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The Journal of Immunology, 2005, 175: 7209-7217.
Copyright © 2005 by The American Association of Immunologists

Identification of the Immunodominant HY H2-Dk Epitope and Evaluation of the Role of Direct and Indirect Antigen Presentation in HY Responses1

Maggie Millrain3,*, Diane Scott3,*, Caroline Addey*, Hamlata Dewchand*, Pamela Ellis4,*, Ingrid Ehrmann5,*, Michael Mitchell{dagger}, Paul Burgoyne{ddagger}, Elizabeth Simpson* and Julian Dyson2,*

* Transplantation Biology Group, Department of Immunology, Imperial College London, Hammersmith Hospital, London, United Kingdom; {dagger} Institut National de la Sante et de la Recherche Medicale Unite 491, Faculté de Médecine, Marseille, France; and {ddagger} National Institute for Medical Research, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Minor histocompatibility Ags derive from self-proteins and provoke allograft rejection and graft-vs-host disease in MHC-matched donor-recipient combinations. In this study, we define the HYDk epitope of the HY minor histocompatibility Ag as the 8mer peptide RRLRKTLL derived from the Smcy gene. Using HY tetramers, the response to this peptide was found to be immunodominant among the four characterized MHC class I-restricted HY epitopes (HYDkSmcy (defined here), HYKkSmcy, HYDbUty, and HYDbSmcy). Indirect presentation stimulated a robust primary HYDkSmcy response. Indirect presentation and priming of HY-specific CD8+ T cells is also operative in the presence of a full MHC mismatch. To determine whether the indirect route of Ag presentation is required for HY priming, female parent into F1 (H2bxk) female recipient bone marrow chimeras were immunized with male cells of the other parental haplotype, limiting presentation to the direct pathway. The dominant H2b HY response (HYDbUty) was dependent on indirect presentation. However, the dominant H2k HY response (HYDkSmcy) could be stimulated efficiently by the direct pathway. In contrast, secondary expansion of both HYDkSmcy and HYDbUty-specific CD8+ T cells was effective only when Ag was presented by the direct route. Transgenic overproduction of Smcy mRNA within the immunizing cells resulted in a corresponding increase in the HYDkSmcy, HYDbSmcy, and HYKkSmcy-specific CD8+ T cell responses when presented via the direct pathway but did not enhance indirect presentation demonstrating the independent regulation of MHC class I-peptide occupancy in the two Ag-processing pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cross-presentation of MHC class I-restricted epitopes can lead to either priming (cross-priming) or the induction of tolerance (cross-tolerance) of CD8+ T cells. This route of Ag presentation involves transfer of material into host APCs where it is processed and presented by MHC class I. Initially described for minor histocompatibility (H)6 Ags (1, 2), the pathway also operates in vivo for tumor cell Ags (3, 4, 5), material introduced into cells via hypotonic shock (6), exosomes (7), viral Ags (5), protein-coated latex beads (8, 9), immune complexes, and Ags associated with chaperones (10, 11, 12, 13). The use of transgenic mouse strains expressing neo-self-Ags has suggested that cross-presentation is a constitutive physiological pathway operating to reinforce peripheral CD8+ T cell tolerance (14, 15, 16).

The cross-presentation pathway is not as well characterized as the classical, direct pathway of class I peptide presentation; however, much attention has recently focused on its definition. Although different dendritic cell (DC) subtypes and macrophages are phagocytic in vivo, cross-presentation of phagocytosed cell-associated material is mediated by a CD8+ DC population (17). Like the classical pathway, the cross-priming pathway appears dependent, at least in vitro, on proteasomal degradation of Ag within recipient cells (9, 18) and transfer of cleavage products by the TAP complex into the endoplasmic reticulum or a phagosome-related compartment. In vitro, macrophages and DCs are able to process and cross-present Ag on MHC class I (19, 20), and in vitro-based analyses have provided insight into the biology of this pathway with the definition of a processing compartment containing endoplasmic reticulum and phagosome components (21, 22, 23). The phagosome imparts an acidic, proteolytic environment and enzyme machinery potentially involved in protein unfolding and degradation before proteasomal digestion. Cross-presentation of Ag by DCs on MHC class I allows Ags not directly expressed within DCs to interface with the CD8 as well as the CD4 T cell compartments. The pathway makes mechanistic sense, eliciting CD8+ T cell immunity toward viruses that do not directly infect DC and for tolerogenic exposure of tissue-specific self-Ags to CD8+ T cells. The maturation status of the presenting DC, effected by CD4+ T cell DC "licensing" and engagement of TLRs, will determine the balance between immunity and tolerance. It has recently been pointed out that cross-presentation may operate only in special experimental conditions and may not act as a general, physiological pathway in vivo (24). It is certainly true that the biological functions of cross-presentation are not fully understood, and its role in physiological presentation of Ag from different sources needs careful evaluation.

Early experiments describing the cross-presentation of minor H Ags have been criticized because they involved multiple, potentially cross-reacting minor H differences that induced weak in vitro cytotoxic responses (24). Recently, we and others (25, 26) have provided direct evidence for efficient cross-presentation by analysis of immunodominant minor H Ags responses ex vivo using MHC class I tetramers, and the functional significance of this pathway in transplantation has been suggested using TCR transgenic recipients (27). To further explore the role of this pathway in minor H responses, we have identified the immunodominant HYDk peptide epitope allowing the use of MHC class I tetramers to directly evaluate the direct and indirect routes of Ag processing in vivo.

