Cross-presentation of tumour antigens by human induced pluripotent stem cell-derived CD141(+)XCR1+ dendritic cells.

Monocyte-derived dendritic cells (moDC) have been widely used in cancer immunotherapy but show significant donor-to-donor variability and low capacity for the cross-presentation of tumour-associated antigens (TAA) to CD8(+) T cells, greatly limiting the success of this approach. Given recent developments in induced pluripotency and the relative ease with which induced pluripotent stem (iPS) cell lines may be generated from individuals, we have succeeded in differentiating dendritic cells (DC) from human leukocyte antigen (HLA)-A(*)0201(+) iPS cells (iPS cell-derived DC (ipDC)), using protocols compliant with their subsequent clinical application. Unlike moDC, a subset of ipDC was found to coexpress CD141 and XCR1 that have been shown previously to define the human equivalent of mouse CD8α(+) DC, in which the capacity for cross-presentation has been shown to reside. Accordingly, ipDC were able to cross-present the TAA, Melan A, to a CD8(+) T-cell clone and stimulate primary Melan A-specific responses among naïve T cells from an HLA-A(*)0201(+) donor. Given that CD141(+)XCR1(+) DC are present in peripheral blood in trace numbers that preclude their clinical application, the ability to generate a potentially unlimited source from iPS cells offers the possibility of harnessing their capacity for cross-priming of cytotoxic T lymphocytes for the induction of tumour-specific immune responses.

The new immunosuppression: intervention at the dendritic cell-T-cell interface.

Dendritic cells (DCs) play an important role in initiating and directing T-cells towards immunity or tolerance. An important aim of emerging immunosuppressive strategies is to ensure that antigen is perceived in a 'tolerogenic context'. This would have obvious benefit in minimising the need for long-term drug maintenance in organ transplantation, hypersensitivity and autoimmune diseases. Here we review the biology of the interplay between the DC and T-cell, with a specific focus on therapeutic drugs targeting molecules that effect their interaction and function.

Directed differentiation of dendritic cells from mouse embryonic stem cells.

Dendritic cells (DCs) are uniquely capable of presenting antigen to naive T cells, either eliciting immunity [1] or ensuring self-tolerance [2]. This property identifies DCs as potential candidates for enhancing responses to foreign [3] and tumour antigens [4], and as targets for immune intervention in the treatment of autoimmunity and allograft rejection [1]. Realisation of their therapeutic potential would be greatly facilitated by a fuller understanding of the function of DC-specific genes, a goal that has frequently proven elusive because of the paucity of stable lines of DCs that retain their unique properties, and the inherent resistance of primary DCs to genetic modification. Protocols for the genetic manipulation of embryonic stem (ES) cells are, by contrast, well established [5], as is their capacity to differentiate into a wide variety of cell types in vitro, including many of hematopoietic origin [6]. Here, we report the establishment, from mouse ES cells, of long-term cultures of immature DCs that share many characteristics with macrophages, but acquire, upon maturation, the allostimulatory capacity and surface phenotype of classical DCs, including expression of CD11c, major histocompatibility complex (MHC) class II and co-stimulatory molecules. This novel source should prove valuable for the generation of primary, untransformed DCs in which candidate genes have been overexpressed or functionally ablated, while providing insights into the earliest stages of DC ontogeny.

Targeting of T lymphocytes to melanoma cells through chimeric anti-GD3 immunoglobulin T-cell receptors.

Immunoglobulin T-cell receptors (IgTCRs) combine the specificity of antibodies with the potency of cellular killing by grafting antibody recognition domains onto TCR signaling chains. IgTCR-modified T cells are thus redirected to kill tumor cells based on their expression of intact antigen on cell surfaces, bypassing the normal mechanism of activation through TCR-peptide-major histocompatibility complex (MHC) recognition. Melanoma is one of the most immunoresponsive of human cancers and has served as a prototype for the development of a number of immunotherapies. The target antigen for this study is the ganglioside GD3, which is highly expressed on metastatic melanoma with only minor immunologic cross-reaction with normal tissues. To determine an optimal configuration for therapy, four combinations of IgTCRs were prepared and studied: sFv-epsilon, sFv-zeta, Fab-epsilon, Fab-zeta. These were expressed on the surface of human T cells by retroviral transduction. IgTCR successfully redirected T-cell effectors in an MHC-unrestricted manner, in this case against a non-T-dependent antigen, with specific binding, activation, and cytotoxicity against GD3+ melanoma cells. Soluble GD3 in concentrations up to 100 microg/ml did not interfere with recognition and binding of membrane-bound antigen. Based on the outcomes of these structural and functional tests, the sFv-zeta construct was selected for clinical development. These results demonstrate key features that emphasize the potential of anti-GD3 IgTCR-modified autologous T cells for melanoma therapies.

