Cynthia McSherry

A multigene family on human chromosome 12 encodes natural killer-cell lectins

We previously isolated a series of cDNA clones designated NKG2-A, B, C, and D from a human natural killer (NK) cell library. These transcripts encode a family of type II integral membrane proteins having an extracellular Ca2+-dependent lectin domain. The predicted peptides share structural similarities and amino acid sequence similarity with known receptor molecules. In this report, the genomic organization and mRNA expression of each of the genes were studied by using transcript-specific probes. Southern blot experiments reveal that the probes cross-hybridize with a maximum of five genes at high stringency. By probing a Southern blot prepared from a series of hamster/human hybrid somatic cell lines, we demonstrated that all of the hybridizing fragments occur on human chromosome 12. No gene rearrangement and little restriction fragment length polymorphism (RFLP) was observed with these probes. mRNA expression of the NKG2 genes occured in NK cells and some T cells but not in other hematopoietic cell types or in other tissues tested. Each of the transcripts occurred in all three of the NK cell lines tested: however, the genes were differentially regulated in T cells. NKG2-D was expressed in nine of fourteen T-cell clones or lines in the panel, whereas NKG2-A/B was expressed in three and NKG2-C was expressed in only one. Expression of each of the transcripts was upregulated following T-cell growth factor (TCGF)-induced activation of a cloned NK cell. The limited distribution of these proteins and their sequence similarity with known receptor molecules suggest that they may function as receptors of human NK cells.

Organ-specific patterns of donor antigen-specific hyporeactivity and peripheral blood allogeneic microchimerism in lung, kidney, and liver transplant recipients

Although their relative importance and interaction are unclear, donor antigen(Ag)*-specific hyporeactivity and allogeneic microchimerism have been associated with improved long-term graft outcome and a lower incidence of chronic rejection in solid organ transplant recipients. We have postulated that a critical level of donor antigen, for a critical time period, is necessary to develop and maintain donor antigen-specific hyporeactivity; both the level and the time may differ by organ transplanted. In our current study, we tested donor antigen-specific hyporeactivity and peripheral blood allogeneic microchimerism in liver and kidney recipients and compared these values with our previous findings in lung recipients. We tested 25 liver recipients at 12 to 29 months posttransplant: 10 (40%) had developed donor antigen-specific hyporeactivity; 5 (20%), peripheral blood allogeneic microchimerism. For all but 1 of the chimeric and hyporeactive recipients, the level of donor cells was very low (< 1:20,000). Five hyporeactive recipients and all 15 donor antigen-responsive recipients did not have detectable levels of peripheral blood microchimerism. No chronic rejection has developed in any of these recipients to date--however, a lower incidence of acute rejection was observed for those recipients with donor antigen-specific hyporeactivity (30% versus 60% without) or with peripheral blood allogeneic microchimerism (20% versus 55% without) (P = ns). These results differ from our previous findings in 19 lung recipients: at 12 to 18 months posttransplant, 35% of them had developed donor antigen-specific hyporeactivity; 47%, peripheral blood allogeneic microchimerism. All donor antigen-specific hyporeactivity recipients as well as some donor antigen-responsive recipients had peripheral blood allogeneic microchimerism. We expanded our current study to include 26 recipients and a quantitative estimate of the level of allogeneic microchimerism. We observed that the hyporesponsive recipients tended to have higher levels of donor cells in their peripheral blood (> 1:6,000) than did the responsive recipients. We previously reported that 22% of kidney recipients had developed donor antigen-specific hyporeactivity at 12 to 18 months posttransplant. In our current study of 33 kidney recipients, we observed peripheral blood allogeneic microchimerism in 7 (21%) at 12 to 18 months posttransplant. The level of donor cells was very low (approximately 1:75,000), with no correlation between donor antigen-specific hyporeactivity and peripheral blood allogeneic microchimerism at the time point tested. These studies emphasize the organ-specific nature of the development of donor antigen-specific hyporeactivity and the persistence of peripheral blood allogeneic microchimerism. Donor antigen-specific hyporeactivity correlates with very low levels of donor cells in liver recipients, while a higher critical level of donor cells is important in lung recipients. Additional sequential early posttransplant studies are necessary to further define the possible interrelationship between donor antigen and the development and maintenance of donor antigen-specific hyporeactivity.

