Detection of autoreactive CD4 T cells using major histocompatibility complex class II dextramers
© Massilamany et al; licensee BioMed Central Ltd. 2011
Received: 28 April 2011
Accepted: 18 July 2011
Published: 18 July 2011
Tetramers are useful tools to enumerate the frequencies of antigen-specific T cells. However, unlike CD8 T cells, CD4 T cells - especially self-reactive cells - are challenging to detect with major histocompatibility complex (MHC) class II tetramers because of low frequencies and low affinities of their T cell receptors to MHC-peptide complexes. Here, we report the use of fluorescent multimers, designated MHC dextramers that contain a large number of peptide-MHC complexes per reagent.
The utility of MHC dextramers was evaluated in three autoimmune disease models: 1) proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis in SJL/J (H-2s) mice; 2) myelin oligodendrocyte glycoprotein (MOG) 35-55-induced experimental autoimmune encephalomyelitis in C57Bl/6 (H-2b) mice; and 3) cardiac myosin heavy chain (Myhc)-α 334-352-induced experimental autoimmune myocarditis in A/J (H-2a) mice. Flow cytometrically, we demonstrate that IAs/PLP 139-151, IAb/MOG 35-55 and IAk/Myhc-α 334-352 dextramers detect the antigen-sensitized cells with specificity, and with a detection sensitivity significantly higher than that achieved with conventional tetramers. Furthermore, we show that binding of dextramers, but not tetramers, is less dependent on the activation status of cells, permitting enumeration of antigen-specific cells ex vivo.
The data suggest that MHC dextramers are useful tools to track the generation and functionalities of self-reactive CD4 cells in various experimental systems.
Traditionally, limiting dilution analysis and cytokine ELISPOT assays have been used to enumerate the frequencies of antigen-specific cells, but low specificity and the tedious nature of these assays limit their use for routine applications [1–3]. To overcome these limitations, tetramer technology has been developed . This approach involves derivation of fluorescent dye-labeled tetramerized complexes containing peptide-assembled major histocompatibility complex (MHC) molecules. The use of tetramers permits easy and rapid detection of antigen-sensitized T cells at a single cell level by flow cytometry (FC) [4, 5]. Additionally, tetramer reagents can be used in conjunction with antibodies to phenotypically characterize the generation and expansion of antigen-specific cells during ensuing immune responses and to study their functionalities by cell sorting.
Unlike with MHC class I tetramers, direct enumeration of antigen-specific cells - in particular, rare and low-affinity autoreactive CD4 T cells - with MHC class II tetramers has been difficult. Under natural conditions, interaction between T cells and antigen-presenting cells involves engagement of MHC molecules with multiple T cell receptors (TCRs), but their binding strength is low. Hence, their stability requires interaction between various adhesion molecules. Tetramers are artificially created peptide-assembled MHC complexes, and their interactions with TCRs occur in the absence of accessory molecules, a limitation that exists with both class I and class II tetramers. In spite of this limitation, class I tetramers can generally bind CD8 T cells, but require participation of a CD8 coreceptor . By comparison, participation of a CD4 coreceptor is not critical for class II tetramers to bind to CD4 cells [7, 8]. This differential requirement of coreceptors may, in part, explain differences in the abilities of tetramers to bind respective T cell populations.
It is generally accepted that class II tetramers preferentially bind activated CD4 cells expressing high amounts of CD4 and CD25 [9–12]. We and others had previously proposed that activation might result in reorientation and configuration of TCR and accessory molecules, leading to enhanced avidity of TCR-MHC binding [11, 13, 14], thus restricting the utility of the reagents for direct enumeration of the frequencies of antigen-sensitized cells ex vivo. Recently, MHC dextramers bearing dextran backbones assembled with peptide-tethered MHC class I molecules have been shown to detect antigen-specific CD8 T cells [15, 16]. Our studies involve the use of a streptavidin (SA)-dextramer conjugate of 270 kDa, which has been validated to detect tumor- and virus-specific cells in various systems [15, 17–19]. To address the utility of dextramers, we used three autoimmune disease models: 1) proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL/J (H-2s) mice; 2) myelin oligodendrocyte glycoprotein (MOG) 35-55-induced EAE in C57Bl/6 (H-2b) mice; and 3) cardiac myosin heavy chain (Myhc)-α 334-352-induced experimental autoimmune myocarditis (EAM) in A/J (H-2a) mice. We demonstrated that MHC class II dextramers detect self-reactive CD4 T cells with specificity and to a greater sensitivity than conventional tetramers. We showed that binding of dextramers, but not tetramers, is less dependent on activation status of the cells, thus permitting the reagents to determine the frequencies of antigen-specific cells ex vivo in immunized mice.
