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Antigen-Specific Cytolysis by Neutrophils and NK Cells Expressing Chimeric Immune Receptors Bearing or Signaling Domains

Cell Genesys Inc., Foster City, CA 94404

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
TCR- and IgG-binding Fc receptors (FcR) mediate a variety of critical biologic activities including cytolysis via the structurally related - and -chains. In previous studies, we have described chimeric immune receptors (CIR) in which the ligand-binding domain of a heterologous receptor or Ab is fused directly to the cytoplasmic domain of the TCR -chain. Such -CIRs efficiently trigger cytotoxic function of both T and NK cells in a target-specific manner. In this report, we compared the ability of both - and -CIRs to activate the cytolytic function of two distinct classes of FcR-bearing effectors, NK cells and neutrophils. Mature neutrophils expressing - and -CIR were generated in vivo from murine hemopoietic stem cells following transplantation of syngeneic mice with retrovirally transduced bone marrow or in vitro from transduced human CD34⁺ progenitors following differentiation. Both - and -based CIRs were capable of activating target-specific cytolysis by both NK cells and neutrophils, although the -CIR was consistently more efficient. The experimental approach described is a powerful one with which to study the role of nonlymphoid effector cells in the host immune system and permits the rational design of immunotherapeutic strategies that rely on harnessing multiple immune cell functions via CIR-modified hemopoietic stem cells or progenitors.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
TCR and FcR are structurally and functionally related immune receptors expressed on T lymphocytes and myeloid/NK cells, respectively, which activate a variety of critical biologic activities including cytolysis, cytokine/inflammatory mediator release, and phagocytosis. One of the primary immune defense mechanisms in the control of disease is killing of malignant and virally infected cells by both TCR and FcR-bearing effectors. TCR and FcR are comprised of two functional components: the first for target cell/Ag recognition via binding to MHC-presented peptides (TCR) or Abs (FcR); the second for activation of signal transduction pathways that culminate in effector function. The latter function is mediated primarily by two structurally related subunits, TCR- and FcRI. Whereas the -chain is primarily associated with the TCR complex, the FcRI subunit () is primarily associated with FcRI expressed on neutrophils and macrophages (1, 2, 3) and the TCR of T cells (4). In addition, both and subunits have been found associated with FcRIIIA expressed on NK cells and macrophages (5, 6). The and subunits contain one and three copies, respectively, of the conserved 18-amino acid Ig tyrosine activation motif (ITAM).⁶ Although the tyrosine and leucine residues present within the ITAM sequence [YXXL-X(7)-YXXL] are conserved between the two subunits, and , the XX and flanking amino acids differ (7). The tyrosine residues of the conserved YXXL are required for signal transduction (8). TCR and FcR cross-linking initiates a signal transduction cascade which begins with activation of Src and Syk family protein tyrosine kinases (PTKs) and phosphorylation of ITAM tyrosine residues and culminates in cellular activation (7, 8, 9, 10, 11, 12, 13, 14).

Chimeric immune receptors (CIRs) have been described in which the ligand-binding domain of a heterologous receptor or single-chain Ab is fused directly to the cytoplasmic domain of a member of the family (15, 16, 17, 18). In this way, MHC-unrestricted CIRs directed against a variety of tumor-associated or viral-derived ligands may be generated. We have previously described an HIV-specific CIR composed of the extracellular domain of CD4 fused to (18). CD4 binds the gp120 moiety of the HIV envelope protein (HIVenv) expressed on the surface of virally infected cells. When CD4 is introduced into mature human CD8⁺ T cells (18) or NK cells (19) via retroviral-mediated transduction, cytotoxic function of the TCR- or FcR-bearing effector cell populations can be efficiently and specifically directed to kill gp120-expressing cells in vitro. More recently, we have described the generation of T cell-independent systemic immunity in SCID mice reconstituted with CD4-expressing myeloid and NK cells following bone marrow transplantation (20). Such Ag-specific CIRs provide an ideal model system with which to investigate immune cell function and develop novel approaches for the treatment of disease. Although much is known about the role of in TCR-bearing cells, the relationship between / structure and FcR-mediated effector function is not well understood. In this report, we compare - and -based CIRs for their ability to activate the cytolytic function of two distinct classes of FcR-bearing effector cells, neutrophils and NK cells. Since fully differentiated NK cells may be propagated in vitro, /-CIRs were introduced into mature human NK cells. In contrast to NK cells, neutrophils are short-lived with a half-life of 18 h in vitro. Mature neutrophils were therefore generated by 1) in vitro differentiation of retrovirally transduced human CD34⁺ cells or 2) in vivo differentiation of retrovirally transduced murine hemopoietic stem cells posttransplantation. Long term expression of both - and -CIRs on reconstituted myeloid and NK cells is observed following murine bone marrow transplantation. The -CIR is consistently more efficient than the -based receptor at mediating target-specific cytolysis by both NK cells and neutrophils, even though the latter primarily utilize the -chain for FcR signaling.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chimeric immune receptors

The CD4 chimeric receptor has been previously described (18), and bears the extracellular and transmembrane (TM) domains of the human CD4 receptor (residues 1–372 and 372–395 of the mature CD4 chain, respectively), fused to the cytoplasmic domain of the human TCR -chain (residues 31 to 142 of the mature -chain). CD4 has the same extracellular and TM domains as CD4, but the cytoplasmic domain is derived from the human FcRI -chain (residues 27–68 of the mature -chain). The CD4^del gene is a truncated form of the CD4 receptor in which the cytoplasmic residues have been deleted (residues 403–433 of the mature CD4 receptor deleted; Gln at position 403 is mutated to a stop codon). Oligonucleotide-directed deletional mutagenesis was used to form specific junctions between gene sequences.