Natural killer lectin-like receptors have divergent carboxyl-termini, distinct from C-type lectins

The mode of recognition of target cells by natural killer (NK) lymphocytes is unknown. Three related groups of NK cell-specific molecules may have receptor function (Ly49, NKR-P1 in rodents and NKG2 in humans). These NK lectin-like receptors (NKLLR) are type II integral membrane proteins with extraeellular domains homologous to Ca2+-dependent animal lectin carbohydrate recognition domains (C-type CRDs; Chambers et al. 1993). The NKG2 family consists of eDNA clones NKG2-A and -C and the more distantly related NKG2-D. Other NKG2 genes may exist (Yabe et aI. 1993). In order to isolate additional gene products related to the NKG2 family, an NK cell eDNA library (Yabe et al. 1993) was screened with the NKG2-C-specific probe at low stringency and with a nearly fulMength NKG2-A probe. Four cDNA clones with a novel sequence designated NKG2-E were isolated and sequenced. Other clones isolated were identified as either NKG2-A, C [including an NKG2-C clone lacking the transmembrane domain, NKG2-C 2 2 0 2 8 4 base pairs (bp)] or E. NKG2-E encodes a protein 261 amino acid long, sufficiently divergent from NKG2-C to be a product of a different gene. NKG2-E is 95% homologous to NKG2-C over the first 191 amino acid residues, then both sequences diverage (76% homologous over the 29 subsequent residues). An Alu repeat encodes the 15 carboxy-terminal residues of NKG2-E, including an arginine instead of a terminal cysteine present in NKLLRs and C-type CRDs. Identical NKG2-Esegments with anAlu repeat ( 600 770 bp) were sequenced from a different cDNA library by TA cloning of nested polymerase chain reactionderived products. Alu repeats have been reported in coding regions of a few other proteins (Claverie 1992). In a northern blot panel, the NKG2-C specific probe (98% bp homology with NKG2-E) hybridized to mRNAs equivalent in size to NKG2-C or E ordy in NK ceils and not other ceil lines (Yabe et al. 1993). C-type lectins have been classified into groups based on CRD sequence similarity. This classification coincides with the type of protein of which the CRDs are a part (i. e., group I: proteoglycans, group n : type I/receptors, group llI: collagen domains, group IV: selectins). Group I and II CRDs are encoded by three exons, whereas a single exon encodes group 111 and IV CRDs. The exon boundaries within these groups are highly conserved (Bezonska et aI. 1991). NKLLRs share several characteristics with group II C-type lectins. Both groups are type 11 integral membrane proteins with a smiliar gene structure. The CRD of Ly49, NKR-P1 and group ]1 CRDs are encoded by three exons with the same boundary positions (Wang et al. 1991; Giorda et aI. 1992; Bezouska et aI. 1991). Despite the apparent similarity between NKLLRs and group lI C-type iectins, a previous computergenerated comparison of lectin domains has demonstrated that NKLLRs constitute a separate group from C-type Iectins (Chambers et al. 1993). The reason for this discrepancy is revealed in a sequence comparison of NKG2-E to related proteins (Fig. 1). The lectin domain of C-type lectins (groups I I V ) and NKLLRs was first divided into two portions. The beginning of the NKG2-E/C divergence (which corresponds to the last coding exon of Ly49, NKR-P1 and group 1I C-type) was used as the dividing point. Only the COOH-terminal segments of NKLLR are clearly distinct from the C-type Iectins. The NH2 segments of NKLLR CRDs are related to groups I and II C-type CRD segments, as expected. Of note, highly conserved calcium and carbohydrate binding residues found in C-type CRDs (Drickamer 1992) are absent from the COOH segment of NKLLRs. In summary, NKLLRs are closely related to group II C-type lectins, although a significant evolutionary divergence seems to have occurred to the portion corresponding to the carboxy terminal coding exon. Should the carboxy terminal segment of NKLLRs serve as the Iigand portion of the molecule, as is the case for C-type lectin (Weis et al. 1992), one can predict different and varied NKLLR binding specificities from C-Type CRDs.

Characterization of a novel gene (NKG7) on human chromosome 19 that is expressed in natural killer cells and T cells

NKG7 is a cDNA clone generated from a human NK-cell clone. The DNA and predicted aa sequence of NKG7 is not homologous with any previously reported genes or peptides. NKG7 mRNA is expressed in activated T cells and in A-LAK cells isolated from the peripheral blood of normal individuals, and in normal human kidney, liver, lung and pancreas. Furthermore, NKG7 mRNA is expressed at high levels in TCR gamma delta-expressing CTL clones, and in some TCR alpha beta-expressing CTL clones (both CD4+ and CD8+), but is not expressed in other TCR alpha beta-expressing CTL clones and in cell lines representing B cells, monocytes, and myeloid cells. NKG7 mRNA is not expressed in normal human brain, heart, or skeletal muscle. Southern hybridization of NKG7 suggests that NKG7 is a single-copy gene localized to chromosome 19. A hydropathicity profile of the predicted 148 aa polypeptide indicates that NKG7 is a type-I integral membrane protein with a 38-aa extracellular domain and a 61-aa cytoplasmic domain. These results indicate that the NKG7 gene encodes a novel cell surface protein expressed in several cell types, including NK cells and T cells.

