Magnetic Bead Depletion Research Articles

Single and Combinational Multiplex Base-Edited 'Universal' CAR T Cells in a Humanised Model of Primary CD7+CD33+ AML

Introduction Universal allogeneic donor CAR T cells are being developed as part of a 'deep conditioning' strategy against AML to secure disease clearance ahead of allogeneic stem cell transplantation (Allo-SCT). CD33 is widely expressed on AML, sometimes on the entire leukemia-associated populations, but is also present on healthy precursors; CD7 may be expressed on AML subpopulations and is present on normal T and NK cells, resulting in problematic fratricide effects during CAR T manufacture. Healthy donor derived CAR T cell banks generated using cytidine deamination base-editing and lentiviral transduction with CARs against both CD33 and CD7 circumvent such issues and could offer cell based anti-leukemic therapy alongside preparative effects ahead of allo-SCT. Here we investigated single and combinational BE-CAR T cells against primary human AML in vivo. Methods: Healthy donor lymphocytes were activated by anti-CD3/28 stimulation ahead of electroporation to deliver codon optimised base editor mRNA and guide RNAs against CD7, CD52 and TRBC, and lentiviral transduction to express either an anti-CD7 or anti-CD33 CAR. Magnetic bead depletion removed residual TCRab T cells, and disruption of CD7 ensured cells avoided fratricide. In vitro, antigen specific function was verified in cytotoxicity and cytokine release assays, and in vivo potency was confirmed against luciferase modified Molm14 and Kasumi lines in NSG mice. Responses against primary AML were investigated in a patient derived xenograft model (PDX) from a KIT mutated AML patient, established by serial transplantation, with full expression of CD33 (100% of blasts) and partial expression of CD7 (30% of blasts). Cells were injected into irradiated NSG mice (n=10 per group). On day 24 at a mean leukemic engraftment of 15% human CD45 positive cells in peripheral blood, mice were treated with either 10x106 BE-CAR33, BE-CAR7 or a combination of both or with anti-CD19-CAR-T cells (CAR19) as a control group (in total 20x106 T cells per mouse). AML engraftment, T cell persistence and expression of checkpoint markers were monitored by multicolour flow cytometric analysis. Results: On days 14 and 28 after CAR-T cell infusion, AML cells were readily detected in peripheral blood of control CAR19 animals (42.8% and 90% respectively) and the BE-CAR7 group (27.8% and 67.6%), but were notably reduced in the BE-CAR33 alone group (2.3% and 8.3%) and the BE-CAR33 and BE-CAR7 combination group (2.5% and 3.4%) (Figure 1A). Survival was significantly prolonged for BE-CAR33 and BE-CAR33/BE-CAR7 treated mice compared to the other groups (P<0.001) (Figure 1B). CD7+ AML cells were depleted to a mean of 1% and 4.5% in the BE-CAR33 group, to 0.6% and 0.7% in the BE-CAR33/BE-CAR7 and to 2.6% and 2.9% in the BE-CAR7 group, while CD7+ AML cells increased to 12% and 30% in the CAR19 group at day 14 and 28 after CAR-T cell infusion. Total CD2+CAR-T cell detection in peripheral blood ranged from 1.6 to 9.8% at day 14, 1 to 3.7% at day 28, and 0.8 to 2.7% at day 42, indicating durable persistence over the investigation period. Interestingly, PD-L1 on AML cells was present in all groups while checkpoint markers PD1, GAL9, TIM3, and LAG3 were significantly upregulated in BE-CAR33 cell treated mice, but not BE-CAR7 or CAR19 cell treated mice (P<0.001 for all comparisons). Conclusions: Lentiviral transduction and multiplexed base editing to remove TCR, CD52 and the shared AML/T lineage antigen CD7, enabled the generation of universal donor CAR T cells for combinational use. This data demonstrates robust activity of BE-CAR33 alone and in combination with BE-CAR7 against human CD7+CD33+AML in mice and highlights the importance of strategies that ensure complete targeting of leukemic populations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TT52CAR19: Phase 1 Trial of CRISPR/Cas9 Edited Allogeneic CAR19 T Cells for Paediatric Relapsed/Refractory B-ALL