Retroviral vectors

A high efficiency retroviral transduction system, kat, was used to generate high titer retroviral supernatants containing the CD4, CD4, or CD4^del gene, from 293 cells transiently transfected with packaging (pkat) and retroviral vector (rkat) plasmids as previously described (21). rkat43.2 is a variant of rkat4 (21) in which Moloney murine leukemia virus (MMLV) Psi sequences (up to the ATG of Gag which has been changed to TAG) have been replaced by the corresponding sequences from Moloney Murine Sarcoma Virus (MMSV). Viral Env sequences between the CIR gene region and the reverse strand binding site have been deleted. SV40 poly(A) and ori sequences have been inserted 3' of the retroviral sequences on a pSK11 (Stratagene, La Jolla, CA) plasmid backbone. Following reverse transcription and integration into target cells, transcription of both unspliced and spliced mRNAs initiates from the viral long terminal repeat (LTR). rkat43.3pgk was generated from rkat43.2 by 1) replacing the XhoI-EcoRI splice acceptor fragment with a PCR-generated 520-bp XhoI-EcoRI fragment encoding the human phosphoglycerate kinase (pgk) promoter/enhancer (nucleotides 5–516, GenBank accession number M11958) and 2) replacing the 3' MMLV LTR with an LTR in which the sequences from PvuII-XbaI (nucleotides 7935–8113, GenBank accession number MLMCG) have been deleted. Following reverse transcription and integration into target cells, transcription of CD4, CD4, or CD4^del is initiated only from the pgk promoter of rkat43.3pgkF3. Previous work in this laboratory has shown that this vector yields stable levels of gene expression over 6 mo in transplanted mice, whereas viral LTR-driven expression diminishes rapidly over 1 to 2 mo (our unpublished data).

Packaging plasmids

pkat2ampacUTd was derived from pkat2ampac (21), by deleting untranslated sequences 3' of the envelope gene: the 172-bp ClaI-NheI fragment of pkat2ampac was replaced with a 100-bp PCR-generated ClaI-NheI fragment containing only the 3' coding regions of MMLV 4070a to give pkat2ampacUTd. pkat2ecopac was derived from pkat2ampacUTd by replacing the 4-kb SalI-ClaI pol-env-encoding fragment of pkat2ampacUTd with the analogous fragment from ecotropic MMLV (GenBank accession number MLMCG).

Transient retroviral production

Retroviral supernatants were generated essentially as previously described (21). Briefly, TSA54 cells were plated at 6.5 x 10⁵/10-cm plate and 48 h later cotransfected with 10 µg of rkat43.2 or rkat43.3pgk retroviral vector encoding the relevant CIR and 5 mg of packaging vector: pkat2ampacUTd for transduction of human CD34⁺ or NK cells; and pkat2ecopac for transduction of murine bone marrow. The medium was changed after 15 to 24 h and replaced with either NK, CD34⁺, or bone marrow culture medium as described below. After an additional 24 h, the culture supernatant was harvested, filtered through a 0.45-µm pore size filter, frozen on dry ice, and stored at -70°C. Retroviral titers on National Institutes of Health 3T3 cells ranged from 6 x 10⁶ to 1 x 10⁷ viral particles/ml.

Human NK cell transduction

Retroviral transduction of NK3.3 cells was conducted with the kat retroviral producer system as previously described (19) except that supernatant instead of coculture transduction was used. NK cells were plated at 1 x 10⁶ cells/well of a 24-well plate in 1 ml of NK medium (19) and exposed to 1 ml of retroviral supernatant and 2 µg/ml Polybrene. After 18 to 24 h, 1 ml of medium was removed and replaced with 1 ml of fresh supernatant and Polybrene. The transduction procedure was then repeated twice more at 18- to 24-h intervals, and the NK3.3 cells were allowed to recover for an additional 48 h in NK medium. Stable expression of CD4 and CD4 chimeric receptors was then analyzed 2 days posttransduction by flow cytometry with FITC-conjugated anti-CD4 mAbs as described below. CD4/-expressing NK cells were subsequently purified by immunoaffinity anti-CD4 mAb-coated flasks (Applied Immune Sciences, Santa Clara, CA).

Retroviral transduction of human CD34⁺ progenitors

Human bone marrow was obtained from consenting healthy adult volunteers by aspiration from the posterior iliac crest. Light density mononuclear cells were isolated using Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density centrifugation within 24 h of harvest. Isolation of CD34⁺ progenitors was performed using the CellPro Ceprate System (CellPro, Bothell, WA). After purification, CD34⁺ cells were prestimulated for 2 days in a CD34⁺ basal medium of Myelocult Long Term Culture Medium (Stem Cell Technologies, Vancouver, British Columbia, Canada) with the addition of 10^-6 M hydrocortisone, human stem cell factor (SCF) (100 ng/ml), human IL-3 (50 ng/ml), and human IL-6 (10 ng/ml). Cells were then cultured at 8 x 10⁵ cells/ml in the presence of freshly collected retroviral supernatant with the addition of 8 µg/ml Polybrene, 100 ng/ml human SCF, 50 ng/ml human IL-3, and 10 ng/ml human IL-6. Transduction was conducted in 10-cm dishes precoated with 5 µg/ml anti-human CD44 and 5 µg/ml anti-human CD49d mAb (Coulter, Westbrook, ME) to increase transduction efficiency. After 4 h, cells were collected, washed, and resuspended in viral supernatant for a second exposure. After 8 h, cells were collected, washed, and resuspended in Myeloid Long Term Culture Medium with the addition of growth factors for liquid expansion into neutrophils. Two days after retroviral infection, relative transduction efficiency was determined by flow cytometric analysis for CD4 expression.

