Research ArticleImmunology

TCR-Ligand koff Rate Correlates with the Protective Capacity of Antigen-Specific CD8+ T Cells for Adoptive Transfer

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Science Translational Medicine  03 Jul 2013:
Vol. 5, Issue 192, pp. 192ra87
DOI: 10.1126/scitranslmed.3005958


Adoptive immunotherapy is a promising therapeutic approach for the treatment of chronic infections and cancer. T cells within a certain range of high avidity for their cognate ligand are believed to be most effective. T cell receptor (TCR) transfer experiments indicate that a major part of avidity is hardwired within the structure of the TCR. Unfortunately, rapid measurement of structural avidity of TCRs is difficult on living T cells. We developed a technology where dissociation (koff rate) of truly monomeric peptide–major histocompatibility complex (pMHC) molecules bound to surface-expressed TCRs can be monitored by real-time microscopy in a highly reliable manner. A first evaluation of this method on distinct human cytomegalovirus (CMV)–specific T cell populations revealed unexpected differences in the koff rates. CMV-specific T cells are currently being evaluated in clinical trials for efficacy in adoptive immunotherapy; therefore, determination of koff rates could guide selection of the most effective donor cells. Indeed, in two different murine infection models, we demonstrate that T cell populations with lower koff rates confer significantly better protection than populations with fast koff rates. These data indicate that koff rate measurements can improve the predictability of adoptive immunotherapy and provide diagnostic information on the in vivo quality of T cells.


Adoptive transfer of antigen-specific CD8+ T cells is a promising approach for the treatment of viral infections (1, 2) and malignancies (35). Effective immunotherapy is believed to be dependent on T cell receptors (TCRs) within a range of high avidities for their cognate peptide epitope–major histocompatibility complex (pMHC) ligands. It has been shown that T cells expressing high-avidity TCRs confer superior efficacy toward their target cells in vitro and in vivo (68) by recognizing their target cells earlier and faster in comparison to low-avidity T cells (6). Thus, interrogating the avidity of T cells used for adoptive transfer or elicited by vaccines might provide important information on the efficacy of immune-based therapies.

TCR avidity is mainly defined by functional readouts such as cytokine production after antigen-specific stimulation or lysis of target cells pulsed with limiting concentrations of peptide (“functional avidity”). The results of these assays can be influenced by many factors including the expression level of TCRs, adhesion molecules or co-receptors, and changes in components of the signaling cascade. Remarkably, not only the specificity for target antigens (911) but also the functional avidity characteristics of specific TCRs could be transferred to newly generated T cells by transgenic expression (12, 13), indicating that the TCR structure is a major determinant of the binding avidity and T cell functionality. TCR gene transfer greatly facilitates clinical applications of adoptive T cell therapy; therefore, it is of growing interest to analyze the “structural avidity” that is defined by the affinity of the TCR to pMHC molecules combined with co-receptor binding via CD8 or CD4 of surface-expressed TCRs.

Most attempts to determine structural TCR binding strength have been performed with surface plasmon resonance (SPR), where pMHCs and TCRs need to be provided as highly purified proteins. Because the expression of correctly folded TCRs is technically challenging, SPR is difficult to use for analysis of a broader spectrum of TCRs. Alternative methods to examine the binding strength between TCR and pMHC are based on pMHC multimer binding and dissociation (14, 15). However, pMHC multimer staining intensity does not necessarily correlate with TCR binding avidity, and current pMHC-multimer dissociation assays monitor the dissociation of a multimeric complex but do not allow accurate analysis of the binding strength between monomeric pMHCs and the TCR. Furthermore, these assays are prone to variability in the degree of pMHC multimerization and in the nature and concentration of blocking reagent, which is used to prevent rebinding of dissociated pMHCs to the TCRs (16).

On the basis of reversible multimers, so-called Streptamers (17), we developed an assay that circumvents the limitations of previous methods and allows the accurate determination of the dissociation of monomeric pMHCs from the TCRs on living T cells. Thereby, the dissociation kinetic is fully independent of the level of multimerization or the nature of a blocking reagent. Using this koff rate assay, we demonstrate that T cell populations relevant for T cell therapy have highly variable koff rates. Furthermore, in vivo experiments using preclinical mouse models for infections demonstrated a strong correlation between the koff rate and the protective capacity of transferred T cells.


Development of a Streptamer-based koff rate assay

Multimeric pMHC dissociation experiments are currently used as a kind of “gold standard” to assess the structural avidity of antigen-specific T cells (fig. S1A). Unfortunately, it is difficult to standardize this assay. For example, pMHC multimer dissociation kinetics strongly depends on the nature and the concentration of blocking reagents used to prevent rebinding of dissociated MHCs to TCRs (16). In addition, the kinetics of pMHC multimer dissociation experiments is very slow, in the range of minutes to hours. When working at low temperatures (4°C), which is a prerequisite to prevent internalization of pMHC molecules bound to the TCR, t1/2 values can even be in the range of days (fig. S1, B and C). Although pMHC multimer dissociation experiments are capable of roughly distinguishing high- and low-avidity T cells within the same experiment, t1/2 values and their differences underlie high interassay variability (fig. S1, B and C). Furthermore, the dissociation of multimeric complexes follows complicated rules: It is unclear how many monomeric MHC molecules are bound to a T cell, and the number of MHC molecules per multimer is difficult to accurately determine.

