Dissecting the Immune Cell Progeny of CAR-Expressing Hematopoietic Stem Cells in a Humanized Mouse Model

Background: Chemotherapy-refractory or recurrent B-lineage leukemias and lymphomas yield less than 50% of chance of cure. Therapy with autologous T-cells expressing chimeric antigen receptors (CAR) have led to complete remissions, but the effector cells may not persist, limiting clinical efficacy. Our hypothesis is the modification of hematopoietic stem cells (HSC) with anti-CD19 CAR will lead to persistent generation of multilineage target-specific immune cells, enhancing graft-versus-cancer activity and leading to development of immunological memory. Design/Methods: We generated second-generation CD28- and 4-1BB-costimulated CD19-specific CAR constructs using third-generation lentiviral vectors for modification of human HSC for assessment in vivo in NSG mice engrafted neonatally with human CD34-positive cells. Cells were harvested from bone marrows, spleens, thymus and peripheral blood at different time points for evaluation by flow cytometry and ddPCR for vector copy numbers. Cohorts of mice received tumor challenge with subcutaneous injection of lymphoma cell lines. Results: Gene modification of HSC with CD19-specific CAR did not impair differentiation or proliferation in humanized mice, leading to CAR-expressing cell progeny in myeloid, NK and T-cells. Humanized NSG engrafted with CAR-modified HSC presented similar humanization rates to non-modified HSC, with multilineage CAR-expressing cells present in all tissues with stable levels up to 44 weeks post-transplant. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. T-cell populations were identified at higher rates in humanized mice with CAR-modified HSC in comparison to mice engrafted with non-modified HSC. CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice engrafted with CAR-modified HSC successfully presented tumor growth inhibition and survival advantage at tumor challenge with lymphoma cell lines, with no difference between both constructs (62.5% survival for CD28-costimulated CAR and 66.6% for 41BB-costimulated CAR). In mice sacrificed due to tumor development, survival post-tumor injection was directly correlated with tumor infiltration by CAR T-cells. Conclusions: CAR modification of human HSC for cancer immunotherapy is feasible and continuously generates CAR-bearing cells in multiple lineages of immune cells. Targeting of different malignancies can be achieved by adjusting target specificity, and this approach can augment the anti-lymphoma activity in autologous HSC recipients. It bears decreased morbidity and mortality and offers alternative therapeutic approach for patients with no available sources for allogeneic transplantation, benefiting ethnic minorities. Disclosures De Oliveira: National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding.

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Absence of Evidence Implicating Hematopoietic Stem Cells As Common Progenitors for DLBCL Mutations

Blood ◽

10.1182/blood.v128.22.4107.4107 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4107-4107

Author(s):

Max Jan ◽

Florian Scherer ◽

David M. Kurtz ◽

Aaron M Newman ◽

Henning Stehr ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

B Cells ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Phylogenetic Trees ◽

