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The Journal of Immunology, 2007, 179, 3588-3595
Copyright © 2007 by The American Association of Immunologists, Inc.
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Xenogeneic beta2-Microglobulin Substitution Affects Functional Binding of MHC Class I Molecules by CD8+ T Cells1

Loralyn A. Benoit* and Rusung Tan2,{dagger}

* Department of Immunology, University of Toronto, Toronto, Ontario, Canada; and {dagger} Department of Pathology and Laboratory Medicine, British Columbia’s Children’s Hospital and University of British Columbia, Vancouver, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
NK cells and CD8+ T cells bind MHC-I molecules using distinct topological interactions. Specifically, murine NK inhibitory receptors bind MHC-I molecules at both the MHC-I H chain regions and beta2-microglobulin (beta2m) while TCR engages MHC-I molecules at a region defined solely by the class I H chain and bound peptide. As such, alterations in beta2m are not predicted to influence functional recognition of MHC-I by TCR. We have tested this hypothesis by assessing the capability of xenogeneic beta2m to modify the interaction between TCR and MHC-I. Using a human beta2m-transgenic C57BL/6 mouse model, we show that human beta2m supports formation and expression of H-2Kb and peptide:H-2Kb complexes at levels nearly equivalent to those in wild-type mice. Despite this finding, the frequencies of CD8+ single-positive thymocytes in the thymus and mature CD8+ T cells in the periphery were significantly reduced and the TCR Vbeta repertoire of peripheral CD8+ T cells was skewed in the human beta2m-transgenic mice. Furthermore, the ability of mouse beta2m-restricted CTL to functionally recognize human beta2m+ target cells was diminished compared with their ability to recognize mouse beta2m+ target cells. Finally, we provide evidence that this effect is achieved through subtle conformational changes occurring in the distal, peptide-binding region of the MHC-I molecule. Our results indicate that alterations in beta2m influence the ability of TCR to engage MHC-I during normal T cell physiology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Conventional T cells express an {alpha}beta T cell Ag receptor (TCR), a heterodimeric complex that provides the principal basis for specificity and clonality in the T cell compartment. The TCR{alpha}beta complex is composed of disulfide-linked {alpha} and beta-chains (1) organized into two separate Ig superfamily domains including a variable (V{alpha} or Vbeta) domain and a constant domain (C{alpha} or Cbeta) (2). During binding of MHC class I (MHC-I),3 the TCR adopts a diagonal orientation above the peptide-MHC-I complex and binds via the TCR variable regions (3, 4). The constant features which govern this association include a constrained topology whereby the TCRV{alpha} chain is positioned over the N terminus of the peptide and the TCRVbeta chain is positioned over the C terminus (5), as well as conformational flexibility that contributes to variation in stability of different TCR-peptide-MHC complexes (6). This flexibility is determined by V{alpha} and Vbeta domains (3, 7) and while limiting the overall affinity of TCR interactions, it enables clonotypic TCR binding to different MHC and peptide molecules, a phenomenon referred to as TCR cross-reactivity (8).

There are several different types of MHC molecules that function as ligands for TCR. In the case of CD8+TCR{alpha}beta+ T cells, the ligand is commonly MHC-I, a heterotrimeric complex composed of a classical MHC class Ia H chain molecule which noncovalently binds the L chain module, beta2-microglobulin (beta2m), and a short peptide (9). The class I H chain is organized into three Ig domains: the {alpha}1 and {alpha}2 modules form the peptide-binding groove and the {alpha}3 module forms the membrane-proximal stalk (9). MHC-I H chain molecules are highly polymorphic (10), with the majority of sequence diversity occurring within the peptide-binding groove of the {alpha}1 and {alpha}2 domains (9, 10). The {alpha}3 domain remains relatively conserved, thus permitting association with the invariant beta2m and CD8 (10).

beta2m is a 11.6-kDa protein consisting of a single constant Ig domain (12). beta2m has limited polymorphism: in humans, beta2m is thought to be monomorphic (13), and although eight alleles have been identified in mice (14), only three alleles exist in common inbred mouse strains, differing at residue 85 (15). Bovine, mouse (m), and human (h) beta2m demonstrate ~70% similarity in amino acid sequence (16), thus enabling xenoforms of beta2m to bind to MHC-I H chains. In the case of h-beta2m binding to H-2Db and H-2Kk, binding affinity is in fact increased by 3-fold (17, 18). The lower affinity of m-beta2m for murine H chain results in lower structural stability and greater molecular flexibility (19). As evidenced by beta2m-deficient mice, binding of beta2m to the H chain is important for stable surface expression of MHC-I (20).