To improve multimer-based koff rate measurements, we designed an assay to determine the dissociation kinetics of monomeric pMHC molecules from surface-expressed TCRs (Fig. 1A) based on reversible Streptamer stainings (17). Multiple pMHC molecules bind to Strep-Tactin via the Strep-tag sequences and stably label epitope-specific T cells (17, 18). The Streptamer complex is disrupted by d-biotin, which binds to the Strep-tag binding sites on Strep-Tactin with higher affinity, leaving monomeric pMHC bound to surface-expressed TCRs. By conjugating the pMHC molecules to a fluorescent dye, the dissociation kinetics of monomerized pMHCs can be observed as the decay in fluorescence intensity.

Fig. 1 Basic principle and experimental realization of the koff rate assay.

(A) Specific CD8+ T cells are stably labeled with dichromatic Streptamers (left). Addition of d-biotin displaces Strep-Tactin, leaving monomeric pMHC molecules bound to surface-expressed TCRs (middle). Subsequent dissociation of monomeric MHC molecules is observed as a decay of the Atto565 fluorescence by real-time microscopy. (B) A cooling device is mounted on the microscope (left). Purified T cells are pipetted into a 4°C cooled reservoir built on a metal insert sealed with a cover slip, arrested by a polycarbonate membrane and a metal shim, and covered with cold buffer (right). (C) Reduced movement of cells covered with a polycarbonate membrane (middle) in comparison to cells not arrested (left) over a time series of 200 pictures after addition of d-biotin. Pores (5 μm) in the membrane allow quick diffusion of buffer (right, dark spots). (D) Validation of Atto565 fluorescence on agarose Strep-Tactin beads in the koff rate setup. Transmitted light and Atto565 fluorescence of unloaded beads (upper row) or beads loaded with Atto565-labeled MHC molecules (lower row) of two representative experiments. Scale bars, 100 μm.

For fluorescence conjugation, a cysteine was inserted into the Strep-tag region and covalently linked to a dye carrying a maleimide group (fig. S2A). We found the bright and small Atto565 best suited for the setup, because it interferes neither with Strep-Tactin binding nor with the binding of the pMHC to the TCR. Further, we replaced a cysteine with tyrosine at position 67 in the β2-microglobulin because this residue is solvent-exposed and freely accessible (19) and allows dye conjugation of different MHC alleles within a conserved substructure. Streptamers conjugated at both conjugation sites stained murine T cells and provided comparable koff rate data (fig. S2B). Fluorochrome conjugation to Cys67 in the β2-microglobulin abrogated human Streptamer stainings for different MHC alleles and peptide specificities. Therefore, all human koff rate data were exclusively obtained with the C-terminal Atto565 dye conjugation.

To prevent internalization of MHC molecules (17), Streptamer-stained cells were constitutively kept at 4°C. Temperature control under the microscope was achieved by a customized cooling device connected to a Peltier cooler. Cells were added into a buffer reservoir, which fits exactly into the cooling device (Fig. 1B), and arrested by a polycarbonate membrane weighed down with a small metal shim to prevent movement (Fig. 1C and movie S1). Fluorescence images were taken before and every 10 s after the addition of d-biotin until complete dissociation of the MHCs.

Fluorescence values were corrected for photobleaching (Supplementary Method 1). The photobleaching rate was monitored by analyzing Strep-Tactin–coated beads multimerized with dye-conjugated MHC molecules with identical settings (Fig. 1D, fig. S3, and movie S2). Addition of d-biotin to Atto565-conjugated pMHCs multimerized on Strep-Tactin beads resulted in a quick loss of fluorescence, demonstrating no interference of the C-terminal dye conjugation at the Strep-tag sequence with the Strep-Tactin binding or dissociation (fig. S4 and movies S3 and S4).

Analysis of individual T cells in the koff rate assay

Human cytomegalovirus (CMV)–specific T cells were purified from healthy blood donor peripheral blood mononuclear cells (PBMCs), stained with Strep-Tactin allophycocyanin (APC; blue) and MHC Atto565 (red) double-labeled Streptamers, and subsequently analyzed in the koff rate assay setup by real-time fluorescence microscopy (Fig. 2A). Surprisingly, in the Streptamer complex, the MHC-Atto565 fluorescence intensity of stained cells was weak (Fig. 2, A and B, 0 s). After addition of d-biotin, Strep-Tactin APC dissociated and its fluorescence quickly decreased. In contrast, the quenched MHC-Atto565 fluorescence reached maximal intensity after Strep-Tactin APC removal (Fig. 2B, 60 s), followed by a fluorescence decrease that reflects the dissociation of monomeric MHCs (Fig. 2B, 120 to 520 s, and movies S5 and S6). The maximal Atto565 fluorescence facilitated the koff rate analysis by identifying the starting point of the dissociation of monomeric MHC molecules. In contrast, simultaneous dissociation of MHC and Strep-Tactin during the first seconds after addition of d-biotin might complicate the analysis. Fluorescence intensities of individual cells were plotted over time, and the koff rate and half-life time (t1/2) of the binding were calculated as described in Materials and Methods (Fig. 2C).

Fig. 2 Analysis of individual antigen-specific T cells in the monomeric Streptamer koff rate assay.

