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Keywords:

  • Antigen presentation;
  • Mass spectrometry;
  • MHC;
  • Self peptidome;
  • T helper (Th) cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

It is generally assumed that the MHC class I antigen (Ag)-processing (CAP) machinery — which supplies peptides for presentation by class I molecules — plays no role in class II–restricted presentation of cytoplasmic Ags. In striking contrast to this assumption, we previously reported that proteasome inhibition, TAP deficiency or ERAAP deficiency led to dramatically altered T helper (Th)-cell responses to allograft (HY) and microbial (Listeria monocytogenes) Ags. Herein, we tested whether altered Ag processing and presentation, altered CD4+ T-cell repertoire, or both underlay the above finding. We found that TAP deficiency and ERAAP deficiency dramatically altered the quality of class II-associated self peptides suggesting that the CAP machinery impacts class II–restricted Ag processing and presentation. Consistent with altered self peptidomes, the CD4+ T-cell receptor repertoire of mice deficient in the CAP machinery substantially differed from that of WT animals resulting in altered CD4+ T-cell Ag recognition patterns. These data suggest that TAP and ERAAP sculpt the class II–restricted peptidome, impacting the CD4+ T-cell repertoire, and ultimately altering Th-cell responses. Together with our previous findings, these data suggest multiple CAP machinery components sequester or degrade MHC class II–restricted epitopes that would otherwise be capable of eliciting functional Th-cell responses.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

CD4+ T helper (Th) cells regulate multiple cellular and humoral responses to pathogenic microbes and parasites to protect against infectious diseases. These cells sense infections by recognizing short microbial peptides presented by MHC class II molecules on the cell surface of APCs. Hence, alterations or deficiencies in factors that control class II–restricted Ag processing and presentation can alter the display of self and microbial peptides by APCs. Alterations in the presented self peptide repertoire (peptidome) can change the CD4+ T-cell repertoire activated in response to an infection, which in turn can affect the host's susceptibility to infectious disease.

Th cells recognize endogenous cytosolic as well as exogenous Ags. The mechanisms controlling exogenous class II–restricted Ag presentation are quite well established [1, 2]. Nonetheless, endogenous cytosolic Ag presentation by class II molecules is less well understood. Endogenous cytosolic Ags existing within professional APCs are presented by class II molecules when they are delivered to the endo/lysosomes. These Ags are delivered to these compartments by various autophagic mechanisms — macro-autophagy [3-7] or chaperone-mediated autophagy [8-10] — and processed therein for presentation to CD4+ T cells [11-17]. Alternatively, cytosolic Ags expressed by class II-negative cells — such as allograft, tumour and infected cells — are acquired by phagocytosis. Professional class II-positive APCs (e.g. DCs and macrophages (MΦs)) phagocytose dying cells and process Ags into short peptides within the phago-lysosomes, assemble with class II molecules and are displayed at the cell surface [18-20]. This process, termed indirect presentation, was originally described to explain solid organ allograft rejection.

Newer data suggest that this dogmatic separation of class I and class II Ag processing and presentation is not so absolute. Interdependence between these two processing pathways has been observed either within the presenting APCs or in damaged neighbouring (donor) cells. As we reported previously, class II–restricted cytosolic Ags are exposed to modification by components of the MHC class I Ag processing (CAP) machinery in both the presenting and donor cells [21]. This modification is evident in animal models deficient in the CAP components TAP and ERAAP where an altered basal class I–restricted peptide repertoire is displayed [22-26]. However, the effect of their absence on the class II–restricted peptide repertoire has not been fully explored. Certain class II–restricted Ags, including several self peptides, that are dependent upon the actions of the CAP machinery have been identified [12-15, 21, 27-31]. Nonetheless, other investigators have not seen a dependence upon this processing machinery for class II–restricted Ag presentation [17, 32-34]. Despite the identification of a few peptides that depend on CAP machinery for presentation, the global impact the CAP machinery has on the self and nonself peptidome remains unknown. Moreover, although previous studies have observed differences in Ag presentation, no notable alterations in the frequencies of TCR Vβ usage in TAP-deficient animals for either CD4+ or CD8+ T cells were observed [35]. It is therefore unclear whether the class II–restricted CD4+ T-cell repertoire is impacted by the CAP machinery.