Four-to-six-week-old female SJL/J (H-2s) and C57Bl/6 (H-2b) mice and six-to-eight-week-old A/J (H-2a) mice were procured from the Jackson Laboratory (Bar Harbor, ME). The mice were maintained according to the animal protocol guidelines of the University of Nebraska-Lincoln, Lincoln, NE.
Peptide synthesis and immunization procedures
PLP 139-151 (HSLGKWLGHPDKF), MOG 35-55 (MEVGWYRSPFSRVVHLYRNGK) and Myhc-α 334-352 (DSAFDVLSFTAEEKAGVYK) were synthesized on 9-fluorenylmethyloxycarbonyl chemistry (Neopeptide, Cambridge, MA). All peptides were HPLC-purified (>90%), identity was confirmed by mass spectroscopy, and the peptides were dissolved in 1 × PBS prior to use. To generate primary T cell cultures for PLP 139-151 and MOG 35-55, SJL and C57Bl/6 mice, respectively, were immunized with the above peptides emulsified in complete Freund's adjuvant (CFA) subcutaneously (s.c.) in multiple sites in the flank and sternal regions (100 μg/mouse) [5, 20]. For immunization of A/J mice with Myhc-α 334-352, the peptide was emulsified in CFA supplemented with Mycobacterium tuberculosis (M. tb; Difco Laboratories, Detroit, MI) extract to a final concentration of 5 mg/ml and injected s.c. in the flank, foot pads and sternal regions (100 μg/mouse). For disease induction, however, regardless of peptides used, emulsions were prepared in CFA containing M. tb extract to a final concentration of 5 mg/ml. To induce EAE with PLP 139-151 or MOG 35-55, peptide emulsions were given as a single dose [11, 20], whereas for EAM induction, two doses of emulsions containing Myhc-α 334-352 were administered with an interval of one week [21, 22]. Nonetheless, all animals involved in the disease-induced protocols received pertussis toxin (List Biological Laboratories, Campbell, CA) intraperitoneally on day 0 and day 2 after the first immunization (PLP 139-151 and Myhc-α 334-352, 100 ng/mouse; MOG 35-55, 200 ng/mouse) [5, 20, 21]. SJL and C57Bl/6 mice were monitored for the appearance of clinical signs of EAE and scored as described [23, 24]: 0 - healthy; 1 - limp tail or hind limb weakness but not both; 2 - limp tail and hind limb weakness; 3 - partial paralysis of hind limbs; 4 - complete paralysis of hind limbs; 5 - moribund or dead.
Generation of antigen-specific primary T cell cultures
Ten days after immunizations with peptides, mice were sacrificed, and the maxillary, mandibular, axillary, inguinal and popliteal draining lymph nodes (LN) were collected. Single cell suspensions were prepared after lysing the erythrocytes with 1 × ammonium chloride potassium buffer (Lonza, Walkersville, MD). Lymph node cells (LNC) were stimulated with the peptides (PLP 139-151, 20 μg/ml; MOG 35-55 and Myhc-α 334-352, 50 μg/ml) at a density of 5 × 106 cells/ml for two days in RPMI medium supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 4 mM L-glutamine, 1 × each of non-essential amino acids and vitamin mixture, 100 U/ml penicillin-streptomycin and 50 μg/ml gentamicin (Lonza). After two days, cultures were supplemented with the above medium containing interleukin (IL)-2 (eBioscience, San Diego, CA), hereafter termed growth medium. Viable lymphoblasts were harvested two to three days later by Ficoll-Hypaque density gradient centrifugation, and the cultures were maintained in growth medium.
Isolation of mononuclear cells (MNC) from CNS tissues and hearts
After euthanization, mice were perfused by injecting 10 ml of 1 × cold PBS into the left ventricle of the hearts; brains were harvested by blunt dissection; and spinal cords were flushed out using 1 × cold PBS. The tissues were homogenized and digested with type IV collagenase (400 U/ml; Worthington, Lakewood, NJ) at 37°C for 1 hour, and MNC were harvested by percoll density gradient centrifugation (70%/30%) as described previously . To harvest MNC from hearts, animals were first perfused as above, and hearts were collected and minced. The tissue suspensions were then incubated with a buffer containing 1 × PBS, collagenase and 2% FBS at 37°C for 15 min in a shaker with continuous agitation. The digested tissues were passed through an 18G needle and incubated for an additional 10 minutes; the cell pellets were obtained by centrifugation and resuspended in 40% buffered percoll. Cell suspensions were overlaid with 75% percoll and centrifuged; the interphase representing MNC was then collected and washed [26, 27].
