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Virus-specific CD4+ T cells contribute to effective virus control through a multiplicity of mechanisms including direct effector functions as well as “help” for B cell and CD8+ T cell responses. Here, we have used the lymphocytic choriomeningitis virus (LCMV) system to assess the minimal constraints of a dominant antiviral CD4+ T cell response. We report that the core epitope derived from the LCMV glycoprotein (GP) is 11 amino acids in length and provides optimal recognition by epitope-specific CD4+ T cells. Surprisingly, this epitope is also recognized by LCMV-specific CD8+ T cells and thus constitutes a unique viral determinant with dual MHC class I- and II-restriction.
Effective control of microbial infections, in particular viruses, relies on the concerted activity of pathogen-specific T and B cells. While the relative contribution of individual immune cell subsets depends on the precise determinants of host-pathogen interaction (nature of the pathogen, route of pathogen entry, pathogen dosage and host immune status), CD8+ T cells and B cells constitute the main effector cell populations in most viral infections (Burton, 2002; Whitton and Oldstone, 2001; Zinkernagel, 2002). In contrast, antiviral CD4+ T cell responses are for the most part significantly smaller than CD8+ T cell responses and may wane over time (Seder and Ahmed, 2003; Sprent and Surh, 2002). Nevertheless, CD4+ T cells play a critical and multifaceted role at all stages of the adaptive immune response by serving as effectors, helpers and/or regulators/supressors.
Direct antiviral effector functions elaborated in vivo by specific CD4+ T cells include the production of cytokines and chemokines and possibly cytotoxic T cell activity (Appay, 2004; Jenkins et al., 2001; Seder and Paul, 1994). The traditional designation of CD4+ T cells as “helper T cells” is based on their importance for enhancement and modulation of specific antibody responses (McHeyzer-Williams et al., 2003), but work over the past few years has made increasingly clear that additional help is also provided to CD8+ T cell responses (reviewed, Bevan, 2004). Here, the relevant molecular interactions remain to be elucidated in detail but it appears that, depending on the in vivo model system under study, induction of primary CD8+ T cell responses, generation and maintenance of CD8+ T cell memory as well as secondary CD8+ T cell immunity are all improved by the activity of specific CD4+ T cells (Bevan, 2004; Homann, 2002). The complex function of antiviral CD4+ T cell immunity is further supported by studies on relevant human diseases such as HIV, EBV, CMV, HBV and HCV infections (Norris and Rosenberg, 2002). Altogether, it may be said that the relative importance of antigen-specific CD4+ T cell responses increases with the extent of viral disease (viral load and associated inflammatory alterations) (Homann, 2002). Finally, in their incarnation as “regulatory T cells”, CD4+ T cells are actively engaged in curtailing immune responses which may have beneficial (limitation of immunopathology) or adversarial (incapacitation of CD8+ T cell responses) effects (Belkaid and Rouse, 2005; Dittmer et al., 2004; Mittrucker and Kaufmann, 2004).
In the present study, we have employed the lymphocytic choriomeningitis virus (LCMV) system to further define the nature of antiviral CD4+ T cell responses (Homann et al., 2001; Kamperschroer and Quinn, 1999; Lenz et al., 2004; Oxenius et al., 1995; Varga and Welsh, 1998a, 1998b, 2000; Whitmire et al., 1998). Originally identified by Oxenius et al. (1995) the immunodominant population of LCMV-specific CD4+ T cells in C57Bl/6J mice targets an epitope derived from the viral glycoprotein (GP61–80) and restricted by I-Ab. Here, we have used several GP61–80-specific CD4+ T cell lines and clones, a battery of truncated peptides from the GP61–80 epitope and surface plasmon resonance to determine the minimal constraints and avidities of a dominant, virus-specific T cell receptor (TCR)–MHC interaction.
