Function and Clonality of T-Cells In Myelodysplasia

Abstract Abstract 4972 Objective: Myelodysplastic (MDS) syndromes are clonal disorders arising from haematopoietic multi-potential stem cells. Research has implied that cells of the immune system play a role in the disease process. The aim of this study was to examine the clonality, response to mitogens, effects of lymphocytes on bone marrow clonogenic progenitors and proliferative capacity of selected marrow progenitors in a well defined cohort of MDS patients. Methodology: Seventeen patients with MDS were accrued. The clonality of the T-cells was studied using the TCR Vβ repertoire kit. In order to assess in vitro function, the lymphocytes were activated using phorbol 12-myristate13 acetate (PMA) and thereafter analysed for expression of the activation antigen CD69 using standard flow cytometry. The results were compared to age matched controls. The effect of the autologous blood mononuclear cell populations on clonogenic growth was studied by culturing Li- selected CD34+ cells with both PMA activated and non-activated autologous lymphocytes using a non-contact double layer culture technique. Results: T-cells did not demonstrate monoclonality, although skewing of the T-cell repertoire was observed. The median percent of CD3+ T-cells expressing CD69 after activation was 30% (9.5 – 95.4%). This was reduced in comparison to age matched controls (p=0.025). The effect of the lymphocytes on clonogenic cell growth was heterogeneous however median values demonstrated a cell dose response in the colony numbers directly related to cultured lymphocyte numbers (p=0.04 compared to no lymphocytes added). In addition, the median CFU-GM scores were higher when cultured with PMA activated lymphocytes (p=0.05). This pattern was not significantly different to normal controls. Background: Our investigations suggest that in this cohort of MDS patients the T-cells were not clonal but had reduced activation capacity when stimulated with the mitogen PMA. However, their ability to stimulate colony growth of autologous bone marrow CD34+ cells was preserved. Furthermore, selected CD34+ MDS cells proliferated well, with clone numbers not different from control. These observations imply that the previously reported abnormal T-cell responses and poor clonogenic growth may be the result of complex interactions between T-cells, the malignant clone and accessory cells in the bone marrow stroma. Further study examining each of these cell populations is required to better understand the mechanism of marrow failure and progression to leukaemia. Disclosures: No relevant conflicts of interest to declare.

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Adoptive Immunotherapy with Tregs Prevents GvHD and Favours Immune Reconstitution After HLA Haploidentical Transplants for Hematological Malignancies.

Blood ◽

10.1182/blood.v114.22.4.4 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4-4 ◽

Cited By ~ 10

Author(s):

