Reversible major histocompatibility complex I-peptide multimers containing Ni(2+)-nitrilotriacetic acid peptides and histidine tags improve analysis and sorting of CD8(+) T cells

MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin (PE) streptavidin (SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell-staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni(2+)-nitrilotriacetic acid (NTA) with oligohistidines. We synthesized biotinylated linear mono-, di-, and tetra-NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His(6), His(12), or 2×His(6) tag by surface plasmon resonance on SA-coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His(6) < His(12) < 2×His(6) and NTA(1) < NTA(2) < NTA(4), respectively, depending on the configuration of the NTA moieties and increased to picomolar K(D) for the combination of a 2×His(6) tag and a 2×Ni(2+)-NTA(2). We demonstrate that HLA-A2-2×His(6)-peptide multimers containing either Ni(2+)-NTA(4)-biotin and PE-SA- or PE-NTA(4)-stained influenza and Melan A-specific CD8+ T cells equal or better than conventional multimers. Although these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells.     

A fast and efficient HLA multimer-based sorting procedure that induces little apoptosis to isolate clinical grade human tumor specific T lymphocytes

HLA multimers are now widely used to stain and sort CD8 T lymphocytes specific for epitopes from viral or tumoral antigens presented in an HLA class I context. However, the transfer of this technology to a clinical setting to obtain clinical grade CD8 T lymphocytes that may be used in adoptive cell transfer (ACT) is hindered by two main obstacles: the first obstacle is the use of streptavidin or derived products that are not available in clinical grade to multimerize HLA/peptide monomers and the second is the reported high degree of apoptosis that eventually occurs when T cell receptors are crosslinked by HLA multimers. In the present report, we describe new HLA multimers composed of immunomagnetic beads covalently coupled to a mAb specific for the AviTag peptide and coated with HLA/peptide monomers bearing the non biotinylated AviTag at the COOH terminus of the HLA heavy chain. Thus, all the components of this new reagent can be obtained in clinical grade. We compared these new multimers with the previously described multimers made with streptavidin beads coated with biotinylated HLA/peptide monomers, in terms of sorting efficiency, recovery of functional T cells, apoptosis and activation. We provide evidence that the new multimers could very efficiently sort pure populations of T lymphocytes specific for three different melanoma antigens (Melan-A, gp100 and NA17-A) after a single peptide stimulation of melanoma patients’ PBMC. The recovered specific T cells were cytotoxic against the relevant melanoma cell-lines and, in most cases, produced cytokines. In addition, in marked contrast with streptavidin-based multimers, our new multimers induced very little apoptosis or activation after binding specific T lymphocytes. Altogether, these new multimers fulfill all the necessary requirements to select clinical grade T lymphocytes and should facilitate the development of ACT protocols in cancer patients.     

CD8 T cells, like CD4 T cells, are triggered by multivalent engagement of TCRs by MHC-peptide ligands but not by monovalent engagement

T cell activation is initiated by recognition of antigenic peptide presented in complex with MHC molecules on the surface of APCs. The mechanism by which this recognition occurs is still unclear, and many models exist in the literature. CD4 T cells have been shown to respond to soluble oligomers of activating class II MHC-peptide complexes, but not to soluble monomers. In determining the reactivity of CD8 T cells to soluble activating class I MHC-peptide complexes, a complicating phenomenon had been observed whereby peptide from soluble complexes was loaded onto cell surface MHCs on the T cells and re-presented to other T cells, clouding the true valency requirement for activation. This study uses soluble allogeneic class I MHC-peptide monomers and oligomers to stimulate murine CD8 T cells without the possible complication of peptide re-presentation. The results show that MHC class I monomers bind to, but do not activate, CD8 T cells whether the cells are in solution or adhered to a surface. Monomeric MHC class I binding can antagonize the stimulation triggered by soluble oligomers, a phenomenon also observed for CD4 T cells. Dimeric engagement is necessary and sufficient to stimulate downstream activation processes including TCR down-regulation, Zap70 phosphorylation, and CD25 and CD69 up-regulation, even in T cells that do not express the MHC coreceptor CD8. Thus, the valency dependence of the response of CD8 T cells to soluble MHC-peptide reagents is the same as previously observed for CD4 T cells.     