Background‘Off-the-shelf’ CAR T cell therapies are being investigated as alternatives to autologous CAR therapy, and can be generated using genome editing from allogeneic donors. Strategies to address HLA barriers include disruption of T cell receptor expression to prevent GVHD and removal of cell surface HLA expression to obviate host mediated rejection, as well as removal of CD52 to create a survival advantage in the presence of Alemtuzumab. In this study, lentiviral mediated CAR19 expression is linked to CRISPR editing through the incorporation of CRISPR guide RNA sequences within the vector 3'Long terminal repeat (LTR). The trial is in progress using pre-manufactured batches of TT52CAR19 T cells.Investigational medicinal product (IMP)Three allogeneic non-HLA matched donor derived CAR19 T cell banks were manufactured from steady state apheresis harvests from registry volunteer donors, using a semi-automated process under compliant conditions. Cells were activated with anti-CD3/CD28 (Transact) reagent and transduced with a lentiviral vector, TT52CAR19, and dual guide sgRNA cassettes targeting the T cell receptor alpha chain (TRAC) and CD52 loci. Next electroporation of Cas9 mRNA elicited transient genome editing, and automated magnetic bead depletion removed remaining TCRαβ+ T cells (mean 0.7%) and enriched CAR19+ T cells (mean 92.8%). Cells were cryopreserved in aliquots suitable for dose-banding and subjected to release testing, including flow cytometry, quantification of copy number (mean 3.83) and exclusion of replication competent lentivirus. Potency of each bank was confirmed in human:murine chimera experiments.Trial sponsorship & approvalsThe study is open at a single site and is sponsored by Great Ormond Street Hospital NHS trust and is supported by the Medical Research Council and National Institute for Health Research. Clinical trial authorisation was awarded by the MHRA after expert review, and ethical approval including gene therapy advisory committee (GTAC) review, and health research authority (HRA) approval. The study is open label, single arm and non-randomised.Study aims and ObjectivesThe study aims to establish the safety and feasibility of TT52CAR19 for the induction of molecular remission in children with relapsed /refractory CD19-positive B-cell acute lymphoblastic leukaemia (B-ALL) within 28 days, ahead of planned allogeneic haematopoietic stem cell transplantation (allo-SCT). Assessments include time to remission, duration of remission, disease-free survival and overall survival. Expansion, persistence and elimination of TT52CAR19 cells and tracking of immune recovery after allo-SCT is monitored. Recording of complications and toxicities, including possible genotoxic side effects from CRISPR/Cas9 modification provides safety profile information.Eligibility, infusion and outcomesThe study plans to treat 10 children aged between 6 months and < 18 years with CD19+ B-ALL quantified at >10 -4 in marrow (by flow or PCR) who are ineligible for autologous therapy. To date 2/4 children screened were found eligible and proceeded to lymphodepletion comprising Fludarabine, Cyclophosphamide and Alemtuzumab followed by a single infusion of 0.8-2.0x10 6 CAR19 T cells and a maximum of 5x10 4/kg TCRαβ T cells. In both cases, there was no GVHD or CRS >grade 1 and recovery of neutrophil counts by d28. Molecular remission was achieved in one child, where TT52CAR19 cells persistence was tracked by chimerism and copy number until further conditioning and allo-SCT. This child remains in remission >6 months later.ConclusionsFeasibility of pre-manufacturing off-the-shelf CRISPR/Cas9 edited CAR19 T cells is demonstrated and the trial has provided first in human safety data and preliminary indications of potent anti-leukaemic activity in one of two subjects dosed. DisclosuresQasim: Autolus: Current equity holder in publicly-traded company; Novartis: Honoraria; Servier: Research Funding; Tessa: Membership on an entity's Board of Directors or advisory committees.

ENHANCED ENRICHMENT OF SMALL CD31+ BLAST-LIKE HUMAN CORD BLOOD CELLS

We previously reported the enrichment and sorting of small CD31+ cells from human umbilical cord blood by magnetic bead depletion of lineage+ and CD34+ cells. We also reported different lineage/CD34+ depletion strategies [combining Lineage depletion and CD34+ UltraPure kits vs Diamond CD34+ kit] results in different input populations for sorting; CD31+ cells could be sorted from either. Here we report adding the missing component from the alternate lineage-depletion cocktail [anti-CD61 microbeads to Lineage depletion kit/anti-CD123 microbeads to Diamond kit] results in identical input populations, with the CD31+ target cells comprising >20% of input population, permitting rapid, high-purity sorting. We have performed sorts with Influx [B-D] and Tyto [Miltenyi] sorters, yielding 125,000 to 750,000 cells [92-98% pure] in 15 minutes. We show purified small CD31+ blast-like cells express the ESC marker alkaline phosphatase, as well as CD4, CD90 and CxCR4. We have been able to successfully isolate our CD31+ cell population from frozen cord blood samples stored in DMSO/FBS after red cell depletion. Frozen storage results in decreased MNC recovery, but CD31+ cell yields appear unaffected, further improving the enrichment of the CD31+ cell population. This is similar to what we have seen with frozen samples for CD34+ HSC isolation. The ability to use frozen samples is greatly improved by the using of Miltenyi's Tyto Running Buffer, a cGMP-compliant wash and transport solution which contains Tytonase to prevent cell clumping. Tyto Running Buffer can be used in the magnetic bead depletion steps without affecting overall yields. These findings permit rapid sorting of highly purified CD31+ cells, which can be used for gene expression profiling and cell culture.

Selective Depletion of Alloreactive Donor T Cells with Adenosine: An Efficient, Scaleable, GMP-Compliant, Low-Cost Method to Prevent Gvhd While Preserving Antiviral and Antileukemic Activity in Haploidentical Stem Cell Transplant