We recently showed that CD4+ T cell recognition of indirectly presented cytosolic, class II–restricted self (HY minor histocompatibility Ag) and non-self (Listeria monocytogenes (Lm)) peptides was enhanced in the absence of the CAP components TAP and ERAAP [21]. Curiously however, the donated HY alloantigen entered the cytosol of acceptor APCs and required LMP2-dependent immunoproteasomes for presentation [21]. Moreover, the effects of CAP components on HY alloantigen presentation were neither due to competition between class I and class II Ags nor due to competition between CD4+ and CD8+ T cells. They were also not caused by enhanced MHC class II, B7.1, B7.2, calreticulin or HSP90 expression nor enhanced macro-autophagy, or enhanced ER-associated degradation. Hence, we concluded from that study that the CAP machinery must regulate the quantity and/or quality of peptides available for presentation by class II molecules. Hence, we hypothesized that by regulating the class II–restricted peptidome, CAP components could alter the robustness of the Th-cell response to class II–restricted Ags [21].

We now report direct evidence that TAP and ERAAP influence the available class II-associated peptide pool. In their absence, a nearly unique self peptidome is displayed by H2Ab molecules. These findings emerged from amino acid sequence analyses of the class II–associated self peptidomes isolated from WT, TAP−/−, or ERAAP−/− splenocytes. As previously described [35], we also found insubstantial alterations in the TCR Vβ usage. Nonetheless, we observed significant changes within the Ag-binding CDR3 of TCR β-chains (CDR3β) expressed by CD4+ T cells. Consistent with altered Ag processing and presentation and an altered TCR diversity, we found that functional Th-cell responses to H2Ab-restricted vaccinia viral (VACV) epitopes were also altered. TAP−/− mice recognized novel epitopes not recognized by WT mice and, conversely, had lost recognition of some epitopes recognized by WT mice. Our in-depth analysis of the self peptidome, mature TCR repertoire and Th-cell responses suggests that the CAP machinery meaningfully sculpts class II–restricted Ag presentation likely through sequestration or degradation of potential epitopes.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

TAP and ERAAP sculpt the class II-restricted self peptidome

Previous reports have documented an altered endogenous class I–associated self class II-restricted peptidome in the absence of the CAP components TAP or ERAAP [22-26]. Recently, increasing interdependence of the class I– and class II–restricted Ag-processing pathways and the identification of several class II–restricted peptides that require the activity of components of the CAP machinery have been reported [12-15, 27-31]. This led us to query whether the basal class II–associated self peptidome might also have a similar dependence on TAP and/or ERAAP. To this end, class II–associated peptides were eluted from affinity purified H2Ab molecules expressed by WT, B6.129-TAP−/−, B6.129-ERAAP−/−, and B6.129-H2Ab−/− splenocytes. Importantly, deficiency in either TAP or ERAAP did not alter the frequency of APCs within the spleen. Nor was the cell surface phenotype (e.g. class II and co-receptor CD80 and CD86 expression) different than WT (data not shown; [24, 25]). The recovered peptides were fractionated by reversed-phase chromatography (RPC) and their sequence deduced by LC-MS/MS tandem mass spectrometry (Fig. 1 and Supporting Information Fig. 1).

image

Figure 1. LC-MS/MS spectra of H2Ab-associated peptides commonly displayed by WT, TAP−/− and ERAAP−/− splenocytes. Peptides eluted from immunoaffinity purified H2Ab molecules expressed by splenocytes from 68 to 70 WT, TAP−/− and ERAAP−/− mice were separated by RPC and their amino acid sequence determined by LC-MS/MS. Representative mass spectra are presented. For each spectrum, the b- and y-ions are indicated along with the Sequest cross-correlation score (Cn) showing the degree of concordance between the observed and expected fragment ions. Within the spectrum, b1, b2, y1 and y2 refer to fragment ions that have mass/charge (m/z) +1 or +2. Below each spectrum are the +1 ion m/z values for each peptide (bold underlined, observed ion masses). Note: the +2 ion mass/charge values are provided in Supporting Information Fig. 1.

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The mass/charge (m/z) pattern generated by MS/MS was compared against a dataset consisting of the m/z patterns of theoretical and known peptide sequences. The degree of concordance between these two patterns was assigned a cross correlation score Xcorr (Cn). Higher Cn values are assigned to those peptides whose m/z pattern showed greater concordance between the observed and expected m/z patterns [36]. Only peptides with a Cn > 1.5 were considered to be possible peptide sequences. However, the larger the Cn value the more confidence is placed in the peptide sequence identification. In addition, greater differences in the Cn values between the top two most likely peptide sequence identifications (ΔCn) provides greater confidence in the identification. Therefore, peptides with a highly confident identification were considered to have a Cn score >3.0 and ΔCn >0.2. Overall, this dataset had an average Cn = 3.536 and ΔCn = 0.324. In addition, 44% of the peptides had only a single possible sequence identification for which no ΔCn can be calculated.

