Epigenetic and in vivo comparison of diverse MSC sources reveals an endochondral signature for human hematopoietic niche formation

In the last decade there has been a rapid expansion in clinical trials using mesenchymal stromal cells (MSCs) from a variety of tissues. However, despite similarities in morphology, immunophenotype, and differentiation behavior in vitro, MSCs sourced from distinct tissues do not necessarily have equivalent biological properties. We performed a genome-wide methylation, transcription, and in vivo evaluation of MSCs from human bone marrow (BM), white adipose tissue, umbilical cord, and skin cultured in humanized media. Surprisingly, only BM-derived MSCs spontaneously formed a BM cavity through a vascularized cartilage intermediate in vivo that was progressively replaced by hematopoietic tissue…

CD8-Depleted Donor Lymphocyte Infusions Convert Mixed into Complete Donor T-Cell Chimerism after T-Cell Depleted Allogeneic Stem Cell Transplantation.

Abstract Donor lymphocyte infusions (DLI) are increasingly used to treat minimal residual disease or mixed hematopoietic donor-recipient chimerism in T-cell depleted allogeneic stem cell transplantation (SCT). In addition, several clinical trials currently investigate the prophylactic application of DLI to promote donor T-cell reconstitution after transplantation. However, DLI carry a substantial risk of inducing graft-versus-host disease (GVHD). We investigate DLI heavily depleted of CD8 T cells using a clinical grade immunomagnetic in vitro procedure in an ongoing clinical study [Meyer et al., Blood2007, 109:374]. These DLI are administered in a prophylactic setting to patients with hemat…

Both mature KIR+ and immature KIR- NK cells control pediatric acute B-cell precursor leukemia in NOD.Cg-Prkdcscid IL2rgtmWjl/Sz mice.

Therapeutic natural killer (NK)-cell-mediated alloreactivity toward acute myeloid leukemia has largely been attributed to mismatches between killer immunoglobulin-like receptors (KIRs) on NK cells and their ligands, HLA class I molecules, on target cells. While adult acute B-cell precursor leukemia (BCP-ALL) appears to be resistant to NK-cell-mediated lysis, recent data indicate that pediatric BCP-ALL might yet be a target of NK cells. In this study, we demonstrate in a donor-patient-specific NOD.Cg-Prkdc(scid) IL2rg(tmWjl)/Sz (NSG) xenotransplantation model that NK cells mediate considerable alloreactivity toward pediatric BCP-ALL in vivo. Notably, both adoptively transferred mature KIR(+)…

HLA-DPB1 mismatch alleles represent powerful leukemia rejection antigens in CD4 T-cell immunotherapy after allogeneic stem-cell transplantation.

Refractory or relapsed acute myeloid leukemia (AML) represents a frequent complication after allogeneic hematopoietic stem-cell transplantation (HSCT). We show herein that primary in vitro stimulation of CD45RA-selected CD4 T cells of stem-cell donors with 10/10 HLA-matched AML blasts results in expansion of cytolytic T-lymphocytes (CTL) that almost all recognize HLA-DPB1 mismatch alleles, which clinically occur in up to 80% of donor-patient pairs. Primary AML blasts were found to strongly express HLA-DPB1, whereas fibroblasts and keratinocytes used as surrogate target cells for graft-versus-host disease did express HLA-DPB1 only upon IFN-γ pre-treatment. Since patients' AML blasts are rare…

Alloreactive and leukemia-reactive T cells are preferentially derived from naive precursors in healthy donors: implications for immunotherapy with memory T cells.

