Abstract
We identified and characterized an HLA-A1 aberrant allele (A*0118N) resulting from a novel molecular mechanism; this allele was present in an unusually informative family with a near identical parental HLA haplotype (c d) differing only by one nucleotide substitution in one HLA-A allele, A*0118N, of the maternal HLA haplotype (c) and not of the paternal HLA haplotype (a). Although serologic HLA typing showed a “blank,” DNA molecular HLA typing detected a HLA-A*0118N allele. Sequence based typing identified the substitution of guanine by cytosine at the nucleotide position 215, which resulted in the replacement of arginine by proline at position 48 of the HLA-A1 H chain. The loss of surface protein expression was also found by FACS analysis. Isoelectric-focusing analysis detected a HLA-A H chain with a unique isoelectric-focusing pattern, which does not associate with the L chain (β2-microglobulin). These results suggest that the residue 48-containing interaction site on the α1 domain plays a critical role in the association between HLA class I H chain and β2-microglobulin. Functional studies showed that the T cells of the propositus (HLA haplotypes c d) carrying this null allele recognized its wild-type counterpart, HLA-A*010101, in her HLA-identical son that carries the HLA-A*0101 heterodimer. This is the first example of the generation of cytotoxic T cells in the absence of proliferation of CD4+ T cells (mixed lymphocyte culture) and the description of an aberrant allele, A*0118N, that may behave as a minor histocompatibility Ag, with implications in allorecognition by cytolytic T cells in solid organ and stem cell transplantation.
Human leukocyte Ag null alleles are characterized by the lack of a serologically detectable molecule (1, 2). Alterations at the DNA level may result in the direct generation of null or variant alleles (3). Nonexpressed alleles could be mistyped as expressed alleles, because not all segments of the genes are being analyzed (2, 3, 4, 5) and some deleterious mutations may go undetected.
In recent years, DNA-based methods have replaced serologic methods for HLA typing (1). The molecular methods may not distinguish between expressed and nonexpressed alleles. As a result, some of the HLA typing results may not be correct (2, 3). Even though HLA class I null alleles are usually not expressed and are uncommon in human populations, they should not be ignored (2, 3). HLA typing errors related to the presence of such alleles may cause complications in the HLA matching of donor/recipient pairs for organ transplant. The World Health Organization Nomenclature Committee has recognized many different nonexpressed alleles of the HLA class I and class II systems (6). Because HLA serology is incomplete, it is possible that some HLA molecules expressed at the cell surface are not detectable (7, 8). Thus, attention has been paid to whether alloreactive T cells may recognize these alleles. Null alleles have been shown to not usually be the targets of alloreactive CTLs. However, the low-expression allele may induce tolerance to the wild-type counterpart. Nevertheless, no study has been able to test whether a nonexpressed allele carrier’s immune system may mount an alloreactive response specifically to products of the wild-type counterpart allele in humans, now named aberrant.
In this report we describe a new variant of HLA-A*01 (A*0118N) that is not expressed at the cell surface due to a heterodimer formation defect induced by an exonic substitution. In addition, by using family members having identical HLA haplotypes we also show the impact of this mutation on the alloreactive T cell repertoire of the subject carrying the allele with the novel mutation. The data to be presented show that this novel allele (aberrant allele) has a role in allotransplantation.
Materials and Methods
Cells
PBMCs were isolated from peripheral blood from the father, mother, and six children of the same family. The EBV-transformed B lymphoblastoid cell lines (B-LCL)4 BY00035, BY00037, BY00039, BY00042, and BY00043 were maintained in RPMI 1640 medium supplemented with 15% FBS (Gemini Bioproducts) and penicillin/streptomycin (Mediatech) at 37°C in a humidified 5% CO2 incubator.
