Antigenicity of peptides comprising the immunosuppressive domain of the retroviral envelope glycoprotein

To achieve persistent infection of the host, viruses often subvert or suppress host immunity through mechanisms that are not entirely understood. The envelope glycoprotein of several retroviruses is thought to possess potent immunosuppressive activity, mapped to a 17-amino acid residue conserved domain. Synthetic peptides corresponding to this immunosuppressive domain can inhibit lymphocyte activation, whereas mutation of key domain residues can increase the lymphocyte response to linked antigenic epitopes. Using three T cell receptors (TCRs) of defined specificity, we examine the effect of the immunosuppressive domain on the T cell response to their respective antigenic peptides. We find that fusion of a T cell epitope to the immunosuppressive domain can greatly modulate its potency. However, the effects heavily depend on the particular combination of TCR and peptide-major histocompatibility complex class II (pMHC II), and are mimicked by sequence-scrambled peptides of similar length, suggesting they operate at the level of pMHC formation or TCR-pMHC interaction. These results offer an alternative explanation for the immunogenicity of T cell epitopes comprising the putative immunosuppressive domain, which is more consistent with an effect on peptide antigenicity than true immunosuppressive activity.


Introduction
Several studies over the last three decades have provided evidence to suggest that the envelope glycoprotein of certain retroviruses is immunosuppressive in vitro and in vivo (Cianciolo et al., 1985;de Parseval et al., 2001;Denner, 2014;Dupressoir et al., 2012;Haraguchi et al., 1997;Kudo-Saito et al., 2014;Mangeney et al., 2007;Schlecht-Louf et al., 2010). Although the precise molecular mechanism remains unresolved, immunosuppressive activity has been pinpointed at a short region of the transmembrane polypeptide that, together with the surface unit (SU), constitutes one part of the trimeric envelope glycoprotein (Cianciolo et al., 1985;Dupressoir et al., 2012;Schlecht-Louf et al., 2010). For example, synthetic peptides corresponding to a region of 17-amino acid residues from the murine leukemia virus (MLV) envelope glycoprotein inhibit immune function in a variety of assays. These assays measure distinct aspects of immune responsiveness. Some measure non-specific lymphocyte or myeloid cell activation in response to mitogens or other generic stimuli (Denner, 2014;Denner et al., 1996;Haraguchi et al., 1993;Haraguchi et al., 1995;Mitani et al., 1987;Tolosa et al., 2012). Others measure antigen-specific T and B cell responses to immunization with synthetic peptides, recombinant envelope domains, and to infection with MLVs (Morozov et al., 2012;Schlecht-Louf et al., 2014;Schlecht-Louf et al., 2010). Use of different assays, many of which are multilayered, has hindered direct comparison of results from different studies, or definition of the specific step in immune function that may be suppressed. Here, we examine the properties of T cell epitopes linked to the immunosuppressive domain and provide an alternative interpretation for the effects.

Mice
All animal experiments were approved by the Ethical Committee of the Francis Crick Institute, and conducted according to local guidelines and UK Home Office regulations under the Animals Scientific Procedures Act 1986. Inbred C57BL/6J and BALB/cJ mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and subsequently maintained at the Francis Crick Institute's animal facilities, under specific pathogen-free conditions. Male or female mice, between 8 and 12 weeks of age, were used for the isolation of macrophages. T cell receptor (TCR)-transgenic OT-II (Barnden et al., 1998) andDO11.10 (Murphy et al., 1990) mice were kept on the C57BL/6J and BALB/cJ genetic backgrounds, respectively, and were additionally crossed to Rag1 -/mice to prevent rearrangement of endogenous TCR loci.

Macrophages
Macrophages were isolated from the peritoneal cavity of naïve euthanized C57BL/6J or BALB/cJ mice (2-6 mice per experiment). Peritoneal cells (containing ~50% macrophages) were plated in flat-bottomed 96 well-plates at 4×10 5 per well the day before their use, in Iscove's Modified Dulbecco's Medium (IMDM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA USA), 2mM L-glutamine, 100U penicillin and 0.1mg/ml streptomycin, and cultured at 37°C in a 5% CO 2 atmosphere. Following overnight culture, macrophages were enriched by the removal of non-adherent cells, and were used for the assays.

T cell hybridomas
The env 124-138 -reactive H5 and H18 CD4 + T cell hybridomas have been previously described (Young et al., 2012). The ova 323-339reactive OT-II and DO11.10 hybridomas were generated by fusion of ova 323-339 -stimulated primary splenic CD4 + T cells from single TCR-transgenic OT-II and DO11.10 mice, respectively, with TCRαβ-negative BW5147 thymoma cells, using established techniques (Young et al., 2012). In at least two experiments, primary CD4 + T cells from TCR-transgenic mice were also used with comparable results.
Peptides encompassing the immunosuppressive domain Peptides were synthesized by Insight Biotechnology Ltd., Wembley, UK, at >98% purity. Scrambled sequences represent permutation of the original peptide sequences and were constructed using the peptide manufacturer's algorithms. All peptides were sufficiently hydrophobic and were dissolved in phosphate-buffered saline prior to use (Table 1).