Background HLA mismatch antigens are major targets of alloreactive T cells in HLA-incompatible stem-cell transplantation, which can trigger severe graft- versus -host disease and reduce survival in transplant recipients. Our objective was to identify T-cell subsets with reduced in vitro reactivity to allogeneic HLA antigens. Design and Methods We sorted CD4 and CD8 T-cell subsets from peripheral blood by flow cytometry according to their expression of naive and memory markers CD45RA, CD45RO, CD62L, and CCR7. Subsets were defined by a single marker to facilitate future establishment of a clinical-grade procedure for reducing alloreactive T-cell precursors and graft- versus -host disease. T c…

Exclusive transduction of human CD4+ T Cells upon systemic delivery of CD4-targeted lentiviral vectors

Abstract Playing a central role in both innate and adaptive immunity, CD4+ T cells are a key target for genetic modifications in basic research and immunotherapy. In this article, we describe novel lentiviral vectors (CD4-LV) that have been rendered selective for human or simian CD4+ cells by surface engineering. When applied to PBMCs, CD4-LV transduced CD4+ but not CD4− cells. Notably, also unstimulated T cells were stably genetically modified. Upon systemic or intrasplenic administration into mice reconstituted with human PBMCs or hematopoietic stem cells, reporter gene expression was predominantly detected in lymphoid organs. Evaluation of GFP expression in organ-derived cells and blood …

Prophylactic transfer of CD8-depleted donor lymphocytes after T-cell–depleted reduced-intensity transplantation

AbstractAllogeneic hematopoietic stem cell transplantation (SCT) regimens incorporating the lymphocytotoxic CD52 antibody alemtuzumab demonstrate efficient engraftment and reduced graft-versus-host disease (GVHD). However, these protocols substantially impair posttransplantation antiviral and antitumor immunity. To accelerate immune reconstitution after alemtuzumab-based reduced-intensity SCT, we administered prophylactic CD8-depleted donor lymphocyte infusions (DLIs) starting on days 60 and 120 after transplantation. DLIs were processed in an immunomagnetic good manufacturing practice depletion procedure resulting in a 2.5- to 6-log reduction in CD8 T cells. Of 23 high-risk patients with h…

Organotypic Epigenetic Signature Predicts Bone and Marrow Niche Forming Capacity of Stromal Progenitors in a Novel Mouse Model in Vivo.

Abstract Abstract 2987 Mesenchymal stem/progenitor cells (MSPCs) from numerous tissues are currently tested in clinical trials despite a limited understanding of their in vivo behavior. In this study we used MSPCs from adult and fetal tissues to select the appropriate source for clinical application. We asked whether MSPCs derived from human bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC), compared to skin fibroblasts, bear an equivalent bone and marrow niche formation potential with of in vivo. Furthermore we evaluated attraction and engraftment of murine as well as human hematopoietic stem/progenitor cells (HSPCs) into newly formed MSPC-derived niches. To elucidate po…

29: Rapid expansion of acute myeloid leukemia-reactive cytotoxic T cells from CD8+CD62L+ blood lymphocytes of HLA-matched healthy donors in vitro

297: Establishment of a chimeric NOD-scid/IL2RγcNull transplantation-model to evaluate graft-vs-host and graft-vs-leukemia immune responses of ex vivo modified human T lymphocyte grafts

Nanoparticles and antigen-specific T-cell therapeutics: A comprehensive study on uptake and release

Aim: T lymphocytes are used as cellular therapeutics in many disease entities including cancer. We investigated the uptake and retention of nanoparticles (NPs) by these nonphagocytic cells. Materials & methods: Uptake, release and toxicity of various polymeric NP preparations were analyzed by flow cytometry, confocal laser scanning microscopy and transmission electron microscopy. T-cell effector functions were measured using IFN-γ-ELISPOT and 51Chromium-release assays. Results: Amino-functionalized NPs were efficiently ingested by antigen-specific T cells without adversely influencing effector functions. NPs were stored in membrane-surrounded vesicles, with major proportions released e…

Selective Depletion of Alloreactive T Lymphocytes Using Patient-Derived Nonhematopoietic Stimulator Cells in Allograft Engineering

Background. Selective depletion of alloreactive T cells in vitro results in efficient graft-versus-host disease prophylaxis in allogeneic hematopoietic stem-cell transplantation, but it is accompanied by increased recurrence of leukemia. To spare donor T-cell-mediated graft-versus-leukemia immunity against hematopoiesis-restricted minor histocompatibility (minor-H) antigens, we explored the use of patient-derived nonhematopoietic antigen-presenting cells (APC) as allogeneic stimulators for selective allodepletion in leukemia-reactive donor T-cell lines. Methods. Primary keratinocytes, dermal fibroblasts, and bone marrow fibroblasts were generated from skin biopsies and diagnostic bone marro…

Monocytes derived from humanized neonatal NOD/SCID/IL2Rγ(null) mice are phenotypically immature and exhibit functional impairments.