The male-specific minor H Ag, HY provides a particularly relevant model for the evaluation of cross-presentation. First, minor H Ags are normal cellular proteins, and the set of HY epitopes have been defined at the molecular level. Second, immune responses to minor H Ags are the cause of the clinical complications associated with graft-vs-host (GvH) disease following bone marrow (bm) transplantation between HLA-matched siblings, and definition of their processing requirements has potential implications for approaches for the control of immune pathology. The importance of host APCs in GvH disease has been established (28).

The first goal of this study was to define the HYDk epitope. This would provide a classical parent into F1 immunization scheme, allowing dissection of the pathways of Ag presentation and their bearing on immunodominance among T cells responding to multiple epitopes of the HY mH Ag. The 8mer HYDk epitope derives from the Smcy gene, already known to encode several mouse and human HY epitopes (29, 30, 31, 32). The primary response to the HYDk epitope is immunodominant among the mouse class I-restricted HY epitopes, even when its presentation is limited to the indirect pathway. In contrast, efficient secondary expansion of HY-specific CD8+ T cells required the direct pathway. The efficiency of indirect presentation during primary, but not secondary, HY CD8+ T cell responses suggested that indirect presentation may be required for priming. This question was evaluated in irradiation bm chimeras, showing that although this was the case for the HYDbUty epitope, indirect presentation of HYDkSmcy was not required for priming CD8+ T cells.

To further explore the direct and indirect pathways of Ag presentation, a Smcy bacterial artificial chromosome (BAC) transgenic mouse was produced. In this strain, Smcy mRNA is expressed at ~8-fold greater than in male tissue. Enhanced mRNA expression led to amplified CD8+ T cell responses specific for all three Smcy-derived epitopes, with maintenance of the immunodominance hierarchy highlighting the close relationship between mRNA production and T cell stimulation in this system. Surprisingly, increased Smcy mRNA production had no discernable effect on responses stimulated through indirect presentation. These data are discussed in the context of current models of MHC class I Ag processing.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cosmid and cDNA transfection and screening

Cosmid MEM 14, containing most of the Smcy gene, was derived from a cosmid contig isolated from an XYSxra library (29). Subcloned cDNA fragments from Smcy and Uty were inserted into pCDNA1 (Invitrogen Life Technologies) downstream of the CMV immediate-early gene promoter (33). A total of 5 x 106 P1Htr cells (34) expressing H2Dk class I (P1Dk) was transfected with cosmid MEM 14 (10 µg) or subcloned cDNA fragment (10 µg) using electroporation or calcium phosphate as described previously (28). Stably transfected cells were assayed using the HYDk-specific clone, K3e (35). A total of 5 x 104 transfectant cells was incubated with 104 K3e cells in 200 µl of RPMI 1640 containing hypoxanthine/aminopterin and 1 IU/ml recombinant human IL-2 in 96-well plates for 72 h. [3H]Thymidine was added for the last 18 h of incubation, and the cells were harvested using a Tomtec 96-well harvester and counted using a Wallac 1450 Trilux Microbeta plate counter (PerkinElmer Wallac).

Quantitative RT-PCR

RNA was prepared from mouse spleen cells using TRIzol reagent (Invitrogen Life Technologies) and treated with RNase-free DNase using RNeasy MinElute spin columns (Qiagen). Concentrations were determined spectrophotometrically by measuring the absorbance at 260 nm. Relative concentrations of 18S ribosomal RNA and Smcy transcripts were determined using the Quantitect SYBR Green RT-PCR kit from Qiagen, following the manufacturer’s recommendations. Reactions were performed on a DNA Engine Opticon (MJ Research) system. Primer pairs were assessed by melting curve analysis and gel electrophoresis to ensure that only specific products were generated. The Smcy primers were chosen from regions with low homology to Smcx, and including one or more introns, and did not produce a PCR product using female mouse RNA. For 18S, the sequences used were as follows: forward primer, 5'- CCGCAGCTAGGAATAATGGAAT-3'; reverse primer, 5'-CGAACCTCCGACTTTCGTTCT-3'. For detection of Smcy, the primers were as follows: forward primer, 5'-CATGTAAAGGAGATAAGGAACT-3'; reverse primer, 5'-ATGAATGCGCTCAGATTGGG-3'. Each primer pair produced a specific product of ~200 bp. Test samples were assayed in triplicate for expression of 18S and Smcy. Expression values were determined from standard curves generated from each RNA preparation, plotting cycle threshold values against log quantity. Normalized Smcy values were obtained by division with the corresponding 18S value.

Peptide synthesis and screening

Peptides were synthesized by the Clinical Sciences Centre Central Research Resources unit using 9-fluorenylmethoxycarbonyl-protected amino acids and (2-(1-H-Benzotriazol-2-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) activation chemistry. After purification by HPLC, the fidelity of synthesis was confirmed by mass spectrometry and made up as filter-sterilized 1-mM stocks in PBS. A total of 104 K3e T cells containing 1 IU/ml recombinant human IL-2 and 5 x 106 irradiated CBA female splenocytes (APCs) in 100 µl of RPMI 1640 were added to triplicate serial 10-fold peptide dilutions (100 µl) in RPMI 1640. The cells were incubated, pulsed with [3H]thymidine, and harvested as described above.