Soluble MHC-peptide complexes containing long rigid linkers abolish CTL-mediated cytotoxicity

Soluble MHC-peptide (pMHC) complexes induce intracellular calcium mobilization, diverse phosphorylation events, and death of CD8+ CTL, given that they are at least dimeric and co-engage CD8. By testing dimeric, tetrameric, and octameric pMHC complexes containing spacers of different lengths, we show that their ability to activate CTL decreases as the distance between their subunit MHC complexes increases. Remarkably, pMHC complexes containing long rigid polyproline spacers (> or =80 A) inhibit target cell killing by cloned S14 CTL in a dose- and valence-dependent manner. Long octameric pMHC complexes abolished target cell lysis, even very strong lysis, at nanomolar concentrations. By contrast, an altered peptide ligand antagonist was only weakly inhibitory and only at high concentrations. Long D(b)-gp33 complexes strongly and specifically inhibited the D(b)-restricted lymphocytic choriomeningitis virus CTL response in vitro and in vivo. We show that complications related to transfer of peptide from soluble to cell-associated MHC molecules can be circumvented by using covalent pMHC complexes. Long pMHC complexes efficiently inhibited CTL target cell conjugate formation by interfering with TCR-mediated activation of LFA-1. Such reagents provide a new and powerful means to inhibit Ag-specific CTL responses and hence should be useful to blunt autoimmune disorders such as diabetes type I.     

Peptide-MHC class I tetrameric complexes display exquisite ligand specificity

The production of synthetic MHC-peptide tetramers has revolutionized cellular immunology by revealing enormous CD8(+) T cell expansions specific for peptides from various pathogens. A feature of these reagents, essential for their staining function, is that they bind T cells with relatively high avidity. This could, theoretically, promote cross-reactivity with irrelevant T cells leading to overestimates of epitope-specific T cell numbers. Therefore, we have investigated the fine specificity of CTL staining with these reagents for comparison with functional data. Using a panel of CTL clones with distinct fine specificity patterns for analogs of an HLA-B8-binding EBV epitope, together with B8 tetramers incorporating these peptides, we show a very good correlation between tetramer staining and peptide activity in cytotoxicity assays. Significant staining only occurred with tetramers that incorporate strong stimulatory agonist peptides and not weak agonists that are unlikely to induce full T cell activation at physiological levels of presentation. In almost every case where a peptide analog had >10-fold less activity than the optimal EBV peptide in cytotoxicity assays, the corresponding tetramer stained with >10-fold less intensity than the EBV epitope tetramer. Furthermore, by examining an EBV-specific clonotypic T cell expansion in EBV-exposed individuals, we show similar fine specificity in tetramer staining of fresh peripheral T cells. Collectively, our data demonstrate the exquisite specificity of class I MHC-peptide tetramers, underlining their accuracy in quantifying only those T cells capable of recognizing the low levels of cell surface peptide presented after endogenous Ag processing.     

Establishment of gp100 and MART-1/Melan-A-specific cytotoxic T lymphocyte clones using in vitro immunization against preselected highly immunogenic melanoma cell clones

The induction of an in vitro T cell response against tumour-associated antigens with subsequent expansion of the individual cytotoxic T lymphocyte (CTL) clones still is not routine and the only tumour-associated antigen that has been found to easily induce the establishment of CTL clones is the MART-1/Melan-A antigen. In this paper, we describe a new approach for in vitro immunization based on the use of preselected melanoma cell clones. The human melanoma cell subline FM3.P was cloned and the immunological properties of individual clones were compared. Melanoma cell clone FM3.29, having a high level of expression of melanoma differentiation antigens, as well as high levels of the HLA class I and class II antigens and adhesion molecules, was used for the establishment of a CTL line that was subsequently cloned. For optimization of the conditions of growth of established CTL clones, a particular melanoma subline FM3.D/40 was selected for supporting the proliferation of CTL clones. The majority of the established CTL clones recognized the melanoma-associated differentiation antigens gp100 and MART-1/Melan-A. Epitope analysis indicated that two different epitopes derived from gp100 (154-162 and 280-288) and a single epitope from MART-1/Melan-A (27 35) were recognized by these CTL clones. The gp100-specific CTL clones were found to be significantly more sensitive to the culture conditions than the MART-1/Melan-A-specific CTL clones. In addition, the presence of excess peptide in the culture medium induced autokilling of the gp100-specific, but not the MART-1/Melan-A-specific CTL clones. Taken together, these results demonstrate that, by careful preselection of melanoma cell lines and clones both for the induction of CTL line from patients' peripheral blood lymphocytes and subsequent cloning, it is possible to obtain a large number of stable CTL clones even against such an inherently "difficult" differentiation antigen as gp100.     