Trials of immune-modulating drugs in septic patients have mostly failed to demonstrate clinical efficacy. Thus, we sought to generate a surrogate model of myelomonocytic lineage differentiation that would potentially allow sepsis induction and preclinical testing of anti-inflammatory drugs. Comparing transplantation of cord blood-derived stem cells in neonatal NOD/SCID/IL2Rγ(null) (neonatal huNSG) mice with transplantation of adult peripheral mobilized stem cells into adult NSG (adult huNSG) recipients, we demonstrate that myelomonocytic lineage differentiation in neonatal huNSG mice is retarded and monocytes are phenotypically immature with respect to HLA-DR expression and the emergence of…

Patient-individualized CD8+cytolytic T-cell therapy effectively combats minimal residual leukemia in immunodeficient mice

Adoptive transfer of donor-derived cytolytic T-lymphocytes (CTL) has evolved as a promising strategy to improve graft-versus-leukemia (GvL) effects in allogeneic hematopoietic stem-cell transplantation. However, durable clinical responses are often hampered by limited capability of transferred T cells to establish effective and sustained antitumor immunity in vivo. We therefore analyzed GvL responses of acute myeloid leukemia (AML)-reactive CD8(+) CTL with central and effector memory phenotype in a new allogeneic donor-patient specific humanized mouse model. CTL lines and clones obtained upon stimulation of naive CD45RA(+) donor CD8(+) T cells with either single HLA antigen-mismatched or HL…

566. Selective and Stable Transduction of Human CD4+ T Cells In Vivo Upon Systemic Administration of CD4-Targeted Lentiviral Vectors

Playing a central role in both innate and adaptive immunity, CD4+ T cells are the key target for genetic modifications in basic research and immunotherapy. Specific and stable delivery of therapeutic genes into these cells is therefore highly desirable. Here, we describe novel lentiviral vectors (CD4-LV) that have been rendered selective for human or simian CD4+ cells by surface engineering. This novel CD4-LV was highly specific and effective in genetic modification of human CD4+ T cells both in vitro and in vivo. When applied to peripheral blood mononuclear cells (PBMC), CD4-LV transduced CD4+ but not CD4− cells. Notably, also unstimulated T cells were stably genetically modified. Upon sys…

Engraftment of low numbers of pediatric acute lymphoid and myeloid leukemias into NOD/SCID/IL2Rcγnull mice reflects individual leukemogenecity and highly correlates with clinical outcome

Although immortalized cell lines have been extensively used to optimize treatment strategies in cancer, the usefulness of such in vitro systems to recapitulate primary disease is limited. Therefore, the design of in vivo models ideally utilizing patient-derived material is of critical importance. In this regard, NOD.Cg-Prkdc(scid) IL2rg(tmWjl) /Sz (NSG) mice have been reported to provide superior engraftment rates. However, limited data exist on the validity of such a model to constitute a surrogate marker for clinical parameters. We studied primary and serial engraftment on more than 200 NSG mice with 54 primary pediatric B cell precursor acute lymphatic leukemia (B-ALL), myeloid leukemia …

Tumor-priming converts NK cells to memory-like NK cells

Fascinating earlier evidence suggests an intrinsic capacity of human natural killer (NK) cells to acquire adaptive immune features in the context of cytomegalovirus (CMV) infection or pro-inflammatory cytokine stimulation. Since the role of memory NK cells in cancer has so far remained elusive and adoptive NK cell transfer in relapsing pediatric acute B cell precursor leukemia (BCP-ALL) patients awaits improvement, we asked the question whether tumor-priming could promote the generation of memory NK cells with enhanced graft-vs.-leukemia (GvL) reactivity. Here, we provide substantial evidence that priming of naive human NK cells with pediatric acute B cell leukemia or acute myeloid leukemia…

Evaluation Of Alloimmune Responses In Humanized NOD/SCID/IL2rgnull Mice Following Human CD34+ Stem Cell Transplantation

Campath-1H-Based Reduced-Intensity Conditioning Followed by Allogeneic Blood Stem-Cell Transplantation and Preemptive CD8-Depleted Donor Lymphocyte Infusions.