Mice

C57BL/6J (B6), CBA/Ca (CBA), BALB/cOla (BALB/c), and (C57BL/6J x CBA/Ca)F1 mice were purchased from Harlan Olac. The Smcy transgenic was produced by injecting 129SV strain BAC clone b49 (CITB-586B12) (36) as closed circles (37) into F1 (C57BL/10 x CBA) oocytes. The single transgenic founder was initially crossed to MF1 random-bred albino females for three generations and then backcrossed to a C57BL/6J background for three generations. A H2b Smcy transgenic male was crossed to CBA to produce H2bxk offspring. Animals were maintained in the Biological Services Unit at the Medical Research Council Clinical Sciences Centre and used at age 6–10 wk. bm chimeras were produced by first depleting NK cells by injection (i.p.) of 100 µg of purified PK136 mAb (American Type Culture Collection; HB-191). Twenty-four hours later, mice were irradiated (900 rad) followed by injection (i.v.) of 107 donor bm after an additional 24 h. Donor reconstitution was assessed after 6 wk by flow cytometric analysis of MHC class I expression on peripheral blood CD11c+ DCs.

Immunization

Spleen cells were prepared by gentle teasing of dissected spleen. Cells were then passed through a cell strainer, washed twice in balanced salt solution, and resuspended in PBS. DCs were prepared from bm cultured with GM-CSF for 7 days. To promote maturation, DC cultures were passaged 24 h before harvesting. The majority of cells in these cultures were CD11c positive, and, of these, >90% were MHC class II and B7-1 positive, but only a minority were positive for B7-2 (data not shown). A total of 5 x 106 cells spleen cells or DCs was used for each immunization.

T cell culture

T cell clones K3e (35) and C6 (29), specific for the HYDkSmcy and HYKkSmcy epitopes, respectively, were cultured in complete medium (RPMI 1640; Invitrogen Life Technologies) supplemented with 10% FCS (Biogen Idec), HEPES (10 mM), penicillin (100 IU/ml), 100 µg/ml streptomycin (Invitrogen Life Technologies), 5 x 10–5 M 2-ME, and 2 mM L-glutamine (Invitrogen Life Technologies) in 2-ml 24-well plates (Linbro; Flow ICN Pharmaceuticals) at 37°C, 5% CO2, and restimulated every 10 days with 5 x 106/ml irradiated syngeneic male spleen cells (APCs) and 20 IU/ml rIL-2.

MLCs were set up using 5 x 106 responder spleen cells and 5 x 106 stimulator (irradiated) spleen cells in complete medium with 20 IU/ml IL-2 in 24-well plates. Cultures were taken at 7 days for analysis with tetramer.

Flow cytometry: cell preparation

PBLs were prepared by direct collection of tail blood into 200 µl of blood buffer (10 mM EDTA, 100 U heparin/ml in PBS) followed by addition of 1 ml of RBC lysis buffer (Puregene; RBC lysis solution). Samples were left at room temperature for 15 min and microfuged (4 min, 3500 rpm). Spleen cell suspensions were depleted of B cells with sheep anti-mouse Ig Dynabeads (Dynal Biotech), according to the manufacturer’s directions, before staining. T cell clones and MLCs were washed twice in PBS before staining.

Staining and analysis

MHC class I-peptide tetramers (HYDkSmcy, HYDbUty, HYDbSmcy, and HYKkSmcy) were supplied by Proimmune. Aliquots of 106 cells were stained in 50 µl of PBS containing 2% FCS (FACS buffer) with 1 µl of tetramer for 10 min at room temperature, then with FITC or PerCP-labeled anti-CD8 Ab (BD Pharmingen) for 15 min at 4°C, followed by two washes in FACS buffer. For assessment of donor APC chimerism, peripheral blood cells were stained with anti-CD11c, anti-H2-Db, and anti-H2-Kk (BD Pharmingen). Samples were acquired on FACScan or FACSCalibur flow cytometers (BD Biosciences). Data were analyzed using CellQuest software (BD Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Definition of the HYDk epitope

The genes encoding HY epitopes are localized in the {Delta}Sxr region of the mouse Y chromosome short arm containing a limited number of ubiquitously expressed genes. Smcy and Uty, which encode the three MHC class I epitopes of the minor H Ag HY that have been identified in mouse and most of the defined class I-restricted human HY epitopes (reviewed in Ref.38), were screened for expression of the HYDk epitope. P1Dk cells were transfected with a Uty cDNA fragment (15.1) previously identified as expressing the immunodominant HYDb epitope (33) or with cosmid MEM14, which contains most of the Smcy gene. A high proportion of the MEM 14 P1Dk transfectants (Fig. 1A), but none of the Uty cDNA transfectants (data not shown), stimulated the HYDk-specific T clone, K3e. Transfection of the subcloned MEM 14 fragments B, C, or E of Smcy into P1Dk cells identified a 1-kb fragment within the MEM 14 B region as encoding the HYDk epitope (Figs. 1B and 2A). The epitope was further mapped using a series of progressively smaller PCR fragments, which identified a 341-bp fragment encoding four candidate peptides that differed in sequence from the X chromosome homologue, Smcx. These were synthesized and tested for stimulation of K3e. Of these peptides, only one (RRLRKTLLEGKI) stimulated but required micromolar concentrations. To define the cognate peptide, a series of N- and C- truncated peptides were synthesized leading to the identification of the 8mer RRLRKTLL, which stimulated K3e at nanomolar concentrations (Fig. 2B). This highly basic peptide contains the anchor residues identified from comparison of defined H2Dk-restricted peptides (39, 40).