Somatic Mutations ◽

Hematopoietic Stem ◽

Tumor Biopsies ◽

Myd88 L265p

Abstract Background: Pre-leukemic hematopoietic stem cells (HSC) have been implicated in AML (Jan et al STM 2012) and also for several lymphoid leukemias including ALL, HCL, and CLL. Separately, relapse of ALL following CD19 CAR-T cell therapy has been associated with lymphomyeloid lineage switch. Finally, healthy persons with clonally expanded HSCs are at increased risk of hematologic malignancies including lymphomas, and in mouse DLBCL models we previously demonstrated the oncogenic sufficiency of BCL6 overexpression in HSC (Green et al 2014 Nat Comm). Nevertheless, the cellular origin of DLBCL in the majority of patients is not definitively known. We sought to investigate the presence of mutations found in DLBCL within matched HSCs. Methods: We deeply genotyped somatic mutations in diagnostic biopsy tissues of 16 patients with DLBCL using CAPP-Seq to a median sequencing depth of 1100x (Newman et al 2014 Nat Med; Scherer et al 2015 ASH). We then profiled each patient for evidence implicating HSCs using somatic mutation lineage tracing, in either direct or indirect fashion. For direct evaluation, we used highly purified, serially FACS-sorted HSCs from grossly uninvolved bone marrow (BM) (n=5; Fig 1a-b). For indirect assessment, we either profiled serial tumor biopsies (n=13), or interrogated sorted cells from terminally differentiated blood lineages (n=7), including peripheral CD3+ T cells, CD14+ Monocytes, and B cells expressing a light-chain discordant to that of tumor isotype. HSCs and differentiated lineages were then interrogated by direct genotyping, using 3 highly sensitive orthogonal quantitative methods, including Myd88 L265P droplet digital PCR (n=6), BCL6 translocation breakpoint qPCR (n=4), and DLBCL CAPP-Seq profiling of 268 genes (n=5). We used the theoretical limit of detection (LOD) genotyping performance for CAPP-Seq (0.001%, Newman et al 2016 Nat Biotech), and established analytical sensitivity of our custom MYD88 ddPCR via limiting dilution (~1%). These LODs met or exceeded the expected limit of sorting impurity by FACS (~1%). For 6 patients experiencing one or more DLBCL relapse, we deeply profiled 13 serial tumor biopsies by CAPP-Seq, and then assessed overlap in somatic mutations and VDJ sequences in biopsy pairs as additional indirect evidence implicating HSCs. Results: We obtained a median of ~2000 sorted HSCs and ~1700 sorted cells from differentiated lineages, and genotyped each population using one or more of the 3 direct genotyping methods described above. Three patients with sufficient cell numbers were profiled both by CAPP-Seq and either ddPCR (n=2) or qPCR (n=1). Surprisingly, we found no evidence implicating HSCs either directly or indirectly in any of the 16 patients, regardless of the assay employed or the cell types/lineages genotyped (e.g., Fig 1b). In 2 patients with MYD88 L265P mutations, we found evidence for MYD88+ B-cells with discordant light chains by ddPCR (~0.1%) potentially implicating common lymphoid precursors (CLPs), but found no evidence for similar involvement of T-cells or monocytes. In 6 DLBCL patients experiencing relapse, tumor pairs profiled by CAPP-Seq (median depth 957) shared 93% of somatic mutations (75-100%, Fig 1c). Such pairs invariably shared clonal IgH VDJ rearrangements (4/4, 100%), thus implicating a common progenitor arising in later stages of B-cell development, not HSCs. Conclusions: We find no evidence to implicate HSCs in the derivation of DLBCL. While formal demonstration of absence of pre-malignant HSCs in DLBCL would require overcoming practical and technical limitations (including number of available HSCs, sorting purity, and genotyping sensitivity), the pattern of shared somatic alterations at relapse makes this highly unlikely. We speculate that unlike lymphoid leukemias, the cell-of-origin for most DLBCLs reside later in B-lymphopoiesis, beyond CLPs. Figure. (a) HSC sorting from BM by FACS (b) Allele frequencies of mutations found by CAPP-Seq in an examplary DLBCL case (x-axis) compared to the same variants in HSCs (y-axis). (c) Phylogenetic trees of DLBCL patients experiencing relapse (n=6) with tumor pairs sequenced by CAPP-Seq. Shown are the evolutionary distances between (i) germline and common inferrable progenitor (CIP) illustrating the fraction of shared mutations between tumor pairs, and (ii) CIP and both diagnostic (tumor 1) and relapse tumors (tumor 2) indicating unique mutations to each tumor. Figure. (a) HSC sorting from BM by FACS (b) Allele frequencies of mutations found by CAPP-Seq in an examplary DLBCL case (x-axis) compared to the same variants in HSCs (y-axis). (c) Phylogenetic trees of DLBCL patients experiencing relapse (n=6) with tumor pairs sequenced by CAPP-Seq. Shown are the evolutionary distances between (i) germline and common inferrable progenitor (CIP) illustrating the fraction of shared mutations between tumor pairs, and (ii) CIP and both diagnostic (tumor 1) and relapse tumors (tumor 2) indicating unique mutations to each tumor. Disclosures Newman: Roche: Consultancy. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Diehn:Novartis: Consultancy; Quanticel Pharmaceuticals: Consultancy; Roche: Consultancy; Varian Medical Systems: Research Funding.