Staining of celiac disease-relevant T cells by peptide-DQ2 multimers.

Gluten-specific T cells in the small intestinal mucosa are thought to play a central role in the pathogenesis of celiac disease (CD). The vast majority of these T cells recognize gluten peptides when presented by HLA-DQ2 (DQA1*05/DQB1*02), a molecule which immunogenetic studies have identified as conferring susceptibility to CD. We have previously identified and characterized three DQ2-restricted gluten epitopes that are recognized by intestinal T cells isolated from CD patients, two of which are immunodominant. Because almost all of the gluten epitopes are restricted by DQ2, and because we have detailed knowledge of several of these epitopes, we chose to develop peptide-DQ2 tetramers as a reagent to further investigate the role of these T cells in CD. In the present study, stable soluble DQ2 was produced such that it contained leucine zipper dimerization motif and a covalently coupled peptide. We have made four different peptide-DQ2 staining reagents, three containing the gluten epitopes and one containing a DQ2-binding self-peptide that provides a negative control for staining. We show in this study that peptide-DQ2 when adhered to plastic specifically stimulates T cell clones and that multimers comprising these molecules specifically stain peptide-specific T cell clones and lines. Interestingly, T cell activation caused severe reduction in staining intensities obtained with the multimers and an Ab to the TCR. The problem of TCR down-modulation must be taken into consideration when using class II multimers to stain T cells that may have been recently activated in vivo.     

Soluble MHC-peptide complexes induce rapid death of CD8+ CTL

Soluble MHC-peptide (pMHC) complexes, commonly referred to as tetramers, are widely used to enumerate and to isolate Ag-specific CD8(+) CTL. It has been noted that such complexes, as well as microsphere- or cell-associated pMHC molecules compromise the functional integrity of CTL, e.g., by inducing apoptosis of CTL, which limits their usefulness for T cell sorting or cloning. By testing well-defined soluble pMHC complexes containing linkers of different length and valence, we find that complexes comprising short linkers (i.e., short pMHC-pMHC distances), but not those containing long linkers, induce rapid death of CTL. This cell death relies on CTL activation, the coreceptor CD8 and cytoskeleton integrity, but is not dependent on death receptors (i.e., Fas, TNFR1, and TRAILR2) or caspases. Within minutes of CTL exposure to pMHC complexes, reactive oxygen species emerged and mitochondrial membrane depolarized, which is reminiscent of caspase-independent T cell death. The morphological changes induced during this rapid CTL death are characteristic of programmed necrosis and not apoptosis. Thus, soluble pMHC complexes containing long linkers are recommended to prevent T cell death, whereas those containing short linkers can be used to eliminate Ag-specific CTL.     

Development of new biotin/streptavidin reagents for pretargeting

The high affinity of biotin for streptavidin has made this pair of molecules very useful for in vivo applications. To optimize reagents for one potential in vivo application, antibody-based pretargeting of cancer, we have prepared a number of new biotin and streptavidin derivatives. The derivatives developed include new radiolabeled biotin reagents, new protein biotinylation reagents, and new biotin multimers for cross-linking and/or polymerization of streptavidin. We have also modified streptavidin by site-directed mutation and chemical modification to improve its in vivo characteristics, and have developed new reagents for cross-linking antibody fragments with streptavidin. A brief overview of these new reagents is provided.     

Oligomeric structure of gp41, the transmembrane protein of human immunodeficiency virus type 1.