(A) Overlay of the APC and the Atto565 fluorescence of Streptamer-stained and fluorescence-activated cell sorting (FACS)–sorted HLA-B7/pp65417–426–specific human T cells before (0 s) and at 30, 60, and 520 s after the addition of d-biotin. (B) Gating on a single cell derived from the images shown in (A) with separated pictures for APC (upper row) and Atto565 fluorescence (lower row). The scale indicates the time after addition of d-biotin. Scale bars, 10 μm. (C) Plot of the fluorescence intensity of APC (blue line) and Atto565 (red line) over time after the addition of d-biotin (left). Fit of a curve with exponential decay into the data points after maximum fluorescence of Atto565 (right). (D) Reproducibility of the analyzed data in different dissociations and independent experiments. The graph shows the t1/2 of a human T cell clone in six individual dissociations of two independent experiments. Numbers in the graph indicate the mean t1/2 ± SD for each measurement. (E) koff rate assay of 2C T cells. The t1/2 values of 51 and 60 single cells stained with H2-Kb/dEV8 or H2-Kb/SIY Streptamers, respectively, each from four independent dissociations are plotted with the means ± SD.

Using Strep-Tactin without fluorochrome conjugation, we measured highly comparable t1/2 of two T cell clones, indicating no influence of the APC on the Atto565 fluorescence decrease (fig. S5).

To compare the Streptamer koff rate assay with conventional multimer dissociation experiments, we analyzed a number of T cell clones with both approaches. In the Streptamer koff rate assay, T cell clone #1 had a mean t1/2 of 200 s and clone #2 had an about fourfold shorter t1/2 of 55 s (fig. S6). Conventional multimer dissociation experiments using the same T cell clones determined very different values in two independent experiments (fig. S1). In contrast, Streptamer koff rate measurements of the T cell clones were much more reproducible, as shown in Fig. 2D and figs. S1 and S6, in different dissociations of two or three independent experiments. Furthermore, the Streptamer koff rate assay was not influenced by pMHC rebinding because off-rate kinetics was not different in the presence of antibodies blocking pMHC rebinding (fig. S7). These data illustrate the specific advantages of the monomeric Streptamer koff rate assay over conventional pMHC multimer dissociation experiments.

Next, we compared the koff rate assay to published data on TCR affinity by analyzing well-characterized 2C TCR transgenic cells that recognize the allogeneic ligand H2-Ld/p2Ca and the syngeneic ligands H2-Kb/dEV8 and H2-Kb/SIY. Similar to results determined by SPR (20), we calculated higher t1/2 for the 2C TCR/H2-Kb/SIY complex (mean t1/2, 64.7 s) compared to the TCR/H2-Kb/dEV8 with a mean t1/2 of 12.7 s (Fig. 2E).

Together, we established an assay that provides reproducible data on MHC koff rates that are comparable for 2C ligands to those determined by SPR.

Correlation between functional avidity and koff rate of two CMV-specific T cell clones

To analyze the correlation between koff rate values and functional avidity of T cells, we analyzed two human T cell clones specific for different epitopes derived from human CMV immediate-early 1 antigen (IE1) (Fig. 3A). HLA-B8/IE188–96–specific clone B had a longer t1/2 (mean, 178.3 s) compared to HLA-B8/IE1199–207K–specific clone A (mean t1/2, 40.6 s) (Fig. 3B). In a chromium release assay and intracellular cytokine staining (ICCS), clone B reached half-maximal specific lysis of peptide-pulsed target cells and half-maximal IFN-γ production (EC50) at lower peptide concentrations in comparison to clone A (Fig. 3, C and D). Functional avidity of the clones correlated well with the differences determined by the koff rate assay.

Fig. 3 koff rate of two human T cell clones is maintained after transgenic expression of TCRs in Jurkat76 cells.

(A) Streptamer staining of T cell clone A (specific for HLA-B8/IE188–96) and clone B (specific for HLA-B8/IE1199–207K). FACS dot plots show CD8 versus Streptamer staining, and cells are pregated on living cells. (B) koff rate assay of clones A and B. The t1/2 values of 7 and 26 single cells from clones A and B, respectively, each from two independent dissociations are plotted with the means ± SD. (C) Cells (1 × 105) from T cell clones A and B were incubated at an effector-to-target (E:T) ratio of 10:1 with target cells from an HLA/B8-expressing lymphoblastoid cell line (LCL), which were labeled with 51Cr and loaded with the indicated amount of IE1199–207K or IE188–96 peptide, respectively. Lysis of target cells was measured in a γ-counter as the amount of radioactivity released into the culture supernatant in triplicates. The peptide concentration for the half-maximal lysis (EC50) was calculated by fitting a nonlinear regression curve. (D) T cells (1 × 106) and HLA/B8-expressing LCLs (1 × 105) were incubated with the indicated amounts of IE1199–207K or IE188–96 peptide, respectively, and IFN-γ production was analyzed by ICCS. The percentage of CD8+ IFN-γ+ living cells was normalized to the maximum, and peptide concentration for half-maximal IFN-γ production (EC50) was calculated after fitting a nonlinear regression curve. (E) Streptamer staining of Jurkat76 cells expressing the TCRs of clones A and B, respectively. Dot plots show CD3 versus Streptamer staining, gated on living cells. (F) Comparison of the koff rate data of clones A and B from (B) to t1/2 of 4 and 27 single cells from Jurkat76 cells transduced with the identical TCR A and B from three and six independent dissociations with the mean t1/2, respectively.