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CD34+ Hematopoietic Stem Cells Exert Accessory Function in Lipopolysaccharide-induced T Cell Stimulation and CD80 Expression on Monocytes

Journal of Experimental Medicine ◽

10.1084/jem.189.4.693 ◽

1999 ◽

Vol 189 (4) ◽

pp. 693-700 ◽

Cited By ~ 15

Author(s):

Taila Mattern ◽

Gundolf Girroleit ◽

Hans-Dieter Flad ◽

Ernst T. Rietschel ◽

Artur J. Ulmer

Keyword(s):

Stem Cells ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cells ◽

Cell Stimulation ◽

Hematopoietic Stem ◽

Accessory Function ◽

T Cell Stimulation ◽

Blood Stem Cells

CD34+ hematopoietic stem cells, which circulate in peripheral blood with very low frequency, exert essential accessory function during lipopolysaccharide (LPS)-induced human T lymphocyte activation, resulting in interferon γ production and proliferation. In contrast, stimulation of T cells by “conventional” recall antigens is not controlled by blood stem cells. These conclusions are based on the observation that depletion of CD34+ blood stem cells results in a loss of LPS-induced T cell stimulation as well as reduced expression of CD80 antigen on monocytes. The addition of CD34-enriched blood stem cells resulted in a recovery of reactivity of T cells and monocytes to LPS. Blood stem cells could be replaced by the hematopoietic stem cell line KG-1a. These findings may be of relevance for high risk patients treated with stem cells or stem cell recruiting compounds and for patients suffering from endotoxin-mediated diseases.

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In Utero Transplantation of Transduced Hematopoietic Stem Cells Results in Deletion of Transgene-Specific T Cells.

Blood ◽

10.1182/blood.v108.11.3266.3266 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3266-3266

Author(s):

Pablo Laje ◽

William H. Peranteau ◽

Masayuki Endo ◽

Philip W. Zoltick ◽

Alan W. Flake

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

Hematopoietic Stem Cells ◽

T Cell Receptor ◽

Peripheral Blood ◽

T Cell Development ◽

Cell Receptor ◽

In Utero ◽

Hematopoietic Stem

Abstract The developing fetal immune system provides a unique opportunity to manipulate normal immunologic development for therapeutic prenatal and anticipated postnatal interventions. In previous studies we have shown that allogeneic in utero hematopoietic cell transplantation (IUHCT) results in donor specific tolerance that can subsequently facilitate non-myeloablative postnatal cellular or organ transplants. It follows that in utero injection of transduced hematopoietic stem cells (HSC) could potentially induce tolerance to a transgene encoded protein. We hypothesized that expression of a transduced antigenic protein by HSC and their progeny would alter thymic T cell development resulting in deletion of antigen specific T-cells. To test this hypothesis, we used the mammary tumor virus (MTV) superantigen system to evaluate the effect of IUHCT of transduced HSC on T cell development. In this system, expression of different MTV oncogenes by different I-E+ strains of mice results in deletion of T cells expressing the relevant Vβ T cell receptor. Specifically, mice which are Mtv7+ delete T cells expressing the Vβ6 T-cell receptor. In this study, CD150+CD48− enriched Balb/c (I-E+ Mtv7−) HSC were transduced with an HIV-based lentivirus expressing MTV7 under an MND promoter. 1.5E+05 transduced cells were injected intravascularly via the vitelline vein into E14 Balb/c fetuses. Non-injected age matched naive Balb/c mice served as the control group. The peripheral blood (PB) and thymuses of injected fetuses and control mice were harvested at day of life (DOL) 10, 20 and 60 and analyzed by flow cytometry for T lymphocyte Vβ6 expression. Additionally, the T cell composition of the thymus was assessed at DOL10 for CD4 and CD8 single positive (SP) and CD4/CD8 double positive (DP) cells. Thymic flow cytometric analysis at DOL10 revealed that greater than 98% of the T cells were CD4CD8 DP, a stage that has not yet undergone negative selection. No significant difference was noted in the percentage of thymic Vβ6+ DP T-cells at this time point or at DOL20 and DOL60. In contrast, there was a significant decrease in the percentage of Vβ6+ peripheral blood SP cells in those mice injected with MTV7 transduced HSC relative to control mice at DOL10, DOL20 and DOL60 (p<0.05) (Fig 1). The current study supports the ability of enriched transduced HSC to induce deletion of transgene specific T cells after IUHCT. In the future, this strategy may be useful to promote tolerance for pre or postnatal cellular or gene therapy. Figure Figure