To ascertain the specificity of the bound peptides, materials eluted from control H2Ab-deficient cells were isolated and analysed by the same methods. We found that only ∼7% of the peptide sequences (Cn > 1.5) identified in WT, TAP−/− and ERAAP−/− samples were also present in the control H2Ab−/− eluates (data not shown). These were largely derived from three sources; (i) Ig – likely representing the Ab used for immunoaffinity purification or splenic Ig that bound to protein A Sepharose used to prepare the immunoaffinity column; (ii) complement – perhaps because they bind Ig; and (iii) fibronectin, fibrinogen and other secreted proteins – likely representing unspecific contaminants of the purification. Few peptides were derived from cytosolic/intracellular proteins. Hence, peptide sequences that matched those isolated from H2Ab−/− splenocytes were considered an artefact of the purification. Such peptide sequences with Cn > 1.5 when present in WT, TAP−/− and ERAAP−/− samples were removed from all downstream analyses.

Analysis of the peptides identified with high confidence (Cn > 3.0 and ΔCn > 0.2) that were eluted from WT, TAP−/− and ERAAP−/− splenocytes surprisingly revealed little overlap between the peptides displayed by WT cells and either TAP−/− or ERAAP−/− cells (Fig. 2 and Supporting Information Table 1). Only 22.5% of the H2Ab-restricted self peptide sequences displayed by WT cells were also presented by TAP−/− or ERAAP−/− cells (Fig. 2A). In a different project, replicate MS samples that consisted of peptides with similar confidence levels eluted from MHC molecules, demonstrated a 63% concordance (SBC, CTS, AJL and SJ, unpublished data). Since class II–associated peptides expressed by WT- and CAP-deficient cells have only 22.5% overlap, the differences in the WT and CAP peptidomes are likely real and not caused by irreproducibility in the experiment. Conversely, 18.4% of self peptide sequences displayed by TAP−/− cells were presented by WT cells, while 33% of self peptide sequences displayed by ERAAP−/− cells were presented by WT cells. This lack of identity was not due to bias in selecting peptides with Cn > 3.0 as datasets which included peptides identified with either moderate (Cn > 2.5 and ΔCn > 0.2; Fig. 2B) or low (Cn > 1.5 and ΔCn > 0.2; Fig. 2C) confidence also demonstrated little overlap in peptide sequence. However, to maintain focus on relevant naturally processed self peptides using this unbiased approach, all downstream analyses were performed on peptides with Cn > 3.0 and ΔCn > 0.2. Importantly, this peptide set was found to have a false discovery rate (FDR; described in the Materials and Methods) of 0, i.e. no peptides were identified by random similarity.

image

Figure 2. TAP and ERAAP deficiency alters the basal H2Ab-restricted self peptidome. The prevalence of H2Ab-restricted self peptide sequences was compared between WT, TAP−/− and ERAAP−/− strains. Venn diagrams indicate the number of unique and common peptide sequences identified amongst the peptidomes displayed by the indicated strains. Cn > 3.0 (A), Cn > 2.5 (B) or Cn > 1.5 (C) indicates decreasing spectral confidence (see Materials and Methods). ΔCn ≥ 0.2 distinguishes between the top two peptide sequences predicted from the spectrum; this criterion allows identification of the best peptide sequence that matches the observed spectrum. (D) Using the LOCATE database, the number of peptides derived from cytosolic and secreted proteins was compared amongst the peptidomes consisting of peptides with Cn > 3.0.