Introduction: The technique of selectively depleting alloreacting T cells from the stem cell transplant (SCT) to prevent graft-versus-host disease (GVHD) while sparing T cells with useful reactivity against viruses and leukemia is commonly termed selective depletion (SD). This approach involves the ex vivo stimulation of donor T cells with host antigen presenting cells (APCs) followed by targeted elimination of the activated alloreactive donor T cell repertoire. SD has been achieved through immunotoxin or magnetic bead depletion targeting T cell activation markers such as CD25 or photodepletion with a rhodamine-like dye. These strategies, while having some success in clinical trials, are limited by poor depletion efficacy or off-target depletion. Here, we present an optimized, novel SD method employing ex vivo treatment of alloactivated T cells with pharmacological concentrations of the purine nucleotide adenosine that preserves quiescent lymphocytes, regulatory T cells (Treg), and T cells with antiviral and antileukemic specificities.Methods: Mature dendritic cell (DC) or irradiated peripheral blood mononuclear cell (PBMCs) "recipient" stimulators were co-cultured with HLA mismatched "donor" PBMC for up to 7 days. Pharmacologic grade adenosine was added at various time points in culture at doses ranging from 100uM-2mM. Depletion efficacy was analyzed by proliferation of the depleted product when challenged separately against stimulator, responder, and 3rd party APCs in 5 day CFSE-dilution assay to measure residual alloreactivity, background proliferation, and response to de novo antigens. Activity against viral antigens was assessed through 6 hour peptide challenge followed by flow-cytometry based intracellular cytokine staining. Leukemia-specific cells were generated from the allodepleted product through two weekly stimulations with autologous DCs transduced to express leukemia associated antigens WT1 and PRAME.Results: Adenosine reduced alloreactive T lymphocytes in HLA-mismatched allogeneic co-cultures to background control frequencies or lower (Figure 1). Optimum allodepletion was achieved with addition of 2mM adenosine on days 1, 2, and 5 after establishing co-culture. Allodepleted lymphocyte products comprised 37±3% of initial cell numbers with 90±1% viability (n=18). Similarly, this method reproducibly achieved SD in four haploidentical donor-recipient pairs, reducing CFSE-tracked proliferation in challenge against haplo APCs below the background of proliferation in challenge against autologous APCs. Adenosine depletion equally affected CD4 and CD8 T cell subsets while sparing NK and B cell populations. Analysis of naïve, effector memory (EM), central memory (CM), and terminal effector (EMRA) T cell compartments revealed no significant differences in depletion of a particular subset in the allodepleted T cell products (n=6). Critically, CD4+CD25+FOXP3+ Treg populations (n=6) and activity against viral antigens (cytomegalovirus, Epstein-Barr virus, and adenovirus) were maintained and in some cases enriched (n=6). Significant activity against leukemia associated antigens (PRAME and WT1) was achieved in allodepleted products from 3 donors.Conclusion: This novel SD technique employing adenosine as a pharmacological agent satisfies the requirements of allodepletion with preservation of viral and leukemia-specific immune responses and thus presents a potentially economical method to deplete alloreacting T cells in SCT products for clinical applications. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reciprocal control of IL-4 and IFN-Lambda (IL-29); mechanisms for the control of Th2 polarization (90.32)

Abstract The newly-described Type III IFN family is comprised of three members: IL-29 (IFN-α), IL-28A (IFN-β) and IL-28B (IFN-γ). All three signal through the same receptor complex, which consists of IL-28Rα (a unique binding and signaling subunit) and IL-10Rβ. While much of the early work describing the activity of IFN-λ1 has focused on its antiviral function, IFN-λ has also been shown to exert powerful immunomodulatory effects, by antagonizing the effect of IL-4 on T cells and inhibiting production of Th2-associated cytokines. To determine whether Th2 cytokines exert reciprocal control over IFN-λ production, PBMC were treated with IL-4 and IL-13 during stimulation. Interestingly, these cytokines significantly enhanced IFN-λ1 production in response to stimuli such as herpes simplex virus (HSV). Intracellular flow cytometry and magnetic bead depletion were used to identify the IL-4-sensitive, IFN-λ -producing cells in peripheral blood. While plasmacytoid DC are a major HSV-responsive cell, they are not responsible for enhanced IFN-λ production. Instead, myeloid cells mediate this response. This identifies a novel mechanism for the regulation of T cell responses.

Gab2 Is a Key Determinant of Mononuclear Phagocyte Development and a Regulator of Macrophage Recruitment in Acute Inflammation

Colony Stimulating Factor-1 (CSF-1) acts through the CSF-1R to regulate proliferation, survival and differentiation of mononuclear phagocytes (MNPs). GAB2, a scaffolding protein, modulates signals from numerous receptors, through recruitment of phosphatidylinositol 3-kinase (PI3K) and SHP2 phosphatase. Previously we reported at this meeting (Lee et al., Blood 110:2197, 2007) that under steady conditions, GAB2−/− mice showed a significant reduction in the number of bone marrow (BM) tissue macrophages (Mφs) and dramatic decreases in the number of CSF-1 dependent colony forming units (CFU-Cs) from total BM and from BM cells that have been induced to commit to the MNP lineage. We also showed by retroviral transduction that GAB2 relies on the recruitment of PI3K and an unidentified pathway to promote CFU-C formation. These findings raised additional questions:What is the identity of the BM progenitor that GAB2 regulates?What is the identity of the second pathway?Does GAB2 have a role in MNP recruitment under in vivo situations of increased MNP demands, e.g. in acute inflammation?To address these questions, we developed methods to isolate BM progenitors committed to the MNP lineage and flow cytometric assays to monitor intracellular phosphorylation in complex BM populations. Based on our CFU-C results, we predicted that GAB2 would have a major role in the expansion of the CD31highLy6C− subset, which should contain CSF-1 responsive early progenitors. We enriched for these cells from ex vivo expanded day 2 BM cultures by magnetic bead depletion of lymphocytes, granulocytes and erythroid cells. The LIN*−CD31highLy6C− subset showed a ≈ 30-fold enrichment of CFU-Cs compared to total BM. Those from Gab2−/− mice gave rise to ≈ 4-fold fewer CFU-Cs and exhibited increased apoptosis and diminished cell cycle progression. To verify that our findings also applied to MNP progenitors isolated directly from the BM, we purified early myeloid progenitors that can proliferate and differentiate in response to CSF- 1. These are lineage depleted (LIN−) cKit+CD31highLy6C− (LK31C) cells and they showed a 51-fold enrichment of CFU-Cs compared to total BM. LK31C cells from GAB2−/− mice also showed a 9-fold reduction in CFU-Cs, relative to GAB2+/+ cells. By CFSE tracking, LK31C cells from GAB2−/− mice demonstrated diminished proliferation. Thus, GAB2 is required for CSF-1 dependent expansion of CD31highLy6C− BM progenitors. To biochemically identify GAB2-regulated signaling pathways that promote expansion of CSF-1 responsive progenitors, we utilized flow cytometry to assay intracellular phosphorylation in day 2 LIN*− cells. We found that the percentage of responding cells as well as the level of phosphorylation of Akt, Erk and S6 were significantly diminished in the LIN*−CD31highLy6C− subset from GAB2−/− cells. We also found that optimal S6 phosphorylation was dependent on both the PI3K and Erk pathway, indicating that Gab2 could play an important role in protein translation in CSF-1 responsive progenitors. Since GAB2's recruitment of SHP2 may be important for optimal Erk activation, we investigated the role of GAB2-SHP2 in CFU-C formation. When a GAB2 mutant that cannot recruit SHP2 was overexpressed in GAB2−/− day 2 BM cells, the number of CFU-Cs was only 40% of that obtained with wildtype GAB2. Together with our previous findings, they indicate that both the PI3K and SHP2-Erk pathways recruited by GAB2 were required for optimal CFU-C formation. Since Gab2−/− mice have a significant defect in CSF-1 dependent MNP development, we reasoned that this deficit could be exacerbated in inflammatory conditions with a prominent MNP infiltration. To test this hypothesis, we used a well-established sterile peritonitis model. GAB2−/− mice showed a 75% (p=0.003) and 67% (p=0.005) reduction in recruited Mφs at 24h and 72h respectively after thioglycollate (TG) challenge. Moreover, there was a 68% increase in CFU-Cs from GAB2+/+ BM cells at 24h (p=0.0001) post TG challenge, whereas GAB2−/− BM cells showed no change. These findings indicate that GAB2−/− mice are much less able to elicit a MNP response after an inflammatory challenge. Altogether, our results reveal that GAB2 utilizes both PI3K-Akt and SHP2-Erk to maximally promote CSF-1 mediated expansion along the MNP lineage by targeting the lineage depleted CD31highLy6C− subset. In the absence of GAB2, the BM is unable to respond maximally to inflammatory challenges.

