, alphavbeta3 integrin is transiently up-regulated (and activated) in GA and may be targeted by RP748 for detection of the proliferative process in early GA.

CCR3-Blocking Antibody Inhibits Allergen-Induced Eosinophil Recruitment in Human Skin Xenografts from Allergic Patients

Eosinophil, basophil, and T helper 2 (TH2) cell recruitment into tissues is a characteristic feature of allergic diseases. These cells have in common the expression of the chemokine receptor CCR3, which may represent a specific pathway for their accumulation in vivo. Although animal models of allergic reactions are available, findings cannot always be extrapolated to man. To overcome these limitations, we have developed a humanized mouse model of allergic cutaneous reaction using severe combined immunodeficiency mice engrafted with skin and autologous peripheral blood mononuclear cells from allergic donors. Intradermal injection of the relevant allergen into human skin xenografts from allergic individuals induced a significant recruitment of human CD4+ T cells, basophils, and TH2-type cytokine mRNA-expressing cells, as well as murine eosinophils. Human skin xenografts, atopic status, and autologous peripheral blood mononuclear cell reconstitution were all mandatory to induce the allergic reaction. Next, we addressed the role of CCR3 in the endogenous mechanisms involved in the inflammatory cell recruitment in this experimental model of allergic cutaneous reaction. In vivo administration of an anti-human CCR3-blocking antibody selectively reduced accumulation of eosinophils but not that of CD4+ cells, basophils, or cells expressing mRNA for TH2-type cytokines. These findings establish a new in vivo model of humanized allergic reaction and suggest that eosinophil migration is mediated mainly through CCR3. Finally, these results suggest that this model might be useful to test human-specific antiallergic modulators.

Immunological Characterization of Human Vaginal Xenografts in Immunocompromised Mice: Development of a Small Animal Model for the Study of Human Immunodeficiency Virus-1 Infection

A small animal model for the in vivo study of human immunodeficiency virus-1 and other fastidious infectious agents in human host target tissues is critical for the advancement of therapeutic and preventative strategies. Our laboratory has developed a human vaginal xenograft model that histologically recapitulates features of the human vaginal epithelial barrier. Vaginal xenografts were surgically implanted into C.B.-Igh-1(b)/IcrTac-Prkdc(scid) (SCID) and NOD/LtSz-scid/scid (NOD/SCID) mice, with and without human peripheral blood mononuclear cell reconstitution. Immunohistochemical staining of vaginal xenografts demonstrated that in the SCID strain healed vaginal xenografts did not retain intrinsic human immune cells at baseline levels, whereas the NOD/SCID strain supported retention of intrinsic human immune cell populations within the xenografts for at least 2 months after engraftment. In peripheral blood mononuclear cell-reconstituted NOD/SCID mice with vaginal xenografts, flow cytometric analyses detected human immune cell populations in the peripheral blood and immunohistochemical methods detected infiltration of human CD45+ cells in the mouse spleens and vaginal xenografts for at least 2 months after reconstitution. This optimized NOD/SCID human vaginal xenograft model may provide a unique small animal in vivo system for the study of human immunodeficiency virus-1 transmission and infection.

The Persistence of Human Peripheral Lymphocytes, Tumor Infiltrating Lymphocytes, and Colon Adenocarcinomas in Immunodeficient Mice

The reconstitution of severely immunodeficient mice with human peripheral blood mononuclear cells (PBMCs) may represent a unique model system to evaluate human antitumor responses. To evaluate this possibility, we studied human PBMC reconstitution, human tumor infiltrating lymphocyte (TIL) persistence, and human colon adenocarcinoma propagation in beige/nude/xid (BNX) and in severe combined immunodeficient (SCID) mice. To evaluate human PBMC reconstitution, 75 mice received 1 x 10(7)-1 x 10(9) human PBMCs i.p. or i.v. and were studied at intervals ranging from 1 to 8 weeks by fluorescence-activated cell sorting (FACS) analysis and by measurement of circulating human immunoglobulin levels. By FACS analyses, only one of 75 mice had evidence of human PBMC persistence at 2 weeks in the spleen. Moreover, liver and peritoneum showed evidence of human cells in only 13 of 56 and 16 of 55 mice, respectively. In these mice, human cells comprised 1-77% of total cells recovered. Human immunoglobulin levels in mouse serum ranged from 0 to 34,000 micrograms/ml and correlated only weakly with evidence of human PBMC reconstitution in peripheral organs, but were generally higher in SCID mice than in BNX mice. Human TIL persistence was evaluated in BNX and SCID mice that were given 3 x 10(7) TILs i.v. (in divided doses) or 1 x 10(8) i.p. TILs along with interleukin-2 administration. At 1, 2, 7, and 14 days following TIL delivery, evidence of human TIL persistence in liver, lung, peritoneum, and spleen was evaluated by FACS analysis. Fresh organ suspensions did not contain human TILs. In mice given cyclophosphamide followed by human TILs i.p., the TILs were demonstrated at 7 days in the SCID peritoneum (leu 4 = 4%) and at 2 days in the SCID spleen (leu 4 = 2%). In BNX mice, 12 of 14 fresh human colon adenocarcinomas were propagated successfully at subcutaneous sites with latency periods ranging from 1 to 13 weeks. Enzymatic disaggregation of tumors greater than 1 cm following one passage yielded 6.5-47 x 10(6) cells with viabilities ranging from 13 to 85%. We conclude that limitations and variability exist in the use of BNX and SCID mice for human PBMC reconstitution, TIL persistence, and propagation of human colon adenocarcinomas.