CD8, expressed as a disulfide-linked dimer, comprises a single Ig domain supported by a stalk region that together function as a coreceptor for TCR (21, 22). In most T cell subsets, CD8 exists as a heterodimer composed of an {alpha}-chain and beta-chain (23). In conjunction with TCR, CD8 associates with MHC-I molecules, stabilizing the overall interaction by modifying the conformation of the multimolecular complex and by contributing to the TCR signaling apparatus (3, 22, 24). Targeted gene disruption of CD8 results in severely impaired (CD8+) CTL development and function (25). Importantly, binding of CD8 to MHC-I occurs independently of MHC isoform and peptide content (22).

The complete TCR-MHC-I complex includes TCR, specific peptide, beta2m, MHC-I, and CD8. The principal TCR{alpha}beta-binding surface is contributed by the H chain {alpha}1-{alpha}2 domain and bound peptide (3, 5, 26). A secondary binding site, implicated in coreceptor CD8 engagement, is supplied by a conserved region of the {alpha}2 domain, the {alpha}3 domain, and beta2m (26, 27, 28, 29, 30, 31). Specific influence of m-beta2m on CD8 binding to MHC-I has not been established, although cocrystal studies (19, 26), mutational analyses (27, 28, 29), and MHC-I tetramer-binding studies (30, 31) suggest that alterations in beta2m could potentially affect the overall association. Importantly, residues glutamine (Q) 6, threonine (T) 28, Q29, and lysine (K) 58 of m-beta2m have been shown to contact directly with CD8{alpha} through a series of hydrogen bonds and salt bridges (26). Of these four residues, Q6, T28, and Q29 are unique to mice, suggesting that mouse-specific beta2m residues may be important during coreceptor engagement of MHC-I. Furthermore, full xenogeneic beta2m substitution appears to affect the MHC-I structure, resulting in a higher affinity association (19) and altered conformation of the MHC-I region that is bound by the TCR (32). We therefore sought to evaluate how xenogeneic beta2m substitution might affect the functional association of TCR with MHC-I molecules. We report that substitution of m-beta2m with h-beta2m influences the association between TCR and MHC-I. In doing so, it appears that beta2m conveys self-identity to the T cell compartment, a novel property that extends our understanding of the TCR-MHC-I interaction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and tissue culture

C57BL/6J (B6, H-2b) mice were purchased from The Jackson Laboratory. H-2Db–/– and H-2Kb–/– B6 mice (33) were donated by Dr. F. Lemonnier (Pasteur Institute, Paris, France). The h-beta2m+/m-beta2m– B6 mice were obtained from Dr. J. W. Chamberlain (Hospital for Sick Children, Toronto, Ontario, Canada) and were generated by breeding m-beta2m–/– B6 mice (20) with h-beta2m+m-beta2m– B6 mice (34). h-beta2m+/m-beta2m–;H-2Db–/– mice were derived by breeding h-beta2m+/m-beta2m+ mice with H-2Db–/– mice. OT-I B6 mice, transgenic (Tg) for the pOVA(SIINFEKL):H-2Kb TCR (35), were donated by Dr. T. Watts (University of Toronto, Toronto, Ontario, Canada). Animals were maintained in the specific pathogen-free vivarium at the Ontario Cancer Institute and used according to institutional guidelines. Tissue was obtained from sex- and age-matched mice. Cells were cultured in complete medium: {alpha}-MEM (Invitrogen Life Technologies) supplemented with 50 µM 2-ME, 10 mM HEPES, and 10% heat-inactivated FBS (Wisent).

Peptides

H-2Kb-binding peptides were pOVA (SIINFEKL) and pVSV (RGYVYQGL). Peptides were purchased from the Alberta Peptide Institute (Edmonton, Alberta, Canada) and were dissolved in PBS at 1 mg/ml and stored at –80°C.

Antibodies

Anti-CD4, -CD8, -B220, -CD28 (37.51), -CD3 (145-2C11), -TCRbeta, and -H-2Db (KH95) were purchased from BD Biosciences. KH95 binds H-2Db irrespective of the beta2m moiety (36). The 2.4G2, 5F1.2, and W6/32 hybridomas were obtained from the American Type Culture Collection. 5F1.2 binds the {alpha}1–{alpha}2 domain of H-2Kb irrespective of the beta2m moiety and peptide (37). W6/32 binds h-beta2m-bound H-2Db (38). S19.8 was provided by Dr. U. Hammerling (Sloan-Kettering, New York, NY) and binds to the B6 alloform of m-beta2m (39). 25D1.16, provided by Dr. R. Germain (National Institute of Allergy and Infectious Diseases, Bethesda, MD), binds pOVA:H-2Kb (40).