Mauro Di Ianni ◽

Franca Falzetti ◽

Alessandra Carotti ◽

Adelmo Terenzi ◽

Elisabetta Bonifacio ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

High Risk ◽

Immune Reconstitution ◽

Nk Cell ◽

Immune Recovery ◽

T Cell Repertoire ◽

Cell Repertoire ◽

Cd34 Cells

Abstract Abstract 4 Transplantation of large numbers of highly purified CD34+ cells from haploidentical relatives is a viable strategy for the cure of acute leukaemia at high risk of relapse (Aversa et al., NEJM 1998; JCO 2005). As extensive T cell depletion is required to prevent GvHD, the very narrow T cell repertoire in the inoculum delays recovery of immune response against pathogens, leading to a high incidence of infection-related deaths. Thus the key challenge is to improve immune recovery by administering allogeneic donor T cells without causing GvHD. Preclinical studies demonstrated that freshly isolated or ex vivo expanded T regulatory cells (Tregs) could be used to control GvHD following bone marrow transplantation. The present phase I/II clinical trial evaluated the impact of early infusion of freshly isolated donor CD4/CD25+ Tregs, followed by an inoculum consisting of donor mature T cells (Tcons) and positively immunoselected CD34+ cells, on GvHD prevention and immunological reconstitution. Twenty-two patients (10 male; 12 female; median age 40.5, range, 21 to 60) with AML (n=17; 8 in CR1 at high risk, 7 in ≥CR2 and 2 in relapse), ALL (n=4; 3 in CR1; 1 in relapse) and 1 with high grade NHL in relapse were enrolled from September 2008 onwards. The conditioning regimen consisted of 8Gy single fraction TBI, thiotepa (4 mg/kg×2), fludarabine (40mg/m2×4), and cyclophosphamide (35 mg/kg×2). All patients received CD4/CD25+ GMP immunoselected Tregs (CliniMACS, Miltenyi Biotec) (21/22 2×106/kg bw; 1/22 1×106/kg bw). Three days later they received positively immunoselected CD34+ cells (median 8.2, range 5.0-19.1) together with Tcons (16/22 1×106/kg bw; 4/22 0.5 ×106/kg bw; 2/22 did not receive Tcons). Immunoselected CD4/CD25+ Tregs (purity 91.5±4.5) consisted of CD25high 25.6%±11.2; CD25int 57.4%±5.9; CD25low 8.5%±6; FoxP3 64%±1; CD127 14.9%±13.7 (mean±SD). As suggested by in vitro immunosuppressive assays and by immunophenotypic analysis, the contaminating cells in the Treg fraction were 50% of the CD25int and 100% of the CD25low, so that the infused Tregs:Tcons ratio was established at 1:1.5. No post-transplant prophylaxis against GvHD was used. 20/22 patients engrafted. Neutrophils reached 1×109/L at a median of 15 days (range, 11 to 39 days). Platelets reached 25×109/L and 50×109/L at median of 13 and 15 days, respectively (range, 11 to 48 days, and 13 to 60 days). All engrafted patients showed persistent full donor-type chimerism in peripheral blood and bone marrow. Strikingly, no GvHD was observed in 17/20 valuable patients, 2/20 developed grade I cutaneous self-limited untreated GvHD and 1/20 developed grade III GvHD. This patient had received the fewest Tregs. Six patients died (1 bacterial sepsis, 2 VOD, 1 fungal pneumonia, 1 CNS aspergillosis and 1 GvHD/systemic toxoplasmosis). In contrast with our previous experience, the speed of immune recovery was enhanced. The CD4 and the CD8 counts reached, respectively, 50/μL medianly on days 34 (range, 19 to 63 days) and 24 (range, 15 to 87); 100/μL medianly on days 47 (range, 28 to 100 days) and 34 (range, 19 to 95); 200/μL on days 70 (range, 41 to 146 days) and 61 (range, 21 to 95). We also observed a rapid development of a wide T-cell repertoire and detection of high frequencies of specific CD4+ and CD8+ for opportunistic pathogens such as Aspergillus, Candida, CMV, ADV, HSV, VZV, Toxoplasma. In KIR ligand-mismatched transplants, speed of NK cell reconstitution/maturation and size of donor vs recipient alloreactive NK cell repertoires were preserved (Ruggeri et al., Science 2002). In conclusion, in the setting of haploidentical transplantation infusion of freshly purified Tregs makes administration of high dose of T cells feasible for the first time. This strategy provides a long-term protection from GvHD and robust immune reconstitution. Treg-based cellular therapy may represent an innovative strategy to improve the outcome of haploidentical transplants. Disclosures: No relevant conflicts of interest to declare.

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Generation of Human T/NK Cell Precursors for Adoptive Transfer To Enhance Immune Reconstitution in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Blood ◽

10.1182/blood.v108.11.3209.3209 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3209-3209

Author(s):

Sonali Chaudhury ◽

Johannes Zakarzewski ◽

Jae-Hung Shieh ◽

Marcel van der Brink ◽

Malcolm A.S. Moore

Keyword(s):

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Hematopoietic Stem ◽

Serum Free ◽

Cd34 Cells

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with significant post-transplant immunoincompetence which affects in particular the T cell lineage and results in an increased susceptibility to infections. Novel strategies to enhance immune recovery after HSCT could prevent malignant relapse and immune deficiency and improve the overall outcome of this therapy. We have established a serum free culture system using murine bone marrow stroma expressing the Notch ligand Delta-like 1 (DL1) to obtain high numbers of human pre-T cells from CD34+ cells. Human cord blood CD34+ cells were plated on OP9 DL1 stroma transduced with adenovirus expressing thrombopoietin (ad-TPO) at an MOI of 30. Media used was QBSF-60 (Serum free media prepared by Quantity Biologicals) supplemented with Flt-3 ligand and IL-7 (10ng/ml). At 4–5 weeks we obtained a 10 5–10 7 fold expansions of cultured cells of which about 70–80% were CD5, CD7 positive pre T cells (Fig 1). We then developed an optimal system to study human lymphohematopoiesis using mouse models (NOD/SCID/IL2rϒnull and NOD/SCIDβ2null) and established an adequate pre T cell number (4 × 10 6) and radiation dose (300 Rads). We injected CD34 and pre-T cells (CD45 +, CD4−, CD5+, CD7+) derived from OP9 DL1 cultures into these mice and achieved ~50%engraftment of NK in the bone marrow and spleen of the mice at 2 weeks following transplant. The thymus from the same mice showed evidence of about 12–15% CD7+ pre T cells. We are currently studying the function of the generated NK and T cells both in vivo and in vitro studies. Figure Figure