Abstract Recent reports have demonstrated the feasibility of using the anti-CD52 antibody Campath-1H for in vivo T-cell depletion (TCD) in the context of a fludarabine/melphalan-based reduced-intensity conditioning regimen (Kottaridis et al. Blood 2000, 96:2419). Major disadvantage of this protocol is the severe immunosuppression which results in an increased rate of opportunistic infections and disease relapses. Donor lymphocyte infusions (DLI) are frequently used to overcome this limitation. The application of DLI, however, is associated with a profound risk of GvHD. Depletion of CD8+ lymphocytes from either the allotransplant or from DLI has proven feasible to reduce the incidence of GvH…

Donor CD4 T cells convert mixed to full donor T-cell chimerism and replenish the CD52-positive T-cell pool after alemtuzumab-based T-cell-depleted allo-transplantation.

Donor lymphocyte infusions (DLI) are used to resolve mixed T-cell chimerism (TCC) after allo-SCT despite a substantial risk of GVHD. We analyzed the impact of prophylactic CD8-depleted (CD8(depl)) DLI in 20 recipients of anti-CD52 alemtuzumab in vivo T-cell-depleted allografts with declining donor TCC after day +60. A total of 13 patients received CD8(depl) DLI and 7 patients did not. All but one of the DLI patients converted to complete donor T-cell chimeras, whereas only one non-DLI patient converted spontaneously. DLI induced transient acute GVHD in five and extensive chronic GVHD in two patients. These data suggest the use of CD8(depl) DLI as an effective treatment for mixed TCC, partic…

Acute myeloid leukemia (AML)-reactive cytotoxic T lymphocyte clones rapidly expanded from CD8(+) CD62L((high)+) T cells of healthy donors prevent AML engraftment in NOD/SCID IL2Rgamma(null) mice.

Objective Current in vitro techniques for isolating leukemia-reactive cytotoxic T lymphocytes (CTLs) from healthy donors are of relatively low efficiency and yield responder populations with unknown biological significance. This study aimed at the development of a more reliable approach, allowing generation and expansion of acute myeloid leukemia (AML)-reactive CTLs using primary in vitro stimulation. Materials and Methods We established allogeneic mini-mixed lymphocyte-leukemia cultures (mini-MLLCs) by stimulating donor CD8 + T cells with human leukocyte antigen (HLA) class I–matched AML blasts in microtiter plates. Before culture, CD8 + T cells were separated into CD62L (high)+ and CD62L …

Human Epidermal Langerhans Cells Replenish Skin Xenografts and Are Depleted by Alloreactive T Cells In Vivo

Abstract Epidermal Langerhans cells (LC) are potent APCs surveying the skin. They are crucial regulators of T cell activation in the context of inflammatory skin disease and graft-versus-host disease (GVHD). In contrast to other dendritic cell subtypes, murine LC are able to reconstitute after local depletion without the need of peripheral blood-derived precursors. In this study, we introduce an experimental model of human skin grafted to NOD-SCID IL2Rγnull mice. In this model, we demonstrate that xenografting leads to the transient loss of LC from the human skin grafts. Despite the lack of a human hematopoietic system, human LC repopulated the xenografts 6 to 9 wk after transplantation. By…

Depletion of Alloreactive Donor T Lymphocytes by CD95-Mediated Activation-Induced Cell Death Retains Antileukemic, Antiviral, and Immunoregulatory T Cell Immunity