Staining and flow cytometry

Approximately 2 x 105 cells were suspended in 100 µl of buffer containing 2.4G2 supernatant, stained for 30 min on ice, washed, and then resuspended in cold buffer for acquisition. Staining for pOVA:H-2Kb complexes involved pulsing ~2 x 105 cells with 1 µg of pOVA on ice, washing, and then staining with 25-D1.16 (40). Samples were analyzed on a FACSCalibur (BD Biosciences). Data were analyzed on CellQuest software (BD Biosciences).

Target cell generation

Lymphoblasts were generated by incubating ~107 splenocytes for 1.5–2 days in complete medium containing 2 µg/ml Con A (ICN Pharmaceuticals). Viable Con A blasts were enriched on Lympholyte-M (Cedarlane Laboratories), washed, and incubated for 2 min in 200 mM methyl {alpha}-D-mannoside/methyl {alpha}-D-glucoside (Sigma-Aldrich).

CTL generation

For polyclonal CTL lines, 2 x 107 lymph node (LN) cells were resuspended in 10 ml of complete medium containing pOVA or pVSV (10 ng/ml) and human rIL-2 (250 U/ml; Chiron). Seven to 9 days afterward, viable CTL were replated in complete medium with IL-2 (250 U/ml) at 2 x 106 cells/ml in flasks containing ~4 x 107 irradiated (20 Gy, 137Cs source, 0.97 Gy/min), plastic-adhered splenocytes prepulsed with pOVA or pVSV (5 ng/ml) for 1 h at 37°C. For monoclonal CTL cultures, LN cells from OT-I mice were B cell-depleted using anti-B220-conjugated magnetic beads (Dynal). In brief, 2 x 106 cells/ml in 100-µl aliquots were plated into 96-wells precoated with anti-CD3 (5 µg/ml; clone 145-2C11) and anti-CD28 (1 µg/ml; clone 37.51). One hundred microliters of complete medium supplemented with 250 IU/ml IL-2 was added and replenished on day 3 or 4. Cells were harvested on days 4–6 for use in assays.

Ag-specific cytotoxicity assays

Con A target cells were labeled for 90 min with 100 mCi of Na51CrO4 (NEN Life Science), washed, and then incubated with pOVA or pVSV (1.0 µg/ml) for 45 min. CTL were serially diluted and plated in 96-well plates as 100-µl aliquots. Target cells (20,000/ml) were added in 100-µl aliquots. Plates were incubated for 4 h, after which 100 µl of supernatant was harvested from each well and counted using a gamma counter. Percent specific lysis was calculated by averaging the gamma emission from five replicate wells and calculated as follows: 100 x (experimental release – spontaneous release)/(maximum release – spontaneous release), where spontaneous release refers to supernatant from target cells cultured alone, maximal release refers to supernatant from targets incubated in 1% glacial acetic acid, and experimental release refers to supernatant obtained at each E:T ratio.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
T cell phenotype is altered in the context of h-beta2m binding to MHC-I

To address whether xenogeneic substitution of beta2m influences TCR recognition of MHC-I, we first examined thymocytes from h-beta2m-Tg mice. H-2Kb–/– mice were included as a control because deletion of H-2Kb reduces the mature CD8+ T cell compartment by ~30–50% (33). Notably, lineage-specific differentiation of CD4+CD8+ double- positive (DP) thymocytes into CD8+ single-positive (SP) thymocytes requires selection on MHC-Ia structures and deletion of MHC-I genes (H-2Kb) results in fewer CD8+ SP cells (41, 42, 43). Inclusion of h-beta2m reduced the CD8+ SP thymocyte frequency to a degree greater than the absence of H-2Kb (cf 64% to 47% reduction; Fig. 1D). As well, a corresponding (3-fold) increase in the CD4 SP compartment was observed within h-beta2m+ thymocytes. Total numbers of thymocyte and double negative (DN) cells were not altered as a consequence of the H-2Kb deletion or h-beta2m expression (data not shown). DN stages were not further investigated since differences in MHC do not affect the DN stage (41). Indeed, the onset of the altered thymocytes phenotype was first detected at the DP stage, where a modest reduction (54.2% DP compared with 62.5% DP; p < 0.10) was observed for h-beta2m+ thymocytes (Fig. 1E).