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Immature T-Cell Populations in Lymph Nodes of Castleman Disease and Angioimmunoblastic T-Cell Lymphoma Suggest Alternate Sites of T-Cell Development,

Blood ◽

10.1182/blood.v118.21.3238.3238 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3238-3238

Author(s):

Robert S. Ohgami ◽

Shuchun Chun ◽

Jane Ohgami ◽

James L. Zehnder ◽

Matthew Van de Rijn ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Castleman Disease ◽

T Cell Lymphoma ◽

Cell Populations ◽

Lymphoid Tissues ◽

Anatomic Site ◽

Angioimmunoblastic T Cell Lymphoma

Abstract Abstract 3238 A central dogma of human immunology is that proliferation of immature T-cells, and their development after release from the bone marrow, occurs in the central thymus. Recently, we identified several patients with aberrant polyclonal immature TdT+ precursor T-cell populations in extra-thymic lymphoid tissues. Here we demonstrate that immature precursor T-cell populations, with a cortical thymocyte phenotype, in fact, are expanded in extra-thymic lymphoid tissues of patients with Castleman disease (P < 0.001; n = 29), and angioimmunoblastic T-cell lymphoma (P = < 0.001; n =31) and increased in cases of Castleman disease in association with follicular dendritic cell sarcoma (Figures 1 and 2). Analysis of the proliferation marker, MiB-1, and the morphologic presence of mitoses reveal that these populations are undergoing extra-thymic proliferation and expansion, arguing against simple release from the central thymus and sequestration in these extra-thymic organs. Finally, these populations of immature T-cells are not associated with a particular anatomic site (i.e. neck or mediastinum). These findings challenge the dogma that proliferation of immature human T-cell populations occurs nearly exclusively in the central thymus and demonstrates that stimulation and significant proliferation of extra-thymic immature T-cells does, and can occur in a subset of patients. Disclosures: No relevant conflicts of interest to declare.

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Comparative Analysis on Cellular Components of Bone Marrow Microenvironment in Normal and Aberrant Hematopoiesis

Blood ◽

10.1182/blood.v118.21.4808.4808 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4808-4808

Author(s):

Young-Ho Lee ◽

Young-hee Kwon ◽

Kyoujung Hwang ◽

Hyunju Jun ◽

Byungbae Park ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Conflicts Of Interest ◽

T Cell Subsets ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Significant Difference ◽

Stromal Cell Derived Factor ◽

And Control ◽

Cellular Components

Abstract Abstract 4808 Background: It is now evident that hematopoietic stem cells (HSCs) reside preferentially at the endosteal region within the bone marrow (BM) where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fate. We investigated the microenvironmental differences including osteoblastic activities and HSC components in myeloproliferative (chronic myeloid leukemia, CML) and hypogenerative disease (aplastic anemia, AA) as well as normal control (NC). Methods: The immunohistochemistry for osteonectin, osteocalcin, stromal cell derived factor (SDF, CXCL12), T cell, T helper/inducer cell, T suppressor/cytotoxic cell, hematopoietic stem/progenitor (CD34, CD117) and megakaryocytes was performed on BM biopsy specimens from 10 AA patients, 10 CML patients and 10 NC (lymphoma without BM involvement). The positive cells for immunohistochemical stainings except osteocalcin on each slide were calculated on 10 high power fields (HPF, ×400), and then corrected by the cellularity. The positive cells for osteocalcin were counted on the peritrabecular line on each slide, and then corrected by the mean length measured. Results: The CD34+ cells (p=0.012) and megakaryocytes (p<0.0001) were significantly lower in AA than in NC, but CD117+ cells was comparable in AA, CML, and control samples. The osteonectin+ cells (p=0.0003) were lower in CML than in AA and NC, however the osteocalcin+ cells showed wide variation (0-903/2035um) and no significant difference. The SDF+ cells (p<0.0001) was significantly higher in AA and very lower in CML, compared with NC. The counts for T cell and T cell subsets were significantly lower in CML than in NC, and higher in AA than in NC (p<0.0001). Conclusions: Cellular components of BM microenvironment in 2 hematologic diseases representative of myeloproliferation (CML) and hyporegeneration (AA) respectively are quite different. Further studies would be required to explore the role of these components for hematopoiesis and the rationale for therapeutic application. Disclosures: No relevant conflicts of interest to declare.

