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The Journal of Immunology, 2006, 177: 3903-3912.
Copyright © 2006 by The American Association of Immunologists, Inc.

Fluorescence-Activated Cell Sorting and Cloning of Bona Fide CD8+ CTL with Reversible MHC-Peptide and Antibody Fab' Conjugates1

Philippe Guillaume*, Petra Baumgaertner{dagger}, Georgi S. Angelov*, Daniel Speiser{dagger} and Immanuel F. Luescher2,*

* Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; and {dagger} Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The isolation of subsets of Ag-specific T cells for in vitro and in vivo studies by FACS is compromised by the fact that the soluble MHC-peptide complexes and Abs used for staining, especially when combined, induce unwanted T cell activation and eventually apoptosis. This is especially a problem for CD8+ CTL, which are susceptible to activation-dependent cell death. In this study, we show that reversible MHC-peptide complexes (tetramers) can be prepared by conjugating MHC-peptide monomers with desthiobiotin (DTB; also called dethiobiotin) and multimerization by reaction with fluorescent streptavidin. While in the cold these reagents are stable and allow good staining, they rapidly dissociate in monomers at elevated temperatures, especially in the presence of free biotin. FACS cloning of Melan-A (MART-1)-specific CTL from a melanoma-infiltrated lymph node with reversible HLA-A2 Melan-A26–35 multimers yielded over two times more clones than when using the conventional biotin-containing multimers. CTL clones obtained by means of reversible multimers killed Melan-A-positive tumor cells more efficiently as compared with clones obtained with the stable multimers. Among the CTL obtained with the reversible multimers, but much less among those obtained with the stable multimers, a high proportion of clones exhibited high functional and physical avidity and died upon incubation with soluble MHC-peptide complexes. Finally, we show that Fab' of an anti-CD8 Ab can be converted in reversible DTB streptavidin conjugates the same way. These DTB reagents efficiently and reversibly stained murine and human CTL without affecting their viability.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Soluble peptide-MHC class I (pMHC)3 complexes, commonly referred to as tetramers or multimers, are widely used to analyze Ag-specific T cells (1). The present study explores the application of these reagents for the isolation of Ag-specific CD8+ CTL by FACS. Multimers are typically prepared by refolding of MHC H and L chains in the presence of a given peptide, enzymatic biotinylation of a C-terminally added biotinylation sequence peptide (BSP), and oligomerized by reacting the monomers with fluorescent streptavidin (1, 2, 3). When monodisperse streptavidin, such as Cy5 streptavidin, is used, tetrameric pMHC complexes are thus obtained (4). However, more commonly, PE-labeled streptavidin is used, which due to the large size of PE contains conjugates of diverse stoichiometry, and hence the resulting pMHC conjugates are heterogeneous, i.e., multimeric (4).

The binding of pMHC complexes to CTL is strengthened, often substantially, by their coordinate engagement of TCR and CD8 (5, 6, 7). Their binding avidity also increases with their valence, mainly by decreasing their dissociation from cells. Although for FACS sorting (or cloning) the dissociation of monomeric pMHC complexes from cells is too fast, it is adequately slow for multimer (or tetrameric) complexes (1, 2, 8). However, the multivalent coengagement of TCR and CD8 induces activation of Src kinases, namely of CD8-associated p56lck, diverse phosphorylation events, intracellular Ca2+ mobilization, and eventually death of CD8+ CTL (4, 7, 9, 10, 11). By means of site-specific alkylation of pMHC monomers containing a free cysteine in position 275 of the H chain with maleimide-containing peptide linkers, we previously prepared different types of pMHC complexes (4, 10, 11, 12). Those containing short linkers, i.e., short pMHC-pMHC distances, induced rapid mitochondrial dysfunction, followed by CTL death (10, 11, 12). By contrast, pMHC complexes containing long rigid linkers failed to activate CTL and to induce CTL death. The value of such complexes for the isolation of Ag-specific CTL in the absence of adverse cell activation is limited by their relatively difficult production.

There are other mechanisms by which pMHC complexes can induce death of CTL. For example, soluble or microsphere-bound pMHC complexes, which due to mutation in their {alpha}3 domain (D227K and T228A) cannot coengage CD8, induce Fas-dependent apoptosis of CTL (4, 13). Because such complexes induce no other CTL activation, they have been proposed to eliminate Ag-specific CTL in the absence of systemic, potentially harmful CTL activation (13). Moreover, it has been shown that on CD8+ T cells, peptides can be transferred from soluble to associated MHC molecules (12, 14, 15). Although the mechanism of this peptide transfer is unclear, it can cause diverse complications, such as sensitization of CTL for fratricide killing (16).

For conclusive in vitro studies and adoptive transfer experiments of Ag-specific T cells, it is necessary to isolate such cells in the absence of unwanted, potentially harmful cell activation. Multimers have been used for FACS sorting and cloning (17, 18, 19). However, in light of previous reports showing that soluble pMHC complexes can induce death, especially of CD8+ effector T cells (4, 11, 14, 15, 16, 20), this strategy harbors the risk that certain cells are lost. This leaves the uncertainty that the cells thus obtained may not correspond to those originally present. Indeed, the efficiencies of tetramer-based CTL cloning are typically poor. To circumvent this caveat different strategies have been used. For example, PBMC or PBL were briefly stimulated with given antigenic peptides and the responding IFN-{gamma}-secreting cells isolated by magnetic cell sorting (21, 22, 23). Alternatively, Ag-specific CD8+ T cells were isolated by FACS (or MACS) by using reversible streptamers that can be dissociated in monomeric pMHC complexes after sorting or cloning by addition of free biotin (20). These reversible staining pMHC multimers are prepared by adding at the C-termini of the H and L chains a streptag peptide sequence, which binds to streptactin, a genetically engineered form of streptavidin, with low avidity to allow easy displacement by free biotin. However, as for the long pMHC complexes (10, 12), these reagents are costly and difficult to prepare, which limits their accessibility. We therefore explored an alternative strategy to prepare reversible staining reagents, which is simple, inexpensive, and also applicable for the production of reversible Ab Fab'.