We characterized the structural forms of the human immunodeficiency virus env-encoded proteins with a panel of monoclonal and polyclonal antibodies. Western blot (immunoblot) assays with antibodies specific for gp41 invariably recognized a major component of 160 kilodaltons and a less intense component of 120 kilodaltons in viral lysates. We demonstrated that these species are noncovalently associated tetramers and trimers of gp41 which represent the native form of this protein in virions. These complexes were stable when boiled in the presence of low concentrations of sodium dodecyl sulfate but were dissociated to gp41 monomers at high sodium dodecyl sulfate concentrations. Moreover, two human monoclonal antibodies preferentially recognized the oligomeric complexes over monomeric gp41 in Western blots, indicating the presence of epitopes recognized by the human immune system on the gp41 multimers which are not efficiently expressed by the dissociated monomers. The demonstration of the existence of multimeric env complexes and the enhanced and altered antigenicity of such multimers may be relevant to the design of subunit and recombinant human immunodeficiency virus env vaccines.     

A novel autocrine pathway of tumor escape from immune recognition: melanoma cell lines produce a soluble protein that diminishes expression of the gene encoding the melanocyte lineage melan-A/MART-1 antigen through down-modulation of its promoter

We have observed that malignant melanoma cells produce a soluble protein factor(s), which down-regulates melanocyte lineage Melan-A/MART-1 Ag expression by melanoma cells with concomitant loss of recognition by Melan-A/MART-1-specific T cells. This down-modulation of Melan-A/MART-1 expression, which we refer to as "Ag silencing," is mediated via its minimal promoter, whereas the promoter for the restricting Ag-presenting HLA-A2 molecule is not affected. Significantly, this Ag silencing is reversible, as removal of factor-containing supernatants from Melan-A/MART-1-expressing cells results in up-regulation of the promoter for the gene encoding this Ag, and renewed expression of the protein. We have evaluated over 20 known factors, none of which accounts for the Ag-silencing activity of the melanoma cell culture supernatants. The existence of this autocrine pathway provides an additional novel explanation for melanoma tumor progression in vivo in the presence of CTL specific for this melanocyte lineage Ag. These observations may have important implications for Melan-A/MART-1-specific CTL-mediated immunotherapy of melanoma tumors.     

Assessment of immunogenicity of human Melan-A peptide analogues in HLA-A*0201/Kb transgenic mice

Previous studies have shown that substitution of single amino acid residues in human Melan-A immunodominant peptides Melan-A27-35 and Melan-A26-35 greatly improved their binding and the stability of peptide/HLA-A*0201 complexes. In particular, one Melan-A peptide analogue was more efficient in the generation of Melan-A peptide-specific and melanoma-reactive CTL than its parental peptide in vitro from human PBL. In this study, we analyzed the in vivo immunogenicity of Melan-A natural peptides and their analogues in HLA-A*0201/Kb transgenic mice. We found that two human Melan-A natural peptides, Melan-A26-35 and Melan-A27-35, were relatively weak immunogens, whereas several Melan-A peptide analogues were potent immunogens for in vivo CTL priming. In addition, induced Melan-A peptide-specific mouse CTL cross-recognized natural Melan-A peptides and their analogues. More interestingly, these mouse CTL were also able to lyse human melanoma cell lines in vitro in a HLA-A*0201-restricted, Melan-A-specific manner. Our results indicate that the HLA-A*0201/Kb transgenic mouse is a useful animal model to perform preclinical testing of potential cancer vaccines, and that Melan-A peptide analogues are attractive candidates for melanoma immunotherapy.     

Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries

We report for the first time the affinity maturation of Fab antibody fragments using fluorescent-activated cell sorting (FACS) of yeast-displayed repertoires. A single yeast display vector which enables the inducible expression of an anchored heavy chain and a soluble light chain has been constructed. The assembly and functional display on the yeast cell surface of Fab antibodies specific for different protein targets has been demonstrated by flow cytometry and immunofluorescence microscopy. We have affinity matured a Fab antibody specific for the tetravalent antigen streptavidin using FACS of yeast-displayed repertoires diversified by error-prone polymerase chain reaction. A panel of variants with up to 10.7-fold improvement in affinity was obtained after selection. Two leading clones, R2H10 (3.2 nM) and R3B1 (5.5 nM), had mutations in light chain complementarity determining region 1 LC-CDR1 (H34R) and LC-CDR3 (Y96H or Y96F) and gave a 10.7-fold and 6.3-fold affinity improvement over the starting antibody, respectively. The ability to efficiently affinity mature Fab antibodies is an important component of the antibody development pipeline and we have shown that yeast display is an efficient method for this purpose.     