Structure and chromosomal location of mouse and human CD52 genes.

Human CD52 (CAMPATH-1 antigen) is an abundant surface molecule on lymphocytes and a favoured target for lymphoma therapy and immunosuppression. It comprises a small glycosylphosphatidylinositol (GPI) anchored peptide to which a large carbohydrate moiety is attached. Structurally similar proteins include the proposed mouse homologue, B7 antigen (B7-Ag; not to be confused with the CD28 ligand), and human and mouse CD24. Sequence similarities between CD52 and B7-Ag precursors are concentrated over the signal peptides and the sequences cleaved during GPI attachment. While the short mature peptides are not apparently homologous, the N-linked glycosylation site is retained in both. We describe similarities in exon-intron organisation, syntenic chromosome positions (human CD52, 1p36; mouse B7-Ag, chromosome 4, between Dsil and D4Nds16) and sequence homology in the promoter regions which strongly suggests that B7-Ag is the mouse homologue of CD52. The structure of these genes is also similar to that of mouse CD24, suggesting a common ancestor. Promoter activities and transcription start sites were also analysed. These results suggest that human CD52 and mouse B7-Ag gene expressions are controlled by TATA-less promoters.

Bypassing immunization: optimized design of "designer T cells" against carcinoembryonic antigen (CEA)-expressing tumors, and lack of suppression by soluble CEA.

Tumor-associated antigens are typically nonimmunogenic in cancer patients, "immune surveillance" having manifestly failed. The fact that most tumor antigens are normal human proteins presents significant obstacles to current cancer immunization approaches that researchers are presently striving to overcome. An alternative strategy bypasses immunization altogether by direct genetic alteration of autologous patient T cells, to create "designer T cells" specific to a particular antigen. Chimeric immunoglobulin-T cell receptors (IgTCR) with a specificity for carcinoembryonic antigen (CEA) were created to evaluate the optimal IgTCR structure for cancer therapy. Antigen-binding domains of a humanized antibody were combined with TCR signaling chains to yield four different chimeric IgTCR: single chain Fv fragment (sFv)-zeta, fragment antigen-binding (Fab)-zeta, sFv-epsilon, and Fab-epsilon. All of the IgTCR were well expressed on T cells, and all showed specific binding and activation, as demonstrated by IL-2 production on contact with immobilized or cellular CEA, excepting sFv-epsilon alone which was inert solely against cellular targets for steric reasons unique to this construct. In contrast to prior studies of isolated TCR chains that related increased tyrosine-based activation motifs in zeta as a reason for superior signaling potency, these tests are the first to show that epsilon and zeta are indistinguishable for T cell signaling when assayed in the context of the intact TCR complex. Further, Fab was equivalent to sFv as an IgTCR component for expression and antigen binding, establishing an important alternative for IgTCR antigen recognition because sFvs may often lose antigen affinity. When IgTCR was expressed on normal human T cells, cytotoxic potency was demonstrated at low E:T ratios, with T cell recycling and progressive tumor cell destruction. Contrary to recent speculations, these observations prove that high affinity TCR interactions are not an impediment to serial target engagement and disengagement by cytotoxic T cells. The multivalent intercellular interactions of target cell binding, activation, and cytotoxicity were resistant to inhibition by soluble CEA. These studies establish a potentially important new immunotherapeutic modality for the treatment of CEA-expressing tumors.

A single-chain immunotoxin against carcinoembryonic antigen that suppresses growth of colorectal carcinoma cells.