To this end we examined biotin derivatives, which bind to streptavidin with reduced avidity, such that rapid and complete displacement with free biotin is possible, yet sufficiently stable complexes can be prepared. Iminobiotin was found unsuitable, because it’s binding to streptavidin is highly pH dependent, which necessitated exposure of cells to harsh pH changes for staining and distaining, respectively (24). For desthiobiotin (DTB; or dethiobiotin), a biotin derivative lacking the sulfur atom, such pH changes were not needed for staining and destaining (25, 26, 27, 28). DTB has the additional advantage that it can be introduced in proteins like biotin by means of the biotin transferase Bir A or by site-specific alkylation with maleimide or iodoacetyl derivatives (29, 30). Because pepsin can cleave Igs in F(ab')2, which can be converted in Fab' by reduction with cysteamine (31), DTB can thus also be introduced in Fab', which then can be conjugated with streptavidin-like DTB-pMHC complexes. While in the cold the binding of DTB to streptavidin is sufficiently stable, it dissociates rapidly at elevated temperatures, especially at 37°C and in the presence of free biotin (26, 28).

In this study, we report that DTB containing pMHC and anti-CD8 Fab multimers allow efficient staining of Ag-specific CTL in the cold and complete destaining in biotin containing medium. When using DTB reversible pMHC complexes for FACS sorting and cloning more than twice, HLA-A2-restricted Melan-A-specific CTL were obtained from a tumor-infiltrated lymph node (TILN). Many of these cloned CTL killed tumor cells more efficiently than those obtained when using conventional stable reagents.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and Abs

S14 CTL and P815 cells were cultured, propagated, and used as described previously (9, 10, 11, 12, 32). Anti-CD8{alpha} mAb 53.6.72 (rat IgG2a) was purified from hybridoma supernatant as described previously (5). TILN from the HLA-A*0201 (A2)-positive melanoma patient Lau 969 (TILN 969) were obtained from a surgery specimen, in accordance with the guidelines of the Medical Ethical Commission of the Cantonal Hospital. Cells isolated from the TILN were cultured for several weeks in 24-well tissue culture plates (Costar) in 2 ml of IMDM (Invitrogen Life Technologies) supplemented with 0.24 mM Asn, 0.55 mM Arg, 1.5 mM Glen, 8% pooled human A+ serum, 100 U/ml IL-2, and 10 ng/ml IL-7. HLA-A2 Melan-A26–35-positive CD8+ T cells obtained from FACS sorting were cloned in Terasaki plates at 0.5 cells/well, and stimulated with PHA and allogenic feeder cells in RPMI 1640 supplemented with nonessential amino acids, sodium-pyruvate, penicillin-streptomycin, 2-ME, 5% human serum, and 150 U/ml hrIL-2. They were restimulated every 3–4 wks with PHA, irradiated feeder cells, and hrIL-2.

Peptides and linkers

All chemicals were obtained from Calbiochem-Novabiochem, Sigma-Aldrich, Pierce, or Amersham Biosciences. The peptides Melan-A26–35 (ELAGIGILTV) and (plasmodium berghei circumsporozoite peptide 252–260) PbCS(ABA; 4-azido-benzoic azide) (SYIPSAEK(ABA)I)) were synthesized on solid phase using Fmoc for transient N-terminal protection and after HPLC purification validated by mass spectrometry (4, 10). The maleimide containing DTB linker maleimide-di-aminopropionic acid (Dap)(DTB)-YEP was obtained by coupling Fmoc-Dap(Dde)-Y(O-2-chlorotrityl)-E({gamma}-2-phenylisopropyl ester)-OH on a proline-coupled 2-chlorotrityl resin. Dde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl) was removed by three 10-min treatments of the resin with 3% hydrazine in dimethylformamide, followed by coupling with DTB in dimethylformamide using 1-hydroxybenzotriazole, di-isopropylethylamine, and di-isopropylcarbodiimide as coupling agents. After removal of Fmoc, beta-alanine maleimide was introduced the same way. The washed and dried resin was incubated in 7% trifluoroacetic acid in methylenechloride containing 5% triethylsilane for 30 min at room temperature and then washed twice with the same solution. The deprotected peptide was precipitated with diethylether, purified on a semipreparative C-4 column (Vydac), which was eluted with a linear gradient of acetonitrile rising in 60 min from 0 to 75% and characterized by MALDI-TOF mass spectrometry.

Preparation of soluble pMHC complexes and 53.6.72 Fab' conjugates

Monomeric pMHC complexes were prepared following published refolding procedures using human beta2-microglobulin and the H chains of A2 and Kd, respectively (2, 3, 4). The H chains contained C-terminally an added BSP or a free cysteine in position 275 (4). For A2 also, H chains were used in which either dodeca proline-cysteine was added after Cys275 or which contained the double mutation D227K,T228A, which ablates CD8 binding (33). As peptides for the refolding of A2 complexes, we used Melan-A26–35 (ELAGIGILTV) or influenza matrix58–66 (GILGFVFTL) as nonspecific controls. For Kd refolding, the PbCS(ABA) (SYIPSAEK(ABA)I or the Cw3 170–179 (RYLENGKETL) were used. Before alkylation, the monomeric pMHC complexes were reduced with 15 mM glutathione and reacted with a 10-fold molar excess of beta N-maleimidoalanine (Mal)-Dap(DTB)YEP maleimide for ≥2 h at room temperature under argon. The alkylated monomers were purified by gel filtration on a Superdex S75 column. Biotin was introduced in BSP containing monomers by means of Bir A, free biotin, and ATP as described previously (2, 4). Multimeric and tetrameric pMHC complexes were obtained by reacting the corresponding biotinylated or desthiobiotinylated monomers with PE-labeled streptavidin (Caltag Laboratories) and Cy5-labeled streptavidin (Amersham Biosciences), respectively (2, 4, 10, 12). Octameric complexes were prepared by alkylation A2 Melan-A26–35 monomer with maleimidoacetoxy-S-Dap(S-maleimidoacetoxy)-Y-P10-K(biotin)-P and by reacting the resulting pMHC dimers with Cy5 streptavidin as described previously (10, 12). DTB-labeled Fab' of the anti-CD8{alpha} mAb 53.6.72 (rat IgG2a) were produced by digestion with pepsin and reduction of the resulting F(ab')2 with cysteamine as described previously (31). The reduced Fab' were alkylated with a 10-fold molar excess of maleimido-Dap(DTB)-YEP under argon at room temperature for ≥2 h. All further procedures were as described for the pMHC multimers.