TCRs from both clones A and B were isolated, expressed on CD8α+ Jurkat cells, and analyzed in the koff rate assay (table S1 and Fig. 3E). Highly comparable t1/2 values of T cell clones and transduced Jurkat cells demonstrate that the TCR is the main determinant of measured koff rates (Fig. 3F).

koff rates of CMV-specific T cell populations derived from different individuals

Enriched CMV-reactive T cells are a promising source for adoptive immunotherapy, and T cell avidity might be a relevant parameter to guide the selection of protective donor cells. Therefore, we performed ex vivo koff rate analyses on four CMV-specific T cell populations derived from three different individuals and found strong variances in koff rates between individual populations (Fig. 4A). For further validation of the koff rate assay, we analyzed T cell clones derived from each of these populations. Indeed, with rare exceptions, the t1/2 of the clones was in the range of values obtained from the respective CMV-specific T cell populations (Fig. 4, B to E; range indicated by dashed line or tables S2 to S5). We found only two clones with a shorter t1/2 than their originating HLA-B8/IE188–96–specific population, which may have been missed in the ex vivo measurement. Sequencing analysis identified identical TCR sequences for the two “outlier” clones with very similar t1/2, emphasizing the high reproducibility of the koff rate assay and its independence of the cellular context. For two TCR identical T cell clones #1 and #2, we compared their koff rates measured early after T cell restimulation (“d12”) and in a resting phase after restimulation (“d21”). In both cases, we obtained very comparable koff rates of the T cell clones (fig. S8), demonstrating that the results of the koff rate assay are not dependent on the activation status of the measured T cells.

Fig. 4 koff rates of polyclonal ex vivo CMV-specific T cell populations show remarkable variability and define the range for the t1/2 of their respective clones.

(A) TCR koff rates (t1/2) of four individual CMV-specific CD8+ T cell populations from three different donors sorted by flow cytometry from purified PBMCs after staining with αCD8 antibody and Streptamers. The t1/2 values of 26, 11, 50, or 15 individual cells of HLA-B8/IE1199–207K–, HLA-B8/IE188–96–, HLA-B7/pp65417–426–, and HLA-A2/pp65495–503–specific T cells, respectively, each from two or six independent dissociations for HLA-B8/IE188–96, are plotted with their mean. (B to E) T cell clones were grown from flow cytometry–sorted CMV-specific ex vivo populations by limiting dilution. t1/2 values of the ex vivo populations and their respective clones (specificity indicated at the top of the diagram) are compared. Mean t1/2 values and number of analyzed cells are summarized in tables S2 to S5.

We analyzed T cell populations of four further donors reactive against the same pMHC (HLA-B8/IE1199–207K) and detected a high variance in the mean t1/2. These data indicate—at least for this pMHC specificity—that neither the human leukocyte antigen (HLA) restriction nor the epitope recognition is a major determinant for a distinct koff rate of CMV-specific populations. The koff rate of these ex vivo populations correlated with the functional avidity (fig. S9).

In summary, by analyzing CMV-specific T cells, we further confirmed the reliability and reproducibility of koff rate measurements with this assay. We discovered remarkable differences in koff rate values of different CMV-specific T cell populations, suggesting the potential for different protective capacity in immunotherapy.

Correlation between koff rate and in vivo protectivity of T cells in preclinical mouse models

Having demonstrated a correlation between the functional avidity and the t1/2 in vitro, we analyzed the correlation between the t1/2 and in vivo functionality of T cells. We generated polyclonal T cell lines A and B specific for the Listeria monocytogenes epitope LLO91–99 by in vitro restimulation with high (10−6 M) and low (10−9 M) peptide concentrations. In line with published data (21), high peptide concentration expanded low-avidity T cell line A that required higher peptide concentrations for half-maximal specific lysis of target cells or half-maximal IFN-γ secretion in comparison to cell line B (Fig. 5, A and B). In the koff rate assay, most cells from cell line A had a short t1/2 below 30 s, whereas cells from cell line B were more heterogeneous with 38% having a high t1/2 between 100 and 340 s (Fig. 5C). In vivo protectivity was tested by adoptively transferring cells from each population into BALB/c mice, infecting the mice with L. monocytogenes, and analyzing bacterial load in spleens and livers 3 days later. Mice that had received cell line A were not protected because they had high bacterial loads in the spleen comparable to the phosphate-buffered saline (PBS) control group, whereas mice that had received cell line B demonstrated a >100-fold reduction of viable bacteria in the spleen (Fig. 5D).

Fig. 5 Long t1/2 correlates with high functional avidity and protectivity of T cell lines in L. monocytogenes and MCMV infection.

(A to D) LLO91–99–specific T cells restimulated with 10−6 M (cell line A) or 10−9 M (cell line B) peptide. (A) Specific killing of LLO91–99–specific T cell lines was tested in a chromium release assay by incubation with 51Cr-labeled and peptide-loaded P815 target cells at an E:T ratio of 10:1 in triplicates. The peptide concentration for the half-maximal lysis (EC50) was calculated after fitting a nonlinear regression curve. (B) IFN-γ production of LLO91–99–specific T cell lines was analyzed by ICCS after incubation with P815 target cells and the indicated amounts of peptide at an E:T ratio of 1:1. Values were normalized to maximal IFN-γ production, and the EC50 was calculated after nonlinear regression. (C) koff rate assay of LLO91–99–specific T cell lines A and B. The t1/2 values of 54 or 84 individual cells of cell line A or B, respectively, each from three independent dissociations, are plotted with the means ± SD. (D) Bacterial load of mice transferred with PBS or 5 × 106 cells from LLO91–99–specific cell line A or B 3 days after L. monocytogenes infection [2 × 104 colony-forming units (CFU)] (n = 3). Groups were compared by one-tailed t test. (E to H) m164257–265–specific T cells restimulated with 10−8 M (cell line A) or 10−10 M (cell line B) peptide. (E) Specific killing of m164257–265–specific T cell lines A and B was tested as described in Materials and Methods. (F) IFN-γ production of m164257–265–specific T cell lines was tested in triplicates by ELISPOT after 18 hours of incubation with peptide-loaded P815 target cells, and frequencies of spot-forming cells were calculated. CTL, cytotoxic T lymphocytes; n.d., not detected. (G) koff rate assay of m164257–265–specific T cell lines. The t1/2 values of 34 or 78 individual cells from five independent dissociations of cell line A or B, respectively, are plotted. (H) Virus titer was quantified 11 days after intravenous injection of PBS or 106 cells from m164257–265–specific T cell line A or B into immunocompromised, MCMV-infected mice (n = 6). Groups were compared by one-tailed t test.