To generate CD4+ T cell lines and clones specific for the I-A b-restricted LCMV GP61–80 epitope, a C57Bl6/J mouse was infected with LCMV Armstrong clone 53b (1.5×105pfu i.p.) to induce LCMV-specific T cell responses and virus clearance. Thirteen days later, spleen cells were isolated, depleted of CD8+ T cells and cultured in the presence of GP61–80 peptide as detailed in Materials and methods. Forty CD4+ T cell lines and/or clones were established and tested for TCR usage and IFNγ production, a hallmark of LCMV-specific CD4+ T cells (Homann et al., 2001; Kamperschroer and Quinn, 1999; Varga and Welsh, 1998a; Whitmire et al., 1998). As about 1/3 of GP61–80-specific effector (data not shown) and memory (Homann et al., 2001) CD4+ T cells utilize Vβ8.1/8.2 TCRs, we selected 6 cell lines/clones for further analysis based on Vβ8.1/8.2 and Vα2 TCR expression as well as efficient IFNγ induction after resting and restimulation with GP61–80 peptide (Figs. 1 and and2B2B).
We subsequently evaluated the selected cell lines/clones for their ability to bind I-AbGP61–80 tetramers. Here, three distinct staining patterns emerged: (1) efficient tetramer binding (clones #3 and #31), (2) partial tetramer binding indicative of more than one TCR specificity present in the cell preparation (cell lines #21 and #32) and (3) absence of tetramer binding (clones/cell lines #8 and #35) (Fig. 2A). To evaluate if effective tetramer staining could be “rescued”, we used a more sensitive staining technique, tetramers deployed on fluorescent liposomes (Mallet-Designé et al., 2003). Although this approach reliably improved the “signal-to-noise ratio” for clones #3 and #31, it demonstrated only slight staining above background levels for clones/cell lines #8, #21 and #35 (Fig. 2A). Furthermore, tetramer binding did not demonstrate any appreciable correlation with the extent of TCR or CD4 co-receptor expression on the cell surface as determined by measuring the mean fluorescent intensity of Vβ8.1/8.2, Vα2 and CD4 staining (Fig. 2B and data not shown).
In spite of their differential ability to bind MHC class II tetramers, the prompt induction of IFNγ production in the selected cell lines/clones (#3, #8, #21, #31, #32, #35) made this functional assay suitable to determine the minimal constraints of I-Ab-restricted GP61–80 epitope presentation and TCR reactivity. Using a panel of truncated peptides and comparing the extent of IFNγ induction to that elicited by the native GP61–80 peptide, we identified a core motif of 11 amino acids (GP67–77) that on average induces 2/3 of the maximal IFNγ response in the 6 cell lines/clones (Fig. 3A). When tested on primary LCMV-specific CD4+ T cells (8 days after LCMV infection), the GP67–77-specific response was consistently of slightly greater magnitude as compared to GP61–80- or GP64–80-specific responses (Fig. 3B). These results indicate that flanking residues of the core epitope present in the GP61–80 and GP64–80 peptides may preclude recognition of the epitope by a subset of virus-specific CD4+ T cells and identify GP67–77 as the immunodominant LCMV-specific, I-Ab-restricted CD4+ T cell core epitope.