Liquid expansion of transduced CD34⁺ cells into neutrophils

CD34⁺ cells were expanded and differentiated into neutrophils utilizing the following schedule of cytokine treatment: during days 1 to 6, the cells were cultured in human SCF (100 ng/ml), human IL-3 (50 ng/ml), and human IL-6 (10 ng/ml). On days 7, 9, and 11, fresh human SCF (10 ng/ml) and human granulocyte CSF (G-CSF) (2 ng/ml) were added to the cultures. On days 14 and 16, cells were fed with G-CSF (10 ng/ml) alone. On days 18 to 22, cells were harvested, analyzed via cytospin preparations and flow cytometry, and utilized in chromium release cytotoxicity assays as described below.

Transplantation of SCID mice with CIR-transduced bone marrow

C.B-17 scid/scid (SCID) mice (Charles River Laboratories, Wilmington, MA) were utilized for bone marrow transplant studies. Bone marrow donors were 8- to 16-wk-old males and females, and recipients were 8- to 12-wk-old males. Animals were housed in sterile laminar airflow hoods and fed ad libitum with sterile food and water. All animal procedures conformed to institutional guidelines. Donor SCID mice were injected via the tail vein with 5-fluorouracil (100 mg/kg) (Roche Laboratories, Nutley, NJ) to enrich for immature hemopoietic progenitors with long term repopulating ability. After 6 days, mice were euthanized by CO₂ asphyxiation. Femurs were harvested and flushed with bone marrow culture medium (DME, 4.5 g/L glucose, 15% FCS, glutamine, penicillin, and streptomycin) and 5 mM EDTA. Low density cells (LDC) were isolated by density gradient separation using Lympholyte-M (Cedar Lane, Hornby, Ontario, Canada). Briefly, bone marrow cells were layered over an equal volume of gradient and spun at 2200 rpm for 20 min at 20°C in a table top centrifuge. Interphase cells were collected, washed, and resuspended in culture medium. LDC were plated at 3 x 10⁶ cells/well of a 6-well plate (Corning Glass, Corning, NY) and exposed to retroviral supernatant in the presence of 6 mg/ml Polybrene. Virus was packaged in ecotropic envelope for efficient transduction of murine cells. After 2 h, the medium was hemidepleted, and fresh viral supernatant was added. Transduced cells were harvested from the plates after 4 h, washed, and resuspended in 0.9% normal saline and 0.1% BSA for injection. Via tail vein injection, 10⁶ transduced LDC/mouse were infused into sublethally irradiated (350 rads) SCID mice.

Immunofluorescence analysis of CD4^-CIR expression posttransplant

Heparinized blood (300 µl) was attained by retroorbital bleeds of CIR transplanted and control mice at various time points posttransplant. RBC were depleted by ammonium chloride lysis. Approximately 2 x 10⁵ cells were incubated with the following murine-specific mAbs conjugated to FITC: anti-GR-1; anti-Mac-3; anti-5E6 (PharMingen, San Diego, CA), in addition to anti-human CD4^- phycoerythrin (PE) (Becton Dickinson, San Jose, CA) according to manufacturer’s instructions. FITC- and PE-conjugated isotype-matched mAbs served as negative controls. Stained cells were analyzed on a FACScan cytometer (Becton Dickinson).

Isolation of murine neutrophils posttransplant

At 3 wk posttransplant, 4 CIR-transduced and 4 control SCID mice were treated with 7 daily s.c. injections of human G-CSF (Amgen, Thousand Oaks, CA) (100 µg/kg/day). Mice were then euthanized, and heparinized blood was recovered by cardiac puncture. Neutrophils were isolated using a modification of the standard "1-step Polymorph" procedure (Accurate Chemical, Westbury, NY) in which 0.8 ml of 1.5% NaCl was added to 10 ml of stock gradient. Murine PB (4 ml) was layered over 4 ml of modified gradient, and tubes were spun for 30 min at 450 x g at 20°C in a table top centrifuge. The neutrophil fraction was removed and washed twice. An aliquot was removed for cytospin analysis and was >95% neutrophils as judged by standard Wright-Giemsa staining techniques. Additional cells were removed for measurement of CIR transduction efficiency by flow cytometric analysis. The remaining cells were used in a chromium release cytotoxicity assay as described below.