Figure 1
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  		FIGURE 1. Effect of h-beta2m on thymocyte CD4/8 phenotype. Representative stains of thymi from Wt (A), H-2Kb–/– (B), and h-beta2m+ (C) mice were stained for CD4 and CD8 and then assessed by FACS. Average CD4:8 ratios (D) and percent DP thymocytes (E). Error bars, SEM. Variance was assessed using the t test, where * is p < 0.10 and ** is p < 0.005. KO, Knockout.

 
We further assessed thymocytes by measuring the surface expression levels of TCRbeta, CD8, and CD3 (Table I). Thymocytes derived from h-beta2m+-Tg mice demonstrated reduced overall density and frequency of profiles compared with control samples. This pattern of expression is consistent with a model of enhanced negative selection (44, 45), whereby only those thymocytes expressing lower TCRbeta and CD3 levels survive beyond this checkpoint (46).


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  		Table I. Phenotypic analysis of thymocytes from Wt and h-beta2m+ B6 mice stained with anti-CD3, CD8, and TCRbeta and then analyzed by flow cytometry

 
Because persistence of TCR{alpha}beta+ cells in the periphery is an active process that depends upon continuous TCR-MHC interactions (42, 47, 48), we therefore additionally assessed the mature T cell compartment. Total cellularity in the spleen was not significantly altered as a consequence of the h-beta2m transgene expression. However, Tg mice demonstrated an ~55% relative reduction of peripheral (LN) CD8+ T cells, while H-2Kb–/– mice exhibited only a 25% reduction (Fig. 2). Because the absolute numbers of peripheral CD8+ T cells are appreciably higher in the periphery than in the thymus, this suggests that once in the periphery, the CD8+ thymocytes that have undergone positive selection in the thymus are then able to interact with MHC-I and expand clonally. Lastly, a moderate (~35%) gain in the number of peripheral (LN) CD4+ T cells was detected in the h-beta2m- Tg mice (data not shown), with a small (~10%) relative reduction of total (LN) TCR{alpha}beta+ cells occurring in the context of a near compensatory (~10%) gain in B cell frequency (Fig. 3D).


Figure 2
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  		FIGURE 2. Effect of h-beta2m on LN T cell CD4/8 phenotype. Representative CD4 and CD8 stains from Wt (A), H-2Kb–/– (B), and h-beta2m+ (C) mice by FACS. D, Average CD4:8 ratios. Error bars, SEM. Variance was assessed using the t test, where * is p < 0.005 and ** is p < 0.001. KO, Knockout.

 

Figure 3
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  		FIGURE 3. Effect of h-beta2m on LN T cell numbers. Representative TCRbeta and B220 stains from Wt (A), H-2Kb–/– (B), and h-beta2m+ (C) mice by FACS. D, Average percent TCRbeta+ and B220+. Error bars, SEM. Variance was assessed using the t test, where * is p < 0.05 and ** is p < 0.0001. KO, Knockout.

 
TCRVbeta repertoire is shaped by thymic selection processes (49) as well as Ag exposure and peripheral maintenance processes (50, 51). To assess the CD8+ variable region repertoire of h-beta2m-Tg mice, we used a panel of eight available Vbeta chain mAbs and compared the values to wild-type (Wt) and H-2Kb–/– controls (Fig. 4A). The V{alpha} repertoire was not assessed because the effect of MHC-I on the mature T cell repertoire is considerably more profound for Vbeta chain selection (43). Comparable to the Vbeta repertoire skewing seen in H-2Kb–/– mice (33), h-beta2m+-Tg mice demonstrated a pattern of decreased Vbeta2 and Vbeta11 usage and expansion of the Vbeta17 subset in the CD8+ T cell population.


Figure 4
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  		FIGURE 4. Effect of h-beta2m on TCR Vbeta repertoire. Vbeta staining of LN cells from Wt, H-2Kb–/–, and h-beta2m+ mice costained with anti-CD8 and then assessed by FACS. Values represent average percent positive staining of CD8+ gated populations and error bars indicate SEM. KO, Knockout.

 
MHC-I cell surface expression is partially altered in h-beta2m mice

Our interpretation of these results rests on the assumption that expression of total MHC-I molecules and individual peptide-MHC-I complexes is not significantly altered as a consequence of h-beta2m expression. However, it has been shown that h-beta2m can alter the expression levels as well as peptide-binding ability of H-2Db (17, 18). We therefore measured the expression levels of MHC-I-dependent epitopes on h-beta2m+ lymphoblasts (Fig. 5, A–D) and found that although h-beta2m enhanced expression of total H-2Db (2- to 3-fold), expression of H-2Kb was not appreciably affected. We therefore focused our studies on H-2Kb.