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Hematopoietic stem cell dose correlates with the speed of immune reconstitution after stem cell transplantation

Blood ◽

10.1182/blood-2003-07-2534 ◽

2004 ◽

Vol 103 (11) ◽

pp. 4344-4352 ◽

Cited By ~ 50

Author(s):

Benny J. Chen ◽

Xiuyu Cui ◽

Gregory D. Sempowski ◽

Jos Domen ◽

Nelson J. Chao

Keyword(s):

Stem Cells ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

B Cells ◽

Hematopoietic Stem Cells ◽

Immune Reconstitution ◽

Hematopoietic Stem ◽

Time Points ◽

Cell Dose

Abstract In the current study, we tested whether higher numbers of hematopoietic stem cells correlate with the speed of immune reconstitution in a congenic transplantation model (C57BL/Ka, CD45.1, Thy1.1→C57BL/6, CD45.2, Thy1.2) using purified hematopoietic stem cells (c-Kit+Thy1.1lowLin-/lowSca-1+). There were 3 different doses of stem cells used (400, 1000, and 5000). Phenotypic analyses in peripheral blood and spleen demonstrated that higher numbers of infused stem cells are associated with more rapid regeneration of T cells (CD4+, CD8+, naive CD4+, naive CD8+) and B cells at early time points. The numbers of T and B cells eventually became equivalent between different dose groups at late time points. Production of interleukin-2 and inter-feron-γ per T cell was similar regardless of stem cell dose even when tested at the time when there were significant differences in peripheral T-cell counts. The improved immune recovery was attributed to a more rapid regeneration of donor-type immune cells. Higher numbers of total thymocytes and signal joint T-cell receptor excision circles were observed in the higher dose stem cell recipients, suggesting that accelerated regeneration of T cells was due to enhanced thymopoiesis. (Blood. 2004;103:4344-4352)

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LRF Regulates Self-Renewal of Hematopoietic Stem Cells by Blocking Notch1-Mediated T Cell Differentiation

Blood ◽

10.1182/blood.v112.11.75.75 ◽

2008 ◽

Vol 112 (11) ◽

pp. 75-75 ◽

Cited By ~ 2

Author(s):

Sung-UK Lee ◽

Manami Maeda ◽

Nagisa Sakurai ◽

Julie Teruya-Feldstein ◽

Freddy Radtke ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Cell Differentiation ◽