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Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽

10.1182/blood.v122.21.1999.1999 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1999-1999

Author(s):

Annie L. Oh ◽

Dolores Mahmud ◽

Benedetta Nicolini ◽

Nadim Mahmud ◽

Elisa Bonetti ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Nsg Mice ◽

Human Cd34 ◽

Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

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Recipient CD8+ DC Delete Alloreactive Donor CTL and Promote Leukemic Relapse after Allogeneic BMT

Blood ◽

10.1182/blood.v126.23.4279.4279 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4279-4279

Author(s):

Kate A Markey ◽

Rachel D Kuns ◽

Renee J Robb ◽

Motoko Koyama ◽

Kate Helen Gartlan ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Antigen Presentation ◽

Median Survival ◽

Conflicts Of Interest ◽

Critical Role ◽

Allogeneic Bone Marrow Transplantation ◽

Allogeneic Bone ◽

Leukemic Relapse

Abstract Allogeneic bone marrow transplantation (BMT) remains the therapy of choice for many haematological malignancies, but despite the curative benefit of the immunological graft-versus-leukemia (GVL) effect, relapse remains a key cause of death. We have investigated the role of recipient dendritic cells (DC) in antigen presentation to donor CD8 cytotoxic T cells (CTL) in a model of BMT where GVHD and GVL are directed to multiple minor histocompatibility antigens (mHA) and survival reflects GVL activity. C3H.Sw bone marrow and purified CD8 T cell grafts were transplanted with B6-derived MLL-AF9 induced primary acute myeloid leukemia (AML) into lethally irradiated B6.CD11c.DOG recipients (diphtheria toxin receptor (DTR), ovalbumin and GFP expression driven off the CD11c promoter) such that recipient DC can be deleted by DT administration. Surprisingly, depletion of recipient DC resulted in improved leukemic control (median survival 43 vs 31 days, P <0.001). The use of IRF8-/- BMT recipients (in which the CD8+ DC subset is absent) confirmed that recipient CD8+ DC were critical for regulating these GVL effects (median survival 43 vs 34 days, P = 0.0005). Conversely, when recipient CD8+ DC were expanded in a B6 to B6D2F1 model with bcr-abl/Nup98-HoxA9 induced primary AML, by using Flt3-L treatment for 10 days prior to BMT, GVL effects were completely eliminated, rendering relapse rate equivalent to that seen in the recipients of T cell depleted (TCD) grafts (median survival 11 days in BM+T and TCD groups where recipients were pre-treated with Flt3-L, vs. >45 days in the saline treated BM+T group). The use of B6.CD11c-Rac1 transgenic BMT recipients (who cannot process and present exogenously acquired antigen) confirmed that this effect was the result of endogenous alloantigen presentation by recipient DC and independent of cross-presentation.Using the same depletion strategies in an antigen-specific model (with donor OT-I T cells and B6.CD11c.DOG x DBA/2 F1 recipients) we confirmed that recipient DC invoked effector donor CTL activation, differentiation (CD25+ CD69+ CD62L-) and subsequent apoptosis (as measured by Annexin V; 52.4% vs. 23.9% in DC replete vs. depleted recipients, P = 0.01). There was a consequent profound contraction of the donor CTL compartment by day 10 in DC replete recipients. This contraction of the CTL compartment was associated with reduced expression of the cytolytic molecule granzyme B (MFI 1922 vs 1097, P = 0.02). Antigen presentation has a critical role in the initiation of donor T cell alloreactivity and GVL after BMT. Here we demonstrate that endogenous alloantigen presentation by recipient CD8+ DC to donor T cells leads to activation induced death of donor CTL early after BMT, which in turn facilitates leukemic relapse. This concept has critical implications for the design of therapies that target DC in the peri-transplant period and confirms that recipient DC regulate GVL effects. Disclosures No relevant conflicts of interest to declare.