Flow cytometry, FACS sorting, and cloning

For staining, cells were resuspended in FACS buffer (1 x 106 cells/ml) (PBS supplemented with 0.5% BSA, 30 mM HEPES, and 5 mM EDTA) and with fluorescent-labeled pMHC complexes for 30–60 min, washed with cold FACS buffer, and analyzed by flow cytometry on a FACSCalibur (BD Biosciences). Unless mentioned otherwise, cells were stained at 0–4°C using 5 nM biotin multimers or 30 nM D227K/T228A multimers. Binding isotherms were measured by incubating cloned CTL with graded concentrations of Cy5-labeled A2 Melan-A tetramer at 4°C for 1 h in FACS buffer. The samples were diluted 10-fold in cold FACS buffer immediately before analysis by flow cytometry. Nonspecific binding was assessed in parallel experiments using A2 flu matrix and Kd Cw3 multimers, respectively. For dissociation kinetic measurements, CTL were incubated with 3.5 nM Cy5-labeled A2 Melan-A26–35 tetramer for 1 h at 4°C. Aliquots were diluted 25-fold in FACS buffer at room temperature containing 3.5 nM unlabeled tetramer and analyzed by flow cytometry. All data were analyzed using the CellQuest software (BD Biosciences).

For FACS sorting, the cells were incubated with PE-labeled A2 Melan-A26–35 multimers containing biotin, mono-DTB, or di-DTB at 0–4°C for 1 h and sorted on a FACSVantage sorter (BD Biosciences) into CTL medium supplemented with 2 mM biotin. After two washes with the same medium, a part of the cells was cloned using Terasaki plates and 0.5 cells/well. Sorted cells were stimulated with PHA and allogenic feeder cells in RPMI 1640 supplemented with nonessential amino acids, sodium-pyruvate, penicillin-streptomycin, 2-ME, 5% human serum, and 150 U/ml hrIL-2. For cell viability assays, cells were transferred in U-bottom plates in DMEM (for S14 CTL) or RPMI 1640 (for human CTL) supplemented with 5% of the respective sera, incubated at 37°C with pMHC complexes, washed once in cold HBSS, and stained with Cy5- or FITC-labeled annexin V or 7-aminoactinomycin D (7AAD) according to the manufacturer’s recommendations (BD Biosciences), and analyzed by flow cytometry.

Cytotoxicity assay

Lytic activity of the cloned CTL was assessed using a standard 4-h 51Cr release assay (12, 32). For S14 CTL, P815 cells were used as target cells that were preincubated with graded concentrations of PbCS(ABA) (YSIPSAEK(ABA)I) peptide at an E:T ratio of 3:1. For the Melan A-specific CTL clones, the Melan-A+, A2+ melanoma line Me 290 was used as target cells and E:T ratios of 3:1 to 1:1. The percentage of specific lysis was calculated as follows: 100 x [(experimental – spontaneous release)/(total – spontaneous release)].


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
DTB Kd-PbCS(ABA) multimers efficiently and reversibly stain S14 CTL

To assess the staining efficiency of DTB containing Kd-PbCS(ABA) multimers, Kd monomers containing a free cysteine in position 275 were alkylated with N{alpha}-maleimido-Dap(DTB)-YEP (Fig. 1). The yield was ~90% and slightly higher than when using for alkylation commercially available DTB DTB PEO iodoacetamide. Kd-PbCS(ABA)-DTB and Kd-PbCS(ABA)-biotin monomers, respectively, were reacted with PE-streptavidin, and the resulting multimers were tested for binding to cloned S14 CTL, which recognize the photoreactive PbCS(ABA) peptide in the context of Kd (9, 10, 11, 12). While at 4°C, the binding of the two multimers was comparable at high concentrations, and the binding of the biotin multimer was higher at low concentrations (Fig. 1, A and B). The binding of the DTB, but not the biotin containing multimers, decreased with increasing temperature. At 15°C, this difference was modest, but at room temperature and especially at 37°C the difference was considerable (~20 and 30% lower, respectively).


Figure 1
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  		FIGURE 1. Binding and dissociation of biotin- and DTB-containing multimers. A, Cloned S14 CTL were incubated at 37°C ({blacktriangleup}), room temperature ({blacksquare}), or 4°C (•) with the indicated concentrations of PE-labeled biotin Kd-PbCS(ABA) multimers for 30 min, diluted 10-fold, and cell-associate fluorescence was assessed by flow cytometry. B, The same experiment was repeated using PE-labeled DTB Kd-PbCS(ABA) multimers. Binding was also assessed at 15°C ({diamondsuit}). In A and B, nonspecific binding was assessed for the corresponding irrelevant Kd-Cw3 170-17 multimers (dashed line) and was subtracted from the binding of the Kd-PbCS(ABA) multimers. The inserts show the structure of biotin and DTB, respectively. C, S14 CTL were incubated at 4°C for 60 min with 20 nM BSP Kd-PbCS(ABA) multimers, and aliquots were diluted 20-fold in prewarmed medium for the indicated periods of time at 37°C ({blacktriangleup}), room temperature ({blacksquare}), or 4°C (•) for the indicated periods of time in medium containing 2 mM biotin; cell-associated fluorescence was assessed by flow cytometry. D, This experiment was repeated in the absence (empty symbols, solid lines) or presence (filled symbols, dashed lines) of 2 mM biotin using DTB multimers. Representative experiments are shown from two or three performed.