During our analyses of peptide-induced IFNγ production by primary murine effector CD4+ T cells (Fig. 3A), we noted unusually high “background staining” in CD4-negative cells obtained from cultures stimulated with GP67 but not GP64 or NP309 peptides (Fig. 4A). We therefore evaluated the possibility that the GP67–77 epitope may also be recognized in an MHC-I-restricted fashion by LCMV-specific CD8+ T cells. In B6 mice, the majority of specific CD8+ T cells recognize two co-dominant epitopes restricted by Db (GP33–41 and NP396–404) as well as two subdominant epitopes restricted by Db and Kb, respectively (GP276–286 and GP34–43) (Gairin et al., 1995; Hudrisier et al., 1997; Klavinskis et al., 1990; Oldstone et al., 1988; Schulz et al., 1989). More recently, several additional subdominant epitopes have been identified and include the epitopes GP92–101 and NP166–175 (Db-restricted) as well as GP118–125, and NP205–212 (Kb-restricted) (van der Most et al., 2003; van der Most et al., 1998). Indeed, we found that the GP67–77 epitope is recognized by both LCMV-specific CD4+ and CD8+ T cells (Fig. 4B) and that Db is the restriction element for specific CD8+ T cell responses (Fig. 4C) Thus, the GP67–77 epitope constitutes a rather unique viral determinant as it is targeted, by virtue of its dual restriction, by both CD4+ and CD8+ T cell populations. Furthermore, since stimulation with the GP64–80 peptide was not associated with IFNγ induction in CD8+ T cells unless provided at excessively high concentrations (50 μg/ml, data not shown), the presence of the 3 additional carboxy-terminal (GP64–66) and amino-terminal(GP78–80) residues apparently interferes with effective Db-restricted presentation.
Finally, the distribution of GP67–77-specific T cells among CD4+ and CD8+ T cell subsets was found to be about similar with 45–50% of GP67–77-reactive T cells expressing either CD4 or CD8 and a minority of T cells specific for this epitope (<10%) expressing both co-receptors (Fig. 4D).
To assess the distribution of TCR usage by GP67–77-restricted CD4+ and CD8+ T cells, we utilized a panel of TCR Vβ-specific antibodies in conjunction with intracellular IFNγ staining. GP67–77-specific CD4+ T cells were preferentially recruited from just two TCR Vβ families (8.1/8.2 and 14) with several other TCR Vβ families contributing less than 5% each to the overall GP67–77-restricted CD4+ T cell response. In contrast, GP67–77-specific CD8+ T cells appeared more diversified as indicated by five TCR Vβ families (2, 8.1/8.2, 8.3, 9, 10) contributing >5% to the overall specific response as well as larger error bars in the bar diagram reflective of differential private TCR Vβ usage among individual mice (Fig. 5A).
We and others have previously reported that the functional avidities of LCMV-specific CD4+ T cells (measured by determining the reciprocal peptide concentration required for IFNγ induction in half of an epitope-specific T cell population) are considerably lower than those of LCMV-specific CD8+ T cells (Homann et al., 2002; 2001; Whitmire et al., 2006). However, a careful interpretation of such data has to consider that induction of effector functions in this assay is dependent on both T cell-intrinsic properties (“functional avidities”) as well as differential binding affinities of synthetic peptides to MHC class I or II molecules (“structural aviditities”). Our observation of virus-specific CD4+ and CD8+ T cells responses specific for the same peptide epitope now allows for a more direct comparison of functional avidities between these cell lineages and indicates comparable functional avidities of GP 67–77-specific CD4+ and CD8+ T cell populations (Fig. 5B).
Based on the identity of naturally processed I-Ab peptides and the recently solved of I-Ab crystal structure (Dongre et al., 2001; Zhu et al., 2003), we proposed a common I-Ab-restricted peptide-binding motif. When applied to the LCMV-GP sequences, we found that no optimal alignment could be detected for the 11-mer GP67–77 peptide or longer variants of the same peptide (Fig. 6A). Attempts to optimize the two most likely sequence alignments (K-Y-F-V and Y-V-Q-S for P1-P4-P6-P9, respectively) by substituting non-optimal residues with canonical residues at the same position failed to produce better agonistic peptides (Fig. 6B and LT/MBAO, unpublished). The absence of an optimum register for the GP peptide suggests that like in the case of I-Ad and I-Ag7, peptides bound to I-Ab can adopt different alternate registers of binding (Corper et al., 2000; Scott et al., 1998). This capacity to use alternate registers has two practical consequences: (i) lower peptide affinity for the MHC and (ii) increase the repertoire of peptides from any given pathogen and therefore increase T cell repertoire against this pathogen. In the case of LCMV, the usage of this strategy to increase the efficiency of the anti-viral response appears very appropriate.