Cytotoxicity assays

Cytotoxicity of transduced neutrophil and NK effectors was evaluated by a standard ⁵¹Cr release method. Raji cells derived from Burkitt’s lymphoma were used as targets, either as parental (Raji-p) or those transfected with HIVenv, Raji-env (19). Raji-p and Raji-env cells were labeled with sodium [⁵¹Cr]chromate (100 mCi/10⁶ cells) for 3 h at 37°C and washed three times. Labeled target cells were then incubated with effector cells as described below. The percentage of specific lysis was calculated from triplicate samples using the following formula: [(CMP-SR)/(MR-SR)] x 100, where cpm is the counts per min released by targets incubated with effector cells, MR is the counts per min released by targets lysed with 100 µl of 1% Triton X-100, and SR is the counts per min released by targets incubated with medium alone. In vitro-differentiated human neutrophils were harvested at day 21 post cytokine treatment and cultured overnight in culture medium with the addition of 10 ng/ml human G-CSF and 500 U/ml human -IFN. The next day, cells were analyzed via cytospin preparations and flow cytometry, and utilized in a ⁵¹Cr release cytotoxicity assay as described above. Briefly, human neutrophils (control and CIR expressing) were resuspended in 100 µl of assay medium (RPMI, 10% FCS, 20 ng/ml human granulocyte-macrophage-CSF) and plated in triplicate in 96-well plates. To each well 10^{5 51}Cr-labeled targets were added in 100 µl of assay medium to establish a range of E:T ratios. To measure Ab-dependent cellular cytotoxicity (ADCC), polyclonal rabbit anti-human lymphocyte serum (Cedar Lane) (5 mg/ml) was added to control cells. The assay was initiated by centrifugation (50 x g, 2 min, 20°C) and allowed to incubate at 37°C for 5 h. From each well, 100 µl of supernatant were removed and counted in a gamma counter for the assessment of ⁵¹Cr release. Freshly isolated murine neutrophils were resuspended in 100 µl of assay medium (RPMI, 10% FCS, 10 ng/ml murine granulocyte-macrophage-CSF) at various concentrations and utilized in a ⁵¹Cr release cytotoxicity assay as described for human neutrophils.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CIR and retroviral vector structure

The structure of the primary immune receptors utilized by T cells and myeloid/NK cells, the TCR and FcR, respectively, is depicted in Figure 1. HIV gp120-specific CIRs were constructed by fusing the cytoplasmic domains of either the TCR- or FcR-signaling chains to the extracellular and TM domains of the human CD4 receptor. Both CD4-CIRs are expressed as monomers since they do not possess the cysteine-bearing TM domain of the - or -chain. Furthermore, by virtue of the CD4 TM domain, the / chimeras are expressed independently of endogenous /-chains (Refs. 15 and 16 and data not shown). Although we used HIV-specific CIRs as a model system in this study, CIRs can be generated against a variety of cell surface Ag on virally infected or malignant cells by utilizing single-chain derivatives of mAbs with the appropriate specificity (17, 18).

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Structure of TCR, Fc receptor (FcR), and chimeric immune receptors CD4 and CD4. Each hatched box located within the TCR, FcR, and CIR cytoplasmic domains represents a copy of the conserved 18-amino acid ITAM.

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Structure of the integrated retroviral vectors. A, Transcription initiates from the MMLV LTR of rkat43.2. B, rkat43.3pgk has a disabled 3'-LTR, and therefore transcription initiates from the internal phosphoglycerate kinase (pgk) promoter. The structures of the resulting CIR transcripts are shown in each case.

/-CIR surface expression by retrovirally transduced NK cells

The human NK clone NK3.3 has the phenotypic and functional characteristics of primary NK cells (22), exhibiting both natural and FcR-mediated ADCC. Such NK clones can be readily expanded ex vivo and are therefore suitable for evaluating the expression and activity of - and -CIRs. NK3.3 cells were transduced with rkat43.2 bearing either CD4 or CD4 as described in Materials and Methods. Although both CIRs were efficiently expressed on the cell surface following transduction, the intensity of expression for CD4 was routinely twofold higher than CD4 despite equivalent levels of gene transfer (data not shown). Subsequent flow cytometric sorting resulted in two CD4 populations: the first with CD4 levels equivalent to the CD4 NK population (CD4^low); the second with CD4 levels approximately twofold higher than the CD4 population (CD4^high) (Fig. 3A). Expression of endogenous FcRIIIA in the transduced populations was unaffected by coexpression of either - or -CIRs (data not shown).

View larger version (32K): [in this window] [in a new window]   FIGURE 3. Surface expression and cytolytic function of CIRs on retrovirally transduced human NK cells. NK3.3 cells were transduced with rkat433.2 retroviral vectors bearing either CD4 or CD4. Transduced NK cells expressing either CD4 or CD4 were subsequently enriched by FACS as described in the text. A, Surface expression of CD4 and CD4. Untransduced and CIR-enriched NK cells were stained with FITC anti-CD4 mAbs, and FITC isotype-matched mAb was used as control. The percentage of CD4+ cells is shown, and numbers in parentheses represent mean intensity of fluorescence. B, Cytolytic function of CD4 and CD4. Parental Raji cells (Raji-p) or HIVgp120-expressing Raji cells (Raji-env) were used as targets in cytotoxicity assays with untransduced or CIR-transduced NK cells as effectors. To compare the efficiency of CIR-mediated cytolytic activity with that of FcRIII-mediated ADCC, all three NK cell populations were also tested for their ability to lyse parental Raji cells in the presence of anti-human lymphocyte serum (Raji-p/ADCC).

/-CIR-mediated cytolysis by human NK cells

We have previously described the ability of CD4-modified NK cells to kill HIVenv-expressing target cells as efficiently as CD4 T cells (19). In this study, we compared the CD4, CD4^low, and CD4^high NK populations shown in Figure 3A for their ability to mediate cytolysis in standard killing assays. Target cells were either unmodified or modified Raji cells expressing low levels of the CIR-specific ligand, HIVgp120. CIR-mediated cytolysis was compared with FcR-mediated ADCC in each case (Fig. 3B). As shown previously (19), the level of CD4 killing exceeded that of ADCC, with 55% cytolysis reached at E:T ratios of 10:1 for CD4 (Fig. 3B) and over 100:1 for ADCC (data not shown). In contrast, the level of cytolytic activity mediated by CD4^low NK cells was similar to ADCC. CD4^low NK cells were also markedly less efficient than CD4 NK cells expressing similar CIR levels, with 20% lysis attained at E:T ratios of 10:1 for CD4^low compared with only 0.07:1 for CD4. Despite the higher CIR levels expressed on the CD4^high NK population, cytolysis was still less efficient than for CD4 effectors, with 20% lysis requiring an E:T ratio of 12.5:1. In summary, the -CIR is markedly more efficient than either endogenous FcRIIIA or the -CIR at mediating NK cytolysis, even when expressed at lower levels than the latter receptor. These data show that the -chain is inherently less active than the -chain at triggering cytolytic function of this NK effector population.