Because of the clinical relevance of CMV-specific T cells for adoptive immunotherapy and the large spectrum of koff rates measured in different epitope-specific human T cell populations, we further tested the correlation of koff rate and protection in a mouse model for CMV infection. We generated cell lines A and B using high (10−8 M) and low (10−10 M) concentrations of the murine CMV (MCMV) epitope m164257–265 for stimulation. Similar to Listeria-specific T cell lines, cell line B showed a higher functional avidity determined by 51Cr release and enzyme-linked immunospot (ELISPOT) assays (Fig. 5, E and F) that correlated with a high proportion of cells with long t1/2 above 50 s (Fig. 5G). At day 11 after transfer of cells from cell line A or B into immunosuppressed and MCMV-infected BALB/c mice, the number of MCMV plaque-forming units (PFU) was determined (Fig. 5H). Only mice that had received cells from cell line B showed a remarkable reduction (>100-fold) in virus titers compared to control mice and mice that received cell line A.

These data strongly support the interpretation that T cell lines composed of cells with long MHC binding t1/2 are superior to protect mice from L. monocytogenes and MCMV infections in adoptive transfer experiments.


Although TCR avidity is believed to be an important component for in vivo T cell efficacy, this parameter is still difficult to determine. Therefore, the aim of this study was to develop a technology for precise acquisition of an important part of the structural avidity of the TCR, the koff rate. By combining reversible MHC multimer staining with real-time microscopy, we succeeded to quantitatively determine koff rates. Using this assay, we demonstrate that CMV-specific T cell populations with different epitope specificities or derived from different donors vary substantially in their koff rates. Adoptive T cell transfer experiments using preclinical mouse models show that T cell lines containing cells with slow koff rates have a higher protective capacity in vivo.

Our new technology overcomes several technical difficulties of current methods used to determine the binding strength between pMHC and TCR. For example, the analysis of conventional MHC multimer-based koff rate assays (16) is complicated by multivalent binding and dissociation rates, whereas the MHC-Streptamers homogenize the system to measure truly monomeric interactions (Fig. 1). This is best illustrated by the large differences in the values of koff rates, which are in the range of minutes to hours for conventional MHC multimers [fig. S1 and reviewed in (16)] and in the range of seconds to minutes for monomeric interactions, determined by SPR or our newly developed koff rate assay (Fig. 2E and fig. S6). In addition, conventional MHC multimer-based koff rate assays require a blocking reagent to prevent rebinding of dissociated MHC molecules, and quality parameters of these blocking reagents (such as affinity and concentration) can further influence the obtained dissociation kinetics. As a consequence, the kinetics of koff rates determined by conventional MHC multimers is influenced by various factors unrelated to monomeric TCR-pMHC interactions, making it difficult to compare results obtained with different T cell populations. In contrast, the monomeric Streptamer koff rate assay does not depend on the addition of a blocking reagent (fig. S7) and is highly reproducible over different experiments (fig. S6).

To determine to what extent results from the Streptamer-based koff rate assays compare to published data, we analyzed transgenic T cells expressing the 2C TCR (22), because this receptor has been well examined with SPR (20). For two cognate epitopes, dEV8 and SIY, which are both presented on H2-Kb, we measured a very similar difference in koff rates of TCR-bound pMHC compared to SPR results (Fig. 2E). The absolute values are slightly higher for koff rates determined by the reversible MHC multimer technology, but this can be explained by the contribution of CD8 co-receptor binding, which was not included in SPR measurements.

A limitation of the current design of the Streptamer-based koff rate assay is that it cannot segregate TCR from CD8 co-receptor binding. However, this might be possible to overcome by the use of mutated MHC molecules that abolish CD8 binding to the α3 domain (23).