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Recovery of immune reactivity after T-cell–depleted bone marrow transplantation depends on thymic activity

Blood ◽

10.1182/blood.v96.6.2299.h8002299_2299_2303 ◽

2000 ◽

Vol 96 (6) ◽

pp. 2299-2303 ◽

Cited By ~ 5

Author(s):

Etienne Roux ◽

Florence Dumont-Girard ◽

Michel Starobinski ◽

Claire-Anne Siegrist ◽

Claudine Helg ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Young Patients ◽

Immune Reactivity ◽

Normal Numbers ◽

Cell Repertoire ◽

Naive T Cells ◽

Naïve T Cells ◽

Thymic Rebound

To evaluate the importance of the thymus for the reconstitution of immunity in recipients of a T-cell–depleted bone marrow, we measured the appearance of CD4+CD45RA+RO−naive T cells (thymic rebound), restoration of the diversity of the T-cell–receptor (TCR) repertoire and the response to vaccinations with tetanus toxoid (TT). Repopulation by CD4+CD45RA+RO− thymic emigrants varied among patients, starting at approximately 6 months after transplantation. Young patients reconstituted swiftly, whereas in older patients, the recovery of normal numbers of naive CD4+ T cells could take several years. Restoration of TCR diversity was correlated with the number of naive CD4+CD45RA+RO− T cells. Moreover, the extent of the thymic rebound correlated with the patient's capacity to respond to vaccinations. Patients without a significant thymic rebound at the moment of vaccination (CD4+CD45RA+RO− T cells less than 30 μL) did not respond, or responded only marginally even after 3 boosts with TT. We conclude that during the first year after transplantation, the absence of an immune response is due mainly to the loss of an adequate T-cell repertoire. Restoration of the repertoire can come only from a thymic rebound that can be monitored by measuring the increase of CD4+CD45RA+RO−naive T cells. This will allow postponing revaccinations to a moment when the patient will be able to respond more effectively. This may be particularly useful in the elderly patient who, owing to low thymic activity, might not yet be able to respond 1 year after transplant when revaccinations are usually scheduled.

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Persistent clonal excess and skewed T-cell repertoire in T cells from patients with hairy cell leukemia

Blood ◽

10.1182/blood.v87.9.3795.bloodjournal8793795 ◽

1996 ◽

Vol 87 (9) ◽

pp. 3795-3802 ◽

Cited By ~ 51

Author(s):

JC Kluin-Nelemans ◽

MG Kester ◽

JJ Melenhorst ◽

JE Landegent ◽

L van de Corput ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Hairy Cell Leukemia ◽

T Cell Subsets ◽

Cell Populations ◽

T Cell Repertoire ◽

Hairy Cell ◽

Cell Leukemia ◽

Cell Repertoire ◽

Blood Samples

Hairy cell leukemia (HCL) is characterized by a severe T-cell-mediated immune deficiency. At the same time, spontaneous T-cell activation is noted when splenic T cells are studied in vivo and in vitro. Therefore, we searched for oligoclonal T-cell populations in the blood and spleens of 25 patients with HCL using a T-cell receptor gamma-polymerase chain reaction (TCR gamma-PCR). Subsequently, in 6 patients, the CDR3 length and conformation from 22 different TCRBV subfamilies were analyzed after PCR amplification of cDNA using TCRBV subfamily-specific primers. T cells from 15 of 25 HCL patients showed clonal excess by the TCR gamma-PCR. In fluorescence-activated cell sorted T-cell subsets, more clonal bands were observed than in the unseparated T cells, with most of these in CD8+ subsets, but also in CD4+, CD3+ gamma/delta+, and a double-negative CD3+ alpha/beta+ subset. In other B-cell malignancies, 6 of 16 samples showed oligoclonal T cells, whereas only 2 of 18 normal spleen and blood samples showed abnormal bands. Analysis of the TCRBV subfamilies disclosed in all 6 HCL patients a markedly abnormal pattern, with many clonal bands in 5 to 15 subfamilies, and absent or abnormal weak patterns in another 1 to 8 subfamilies. In comparison, 6 normal samples (2 spleens and 4 blood samples) showed in only 1 blood donor 1 clonal band. Two patients with active HCL but without infections or treatment were tested several times during the course of the disease and showed a complete identical skewed T-cell repertoire with the same oligoclonal T-cell populations. In conclusion, T cells in the blood and spleen of HCL patients show impressive abnormalities with many oligoclonal T-cell populations and a very restricted and skewed TCRBV repertoire.