In allogeneic hematopoietic stem cell transplantation (AHSCT) graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect are closely but not invariably linked. Thus, harnessing donor lymphocyte mediated GVL immunity and separating it from GVHD is of particular interest. Based on results obtained in murine models we have explored the CD95-mediated activation-induced cell death (AICD) strategy to selectively deplete alloreactivity in human donor T lymphocytes in vitro. Following stimulation of CD3(+) T cells isolated from HLA-A* 0201-positive donors with HLA or minor histocompatibility antigen mismatched hematopoietic or nonhematopoietic cells in the presence of agonistic anti-CD…

Functional Analyses of Human T Cell Extravasation in a Humanized NOD/SCID/IL2Rγcnull Transplantation Model.

Abstract Abstract 2448 Poster Board II-425 Donor lymphocyte graft engineering to avoid graft-versus-host (GVH) reactivity while improving graft-versus-leukemia (GVL) immunity remains of central interest in allogeneic hematopoietic stem cell transplantation (HSCT). However, appropriate models to evaluate experimental concepts of donor lymphocyte allograft engineering in vivo are missing. We, therefore, established a human-murine chimeric transplantation model using immunodeficient NOD/SCID/IL2Rγcnull (NSG) mice to evaluate GVH reactivity of human T cell grafts in vivo. Moreover, since mechanisms of immune functions resembling human GVH immunity have not yet been addressed in detail in these …

Rapid identification and sorting of viable virus-reactive CD4+ and CD8+ T cells based on antigen-triggered CD137 expression

Abstract Current methods for the detection and isolation of antigen-specific CD4 + and CD8 + T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. Here we show that de novo cell surface expression of the TNF-receptor family member CD137 (4-1BB) identifies recently activated, but not resting, human CD4 + and CD8 + memory T cells. Maximum CD137 expression level is uniformly observed in both T-cell subsets at 24h after stimulation with antigen. In experiments with CMV and EBV-reactive T cells, we confirmed the specificity of CD137 expression by co-staining with peptide/HLA tetramers. Substantial proportions of CD137 +…

Long-Term Human CD34+ Stem Cell-Engrafted Nonobese Diabetic/SCID/IL-2Rγnull Mice Show Impaired CD8+ T Cell Maintenance and a Functional Arrest of Immature NK Cells

Abstract Allogeneic hematopoietic stem cell transplantation represents the most effective form of immunotherapy for chemorefractory diseases. However, animal models have been missing that allow evaluation of donor-patient–specific graft-versus-leukemia effects. Thus, we sought to establish a patient-tailored humanized mouse model that would result in long-term engraftment of various lymphocytic lineages and would serve as a donor-specific surrogate. Following transfer of donor-derived peripheral blood stem cells into NOD/SCID/IL-2Rγnull (NSG) mice with supplementation of human IL-7, we could demonstrate robust engraftment and multilineage differentiation comparable to earlier studies using …

Tetracycline-controlled transgenic targeting from the SCL locus directs conditional expression to erythrocytes, megakaryocytes, granulocytes, and c-kit-expressing lineage-negative hematopoietic cells

The stem cell leukemia gene SCL, also known as TAL-1, encodes a basic helix-loop-helix transcription factor expressed in erythroid, myeloid, megakaryocytic, and hematopoietic stem cells. To be able to make use of the unique tissue-restricted and spatio-temporal expression pattern of the SCL gene, we have generated a knock-in mouse line containing the tTA-2S tetracycline transactivator under the control of SCL regulatory elements. Analysis of this mouse using different tetracycline-dependent reporter strains demonstrated that switchable transgene expression was restricted to erythrocytes, megakaryocytes, granulocytes, and, importantly, to the c-kit-expressing and lineage-negative cell fracti…

Definitive evidence for Club cells as progenitors for mutantKras/Trp53‐deficient lung cancer