We have engineered an anti-carcinoembryonic antigen (CEA) single-chain immunotoxin derived from humanized anti-CEA antibody (hMN14) and a truncated Pseudomonas exotoxin (PE), PE40. The purified anti-CEA immunotoxin (hMN14(Fv)-PE40) was first measured for binding affinity against a CEA-positive colorectal carcinoma cell line and compared with its parental IgG and the monovalent Fab fragment. The Ka of sFv-PE40, Fab, and IgG were 5 x 10(7), 6 x 10(7), and 3 x 10(8) M(-1), respectively. There was no significant affinity loss by conversion of Fab to the single-chain Fv, but these monovalent forms were 5-6-fold reduced in affinity compared with the parental IgG. In cytotoxicity assays, the hMN14(Fv)-PE40 showed specific growth suppression of CEA-expressing colon cancer cell lines MIP-CEA (high CEA) and LS174T (moderate CEA) with IC50s of 12 ng/ml (0.2 nM) and 69 ng/ml (1.1 nM). These IC50s correlated inversely with the surface expression of CEA, such that 50% killing was equivalent for each cell type when expressed in toxin molecules bound/cell (3000-5000). The presence of soluble CEA up to 1000 ng/ml did not affect the cytotoxicity against CEA-expressing cells, with 50% suppression only at 4000 ng/ml that correlated with the binding Kd of the single-chain Fv. The stability of the hMN14(Fv)-PE40 molecule at 37 degrees C was confirmed by bioassay and by lack of aggregation. Our hMN14(Fv)-PE40 may be clinically useful for tumors with high CEA expression without affecting normal tissues with low or absent CEA, even in patients with high soluble antigen levels.

Understanding the business of physician practices.

Acquiring physician practices requires knowledge of the business of managing a group practice. Before establishing revenue expectations for the acquired practice, the purchaser should understand the nature of the product being sold, the change in cost behavior patterns brought about by the acquisition, components of physician compensation, and the accounting impact of writing off goodwill.

Structure and chromosomal location of the mouse interleukin-12 p35 and p40 subunit genes.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of p35 and p40 subunits and is required for induction of T helper 1 (Th1) responses. Knowledge of how the IL-12 gene is regulated will permit an understanding of susceptibility and resistance to pathogenic microbes and to autoiummune diseases. In this report, we provide the gene structures, nucleotide sequences and chromosomal assignment for the p35 and p40 subunits of mouse IL-12. The p35 and p40 subunit genes are distributed over 8 kb and 14 kb, and map to chromosomes 3 and 11, respectively. The p35 subunit gene consists of eight exons, including a 5'-noncoding exon that was defined by sequence comparison of genomic DNA with the 5'ends of novel cDNA molecules. Transcription of p35 mRNA can start from the first exon but can also initiate further downstream. Potential transcription regulatory elements, AP1, AP2, AP3, NF-kB and GATA recognition sequences, are located within 523 bp upstream of the p35 gene; however, no TATA box was identified. The p40 subunit gene consists of eight exons. A TATA box is located 30 bp upstream from the transcription start site, and AP1, AP3, GATA, and Pu.1 recognition sequences are located within 690 bp upstream of the p40 gene. An AGTTTCTACTTT sequence, which acts as an interferon-gamma response element in the promoter of the major histocompatibility complex class I gene, was also found upstream of the p40 gene.

Characterization of the human properdin gene.

A cosmid clone containing the complete coding sequence of the human properdin gene has been characterized. The gene is located at one end of the approximately 40 kb cosmid insert and approximately 8.2 kb of the sequence data have been obtained from this region. Two discrepancies with the published cDNA sequence [Nolan, Schwaeble, Kaluz, Dierich & Reid (1991) Eur. J. Immunol. 21, 771-776] have been resolved. Properdin has previously been described as a modular protein, with the majority of its sequence composed of six tandem repeats of a sequence motif of approximately 60 amino acids which is related to the type-I repeat sequence (TSR), initially described in thrombospondin [Lawler & Hynes (1986) J. Cell Biol. 103, 1635-1648; Goundis & Reid (1988), Nature (London) 335, 82-85]. Analysis of the genomic sequence data indicates that the human properdin gene is organized into ten exons which span approximately 6 kb of the genome. TSRs 2-5 are coded for by discrete, symmetrical exons (phase 1-1), which supports the hypothesis that modular proteins evolved by a process involving exon shuffling. TSR1 is also coded for by a discrete exon, but the boundaries are asymmetrical (phase 2-1). The sequence coding for the sixth TSR is split across the final two exons of the gene with the first 38 amino acids of the repeat coded for by an asymmetric exon (phase 1-2). This split at the genomic level has been shown, by alignment analysis, to be reflected at the protein level with the division of repeat 6 into TSR-like and TSR-unlike sequences.

Genetic and physical mapping around the properdin P gene.