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Notably, the average length of H2Ab-associated peptides increased from 14–16 amino acid residues in WT cells to 18–20 amino acids in TAP−/− and ERAAP−/− cells (Supporting Information Table 1 and Fig. 2). This was consistent with peptide length changes previously observed for class I–associated peptides displayed by ERAAP−/− cells [22]. In addition, we observed numerous groups of nested peptides arising from the same protein (Supporting Information Table 2) as would be expected from class II–associated peptides expressed by WT cells [37, 38]. These nested peptides contained both N- and C-terminal extensions, consistent with previous reports on class II–associated peptides expressed by WT cells [37, 38]. Moreover, only two peptides identified in this study have been previously reported (Supporting Information Table 1) [37, 38]. The lack of overlap in peptides identified in previous studies and this one may have resulted from the analysis of different cell populations. We used unmanipulated APCs isolated directly ex vivo in this study compared with B-cell lymphomas, LPS-induced B-cell blasts, IFN-γ–induced BMC2.3 cell line and Flt3-induced cells used in the earlier reports [37, 38]. In addition, although we found thousands of peptides by LC-MS/MS, we have focused solely on those with the highest Cn values. It is conceivable that the few hundred peptides previously reported were excluded based on the criteria used for sequence determination and validation and may be present in the larger dataset. Hence the differences observed in the different reports do not detract from the novel peptides reported herein as similar results were observed with the larger datasets as well (Fig. 2B and C).

H2Ab-associated peptides were derived from both secreted/extracellular and cytosolic/intracellular proteins as defined in the LOCATE database [39]. However, the majority (∼70%) were processed from cytosolic/intracellular proteins (Fig. 2D), including proteins associated with endosomes. Comparing individual genotypes, the presentation of cytoplasmic/intracellular protein-derived peptides was increased in TAP−/− and ERAAP−/− splenocytes. Consistent with previous reports [40], ∼63% of the H2Ab-associated self peptidome presented by WT cells were generated from cytosolic/intracellular proteins. In contrast, 87.5% and 80.2% of the H2Ab-associated peptides displayed by TAP−/− and ERAAP−/− splenocytes, respectively, were derived from cytosolic/intracellular proteins (Fig. 2D). These data demonstrate that numerous cytoplasmic/intracellular proteins, including endosomal proteins, are processed and presented by H2Ab in TAP−/− and ERAAP−/− mice. From these analyses, we conclude that CAP components can impact the H2Ab-associated self peptidome.

TAP and ERAAP deficiency alter the CD4+ TCR repertoire

As the self peptidome instructs the developing TCR repertoire, we compared TCR Vβ usage by CD4+ CD62LhiCD44lo naive T (Tn) cells between WT mice and for TAP−/− or ERAAP−/− animals using a panel of Vβ-specific antibodies. As previously reported [35], the frequencies of TCR Vβ usage between WT-, TAP−/−- or ERAAP−/−-derived CD4+ Tn cells were quite similar, although not identical (Fig. 3A). Likewise, TCR Vβ usage within Lm-reactive CD4+ CD62LloCD44hi effector T (Teff) cells of WT, TAP−/− or ERAAP−/− mice were similar as well (Fig. 3B).

image

Figure 3. Differential self peptidome display has little impact on the TCR Vβ usage. WT, TAP−/− and ERAAP−/− mice were inoculated with Lm or not and the TCR Vβ usage of the indicated CD4+ T-cell population was determined by flow cytometry after staining with a panel of Vβ-specific antibodies. The cumulative bar graphs indicate the proportion of each Vβ segment present within the (A) CD4+ Tn (CD44loCD62Lhi) or (B) Lm-immune Teff (CD44hiCD62Llo) population in replicate experiments.

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Since Ag recognition is mediated by the highly variable CDR3, we specifically examined this region of the TCR β-chains. CDR3β sequence diversity can be estimated by analysing the number of amino acids spanning the V-D-J recombination site by spectratyping the nucleotides that encode them [41, 42]. Although different sequences may have equivalent lengths, thereby underestimating the true diversity, differences in the number of amino acids, nonetheless, provide a high throughput estimate of Ag receptor diversity. The diversity of the TCR of flow sorted CD4+ Tn cells were analysed by spectratyping 52 - pairings. This analysis revealed extensive alterations in some but not all CDR3β length profiles in the naive TCR β-chain repertoire expressed by WT, TAP−/− or ERAAP−/− mice (Fig. 4 and Supporting Information Fig. 3A). Similar analysis of flow sorted Lm-responsive CD4+ Teff cells revealed extensive differences in the CDR3β length profiles between WT and TAP- or ERAAP-deficient CD4+ Teff cells (Fig. 5 and Supporting Information Fig. 3B). These data suggest that, despite similarities in Vβ usage, which was serologically determined, CD4+ T cells utilize different CDR3β sequences in the absence of the CAP machinery. Since the CDR3β region of the TCR is predominantly involved in Ag recognition, sequence differences in this region could potentially lead to alterations in the CD4+ T-cell responses to microbial challenge.