T Cell ◽

Hematopoietic Stem Cells ◽

B Cell ◽

Knockout Mice ◽

Cell Development ◽

B Cell Development ◽

Hematopoietic Stem

Abstract The proto-oncogene LRF, encoded by the Zbtb7a gene, is a transcriptional repressor that belongs to the POK (POZ/BTB and KrŸppel) protein family. Along with its oncogenic property, recent evidence has shown that POK proteins play distinct roles in hematopoiesis and immune system development. Conditional inactivation of the LRF gene in mouse hematopoietic stem cells (HSCs) results in the development of CD4/8 double positive (DP) T cells in bone marrow (BM) at the expense of B cell development (Maeda et al. Science 2007). While LRF acts as a master regulator of B versus T lymphoid lineage fate decision by suppressing Notch-mediated signals, it is unclear as to which Notch genes LRF targets and whether LRF is required for the maintenance of HSCs per se. To address these questions, we analyzed HSC/progenitor population of conditional LRF knockout mice (LRFF/FMx1-Cre) as well as LRF/Notch1 double conditional knockout mice (LRFF/FNotch1F/FMx1-Cre). In the absence of Notch1, LRF deficient HSCs/lymphoid progenitors (LRFF/FNotch1F/FMx1-Cre) could successfully give rise to early B cells (Pro B, Pre B and immature B). There were no abnormal DP-T cells seen in the BM, suggesting that LRF primarily targets Notch1 at the HSC/progenitor stages to maintain normal lymphoid development. However the loss of the LRF gene did not rescue the phenotype of Notch1F/FMx1-Cre mice (Radtke et al. Immunity 1999). Immature B cell development in the thymus was still observed in LRFF/FNotch1F/FMx1-Cre mice, suggesting that LRF acts genetically upstream of Notch1 during the early lymphocyte development. Notably, LRFF/FNotch1F/FMx1-Cre mice still exhibit a block of terminal erythroid differentiation and macrocytic anemia as seen in LRFF/FMx1-Cre mice. Thus, LRF is required for erythropoiesis via Notch-independent mechanisms. To further identify distinct HSC/progenitor compartments, we performed multicolor-FACS analysis utilizing antibodies for SLAM family members (CD41, CD48 and CD150), c-Kit, Sca-1, Flt3, IL7R-α, Vcam-1 and lineage markers (Lin). Remarkably, no Flt3 positive HSC/progenitors were observed in LRFF/FMx1-Cre mice. While IL7R-α+ T cell precursors (IL7Rα+Lin-Sca1+c-Kit+Flt3-), which were previously reported as common lymphoid progenitors (Maeda et al. Science 2007), existed abundantly. Absolute numbers of the long-term HSCs (LT-HSCs), defined as CD150+CD48-Flt3-Vcam-1+IL7Rα-LSK (Lin-Sca1+c-Kit+), were significantly reduced in LRFF/FMx1-Cre mice one month after pIpC injection. At the same time, CD150+CD48high+Flt3-Vcam-1-IL7Rα-LSK cells, which are likely T-committed lymphoid precursors, are increased in LRFF/FMx1-Cre mice. To investigate the presence of a population of quiescent HSC/progenitors, we treated LRFF/FMx1-Cre mice with 5-fluorouracil (5-FU), a S phase-specific cytotoxic chemotherapeutic agent, and examined recovery of HSCs in BM. LT-HSCs in LRFF/FMx1-Cre mice did not repopulate as many as their counterpart one month after 5-FU treatment. Our data indicates that LRF deficient HSCs are unable to maintain its quiescent status and are on the state of cell differentiation toward T cells due to the high Notch activity. In fact, loss of the Notch1 gene partially rescued reduced LT-HSCs numbers seen in LRFF/FMx1-Cre mice.

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GPI-AP Deficient Cells Detected Exclusively in T Cells in Patients with Aplastic Anemia: Evidence against An Escape Mechanism for the Initial Proliferation of PIG-AMutant Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.3197.3197 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3197-3197

Author(s):

Takamasa Katagiri ◽

Zhirong Qi ◽

Yu Kiyu ◽

Naomi Sugimori ◽

J. Luis Espinoza ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Immune System ◽

T Cell ◽

Hematopoietic Stem Cells ◽

Aplastic Anemia ◽

Effector Memory ◽

Hematopoietic Stem ◽

Escape Theory ◽

Effector Memory Cells

Abstract Abstract 3197 Poster Board III-134 Small populations of glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient blood cells are often detectable in the peripheral blood (PB) of patients with aplastic anemia (AA) and refractory anemia (RA) of myelodysplastic syndromes defined by the FAB classification. Such PNH-type cells are thought to be derived from PIG-A mutant hematopoietic stem cells (HSCs) that avoid the immunological attack against HSCs. Inefficient T cell responses to PNH-type cells were indeed demonstrated by a murine study. However, there is no direct evidence in support of the escape theory concerning the expansion of PIG-A mutant HSCs in such patients with bone marrow (BM) failure. If the escape theory is true, the PNH-type cells should be detected in myeloid cells derived from HSCs that are targeted by the immune system attack. The PB of 527 patients with BM failure was examined for the presence of GPI-AP deficient cells in various lineages of cells including granulocytes, erythrocytes, monocytes, T cells, B cells, and NK cells using high sensitivity flow cytometry to verify this hypothesis. PNH-type cells were detectable in at least one lineage of cells from 228 (43%) patients. Although most of the positive patients showed PNH-type cells in two or more lineages including granulocytes or monocytes, 14 patients (13 with AA and 1 with amegakaryocytic thrombocytopenia) displayed PNH-type CD48-CD55-CD59- cells only in T cells at a frequency of 0.003-0.3% of the total T cells (Figure). The PNH-type T cells were undetectable in any of 25 healthy individuals. The CD48-CD55-CD59- T cells consisted of predominantly effector memory and terminal effector memory cells with naïve phenotype cells. The phenotypic pattern of the PNH-type T cells was very similar to that of CD48-CD55-CD59- T cells from 11 patients with florid PNH but was different from that of CD48-CD55-CD59- T cells (central and effector memory cells alone) detected in 4 marrow transplant recipients who received anti-CD52 antibody (alemtuzumab) therapy as conditioning. PIG-A gene analyses of CD48-CD55-CD59- T cells revealed a single mutation in 2 patients with PNH-type T cells alone, while two different mutations were revealed in 2 patients treated with alemtuzumab. BM failure patients with PNH-type T cells alone and other BM failure patients possessing PNH-type granulocytes or monocytes showed similar clinical features characterized by predominant thrombocytopenia and good response to immunosuppressive therapy, thus suggesting an increase in the number of PNH-type cells in both groups to be associated with a similar immune pathophysiology. The escape theory cannot account for the presence of PNH-type cells exclusively in T cells in immune-mediated BM failure because T cell precursors are not the target of the immune system attack in AA. Therefore, mechanisms other than the escape theory must be considered for the initial proliferation of PIG-A mutant HSCs associated with the development of AA, such as preferential activation of dormant PIG-A mutant HSCs or T cell precursors due to the deficiency of GPI-APs that transmit negative signal Disclosures No relevant conflicts of interest to declare.