Having determined the minimal requirements for GP61–80 epitope binding and recognition, we proceeded to investigate in more detail the binding affinity of selected CD4+ T cell clones. To this extent, PCR products from clones #3 and #31 were inserted into the pCR2.1-TOPO cloning vector and sequenced. Both I-Ab tetramer-binding CD4+ T cell clones exhibited identical CDR3 amino acid sequences although we noted small differences at the level of nucleotide sequences (not shown). For direct measurement of TCR binding affinity, we generated recombinant TCRs from clone #3 and evaluated binding to I-AbGP61–80 complexes by surface plasmon resonance (SPR) as detailed in Materials and methods (Fig. 7). SPR measurements at equilibrium demonstrated a KD of 4.2 μM indicating a relatively high binding affinity as compared to other published CD4+ TCR affinities (Krummel et al., 2000).
In this study, we have defined the minimal constraints for the recognition of a dominant, MHC class II-restricted viral determinant. Naturally processed peptides selected by MHC class II molecules demonstrate considerable variability of length (usually 12–26 amino acids) but tend to cluster into families that share a 9 amino acid core segment that interacts with the four major binding pockets of the MHC II moleculev (p1, p4, p6 and p9). Flanking residues of natural peptides can modulate binding affinities and also serve as direct contact residues for TCR recognition (Suri et al., 2006). A recent study has discerned 128 naturally processed I-Ab-restricted peptides that are 11–21 amino acids in length and derived from both endocytic and cytosolic compartments. Although some studies have begun to employ similar techniques based on tandem mass-spectrometry for the identification of microbial determinants that are processed and presented by MHC II-bearing cells in vivo (Meiring et al., 2005; Ovsyannikova et al., 2003), the majority of studies performed to date have relied on measuring the binding affinity of synthetic microbial peptides to recombinant MHC molecules as well as the induction of effector functions in specific responder T cells. The latter approach has been guided, in particular for the mapping of MHC I-restricted epitopes, by the identification of common binding motifs and is usually less effective for prediction of MHC II-restricted determinants. Nevertheless, information about the naturally processed I-Ab-restricted peptide sequences together with the recently solved crystal structure of I-Ab has allowed for the development of a simple scoring matrix (Dongre et al., 2001; Zhu et al., 2003). However, retroactive application of this scoring matrix to the core and related LCMV-GP epitopes identified in this study failed to reveal optimal binding sequences. These observations emphasize that in spite of the comparatively high binding affinities of the dominant LCMV-GP epitope (Krummel et al., 2000), any attempts to predict optimal MHC class II-binding epitope sequences are impaired by the likely usage of alternating peptide binding registers.
The LCMV GP61–80 epitope was originally identified by Oxenius et al. (1995) as an immunodominant, I-Ab-restricted determinant derived from the LCMV WE strain. Although LCMV Armstrong differs from LCMV WE by a single N → K substitution in position GP63 of this determinant, subsequent work by other groups established that the GP61–80 epitope is also dominant for CD4+ T cell responses directed against LCMV Armstrong (Homann et al., 2001; Kamperschroer and Quinn, 1999; Varga and Welsh, 1998a; Varga and Welsh, 1998b; Whitmire et al., 1998). Indeed, we and others have estimated that more than half of the I-Ab-restricted, LCMV Armstrong-specific CD4+ T cell response is directed against this epitope (Homann et al., 2001; Kamperschroer and Quinn, 1999). More recently, Oxenius’ group has utilized a truncated GP peptide that lacks the first three C-terminal amino acids (GP64–80) to effectively stimulate TCR transgenic CD4+ T cells originally derived from a clone specific for the GP61–80 epitope (Wolint et al., 2004). This finding is in agreement with our observation that residues GP61–63 are completely dispensable for recognition by GP61–80 specific CD4+ T cell clones and also supported by unpublished findings that the GP-specific CD4+ T cell response is unaltered after infection with a variant virus lacking GP residues 59–63 (virus obtained, cloned and sequenced from persistently LCMV Armstrong-infected Db−/−×Kb−/− mice).