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Figure 4. The TCR repertoire of naïve CD4+ T cells in both TAP−/− and ERAAP−/− mice is substantially different from that of WT mice. Total RNA was isolated from purified CD4+ Tn cells from naive, uninfected mice and amplified by RT-PCR using primers specific for the indicated [RIGHTWARDS ARROW] rearrangements. CDR3β length diversity was detected by capillary-gel electrophoresis and quantified by calculating the area under each peak. Representative spectrograms of four TCR [RIGHTWARDS ARROW] CDR3 length distributions are shown from WT, TAP−/− and ERAAP−/− derived CD4+ Tn cells. Bar graphs depict the fraction of specific CDR3β lengths present in the total population. Data presented are from one experiment, which is a representative of two performed (n = 3–5 mice per strain per experiment). Replicates sometimes displayed minor alterations in the absolute frequencies of CDR3 lengths but no alteration in their presence or relative frequencies were observed within a sample.

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image

Figure 5. The Listeria-immune TCR repertoire is altered in TAP−/− and ERAAP−/− CD4+ Teff cells when compared with WT Teff cells. WT, TAP−/− and ERAAP−/− mice were inoculated with Lm. After 7 days, CD4+ Teff cells were flow sorted to ≥95% purity and total RNA was isolated, processed and analysed as described in Figure 4. Representative spectrograms of four TCR [RIGHTWARDS ARROW] CDR3 length distributions are shown for WT, ERAAP−/− and TAP−/− CD4+ Teff cells. Bar graphs depict the fraction of specific CDR3β lengths present in the total population. Data presented are from one experiment, which is a representative of two performed (n = 3–5 mice per strain per experiment). Replicates sometimes displayed minor alterations in the absolute frequencies of CDR3 lengths but no alteration in their presence or relative frequencies were observed within a sample.

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TAP-deficiency alters class II–restricted microbial Ag recognition

Previously, we reported that the magnitude of the CD4+ T cell response to minor histocompatibility Ag HY and Lm-derived LLO and p60 peptides were increased in animals deficient in TAP or ERAAP [21]. Here, we have shown that TAP and ERAAP impact the quality of the H2Ab-restricted self peptidome and alter the TCR repertoire. Therefore, we queried whether the CAP machinery could destroy and/or create class II–restricted microbial peptides recognized by CD4+ T cells. To this end, WT, H2Ab−/− and TAP−/− mice were inoculated with VACV and, 7 days later, the Th response tested against a panel of 448 15-mer peptides. This panel consisted of putative H2Ab-restricted peptides from VACV ORFs [43]. An initial screen of these peptides revealed few shared specificities and significant alterations in the magnitude of CD4+ T-cell responses to these shared peptides in TAP−/− mice when compared to WT animals (data not shown). In addition, the loss of response to some peptides and novel responses to others was suggested (data not shown). To confirm these results, WT, TAP−/− and H2Ab−/− mice were inoculated with VACV. After 7 days, splenocytes were restimulated in vitro with increasing amounts of select peptides identified from the initial screen. This interrogation confirmed our previous observation [21] that TAP−/− Th cells responded to certain peptides with increased magnitude (Fig. 6A). In addition, the reactivity against other peptides was lost when compared to the response elicited in WT mice, suggesting they are dependent on the activity of TAP (Fig. 6B). Still other peptides were uniquely recognized only by TAP−/− Th cells and not WT Th cells (Fig. 6C) suggesting that in WT animals those epitopes are destroyed by the action of TAP. Importantly, VACV-immune spleen cells from H2Ab−/− mice recognized none of the peptides tested (Fig. 6) indicating H2Ab-restricted recognition of these epitopes by Th cells and not CD8+ T cells. Hence, these data demonstrate that the CAP machinery profoundly affected the Th-cell response. The altered T-cell response is a reflection of both altered Ag processing and presentation as well as an altered CD4+ T-cell repertoire.

image

Figure 6. WT and TAP−/− CD4+ T cells recognize a different subset of vaccinia viral epitopes. WT, TAP−/− or H2Ab-/− mice were inoculated with 5 × 105 pfu VACV. After 7 days, 106 splenocytes were restimulated in vitro with the indicated class II–restricted VACV peptides and the number of IFN-γ–producing cells determined by ELISPOT. Peptide recognition was either (A) enhanced, (B) lost or (C) uniquely generated in TAP−/− animals compared with WT responses. Data are shown as mean ± SD of n = 3–5 mice and are from one experiment representative of two performed. Peptides were derived from (A) I1L, (B) K ORF B and (C) D13L and E ORF B, respectively.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