Follicular Lymphoma Intratumoral CD4+CD25+GITR+ Regulatory T Cells Potently Suppress CD3/CD28-Costimulated Autologous and Allogeneic CD8+CD25− and CD4+CD25− T Cells

Regulatory T cells (T(R)) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor-reactive effector T cells. In this study, we demonstrate that follicular lymphoma (FL)-infiltrating CD8+ and CD4+ T cells are hyporesponsive to CD3/CD28 costimulation. We further identify a population of FL-infiltrating CD4+CD25+GITR+ T(R) that are significantly overrepresented within FL nodes (FLN) compared with that seen in normal (nonmalignant, nonlymphoid hyperplastic) or reactive (nonmalignant, lymphoid hyperplastic) nodes. These T(R) actively suppress both the proliferation of autologous nodal CD8+CD25- and CD4+CD25- T cells, as well as cytokine production (IFN-gamma, TNF-alpha and IL-2), after CD3/CD28 costimulation. Removal of these cells in vitro by CD25+ magnetic bead depletion restores both the proliferation and cytokine production of the remaining T cells, demonstrating that FLN T cell hyporesponsiveness is reversible. In addition to suppressing autologous nodal T cells, these T(R) are also capable of suppressing the proliferation of allogeneic CD8+CD25- and CD4+CD25- T cells from normal lymph nodes as well as normal donor PBL, regardless of very robust stimulation of the target cells with plate-bound anti-CD3 and anti-CD28 Abs. The allogeneic suppression is not reciprocal, as equivalent numbers of CD25+FOXP3+ cells derived from either normal lymph nodes or PBL are not capable of suppressing allogeneic CD8+CD25- and CD4+CD25- T cells, suggesting that FLN T(R) are more suppressive than those derived from nonmalignant sources. Lastly, we demonstrate that inhibition of TGF-beta signaling partially restores FLN T cell proliferation suggesting a mechanistic role for TGF-beta in FLN T(R)-mediated suppression.

Follicular Lymphoma Infiltrating CD4+CD25+GITR+ Regulatory T-Cells Are Potent Suppressors of CD3/CD28 Co-Stimulated Autologous and Allogeneic CD8+CD25− and CD4+CD25− T-Cells.