Our first direct ex vivo analyses on human CMV epitope–specific T cell populations demonstrate that cell populations with specificity to the same pathogen can differ substantially in the ligand-binding strength of recruited TCRs (Fig. 4A and fig. S9). This is likely to be clinically important because enriched CMV-reactive T cells are currently used for adoptive immunotherapy in immunocompromised patients (1, 2), in particular patients who have received allogeneic hematopoietic stem cell transplantation (HSCT). Surprisingly, the analysis of just a few CMV epitope–specific CD8+ T cell populations revealed marked differences in clonal composition as well as koff rates of individual TCRs. Thereby, the largest CMV-specific T cell population analyzed in our study (HLA-B8/IE1199–207K from donor #1, Fig. 4) was characterized by a very short pMHC-binding half-life and low functional avidity, as determined ex vivo as well as on in vitro–expanded T cell clones. We are currently performing several clinical trials on adoptive transfer of MHC Streptamer-sorted CMV epitope–specific CD8+ T cell populations for the treatment of therapy-resistant CMV disease upon allogeneic HSCT [EudraCT-Nr.: 2006-006146-34 and IMPACT— Identifier: NCT01077908; first case reports of this promising approach have been recently published (1)]. In this setting, we are planning a detailed analysis of koff rates for transferred donor T cell populations, which will allow correlation of this parameter with the therapeutic effects of immunotherapy. In the example presented in this report, the largest CMV-reactive population was characterized by the lowest (functional and structural) avidity. There are other reports on low functional avidity of large CMV-specific CD8+ T cell populations (24, 25), but whether these are exceptions or a common characteristic of the CD8+ T cell response to CMV has to be determined. On the basis of the results from preclinical protection models (Fig. 5), we suggest that determining koff rates might help to select the most protective antigen-specific T cell populations for adoptive immunotherapy; thereby, the largest cell populations, which are easiest to detect and to purify, might not always be the best choice (26, 27).

In conclusion, we demonstrate that the development of a technology based on reversible MHC multimer staining provides reliable access to the koff rate of TCR-ligand interactions and a new parameter for measuring the quality of T cells. This parameter might be useful not only to choose best suitable T cells for adoptive transfer but also to assess the quality of induced or existing immunity, such as after vaccination or because of permanent exposition to antigen as in chronic infections or tumors.

Materials and Methods

Blood samples

Peripheral blood was obtained from healthy adult donors of both sexes. Written informed consent was obtained from the donors, and usage of the blood samples was approved according to national law by the local Institutional Review Board (Ethikkommission der Medizinischen Fakultät der Technischen Universität München) in accordance with the Declaration of Helsinki. Three individuals were selected for in-depth analysis of human CMV–specific T cell clones. No further randomization was used.

Human T cell clones

Sort-purified Streptamer+ cells (HLA-B8/IE1199–207K, HLA-B8/IE188–96, HLA-A2/pp65495–503, or HLA-B7/pp65417–426) from CMV+ healthy donors were plated by limiting dilution (0.6 cells per well) and cocultured with 1 × 104 γ-irradiated allogeneic LCLs (50 Gy) and 7.5 × 104 PBMCs (35 Gy) in h-RP10+ (RPMI 1640, 10% human serum, 0.025% l-glutamine, 0.1% Hepes, 0.001% gentamicin, 0.002% streptomycin, 0.002% penicillin) supplemented with anti-CD3 monoclonal antibody (mAb) (OKT-3, 30 ng/ml) and interleukin-2 (IL-2) (50 U/ml). Cells (5 × 104) were restimulated every 14 days with 1 × 106 γ-irradiated allogeneic LCLs and 5 × 106 PBMCs in h-RP+ supplemented with anti-CD3 mAb. IL-2 (50 U/ml) was added 1 day later. Cells were analyzed between days 10 and 21 after restimulation.

Murine T cell lines

LLO91–99–specific T cells were isolated from spleens of BALB/c mice 7 days after L. monocytogenes infection and stimulated weekly by addition of 3 × 107 γ-irradiated (25 Gy) splenocytes of syngeneic, naïve mice, loaded with the appropriate concentrations of LLO91–99 peptide (10−6 and 10−9 M) for 1 hour. RP10+ (RPMI 1640, 10% fetal calf serum, 0.025% l-glutamine, 0.1% Hepes, 0.001% gentamicin, 0.002% streptomycin, 0.002% penicillin) was supplemented with rat concanavalin A (ConA) supernatant (5% T-Stim, inactivation of ConA by addition of 5% α-methyl-d-mannoside) from the second in vitro restimulation.

MCMV-specific T cell lines were generated and restimulated similarly with 10−8 or 10−10 M of the m164257–265 peptide (27).

TCR transduction

TCRs of the T cell clones were amplified by RACE-PCR (rapid amplification of complementary DNA ends–polymerase chain reaction) and subsequently sequenced. The TCR transduction was performed as described (28). Briefly, TCR α and β chains were separately cloned in the retroviral vector MP71. Packaging was performed by triplasmid CaCl2 transfection with the retroviral vector plasmid and the expression plasmids encoding the Moloney murine leukemia virus (MLV) gag/pol genes (pcDNA3.1MLVg/p, provided by C. Baum, Hannover, Germany) and the MLV-10A1 env gene (pALF-10A1) (29) in human embryonic kidney (HEK) 293T cells. Viral supernatant was spun down (800g) in RetroNectin-coated 24-well plates together with Jurkat76 cells for 90 min at 32°C. Five days after transduction, cells were analyzed for TCR expression by FACS.

MHC class I molecules

A cysteine on a glycine serine linker was inserted by site-directed mutagenesis into DNA vectors containing the respective MHC I molecule and the Strep-tag sequence. Vectors were expressed in Escherichia coli strains and subsequently refolded with β2-microglobulin at high dilution. Correctly folded MHC I molecules were purified by gel filtration (Superdex 200HR), pooled, and incubated overnight in a buffer containing NaN3, protease inhibitors (1 mM Na-EDTA, leupeptin, and pepstatin), and 0.1 mM dithiothreitol. The buffer was exchanged against PBS (pH 7.3) to allow for conjugation with activated fluorescent dyes Alexa Fluor 488–maleimide or Atto565-maleimide in a molar ratio of 10:1 at room temperature for 2 hours. Conjugated proteins were separated from unbound dye with gravity flow columns (illustra NAP-25 Columns, GE Healthcare), the buffer was exchanged against PBS (pH 8.0) containing NaN3 and protease inhibitors (1 mM Na-EDTA, leupeptin, and pepstatin), and the protein was stored in liquid nitrogen.