Monoclonal Abs
The mAb W6/32 recognizes a monomorphic epitope shared by the gene products of the HLA-A, HLA-B, HLA-C, HLA-E, and HLA-G loci and immunoprecipitates both 43-kDa and 11- to 12-kDa chains (9), mAb 4E recognizes a determinant expressed by β2-microglobulin (β2m)-associated HLA-B and HLA-C H chains (8), and mAb HC-A2 (a gift from Dr. H. Ploegh, Harvard Medical School, Boston, MA) recognizes a determinant expressed by β2m-free HLA-A (excluding A*24), -B*7301, and -G heavy chains (10, 11). The mAb 13th International Histocompatibility Workshop and Conference (IHWC) 0003 (mAb VDK 1D12 29824.10 was kindly provided by Dr. A. Mulder from Leiden University, Leiden, The Netherlands) and mAbs IHWC 0004 and 0048 (mAbs H599 and H1160 were kindly provided by Dr. J. H. Lee from One Lamda Inc.) to the 13th International Histocompatibility Workshop Serology Component (12). The specificity of these Abs has been determined by extensive testing with a lymphocytotoxicity assay. It has been determined that mAb 13th IHWC 0003 reacts with all common alleles of the HLA-A*01 and A*36 serotypes; HLA-A alleles carrying the amino acids lysine at position 44, methionine at residue 67, and valine at position 150 (K44∼M67∼V150) are predicted to be reactive with this mAb. The mAb IHWC 0048 reacts with all of the common alleles related to the specificity HLA-A*24; all HLA-A alleles carrying the combination of residues K144∼R79∼I80∼A81∼L82∼R83 or E76∼K144 are predicted to react with this mAb. The mAb IHWC 0004 reacts with all of the common alleles related to the specificities HLA-A*01, A*23, A*24 (excluding A*2403), and A*80; all HLA-A alleles carrying the combination of residues D166∼G167∼N77 or D166∼G167∼I142∼R145 or D166∼G167∼G107 are predicted to react with this mAb. Rabbit anti-mouse IgG (H+L) Ab was purchased from Jackson ImmunoResearch Laboratories.
Indirect immunofluorescence staining
PBMCs were resuspended at 5 × 105 cells/tube in staining buffer (PBS, 0.1% BSA, and 0.05% NaN3), cells were incubated with mAb IHWC 0003 (13WS0003), IHWC 0004 (13WS0004), IHWC 0048 (13WS0048), IHWC 0033 (13WS0033), IHWC 0038 (13WS0188), and IHWC 0016 (13WS0016) for 30 min at 4°C. mAbs were directly labeled with the fluorescent dye Alexa Fluor 488 (Molecular Probes). The fluorescent intensity of Alexa 488 was measured in median channel values (0–1024). A protein labeling kit (Molecular Probes) was used to conjugate the Alexa Fluor 488 to each mAb following the manufacturer’s instructions. Protein concentration and degree of labeling were estimated by measuring the absorbance of the Alexa 488-labeled mAb at 280 and 494 nm with a flow cytometric three-color (FC3C) test. To measure HLA-A cell surface expression, B-LCL, PHA blasts, and PBMCs were washed and resuspended in a flow washing solution (FWS) consisting of PBS with 0.1% sodium azide (Sigma-Aldrich) and 2% FBS. Aliquots (15–20 ml with 3–5 × 105 cells) were mixed with 5 ml of Alexa 488-labeled HLA mAbs, incubated for 20 min on ice, and washed twice with FWS. If the characterization of lymphocyte phenotype was also necessary, 5 ml of anti-CD19-PE and 5 ml of anti-CD3-PerCP mAbs (BD Immunocytometry Systems) with 10 ml of FWS were added at this point, and the same procedure as described above for HLA-specific mAb was followed. Finally, cell pellets were fixed with 0.5 ml of 2% paraformaldehyde in PBS. Samples were analyzed with CellQuest software in a FACSCalibur dual laser flow cytometer (BD Immunocytometry Systems). To evaluate the ability of Alexa Fluor reagents to discriminate between positive and negative reactions by flow cytometry, each mAb was tested with 37–50 cell lines. Cell samples were assayed with 23 HLA-A-specific mAbs whose fluorescence intensity (Alexa Fluor 488) was obtained from the histogram statistics expressed as median channel values ± SD. The Daudi cell line was assayed to determine the fluorescence background for each mAb labeled with Alexa Fluor 488. Reactions were considered positive if they were above the median of all negative reactions +2 SD.