Figure 5
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  		FIGURE 5. Effect of h-beta2m on target cell MHC-I expression. Con A blasts derived from Wt and h-beta2m+mice were stained for m-beta2m (A), h-beta2m (B), total H-2Db (C), total H-2Kb (D), and pOVA-Kb (E) and then assessed by FACS. F, Total H-2Kb MFI values on the surface of Wt and h-beta2m+Con A blasts following no treatment, pOVA 90-min pulsing, and pOVA overnight (O/N) pulsing.

 
The ability of h-beta2m to influence expression of specific peptide-MHC-I complexes was next assessed (Fig. 5, E and F). Lymphoblasts derived from either m-beta2m+ or h-beta2m+ mice were pulsed for 90 min with saturating quantities of pOVA (which binds to H-2Kb with high affinity), then stained with the mAb 25D1.16 (which has specificity for pOVA:H-2Kb complexes (40)). Nearly comparable levels of pOVA:H-2Kb expression were detected in each cohort (Fig. 5, E and F). As well, total H-2Kb surface expression generated as a consequence of long-term incubation with saturating quantities of peptide (Fig. 5F) demonstrated only a nominal (~5%) increase in H-2Kb expression in h-beta2m+ mice. Thus, it appears that h-beta2m does not significantly alter the ability of H-2Kb to bind exogenous pOVA.

The epitope recognized by the mAb 25D1.16 binds the C terminus of the bound peptide and the corresponding {alpha}1-{alpha}2 helical domain in a region that is distal to beta2m and which partially overlaps with the OT-1(42.12) TCRbeta chain binding site (52). Binding of 25D1.16 should therefore not be directly affected by the moiety of beta2m that is bound to H-2Kb. To test this reasoning, Con A blasts derived from H-2Db–/– and h-beta2m+ H-2Db–/– mice were pulsed with pOVA for 60 min and then stained with increasing concentrations of 25D1.16-FITC. Surprisingly, expression of h-beta2m resulted in a window of increased mAb 25D1.16 binding to the pOVA:H-2Kb epitope (Fig. 6). This finding suggests that the number of ligand structures is not altered as a consequence of h-beta2m binding to H-2Kb, but rather that the binding surface used by 25D1.16, and by extension the OT-1(42.12) TCR beta-chain, is (slightly) modified resulting in increased mAb-binding affinity.


Figure 6
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  		FIGURE 6. Effect of h-beta2m binding to H-2Kb on the 25D-1.16 epitope. Con A blasts derived from H-2Kb–/–, h-beta2m+ H-2Db–/–, and H-2Db–/– mice were incubated with pOVA (1.0 µg/ml) for 60 min, washed, stained with increasing amounts of 25D-1.16, and assessed by FACS.

 
CD8+ T effector functions are altered as a consequence of h-beta2m expression

We next assessed whether this small conformational change detected by mAb staining could directly influence the affinity of the TCR-MHC-I interaction. Bulk CTL lines specific for pOVA and pVSV were generated in vitro from H-2Db–/– and h-beta2m+ H-2Db–/– mice. Peptide-specific CTL derived from m-beta2m+ mice demonstrated impaired killing of h-beta2m+ targets, compared with the killing of m-beta2m+ targets (Table II). In contrast, peptide-specific CTL derived from h-beta2m-Tg mice did not discriminate between target cells (Table II). These data suggest that although h-beta2m+CD8+ CTL cells are able to recognize H-2Kb+ syngeneic target cells pulsed with pOVA as well as cells from Wt mice, TCR selection in m-beta2m+ mice produces pOVA:H-2Kb-specific clonotypes that are inefficient at recognizing epitopes bound by h-beta2m.


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  		Table II. Effect of h-beta2m on polyclonal CTL recognition of MHC-Ia

 
To study this phenomenon at the level of a single pOVA:H-2Kb-specific TCR clone, a monoclonal CTL line was generated using OT-1 mice. The OT-1-Tg TCR derived from the B6 CTL clone 42.12 uses Vbeta5 (35) and because the frequency of Vbeta5 was not altered in the h-beta2m+ mice (Fig. 4A), this clone seemed suitable for testing the ability of the m-beta2m-restricted CTL clone to recognize h-beta2m+ targets. We found that although OT-1 CTL efficiently lysed m-beta2m+ H-2Db–/– targets pulsed with pOVA (Fig. 7A), they failed to kill pOVA-pulsed h-beta2m+ H-2Dd–/– targets (Fig. 7B). Thus, we conclude that xenogeneic substitution of beta2m reduces the ability of peptide-specific TCR to bind to MHC-I by altering the epitope shared by a clonotypic TCR and the mAb 25D1.6.