Peripheral blood allogeneic microchimerism in lung and cardiac allograft recipients

We have been investigating two parameters, donor antigen‐specific hyporeactivity and peripheral blood allogeneic microchimerism, to determine whether these parameters will predict a chronic rejection‐free state and which recipients may be candidates for steroid withdrawal. We have identified donor antigen‐specific hyporeactivity for 33% (16/48) of lung and 23% (11/47) of heart recipients. For both organ groups, the hyporeactive subgroup experienced a lower incidence of chronic rejection. The probability of donor antigen‐specific hyporeactivity predicting a chronic rejection‐free state is 100% for lung and 91% for heart recipients. We have identified peripheral blood allogeneic microchimerism for 77% (20/26) of lung and 36% (9/25) of heart recipients tested at 12–18 months posttransplant. Donor antigen‐specific hyporeactivity correlates with a critical level of donor cells in lung recipients; the probability of high peripheral blood allogeneic microchimerism levels predicting a chronic rejection‐free state in lung recipients is 100%. The results in heart recipients are not as clear with a short‐, but not long‐term, trend of higher chimerism levels correlating with the development of donor antigen‐specific hyporeactivity. These results illustrate the usefulness of immune parameters to predict long‐term graft outcome in an organ‐specific manner. J. Leukoc. Biol. 66: 306–309; 1999.

Correlation of donor antigen-specific hyporeactivity with allogeneic microchimerism in kidney and lung recipients

Our previous studies indicate donor antigenspecific hyporeactivity is a useful marker for identifying solid organ transplant recipients at low risk for immunological complications; the hyporeactive subgroup experiences a lower incidence of chronic rejection. One purpose of the current study was to determine whether hyporeactivity could be identified in pediatric kidney recipients and whether it correlated with improved graft outcome. Of 18 pediatric kidney recipients tested, 6 (33%) had developed donor antigen-specific hyporeactivity. All 18 experienced good graft outcome. Second, we determined whether donor antigen-specific hyporeactivity correlates with peripheral blood microchimerism and outcome in adult kidney recipients. Our previous studies of lung recipients demonstrated development of obliterative bronchiolitis in recipients with microchimerism who remain responsive, but not in recipients who had become hyporesponsive to donor antigen. Preliminary results in our current study of 23 adult kidney recipients indicate microchimerism for 6 (26%): 4 hyporesponsive and 2 responsive to donor antigen. Microchimerism was not detected for 17 recipients: 6 hyporesponsive and 11 responsive to donor antigen. One hyporesponsive/chimeric patient and 4 recipients negative for both parameters have been diagnosed with biopsy-proven chronic rejection. In summary, both hyporeactivity and chimerism are found at a higher frequency in lung than kidney recipients. Unlike lung recipients, not all hyporesponsive kidney recipients had peripheral blood chimerism. Additional numbers are needed to determine if microchimerism correlates with donor antigen-specific hyporeactivity or graft outcome.

Pretransplant Exposure to Donor HLA-DR Antigen in Random Transfusion Units and the Development of Donor Antigen-Specific Hyporeactivity

Our previous studies have shown that the in vitro assay of donor antigen-specific hyporeactivity is a useful marker for identifying solid organ transplant recipients (kidney, lung and heart) at low risk for immunologic complications (i.e., late acute rejection episodes and chronic rejection). Donor antigen-specific hyporeactivity is defined as a significantly decreased post- vs. pretransplant proliferative response to donor antigens while response to third-party controls remains unchanged. We analyzed whether exposure to the same HLA-DR antigen pretransplant via random blood transfusion and posttransplant via the transplanted organ influenced the development of hyporeactivity. Thirty previously nontransfused recipients, each receiving two 150 ml pretransplant random blood transfusions, were assessed for hyporeactivity at 1 year posttransplant. Of the 12 recipients with pretransplant exposure to kidney HLA-DR via transfusions, 6 (50%) developed hyporesponsiveness; in contrast, of the 18 recipients who were not preexposed, only 3 (15%) exhibited this form of immunomodulation. Of interest, 2 of the 3 hyporesponsive recipients who were not preexposed, received units containing HLA-DR antigens previously shown to share crossreactive epitopes with the kidney HLA-DR. In conclusion, these results suggest a increased incidence in the development of hyporeactivity in patients receiving pretransplant transfusions which share an HLA-DR antigen with the transplanted kidney.