Immunoprecipitation and one-dimensional isoelectric focusing (IEF) gel analysis
HLA class I Ags were immunoprecipitated with mAb from [35S]methionine-labeled cells and analyses by one-dimensional IEF gel electrophoresis as described (5). Briefly, 5–10 × 106 cells were re-suspended in RPMI 1640 medium without methionine and subsequently labeled with 100 μCi/ml 35S]methionine for an additional 4 h. Ampholine ampholytes (Bio-Rad) of pH 3.5–10.0, pH 5.0–7.0, and pH 7.0–9.0 were used for the one-dimensional IEF gel electrophoresis. Gels were processed for fluorography as described (5)
Genetic studies
HLA serology, HLA class I and class II intermediate resolution molecular typing, and sequence-based DNA typing of the individual carrying the new allele and of family members have been reported in an earlier study (13). Briefly, sequence-based DNA typing analysis of the A*0118N allele was performed using the ABI BigDye Terminator cycle sequencing kit (Applied Biosystems). PCR amplification of exons 1, 2, and 3 was performed using cloned PfuDNA polymerase (Stratagene); PCR amplification of exons 4–7 was performed using TaqDNA polymerase (Promega).
Mixed lymphocyte culture
MLC was performed as previously described (14). Stimulator and effector cells were cocultured for 6 days, the cells were labeled with tritiated thymidine, and the degree of proliferation of CD4+ T lymphocytes from the responder individual was determined. Results are expressed as the relative response, which is defined as the ratio of thymidine uptake by the responder cells in response to exposure to the irradiated stimulator cells as compared with the exposure to control cells.
Cell-mediated lysis (CML)
Cells from the probands were cultured for 72 h at 37°C with irradiated target cells to create primed effector cells. 51Cr-labeled target cells were added to the effector cells at various E:T cell ratios. After a 4-h incubation at room temperature, supernatants were removed and released amounts of 51Cr from target cells were analyzed. Results are expressed as the percentage of specific cytotoxicity, defined as the amount of 51Cr released in comparison to the total cell-associated 51Cr release assay (14). Cytotoxicity was determined in triplicate and repeated for our E:T ratios of 6.25, 12.5, 25, and 50. Spontaneous release was determined by incubating target cells alone and measuring 51Cr release in the supernatants. The percentage of cytotoxicity was calculated as follows: percentage of specific lysis = (experimental cpm − spontaneous cpm/total cpm − spontaneous cpm) × 100.
Detection of HLA-A1 H chain mRNA by RT-PCR
The HLA-A*01 H chain mRNA was detected by a RT-PCR procedure as described previously (15). Reverse transcription was oligo(dT) primed and PCR was conducted using HLA-A*01-specific primers. Poly(A)+ mRNA was isolated from PBMCs using the Oligotex mRNA Mini Kit (Qiagen). Each experiment was performed at least twice to confirm the results.
Prediction of proteosomal cleavage sites of the HLA alleles A*0101 and A*0118N
This analysis was performed utilizing the program PAProC (16, 17). This program is a prediction tool for cleavages by human and yeast 20S proteasomes based on experimental cleavage data and is available online (〈http://www.paproc.de〉); the sequences of the HLA alleles were obtained from the IMGT/HLA Database (〈http://www.ebi.ac.uk/imgt/hla/〉) (18). The program reports cleavage sites. In this report we looked for C-terminal cleavage sites that might be dominant, whereas those within the epitope might be weak. Furthermore, in vitro determined intraepitope proteasomal cleavage sites have been reported not to abolish epitope presentation in vivo (19).
Results
Previous studies (13) have shown the occurrence of a novel allele of HLA-A, in this report designated as the HLA-A*0101 aberrant allele (A*0118N) in the mother of family H (Fig. 1⇓). The mixed lymphocyte cultures were performed to test the reactivity of the cells of the mother (HLA haplotypes c d) and her son (HLA haplotypes a d). These two individuals are HLA identical except for the fact that the mother carries the HLA-A*01 mutation. The HLA class II alleles are identical and are not expected to produce an alloreactive reaction. Indeed, Table I⇓ shows the lack of mutual alloreactivity among the cells of these individuals. By contrast, her husband’s cells and the cells of two offspring carrying (a) and (d) HLA haplotypes produced significant alloreactivity consistent with the one or two HLA haplotype differences among these three family members. The HLA haplotype (c) of the mother and the haplotype (a) of the son differ only in one allele of the HLA-A locus. Haplotype (c) includes a novel allele of HLA-A*1, A*0101 N, that differs from the HLA-A*0101 allele by one amino acid substitution at residue 48. The HLA-A, -B, -Cw, DRB1, DQB1, and DPB1 haplotypes of this family are shown in the legend of Table I⇓.