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The Liver Can Host Donor Hematopoiesis and Maintains a Strong Population of Innate Immune Cells That Contributes to Host Protection After Transplantation of Purified Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.3545.3545 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3545-3545

Author(s):

Pelu Tran ◽

Antonia MS Mueller ◽

Judith Shizuru

Keyword(s):

Stem Cells ◽

Innate Immunity ◽

T Cells ◽

Hematopoietic Stem Cells ◽

Nk Cells ◽

Immune Cells ◽

Lymphoid Cells ◽

Hematopoietic Stem ◽

Host Protection

Abstract Abstract 3545 Poster Board III-482 Standing in the line of first defense, the liver is a critical immunocompetent organ. It is armed with lymphocytes, including T cells (TC), natural killer (NK) cells, NK T cells, and a variety of antigen-presenting cells (APC), such as dendritic cells and resident macrophages (Mph), called Kupffer cells. Because it is exposed to large amounts of toxins and antigens, both destructive and harmless, liver immunity must provide immunogenic and tolerogenic mechanisms. Moreover, as the organ of fetal blood production the liver can, if required, resume its hematopoietic function. Here, we studied the role of the liver as a hematopoietic and lymphatic organ after hematopoietic cell transplantation (HCT). Lethally irradiated BALB.K and BALB.B mice were given MHC-matched, FACS purified hematopoietic stem cells (HSC; cKit+Sca1+Thy1.1loLin-) from AKR/J and C57BL/6 donors, respectively, alone or supplemented with 10∧7 splenocytes (SP) for GVHD induction. Mononuclear cells (MNC) were Ficoll-separated from flushed livers 1 to 6 weeks (w) post transplant (pTX) and FACS analyzed. In recipients of TC-containing grafts, the liver was a major target organ of acute graft-vs-host disease (GVHD) with prominent donor lymphocyte expansion causing destruction of the hepatic portal morphology. Rare HSC-derived cells were observed in the livers. In contrast, mice given purified HSC showed no clinical or histological signs of GVHD, yet early pTX a high proportion of donor HSC-derived MNC was observed within the livers, comprising ∼75% of the MNC at 2w. Phenotype analysis revealed that these HSC-derived MNC were primarily NK cells (DX5+CD122+) or Mph (Mac1+F4/80+). In fact, amongst all nucleated cells, NK cells represented >10% and were mixed donor/host type. Interestingly, the Mph were all donor derived. This observation of over-representation by cells of innate immunity (including NK cells and Mph) in livers of recipients of HSC alone led us to hypothesize that these cells might exert protective functions against increased amounts of pathogens and toxins entering the circulation from irradiation-damaged intestines. Thus, to suppress donor Mph reconstitution pTX, silica was injected intraperitoneally on d-1, and every 3d thereafter. All recipients of HSC alone recovered rapidly after irradiation (d5-7), while at this time point recipients of HSC plus silica showed severe weight loss, hunched posture, ruffled fur, diarrhea, with <50% (7/15) survival. These survivors clinically stabilized around d12, suggesting that the intestines recovered from injury. To test if the presence of the HSC derived NK cells and APC could contribute to host protection from GVHD, a lethal dose of SP (10∧7) was injected simultaneously with HSC, or with a delay of 7d or 9d. All mice given SP on d0 died within 9d and 3/5 of those receiving SP on d7 died by d12. However, all mice given SP on d9 recovered fully and showed no signs of GVHD, despite the lymphopenic host environment that usually promotes homeostatic expansion of mature donor TC. In conclusion, the role of the liver as an immunologically active organ after ‘conventional’ HCT is often masked by donor TC expansion with subsequent GVHD. Here, we provide evidence that if grafts are devoid of mature lymphoid cells, innate immunity recovers rapidly, and in fact exceeds unmanipulated controls. Donor Mph may protect the host from pathogens and endotoxemia. Moreover, they may neutralize activated donor TC and thereby mediate tolerance between donor and host. Likewise, the elevated proportion of donor and host NK cells, which is lacking in GVHD affected mice, suggest another beneficial mechanism of protection, as NK cells have been reported to be capable of reducing GVHD. Immunohistochemical studies for a better quantitative assessment of resident immune cells in the liver pTX are underway. Disclosures: No relevant conflicts of interest to declare.