Figure 7
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  		FIGURE 7. Effect of h-beta2m on clonal CTL recognition of MHC-I. OT-1 T cells were used against Con A blasts from H-2Db–/– (A) and h-beta2m+ H-2Db–/– (B) mice pulsed with pOVA or pVSV. Error bars, SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The cellular arm of the immune system has evolved various T cell and NK cell receptors that bind the conserved MHC fold, albeit using unique topological means. For example, TCR{alpha}beta and (for the most part) the killer cell Ig-like receptors have been shown to bind MHC-I molecules diagonally along the {alpha}1–{alpha}2 surface independently of beta2m (4, 53). In comparison, Ly49 inhibitory receptors and leukocyte Ig-like receptors associate with MHC-I molecules using a more intricate orientation (54, 55, 56, 57, 58), as with leukocyte Ig-like receptor 1, Ly49A, and Ly49C, involving direct partnering with beta2m (54, 55, 56, 57, 58). In this way, changes in beta2m, including point mutations at species-specific residues (57) as well as xenogeneic substitutions (54), have been shown to interfere with the ability of Ly49A and Ly49C to bind to cognate MHC-I molecules.

Inasmuch as structural analysis indicates that TCR{alpha}beta binds MHC-I independently of beta2m (3, 11), changes in beta2m are not predicted to affect the TCR binding. However, there is some evidence that beta2m may in fact influence TCR recognition. One notable example is the allelic change in alanine (A) to aspartic acid (D) at position 85 that restores diabetes susceptibility to disease-resistant NOD.beta2mnull or b allele mice (59). This A85->D conversion induces a subtle conformational change in H-2Db that modifies the hydrogen bond networks along the binding interface (60). The diabetes susceptibility of this single amino acid change may be explained using several different models: altered CTL specificity (as seen for H-2Kb-restricted CTL) (61, 62), an altered peptide-binding repertoire (as seen for H-2Kd-restricted CTL) (63), or lastly, that specific residues of beta2m may influence the strength of TCR recognition by affecting CD8 binding (19).

With these possible mechanisms in mind, we sought to functionally assess the role of species-specific beta2m on recognition of MHC-I by TCR{alpha}beta in vivo using a h-beta2m-Tg mouse model. To begin with, we have shown that the CD8+ lineage in these mice was markedly reduced such that CD8+ SP thymocytes and mature CD8+ T cells were ~50% of Wt values, with compensatory increases in the CD4+ T cell lineage (Figs. 1–3). Importantly, the reduction in the CD8+ lineage occurred in the context of nearly equivalent MHC-I expression levels (Fig. 5, C and D). These findings can be rationalized using two potential negative selection models: 1) h-beta2m binding to mouse MHC-I molecules adversely influences the self-peptide/MHC-I structure presented on the thymic epithelium such that it is poorly recognized by mouse TCR{alpha}beta (resulting in increased death by neglect) or 2) enhanced TCR/self-peptide/MHC-I affinity (and decreased intermolecular flexibility) induced by h-beta2m results in excessive, mortal signaling (and increased negative selection). Although the data presented herein cannot categorically distinguish between the two models, in Fig. 1, the pattern of reduction in both the DP and SP thymocyte populations is distinct from death by neglect, which is marked by decreased SP frequency alone (44, 45), as capitulated by the H-2Kb–/– control mouse. Rather, the phenotype we have described is consistent with a negative selection model whereby high-affinity binding thymocytes are eliminated as early as the DP stage (44, 45, 64) and where thymocytes with lower CD3, CD8, and TCR{alpha}beta expression levels (Table I) are permitted to undergo maturation and expansion (44, 45, 46). We therefore tentatively conclude that h-beta2m substitution alters the MHC-I surface that is recognized by TCR{alpha}beta, resulting in enhanced affinity (or rigidity) of the intermolecular association and increased negative selection of thymocytes in the h-beta2m-Tg mouse.