Regulatory T-cells (Tregs) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor reactive effector T-cells. We demonstrate that follicular lymphoma (FL) T-cells are hypo-responsive to CD3/CD28 costimulation, as assessed by proliferation of CFSE (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester) labeled cells, with only 3.11% ± 2.38 and 2.26% ± 1.76 of the CD8+ and CD4+ T-cells proliferating upon stimulation, respectively (n=7). In contrast, both normal lymph node (NLN), and reactive lymph node (RLN, lymphoid hyperplasia) T-cells proliferate significantly in response to costimulation. Specifically, NLN CD8+ and CD4+ T-cells demonstrate 35.2% ± 31.1 and 18.1% ± 15.9 cells proliferating upon stimulation, respectively (n=7). Similarly, upon stimulation, RLN CD8+ and CD4+ T-cells demonstrate 40.6% ± 22.6 and 40.3% ± 30.3 cells proliferating, respectively (n=5). We identify a population of FL infiltrating CD4+CD25+GITR+ T-cells that are significantly overrepresented within FL, 9.86% ± 6.70 (n=11) of the CD4+ T-cells, as compared to that seen in NLN, 0.70% ± 0.29 (n=13), or RLN, 1.40% ± 1.04 (n=5). These cells actively suppress the proliferation of autologous nodal CD8+ and CD4+ T-cells after costimulation, as CD25+ magnetic bead depletion of these cells in vitro restores proliferation of the remaining CD25− T-cells. Specifically, proliferation of FL CD8+CD25− and CD4+CD25− T-cells increases to 24.05% ± 11.46 and 10.53% ± 6.47, respectively, upon costimulation (n=4). The CD25+ enriched cell fraction contains functionally suppressive cells since add back of unlabelled CD25+ enriched cells to CFSE labeled CD25− cells results in a decrease in proliferation of the costimulated CD8+CD25− and CD4+CD25− T-cells, namely 7.59% ± 3.86 and 4.16% ± 1.79, respectively (n=4). These cells also suppress cytokine production (IFN-g, TNF-a and IL-2) from autologous nodal T-cells as assessed by multiplex analysis of culture supernatants. In addition to suppressing autologous nodal T-cells, the FL CD25+ enriched cells are also capable of suppressing proliferation of allogeneic CD8+CD25− and CD4+CD25− T-cells from NLN as well as normal donor peripheral blood lymphocytes (PBL), regardless of very robust stimulation of the target cells with plate bound anti-CD3 and anti-CD28 antibodies. The allogeneic suppression is not reciprocal, since CD25+ enriched cells derived from either NLN or normal donor PBL, used at the same ratio, are not capable of suppressing allogeneic CD8+CD25− and CD4+CD25− T-cells derived from FL and in fact, are less suppressive against autologous T-cells than are the FL derived CD4+CD25+ cells. Whether this is due to a higher proportion of functionally suppressive T cells within the FL derived CD25+ enriched cells, compared to that of NLN or normal donor PBL, or to an increased suppressive capacity of the FL derived CD25+ T cells is currently being investigated. These data show that FL infiltrating CD4+CD25+GITR+ T-cells have a phenotype and function consistent with Tregs and are very potent suppressors of lymphoma associated-CD8+ and CD4+ T-cells, and therefore may play an important role in lymphoma development, progression and response to treatment.

Type I Interferon Production by Tertiary Lymphoid Tissue Developing in Response to 2,6,10,14-Tetramethyl-Pentadecane (Pristane)

Lymphoid neogenesis is associated with antibody-mediated autoimmune diseases such as Sjogren's syndrome and rheumatoid arthritis. Although systemic lupus erythematosus is the prototypical B-cell-mediated autoimmune disease, the role of lymphoid neogenesis in its pathogenesis is unknown. Intraperitoneal injection of 2,6,10,14-tetramethyl-pentadecane (TMPD, pristane) or mineral oil causes lipogranuloma formation in mice, but only TMPD-treated mice develop lupus. We report that lipogranulomas are a form of lymphoid neogenesis. Immunoperoxidase staining of lipogranulomas revealed B cells, CD4(+) T cells, and dendritic cells and in some cases organization into T- and B-cell zones. Lipogranulomas also expressed the lymphoid chemokines CCL21, CCL19, CXCL13, CXCL12, and CCL22. Expression of the type I interferon (IFN-I)-inducible genes Mx1, IRF7, IP-10, and ISG-15 was greatly increased in TMPD- versus mineral oil-induced lipogranulomas. Dendritic cells from TMPD lipogranulomas underwent activation/maturation with high CD86 and interleukin-12 expression. Magnetic bead depletion of dendritic cells markedly diminished IFN-inducible gene (Mx1) expression. We conclude that TMPD-induced lupus is associated with the formation of ectopic lymphoid tissue containing activated dendritic cells producing IFN-I and interleukin-12. In view of the increased IFN-I production in systemic lupus erythematosus, these studies suggest that IFN-I from ectopic lymphoid tissue could play a role in the pathogenesis of experimental lupus in mice.

CT7 (MAGE-C1)-Specific Cellular Immune Responses in the Bone Marrow Microenvironment of Multiple Myeloma Patients.

In previous studies we demonstrated that CT7 (MAGE-C1), a member of the type I MAGE family of Cancer-Testis antigens, was expressed in 82% of primary multiple myeloma specimens. Higher percentages of CT7-expressing cells were correlated with abnormally elevated plasma cell proliferation in these patients, and CT7 was co-expressed in proliferating myeloma cells. Therefore, a tumor vaccine targeting CT7 may preferentially eliminate proliferating myeloma cells, resulting in long-term cures. The type I MAGE were originally identified because they elicited cellular immune responses in melanoma patients whose tumors expressed these antigens. These data suggest the hypothesis that CT7 expression in myeloma cells may similarly elicit T cell immunity in myeloma patients. To test this hypothesis, we examined lymphocytes from myeloma patient bone marrow, the tumor microenvironment. Lymphocytes were isolated from myeloma patient bone marrow aspirates by magnetic bead depletion of CD138 plasma cells followed by plating to remove adherent monocyte. Bone marrow lymphocytes were then expanded by two rounds of stimulation with anti-CD3, anti-CD28 monoclonal antibody (mAb)-conjugated beads and 50 IU/ml recombinant human (rhu) IL-2. Autologous myeloid dendritic cells (mDC) served as antigen-presenting cells for this analysis; they were prepared from peripheral blood monocytes by cultivation with rhu-IL-4 and GM-CSF, then matured with a cytokine cocktail consisting of TNFα , rhu-IL-1β , rhu IL-6, and prostaglandin E2. Myeloid DC were then transduced with in vitro transcribed CT7 mRNA or positive control antigen flu matrix protein (MP) mRNA by electroporation. Negative control mDC were also mock electroporated without mRNA. The dendritic cells were then co-cultured with the expanded bone marrow lymphocytes at 1:1 and 1:20 ratios in IFNγ ELISpot assays. Positive control stimulation with the T cell superantigen Staphylococcal enterotoxin B (SEB) served to assess overall responsiveness of the polyclonal lymphocyte population. Results from four patients are shown in Table 1. Two of four patients, both diagnosed with stage IIIa disease and both expressing CT7 mRNA by RT-PCR, exhibited increases in IFNγ-secreting lymphocytes when co-incubated with CT7-transduced mDC compared to mock electroporated mDC. In contrast, one stage I patient who did not express CT7 mRNA and one stage IIIb, CT7+ patient did not show any increase in IFNγ-secreting cells in response to CT7-transduced mDC stimulation. These data indicated that CT7 expression in myeloma cells can provoke cellular immunity, and suggests that CT7-specific vaccines may boost this immunity. These ongoing studies will also characterize the antigenic T cell epitopes of CT7.Table 1CT7-specific bone marrow lymphocytesPatientStageCT7 mRNA expressionMockCT7Flu MPSEB1IIIa+28.0 ± 7.157.7 ± 5.9nd288 ± 42.12IIIa+6.3 ± 2.327.7 ± 10.117.7 ± 5.390.0 ± 50.33Ia−6.0 ± 1.08.5 ± 0.5nd91.5 ± 54.54IIIb+1.5 ± 0.54.5 ± 0.5nd72.0 ± 43.0IFNγ-secreting cells/50,000 bone marrow lymphocytes