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  		FIGURE 1. Smcy encodes the HYDk epitope. A, P1Dk cells transfected with cosmid MEM 14, containing most of the Smcy gene, stimulated the HYDk-specific T cell clone (K3e) as well or better than male splenocytes. B, Transfection of subcloned fragments of MEM 14 into P1Dk cells shows that the epitope is located in the B fragment.

 


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  		FIGURE 2. The HYDk epitope is identified as the octamer peptide RRLRKTLL. A, Diagram indicating HYDk expression by P1Dk cells transfected with cDNA fragments (3-kb to 341-bp) derived from the regions of cosmid MEM 14 indicted by the bars. HYDk expression was assessed by the proliferative response of K3e. B, Ability of peptides derived from the 341-bp fragment to stimulate K3e after pulsing onto female splenocytes. Peptide 2a(iv) RRLRKTLL is able to stimulate maximal proliferation at 10 nM concentration.

 
Staining of H2k-restricted HY-specific CD8+ T cells with MHC class I tetramers

This H2-Dk-restricted peptide and the previously described H2-Kk-restricted peptide, TENSGKDI (29), define the MHC class I HY epitopes of the H2k haplotype. The two epitopes were produced as recombinant MHC class I tetramers, and their specificity was confirmed by staining the T cell clones used for identification of the cognate peptides (Fig. 3). These reagents, together with HYDbUty and HYDbSmcy tetramers (25) can be used to quantitate CD8+ T cell responses to each of the four HY peptide class I-associated epitopes in (H2bxk)F1 mice.



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  		FIGURE 3. HYDk and HYKk tetramers specifically stain the corresponding T cell clone. T cell clones K3e and C6 specific for HYDk and HYKkSmcy, respectively, are stained only with the tetramer of the corresponding specificity.

 
Immunodominance and efficient indirect presentation of HYDkSmcy

We first investigated the hierarchy of immunodominance among CD8+ T cells responding to the four HY epitopes (HYDkSmcy, HYDbSmcy, HYKkSmcy, and HYDbUty) by immunization (i.v.) of female F1 H2bxk (B6 x CBA) recipients with syngeneic male F1 splenocytes. Two weeks after immunization, the four HY tetramers were used to stain PBL. CD8+ T cells recognizing the HYDk epitope dominated in all recipients, representing 3–5% of the CD8 T cell compartment (Fig. 4A). Responses to HYDbSmcy, HYKkSmcy, and HYDbUty were similar, generally representing <1% of the peripheral blood CD8 T cell compartment. The HYDbSmcy and HYDbUty responses are similar to those seen in B6 mice, although HYDbUty becomes immunodominant in secondary responses (25). HYDkSmcy responses also dominated at 4 and 6 wk after immunization (data not shown). These data are in agreement with in vitro analysis of haplotype preferences of CTL from similar F1 H2bxk male into female immunization (41).



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  		FIGURE 4. The HYDk response is immunodominant in F1 hybrid mice. (CBA x C57BL/6)F1 (H2bxk) female hybrid mice were immunized (i.v.) with male F1 (A), male C57BL/6 (B), and male CBA (C) spleen cells as indicated. The four HY- specific MHC class I responses were assessed after 2-wk staining of PBLs with the four HY MHC class I tetramers. For each immunization, eight mice were analyzed, and the HY CD8+ T cell responses are shown. The four HY responses are indicated by different symbols, as indicated. Filled symbols indicate Ag presentation by both direct and indirect presentation, whereas open symbols indicate indirect MHC class I presentation only. For all three immunization schedules, the DkSmcy responses were significantly greater than the DbSmcy, KkSmcy, and DbUty responses (p ≤ 0.002, Student’s t test).

 
The robust response made to the HYDkSmcy epitope in F1 mice provides a system for evaluating the relative contribution of the indirect and direct pathways of class I Ag presentation. To limit presentation of HY epitopes to the indirect pathway of presentation, F1 females were immunized with male B6 (H2b) or CBA (H2k) splenocytes, respectively (Fig. 4, B and C). The HYDkSmcy response remained dominant even when presentation was limited to the indirect pathway (male B6 immunization; Fig. 4B), clearly demonstrating the efficiency of indirect presentation for processing cellular material. Cross-presentation of the other HY epitopes was also detectable in some recipients (Fig. 4, B and C).

Indirect presentation of Smcy and Uty during allogeneic immunization

The efficient indirect presentation of HY epitopes derived from the Smcy and Uty proteins suggests they may have preferential access to the indirect processing pathway. To address this question, we asked whether cross-presented HYDkSmcy and DbUty epitopes are able to prime in the context of a full MHC and minor H mismatch. In this setting, the overall T cell response to cross-presented peptides would be increased through recognition of epitopes derived from multiple minor H differences and from polymorphic MHC class I and II molecules. In contrast to the parent into F1 setting, HY-specific CD8+ T cells will be competing for access to the APC cell surface and would therefore require robust cross-presentation of their cognate peptide species to elicit an HY-specific response. Fig. 5 shows representative results from a series of fully allogeneic male into female immunizations. Priming to HY was assessed ex vivo by tetramer staining of PBL from blood taken 2 wk after immunization or following one in vitro stimulation of blood lymphocytes with syngeneic male cells. In some experiments, an indirectly primed HYDbUty response was detectable ex vivo in B6 females immunized with CBA male spleen cells (Fig. 5A). F1H2bxk (B6 x CBA) females immunized with BALB/c male (H2d) spleen cells did not develop HY-specific CD8+ T cells detectable ex vivo, but HYDbUty and HYDkSmcy-specific cells were detectable after one in vitro restimulation (Fig. 5B), demonstrating that cross-priming had taken place in vivo. Some allogeneic combinations, for example immunization of CBA females with B6 male spleen cells, failed to provoke detectable HY-specific CD8+ T cell expansion, even after in vitro restimulation (data not shown). These data show that HY-specific CD8+ T cells are often cross-primed during an allogeneic response, despite competing CD8+ and CD4+ T cells specific for other alloantigens. The general failure to detect these responses directly ex vivo may reflect the effect of immunodominance within the resulting complex T cell response.