In vitro differentiation of /-CIR-expressing neutrophils from retrovirally transduced human CD34⁺ progenitors

Neutrophils are the most numerous immune cell type, constituting approximately one-half to two-thirds of all circulating human white blood cells. The bone marrow is responsible for maintaining a constant supply of neutrophils at the remarkable rate of 1 x 10¹¹ per day (23), each of which is terminally differentiated and programmed to die within 1 to 2 days. To evaluate the ability of - and -bearing immune receptors to initiate and specifically direct human neutrophil cytolytic activity, CD4 or CD4 CIRs were introduced into human CD34⁺ cells in vitro by retroviral transduction with rkat43.2. Transduced progenitors were analyzed by two-color flow cytometry with mAbs specific for CD4 (to detect CIR expression) and the pan-leukocyte marker, CD45. As shown in Figure 4A, 70 and 49% of CD45⁺ cells expressed CD4 or CD4, respectively. The transduced populations were subsequently expanded and differentiated into neutrophils in liquid culture containing appropriate cytokines. After 16 days of exposure to cytokines, 25 and 8% of CD15⁺ neutrophils derived from the transduced progenitor population expressed CD4 or CD4, respectively (Fig. 4B). In addition to the decrease in the percentage of CD4⁺ cells detected, the intensity of CIR expression per cell decreased during differentiation. Southern analysis of the CD4- and CD4-transduced neutrophil populations revealed that the frequency of CIR-marked cells was similar in both cases (approximately three copies per cell; data not shown), suggesting that the differential change in CD4 and CD4 surface expression observed during neutrophil maturation from progenitors is most likely posttranscriptional in origin.

View larger version (51K): [in this window] [in a new window]   FIGURE 4. Surface expression of - and -CIRs on human neutrophils derived from retrovirally transduced CD34+ progenitors. CD34+ cells isolated from human bone marrow were prestimulated for 2 days and subsequently exposed to rkat43.2 retroviral supernatant encoding either CD4 or CD4 as described in Materials and Methods. A, Two days posttransduction, cells were stained with PE anti-CD4 and FITC anti-CD45 mAbs to detect the level of CD4 or CD4 expression. The percentage of CD45+ cells expressing CD4 is shown. B, After 16 days of liquid expansion and differentiation into neutrophils (by exposure to human G-CSF), cells were stained with PE anti-CD4 and FITC anti-CD15 mAbs to detect the level of CD4 or CD4 expression in mature neutrophils. The percentage of CD15+ cells expressing CD4 is shown. Numbers in parentheses represent mean intensity of fluorescence for A and B. Untransduced cells were used as controls for A and B.

/-CIR-mediated cytolysis by human neutrophils derived in vitro

The transduced and unmodified neutrophils shown in Figure 4B were subsequently tested in cytotoxicity assays against parental Raji cells and Raji cells expressing gp120. Figure 5 shows the relative cytolytic activity of the CD4- and CD4-expressing populations, with the E:T ratio corrected for differences in the absolute percentage of CIR-expressing neutrophils in each case. The data show that the CD4 receptor in particular was able to efficiently activate cytolysis by the human neutrophil effector population against the Raji-env targets. The level of CD4-mediated lysis was very impressive (specific lysis above background was seen at E:T ratios as low as 1.5:1) and was even more efficient than ADCC at E:T ratios of <25:1. /-CIR-mediated killing was highly ligand specific as shown by the absence of detectable Raji-p killing. Unmodified neutrophils exhibited no significant activity above background against Raji-env or Raji-p cells. CD4 neutrophils exhibited higher levels of target-specific lysis than the CD4 population, with 25% maximal lysis by CD4 neutrophils at E:T ratios of 25:1 as compared with only 5% maximal lysis by CD4 neutrophils at E:T ratios of 5:1. In summary, the -CIR is able to direct the cytolytic activity of human neutrophils in a highly target-specific manner and with impressive efficiency and is more efficient than the -CIR. However, correction of E:T ratios for differences in the absolute percentage of CD4 and CD4 expression may still underestimate CD4 activity. We therefore went on to compare both CD4 and CD4 expression and function in the murine transplant system described below.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. Cytolytic function of CD4 and CD4 expressed on human neutrophils. Parental Raji cells (Raji-p) or HIVgp120-expressing Raji cells (Raji-env) were used as targets in cytotoxicity assays with untransduced (control), CD4-transduced, or CD4-transduced human neutrophils shown in Figure 4B. E:T ratios were corrected for the percentage of the bulk population expressing CD4 or CD4 by flow cytometric analysis. FcR-mediated ADCC was measured by incubation of neutrophils with Raji-p cells in the presence of anti-human lymphocyte serum (ADCC/Raji-p).