Identification of T-cell epitopes using ELISpot and peptide pool arrays.

Here we describe a method for T-cell epitope identification using a modified ELISpot assay that is both simple and efficient. By using a carefully constructed array of pools of overlapping peptides spanning the entire antigen sequence to stimulate T-cell responses, we are able to detect antigen-specific cytokine responses by both CD8+ and CD4+ T cells and identify the specific peptides to which the cells are responding. Additionally, by performing magnetic bead depletion of either CD8+ or CD4+ cells prior to the assay, we are able to determine the phenotype of the responding cells to each of the peptide epitopes identified. Use of this method will allow the identification of both CD4+ and CD8+ T-cell epitopes without the need for MHC allele-matched reagents and without the need for highly specialized instrumentation. By using an array of peptide pools, this method also dramatically reduces the number of immune cells required to test the entire antigen sequence, often a limiting factor in vaccine testing and other studies.

Competitive antibody binding to soluble CD16B antigen and CD16B antigen on neutrophils in whole blood by flow cytometry.

Shed receptors from the surface of white blood cells in whole blood have been quantitated using the long and tedious enzyme-linked immunosorbent assay (ELISA) method. A simple rapid flow cytometric method of analysis for shed antigen in the presence of cell-bound antigen can be advantageous. Magnetic bead depletion of neutrophils in whole blood with CD16b antibody-conjugated beads as measured by flow cytometric analysis of the remaining cell suspension was inhibited by the presence of soluble CD16b antigen in the blood plasma of normal donors. We describe a competitive binding assay between labeled and unlabeled CD16b antibody for receptors shed from the surface of formed bodies (cells) into solution. Also presented is a new method of obtaining the amount of soluble antigen in a sample. We determine the total unlabeled and labeled ligand concentration at which the fluorescence intensity of the labeled ligand matches the fluorescence intensity in a control run with only the labeled ligand. Normal blood donors showed serum concentration levels of shed CD16b antigen in the range of 1-50 nM as determined by a flow cytometric competitive binding assay. These figures compared favorably with parallel determinations using magnetic bead depletion of targeted neutrophils for washed and unwashed whole blood samples to evaluate the concentration of shed CD16b antigen. The competitive antibody binding assay for shed and cell-bound CD16b antigen can be applied to similar GPI-linked antigens, for which purified antibody and fluorescent antibody against the same antigenic receptor are available.

Selection and expansion of T cells from untreated patients with CLL: source of cells for immune reconstitution?

Lymphocyte-derived malignancies can be treated with combinations of drugs that efficiently eradicate normal and malignant lymphocytes. Lack of T lymphocytes after treatment of B lymphocyte CLL (B-CLL) makes the patients susceptible to serious infections and may limit the benefit of the therapy. The aim of the study was to purify and culture-expand normal T lymphocytes from B-CLL patients prior to therapy. These cells could be frozen and given to the patients in the lymphopenic period post-chemotherapy. T lymphocytes were isolated from the mononuclear cell apheresis products from five patients with previously untreated B-CLL. The apheresis products were red-cell depleted by density gradient centrifugation. B-lymphocyte purging was performed by incubating with MAbs to four different B-cell epitopes, followed by magnetic-bead depletion. One round of negative selection removed >90% of the B lymphocytes. The T-lymphocyte enriched cell suspension was cultured for 10/11 days in the presence of IL-2 and the anti-T cell receptor Ab OKT3. In addition, in some cultures anti-CD22 ricin immunotoxin was added. T cells from CLL patients expanded 4.7-21-fold over a 10/11 days culture interval. After culture, CLL cells could no longer be identified by flow cytometric evaluation. The cultured T lymphocytes were predominantly CD8(+), and were capable of lysing autologous CLL cells through a fas-dependent mechanism. Selection and expansion of T lymphocytes by this method may represent a strategy for enhancing immunity in the lymphopenic period following CLL treatment.

Selective deletion of antigen-specific, activated T cells by a humanized MAB to CD2 (MEDI-507) is mediated by NK cells.