Accumulating evidence suggests that both the nature of oncogenic lesions and the cell-of-origin can strongly influence cancer histopathology, tumor aggressiveness and response to therapy. Although oncogenic Kras expression and loss of Trp53 tumor suppressor gene function have been demonstrated to initiate murine lung adenocarcinomas (LUADs) in alveolar type II (AT2) cells, clear evidence that Club cells, representing the second major subset of lung epithelial cells, can also act as cells-of-origin for LUAD is lacking. Equally, the exact anatomic location of Club cells that are susceptible to Kras transformation and the resulting tumor histotype remains to be established. Here, we provide de…

Towards More Specificity and Effectivity in the Antileukemia Immune Response

Experimental and clinical studies have shown that alloreactive T-cell responses derived from donor lymphocytes can effectively eliminate leukemia cells after allogeneic hematopoietic stem cell transplantation. However, there are still too many patients in whom this graft-versus-leukemia reactivity is insufficient to prevent leukemia relapse or who suffer from severe alloreactivity to nonmalignant host tissues also mediated by donor-derived T cells. Therefore, various conceptually different approaches have been developed at the level of donor T cells in order to improve the efficacy of leukemia-directed immunity while reducing the incidence of unwanted graft-versus-host disease. As outlined …

Zebrafish xenografts as a tool for in vivo studies on human cancer

The zebrafish has become a powerful vertebrate model for genetic studies of embryonic development and organogenesis and increasingly for studies in cancer biology. Zebrafish facilitate the performance of reverse and forward genetic approaches, including mutagenesis and small molecule screens. Moreover, several studies report the feasibility of xenotransplanting human cells into zebrafish embryos and adult fish. This model provides a unique opportunity to monitor tumor-induced angiogenesis, invasiveness, and response to a range of treatments in vivo and in real time. Despite the high conservation of gene function between fish and humans, concern remains that potential differences in zebrafis…

Depletion of alloreactive T cells via CD69: implications on antiviral, antileukemic and immunoregulatory T lymphocytes

Selective depletion of alloreactive T cells from stem-cell allografts should abrogate graft-versus-host disease while preserving beneficial T cell specificities to facilitate engraftment and immune reconstitution. We therefore explored a refined immunomagnetic separation strategy to effectively deplete alloreactive donor lymphocytes expressing the activation antigen CD69 upon stimulation, and examined the retainment of antiviral, antileukemic, and immunoregulatory T cells. In addition to the CD69high T cell fraction, our studies retrieved two T cell subsets based on residual CD69 expression. Whereas, truly CD69(neg) cells were devoid of detectable alloresponses to original stimulators, CD69…

Regulation of hematopoietic growth factor production by genetically modified human bone marrow stromal cells expressing interleukin-1beta antisense RNA.

Interleukin-1 (IL-1) plays a major role in the regulation of bone marrow stromal cell function and hematopoiesis. It is known to induce secretion of the hematopoietic growth factors granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), IL-6, and IL-8 as well as IL-1 itself in stromal cells. We investigated the role of IL-1beta-mediated growth factor production in the human stromal cell line L88/5. Using liposome-mediated DNA transfer, two stromal cell transfectants that constitutively express IL-1beta antisense (AS) RNA were generated. Expression of IL-1beta AS RNA and IL-1beta RNA was determined by RT-PCR. The stromal cell transfectants were strongly impaired …

Redirected EBV-Specific Stem Cell-Memory and Central-Memory CD8+ T Lymphocytes Exhibit Effective and Sustained Antileukemic Immunity to Acute Myeloid Leukemia in a Patient-Tailored Humanized NSG Mouse Model

Abstract Introduction: Adoptive cellular therapy (ACT) of donor-derived cytolytic T lymphocytes (CTL) directed to leukemia or herpesvirus has proven promising to improve antiviral and antileukemic immunity in patients following allogeneic hematopoietic stem cell transplantation (AHSCT). However, durable clinical responses are often hampered by detrimental graft-versus host (GvH) reactivity and limited persistence of transferred, fully differentiated antileukemic effector T cells (TEFF). We thus explored memory and tumoricidal features of in vitro generated EBV-specific stem cell-memory T cells (TSCM) and central-memory T (TCM) cells, T cell-receptor (TCR) redirected to primary acute myeloid…

Targeting the activation-induced antigen CD137 can selectively deplete alloreactive T cells from antileukemic and antitumor donor T-cell lines.