 
As assessed in dissociation experiments, the staining of the DTB multimers was thermolabile. Half of the bound DTB multimer decayed within ~20 min of incubation at 4°C (Fig. 1D). This spontaneous dissociation increased dramatically with the temperature. Half-maximal dissociation was observed after 8 min at room temperature and after only ~3 min at 37°C. Addition of free biotin greatly accelerated the dissociation. At 37°C the dissociation was essentially complete after 2 min, i.e., at the first time point measured. At room temperature, half-maximal dissociation was observed after 3–4 min and at 4°C after ~12 min. By comparison, the dissociation of the biotin-containing multimers was very slow at all temperatures measured, irrespective of whether or not free biotin was present (Figs. 1C and data not shown).

Stable, but not reversible Kd-PbCS(ABA) multimers activate and damage S14 CTL

One of the earliest events in CTL activation is elevation of intracellular calcium. As assessed by flow cytometry, S14 CTL exhibited rapid and concentration-dependent intracellular calcium mobilization upon incubation with biotin-containing multimers, which reached a transient maximum after ~90 s (Fig. 2A). By contrast, DTB multimers induced only scant calcium flux at the highest concentrations tested and at later time points (Fig. 2B). These results demonstrate that DTB multimers at 37°C in the presence of biotin dissociate before they are able to trigger significant calcium flux.


Figure 2
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  		FIGURE 2. Stable, but not reversible Kd-PbCS(ABA) multimers activate and damage S14 CTL. A and B, Indo-1-labeled S14 CTL were incubated at 37°C in biotin-containing medium with 20 nM (•), 10 nM ({blacksquare}), and 5 nM ({blacktriangleup}) or 1 nM ({diamondsuit}) of Kd-PbCS(ABA) biotin (A), or DTB (B) multimers, and calcium-dependent Indo-1 fluorescence was measured by flow cytometry over a period of 4.5 min. One of three experiments is shown. C, S14 CTL were incubated at 4°C for 30 min with 20 nM biotin ({blacksquare}), DTB ({blacktriangleup}) Kd-PbCS(ABA) multimers or left untreated (•), washed, and incubated with 51Cr-labeled P815 cells (E:T = 3:1) in the presence of the indicated concentrations of PbCS(ABA) peptide. After 4 h of incubation at 37°C, the lysis was determined from the released chromium. D, S14 CTL were incubated for 30 min at 4°C, 15°C, room temperature (RT), or 37°C with 20 nM Kd-PbCS(ABA) biotin multimers (Figure 2), DTB multimers ({blacksquare}), or left untreated ({square}). After washing in biotin-containing medium, the cells were incubated at 37°C, and after 3, 6, and 16 h cells were stained with annexin V and analyzed by flow cytometry.

 
Furthermore, brief incubation of S14 CTL with biotin Kd-PbCS(ABA) multimers substantially reduced their ability to kill PbCS(ABA)-sensitized P815 cells (Fig. 2C). Conversely, S14 CTL pretreated likewise with DTB multimers exhibited lysis only slightly reduced as compared with untreated CTL. This reduction of cytotoxicity was caused in part by biotin Kd-PbCS(ABA) multimer-induced TCR down-modulation and in part by multimer-induced apoptosis (Fig. 2D and data not shown). Although S14 CTL that were preincubated with biotin Kd-PbCS(ABA) multimers progressively became annexin V positive upon incubation at 37°C, this was observed for DTB Kd-PbCS(ABA) multimer-pretreated cells. This difference was particularly striking when the preincubation was performed in the cold and the annexin V staining after 16 h (Fig. 2D).

FACS sorting of Melan-A-specific CTL using reversible and stable multimers

To further test the reversible DTB multimers, we examined their binding on various A2-restricted, Melan-A-specific CD8+ T cell clones. Although on many clones binding was similar as on S14 CTL (Fig. 1, A and B), DTB A2 Melan-A26–35 multimers often stained low-avidity cells, namely clones derived from naive CD8+ T cells, less efficiently than biotin multimers (data not shown). For example, on the N2.24 clone, the staining with DTB multimers in the cold was significantly weaker as compared with the biotin multimers at 4°C and even more so at room temperature or at 37°C (Fig. 3, A and B). Being concerned that FACS sorting or cloning with DTB multimers may result in preferential selection of high-avidity cells, we prepared reversible multimers containing two DTB groups spaced by a dodeca proline spacer. The 37 Å long, rigid proline spacer prevented the two DTB moieties to bind to the same streptavidin molecule, thereby enforcing the formation of larger conjugates (11, 34). The binding of these di-DTB multimers at room temperature or at 37°C was significantly increased as compared with the mono-DTB multimers and at 4°C reached slightly higher levels than the biotin multimers (Fig. 3, A and B). At room temperature and especially at 37°C, the binding of the di-DTB multimers decreased, but less pronounced as compared with the mono-DTB multimers. The dissociation of the mono- and di-DTB complexes in the presence of free biotin was as rapid as observed on S14 CTL (Fig. 1D and data not shown).