CD2 is a 50-kDa transmembrane glycoprotein that plays an important role in T and natural killer (NT) lymphocyte functions. CD2 serves as both an adhesion molecule and as a costimulatory molecule through interactions with its ligand, CD58, on antigen presenting or target cells. Consistent with earlier studies using a rat anti-CD2 mAb, we have shown that treatment of alloantigen stimulated T lymphocytes with a humanized mAb, MEDI-507 (IgG1, kappa), induced hyporesponsiveness to subsequent stimulation with alloantigen but not to mitogen (phytohemagglutinin). Fluorescence-activated cell sorting analysis of cells from mixed lymphocyte reaction (MLR) treated with MEDI-507 revealed pronounced deletion of T and NK cells, consistent with lack of proliferation in the MLR. MEDI-507 F(ab')2 fragments did not have inhibitory activity or induce deletion of lymphocytes in the MLR. Removal of the NK cell subset by magnetic bead depletion using anti-CD16 and anti-CD56 mAbs eliminated both the T cell deletion and the inhibitory effect. Reconstitution of NK depleted responder populations using autologous NK cells restored the MEDI-507-mediated deletion activity to levels measured in the original MLR. Formaldehyde-fixed NK cells failed to mediate the MEDI-507-induced deletion effect. Altogether, our studies indicate that activated T cells with MEDI-507 bound to CD2 are preferential targets for autologous NK cells through a nonapoptotic cytotoxic mechanism.

Both memory and CD45RA+/CD62L+ naive CD4(+) T cells are infected in human immunodeficiency virus type 1-infected individuals.

Cellular activation is critical for the propagation of human immunodeficiency virus type 1 (HIV-1) infection. It has been suggested that truly naive CD4(+) T cells are resistant to productive HIV-1 infection because of their constitutive resting state. Memory and naive CD4(+) T-cell subsets from 11 HIV-1-infected individuals were isolated ex vivo by a combination of magnetic bead depletion and fluorescence-activated cell sorting techniques with stringent criteria of combined expression of CD45RA and CD62L to identify naive CD4(+) T-cell subsets. In all patients HIV-1 provirus could be detected within naive CD45RA+/CD62L+ CD4(+) T cells; in addition, replication-competent HIV-1 was isolated from these cells upon CD4(+) T-cell stimulation in tissue cultures. Memory CD4(+) T cells had a median of fourfold more replication-competent virus and a median of sixfold more provirus than naive CD4(+) T cells. Overall, there was a median of 16-fold more integrated provirus identified in memory CD4(+) T cells than in naive CD4(+) T cells within a given patient. Interestingly, there was a trend toward equalization of viral loads in memory and naive CD4(+) T-cell subsets in those patients who harbored CXCR4-using (syncytium-inducing) viruses. Within any given patient, there was no selective usage of a particular coreceptor by virus isolated from memory versus naive CD4(+) T cells. Our findings suggest that naive CD4(+) T cells may be a significant viral reservoir for HIV, particularly in those patients harboring CXCR4-using viruses.

MIXED LYMPHOCYTE ISLET CELL CULTURE ANALYSIS OF CD45-DEPLETED HUMAN FETAL PANCREAS.

553 Human fetal pancreas (HFP) undergoes further growth and differentiation following transplantation and should be considered as an alternative source of transplantable is lets for the treatment of insulin dependent diabetes mellitus. Our goal is to identify a preparation of HFP which has reduced immunogenicity for transplantation. We have previously reported that HFP contains a significant proportion of passenger leukocytes (8 ± 2% CD45+ cells) potentially capable of stimulating a vigorous immune response via the direct pathway of antigen presentation there by contributing to graft loss. We demonstrated that endocrine-enriched "proislets" prepared by collagenase digestion have fewer passenger leukocytes (6 ± 4% CD45+) than the exocrine-enriched population (20 ± 5% CD45+) released through the digestion procedure. Correspondingly, mixed lymphocyte is let cultures (MLIC) stimulated with endocrine-enriched cells had significantly lower IFNγ production (p=.003 on d4, p=.02 on d7, n=5), than MLIC stimulated with exocrine-enriched cells. While simple digestion procedures yielding endocrine-enriched "proislets" significantly reduce the passenger leukocyte component, all of the cells capable of functioning as direct APC's are not removed. Therefore, we used antibody-mediated magnetic bead depletion using antibody specific for human CD45. Single cell suspensions of HFP were prepared following dispase digestion (1 unit dispase/ml for approximately 1 hour). The efficiency of depletion was confirmed by flow cytometry (7 ± 1.8% CD45+ cells pre-depletion to 0.2 ± 0.2 post-depletion, n=3). MLIC cultures were established using either CD45 depleted or non-depleted HFP preparations and tested both for cyokine production and for development of donor-specific CTL activity (measured by lysis of splenocytes derived from the HFP donor). MLIC stimulated with CD45-depleted HFP produced 918 ± 58 pg/ml IFNγ in contrast to 1483 ± 340 pg/ml in corresponding MLIC cultures stimulated with non-CD45-depleted HFP. CTL activity was inhibited 50-100% in MLIC cultures stimulated with CD45-depleted HFP preparations even after the addition of IL2 to the culture (10 units/ml IL2 added on day 7, CTL determined on d10). These results suggest that indirect antigen presentation occurred in CD45-depleted cultures, but that direct pathway is more effective at generating CTL activity. These data are important to our understanding of the contribution of direct and indirect antigen presentation pathways in immune responses to HFP. Because dispersed HFP retains in vitro capacity to release insulin in response to challenge this a viable approach to the establishment of an optimal preparation of HFP tissue for transplantation with reduced immunogenicity.

T cell subset involvement in immune responses to Fasciola hepatica infection in cattle.