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Development of Novel Stem Cell Transplantation and Gene-Immunotherapy Using WT1-Specific T-Cell Receptor Gene.

Blood ◽

10.1182/blood.v114.22.3028.3028 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3028-3028

Author(s):

Toshiki Ochi ◽

Hiroshi Fujiwara ◽

Kozo Nagai ◽

Toshiaki Shirakata ◽

Kiyotaka Kuzushima ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Hematopoietic Stem Cells ◽

Cell Transplantation ◽

Clinical Efficacy ◽

Cd8 T Cells ◽

Hematopoietic Stem

Abstract Abstract 3028 Poster Board II-1004 Purpose The Wilms' tumor 1 (WT1) is one of the zinc-finger transcriptional regulators, and its expression level is very low in most tissues of adults. In contrast, various kinds of leukemia and solid tumors express WT1 abundantly, and high expression level of WT1 is correlated with disease aggressiveness and poor prognosis. These findings indicate that WT1 is a promising target antigen for anti-cancer cellular immunotherapy. Following identification of immunogenic epitopes derived from WT1 which are recognized by HLA class I-restricted and HLA class II-restricted T cells, phase I/II WT1 peptide vaccination trials have been conducted. Although the positive correlation between the clinical efficacy and vaccine-induced WT1-specific T-cell response has been reported, the clinical efficacy is not satisfactory. Adoptive transfer of WT1-specific T cells seems to be the promising approach to achieve marked improvement in clinical efficacy of WT1-targeting immunotherapy, however, it still remains difficult to expand WT1-specific T cells sufficiently ex vivo. To overcome these problems, we attempted to establish gene-immunotherapy targeting WT1 using T-cell receptor (TCR) gene isolated from the WT1-specific T-cell clone. We also verified the feasibility of novel stem cell transplantation by transducing WT1-specific TCR gene into hematopoietic stem cells. Methods We cloned the full length TCR-αa and -β genes from a WT1235-243-specific and HLA-A*2402-restricted cytotoxic T lymphocyte (CTL) clone. The WT1-specific TCR gene-repressing retroviral and lentiviral vectors were constructed. Retroviral vector was transduced to human peripheral T cells in retronectin-coated plate. WT1-specific functions of TCR gene-transduced CD8+ T cells and CD4+ T cells were examined by evaluating WT1 peptide-specific cytotoxicity by 51Cr-release assay and WT1 peptide-specific Th1 cytokine production, respectively. To improve the efficacy of WT1-specific TCR expression, we developed the novel retroviral vector which can inhibit selectively intrinsic TCR expression (si-TCR vector). Finally, we transduced the WT1-specific TCR lentiviral vector into human cord blood CD34+ cells, and transplanted them to NOD/SCID/common-γnull mice. Then, we examined whether WT1-specific human mature T cells can differentiate in mice. The presence of WT1-specific human T cells in mice was determined by tetramer assay and IFN-γ production in response to stimulation with WT1 peptide. Results Following transfer of WT1-specific TCR gene into peripheral blood lymphocytes, WT1 peptide-specific CD8+ and CD4+ T cells could be expanded easily in vitro. TCR gene-transduced CD8+ T cells exerted cytotoxicity against WT1 peptide-pulsed target cells and human leukemia cells in an HLA-A*2402-restricted manner. Similarly, TCR gene-transduced CD4+ T cells showed WT1-specific Th1 cytokine production in response to stimulation with human leukemia cells in HLA-A*2402-restricted fashion depending on the interaction of CD4 and HLA class II molecules. The newly developed si-TCR vector appeared to inhibit expression of endogenous TCR efficiently and improved the efficacy of WT1-specific TCR expression 3 to 5-fold higher as compared to the conventional vector. Three months after transplantation of WT1-specific TCR gene-transduced human hematopoietic stem cells in NOD/SCID/common-γnull mice, differentiation of WT1-specific human T cells in murine spleen was evaluated. Tetramer assay revealed that human mature T cells expressing WT1-specific TCR on their cell surface were clearly detected. Furthermore, these WT1-specific CD8+ T cells appeared to produce IFN-γ in response to stimulation with WT1 peptide-loaded HLA-A*2402-positive cells. Conclusion The adoptive gene-immunotherpay using WT1-specific TCR gene against leukemia seems to be promising. Moreover, the novel stem cell transplantation using WT1-specific TCR gene-transduced hematopoietic stem cells might open the door to induce long-lasting anti-leukemic cellular immunity in patients with leukemia. Disclosures No relevant conflicts of interest to declare.