HLA family typing and molecular DNA. Males are represented by squares and females by circles.
Results of the MLC assaya
CML tests at the E:T ratio of 50:1 showed that the mother’s effector cells had low but significant killing of target cells from her son (mother E:T ratio of 50:1 = 10.37%) carrying the HLA haplotypes (a) and (d); this specific lysis can be attributed to the recognition of the allele HLA-A*0101 present in the son and absent in the mother (Table II⇓). Control experiments demonstrated that the mother’s effector cells could be raised against and kill target cells from the husband and her daughter. The mother’s CTLs raised against her husband’s cells, her daughter’ cells, and a pool of unrelated targets showed significant cytotoxicity values. In previous studies using this technique in our laboratory (14), an analysis of 16 HLA different, 38 haploidentical, and eight HLA-identical siblings that did not include the transplant candidate demonstrated that the 51Cr released, measured as percentage of cytotoxicity, ranged between 43.3 and 16.50% (mean 28.4% ± 5.3), 28.8 and 13.6% (mean 18.3 ± 4.1), and 0 to 6.8% (mean 2.3 ± 1.4), respectively.
CML between lymphocytes of HLA identical mother c d (HLA-A*01 aberrant allele) and son a d (HLA-A*01)
The reverse priming of lymphocytes from the offspring carrying the HLA haplotypes (a) and (d) by stimulators from his mother did not raise the significant killing of target cells from his mother. The offspring’s lymphocytes displayed significant responses against cells from the father, a sibling, and a third party.
The results of the flow cytometric analysis of cells stained with seven mAbs tested during the 13th International Histocompatibility Workshop are shown in Table III⇓. The mAb 13WS0003, reactive with the specificities HLA-A*01 and A*36, stained all cells of the family members carrying the allele A*0101 (haplotype (a)) but failed to stain cells from the mother. The mAb anti-A*24 (13WS0048) stained cells from the mother and cells from children with an HLA haplotype (d). The mother’s cells and those of the son (a d) are HLA identical except for one HLA-A allele. It appears that the novel HLA-A allele is not expressed on the membrane of the mother’s lymphocytes. Representative results of the flow cytometric analysis of cells stained with HLA class I Ag-specific mAbs are shown in Table III⇓ and in Fig. 2⇓.
Flow cytometric testing of the mother and the offspring were conducted with the “aberrant” variant to display cell surface staining; hence, anti-HLA-A1 activity in the presence of haplotype a but not of c. A, Fluorescence intensity of mAb 13th IHWC 0003 (HLA-A1, 36) to B-LCL BY00037 (green line) and BY00039 (red line). B, Fluorescence intensity of mAb 13th IHWC 0048 (HLA-A24 full) to B-LCL BY00037 (green line) and BY00039 (red line). Blue lines in both histograms represent negative controls with irrelevant mAb HLA-A3 (13th IHWC 0033).
HLA-A alleles (including HLA-A*01 and HLA-A*01 aberrant allele (A*0118N) reactivity patterns by flow cytometrya
IEF analysis shows that the HLA-A H chains in the mother’s cells are not associated with β2m, because the HLA-A*01 H chain was not immunoprecipitated from mother’s cell lysate by the mAb W6/32. In contrast, we detected the presence of an Ag immunoprecipitated by the mAb HC-A2 that reacts only with the free H chain (Fig. 3⇓). The IEF of this Ag shifts a little more acidic as compared with the wild-type HLA-A*01 H chain. This Ag is an unlikely HLA-G H chain because the mAb W6/32, which also recognizes HLA-G, does not precipitate this component from the mother’s cells. Thus, the HLA-A*01 aberrant allele H chains are produced in the mother’s cells but fail to associate with β2m and be expressed on the cell surface.