Interestingly, our analyses of primary LCMV-specific T cell responses directed against the GP67–77 epitope demonstrated the presence of CD8+ T cells that recognize this epitope in the context of Db. Although a previous publication, using an in vitro peptide binding assay, has described a truncated version of the GP67–77 epitope (GP70–77) as a peptide with high binding affinity to Kb in (van der Most et al., 1998), the use of Kb as a restriction element for the shorter GP70–77 epitope has, to the best of our knowledge, not been validated in ex vivo experiments. It thus remains possible that the GP67–77 and GP70–77 epitopes indeed demonstrate restriction by different MHC molecules (Db vs. Kb, respectively) or that GP70–77, similar to the GP33–41 epitope, is restricted by both Db and Kb. Several features are noteworthy in considering binding of GP67–77 to H-2Db, a restriction element that has been investigated in great detail. The signature motif has an Asn at P5 and an hydrophobic amino acid at P9 (Meth, Ileu or Leu) (Falk et al., 1991). In the present case, GP67–77 is missing the P5 anchor and will have to bind to Db in a non-classical way (Ostrov et al., 2002). It is likely that Valine77 anchors the C-terminus of the peptide, whereas the middle part bulges out, like most 11 mer peptides bound to MHC class I do (Apostolopoulos et al., 2002; Ostrov et al., 2002), and is exposed for T cell recognition. The recognition of one and the same viral determinant by both CD8+ and CD4+ T cells constitutes a unique feature within the LCMV system and should be taken into consideration for the interpretation of differential CD4+ and CD8+ T cell activities in this model system. For example, CD8+ T cells that recognize the I-Ab-restricted GP61–80 epitope have been described in CD4-deficient mice (Pearce et al., 2004; Tyznik et al., 2004). However, this CD8+ T cell response is restricted by I-Ab and its existence was attributed to abnormal T cell development in CD4+ T cell-deficient mice since further experiments indicated that neither wild-type nor acutely CD4-depleted mice mounted an MHC II-restricted CD8+ T cell response following infection with recombinant L. monocytogenes expressing a secreted form of chicken ovalbumin (Tyznik et al., 2004).
Finally, recognition of the same viral determinant by CD4+ and CD8+ T cells may offer an opportunity to optimize immunization strategies utilizing small, defined peptide sequences. As shown by van der Most and colleagues, protection of mice against chronic infection can be achieved even through induction of subdominant CD8+ T cell responses (van der Most et al., 1998) that constitute only a very small fraction of the antiviral CD8+ Tcell population in a natural infection (~2%). To improve the overall effectiveness of these CD8+ T cell responses, the authors utilized a lipidated LCMV GP92–101 peptide conjugated to an ovalbumin “helper” epitope (van der Most et al., 1998), a strategy that may be simplified by the use of the GP67–77 core peptide and the concurrent induction of genuine virus-specific CD8+ and CD4+ T cell responses.
C57BL/6 (H-2b, Thy1.2+) and congenic B6.PL (Thy1.1+) mice were purchased from the closed vivarium breeding colony at The Scripps Research Institute. Mice lacking Db, Kb or Db ×Kb expression were obtained from Taconic, Hudson NY. Origin, growth and titration of lymphocytic choriomeningitis virus (LCMV) Armstrong clone 53b have been described (Homann et al., 1998). Eight to ten week old C57BL/6 mice were infected with a single intraperitoneal dose of 105 plaque forming units LCMV Armstrong.