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In vivo depletion of hematopoietic stem cells in the rat by an anti-CD45 (RT7) antibody

Blood ◽

10.1182/blood.v99.10.3566 ◽

2002 ◽

Vol 99 (10) ◽

pp. 3566-3572 ◽

Cited By ~ 18

Author(s):

Marc H. Dahlke ◽

Oliver S. Lauth ◽

Mark D. Jäger ◽

Till Roeseler ◽

Kai Timrott ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

B Cells ◽

Hematopoietic Stem Cells ◽

Precursor Cells ◽

Severe Thrombocytopenia ◽

Antibody Treatment ◽

Hematopoietic Stem

Anti-CD45 monoclonal antibodies (mAbs) are potentially powerful tools for the depletion of mature leukocytes. As their application for immunotherapy also depends on their effects on bone marrow (BM) progeny, the in vivo effects of an anti-CD45 mAb (anti-RT7a mAb) on BM precursor cells were analyzed in a rat model. Anti-RT7a mAb treatment was performed in LEW.1W (RT1u RT7a) rats with the use of different dosages. In addition, major histocompatibility complex (MHC)–congenic BM transplantation making use of a diallelic polymorphism (RT7a/RT7b) of rat CD45 was applied. Following injection of anti-RT7a mAb into normal LEW.1W rats, T cells were profoundly depleted in blood, lymph nodes, and spleen, whereas B cells were coated only by the antibody. Single injection of anti-RT7a mAb in a high dose induced a lethal aplastic syndrome with severe thrombocytopenia. Rescue of antibody-treated animals with BM from congenic LEW.1W-7B rats (RT1u RT7b) and transplantation of BM from LEW.1W rats pretreated with anti-RT7a mAb into sublethally irradiated LEW.1W-7B recipients revealed a profound effect of the mAb on progeny of myeloid and T-cell lineage. Following repeated antibody treatment of stable mixed chimeras (RT7b/RT7a), very few RT7a-positive B cells were still detectable after 6 months and their number declined during the subsequent year. These observations show that this anti-RT7a mAb effectively depletes mature T cells as well as BM precursor cells of myeloid, T-cell, and thrombocytic lineage after in vivo application. In contrast, mature B cells are not depleted, but precursors also appear to be eliminated. Overall, the findings suggest that the anti-RT7a mAb efficiently depletes early rat hematopoietic stem cells.

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