CD4+ T cells regulate the adaptive cellular- and Ab-mediated responses to numerous microbial pathogens as well as cancers and autoantigens. Hence, it is critical to understand the processes regulating CD4+ T-cell development and activation. The results presented herein provide direct evidence that components of the CAP machinery sculpt the self peptidome displayed by H2Ab molecules. Alterations in the displayed peptidome subsequently impact both the CD4+ T-cell repertoire and Ag-specific T-cell responses. Though altered CD4+ T-cell repertoire and Ag-specific T-cell responses would be expected from an altered peptidome, these data imply that interference with the CAP machinery could profoundly affect anti-microbial T-cell responses. Many viruses and oncogenic mutations result in downregulation of TAP expression [44-49]. This downregulation is triggered to prevent class I–restricted peptide presentation. However, our data suggest that this downregulation would also alter class II–restricted self and viral peptide presentation and the subsequent T-cell response. Furthermore, the results presented herein enhance our understanding of CD4+ T-cell responses in those individuals who lack TAP expression or express natural genetic variants of TAP or ERAAP [50-59]. The altered CD4+ T-cell repertoire and the recognition of a different antigenic peptidome may help explain the recurrence of bacterial infections and tumours in individuals that lack TAP function [54, 57, 58].

With the discoveries of class I–restricted Ag cross-presentation and class II–restricted cytosolic Ag presentation, the division of the class I and class II Ag-processing pathways is becoming blurred. It becomes important, therefore, to understand the effect(s) that components of the CAP machinery may have on cytosolic Ags presented by class II molecules. We have shown that activities of CAP components profoundly alter the class II–restricted self peptidome. Therefore, not only is class I–restricted Ag presentation affected by the CAP machinery [22-26, 59], but class II–restricted peptide presentation is altered as well [21]. By manipulating the expression of CAP components, therefore, pathogenic microbes can both block class I– and skew class II–restricted peptide presentation. By skewing the Th-cell response, microbes could potentially evade sterilizing immunity or cause immunopathologic responses. Furthermore, these data have implications for next generation subunit vaccines and immunotherapies targeting Ag-specific T cells. Epitopes inducing protective immunity against microbes capable of manipulating the CAP machinery may only be presented in the absence of fully functional CAP components. In the absence of CAP suppression, e.g. peptide-pulsed APCs, these protective epitopes may not be processed and presented rendering such vaccines ineffective. Therefore, our data suggest that studies utilizing the live pathogen capable of manipulating the CAP machinery would be most likely to identify protective epitopes processed and presented during a natural infection.

Selection of CD4+ T cells with an altered self peptidome appeared to generate a distinct CD4+ TCR repertoire in CAP-deficient mice compared with that of the WT animals. Consistent with previous reports [35], this altered repertoire was not obvious when Vβ usage was queried. However, analysis of the CDR3β length variation revealed clear differences between WT- and CAP-deficient repertoires. Functionally, TAP deficiency led to the enhanced recognition of certain peptides by CD4+ T cells compared with recognition in WT animals. In addition, the recognition of some epitopes in WT mice was lost while at least two novel epitopes were recognized solely in the absence of TAP. This altered recognition pattern represents the combined effects of an altered T-cell repertoire and alterations in viral Ag processing and presentation. This implies that, in WT mice, the novel epitopes identified here were perhaps degraded by the CAP machinery within VACV infected cells and, hence, did not generate a CD4+ T-cell response. Alternatively, the TCRs specific for these epitopes normally may not be selected during development on the WT self peptidome, leaving a hole in the CD4+ T-cell repertoire.

By extension, humans deficient in TAP expression or those that express genetic variants of TAP or ERAAP might have similar alterations in their CD4+ T-cell repertoires [50-61]. This could result in altered recognition of microbial peptides leading to either limited immunogenicity or enhanced immunopathology. In this regard, it is noteworthy that herpetic stromal keratitis (HSK) — a leading cause of blindness that has an infectious etiology [62] — evolves as a consequence of chronic HSV infection. HSK is a chronic inflammatory disease that is mediated by CD4+ T cells [63]. As ICP47 of HSV blocks TAP function [48], one might predict that the display of an altered peptidome by HSV-infected cells might lead to CD4+ T-cell–mediated inflammation resulting in HSK. Further investigations will be needed to understand the clinical outcome of CAP deficiencies in humans.