Derivation of IAs, IAb and IAk tetramers and dextramers
IAs/PLP 139-151 and IAs/Theiler's murine encephalomyelitis virus (TMEV) viral capsid protein2 70-86, IAb/MOG 35-55, and IAk/Myhc-α 334-352 and IAk/Bovine ribonuclease (RNase) 43-56 tetramers were generated as previously described [5, 11, 25, 28]. While we generated the IAk constructs [11, 29], the IAs and IAb constructs were kindly gifted by Dr. Vijay Kuchroo, Harvard University, Boston, MA. Briefly, α and β constructs for each IA allele containing the sequences of the respective peptides were expressed in a Baculovirus system using Sf9 insect cells (Invitrogen, Carlsbad, CA) and soluble MHC molecules were obtained [5, 11]. IAs, IAb and IAk monomers were purified on antibody columns prepared using MKS4, M5114, and 10-2.16 (Bio × Cell, West Lebanon, NH), respectively and the protein yield generally ranged from 0.5 mg to 1 mg/L [5, 11, 25, 28]. After concentrating, the soluble MHC proteins were biotinylated using biotin protein ligase at an optimized concentration of 25 μg/10 nmol of substrate as recommended by the manufacturer (Avidity, Denver, CO). The biotinylated proteins were then incubated with SA conjugated with a fluorescent dye - fluorescein isothiocyanate (FITC), phycoerythrin (PE) or allophycocyanin (APC) - at a 4:1 ratio for one hour on ice. The reagents thus prepared are referred to as tetramers. To prepare dextramers, biotinylated soluble monomers of all three IA alleles (IAs, IAb and IAk) containing the peptides were coupled to activated dextran backbones (kindly provided by Immudex Aps, Copenhagen, Denmark) at various molar ratios in 1 × Tris Hcl 0.05 M, pH 7.2 for 30 minutes at room temperature (RT) and the preparation of fluorochrome-labeled dextran backbone has been previously described . Fluorochrome-labeled dextramers were prepared by mixing biotinylated IA monomers with dextran molecules. For example, dextramer-PE reagents were prepared by mixing 1.6 × 10-11 moles of dextran-PE molecules with 3.17 × 10-10 moles of IAs monomers which resulted a molar ratio of 1:20. All the reagents were aliquoted and stored at -80°C or 4°C until further use.
Tetramer or dextramer staining
(a) Cells activated in vitro
To enumerate the frequencies of tetramer (tet+) or dextramer (dext+) cells, viable lymphoblasts harvested from cultures prepared from immunized SJL, C57Bl/6 or A/J mice were used for tet or dext staining as described previously [5, 30]. Briefly, cells were stained with tetramers or dextramers in growth medium, pH 7.6, containing 2.5% FBS at RT for up to 3 hours, followed by staining with anti-CD4 (eBioscience, San Diego, CA) and 7-aminoactinomycin D (7-AAD; Invitrogen). After washing, cells were analyzed by flow cytometry (FC; FACSCalibur, BD Biosciences, San Diego, CA). Percentages of tet+ or dext+ cells were then determined in the live (7-AAD-) CD4 subset using Flow Jo software (Tree Star, Ashland, OR). In some experiments, antigen-sensitized cells were rested for two weeks in growth medium and treated with or without neuraminidase (NASE) (0.7 U/ml) (Type X from Clostridium perfringens; Sigma-Aldrich, St. Louis, MO) prior to tet or dext staining .