Streptamer staining

For MHC multimerization, 1 μg of Atto565 or Alexa Fluor 488–conjugated MHC I and 0.75 μg of Strep-Tactin APC or Strep-Tactin-PE (phycoerythrin) (IBA) were incubated for 45 min in 50 μl of FACS buffer following the manufacturer’s instructions. Cells were rested for 30 min on ice before staining, and antibodies were added for the last 20 min of the staining.

Bulk analysis of TCR avidity

All koff rate assays were performed on a Zeiss LSM 510 or a Leica SP5 confocal laser scanning microscope. Cells were concentrated to 1 × 104/μl, and 1 μl of cells was pipetted into the cooling reservoir as described. To test the influence of an MHC blocking antibody in our system, the koff rate measurements were performed similarly but in the presence of 10 μM W6/32 antibody that was added in combination with d-biotin.

Data analysis

Images were analyzed with MetaMorph Offline image analysis software (Molecular Devices). Integrated fluorescence intensity inside a gate containing an individual cell was measured over the whole time series acquired. The fluorescence intensity of an identical gate in close proximity to the cell, but not containing a cell, was acquired for measurement of background fluorescence intensity. Data were logged into Microsoft Excel. With the analyzer software “analyzer.nt,” the background correction of the fluorescence intensity and the plotting of the corrected values over time were performed in an automated manner. To ensure optimal fitting of the data points to an exponential curve (Supplementary Method 1), the software allows for the adaption of the area of data points included for fitting by specifying the first and last valid data point. Thereby, fitting of each cell is controlled directly, and incorrect fittings, for example, because of variations at the end of the assay, can be avoided.

Photobleaching measurements

One microgram of either pMHC I molecules conjugated to Alexa Fluor 488 or Atto565 or biotinylated APC or PE was multimerized with 7 μl of Strep-Tactin agarose beads (Superflow, 50% suspension, IBA) in a total volume of 50 μl for 45 min. Beads were washed and analyzed for fluorescence decay under the microscope with the same setup and settings used for cells (pictures, 200). Photobleaching values were obtained from the exponent of an exponential decay fitting curve of the background-corrected raw data and used for correction of the koff rate data (Supplementary Method 1).

Conventional multimer dissociation experiment

T cell clones were analyzed according to (16). Briefly, T cell clones were rested for 30 min on ice before staining with conventional HLA-B8/IE188–96 multimer PE for 45 min. After washing, cells were resuspended in 200 μl of FACS buffer in the presence or absence of 10 μM W6/32 blocking antibody (anti–HLA-A, anti–HLA-B, and anti–HLA-C). Cells were kept at 4°C for 10 hours, and aliquots were taken at the indicated time points. The maximal mean fluorescence intensity of living multimer PE+ cells was normalized to 100%, and the normalized data were plotted over the time. An exponential decay was fitted into the data and used to calculate the t1/2.

Intracellular cytokine staining

T cells were incubated in the presence of the indicated amount of the respective peptides for 5 hours in RP10+ supplemented with anti-CD28 and anti-CD49d (BD Biosciences). Brefeldin A (2 μg per well) (Sigma) was added after 1 hour (human cells) or 2 hours (murine cells). After stimulation, cells were kept at 4°C and stained with ethidium monoazide (EMA) (0.1 μg in 50 μl of FACS buffer) for 20 min under light. For surface staining, cells were incubated for 20 min in the dark with anti-CD8α mAb before lysis in Cytofix/Cytoperm (BD Biosciences) for 20 min and for intracellular staining against IFN-γ (30 min, in the dark, on ice in BD Biosciences Perm/Wash buffer). The maximal percentage of IFN-γ+ CD8+ was normalized to 100%, and a nonlinear curve was fitted into the normalized data in response to each peptide concentration.

For CMV-specific T cell populations, 2 × 106 PBMCs from different donors were incubated per sample. Human T cell clones were cocultured with HLA/B8-expressing LCLs at an E:T ratio of 10:1, and murine LLO91–99–specific T cell lines were cocultured with P815 cells at an E:T ratio of 1:1.

IFN-γ–based ELISPOT assay

Titrated numbers of m164-specific T cells (50, 100, and 200) were stimulated for 18 hours with 1 × 105 P815 cells that were loaded for 1 hour at 37°C with the indicated concentrations of m164 peptide as described previously (30). All titration steps were performed in triplicates. Thereafter, plates were developed, spot numbers were counted, and the frequencies of IFN-γ–secreting spot-forming cells with the corresponding 95% confidence intervals were calculated by intercept-free linear regression analysis.

51Cr release assay

For the analysis of human T cell clones, 1 × 106 HLA/B8-expressing LCLs were loaded with the indicated amounts of IE188–96 or IE1199–207K peptide (10−11 to 10−5 M) and 1.85 MBq of 51Cr for 1 hour at 37°C, and 104 cells were used as target cells for 1 × 105 T cell clones (E:T ratio = 10:1). To determine the spontaneous and maximal lysis, RP10+ or 5% Triton X-100 was used, respectively, instead of effector cells. All titration steps were performed in triplicates. The cells were incubated for 4 to 5 hours at 37°C and spun down, and the supernatants were transferred to counting tubes. The amount of radioactivity in the supernatants was measured in a γ-counter, and the specific lysis for each sample was calculated.