Figure 3
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  		FIGURE 3. Multimer staining and FACS sorting of TILN. A and B, Low-avidity CD8+ T cell clone N2.24 (A2-restricted, Melan-A-reactive) was incubated at 4°C (•), at room temperature ({blacksquare}) or at 37°C ({blacktriangleup}) with the indicated concentrations of A2 Melan-A26–35 multimers containing biotin (A), DTB (open symbols in B), or di-DTB (filled symbols in B), and cell-associated fluorescence was determined by flow cytometry. Nonspecific binding, as assessed by A2 flu multimers, was subtracted. C–E, TILN of melanoma patient Lau 969 were incubated at 4°C for 30 min with A2 Melan-A26–35 multimers containing biotin (C) (5 nM), mono-DTB (D) (50 nM), or di-DTB (E) (10 nM), and were sorted in biotin (2 mM)-containing medium using the indicated gates.

 
We next FACS sorted Melan-specific cells from TILN 969 upon staining in the cold with the A2 Melan-A26–35 multimers containing one or two DTB and biotin, respectively. The percentages of the multimer-positive cells were very similar (Fig. 3, C–E). Restaining of the sorted cells with normal and CD8 binding-deficient A2D227K,T228A Melan-A multimers showed an interesting difference. Although normal multimers stained DTB and biotin multimer-sorted cells equally well, the CD8 binding-deficient multimers stained twice as many cells in the former than in the latter population (Fig. 4A). This was also true for the cells sorted with the di-DTB multimers, although the difference was slightly smaller (data not shown). Because CD8 strengthens the pMHC binding avidity, the CD8 binding-deficient A2D227K,T228A multimers stain preferentially high-avidity CTL (35). These results thus argue that upon sorting with biotin-containing multimers, high-avidity CTL were selectively lost. To test this, we briefly exposed the DTB multimer-sorted cells to biotin multimers and repeated the analysis 1 wk later. This treatment decreased the biotin multimer staining by only 5%, but diminished the A2D227K,T228A multimers binding by nearly 20% (Fig. 4B). This corroborates the view that biotin-containing multimers cause the loss of high-avidity CTL.


Figure 4
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  		FIGURE 4. Multimer staining and cloning of sorted A2/Melan A-specific TILN. A, TILN from patient Lau 969 were sorted as described for Fig. 3, C–E, with A2 Melan-A multimers containing biotin or DTB were stained at 4°C for 30 min with 5 nM PE-labeled A2 Melan-A26–35 multimers (A2 Melan-A) or 30 nM A2D277K/T228A Melan-A26–35 multimers (A2 227/228 Melan-A) and analyzed by flow cytometry. B, The cells sorted with DTB multimers were incubated for 30 min at 4°C with 5 nM A2 Melan-A multimers, washed in biotin-containing medium, stimulated, and 10 days later analyzed as described for A. C, Two hundred of the sorted cells from A were cloned by limiting dilution, and after two stimulations the number of expanding clones was counted. The number of the clones obtained with the biotin A2 Melan-A multimers, in average 21.4 clones, was referred to as 100%. Mean values and SD were calculated from three experiments.

 
Cloning of Melan-A-specific CTL

For further analysis, we cloned by limiting dilution the different sorted Melan-A-specific T populations (Fig. 3, C–E). Approximately 2.3 and 2.4 times more clones were obtained from the populations sorted with the DTB and di-DTB multimers, respectively (Fig. 4C). The percentage of cells that yielded clones was in average 10.7% for the cells sorted with biotin multimer, and 24.0 and 25.2% for the DTB and di-DTB multimer-sorted cells, respectively. These values varied more from experiment to experiment than the ratios between the groups.

Staining of randomly chosen CTL clones from each group with A2 Melan-A26–35 multimers showed that there were considerable variations among the different clones (Fig. 5A). The brightest staining was observed for clones derived from DTB-sorted TILN. When the staining was performed with the CD8 binding-deficient multimers, all clones, except one that stained brightly, were derived from DTB multimer-sorted cells (Fig. 5B). This is in accordance with the finding that the A2 D227K,T228A Melan A26–35 multimers stained more efficiently the cells obtained by sorting with DTB multimers (Fig. 4A).


Figure 5
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  		FIGURE 5. Reversible multimer-sorted CTL clones exhibit high avidity and susceptibility for apoptosis. A and B, Clones obtained after sorting with A2 Melan-A multimers containing biotin ({blacksquare}), DTB ({square}), or di-DTB (Figure 5) (Fig. 3, C–E) were incubated at room temperature for 30 min with 5 nM PE-labeled A2 Melan-A26–35 multimers (A) or 30 nM A2D227K/T228A Melan-A26–35 multimers (B), washed, and analyzed by flow cytometry. Unspecific staining was assessed by using the A2 influenza hemagglutinin (GILGFVFTL) multimers and was subtracted. C, The cloned cells were incubated for 30 min at 37°C with 5 nM unlabeled A2 Melan-A26–35 octamer, stained with Cy5-labeled annexin V, and cell-associated fluorescence was measured by flow cytometry. The dashed lines indicate arbitrary threshold values. In A, the line indicates the mean fluorescence intensity (MFI) of 1500, in B the MFI of 20, and in C 15% annexin V-positive cells. Mean values and SD of the MFI were calculated from three to five experiments.

 
We then tested the different clones for annexin V staining after 30 min of incubation with A2 Melan-A26–35 octamer, which on susceptible CTL clones induces rapid phosphatidylserine externalization and cell death (10). Although many clones exhibited large variations in annexin V staining from experiment to experiment, on several clones reproducibly 15–35% of the cells became annexin V positive (Fig. 5C). Similar results were obtained when multimeric A2 Melan-A26–35 complexes were used and the incubation periods were prolonged (data not shown and Ref. 10). These results indicate that some, but not all, clones are susceptible to rapid, pMHC-induced cell death.