(b) Ex vivo staining
LNC were obtained from a pool of draining LN harvested from groups of mice immunized with various peptides. CD4 cells were enriched from LNC to a purity of more than 95% by negative selection based on magnetic separation using IMAG as recommended by the manufacturer (BD Biosciences). After treating the cells with or without NASE as above, cells were washed in 5 ml of 1 × PBS; incubated with tetramers or dextramers for two hours at RT in growth medium, pH 7.6, containing FBS (2.5%). Cells were washed twice with 5 ml of 1 × PBS and stained with anti-CD4, anti-CD25, anti-CD44 (all from eBioscience) and 7-AAD and analyzed by FC. The percentages of tet+ or dext+ cells were determined in the live (7-AAD-) cells corresponding to CD4, CD25 and CD44 subsets. To stain MNC obtained from CNS tissues, cells were incubated with tetramers or dextramers directly without NASE treatment, and the percentages of tet+ or dext+ cells were determined as described above.
Sorting of tet+ or dext+ cells by flow cytometry
LNC isolated from immunized mice were stimulated with PLP 139-151 at a density of 5 × 106 cells/ml and viable lymphoblasts were harvested on day 6 poststimulation and stained with tetramers or dextramers for two hours at RT. After washing, cells were stained with anti-CD4 and 7-AAD; the tet-CD4+ and dext-CD4+ cells were sorted by FC (FACSAria, BD Biosciences, San Jose, CA), and each fraction was divided into two aliquots. One aliquot of cells was immediately stained with APC-labeled PLP 139-151 or control dextramers for two hours, followed by 7-AAD. After acquiring the data by FC, dext+ cells were analyzed in the live (7-AAD-) subset. The second aliquot (0.75 × 106 cells/ml) was rested in growth medium for two weeks and stimulated with PLP 139-151 (10 μg/ml) in the presence of irradiated antigen-presenting cells for two days; growth medium was then supplemented. Viable lymphoblasts were harvested two days later, and on day 6 poststimulation, PLP 139-151 or control tet or dext staining was performed followed by anti-CD4 and 7-AAD and the percentages of live tet+ or dext+ cells were enumerated as above.
Differences between specific and control tet or dext and also between the groups were analyzed by student's t. P ≤ 0.05 values were considered significant.
Optimization of reaction conditions for dextramer staining
FITC-labeled dextramers but not tetramers detect antigen-reactive cells
In spite of high sensitivity, dextramers fail to detect all antigen-reactive cells
Activation dependency is less stringent for dextramer binding
Dextramers are useful tools to enumerate the frequencies of antigen-specific cells ex vivo
Using three different murine autoimmune disease models, we have demonstrated that MHC class II dextramers permit ex vivo enumeration of self-reactive CD4 T cells with specificity. Tetramers, but not monomers or dimers, are useful in detecting CD4 T cells by FC [4, 12, 33]. In spite of this technical advancement, detection of autoreactive CD4 cells is not straightforward because of their rarity in the peripheral repertoires, and their detection often requires in vitro expansion and/or enrichment . In this scenario, however, while antigen specificity of cells can still be confirmed, accurate estimation of their frequencies can be prone to error because of the need to activate cells prior to tetramer staining. Structurally, dextramers contain dextran backbones, which are polymers of glucose molecules attached through 1-6 and 1-3 linkages . Each dextran molecule carries multiple moieties of SA to which biotinylated peptide-tethered MHC molecules can be assembled . Therefore, MHC dextramers can yield MHC-peptide complexes of large molecular weight, allowing them to engage multiple TCRs - more than that could be achieved with tetramers. To address this hypothesis, we created MHC dextramers for three mouse IA alleles, IAs, IAb and IAk for PLP 139-151, MOG 35-55 and Myhc-α 334-352, respectively, and showed that the reagents detect corresponding antigen-sensitized cells with specificity and also to a higher sensitivity than conventional tetramers.
We optimized the conditions for dextramer staining using PLP-reactive cells generated from SJL mice immunized with PLP 139-151. Essentially, the conditions previously defined for conventional tetramers were also optimum for dextramers, and the staining was performed in growth medium supplemented with 2.5% FBS, pH 7.6 [5, 11]. However, we noted some important advantages with dextramers, including enhanced sensitivity, shortened staining duration and the ability to create dextramers with a low amount of MHC/peptide complexes. We found that dextramers prepared at a 1:20 molar ratio of SA-dextran molecules to biotinylated MHC proteins were optimum to detect PLP-reactive cells, and their detection sensitivity was ~4-fold higher than that of tetramers. Importantly, the use of dextramers allowed us to capture antigen-specific cells by FC unambiguously as observed by an increase in their mean fluorescent intensities in relation to tetramers (Figure 1). This is an important consideration in flow cytometric analysis, especially when FITC-labeled reagents are used. In support of this notion, PLP 139-151 dextramers, but not tetramers, labeled with FITC detect nearly equivalent percentages of PLP-reactive cells similar to percentages obtained with PE- or APC-labeled dextramers. Furthermore, the enhanced detection of antigen-reactive cells could be achieved as early as 30 minutes postincubation with dextramers, whereas with tetramers, the staining intensity was only marginally improved even when extending the incubation time up to 3 hours. On a reaction basis, we calculated the amount of MHC protein required to prepare tetramers to be 2- and 4-fold higher than the amount of protein required to make dextramers labeled with PE and APC, respectively. The marginal reduction in the staining intensity obtained with FITC-dextramers was expected because signals generated from FITC are weaker than other fluorescent dyes. The fact that FITC-labeled dextramers, but not tetramers, bound PLP-reactive cells provides opportunities to include this dye in the FC-based multicolor analysis of antigen-specific T cell populations.