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  		FIGURE 5. Priming of HY responses in the presence of full MHC and minor Ag mismatch. A, Representative HYDbUty tetramer staining of PBL from a B6 female recipient taken 2 wk after immunization (i.v.) with male CBA spleen cells. Similar ex vivo responses were seen in 2 of 8 B6 females. B, Female (B6 x CBA)F1 recipients were immunized (i.v.) with BALB/c male spleen cells, and PBL were collected 2 wk later and put into a MLC with either male B6 or male CBA APCs. DbUty and HYDkSmcy tetramers were used to stain the cultures after 7 days. These data are representative of several experiments.

 
Variable dependence on cross-presentation for priming epitope-specific HY responses

Given the efficiency of the indirect route of Ag presentation for priming HY-specific CD8+ T cell responses, we asked whether indirect presentation is a requirement for priming. If this were the case, direct presentation of HY would fail to prime a CD8+ T cell response. To limit HY presentation to the direct pathway, two sets of bm chimeras were produced. F1 H2bxk (B6 x CBA) female recipients were lethally irradiated and reconstituted with either parental female B6 (H2b) or female CBA (H2k) bm. After reconstitution, peripheral blood CD11c+ DCs were >98% donor derived in most chimeras (Fig. 6, A and B). The chimeras were then injected (i.v.) with bm-derived male DCs or male spleen cells of the nondonor parental type to limit Ag presentation to the direct pathway. All H2b-reconstituted chimeras gave robust primary HYDkSmcy responses, demonstrating that this response can be initiated by direct Ag presentation on CBA bm-derived DCs or spleen cell APCs (Fig. 6, C and E) and does not have an absolute requirement for indirect Ag presentation via host APCs. By contrast, the reciprocal chimeras (reconstituted with CBA female) immunized with male B6 DCs or spleen cells did not develop detectable CD8+ T cells specific for the immunodominant H2b HY epitope DbUty (Fig. 6, D and F). To test whether a limited primary response to HYDbUty had been primed, spleen cells from all of the chimeras were restimulated in vitro with B6 male, but, again, did not develop HYDbUty tetramer-binding populations (data not shown).



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  		FIGURE 6. Direct presentation of HYDkSmcy, but not HYDbUty, primes CD8+ T cells. Representative female B6 (A) and female CBA (B) reconstitution of female (B6 x CBA)F1 (H2bxk) recipients. PBL were taken 6 wk after bm transfer and stained for H2-Kk and -Db. Gated CD11c+ cells are shown. Representative, directly primed HY responses detected in PBL in bm chimeras immunized (i.v.) with male CBA DCs (C) and male B6 DCs (D). Summary of HY responses: four chimeras of each type were immunized with male DCs and four with male spleen cells. Symbols represent responses of individual mice, detected in PBL (E and F), 2 wk after immunization. Data are representative of three similar experiments.

 
The direct route of presentation dominates the secondary HY response

The F1 H2bxk (B6 x CBA) females immunized with parent or F1 male cells (Fig. 4, A–C) were left for 3 mo to allow the HY responses to decline to <1% of peripheral blood CD8+ T cells. All of the mice were then randomly assigned to three groups for secondary immunization (i.v.) with parental (CBA, B6) or F1 male spleen cells. As for the primary immunization, use of parental strains allows the contribution of the direct and indirect pathways of Ag presentation to be evaluated. In contrast to the primary response, effective secondary HYDkSmcy and HYDbUty responses were only observed when Ag presentation could use the direct route (Fig. 7). Limiting Ag presentation to the indirect route at the priming stage did not influence the extent of (directly stimulated) secondary expansion, suggesting that CD8+ T cell function was not significantly affected by the route of Ag presentation during priming (data not shown). Interestingly, syngeneic F1 male spleen cells were less effective than parental cells of the appropriate haplotype at stimulating HYDkSmcy and HYDbUty-specific CD8+ T cells (data not shown). One explanation for this observation is the reduction in parental MHC class I expression by F1 hybrid spleen cells, leading to less effective direct stimulation in comparison with MHC homozygous cells. To test whether affinity maturation may play a role in establishing immunodominance, a third immunization of CBA (H2k) male together with (B6) H2b male spleen cells was given, after a further rest period, to all recipients shown in Fig. 7. This provoked dominant HYDkSmcy and/or DbUty and/or DbSmcy responses, which were not influenced by the skew of the earlier responses (data not shown). This suggests that after repeated stimulation in the absence of competition for APCs, affinity maturation may influence immunodominance in this system.



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  		FIGURE 7. Direct Ag presentation of MHC class I HY epitopes in secondary responses. (B6 x CBA) F1 (H2bxk) female mice were primed for HY and then rested until the HYDkSmcy and HYDbUty HY-specific CD8+ T cell populations comprised <1% of the CD8 T cell compartment. They were then immunized (i.v.) with CBA or C57BL/6 male spleen cells. Each symbol represents epitope-specific CD8+ T cell HY responses in individual mice determined ex vivo, 2 wk after immunization, by HY tetramer analysis of PBLs. Closed symbols represent direct/indirect Ag presentation, and open symbols represent direct Ag presentation only. Direct Ag presentation produced significantly larger HYDkSmcy (p < 0.001, Student’s t test) and HYDbUty (p < 0.02, Student’s t test) responses.{dagger}, Recipients (7 of 9) had ≤0.1% tetramer-positive CD8+ T cells.