Final antigenic Melan-A peptides produced directly by the proteasomes are preferentially selected for presentation by HLA-A*0201 in melanoma cells

The melanoma-associated protein Melan-A contains the immunodominant CTL epitope Melan-A(26/27-35)/HLA-A*0201 against which a high frequency of T lymphocytes has been detected in many melanoma patients. In this study we show that the in vitro degradation of a polypeptide encompassing Melan-A(26/27-35) by proteasomes produces both the final antigenic peptide and N-terminally extended intermediates. When human melanoma cells expressing the corresponding fragments were exposed to specific CTL, those expressing the minimal antigenic sequence were recognized more efficiently than those expressing the N-terminally extended intermediates. Using a tumor-reactive CTL clone, we confirmed that the recognition of melanoma cells expressing an N-terminally extended intermediate of Melan-A is inefficient. We demonstrated that the inefficient cytosolic trimming of N-terminally extended intermediates could offer a selective advantage for the preferred presentation of Melan-A peptides directly produced by the proteasomes. These results imply that both the proteasomes and postproteasomal peptidases limit the availability of antigenic peptides and that the efficiency of presentation may be affected by conditions that alter the ratio between fully and partially processed proteasomal products.     

A new generation of Melan-A/MART-1 peptides that fulfill both increased immunogenicity and high resistance to biodegradation: implication for molecular anti-melanoma immunotherapy.

Intense efforts of research are made for developing antitumor vaccines that stimulate T cell-mediated immunity. Tumor cells specifically express at their surfaces antigenic peptides presented by MHC class I and recognized by CTL. Tumor antigenic peptides hold promise for the development of novel cancer immunotherapies. However, peptide-based vaccines face two major limitations: the weak immunogenicity of tumor Ags and their low metabolic stability in biological fluids. These two hurdles, for which separate solutions exist, must, however, be solved simultaneously for developing improved vaccines. Unfortunately, attempts made to combine increased immunogenicity and stability of tumor Ags have failed until now. Here we report the successful design of synthetic derivatives of the human tumor Ag Melan-A/MART-1 that combine for the first time both higher immunogenicity and high peptidase resistance. A series of 36 nonnatural peptide derivatives was rationally designed on the basis of knowledge of the mechanism of degradation of Melan-A peptides in human serum and synthesized. Eight of them were efficiently protected against proteolysis and retained the antigenic properties of the parental peptide. Three of the eight analogs were twice as potent as the parental peptide in stimulating in vitro Melan-specific CTL responses in PBMC from normal donors. We isolated these CTL by tetramer-guided cell sorting and expanded them in vitro. The resulting CTL efficiently lysed tumor cells expressing Melan-A Ag. These Melan-A/MART-1 Ag derivatives should be considered as a new generation of potential immunogens in the development of molecular anti-melanoma vaccines.     

Multimer technologies for detection and adoptive transfer of antigen-specific T cells

Identification and purification of antigen-specific T cells without altering their functional status are of high scientific and clinical interest. Staining with major histocompatibility complex (MHC)-peptide multimers constitutes a very powerful method to study antigen-specific T-cell subpopulations, allowing their direct visualization and quantification. MHC-peptide multimers, such as dimers, tetramers, pentamers, streptamers, dextramers and octamers have been used to evaluate the frequency of CD8⁺ T cells, specific for tumor/leukemia-associated antigens as well as for viral antigens, e.g., CMVpp65 and EBV-EBNA. Moreover, MHC-peptide multimers have been used for rapid and efficient ex vivo isolation and expansion of T cells. A recent development in the field of MHC-peptide multimers led to the purification of CD8⁺ T cells specific for leukemia antigens. This might help to select leukemia-specific donor lymphocyte infusions (DLIs), thus allowing dissection of the noxious graft-versus-host disease (GvHD) from beneficial anti-viral and even anti-leukemic effects. This review covers different types of MHC-peptide multimers and their applications, as well as the impact that multimers might have on further development of DLIs.     