In vivo differentiation of /-CIR-expressing neutrophils from retrovirally transduced murine bone marrow

The data described above demonstrate that CIRs are expressed and functionally active in neutrophils differentiated in vitro from retrovirally transduced human CD34⁺ progenitors. To confirm that such observations are also relevant to mature neutrophils derived in vivo, we went on to evaluate CIR expression and function following transplantation of SCID mice with CIR-transduced syngeneic bone marrow. As in humans, SCID mice rapidly reconstitute donor-derived myeloid and NK lineages posttransplant. We have previously shown that SCID bone marrow/hemopoietic stem cells (HSC) transduced with -CIR successfully differentiate into CIR-expressing myeloid and NK cells in vivo, giving rise to murine neutrophils with -CIR-directed cytolytic function (20). In the current study, we have extended this observation to include -bearing CIRs, thereby enabling subsequent comparison of CD4 and CD4 expression and neutrophil-mediated cytotoxicity.

High titer retroviral supernatants bearing CD4, CD4, or a "silent" CD4 receptor in which the cytoplasmic domain is deleted, CD4^del, were generated using the kat retroviral system as described in Materials and Methods. Transduction of mouse bone marrow stem cells was accomplished as described in Materials and Methods using chemotherapy plus cytokine incubation to induce stem cell cycling before transduction. Surface expression of each CD4- CIR on transduced cells preinfusion is shown in Figure 6A. Although the total number of cells expressing CD4 and CD4 was similar (78 and 62%, respectively), the mean intensity of expression was considerably higher for the -CIR and CD4^del receptor. Quantitative-competitive PCR analysis confirmed the immunofluorescence data and showed similar levels of integrated provirus for the CIR-transduced populations (data not shown). The transduced bone marrow was subsequently transplanted into sublethally irradiated syngeneic mice via tail vein injection.

View larger version (35K): [in this window] [in a new window]   FIGURE 6. Surface expression of - and -CIRs on murine neutrophils derived in vivo following bone marrow transplantation. Bone marrow cells isolated from 5-fluorouracil-treated SCID mice were transduced with rkat43.3pgk retroviral supernatant encoding CD4, CD4, or CD4del (CD4), as described in Materials and Methods. A, Before infusion, transduced cells were stained with PE anti-huCD4 mAb to detect the level of CD4, CD4, or CD4del (CD4) expression. The percentage of bone marrow cells expressing CD4 is shown. Numbers in parentheses represent the mean intensity of fluorescence. B, Transduced marrow was subsequently transplanted into sublethally irradiated SCID mice. Three weeks posttransplant, mice were treated with G-CSF and neutrophils isolated from peripheral blood as described in Materials and Methods. The percentage of neutrophils expressing human CD4 is shown. Numbers in parentheses represent the mean intensity of fluorescence.

/-CIR mediated cytolysis by murine neutrophils derived in vivo

The CD4, CD4, and CD4^del neutrophil populations (Fig. 6B) were subsequently tested in cytotoxicity assays for their ability to kill Raji-p and Raji-env cells over a range of E:T ratios. Although the absolute level of CD4-mediated cytotoxicity is relatively low in the murine as compared with the human system, it is absolutely specific for Raji cells expressing gp120 (Fig. 7A). Efficient killing via ADCC was observed for each of the three CD4-CIR neutrophils, with specific lysis of >50% target cells at E:T ratios of >25:1 (data not shown). Although the murine and human -chains exhibit a high degree of homology (25), the human -chain may be less active in murine neutrophils. Furthermore, human cells may be less efficient targets for murine than for human effectors. Alternatively, differences in the absolute levels of CD4/ activity between the human and murine systems may not be species specific but may correlate with ex vivo vs in vivo modes of neutrophil generation, respectively. A previous study with CD4-transplanted C3H mice yielded even higher levels of Raji-env specific lysis, however, with 17% lysis attained at an E:T ratio of 40:1 (Fig. 7B). Although some specific CD4-mediated cytolysis against Raji-env cells was observed over background, the efficiency was much lower than for CD4 and rapidly decreased with E:T ratio. The lower level of CD4-mediated cytolytic activity was confirmed in two subsequent experiments (data not shown). As expected, CD4^del neutrophils exhibited no specific cytolysis in these assays. In summary, these data confirm and extend the results described above for CD34-derived neutrophils.

View larger version (30K): [in this window] [in a new window]   FIGURE 7. Cytolytic function of CD4 or CD4 expressed on murine neutrophils. A, Raji-p or Raji-env cells were used as targets in cytotoxicity assays with the CD4del-(CD4), CD4-, or CD4-transduced SCID neutrophils shown in Figure 6B. B, Raji-p or Raji-env cells were used as targets in cytotoxicity assays with CD4-transduced or untransduced (control) murine C3H neutrophils as described in the text. FcR-mediated ADCC was measured by incubation of control neutrophils with Raji-env cells in the presence of anti-human lymphocyte serum (ADCC). Background cytolysis by controls was always <1% even at E:T ratios >100:1 (A and B).

Relative expression of /-CIR following bone marrow transplantation

SCID mice transplanted with each of the 3 CIRs were subjected to more extensive immunophenotyping to determine the relative efficiency of - vs - expression in both myeloid and NK cell compartments over time. SCID mice possess a higher percentage of NK cells than their immunocompetent counterparts, thereby facilitating phenotypic analysis of this relatively small effector population. Peripheral blood was isolated from reconstituted animals at various times posttransplant and analyzed by two-color flow cytometry using mAbs against human CD4 and the following mouse blood cell lineage markers: GR-1 (expressed on all granulocytes including neutrophils and basophils); Mac-1 (expressed on monocyte/macrophages and neutrophils); or NK-1.1 (expressed on NK cells). Expression of CD4, CD4, and CD4^del was detected in granulocytes, monocytes, and NK cells in the peripheral blood of transplanted animals at 16 weeks posttransplant (Fig. 8). The percentage of myeloid and NK cells expressing CD4 and CD4^del was similarly high, with averages of 30% for CD4 and 40% for CD4^del. In contrast, surface expression of CD4 was significantly lower, with an average of 12% of myeloid and NK cells expressing this receptor. In addition to the lower number of cells expressing detectable CD4, the lower intensity of CD4 receptors parallels that observed for the transduced human NK- and CD34-derived neutrophil populations described above (Figs. 3 and 4). It is striking that the percentages of GR-1-, Mac-1-, and NK-1.1-positive populations expressing a particular CD4-CIR are almost identical. Since the "neutral" receptor, CD4^del, gives a similar expression profile to CD4/, it appears that expression of - or -bearing receptors in primitive progenitors early in development does not negatively impact subsequent maturation of myeloid and NK cells. Furthermore, although the overall level of expression is lower for CD4 than for CD4, no major qualitative differences in expression are seen for - as compared with -bearing receptors. Finally, expression of all three CD4-CIRs was stable over at least a 7-month period (data not shown).