 
Independent regulation of Smcy entry into the direct and indirect pathways

Use of F1 (H2bxk) female responders allows simultaneous evaluation of the direct and indirect pathways of Smcy presentation in vivo. The two pathways have the potential for independent regulation because they are likely to use distinct sources of protein. We therefore assessed the efficiency of HY presentation via the two Ag-processing pathways following transgenic manipulation of the rate of Smcy mRNA synthesis. Smcy transgenic mice were produced using a 136-kb BAC carrying the entire Smcy gene and flanking regulatory elements but none of the flanking genes Ube1y or Eif2s3y (Fig. 8A). The positions of the three HY MHC class I epitopes within the predicted Smcy translation product are shown in Fig. 8B. Smcy mRNA expression from the transgene was assessed by quantitative PCR in whole spleen cell cDNA, which showed that the transgene expressed ~8-fold more Smcy mRNA than the endogenous Smcy gene (Fig. 8C).



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  		FIGURE 8. Characterization of Smcy BAC transgenic mice. A, The location of the {Delta}Sxr region on the mouse Y chromosome short arm is indicated. A genetic map of the {Delta}Sxr region and location of the 136-kb BAC containing the Smcy gene used to make the transgenic strain is shown. B, Location of the three HY epitopes within the predicted Smcy translation product. C, Quantitative PCR analysis of Smcy mRNA in transgenic and nontransgenic spleen cells. The y axis represents an arbitrary linear scale of the Smcy PCR product.

 
Male H2bxk F1 Smcy transgenic and nontransgenic littermate spleen cells were injected (i.v.) into F1 H2bxk (B6 x CBA) female recipients, and their HY CD8+ T cell responses were evaluated after 14 days. Strikingly, responses to all three Smcy- encoded peptides, each presented on a different class I allele (HYDkSmcy, HYDbSmcy, HYKkSmcy), were significantly amplified in recipients of Smcy transgenic male cells (Fig. 9A). The response to HYDkSmcy now constituted up to 17% (mean 11%) of the CD8 compartment, comparable with the response to H60 that dominates among defined H2b MHC class I-restricted minor H Ags (42). Although the subdominant HYDbSmcy, HYKkSmcy, and HYDbUty are not significantly different to each other when stimulated with nontransgenic F1 cells (Figs. 4A and 9A), both Smcy responses are significantly stronger than HYDbUty after immunization with Smcy transgenic male cells (Fig. 9A).



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  		FIGURE 9. Enhanced Smcy synthesis promotes direct but not indirect Ag presentation. A, Eight (B6 x CBA) F1 (H2bxk) female mice were immunized with spleen cells of male H2bxk Smcy transgenic or nontransgenic male littermate spleen cells. Two weeks later, HY-specific CD8+ T cells in peripheral blood were quantified. In comparison with nontransgenic spleen cells, Smcy transgenic spleen cells stimulated significantly larger DkSmcy, DbSmcy, and KkSmcy responses (p < 0.001, p < 0.01, p < 0.004, respectively; Student’s t test) but significantly smaller DbUty responses (p < 0.004, Student’s t test). Smcy transgenic spleen cell immunization produced significantly greater DbSmcy and KkSmcy responses than DbUty response (p < 0.001, Student’s t test). B, Eight (B6 x CBA) F1 (H2bxk) female mice were immunized with spleen cells of male H2b Smcy transgenic spleen cells. Two weeks later, HYDkSmcy and HYDbSmcy HY-specific CD8+ T cell populations were quantified in PBLs. The four HY responses are indicated by different symbols, as indicated. Filled symbols indicate Ag presentation by direct and indirect presentation, whereas open symbols indicate indirect MHC class I presentation only. {dagger}, Recipients (6 of 8) had ≤0.1% tetramer-positive CD8+ T cells.

 
We next examined whether indirect presentation of Smcy would also be enhanced by increased mRNA expression within the immunizing spleen cells. To assess the consequence of Smcy overexpression on indirect presentation of the HYDkSmcy epitope, H2b male Smcy transgenic spleen cells were injected (i.v.) into F1 H2bxk (B6 x CBA) female recipients. In all recipients, the indirectly stimulated DkSmcy response returned to the nontransgenic range (Fig. 9B). HYKkSmcy, presented only by the indirect pathway in these recipients, also stimulated poor responses. By contrast, HYDbSmcy, which can be presented by both the direct and indirect pathways, again stimulated enhanced responses in all recipients, leading to a new immunodominance ranking. These data show that enhanced Smcy mRNA expression in the immunizing cells amplifies the T cell response to all three Smcy-derived epitopes when presented through the direct pathway but has no detectable impact on in vivo indirect presentation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The HYDk epitope RRLRKTLL conforms to the motif defined by Lukacher and Wilson (39) of basic residues at P2, P5, and a hydrophobic residue (usually a leucine) at the carboxy terminus. Previously defined Dk epitopes are 9-mer, whereas the HYDk epitope is an 8-mer. The sequence of the HYDk peptide allows for alternative anchor residues because P1/P2, P4/5, and P7/8 meet the requirements for the three anchor residues. Given the importance of the amino- and carboxyl-terminal peptide/MHC H-bond networks (43), and the sharing of amino-terminal RR motif and carboxyl-terminal LL motif in HYDkSmcy with two of the other five defined Dk epitopes, a likely configuration of the peptide is in extended form with P2 and P8 serving as the terminal anchor residues. P4 or P5 may serve as the central anchor residue. Because the peptide sequence (underlined) between anchor residues (bold) in a Dk 9-mer is XA1XXA2XXXA3, lysine at P5 is more likely as the central anchor because a HYDk configuration of RRLRKTLL would impose less stress on the interanchor sequence than the alternative RRLRKTLL because the relative truncations are 0, 33, and 50, 0%, respectively. The homologous peptide, expressed from the Smcx gene, retains the Dk-binding motif and has a singleA substitution of alanine at P7. The increased size of the P7 side chain in the male version is sufficient to explain specific HYDkSmcy recognition by the TCR but could additionally involve the P7 substitution repositioning neighboring side chains.