AbstractIn HLA-incompatible hematopoietic stem cell transplantation, alloreactive donor T cells recognizing recipient mismatch HLA cause severe graft-versus-host disease (GVHD). Strategies allowing the selective depletion of alloreactive T cells as well as the enhancement of graft-versus-malignancy immunity would be beneficial. We generated donor CD8 T-cell lines in vitro using allogeneic recipient cells mismatched at a single HLA class I allele or haplotype as stimulators. Recipient cells were obtained from acute myeloid leukemias, renal-cell carcinomas, and CD40L-induced B lymphoblasts. Resulting alloreactive T cells were activated by incubating day 21 T-cell cultures with HLA-mismatch tr…

Preemptive Transfer of GMP-Grade CD8-Depleted Donor Lymphocytes after T-Cell Depleted Reduced-Intensity Transplantation.

Abstract The combination of fludarabin (150mg/m2) / melphalan (140mg/m2) based reduced-intensity allo-transplantation (RIT) with in vivo T-cell depletion (TCD) by the anti-CD52 antibody alemtuzumab (100mg) has demonstrated efficient engraftment and reduced graft-versus-host disease (GVHD) (Kottaridis et al., Blood 2000). However, the slow lymphocyte recovery following this protocol is associated with reduced anti-infectious and antitumor immunity. In an attempt to improve immune-reconstitution after TCD / RIT, we investigated the early preemptive use of CD8-depleted donor lymphocyte infusions (DLIs). For CD8 depletion of donor leukaphereses, a new depletion protocol using clinical grade CD8…

Maintenance and Function of Human CD8+ T Cells and NK Cells in Humanized Mice

Human CD8+ T lymphocytes and NK cells can be successfully engrafted in highly immuno-deficient mouse strains such as NOD/shi-SCID/γgcnull (NOG), NOD/SCID/IL2Rγnull (NSG), NOD/Rag1KO/γcnull (NRG), and BALB/c-Rag2KO/γcnull (BRG) mice following reconstitution with human CD34+ hematopoietic stem cells (HSCs) or, alternatively, upon adoptive transfer of peripheral blood mononuclear cells (PBMC). These humanized immune system (HIS) mice have evolved as a promising tool to study human CD8+ T cell and NK cell-mediated immune responses to cancer and infectious diseases and to explore new approaches in adoptive immunotherapy and vaccination. However, long-term generation of CD8+ T lymphocytes and NK …

Major histocompatibility complex class II binding site for streptococcal pyrogenic (erythrogenic) toxin A.

Streptococcal pyrogenic exotoxin A (SPEA) is an important pathogenicity factor of group A streptococci. It is a member of the family of „superantigens” produced by Staphylococcus aureus and Streptococcus pyogenes and its T lymphocyte stimulating activity is involved into the pathogenesis of certain diseases caused by pyogenic streptococci. In this study we have produced and characterized recombinant SPEA molecules in Escherichia coli. These molecules are indistinguishable from natural SPEA in both T cell stimulatory and HLA class II binding activities. Human class II molecules are more efficient than mouse class II molecules in presenting SPEA to T cells. In binding tests to major histocomp…

Mutations affecting MHC class II binding of the superantigen streptococcal erythrogenic toxin A.

Streptococcal pyrogenic exotoxin A (SPEA) is an important pathogenicity factor of group A streptococci. It is a member of the family of 'superantigens' produced by Staphylococcus aureus and Streptococcus pyogenes, and its T lymphocyte stimulating activity is involved in the pathogenesis of certain diseases caused by pyogenic streptococci. In this study we have generated nine mutant SPEA molecules by substituting amino acids in the regions of homology between different streptococcal and staphylococcal superantigens. An additional mutant was created by deletion of the 10 N-terminal amino acids. The mutants were expressed as fusion proteins. Several mutations led to a loss of function due to a…