View larger version (58K): [in this window] [in a new window]   FIGURE 8. Efficient long term reconstitution of SCID mice with myeloid and NK cells expressing - or -CIR following bone marrow transplantation. Peripheral blood was isolated from SCID mice at 16 wk following transplantation with CD4-, CD4-, or CD4del (CD4)-transduced bone marrow. Peripheral blood was analyzed by two-color flow cytometry using PE-conjugated anti-human CD4 mAb and FITC-conjugated anti-GR-1, anti-Mac-1, or anti-NK-1.1 mAb. Specific staining was measured on gated populations containing myeloid cells (GR-1 and Mac-3) or lymphoid cells (NK-1.1) as determined by forward and side scatter characteristics. The percentage of GR-1-, Mac-1-, or NK-1.1-positive cells expressing human CD4 is shown in each case and is representative of 20 additional mice. Expression of all three CD4 receptors was stable over several months.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

/-CIR- and FcR-mediated cytolysis

FcRIIIA is the primary FcR utilized for by NK cells for ADCC (26) and requires associated homo- and heterodimers of - and -chains for both surface expression and effector function (5, 6). We have previously shown that CIRs bearing the cytoplasmic domain can efficiently activate NK cells (19). In this report, we have extended these studies to compare the cytolytic capacity of - and -CIRs in NK cells, and we show that CIRs possessing the cytoplasmic domain also redirect the cytolytic activity of NK cells in an Ag-dependent manner. Presumably, both - and -receptors mediate NK cytolytic activity by coupling to the endogenous FcRIIIA signaling pathway. However, our results reveal that -CIR-mediated cytotoxicity is far more efficient than that triggered by either -CIR or FcRIIIA (ADCC), even when expressed at lower levels than -CIR. These results suggest that in NK cells, the -chain has intrinsically more cytolytic activity than .

The role of the signaling chain in facilitating FcR-mediated ADCC in neutrophils formed the basis for the studies described in this report in which we compare /-CIRs for their ability to activate neutrophil cytolytic function. Of the three classes of FcRs that neutrophils use to trigger the lytic machinery, FcRI appears to be the only one that utilizes a member of the / family for signal transduction. Specifically, FcRI requires the -chain for assembly and effector functions such as phagocytosis and presumably ADCC (27, 28). In contrast, FcRIIA possesses an ITAM-like motif within the cytoplasmic domain that mediates cytolysis in the absence of the -chain (29), and does not require for assembly. The third FcR expressed on human neutrophils, FcRIIIB, is anchored to the plasma membrane through a glycosylphosphatidylinositol linkage and is not associated with / subunits. Although FcRIIIB cannot mediate ADCC independently, it may function synergistically with other FcR (30, 31). Since there is no homologous glycosylphosphatidylinositol-anchored FcRIIIB in mice, however, murine neutrophils express FcRIIIA. We show in this report that CIRs bearing the signal transduction domain of the subunit efficiently and specifically direct the cytolytic activity of neutrophils. This finding is intriguing given the fact that human neutrophils primarily utilize the subunit for FcR-mediated cytolysis. To understand the relationship between signal transduction pathways initiated by FcRs and -chimeras to induce neutrophil cytotoxicity, a number of issues require further clarification including the impact of and ITAM structure on PTK binding and activation, the role of individual classes of FcR in ADCC, and the influence of ligand identity on function.

Although CD4-mediated killing was marginally more efficient than FcR-driven ADCC by human neutrophils (e.g., twofold difference based on E:T ratio required to achieve 20% lysis), CD4-mediated cytolysis by human NK cells routinely exceeded that of FcRIIIA-mediated ADCC (e.g., 14-fold difference based on E:T ratio required to achieve 20% lysis). The consistently smaller CIR/FcR "cytolytic ratio" for human neutrophils as compared with NK cells does not appear to result from major differences in ADCC efficiency between the two effector cell types. Despite the potential for the direct and synergistic contribution of all three classes of neutrophil FcR in harnessing both /-dependent and -independent pathways, ADCC by human neutrophils was equal to or somewhat less efficient than FcRIIIA-mediated ADCC by human NK cells in our system. The observation that the -CIR functions less efficiently relative to endogenous FcR for human neutrophils than do NK cells is intriguing. An interesting report potentially relevant to this observation describes how neutrophil-ADCC but not NK-ADCC against Raji cells shows a striking dependency on target ligand identity rather than expression level per se (32).