Definition of the immunodominant HYDk epitope facilitated classical parent into F1 experiments in which both direct and indirect MHC class I Ag-processing pathways could be evaluated. During priming, presentation of HYDkSmcy, despite being limited to the indirect pathway, was efficient, and the response to this epitope was dominant, even when HYDbUty could access both the direct and indirect pathways (B6 male spleen into F1 (B6 x CBA) female). Although the dominance of HYDkSmcy is apparent using tetramers, by this stage extensive cell division has already taken place, and a different hierarchy may exist at earlier stages. The four tetramers bound with similar MFIs to ex vivo Ag-specific CD8+ T cell populations, indicating that gross differences in TCR affinity are not responsible for immunodominance. Subtle differences in the on and off rates of TCRs with MHC peptide may well contribute to establishing the immunodominance hierarchy, but analysis of these parameters would require extensive biophysical measurements on multiple TCRs. Indirect presentation of HY and CD8+ T cell priming also occurred in the presence of a full MHC and minor H mismatch. This observation explains accelerated rejection of male grafts after immunization of females with MHC-mismatched male cells (44).

In view of the efficiency of minor H Ag presentation via the indirect pathway and the potential requirement for a particular, localized DC population for presentation (17), we asked whether the indirect route of Ag presentation of HY class I epitopes is crucial for T cell priming. These experiments, using bm chimeras, revealed a differential requirement for processing depending on the epitope examined. HYDkSmcy responses were primed by direct Ag presentation by male bm-derived DCs and spleen cells, whereas HYDbUty responses required indirect presentation. Expression of Smcy and Uty in DCs has not been directly assessed, but DCs have been shown to be poor stimulators of HYDbUty-specific T cells (45), which may account for their dependence on indirect presentation by host APCs. A second possibility is that the surface density of the HYDbUty epitope may be limiting on nonprofessional APCs, although this seems unlikely in view of the efficient secondary stimulation via the direct route (Fig. 7). HYDkSmcy-specific CD8+ T cells were primed more efficiently with male spleen cells than with male bm-derived DCs, suggesting either more efficient direct priming with splenic DCs or reduced requirements for activation. One possibility is that an element of the HYDkSmcy response derives from a cross-reactive memory CD8+ T cell pool with a less stringent requirement for activation. Cross-protection among virus-specific CD8+ T cells indicates the memory pool may have sufficient diversity to react to structurally unrelated peptide Ags (46). The relative radioresistance of memory T cells (47) will enrich for this subset among surviving recipient F1 cells in reconstituted chimeras. Consistent with the notion that the HYDkSmcy response can use memory T cells, recipient F1 CD8+ T cells were highly overrepresented within the responding HYDkSmcy-specific population in some chimeras (data not shown). In the context of GvH disease, the variable dependence on cross-presentation observed in this study may correspond to the requirement for professional host APCs for the development of severe but not mild disease in a minor H Ag-mismatched setting (28). Furthermore, strategies aimed at depleting host APCs may have a beneficial outcome on host-vs-graft as well as GvH disease.

For secondary HY responses, a very different picture was seen, in which the direct route of Ag presentation dominates. We have not, however, excluded a component of indirect presentation during the secondary stage. The switch to dominant direct presentation is consistent with studies on viral immunity in which, after priming by DCs, effector CD8+ T cells migrate and can be activated by MHC class I/peptide complexes on nonprofessional APCs located in nonlymphoid tissues (48).

Recent reports (49) indicate that in vivo cross-presentation of cellular material involves processing of protein within the cross-presenting cell rather than presentation of pre-existing processed peptide within the phagocytosed cell. This is likely to apply in the parent into F1 system used in this study (Fig. 4), because the parental male donor cells lack the relevant MHC class I molecule required for stabilization of the antigenic peptide. A crucial difference between the direct and indirect routes of MHC class I presentation is that in the direct route, the dominant source of peptides is thought to come from defective ribosomal products (DRiPs), which derive from misfolded polypeptides generated at the point of synthesis and which are rapidly degraded by the proteasome (50, 51, 52). The supply of peptides for the direct route will therefore largely correspond with the profile of protein synthesis. In contrast, material accessing the indirect pathway derives from an intact cell or cell fragments that are degraded after endocytosis or phagocytosis by host APCs. Entry into the processing machinery of host APCs is likely to be highly selective because there is no mechanism, like the DRiP process, allowing representation of all protein species. Stability, location, and abundance have recently been identified as factors influencing indirect presentation (53, 54), potentially skewing peptide representation from that initiated by DRiPs. The parameters influencing access to the cross-presenting machinery for a given protein are likely to be complex and difficult to predict. With highly complex structures such as whole and fragmented cells, entry into the indirect pathway and Ag presentation is likely to be highly selective. Cross-priming by HY epitopes derived from injected spleen cells, even when they comprise a relatively small component of an allogeneic response, implies that the Smcy and Uty proteins may have properties, such as stability, that favor their entry into the indirect pathway. Predictive structural analysis of Smcy and Uty have defined domains involved in transcriptional regulation, suggesting nuclear localization (55). Smcy (1548 aas) and Uty (1212 aas) are both large proteins, a factor that may delay proteolysis and would exclude them as primary substrates for transport into the cytosol by the Sec61 translocator (56).