One-dimensional IEF gel. Cells from the mother (lanes M) and two sons (lanes 1 and 5) were metabolically labeled with [35S]methionine and solubilized with 1% Nonidet P-40. Ags immunoprecipitated by the indicated mAbs were subjected to one-dimensional IEF gel electrophoresis using a vertical slab gel apparatus. Gels were then processed for fluorography.
Discussion
In this report we have used several mAbs studied during the 13th International Histocompatibility Workshop to confirm the previous serological findings obtained with HLA class I Ag-specific alloantisera. FACS analysis of cells stained with HLA class I Ag-specific mAbs demonstrated that the HLA-A*01 was undetectable on cells from the mother and two children carrying the allele with the novel nucleotide substitution, indicating that the A*01 aberrant allele is not expressed on the cell surface (A*0118N). As reported previously (13), this family was also unusual because the paternal (a) and the maternal (c) HLA haplotypes, although identical in all MHC class I and class II alleles, demonstrated a lack of serological reactivity of the cells of the mother and two of her children with HLA-A*01 Ag-specific alloantibodies. DNA typing techniques showed that the HLA-A*01 aberrant allele gene was detectable in these individuals. DNA sequencing demonstrated a substitution of guanine by cytosine at the nucleotide position 215 that resulted in a substitution of arginine (CGG) by proline (CCG) at codon 48 (R48P). Other mutations in the coding region sequence of the HLA-A*0118N allele (exons 4–7; data not shown) were not detected. The shifting of the products of this aberrant allele to a more acidic position, as shown by IEF analysis, is consistent with the substitution of the positively charged arginine by the nonpolar proline. The mAb W6/32 (IgG2a) recognizes HLA-A, -B, -C, and -G complexes and does not react with free H chains. As expected, in the IEF analysis this mAb did not precipitate this Ag (HLA-A free H chain), the Ag precipitated by mAb HC-A2 reacting to HLA-A free H chains (HLA-A1 aberrant allele product) and very unlikely to be HLA-G.
It is important to mention that the association of the β2m and the HLA class I H chain is stabilized by numerous interactions involving residues of the β2m (20, 21). The most significant interaction site is Asp53, which is located in the β bulge of β2m and forms hydrogen bonds to Gln32, Arg35, and Arg48 in the α1 domain of the HLA class I H chain (22). These three residues site as a triangle (Fig. 4⇓a) that provides a very stable interaction with Asp53 on β2m. The substitution of arginine, a positively charged and very hydrophilic residue, by proline, a nonpolar hydrophobic residue, induces important changes in the physicochemical properties of one of the most significant HLA class I H chain-β2m interacting sites (Fig. 4⇓b) that may inhibit heterodimerization of the HLA class I H chain with β2m (13). In addition, we also demonstrate the transcriptional activity of the HLA-A1 H chain by the detection of H chain mRNA by an allele-specific RT-PCR assay (data not shown).
Schematic representation of the residues at the HLA heavy α-chain involved in the contact with β2m (modified from reference 17 ). A, The contact residues in the α1 chain are represented by black spheres (•). The replacement at position 48 (arginine by proline) is shown as a white sphere (○). B, The substitution R48P causes significant physicochemical changes (positively charged and hydrophilic Arg by the nonpolar, hydrophobic Pro) in the interaction of residue complex (Gln32, Arg35, and Arg48) at the α1 chain with Arg53 of β2m.