Structural and functional relationship between and

CD4 mediates significantly higher levels of cytolytic activity than CD4 when expressed in NK cells, even when expressed at lower levels than the latter receptor. Therefore, the signaling chain is inherently more active than at harnessing the cytolytic pathway in NK effectors. For neutrophils, however, the significantly lower intensity of CD4 as compared with CD4 expression (e.g. eightfold for murine neutrophils) may also contribute to the lower cytolytic function of CD4 observed. The structure of the and subunits differs in two major ways: 1) quantitatively: and contain three and one copies, respectively, of the ITAM; and 2) qualitatively: specific internal and flanking amino acids differ between and ITAMs, although the tyrosine and leucine residues are conserved. Therefore, -CIR may be more effective than -CIR at NK-mediated cytolysis for quantitative and/or qualitative reasons. Quantitative differences between - and -based chimeras are supported by studies showing a direct relationship between the number of -chain ITAMs and the efficiency of cytokine release (33), apoptosis (34), and thymocyte selection (35). However, microheterogeneity in the amino acid sequence between - and -chain ITAMs can also result in qualitative differences in signal transduction and effector function. For example, differential binding and activation of Src and Syk family PTKs have been reported for - and -chain ITAMs (7, 36, 37, 38, 39), and the subunit is approximately six times more efficient than the subunit at mediating phagocytosis (7). Indeed, recent data from our laboratory reveal that CD4 is far more efficient than CD4 at mediating phagocytosis despite the lower level of CD4 surface expression (M. Wang and M. R. Roberts, unpublished data). Further studies will be necessary to dissect the relationship between - and -ITAM structure, Src/Syk family PTK interaction, and cytolytic function.

Relative expression of /-CIRs on myeloid and NK cells in vivo following bone marrow transplantation

We have previously shown that -CIR are efficiently expressed on myeloid and NK cells following transplantation of CIR-modified bone marrow (20). In this report, we show that bone marrow modified with a CIR bearing the signaling domain of the FcRI -chain can also give rise to mature FcR⁺ effector cells expressing -CIR in vivo. However, -CIR are always expressed more efficiently on the cell surface than their -bearing counterparts. This observation is consistent with a study of knockout mice expressing the -chain that showed that TCR complexes associated with rather than are expressed less well on T cells (40). The reasons for this interesting phenomenon are unclear, but they may involve differences in the half-life of - and -derived receptors and/or differences in PTKs associated with them. For example, activation of Lck maintains low TCR expression in immature thymocytes by causing retention and degradation of TCR components (41). Future experiments will test this hypothesis for -CIR expression in NK cells and neutrophils.

Concluding remarks

In a previous study, we showed that CD4-transplanted SCID mice were protected from challenge with a lethal dose of a human B cell leukemia (Raji) expressing HIVenv (20). Cytolysis must clearly be an essential component of this in vivo antitumor effect, although the relative contribution of different effector functions (phagocytosis, for example) and classes of myeloid and NK cells remains to be determined. In the current report, we show that -CIR is more effective than -CIR at triggering NK- and neutrophil-mediated cytolysis in vitro. However, -CIR may be more effective at mediating other therapeutically relevant effector functions. For example, the subunit is more efficient than in mediating phagocytosis (7), a finding consistent with preliminary data from our laboratory comparing phagocytic signaling by - and -CIR (M. Wang and M. R. Roberts, unpublished data). It will therefore be of interest in future studies to compare - and -CIRs for their ability to generate T cell-independent systemic immunity in vivo.

The pleiotropic biologic responses triggered via FcR make them potentially attractive candidates for directed immunotherapy. Indeed, FcR-directed bispecific Abs are currently being clinically evaluated for treating cancer and infectious disease (42, 43). Introduction of disease-specific chimeric immune receptors into bone marrow progenitor cells may have significant therapeutic potential for malignant and infectious diseases. Such an approach would provide a constant supply of Ag-specific myeloid and NK cells from gene-marked HSC in vivo and therefore may have advantages over ex vivo modification of terminally differentiated effector cells such as T lymphocytes or systemic delivery of FcR-directed bispecific Abs. Finally, the ability to readily harness myeloid and NK cell function by genetically modifying HSC and progenitors with Ag-specific chimeric receptors provides a powerful experimental approach with which to study the role of nonlymphoid effector cells in the host immune system.

	Acknowledgments

 
We thank Drs. Ronald Germain, Harold Malech, and Arthur Weiss for a critical reading of the manuscript and Jacqueline Rothwell for preparation of the manuscript.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Margo R. Roberts, Cell Genesys Inc., 342 Lakeside Drive, Foster City, CA 94404. E-mail address:

² Current address: Amgen, Thousand Oaks, CA 94132.

³ Current address: Scripps Research Institute, La Jolla, CA 92037.

⁴ Current address: Systemix, Palo Alto, CA 94304.

⁵ Current address: Biovest Consulting, Cupertino, CA 95015.

⁶ Abbreviations used in this paper: ITAM, immunoglobulin tyrosine activation motif; CIR, chimeric immune receptors; HSC, hemopoietic stem cells; , FcRI subunit; PTK, protein tyrosine kinase; CD4^del gene, truncated form of the CD4 receptor in which the cytoplasmic residues have been deleted; HIVenv, HIV envelope protein; MMLV, Moloney murine leukemia virus; LTR, long terminal repeat; hu, human; SCF, stem cell factor; G-CSF, granulocyte-colony-stimulating factor; LDC, low density cells; PE, phycoerythrin; ADCC, antibody-dependent cellular cytotoxicity; TM, transmembrane; Raji-p, parental Raji cells derived from Burkitt’s lymphoma; Raji-env, Raji cells derived from Burkitt’s lymphoma and transfected with HIVenv.

Received for publication October 8, 1997. Accepted for publication March 3, 1998.

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