In sum, it is becoming clearer that many Th-cell epitopes are being processed by components of both cytosolic and endo/lysosomal Ag-processing pathways [11-15, 21, 27-31, 61]. Data obtained from tagged Ags have suggested that the subcellular localization of the Ag may be critical for its presentation [15, 31, 34, 64-66]. Proteasomes and endo/lysosomal proteases may degrade proteins at the point of Ag entry, endogenous versus exogenous, respectively. Subsequently, peptides may then be shared between the two Ag presentation pathways depending on the efficiency of molecular components that transport processed Ags. While some peptides can be presented by both pathways [11-15, 27-31], it is evident that other peptides are restricted to a single presentation pathway [32, 34]. This is likely due to an as yet undefined biochemical mechanism(s) by which partially processed Ags are targeted from the cytosol to the endo/lysosome. Understanding the underlying mechanism will impact how T-cell biology is harnessed for vaccinations and immunotherapies as well as in treating autoimmune disorders that have a microbial etiology (e.g. HSK).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

Animals

C57BL/6J mice were purchased from The Jackson Laboratory. B6.129-TAP−/−, B6.129-ERAAP−/− and B6.129-Ab−/− mice [21] were bred, maintained and used in experiments in compliance with Vanderbilt University's Institutional Animal Care and Use Committee regulations and approval. B6.129-TAP−/−, B6.129-ERAAP−/− and B6.129-Ab−/− mice had been backcrossed to the C57BL/6 strain 8–10 generations before use.

Isolation of naturally processed H2Ab-associated self peptides

RBC-depleted single cell suspensions of splenocytes pooled from 68 to 70 mice per strain were solubilized, clarified and pre-cleared with normal mouse serum by previously described methods [67, 68]. Pre-cleared lysates were passed twice over protein A Sepharose (Repligen)-bound W6/32 (anti-HLA class I, an irrelevant Ab; Cedarlane) column followed by bead-bound H2Ab-specific Ab column (NYRmI-A, Cedarlane) at 4°C. After extensive washes, columns were eluted with 0.2N acetic acid. The eluates were adjusted to 2N acetic acid, incubated for 20 min in a boiling water bath and cooled on ice [68]. Eluted peptides were enriched by Centricon 10 ultrafiltration (Millipore), freeze dried, resuspended in ∼0.1 mL deionized distilled water (Sigma) and fractionated by RPC (HP1090, Hewlett-Packard) as described [68]. Approximately 150 fractions were collected and lyophilized to dryness.

MS-ESI sequencing of naturally processed H2Ab-associated self peptides

Each lyophilized RPC fraction was resuspended in 0.1% formic acid and subjected to reversed-phase microcapillary LC-MS/MS analysis using an LTQ linear ion trap mass spectrometer (Thermofisher). A fritless, microcapillary column (100 μm inner diameter) was packed with 10 cm of 5-μm C18 reversed-phase material (Synergi 4u Hydro RP80a, Phenomenex). RPC fractionated peptides were loaded onto the column equilibrated in buffer A (0.1% formic acid, 5% acetonitrile) using the LCPacking autosampler. The column was placed in line with an LTQ mass spectrometer. Peptides were eluted using a 60 min linear gradient from 0 to 60% buffer B (0.1% formic acid, 80% acetonitrile) at a flow rate of 0.3 μL/min. During the gradient, the eluted ions were analyzed by one full precursor MS scan (400–2000 m/z) followed by five MS/MS scans of the five most abundant ions detected in the precursor MS scan while operating under dynamic exclusion. Extractms2 program was used to generate the ASCII peak list and to identify +1 or multiply charged precursor ions from the native mass spectrometry data file [69]. Tandem spectra were searched with no protease specificity using SEQUEST-PVM against a RefSeq murine protein database [36]. For multiply charged precursor ions (z ≥ +2), an independent search was performed on both the +2 and +3 mass of the parent ion. Data were processed and organized using the BIGCAT software analysis suite with a weighted scoring matrix used to select the most likely charge state of multiply charged precursor ions [70]. Fragmentation/ionization patterns were compared against a dataset consisting of the fragmentation/ionization patterns of theoretical and known peptide sequences. The degree of concordance between these two patterns was assigned a cross correlation score Xcorr (Cn) with higher values representing greater concordance between the observed and expected fragmentation/ionization patterns [36]. Peptides with a Sequest Cn score >3.0 and ΔCn > 0.2 compared with the second most likely assignment were considered highly concordant (see Supporting Information Fig. 1).