The lymphocyte response to F. hepatica during a primary infection in cattle was analysed to define the role of T cell subsets in the immune response. Blood lymphocytes were isolated from eight cattle infected with F. hepatica via trickle infection over a ten-day period and from two non-infected controls. CD4+, CD8+ and gamma delta + T cells were depleted from whole lymphocyte populations by magnetic bead depletion. Lymphocytes from infected animals demonstrated a transient, but marked elevation in responsiveness to F. hepatica antigen between weeks 3 and 8 post-infection. Responses were attenuated by depletion of CD4+ and CD8+ T cells during this period. Depletion of gamma delta + T cells attenuated antigen responses at one time point only, and at an earlier stage post-infection than when alpha beta + T cells were depleted. Responses to antigen correlated positively with both hepatic fluke burden and with the degree of hepatic damage. This suggests that the cellular immune response was not protective. Antigen responses in gamma delta + T cell-depleted populations were also associated with post-mortem fluke burden and with hepatic damage. This suggests that gamma delta + T cells are involved in down regulating alpha beta + lymphocytes which may have a role in a non-protective or immunopathological immune responses.

Concanavalin A-stimulated proliferation of T cell subset-depleted lymphocyte populations isolated from Fasciola hepatica-infected cattle

Over 14 weeks, peripheral blood lymphocytes (PBL) were isolated from eight adult cattle which had been orally infected with Fasciola hepatica via trickle infection over a 10-day period. Two age, breed and sex-matched cattle served as controls. CD4 +, CD8 + and γδ + T cells were depleted from whole PBL populations by magnetic bead depletion. Lymphocyte proliferation assays demonstrated a transient, but marked elevation in responsiveness to Concanavalin A (Con A) between weeks 2 and 4 post-infection in PBL from infected animals. Proliferative responses to Con A were significantly greater in PBL from infected cattle than uninfected/control cattle over the initial period of the experiment. Con A-stimulated proliferation of PBL isolated from infected cattle followed a similar pattern to PBL responses to F. hepatica antigen. In both whole and subset-depleted lymphocyte populations from infected cattle, proliferative responses to Con A decreased from day 28 post-infection. Depletion of CD4 +, CD8 + and γδ + T cell subpopulations significantly augmented responses soon after infection. These findings suggest that the capacity of bovine PBL to proliferate in response to Con A stimulation, was in some way attenuated by F. hepatica infection and proliferative responses due to non-specific activation was suppressed by the coordinated activities of various lymphocyte subsets.

Expressed genes in interleukin-4 treated Bcellsidentified by cDNA representational differenceanalysis

Interleukin-4 (IL-4) stimulates B cell growth and differentiation, such asinducingmature B cells to switch to IgG1 and IgE production. To further characterize IL-4effects on Bcells, we used a sensitive PCR-based subtraction approach to isolate genes expressedin IL-4treated cells. Our approach combined an adaptation of the genomic representationaldifferenceanalysis (RDA) method to cDNA analysis with a physical separation method(magnetic beaddepletion). This cDNA RDA technique allowed us to perform subtraction on therelatively smallnumber of highly, characterized, purified B cells that can be convenientlyprepared. In the hopesof removing genes responsible for general cell growth, we subtractedcDNA made fromlipopolysaccharide (LPS)-stimulated B cells from cDNA from LPS+IL-4stimulated B cells.Two rounds of subtraction resulted in greater than 100-fold enhancement ofexpectedIL-4-induced Cγ1 cDNA. At that point, we cloned this subtraction library and analysed154randomly picked clones for sequence similarities. From these clones, 37 individual geneswereobtained. Most of these genes (30) could be functionally identified by sequence similarity.Theseincluded genes encoding Cγ1 (1), cytoskeletal components (4) and products involved inDNAreplication (3), metabolism (5), signal transduction (4), transcription (4), translation (6)andtransport (3). Only 7 genes had no similarity to known sequences in the GenBank, EMBLorSwiss Prot databases. One unknown gene (designated Fig1 for IL-Four InducedGene1) and one gene with homology to the human transcription factor E4BP4 were confirmedbyNorthern blot analysis to be induced 10–20-fold by IL-4 treatment. This list of expressedgenesin LPS+IL-4 treated B cells may shed further insight on the action and mechanism ofIL-4stimulation of cells.

Rare event selection of fetal nucleated erythrocytes in maternal blood by flow cytometry.

A noninvasive method of prenatal genetic diagnosis requires fetal cell selection from the maternal circulation that allows efficient recovery for analysis by fluorescence in situ hybridization (FISH). We have solved several problems that negatively affect the isolation and FISH analysis of fetal nucleated red blood cells (nRBCs) in the maternal circulation. The use of glycophorin A (Gly A) antibodies (Abs) for selection is problematic because all five monoclonal antibodies (mAbs) tested caused agglutination of non-nRBCs, thereby changing both light scatter and fluorescence properties of cells by flow cytometry. Because the number of non-nRBCs is variable after Ficoll separation, isolation of nRBCs could be compromised severely by agglutination of nucleated cells with nonnucleated cells, causing them to shift light scatter and fluorescence properties. Several methods for the removal of unwanted maternal white blood cells with CD45 mAbs were also evaluated. Magnetic bead depletion was found to interfere with FISH detection because of residual bead debris after sorting. By contrast, removal of CD45+ cells by a panning technique eliminated this problem. Positive selection methods based on CD71, CD45, and LDS-751 staining and detection of fetal cells by gamma globin expression were also analyzed. Fetal cells were detected by FISH in 11 of 19 (CD71 selection) and in 13 of 15 (gamma selection) random pregnancies. These data support the possibility of a noninvasive method for isolation and analysis of fetal cells for prenatal diagnosis.