DTB-sorted CTL clones efficiently kill Melan-A-positive melanoma cells

We next assessed the cytolytic capacity of randomly selected CTL clones of the three sorted populations for killing of the Melan-A+ melanoma line Me 290. From the eight clones selected from the biotin A2 Melan-A multimer-sorted cells, six lysed the target cells with intermediate efficiency (maximal lysis between 50 and 70%) (Fig. 6A). One clone (no. 8) was essentially not lytic, and another one (no. 18) exhibited strong lysis. From the eight DTB-derived clones, six exhibited strong lysis (75- 96%) (Fig. 6B). Again one clone (no. 15) was not lytic, and one (no. 30) displayed weak lysis. Finally, from the seven di-DTB-derived clones, four exhibited very strong lysis (up to 100%), two slightly less (nos. 31 and 37), and one (no. 24) considerably less (Fig. 6C). Taken together, these results indicate that upon sorting of TILN 969-derived Melan-A+, CD8+ T cells with the stable multimers, a substantial fraction of the cells was lost, which could be recovered when using the reversible DTB multimers (Fig. 4C). These cells bind pMHC multimers, namely the CD8 binding-deficient ones, with increased avidity and kill efficiently Melan-A+ tumor cells (Figs. 5 and 6).


Figure 6
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  		FIGURE 6. CTL clones derived from reversible multimers more efficiently kill melanoma cells than those derived from stable multimers. Cloned CTL derived from sorting of TILN 969 cells with multimers containing biotin (A), DTB (B), or di-DTB (C) were incubated at 37°C for 4 h with 51Cr-labeled melanoma Me 290 cells at the E:T cell ratios of 30:1, 10:1, 3:1, and 1:1. Specific lysis was calculated from the released chromium. Representative experiments were selected from two to three repeats.

 
Correlation between functional and physical avidity and CTL death

We next investigated why CTL of high physical and functional avidity were preferentially lost upon exposure to stable multimers. To this end, we randomly selected eight CTL clones derived from biotin multimers and 13 from DTB multimer-sorted cells and performed detailed A2 Melan-A26–35 tetramer binding and dissociation studies. For better comparison, we expressed the lysis of Me 290 tumor cells by the different clones in a bar graph representing the average lysis measured at the different E:T ratios (Figs. 6 and 7A). From these 21 CTL clones, 12 displayed strong lysis (> 60%). Only one of these clones, clone 18, was derived from biotin multimer-sorted cells. From the 11 strongly lytic, DTB-derived clones, six exhibited strong (≥20%) phosphatidylserine externalization upon short incubation with A2 Melan-A octamer (the DTB clones 9, 20, 32, and the di-DTB clones 17, 31, and 34) (Figs. 5C and 7, A and B, red bars). We argue that the clones exhibiting high functional avidity, i.e., nearly one-third of the clones examined, were lost upon exposure to biotin A2 Melan-A multimers due to activation-induced mitochondrial dysfunction, as shown for S14 CTL (10). As seen for the di-DTB-derived clone 17, high susceptibility to pMHC cell death does not require strong and stable A2 Melan-A multimer binding (Figs. 5 and 7).


Figure 7
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  		FIGURE 7. CTL clones derived from reversible multimers exhibit increased susceptibility to apoptosis and/or more stably bind A2 Melan-A complexes. A, Mean values of the lysis at the different E:T ratios as shown in Fig. 6. B, CTL clones were incubated for 30 min with A2 Melan-A26–35 octamer, and the percentage of annexin-positive cells was measured as described for Fig. 5C. C, Cloned CTL were incubated with 3.5 nM Cy5-labeled A2 Melan-A26–35 tetramer for 1 h at 4°C, and aliquots were diluted 25-fold in FACS buffer containing 3.5 nM unlabeled tetramer, incubated at room temperature for the indicated periods of time, and analyzed by flow cytometry. The half-life designates the time in minutes, after which dissociation was half-maximal. D, Cloned CTL were incubated at room temperature for 30 min with graded concentrations of Cy5-labeled A2-Melan-A26–35 tetramer, and from the binding isotherms the concentrations were determined for which binding was half-maximal. The dashed lines indicate arbitrary threshold values. In A, it indicates 60% of lysis, in B, 15% annexin-positive cells, in C, a half-life of 4 min, and in D, KD, of 2 nM. Red bars indicate clones that exhibited >15% annexin V-positive cells upon incubation with A2 Melan-A ocatmers; blue bars indicate clones that displayed >60% specific lysis, but <15% annexin V-positive cells; {blacksquare}, {square}, and Figure 7, indicate the remaining biotin, DTB, and di-DTB clones, respectively. Experiments were repeated at least once or twice.

 
The other clones, i.e., the DTB clones 7 and 8, as well as the di-DTB clones 5, 11, and 37 had in common that they avidly and/or stably bound A2 Melan-A tetramers (Figs. 7, C and D, bars in blue). For example, the DTB clone 8 and the di-DTB clones 5 and 37 exhibited slow dissociation of A2 Melan-A tetramers (half-lives of ≥4 min) (Fig. 7C, in blue). Also, these clones exhibited avid A2 Melan-A tetramer binding, with half-maximal binding occurring between 0.1 and 2.2 nM (Fig. 7D, in blue). By comparison, for the less cytotoxic biotin clones, the A2 Melan-A tetramer dissociation half-lives were typically shorter, and the concentrations for half-maximal binding were higher (Fig. 7, C and D). Taken together, these results indicate that the strongly cytotoxic CTL clones derived from mono- and di-DTB-sorted TILN 969 were either susceptible to rapid pMHC-induced cell death or exhibited stable and/or avid tetramer binding (Fig. 7); the resulting increased exposure of CTL to soluble pMHC complexes may put them at risk to other pMHC-induced cell death mechanisms (4, 13, 14, 15, 16).