To test murine LLO91–99– and m164257–265–specific T cell lines, we labeled P815 target cells with 18.5 MBq of 51Cr for 1 hour at 37°C and loaded with their respective peptide in concentrations of 10−6 to 10−13 M (LLO91–99) or 10−6 to 10−13 M (m164257–265). Effector cells were cocultivated and analyzed in an E:T ratio of 10:1 as described above.

Infection and adoptive cell transfer

For Listeria monocytogenes infection of BALB/c mice, the isolate L. monocytogenes 10403s [American Type Culture Collection (ATCC)] was used. Mice were infected intravenously with 2 × 104 CFU of bacteria in 200 μl of PBS 1 hour after intravenous transfer of 5 × 106 LLO91–99–specific T cells. The organs were harvested at day 3 after infection, homogenized, and resuspended in 5 ml of sterile PBS. A serial dilution of the cells in 0.1% Triton X-100 was plated out on brain heart infusion plates, and the CFU were counted after one night of incubation at 37°C.

For the MCMV protection assay (26, 27, 30), 106 cells from the indicated cell line were transferred intravenously into 8- to 10-week-old female BALB/c recipients, which were immunocompromised by total body γ-irradiation with a single dose of 6.5 Gy before transfer. Subsequently, subcutaneous, intraplantar infection was performed at the left hind footpad with 105 PFU of cell culture–propagated and then purified MCMV, strain Smith (ATCC VR-194/1981), in 25 μl of physiological saline. Infectious virus was quantified on day 11 after transfer in homogenates of spleens by a virus plaque assay under conditions of centrifugal enhancement of infectivity.

Supplementary Materials


Method 1. Model function and correction for photobleaching.

Fig. S1. t1/2 values obtained from conventional multimer MHC dissociation experiments are difficult to reproduce.

Fig. S2. t1/2 values measured by the Streptamer koff rate assay are comparable with different combinations of MHC and Strep-Tactin.

Fig. S3. Photobleaching measurement of the fluorescent dyes APC and Atto565 in the koff rate setup.

Fig. S4. Reversible interaction of the Strep-tag III sequence of fluorochrome-conjugated MHC I molecules and Strep-Tactin on beads.

Fig. S5. Streptamer koff rates are not influenced by the fluorochrome on the Strep-Tactin backbone.

Fig. S6. Measurement of the dissociation of monomeric MHC molecules in the koff rate assay is highly reproducible.

Fig. S7. Dissociation rates of monomeric MHC molecules in the koff rate assay are not substantially affected by MHC rebinding.

Fig. S8. t1/2 values analyzed by the Streptamer koff rate assay are not influenced by the activation status of analyzed cells.

Fig. S9. t1/2 values determined by the Streptamer koff rate assay correlate with functional avidity of CMV-specific T cell populations with identical HLA restriction and epitope recognition from different donors.

Table S1. TCR sequences of human clones A and B.

Table S2. t1/2 of HLA-B8/IE1199–207K–specific T cell clones.

Table S3. t1/2 of HLA-B8/IE188–96–specific T cell clones.

Table S4. t1/2 of HLA-B7/pp65417–426–specific T cell clones.

Table S5. t1/2 of HLA-A2/pp65495–503–specific T cell clones.

Movie S1. Observation of living cells arrested in a cooled buffer reservoir.

Movie S2. Determination of the photobleaching rate on beads.

Movies S3 and S4. Reversible interaction of the Strep-tag III sequence of fluorochrome-conjugated MHC I molecules and Strep-Tactin on beads.

Movie S5. Dissociation of Strep-Tactin and monomerized MHC from individual living T cells in overlaid fluorescence channels.

Movie S6. Dissociation of Strep-Tactin and monomerized MHC from individual living T cells in separate fluorescence channels.

References and Notes

  1. Acknowledgments: We thank L. Räty, L. Frimmer, and A. Hochholzer for support in performing experiments; J. Mages and S. Nauerth for support in the development of analyzer software used for automated fitting of fluorescence data; L. Henkel, M. Schiemann, I. Andrä, and K. Wild for flow cytometry–based cell sorting; and P. Gräf, C. Stemberger, and V. Buchholz for critical discussion of the manuscript. Funding: This work was supported by the SFB TR36 (TP-B10/13), SFB 1054 (TP-B09), SFB 490 (TP-E3), and a German Research Foundation grant from the Clinical Research Group KFO 183, individual project TP8; NIH CA114536; and CA136551. Author contributions: M.N., B.W., R.K., G.D., L.P., T.F., D.G.-M., M.B., and R.H. did experiments; J.B., P.J.P., M.N., R.H., and S.R.R. supplied cells; S.R.R. helped with the cell culture of human CMV–specific T cell clones; A.K. supplied blocking antibody and helped with the cell culture of human T cells; M.B. and W.U. supplied retroviral vectors and cells; D.H.B. conceived the study; R.K., M.N., and B.W. analyzed the data; M.J.R. designed the MCMV protection experiments; D.H.B. planned most of the experiments and supervised the study; and M.N., B.W., and D.H.B. wrote the paper. Competing interests: The authors declare no that they have no competing interests.
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