Amendments from Version 1
The following changes were made in response to the Reviewers' comments: 1. The following sentence in the Abstract: "However, the effects heavily depend on the particular combination of TCR and peptide-major histocompatibility complex class II (pMHC II), and are mimicked by sequence-scrambled peptides of similar length, suggesting they operate at the level of TCR-pMHC interaction." now reads: However, the effects heavily depend on the particular combination of TCR and peptide-major histocompatibility complex class II (pMHC II), and are mimicked by sequence-scrambled peptides of similar length, suggesting they operate at the level of pMHC formation or TCR-pMHC interaction.
2. The following sentence was added to the Methods/Peptides encompassing the immunosuppressive domain: "Scrambled sequences represent permutation of the original peptide sequences and were constructed using the peptide manufacturer's algorithms. All peptides were sufficiently hydrophobic…" 3. Shaded parts of Table 1 were replaced with red colour text for enhanced contrast. This is also indicated in the Table 1 legend.
4. The following sentence in the last paragraph of Results and Discussion: "In contrast, neither peptide fusion affected the response of the DO11.10 clone ( Figure 2B), indicating that effects of the immunosuppressive peptides can be observed in some TCR-pMHC pairs, but not others." now reads: "In contrast, neither peptide fusion affected the response of the DO11.10 clone ( Figure 2B), indicating that effects of the immunosuppressive peptides can be observed in some pMHC combinations or TCR-pMHC pairs, but not others."

REVISED
T cell stimulation T cell hybridomas were stimulated by the indicated peptides presented by primary macrophages. T cell clones restricted by H2-A b (H5, H18 and OT-II) were stimulated by C57BL/6J-derived macrophages, whereas the H2-A d -restricted clone (DO11.10) was stimulated by BALB/cJ-derived macrophages. Similar results were also obtained when primary splenic B cells or bonemarrow-derived dendritic cells were used for presentation.

Statistical analysis
Statistical comparisons were made using SigmaPlot 13.0 (Systat Software Inc., Germany). Parametric comparisons of normallydistributed values that satisfied the variance criteria were made by unpaired Student's t-tests. Data that did not pass the variance test were compared with non-parametric two-tailed Mann-Whitney Rank Sum tests. P<0.05 were considered significant.