This family included two similar HLA haplotypes with different paternal origins: (a), A*010101-B*07021/26-Cw*07021-DRB1*1501-DRB5*0101-DQB1*0602-DPB1*0401; (b), A*3001-B*1402/03-Cw*0802-DRB1*0101-DQB1*0501-DPB1*1301; (c), A*0101nv-B*07021-Cw*07021-DRB1*1501-DRB5*0101-DPB1*0401; and (d), A*2402-B*39062-Cw*07021-DRB1*0801-DQB1*0402-DPB1*0301. It is rare to find families, such as the family described in this report, in which the parents share an HLA identical haplotype. In such families, there is a 25% probability of finding children with haplotype homozygosity. This unusual family permitted the study of alloreactivity in two family members that showed a lack of mixed lymphocyte reaction and alloreactivity by a CML assay. Unidirectional CML assays between the lymphocytes of the propositus (the mother, A*0118N) and those of her HLA identical son (A*0101 allele carrier) showed that the aberrant allele lacks functional expression because the T cells of the son cannot recognize the aberrant allele. In contrast, the mother’s T lymphocytes recognize the HLA-A*0101 allele expressed on the cells of one (sibling 3) of two of her children tested who were HLA identical to her but carried the HLA-A*010101. In addition, two of her children were homozygous for HLA-A*01, HLA-A*010101 and HLA-*0118N, her siblings 5 and 6. As expected, these two family members are MHC identical and nonreactive in a MLC, which primarily detects differences in HLA class II alleles (14). In this regard, it is well known that cell-mediated lysis requires activation of CD8+ T cells together with activation of CD4+ T cells to produce significant MLC reactivity to influence cytotoxic CD8+ T cells. However, in this report we documented for the first time that a weak but significant CML can be detected in the absence of MLC reactivity. We have been able to demonstrate that the percentage of cytotoxicity between the reaction of the lymphocytes of the propositus, the mother reacting with two of her children carrying the HLA-A*010101, was significant based on comparison with a large number of CML assays performed in our laboratory (14). Our results showed weak but significant cytotoxicity compared with the highest ratios found in HLA-identical siblings. In the present study we demonstrated that the carrier of the allele A*0118N can elicit an alloreactive T cell response against the allele A*0101. Therefore, it appears that the sole presence of a H chain protein in the carrier of the allele A*0118N does not tolerize against the intact heterodimer formed by the H chain of A*0101 and β2m. Because of a single amino acid difference between the alleles A*0118N and A*0101, a T cell response was generated. Also, the finding of an alloreactive T cell response against the allele HLA-A*0101 in the mother of this family does not allow us to determine whether the detectable levels of this response resulted from “natural alloreactivity” or if it was the result of previous sensitization from multiple exposures against HLA-A*0101 through pregnancy; some cytotoxic T cells may recognize HLA molecules encoded by other alleles by direct recognition, loaded with antigenic peptides including the amino acid Arg48 peptide derived from the H chain of HLA-A*0101 in the absence of lymphocyte priming such as that provided by the MLC. But cytokines such as IL-12 and IFN-γ are produced by Th cells to influence CD8+ T cell activity (23). The weak response against the mismatch in HLA-A*01, in the absence of incompatibility in HLA class II alleles between stimulator and responder individuals, could result in stimulation by minor histocompatibility Ags that may have induced weak T cell responses (24, 25). In vivo sensitization through pregnancy and transplantation is required to reveal significant T cell responses to minor histocompatibility Ags (26). Also, it is possible that the low response may also have resulted from the tolerizing effect caused by the presence of a self-protein (A*0118N) in the responder individual that has high homology with the stimulating mismatched allele, *A*0101.
A T cell response such as that produced by the peptides of the H chain of A*118N has implications for understanding the thymic selection of the alloreactive T cell repertoire; the presentation of the peptide sequences of the aberrant allele on self-MHC class I molecules might induce thymic positive selection of thymocytes capable of binding with aberrant allele-derived peptide-MHC complexes with sufficient affinity for self-MHC for positive selection and peripheral survival (27).
Also, the relevance of mismatching for aberrant alleles such as that described in this study could have adverse results in the allogeneic transplantation of organs and tissues. In this context, a recipient carrying the wild-type allele receiving organs carrying the aberrant allele could result in graft rejection mediated by cytotoxic T cells. In the case of an organ donor carrying the allele A*0118N, the responses against peptides derived from this allele would rather be weaker or delayed, involving direct T cell recognition as minor histocompatibility. Less likely, but with lower impact in clinical outcome, the role of indirect allorecognition can play a role in mismatches involving the allele A*0118*N. It is possible that the aberrant protein encoded by the allele A*0118N could be shed to the extracellular compartments and, in subsequent stages, be captured by host APCs and presented to CD4+ T cells through the indirect presentation pathway inducing proliferative responses (26). Indirect recognition could also be involved in the mechanisms causing graft vs host disease in transplants, including allogeneic immunocompetent cells.