We believe that the higher detection sensitivity of dextramers is due to the presence of multiple SA moieties, allowing the formation of large clusters of MHC-peptide complexes, which can interact with multiple TCRs, thus enhancing the avidity of their interactions [15, 33]. Conversely, each tetramer molecule contains a single SA moiety to which only up to four MHC-peptide complexes can bind, limiting the availability of MHC-peptide complexes to engage with multiple TCRs. As a result, tetramers might bind T cells bearing high- but not low-affinity TCRs. This appears to be the case because a proportion of tet- cells sorted by FC could still be detected by dextramers (Figure 4A). Nevertheless, it should be noted that dextramers do not detect all antigen-sensitized cells, as indicated by the fraction of PLP-reactive cells previously negative for PLP dextramers that can become detectable after a second restimulation with PLP 139-151 (Figure 4B). Presently the significance of these cell populations is not known with respect to their TCR-affinities or functionalities, such as cytokine synthesis or encephalitogenicity. Alternatively, failure to detect all antigen-reactive cells with dextramers might be directly related to the density of TCRs expressed on the surface of T cells such that low TCR-expressed cells may escape detection. From a technical point of view, formation of stable MHC-peptide-TCR interactions with dextramers can help cells withstand harsh steps such as centrifugation and washing, thereby enhancing specificity by minimizing the background staining. Overall, the fact that dextramers permit quick determination of antigen-specific cells with enhanced sensitivity and because their creation requires less starting material, they have proven to be superior to conventional tetramers.
One of the limitations of class II tetramer binding is activation dependency [9–12]. Various strategies have been employed to enhance the detection sensitivity of class II tetramers by promoting interactions with multiple TCRs. These include antibody-mediated TCR clustering, MHC-peptide-coated liposomes and immunoglobulin molecules [14, 34–36]. We had previously shown that, upon resting, antigen-sensitized cells that have expanded in vitro become undetectable with tetramers, but can be detected through NASE treatment prior to staining . We asked whether the use of dextramers can overcome as the need for NASE treatment or activation. Expectedly, dextramers could bind rested cells in the absence of NASE treatment to a significantly greater proportion than that achieved with tetramers (Figure 5). The data suggest that activation dependency is not a critical factor for dextramer staining, and we verified this phenomenon further by ex vivo enumeration of PLP-specific cells in SJL mice immunized with PLP 139-151 (Figure 6). While, similar results were obtained with IAb/MOG 35-55 and IAk/Myhc-α 334-352 dextramers, NASE treatment added the advantage of enhancing their detection sensitivity as compared with tetramers (Figures 7 and 8). The data prove that MHC dextramers are useful tools for enumerating the frequencies of antigen-specific T cells ex vivo in various experimental systems.
We have shown that dextramers are superior to tetramers in detecting autoreactive cells, which otherwise can be technically challenging. While activation dependency is less important for dextramer binding, detection sensitivity can be enhanced by NASE treatment without compromising the specificity. Although dextramers cannot detect all cells in spite of being antigen specific, their greater detection sensitivity makes them first-choice reagents over conventional tetramers. Additionally, the inherent variation in the detection abilities of these reagents is to be considered while interpreting the results because frequencies of antigen-specific cells identified by each reagent are expected to be different.
We thank Immudex, Denmark for providing dextramer reagents (contact person: Tina Jakobsen, firstname.lastname@example.org), and Dr. Jean-Jack Riethoven for statistical analysis. This work was co-funded by the American Heart Association and the Children's Cardiomyopathy Foundation (SDG2462390204001).
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