Results and discussion
We used synthetic peptides corresponding to the conserved env 548-567 region of the Friend MLV envelope precursor gPr80 (LQNRRGLDLLFLKEGGLCAA), which contains the originally described 17-amino acid residue immunosuppressive peptide (Cianciolo et al., 1985). As controls, we introduced two amino acid substitutions (E561R and A567F), previously shown to abrogate the immunosuppressive activity of this region (Schlecht-Louf et al., 2010), and also used scrambled sequences for both peptides ( Figure 1). These peptides were tested for their effects on the antigenicity of the H2-A b -restricted env 124-138 CD4 + T cell epitope in the SU of F-MLV (PLTSLTPRCNTAWNR). As immunosuppressive peptides have generally been found active only as part of larger polypeptides (Denner, 2014), we used peptide fusion of the T cell epitope and the immunosuppressive peptide into a single molecule (env 124-138 -env 548-567 ). Fusion with the immunosuppressive peptide env 548-567 had no measureable effect on the ability of the env 124-138 epitope to stimulate two env 124-138 -specific CD4 + T cell  Figure 1A and F) that differ in functional avidity (Young et al., 2012). CD4 + T cell responses to env 124-138 were similarly unaffected by the addition in the cultures of the immunosuppressive peptide env 548-567, either as a separate entity or fused with the unrelated H2-A b -restricted ova 323-339 epitope from chicken egg ovalbumin (ISQAVHAAHAEINEAGR) (Supplementary Figure 1). In contrast, fusion with either the doubly point-mutated ( Figure 1B and G; p=0.003 and p<0.001 for clones H5 and H18, respectively) or sequence-scrambled immunosuppressive peptide ( Figure 1C and H; p=0.005 and p=0.021 for clones H5 and H18, respectively) significantly increased the antigenicity of the env 124-138 epitope for both clones by ~10-fold. Increased antigenicity of the env 124-138 epitope as a result of mutations at the two residues or sequence scrambling of the fused immunosuppressive peptide could be interpreted as reversal of immunosuppression due to these modifications. However, comparison with the env 124-138 epitope on its own revealed that fusion with the index immunosuppressive peptide was in fact neutral ( Figure 1A and F), whereas fusion with the modified immunosuppressive peptides improved antigenicity.
Peptide length is an important contributor to the antigenicity of MHC II-restricted epitopes. Indeed, N-terminal extensions of the env 124-138 epitope have been shown to increase antigenicity (Young et al., 2012), in line with findings in other systems (Carson et al., 1997;Holland et al., 2013). The open structure of the MHC II groove allows presentation of peptides that extend at either end of the core epitope, and these epitope-flanking residues can contribute to T cell stimulation (Holland et al., 2013). Importantly, the involvement of epitope-flanking residues critically depends on the particular pair of TCR and peptide-MHC II (pMHC) complex, as contact between particular epitope-flanking residues and residues in the TCR is necessary (Holland et al., 2013). To examine whether the observed effect of the index or modified immunosuppressive peptides was TCR-and peptide sequence-dependent, we used additional fusion peptides. Notably, a peptide that carried the E561R/A567F substitutions and was additionally sequencedscrambled enhanced antigenicity of the env 124-138 epitope for the H5 clone (p=0.004), but not the H18 clone ( Figure 1D and I).
Moreover, extending the length of the env 124-138 epitope with the sequence that naturally occurs in the F-MLV SU (env 124-158 ) enhanced activation of the H18 clone (p=0.007), but not of the H5 clone ( Figure 1E and J). These two clones share the same TCRβ chain, but use different TCRα chains, which are responsible for the difference in functional avidity (Young et al., 2012). The disparate behaviour of the two clones in response to the last two fusion peptides demonstrate that changes in antigenicity depend on the particular TCR. This finding argues against general immunosuppression caused by the env 548-567 peptide.
Also arguing against immunosuppressive ability, env 124-138 fusion peptides containing the index immunosuppressive peptide or the E561R/A567F mutant induced comparable transcriptional changes to the APCs used in these assays ( Figure 2A). Incubation of primary macrophages with either of the two env 124-138 fusion peptides altered their transcriptional signature in comparison with the absence of peptide, with Tnfsf14 (also known as HVEM-L or LIGHT) most strongly induced (between 3-and 4-fold) ( Figure 2A). However, the transcriptional signatures induced by the two peptides were effectively identical ( Figure 2A).
We next examined if our findings with env 124-138 -specific CD4 + T cell clones extended to other combinations of TCR and pMHC.
To this end, we used the OT-II and DO11.10 clones, which react with the same ova 323-339 epitope presented by H2-A b and H2-A d , respectively. Fusion of the ova 323-339 epitope with the env 548-567 E561R/A567F mutant, but not the index immunosuppressive peptide enhanced the response of the OT-II clone ( Figure 2B) (p=0.04). In contrast, neither peptide fusion affected the response of the DO11.10 clone ( Figure 2B), indicating that effects of the immunosuppressive peptides can be observed in some pMHC combinations or TCR-pMHC pairs, but not others. Thus, this pattern of changes in T cell activation, depending on the particular TCR-pMHC pair, is more consistent with an effect mediated by epitope-flanking residues than genuine immunosuppression. The potential of the immunosuppressive peptide, and modifications thereof, to alter TCR or BCR recognition of linked epitopes should, therefore, be considered when interpreting the antigenicity or immunogenicity of the retroviral envelope glycoprotein.
Raw values for T cell responses shown in Figure 1, Figure 2B and Supplementary Figure 1  1.

2.
3. This work gives insights into the immunosuppressive role of the envelope glycoprotein of certain retrovirus. They present the T cell response of 3 TCRs stimulated with different peptides encompassing both the T cell epitope and the immunosuppressive domain. The research note is of interest, clear and well written, and the final conclusion seems appropriate.

Some section could be developed or clarify:
Abstract: "The effects heavily depend on the particular combination of TCR and peotide-major histocompatibility complex class II, and are mimicked by sequence-scrambled peptides of similar length, suggesting they operate at the level of TCR-pMHC interaction". The addition/substitution within the peptides could also influence the peptide stability within the MHC-II, or half-life of the pMHC-II complex, and therefore change the T cell stimulation, this should probably be discuss, or acknowledge that the TCR-pMHC-II interactions might not explain everything.

Methods.
A brief explanation about "scrambled" peptides would be helpful, as would a sentence explaining why peptides were reconstituted in PBS rather then DMSO.
Conclusion. I would like a bit more explanation for the unexpected results. As well as maybe considering taking into account the comment from the abstract to re-word the strong conclusion "In contrast, neither peptide fusion affected the response of the DO11.10 clone, indicating that effects of the immunosuppressive peptides can be observed in some TCR-pMHC pairs, but not others".

Minor comments:
Title: I would suggest changing "comprising" by "within". Table 1: the shade section is not visible, either the colour needs to be darker or maybe bold the section.
The peptides env548-567 and env548-567-E561R-A567F are in Table 1 but don't seem to be used