In bone marrow transplantation, if an HLA-identical donor cannot be identified for a patient carrying the allele A*0118, the unrelated donors homozygous for the other HLA-A allele of the patient could perhaps be the best candidates. In this scenario the peptides derived from the allele A*0118N could potentially be recognized as minor histocompatibility Ags that could target for graft vs host disease. The presence of multiple cleavage sites by human proteasomes in the sequences of the mature protein of the A*0118N allele and the generation of peptides from the H chain of A*0118N by a web-based available program (16, 17) indicated that peptides spanning residue 48 could be generated by the human proteasomes. Therefore, it is conceivable that one or more peptides derived from the intracellular protein A*0118N could be processed through the intracellular Ag processing pathway and be presented in the context of HLA class I and possibly by HLA class II molecules acting as minor histocompatibility Ags (28). The presentation of peptides derived from the H chain of HLA-A*0118N would depend on the peptide-binding capabilities of the other HLA alleles matched between the patient and donor. Therefore the mismatch in HLA-A*0118N would not be relevant in transplant pairs where the matched HLA alleles do not have the capacity of binding peptides derived from A*0118N.
Furthermore, it is important to point out that the assembly of HLA class I molecules is the result of a coordinated and regulated series of interactions with chaperones and accessory molecules residing within the endoplasmic reticulum (29). Some multiprotein interactions, such as those with calnexin, calreticulin, tapasin, and the thiol-oxidoreductase Erp57, participate in the pathway for assembly of asparagine-glycosylated proteins in the endoplasmic reticulum (29, 30). The peptide-loading complex is formed during the later stages of assembly and includes tapasin and TAP (30). These processes do not occur when the alleles are not expressed on the surface of nucleated cells that produce “null alleles”. The causes of the nonexpression of the “null alleles” reported thus far include several mechanisms such as: 1) exonic substitution; 2) exonic deletion; 3) exonic insertion; and 4) splicing modification (reviewed in Ref. 3). Previous studies have described mutations in the HLA-A*01 gene that causes the generation of an early stop codon in the fourth exon (31, 32).
In summary, we described an aberrant HLA-A allele whose causes of nonexpression include an exonic substitution that result in the defect of heterodimer formation between the HLA class I H chain and β2m, as demonstrated by IEF (11) using an Ab that reacts with the free H chain of HLA-A alleles. Family studies of this aberrant allele were unusual, because the carrier (mother) and one of her sons were HLA identical except for the presence of the HLA-A heterodimer in her son’s cells. The lymphocyte of the carrier of the aberrant allele produced CD8 cytotoxicity against the lymphocyte of her son carrying the heterodimer. It is possible that the aberrant allele A*0118N may behave as a minor histocompatibility Ag presented by APCs, inducing direct allorecognition by cytolytic T cells. This report provides the first example of the generation of CTLs in the absence of the proliferation of CD4+ T cells (MLC) and the possible implications of HLA aberrant alleles in solid organ and stem cell transplantation in humans.
Acknowledgments
We thank Dr. H. Ploegh, Harvard Medical School, Dr. Arend Mulder, Leiden University, and Dr. J. H. Lee, One λ Inc. for generously providing anti-HLA mAbs.
Disclosures
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 supported by Public Health Service Grants HL29583 and HL59838 from the National Heart, Lung and Blood Institute and CA67108 from the National Cancer Institute, National Institutes of Health..
↵2 I.A., Z.C.W., J.Z., and M.F.-V. contributed equally to this work.
↵3 Address correspondence and reprint requests to Dr. Edmond J. Yunis, Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. E-mail address: edmond_yunis{at}dfci.harvard.edu or Dr. Marcelo Fernandez-Vina, Division of Pathology and Laboratory Medicine, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030. E-mail: mfernand{at}mdanderson.org
↵4 Abbreviations used in this paper: B-LCL, B lymphoblastoid cell line; β2m, β2-microglobulin; CML, cell-mediated lysis; FWS, flow washing system; IEF, isoelectric focusing.
- Received July 13, 2006.
- Accepted September 29, 2006.
- Copyright © 2006 by The American Association of Immunologists
References
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