Efficient cross-presentation of cellular material is perhaps not surprising because phagocytosis of apoptotic cells within multicellular organisms is an ancient evolutionary development. Adaptive immunity has interfaced with this process to sample the intracellular contents of both normal and infected cells. Cross-tolerance mechanisms reinforce peripheral tolerance, and cross-priming with infected cells is essential for initiating immunity to viruses that do not directly infect DCs. Our observations on the efficiency of indirect priming for the HY minor H Ag demonstrate the importance of this pathway in transplantation. Although transplantation is a nonphysiological process, the graft’s engagement with host tissue sampling and clearance mechanisms is entirely physiological. Minor H Ags were originally selected for their ability to provoke skin graft rejection. Effective rejection of skin may well require cross-presentation of donor Ag by host DCs (28) imposing a filter on the proteins that can contribute to the generation of minor Ags. Selective entry into the indirect pathway has important implications for cross-tolerance and tumor immunology. First, cross-tolerance by DC immune sampling may be restricted to efficiently cross-presented proteins. Second, T cell priming and tolerance to tumors may also be restricted to those proteins that are efficiently cross-presented. Third, potential tumor Ags that are not cross-presented, and for which responsive T cells may escape cross-tolerance, could provide attractive targets for tumor immunotherapy.

The consequence of increasing the synthesis of the Smcy gene product on presentation via the direct and indirect pathways was assessed using Smcy transgenic spleen cells that express ~8-fold more Smcy mRNA than male cells. Immunization of H2bxk females with H2bxk Smcy transgenic male cells led to enhancement of all three Smcy responses, which became immunodominant over DbUty in the primary response (Fig. 9A). These data support the view of a close correlation between mRNA synthesis, DRiP production, MHC occupancy, and the resulting CD8+ T cell response, and underline the importance of peptide density in determining immunodominance. In this study, the correlation extends to three epitopes that involve peptide loading and presentation by three different MHC class I molecules. Interestingly, although the DkSmcy response represented almost 1 in 5 CD8+ T cells in some mice, competition at the level of APCs did not lead to suppression of the other Smcy responses. When H2b Smcy transgenic cells were used to immunize H2bxk females, the response to the H2DkSmcy epitope, which will be presented only by the indirect route, returned to the nontransgenic level; the normally immunorecessive DbSmcy response (Fig. 4), which can be presented both directly and indirectly, became immunodominant (Fig. 9B). Transgenic Smcy production thus promotes CD8 T cell immunity only through direct presentation. Why might the indirect route not be enhanced despite increased Smcy production in the cross-presented cells? One possibility is that the cellular pool of Smcy protein that enters the cross-presenting pathway is already saturated by the endogenous steady-state level of Smcy (and the related X chromosome product Smcx). The Smcy and Smcx products are predicted to be transcriptional regulators, which may be present in multicomponent complexes with other transcription factors(s). Degradation motifs can be masked by protein-protein interactions within multicomponent complexes, providing a mechanism for limiting the steady-state level of a protein independently of its rate of transcription (57). We are presently exploring this possibility. In vivo, cross-presentation can be mediated by transfer of peptides between APCs and T cells via gap junctions (58). Because the production of Smcy-derived peptides is increased in Smcy transgenic APCs without enhancing cross-presentation, it is unlikely that this mechanism has a significant role during in vivo cross-priming with cell-associated substrates.

The ability of a peptide to be efficiently processed and presented by MHC class I via the indirect route may be a crucial factor in immunodominance, particularly at the priming stage where this pathway operates most efficiently. This report highlights the efficiency of the indirect pathway and clearly shows its independent sourcing of peptide. Characterization of the mechanisms governing cross-presentation will be important for the design of vaccines against pathogens and for enhancing T cell responses to tumors.


   Acknowledgments
 
We thank Prof. Enzo Cerundolo and Dr. Michael Palmowski for valuable discussions and Dr. Oro Rufian for BAC oocyte injections.


   Disclosures
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 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 This work was funded by the Medical Research Council. Back

2 Address correspondence and reprint requests Dr. Julian Dyson, Transplantation Biology Group, Department of Immunology, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, U.K. E-mail address: peter.dyson{at}imperial.ac.uk Back

3 M.M. and D.S. contributed equally to this work. Back

4 Current address: Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield S10 2TN, U.K. Back

5 Current address: Institute of Human Genetics, The International Centre for Life, University of Newcastle upon Tyne, Newcastle upon Tyne NE13BZ, U.K. Back

6 Abbreviations used in this paper: H, histocompatibility; DC, dendritic cell; GvH, graft-vs-host; bm, bone marrow; BAC, bacterial artificial chromosome; DRiP, defective ribosomal product. Back

Received for publication May 27, 2005. Accepted for publication September 1, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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