The few CD8+ T cells that were selected for in the thymus of h-beta2m+ mice underwent partial expansion in the periphery (cf Fig. 1C with Fig. 2C) with only very minor skewing of the Vbeta repertoire (Fig. 4), thereby suggesting that mouse TCR{alpha}beta+ T cells are capable of binding to h-beta2m:MHC-I molecules in both the thymus and the periphery without gross changes in overall binding affinities (42, 47, 48). However, further conclusions regarding the minor Vbeta repertoire alterations are limited by the fact that the CD4+ subset unexpectedly demonstrated mild repertoire variability as well, with decreased Vbeta2, 11, and 14 usage detected in both the h-beta2m-Tg and H-2Kb–/– CD4+ T cell cohorts (data not shown). It is unlikely that this minor variability is the result of embryonic stem cell (ES) contaminants since the H-2Kb–/– mice were generated using ES cells of H-2b origin and h-beta2m+ mice were derived in part from ES cells of H-2S2 origin, yet both the strains exhibited similar Vbeta results. Therefore, it seems more likely that the changes in the Vbeta repertoire seen in the h-beta2m+ (and H-2Kb–/–) T cell compartments are a consequence of either altered lineage commitment (occurring before positive selection events) or altered peripheral maintenance/persistence. Possible explanations that could account for this finding include 1) altered (or lack of) specific peptide/MHC-I structures in the thymus or periphery, 2) modified minor histocompatibility Ags (e.g., beta2m-derived fragments), and/or 3) differences of flora in the MHC-I mutant mice maintained in our animal colony vs in controls that were used shortly after arrival from the vendor.

We next undertook functional analysis to determine whether there might be differences in how h-beta2m-resticted CD8+ T cells recognize MHC-I molecules loaded with exogenous peptide. The experiment was designed using H-2Kb-restricted CTL since the expression of H-2Kb, unlike H-2Db, is not significantly affected by inclusion of h-beta2m. Furthermore, both the effector and target cell preparations were derived using H-2Db–/– mice so as to eliminate background effects of H-2Db in our system. Lastly, H-2Kb-restricted pOVA was chosen as one of the antigenic peptides because the Vbeta5 frequency, which is known to bind pOVA:H-2Kb (35), was not altered in the h-beta2m+ mouse (Fig. 4A) and because both forms of beta2m support equal pOVA loading of H-2Kb (Fig. 5F). In our hands, CTL lines from both strains were equally capable of proliferating in response to pVSV- and pOVA-pulsed syngeneic target cells (data not shown). Moreover, we found that h-beta2m+ and m-beta2m+ CTL lines were equally competent at killing target cells expressing cognate ligand in the context of syngeneic beta2m (Table II). From this we concluded that the overall affinity of the polyclonal h-beta2m+ TCR{alpha}beta for h-beta2m:peptide:MHC-I is comparable to the overall affinity of polyclonal m-beta2m+ TCR{alpha}beta for m-beta2m:peptide:MHC-I.

However, using these same polyclonal CTL lines, we found that m-beta2m-restricted polyclonal TCR{alpha}beta exhibited moderately reduced (approximately one-third) affinity for h-beta2m+ targets as compared with syngeneic targets regardless of peptide used (Table II). In contrast, h-beta2m-restricted TCR{alpha}beta demonstrated only mildly reduced killing of m-beta2m+ targets (in the context of pOVA). Since the CTL lines used in this experiment were polyclonal, we therefore interpreted this to mean that distinct forms of beta2m could prevent TCR recognition of MHC-I in some instances but not all. Although h-beta2m-induced ablation of CD8 binding to H-2Db, as proposed by Achour et al. (19), could be used to explain in part this pattern of reactivity toward H-2Kb, we disfavor this interpretation because it would follow that for this to be true, few if any CD8+ thymocytes would be positively selected in h-beta2m+ mice given these restrictions. Moreover, according to this model, h-beta2m would be expected to drastically influence the Vbeta repertoire as well as to influence functional binding, and, again, this was not the case: h-beta2m-restricted CTL were as capable as m-beta2m-restricted CTL in killing syngeneic targets and only minor shifts in Vbeta usage were detected. In fact, it has been shown using COS-7 cells transfected with both mouse CD8{alpha} and CD8beta that H-2Kb tetramers refolded with h-beta2m can efficiently bind despite the xenogeneic substitution (65), thereby signifying that h-beta2m does not ablate the interaction between CD8 and H-2Kb. Instead, by applying the 25D-1.16 mean fluorescence intensity (MFI) staining results of Fig. 6, we maintain that binding of h-beta2m to H-2Kb alters the overall conformation (19), particularly in the TCR binding site and possibly enhances the overall intermolecular rigidity of the MHC-I complex (19, 60) and in doing so alters the ability of CTL TCR to bind MHC-I molecules (61, 62). We emphasize that this effect on CTL recognition is indirect, i.e., is acting distally, and extrapolate from the structural H-2Db data (19, 60) that binding of h-beta2m to H-2Kb likely induces a conversion in H chain structure that translates to the distal peptide-binding region. However, it remains possible that thymic-associated processes might also contribute to the differential ability of m-beta2m-restricted TCR{alpha}beta to recognize the two distinct MHC-I structures. Indeed, the results of Table II imply that TCR molecules restricted on h-beta2m have altered specificity compared with TCR restricted on m-beta2m. However, without applying a suitable model of negative selection, we cannot knowingly attribute this altered affinity to differences in TCR education.