LCMV GP61–80-specific, I-Ab-restricted CD4+ T cell lines and clones were obtained from C57BL/6 mice 13 days after infection with 1.5×105 pfu LCMVArmstrong i.p. Spleens were harvested, single cell suspensions prepared, red blood cell lysed and CD8+ T cells depleted with antibody-coated magnetic beads as described (Berger et al., 2000). Cell counts were adjusted to 5×106 cells/ml in cloning medium (RPMI 1640 supplemented with 8% FCS, 1% penicillin/streptomycin, 1 mM L-glutamine and 50 units of human IL-2) supplemented with 1 μg/ml GP61–80 peptide, and plated in 24-well tissue culture plates. After 1 week, cultures were supplemented regularly with “feeder cells” (irradiated C57BL/6 spleen cells) as needed, intermittently rested by withdrawal of LCMV GP61–80 peptide and specific CD4+ T cell lines and clones established by limiting dilution techniques (Anderson et al., 1985).
For detection of intracellular IFNγ, rested cell lines and clones as well as primary T cells were restimulated for 5 h with peptides (1–5 μg/ml) prior to surface and intracellular stains as detailed elsewhere (Homann et al., 2001). Preparation and use of I-Ab tetramers and tetramer-liposomes have been described (Homann et al., 2001; Mallet-Designé et al., 2003). In this study, tetramers (20 μg/ml; 40 μl volume) were incubated with cells at room temperature for 45–75 min prior to addition of antibody reagents, further incubation (20 min), washes and immediate acquisition on a BD FACSCalibur flow cytometer. For quantitation of expression levels, we used the normalized geometric mean of fluorescence intensity (GMFI, calculated by dividing GMFI of the experimental stain by GMFI of corresponding control stain). Functional avidities were determined as described (Homann et al., 2006; Homann et al., 2002). For determination of TCR Vβ usage, we employed a kit comprising a panel of available TCR Vβ-specific antibodies (BDPharmingen).
Direct ex vivo CTL activity was analyzed 7 days after LCMV infection in a standard 51Cr release assay as described (Homann et al., 1998).
The cDNA for the α and β chain of TCR for CD4 T cell clones #3 and #31 was obtained by RT-PCR using Ready-To-Go RT-PCR beads (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instructions. In brief, RNA isolated from clones was used for cDNA synthesis and PCR amplification. PCR products were subcloned into pCR2.1-TOPO cloning vector (Invitrogen, Carlsbad, CA), sequenced and subcloned into the metallothionein promoter-based fly expression vector pRMHa3 (Fremont et al., 1992). The final constructs coded for the α1α2 and β1β2 domains, respectively, followed by a linker sequence (SSADL), a thrombin site (LVPRGS), a leucine zipper (acidic for the α chain, basic for the β chain) (Scott et al., 1996) and a hexahistidine tag. Vectors were transfected into SC2 cells and stable cell lines and clones were established. Soluble TCRs were purified from culture supernatants as described (Garcia et al., 1997; Matsumura et al., 1992; Scott et al., 1998).
A BIACORE 2000 instrument was used to determine interactions between purified pMHC (peptide:MHC) complexes and recombinant TCR molecules as described (Mallet-Designé et al., 2003). In brief, pMHC complexes or TCRs were immobilized by amine coupling chemistry on a CM5 research grade sensor chip. Successive injections of pMHC complexes or TCRs were performed in filtered and degassed PBS buffer at a flow rate of 20 μl/min at concentrations of 20, 10, 5, 2.5, and 1.25 μM. In all experiments, irrelevant TCR molecules or irrelevant pMHC (I-Ab/ova) were used as negative control and subtracted from experimental sensorgrams. On- and off- rates were obtained by non-linear curve fitting of subtracted curves using the 1:1 Langmuir binding model using the BIAevaluation program (version 3.0.2).
Data handling, analysis and graphic representation were performed using Prism 4.0a (GraphPad Software, San Diego, California); p values were calculated by Student’s t-test.
This is publication number 17545-NP from the Molecular and Integrative Neurosciences Department and Department of Infectology, The Scripps Research Institute, La Jolla, CA. This work was supported in part by NIH 1R21 AG026518-01 (D.H), AI009484 (M.B.A.O.), NIH training grant 5T32 AI007244-23 (D.B.) and NIH DK055037 (L.T.).