Reversible DTB anti-CD8 Fab conjugates allow FACS sorting of bona fide CTL

Based on the findings that DTB containing pMHC multimers allow efficient and reversible staining of Ag-specific CTL, we examined whether this strategy is also applicable to Ab conjugates. To this end, we digested the anti-CD8{alpha} mAb 53.6.72 with pepsin and reduced the resulting F(ab') 2 with cysteamine (Fig. 8A and Ref. 31). The resulting Fab' containing free C-terminal cysteines were then alkylated with DTB-maleimide, and the Fab'-DTB conjugates reacted with PE/Cy5 streptavidin. The 53.6.72 DTB Fab' multimers stained S14 CTL more avidly than PE/Cy5-labeled 53.6.72 Ab, which is consistent with the fact that the former have four and the latter two binding sites (data not shown). S14 CTL stained equally well with Kd-PbCS(ABA) multimers containing DTB or biotin and 53.6.72 Ab or 53.6.72 Fab' conjugates, respectively (Fig. 8B). However, upon washing with biotin-containing buffer, the staining with the DTB-containing Kd-PbCS(ABA) multimers and anti-CD8{alpha} Fab' conjugates was lost, whereas the staining with the biotin-containing multimer and the anti-CD8 Ab remained unchanged. We then incubated S14 CTL in the cold with biotin-containing Kd-PbCS(ABA) multimers, washed with biotin-containing buffer, and after reincubation for 12 h at 37°C enumerated the living cells by flow cytometry using 7AAD staining to exclude dead cells. Consistent with previous findings, S14 CTL thus treated exhibited greatly impaired viability (Fig. 8C and Ref. 10). Although the same treatment with anti-CD8 Ab 53.6.72 alone impaired cell viability only modestly, costaining with Kd-PbCS(ABA) multimers and anti-CD8 Ab provoked extensive apoptosis. By contrast, when costaining was performed with the reversible reagents, CTL viability was affected by <10%. Taken collectively, these findings show that the DTB streptavidin binding can also be used for the preparation of reversible Ab Fab' conjugates, which can be used in combination with DTB pMHC complexes without harming CTL.


Figure 8
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  		FIGURE 8. Reversible Kd PbCS(ABA) and anti-CD8 Fab' multimers allow FACS sorting of bona fide CTL. A, Cartoon of reversible Fab staining reagents. Anti-CD8 mAb 53.6.72 was digested with pepsin, and the resulting (Fab)'2 was reduced with cysteamine. The resulting Fab' were alkylated with maleimide DTB, and the Fab'-DTB conjugates reacted with PE/Cy5 streptavidin. B, S14 CTL were incubated for 30 min at 4°C as indicated with 30 nM PE/Cy5-labeled 53.6.62 mAb or 53.6.72 Fab' multimers and PE-labeled Kd-PbCS(ABA) multimers containing biotin or DTB. After washing the cells with FACS buffer (upper row) or with FACS buffer containing 2 mM biotin (lower row), they were analyzed by flow cytometry. C, Equal numbers of S14 CTL were left untreated (1) or stained likewise with stable (2 and 4) or reversible (5) Kd-PbCS(ABA) multimers in the absence (2) or presence of anti-CD8{alpha} mAb 53.6.72 (3 and 4) or 53.6.72 Fab' multimers (5), washed twice at room temperature with biotin-containing medium. After 12 h of incubation at 37°C, the cells were stained with 7AAD, and the viable cells were enumerated by flow cytometry. Mean values and SD were calculated from two experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The key finding of the present study is that activated effector CD8+ T cells can be isolated by FACS using reversible DTB pMHC multimers in the absence of undesired cell activation and cell death. Our staining experiments with DTB multimers showed that the staining of CTL with DTB multimers at low concentrations was markedly less efficient as compared with the biotin-containing multimers, a difference that decreased at higher concentrations (Figs. 1, A and B, and 3, A and B). The most likely explanation for this is that at the low pMHC concentrations (nanomolar) used in these experiments, some dissociation of DTB from streptavidin takes place. In agreement with this is the observation that DTB-multimer stained cells, destained upon washing in cold FACS buffer (data not shown). Because of this result, for efficient staining with DTB multimers typically higher concentrations have to be used, and the samples are simply diluted, but not washed, before flow cytometric analysis.

Another feature of DTB multimers is that they are thermolabile. The staining efficiency of DTB multimers substantially decreases at elevated temperatures, and the spontaneous destaining increased dramatically with the temperature (Figs. 1, A and B, and 3, A and B). The binding of biotin to streptavidin is very strong (KD of ~10–15 M) and induces structural changes in the subunits, which greatly strengthens their tetramer formation and biotin binding (27). For the much less avid binding of DTB to streptavidin, no such structural changes and cooperative effects were observed (27). For the isolation of CTL by means of DTB pMHC multimers, this thermolabile property has the advantage that it protects the cells from unwanted, potentially harmful activation upon reculturing at 37°C. In the presence of biotin, the dissociation of DTB pMHC multimers is so rapid that they are unable to elicit calcium mobilization in CTL, which occurs very rapidly after addition of stable pMHC complexes (Fig. 2, A and B, and Refs. 4, 12).

The difference in staining between biotin and DTB-containing pMHC multimers also depends on the avidity of the T cells under study. For the high-avidity S14 CTL clone, the difference in staining with the DTB vs the biotin-containing multimers was smaller as compared with low-avidity N2.24 CD8+ T cell clone (Figs. 1, A and B, and 3, A and B). This difference could be overcome by using di-DTB pMHC multimers, which contain larger conjugates and hence have an increased binding avidity. A striking finding was that the cells obtained from sorting with DTB A2 Melan-A multimers typically exhibited higher avidity than those sorted with biotin-containing multimers (Figs. 4, A and B, and 5, A and B). To test whether this was so, because the DTB multimers stained selectively high-avidity CTL, we also performed sorting with di-DTB multimers. The percentages of A2 Melan-A-positive T cells obtained by sorting using the three different multimers were very similar (Fig. 3, C–E). This argues that the differences in staining observed were accounted for mainly by the fluorescence intensity of the staining and not the number of cells binding the multimers. However, from our data some preferential sorting of high-avidity cells with the DTB multimers cannot be ruled out. For example, the cells sorted with DTB multimers stained slightly brighter with the CD8 binding-deficient A2D227K,T228A/Melan A multimers than those sorted with the di-DTB multimers (our unpublished data).