With regard to the polyclonal CTL data, it is important to note that during the induction phase of CTL, it is possible to generate multiple peptide-specific clonotypes which can bind to the same ligand structure, albeit with altered thermodynamics (66). As a consequence, the cytotoxicity imparted by the bulk cultures is an integration of the peptide:MHC-I-binding potential and frequency of each representative clone. Furthermore, bulk CTL cultures can include TCR-mediated responses that are not directed against the peptide of interest. Therefore, the results of Table II do not predict the outcome of an individual peptide-specific clone or peptide-specific response for that matter. To circumvent these limitations, we therefore tested a single clonotype using a CTL line derived from the OT-1 B6 mouse. Importantly, the OT-1 TCR is restricted against pOVA:H-2Kb that is bound by m-beta2mb (35) and its MHC-I -binding print partially overlaps with the 25D1.16 Ag binding site (52), making this a suitable choice of TCR with which to extend our findings. Using this defined system, we found that OT-1 CTL demonstrated very poor affinity for the h-beta2m-bound pOVA:H-2Kb structure, relative to the m-beta2m-bound pOVA:H-2Kb structure. Note, the minor background killing that was detected above control killing values can be explained by beta2m replacement (either from CTL present in the coculture or from the serum) that allowed for formation of OT-I TCR-recognizable structures. From these data, we have concluded that there is significantly diminished TCR-binding capacity of the m-beta2m-derived CTL toward h-beta2m+ targets as a result of conformational changes on the MHC-I molecule at the TCR recognition surface. However, given our mixed polyclonal results from Table II, it is important to keep in mind that this affect is not absolute, that some TCR molecules are unable to discriminate between allogeneic/xenogeneic forms of beta2m because some m-beta2m-restricted TCR are still able to bind h-beta2m-bound MHC-I.

The interpretation of our functional results is supported by considerable evidence that minimal to modest changes in beta2m can affect the association between MHC-I and TCR. Indeed, h-beta2m binding to H-2Db rotates the core of each component by 10 degrees, thus creating two new regions of stabilizing bonds that in turn enhance the affinity of the intermolecular association between the two molecules (19). Furthermore, serological-based analysis indicates that binding of h-beta2m to H-2Ld results in distal structural alterations in the {alpha}1–{alpha}2 helices (32). Moreover, even very subtle changes imparted by allelic substitutions, such as the aspartic acid (D) in beta2ma and alanine (A) in beta2mb at position 85, are sufficient to cause a modest conformational alteration in the ternary structures of mouse MHC-I H chains H-2Db and H-2Kd (60, 67) and effect a discontinuous epitope as defined by the mAb S19.8 (68). We have herein extended this body of evidence by demonstrating that gross, xenogeneic substitution of beta2m can affect recognition of peptide:H-2Kb by peptide-specific TCR{alpha}beta in vitro as well as alter normal CD8+ T cell immunology in vivo. We have attributed this to conformational change and/or chemistry and not to altered CD8 binding. Thus, beta2m appears to define T cell responses. In that there are numerous implications from this finding, further work is recommended to determine whether the beta2m isoform can affect T cell immunology using other peptides and MHC-I molecules as well.


   Disclosures
Top
 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 R.T. is a Michael Smith Senior Scientist and is funded by the Canadian Institutes of Health Research. Back

2 Address correspondence and reprint requests to Dr. Rusung Tan, Department of Pathology and Laboratory Medicine, British Columbia’s Children’s Hospital, 4480 Oak Street, Vancouver, British Columbia, Canada. E-mail address: roo{at}interchange.ubc.ca Back

3 Abbreviations used in this paper: MHC-I, MHC class I; beta2m, beta2-microglobulin; h, human; m, mouse; MFI, mean fluorescence intensity; DP, double positive; DN, double negative; SP, single positive; LN, lymph node; ES, embryonic stem cell; Tg, transgenic. Back

Received for publication February 22, 2007. Accepted for publication July 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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