Subcellular localization of the melanoma-associated protein Melan-AMART-1 influences the processing of its HLA-A2-restricted epitope

The peptide derived from the melanoma-associated protein Melan-A (Melan-A(26-35)/HLA-A2) is an attractive candidate for tumor immunotherapy but little is known about the intracellular processing of this antigen. Here we show that Melan-A is a single-pass membrane protein with an NH(2) terminus exposed to the lumen of the exocytic compartment. In transfected melanoma cells, Melan-A accumulates in the Golgi region. Inversion of the membrane topology leads to the retention of Melan-A in the endoplasmic reticulum. Most strikingly, melanoma cells expressing this form of Melan-A are more effectively recognized by specific CTL than those expressing either Melan-A in its native membrane orientation or Melan-A artificially localized in the cytosol. Our data are compatible with the notion that proteins retained in the endoplasmic reticulum are more efficiently degraded and produce more antigenic peptides.     

Induction of anomalous killing activity from antigen-specific CTL clones by adding high doses of human recombinant interleukin 2

Four out of six long-term murine cytotoxic T lymphocyte (CTL) clones specific for trinitrophenyl (TNP)-modified spleen cells could develop an anomalous cytotoxicity against syngeneic and allogeneic tumor cells upon stimulation with TNP-modified spleen cells and high doses of human recombinant interleukin 2 (rIL-2). On FACS analysis, hyperactivated CTLs were positive for Thy-1, Ly 2 and LFA-1, but negative for L3T4 and asialo GM1. The staining profile of the cells with each antibody indicated that the CTL clones consisted of just one cell type. Monoclonal anti-Ly 2.2 and anti-LAA (lymphokine-activated cell-associated antigen) antibodies inhibited cytolysis of CTL and hyperactivated CTL clones against TNP-modified spleen cells, but failed to inhibit the anomalous killing of the hyperactivated CTL. The cold target competition test suggested the degeneracy of antigen specificity. The present study demonstrated that the CTL clone acquired a new specificity for tumor target cells upon stimulation with a high dose of rIL-2.     

Potent costimulation of human CD8 T cells by anti-4-1BB and anti-CD28 on synthetic artificial antigen presenting cells

The in vitro generation of cytotoxic T lymphocytes (CTLs) for anticancer immunotherapy is a promising approach to take patient-specific therapy from the bench to the bedside. Two criteria must be met by protocols for the expansion of CTLs: high yield of functional cells and suitability for good manufacturing practice (GMP). The antigen presenting cells (APCs) used to expand the CTLs are the key to achieving both targets but they pose a challenge: Unspecific stimulation is not feasible because only memory T cells are expanded and not rare naïve CTL precursors; in addition, antigen-specific stimulation by cell-based APCs is cumbersome and problematic in a clinical setting. However, synthetic artificial APCs which can be loaded reproducibly with MHC-peptide monomers and antibodies specific for costimulatory molecules could resolve these problems. The purpose of this study was to investigate the potential of complex synthetic artificial APCs in triggering the costimulatory molecules CD28 and 4-1BB on the T cell. Anti-4-1BB antibodies were added to an established system of microbeads coated with MHC-peptide monomers and anti-CD28. Triggering via CD28 and 4-1BB resulted in strong costimulatory synergy. The quantitative ratio between these signals determined the outcome of the stimulation with optimal results when anti-4-1BB and anti-CD28 were applied in a 3:1 ratio. Functional CTLs of an effector memory subtype (CD45RA^? CCR7^?) were generated in high numbers. We present a highly defined APC platform using off-the-shelf reagents for the convenient generation of large numbers of antigen-specific CTLs.     

Multimerization of high copy number plasmids causes instability: CoIE1 encodes a determinant essential for plasmid monomerization and stability

Although the natural multicopy plasmid CoIE1 is maintained stably under most growth conditions, plasmid cloning vectors related to it are relatively unstable, being lost at frequencies of 10(-2)-10(-5) per cell per generation. Evidence suggests that CoIE1 and related plasmids are partitioned randomly at cell division and that plasmid stability is correlated inversely with plasmid multimerization; factors or conditions that reduce multimerization increase stability. Cells containing plasmid multimers segregate plasmid-free cells because the multimers are maintained at lower copy numbers than monomers, as predicted by origin-counting models for copy number control. CoIE1 is stable because it encodes a determinant, cer, that is necessary for recA-, recF-, and recE-independent recombination events that efficiently convert any multimers to monomers. We have localized monomerizing and stability determinants of CoIE1 to within a 0.38 kb region that, when cloned into plasmid vectors, greatly increases their stability.