The ion fragments were also searched against the reversed mouse proteome database to determine the false detection rate FDR. FDR was calculated as (2 × # reverse hits)/(# reverse hits + # forward hits). This generated an overall FDR of 7%. Whereas a search of only the highly concordant peptide spectra (Cn > 3.0 and ΔCn > 0.2) generated an FDR of 0, i.e. no peptides were identified in the reversed database. The parental ions representing peptides eluted from class II molecules of only two genotypes were manually searched against the database of parental ions of the third genotype. Of the 62 overlapping peptide sequences, only 2 (3.2%) were identified in the third genotype within 10 HPLC fractions and 10 min of LC elution of the same fraction number/retention time. Of these, one was inappropriately identified by the tandem MS and the other was not analysed by tandem MS for identification. From this analysis, we conclude that 96.8% of peptides presented by class II molecules of only two genotypes were correctly identified and were not presented by that of the third genotype.

Immunization, T-cell purification and functional analysis

The indicated mouse strains were inoculated either retro-orbitally with ∼5 × 104 cfu WT Lm or i.p. with 2 × 105 pfu VACV WR strain. After 7 days, splenocytes were harvested and either stained for flow cytometric characterization or restimulated for functional analyses. Lm-immune splenocytes were stained with mAb against mouse CD62L and CD44 for flow sorting of naive (Tn) and effector (Teff) CD4+ T-cell populations (FACS Aria, BD Bioscience). Post-sort purity was ascertained by flow cytometry and found to be >98% (data not shown). A separate aliquot of CD4+ T cells were analysed for Vβ usage with a panel of 15 anti-Vβ antibodies (BD Bioscience) within the Tn (CD44loCD62Lhi) or Lm-immune Teff (CD44hiCD62Llo) subsets.

Co-culture of total VACV-immune splenocytes with H2Ab-restricted peptides derived from VACV [43] for IFN-γ ELISPOT was performed as previously described [21].

TCR spectratyping

Total RNA was isolated from flow sorted non-immune CD4+ T cells or flow sorted CD62LhiCD44loCD4+ Tn cells and activated, effector CD62LloCD44hiCD4+ Teff cells and converted to cDNA as described [71]. PCR amplification of individual Vβ-Cβ junctions and -specific run-off was performed using previously reported primer pairs [72] and Supermix (Invitrogen). The run-off primers were end-modified with WellRED D2, D3 or D4 fluorescent dyes (Sigma-Genosys) to detect products using capillary gel electrophoresis (CEQ8000; Beckman Coutler). CDR3β fragment sizes were determined by correlation against a size standard consisting of WellRED D1 fluorescent DNA strands of incremental 20 nt residues (Beckman-Coulter) and the frequency within the population was determined by integration of the peak area. CDR3β length was calculated as the number of amino acids between the conserved last germline encoded Vβ Cys to the Jβ Gly-X-Gly motif.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

This work was supported by grant G12MD007592 from the National Institutes on Minority Health and Health Disparities (NIMHD), a component of the National Institutes of Health (NIH) as well as training (HL069765), research (HL054977 and AI040079 to S.J. and AI040024 to A.S.) and core (CA068485 & DK058404) grants from the NIH.

References

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information
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Abbreviations
CAP

MHC class I antigen processing

FDR

false detection rate

LM

Listeria monocytogenes

RPC

reversed-phase chromatography

VACV

vaccinia viral

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

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Table S1. Sequence and spectral characteristics of the H2Ab-associated peptides eluted from wild type, TAP-/- and ERAAP-/- splenocytes.

Table S2. Nested H2Ab-associated peptides eluted from wild type, TAP-/- and ERAAP-/- splenocytes.

Figure S1. ESI-MS/MS spectra of H2Ab-associated peptides displayed by wild type, TAP-/- and ERAAP-/- splenocytes.

Figure S2. ERAAP- and TAP-deficient MHC class II molecules present longer peptides.

Figure S3. Altered TCR β-chain repertoire of CAP-deficient CD4+ T cells.

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