A CA repeat has been found on the human X chromosome within 16 kb of the gene encoding properdin P factor (PFC) and has been shown to be a highly informative marker. Two more polymorphic CA repeats were found in a cosmid containing DXS228. The CA repeats, and other markers from proximal Xp, were mapped genetically in CEPH families and the likely order of markers was established as Xpter-(DXS7, MAO-A, DXS228)-(PFC, DXS426)-(TIMP, OATL1)-DXS255-Xcen. This places PFC in the region Xp11.3-Xp11.23, thus refining previous in situ hybridization data. Two yeast artificial chromosomes (YACs) (440 and 390 kb) contain both PFC and DXS426, and one of them (440 kb) also contains TIMP. This confirms the genetic order TIMP-(PFC, DXS426). PFC and TIMP are located on the same 100-kb SalI/PvuI fragment of the 440-kb YAC. Given the genetic orientation of TIMP and (PFC, DXS426), this YAC can now serve as a starting point for directional walking toward disease genes located in Xp11.3-Xp11.2 such as retinitis pigmentosa (RP2) and Wiskott-Aldrich syndrome.

Neutron and X-ray scattering studies on the human complement protein properdin provide an analysis of the thrombospondin repeat.

Properdin is a regulatory glycoprotein of the alternative pathway of the complement system of immune defense. It is responsible for the stabilization of the C3 convertase complex formed between C3b and the Bb fragment of factor B. Neutron and X-ray solution scattering experiments were performed on the dimeric and trimeric forms of properdin. These have RG values of 9.1 and 10.7 nm, respectively. The scattering curves were compared with Debye sphere modeling simulations for properdin. Good agreements were obtained for models similar to published electron micrographs showing that the properdin trimer has a triangular structure with sides of 26 nm. Such a structure also accounted for sedimentation coefficient data on properdin. Primary structure analyses for mouse and human properdin have shown that this contains six homologous motifs known as the thrombospondin repeat (TSR), which is the second most abundant domain type found in the complement proteins. Sequences for these 12 TSRs were aligned with 19 others found in thrombospondin and the late complement components. Three distinct groups of TSRs were identified, namely, the TSRs found in thrombospondin and properdin, the TSRs mostly found at the N-terminus of the late complement components, and the TSRs found at the C-terminus of the late components. Averaged secondary structure predictions suggested that all three groups contain similar backbone structures with two amphipathic turn regions and one hydrophilic beta-strand region. The mean dimensions of the TSRs of properdin in solution were determined to be approximately 4 nm X 1.7 nm X 1.7 nm, showing that these are elongated in structure.

Molecular cloning of the cDNA coding for properdin, a positive regulator of the alternative pathway of human complement.

Northern blot analysis indicated that the mRNA for human properdin is approximately 1.5 kb long and that its level in U-937 cells is increased by pretreating the cells with phorbol 12-myristate 13-acetate (PMA). Using a human genomic probe clones coding for human properdin were isolated from a lambda gt10 cDNA library derived from PMA-treated U-937 cells. The sequence of the 1474-bp cDNA insert of the longest clone revealed an open reading fram of 1326 bp coding for the entire 442 amino acids of the mature form of human properdin and 67 bp coding for 22 amino acids of typical, but incomplete leader sequence. Polymerase chain reaction "RACE" experiments identified the start site ATG and revealed the complete, 27-amino acid-long, leader peptide sequence. Within the 81-bp 3' non-translated extension a polyadenylation signal was identified 41 bp downstream from the stop codon, TAA, and 12 bp upstream of a 19 nucleotide long poly(A) tail. The amino acid sequence of human properdin is clearly divided into three distinct regions: a 49 residue-long N-terminal region, a 32 residue-long C-terminal region and a middle region, covering residues 50 to 411, composed of six tandemly repeated thrombospondin repeat (TSR) motifs of the type first described in the adhesive glycoprotein thrombospondin and also known to be present in the C6, C7, C8 alpha, C8 beta and C9 terminal components of complement. Human and mouse properdin sequences show a high (approximately 76%) degree of identity with almost complete conservation of the relatively large number of Cys (44) and Trp (20) residues.

The design and use of specific genetic probes to identify closely related bunyaviruses and to determine the genotype of their recombinants.

Viruses that are very closely related to each other at the genetic and gene product level can prove difficult to distinguish, although they may differ in phenotype (for example in their virulence or vector preferences). A chimeric genetic probe has been developed and tested to distinguish the S RNAs of two closely related bunyaviruses, snowshoe hare and La Crosse viruses. The technique is applicable to other RNA species of these two bunyaviruses.