CD8 binding-deficient multimers have been shown to preferentially stain high-avidity cells and have been used to sort and clone high-avidity Ag-specific CTL (35, 36). The majority of Melan-A-specific CTL isolated with A2D227K,T228A Melan-A multimers exhibited high functional avidity and superior killing of Melan-A-positive tumor cells (36). Interestingly, we also found that the CTL clones derived from DTB or di-DTB multimer-sorted cells typically killed Me 290 melanoma cells more efficiently (Fig. 6). The finding that sorting (and cloning) of Ag-specific CTL with CD8 binding-deficient multimers and reversible DTB multimers both yield high-avidity CTL may not be surprising. Indeed, a positive correlation between the functional and physical avidity of CTL is expected, i.e., CTL that efficiently recognize Ag need to have highly sensitive pMHC binding (37, 38, 39, 40).

One mechanism by which soluble pMHC complexes elicit death of CD8+ effector T cells is via activation-induced mitochondrial dysfunction (10, 41). Indeed, a considerable fraction of the DTB and di-DTB multimer-derived CTL clones stained annexin V positive upon brief exposure to soluble A2 Melan-A complexes (Fig. 5C). Because this was not so for biotin multimer-derived CTL clones, we suggest that a significant fraction of the CTL contained in the TILN 969 were lost upon sorting with stable multimers via rapid activation-induced cell death (10). For other CTL clones, however, annexin V staining upon brief exposure to pMHC complexes was modest and considerably variable (Fig. 5C). For these clones, our limited analysis showed that pMHC multimer binding was strong and stable (Fig. 7, shown in blue), suggesting that their extended exposure to these complexes favors cell death by other mechanisms. For example, it has been shown that on CD8+ T cells, peptide is transferred from soluble to cell-associated MHC molecules, which renders the CTL susceptible to fratricide (Refs. 14, 15, 16 and our unpublished data). Moreover, pMHC complexes can induce Fas-dependent apoptosis of CTL (4, 13). Both death pathways occur with a slower kinetic than the activation-induced mitochondrial dysfunction caused cell death (4, 10, 13, 41). It must be noted, however, that although avid and stable pMHC binding increases the risk of CTL to succumb to these mechanisms, other factors are to be considered as well. For example, the susceptibility of CTL (and T cells in general) to apoptosis is determined to a large degree by their expression of molecules that promote (e.g., BIM, Bax, or BNIP3) or prevent T cell death (e.g., Bcl-2 or Bcl-x) (41, 42, 43). The advantage of the reversible DTB staining reagents is that the pMHC complexes rapidly dissociate in pMHC monomers, which in turn rapidly dissociate from the T cells and hence are lost upon washing. The complete removal of pMHC complexes from the system thus prevents all pMHC-induced cell death mechanisms.

The reversible binding of DTB to streptavidin is equally applicable to Abs. The production of DTB Fab' streptavidin conjugates follows well the established procedures of pepsin digestion of Igs, reduction of the resulting F(ab')2 in Fab', and their alkylation (31). All reagents needed are commercially available, including ready to use kits. The DTB-derivatized Fab' conjugated with fluorescent streptavidin can be used the same way as pMHC complexes. Because the DTB-streptavidin linkage is the same, the dissociation of both complexes is the same. However, because the binding of Fab' to Ag typically is more stable than pMHC monomers binding to TCR, they persist on the cells for some time. Because the adverse effects that Abs have on T cells involve cross-linking of surface molecules and surface expression of Fc portions, this is of no further consequence. Although Fab' of Igs exhibit reduced, often dramatically reduced, Ag binding, the tetrameric Fab' streptavidin conjugates exhibit equal or better staining than the parental Ab (Fig. 8 and unpublished data). Moreover, because streptavidins are commercially available in fluorescent-labeled forms, there is no need to fluorescent label Abs or their Fab.

In conclusion, the reversible binding of DTB to streptavidin allows the production of reversible pMHC-staining reagents that can be removed from cells simply by washing with biotin-containing medium at ambient temperature. DTB can be introduced in MHC class I (and class II) molecules by site-specific alkylation with commercial or easy to prepare DTB alkylation reagents or by means of Bir A as commonly used for biotin (4, 10, 11, 29). The same strategy is equally applicable for the production of reversible Ab conjugates (Fig. 8 and Ref. 31). Both types of reagents are simple and inexpensive to prepare and can also be used in combinations, thus allowing full use of powerful polychrome FACS for the isolation of specific subsets of Ag-specific T cells.


   Acknowledgments
 
We are most thankful to G. Bosshard for expert technical assistance and to Dr. P. Romero for helpful discussions.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by grants from Swiss National Foundation (no. 310000-108251) and the Swiss Cancer League (OCS 01421-08-2003). Back

2 Address correspondence and reprint requests to Dr. Immanuel F. Luescher, Ludwig Institute for Cancer Research, Lausanne Branch, 1066 Epalinges, Switzerland. E-mail address: immanuel.luescher{at}isrec.unil.ch Back

3 Abbreviations used in this paper: pMHC, peptide-MHC class I; BSP, biotinylation sequence peptide; DTB, desthiobiotin (or dethiobiotin); TILN, tumor-infiltrated lymph node; ABA, 4-azido-benzoic azide; Dap, di-aminopropionic acid; 7AAD, 7-aminoactinomycin D; MFI, mean fluorescence intensity. Back

Received for publication April 14, 2006. Accepted for publication June 16, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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