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Generation of T cells from cytokine-mobilized peripheral blood and adult bone marrow CD34+ cells

Blood ◽

10.1182/blood.v84.1.104.104 ◽

1994 ◽

Vol 84 (1) ◽

pp. 104-110 ◽

Cited By ~ 14

Author(s):

AH Galy ◽

S Webb ◽

D Cen ◽

LJ Murray ◽

J Condino ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Cell Activity ◽

Immunodeficient Mice ◽

Adult Bone Marrow ◽

Cd34 Cells ◽

Adult Bone ◽

Mobilized Peripheral Blood

Abstract The present study compared the T-cell progenitor content of CD34+ lineage (Lin)- cells isolated from normal adult bone marrow (ABM) and mobilized peripheral blood (MPB). Both cell populations were found to differentiate into T cells when injected into human fetal thymi implanted into severe combined immunodeficient mice. Cytokine-MPB cells were less efficient than ABM cells in engrafting in the fetal human thymus, although both gave rise to thymocytes with identical phenotypes based on the analysis of CD1a, CD3, CD4, and CD8 expression. Thymocytes derived from adult CD34+ Lin- cells were capable of fully differentiating into mature CD3+ T cells expressing either the T-cell receptor (TCR) gamma delta or the TCR alpha beta (the later associated with CD4 or CD8), showing that the T-cell progenies of adult CD34+ cells were polyclonal and functional. Our data indicate that human MPB CD34+ cells are qualitatively identical to their BM counterparts, and demonstrate the existence of T-lymphoid progenitor cell activity in MPB.

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The Rapid Ex Vivo Generation of an Immunodominant Antigen-Specific Memory CD8 Population and Its Subsequent Homeostatic Expansion in Ablatively-Conditioned HCT Recipients.

Blood ◽

10.1182/blood.v108.11.3172.3172 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3172-3172

Author(s):

Melinda Roskos ◽

Robert B. Levy

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Ex Vivo ◽

Cell Populations ◽

Cd8 Cells ◽

Immunodominant Antigen ◽

Transfer Strategies

Abstract There is currently significant interest in the transplant field to develop adoptive-transfer strategies utilizing T cell populations to provide immediate immune function as well as long-term immune reconstitution following hematopoietic cell transplantation (HCT). Presumably, these pre-selected T cell populations could then be further expanded in the transplant recipient as a consequence of lymphopenia-induced proliferation. However, clinical application of adoptive transfer strategies has been limited by practical (time, expense) and technical (isolation and expansion of antigen-specific T cell populations) difficulties, hence more efficient approaches need to be identified. Recent reports have demonstrated the feasibility for the rapid ex vivo generation of transgenic memory CD8 populations. We investigated the potential of applying this ex vivo approach to generate and expand an immunodominant antigen-specific memory population from primary CD8 T cells. CD8 cells recognizing the mouse H60 epitope were selected as the antigen-specific CD8 population. The H60 glycoprotein is the ligand for NKG2D and the LTFNYRNL peptide is an immunodominant minor transplantation antigen. H60 is expressed by BALB.B (H2b) hematopoietic cells and recognized by C57BL/6 (B6) CD8 cells within the context of the H2Kb molecule. CD8 T cells from normal B6 spleens were positively selected using Miltenyi beads. The purified CD8 cells (97%) were then cultured with bone marrow-derived B6 DC, rmIL-2, and H60 peptide (1μM) for 3 days. Cells were harvested and re-cultured with rmIL-15 for 2–4 days. The resultant CD8 population was enriched 10 fold for tetramer-stained H60+ CD8 T cells (average: 3.0% of CD8 T cells). The H60+ CD8 cells displayed a memory phenotype as characterized by CD44+, Ly6C+, CD62Lintermed, and CD25lo expression. We hypothesized these H60+ CD8 T cells could be further expanded in transplant recipients by lymphopenia-induced proliferation. To determine the expansion and persistence of H60+ TM post-HCT, H60+-enriched CD8 cells were co-transplanted with T cell-depleted B6 bone marrow into 9.0Gy-conditioned syngeneic recipients. The phenotype and number of H60+ cells in recipient spleens and bone marrow were assessed beginning 3 days post-HCT. Notably, the H60+ CD8 cells maintained their memory phenotype and persisted at least 2 months post-transplant. The ex vivo-generated H60+ TM underwent a relative expansion of 1.5–2 fold as assessed in recipient spleens, similar to the post-transplant expansion of H60+ CD8 TM derived in vivo from B6 mice primed to BALB.B cells. Moreover, this post-HCT expansion was also similar to that by an ex vivo-generated, transgenic CD8 TM population. Both (ex vivo and in vivo generated) H60+ TM populations also exhibited expansion (1.5–2 fold) in the bone marrow. In total, an immunodominant antigen-specific CD8 TM population was selectively generated and enriched ex vivo and found to undergo expansion following transplant into ablatively conditioned HCT recipients. The similarities in expansion and persistence between ex vivo generated H60 and in vivo primed H60 populations suggest this approach may have useful applications towards the development of successful adoptive transfer strategies.

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