Influence of killer immunoglobulin-like receptor/HLA ligand matching on achievement of T-cell complete donor chimerism in related donor nonmyeloablative allogeneic hematopoietic stem cell transplantation.

Achievement of complete donor chimerism (CDC) after allogeneic nonmyeloablative hematopoietic stem cell transplantation (NMHSCT) is important for preventing graft rejection and for generating a graft-vs-malignancy effect. The alloreactivity of NK cells and some T-cell subsets is mediated through the interaction of their killer immunoglobulin-like receptors (KIRs) with target cell HLA/KIR ligands. The influence of KIR matching on the achievement of T-cell CDC after NMHSCT has not been previously described. We analyzed 31 patients undergoing T-cell replete related donor NMHSCT following fludarabine and 200 cGy TBI. Recipient inhibitory KIR genotype and donor HLA/KIR ligand matches were used to generate an inhibitory KIR score from 1 to 4 based upon the potential number of recipient inhibitory KIRs that could be engaged with donor HLA/KIR ligands. Patients with a score of 1 were less likely to achieve T-cell CDC (P=0.016) and more likely to develop graft rejection (P=0.011) than those with scores greater than 1. Thus, patients with lower inhibitory KIR scores may have more active anti-donor immune effector cells that may reduce donor chimerism. Conversely, patients with greater inhibitory KIR scores may have less active NK cell and T-cell populations, which may make them more likely to achieve CDC.

Survival of AML patients receiving HLA-matched sibling donor allogeneic bone marrow transplantation correlates with HLA-Cw ligand groups for killer immunoglobulin-like receptors.

The reactivity of natural killer cells and some T-cell populations is regulated by killer immunoglobulin-like receptors (KIR) interactions with target cell HLA class I molecules. Such interactions have been suggested to influence outcomes after allogeneic hematopoietic stem cell transplantation, particularly for myeloid malignancies and with T-cell depletion. Donor KIR genotypes and recipient HLA KIR ligands were analyzed in 60 AML patients receiving T-cell replete, HLA-matched-related donor allogeneic bone marrow transplants. Patients were categorized according to their HLA inhibitory KIR ligand groups by determining whether or not they expressed: HLA-A3 or -A11; HLA-Bw4 and HLA-Cw groups (homozygous C1, homozygous C2 or heterozygous C1/C2). Heterozygous C1/C2 patients had significantly worse survival than those homozygous for C1 or C2 (5.8 vs 43.5 months, respectively, P=0.018) and the C1/C2 group had a higher relapse rate (47 vs 31%, respectively, P=0.048). Multivariate analysis found C1/C2 status to be an independent predictor for mortality (P=0.007, HR 2.54, confidence interval 1.29-5.00). C1/C2 heterozygosity was also associated with a delayed time to platelet engraftment, particularly for those with concurrent HLA-Bw4 expression (P=0.003). Since C1/C2 heterozygotes have a greater opportunity to engage inhibitory KIRs than do C1 or C2 homozygotes, they may more effectively inhibit KIR-positive NK- and T-cell populations involved in graft vs leukemia responses.

Total energy expenditure, body fatness, and physical activity in children aged 6-9 y.

BACKGROUND: The recent worldwide increase in the prevalence of childhood obesity may be due in part to a decrease in children's physical activity levels. OBJECTIVE: The current study of children in the years just before puberty aimed to 1) measure total energy expenditure (TEE) by use of the doubly labeled water (DLW) method, 2) determine the proportion of TEE related to physical activity, 3) investigate the relations between measures of physical activity and body fatness, and 4) investigate possible sex differences in these relations. DESIGN: The DLW technique was used to measure TEE over 10 d in 106 healthy children (52 boys) aged 7.8 +/- 0.9 y (x +/- SD). Fat-free mass, and hence fat mass, was derived from the (18)O dilution space. Resting energy expenditure (REE) was calculated with use of the Schofield equations. Physical activity level was calculated as TEE/REE. RESULTS: Mean TEE in both boys (7871 +/- 1135 kJ/d) and girls (7512 +/- 1195 kJ/d) was significantly different (P < 0.0001) from FAO/WHO/UNU recommendations (13% and 9% lower, respectively). There was no significant difference in physical activity level between boys (1.69 +/- 0.22) and girls (1.71 +/- 0.23). In boys but not girls, physical activity level was inversely correlated with BMI (r = -0.37, P < 0.01), fat mass (r = -0.46, P < 0.005), and percentage of body fat (r = -0.50, P < 0.0001). CONCLUSIONS: In boys but not girls, percentage of body fat is inversely associated with physical activity level. Physical activity is one factor contributing to body fatness in boys, but additional factors may influence the size of the fat stores in girls.

Comparison of total energy expenditure and energy intake in children aged 6-9 y.

BACKGROUND: The accurate measurement of food intake in children is important for assessing nutritional status. OBJECTIVE: We sought to both compare measurements of energy intake (EI) from diet records and of total energy expenditure (TEE) by the doubly labeled water (DLW) method and to investigate misreporting of EI. DESIGN: Forty-seven children (22 boys and 25 girls) aged 7.4 +/- 0.8 y ( +/- SD) were recruited from 25 schools in western Sydney. TEE was measured by DLW over 10 d and EI by use of 3-d food records. Misreporting was defined as [(EI - TEE)/TEE] x 100%. RESULTS: Girls had a higher (P = 0.02) percentage of body fat (28.2 +/- 7.0%) than did boys (22.9 +/- 8.0%); otherwise there were no differences among sex. Although mean (+/-SD) values for EI (7514 +/- 1260 kJ/d) and TEE (7396 +/- 1281 kJ/d) were not significantly different, there was no significant correlation between EI and TEE. EI and TEE were 9% and 11% lower, respectively, than current World Health Organization recommendations for EI. The relative bias (mean difference, EI - TEE) was low at 118 kJ/d, but the limits of agreement (bias +/- 2 SD of the difference) were wide at 118 +/- 3345 kJ/d. Although the mean percentage of misreporting was low (4 +/- 23%), the high SD indicates large intraindividual differences between EI and TEE. The most significant predictor of misreporting was dietary fat intake (r(2) = 0.45, P < 0.0001). Misreporting was not associated with sex or body composition. CONCLUSIONS: In this age group, reported EI is not representative of TEE at the individual level. However, at the population level, 3-d food records may be used for surveys of EI by 6-9-y-old children.

Class II human leukocyte antigen genes and T cell receptor polymorphisms in patients with rheumatoid arthritis.

Human leukocyte antigen (HLA) typing was performed in 174 patients with rheumatoid arthritis and 222 white control subjects. Increases in HLA-DR4 and HLA-DR1 were observed as in previous studies. Each of these appeared to be inherited as dominant risk factors. Southern blotting with a DR-beta probe after digestion of genomic DNA with the restriction enzyme Bam HI showed seven bands. Three of them correlated with DR4 and were increased in rheumatoid arthritis patients. Subsets of DR4 were determined by polymerase chain reaction amplification followed by hybridization with oligonucleotide probes. Dw4 was increased in rheumatoid arthritis patients, and the frequency of the other subsets appeared to be similar in rheumatoid arthritis patients and control subjects. A polymorphism associated with the T cell receptor V-beta-8 gene family was significantly increased in rheumatoid arthritis patients.

Allogeneic lymphocyte stimulation by human spleen. Identification of cells that are very effective for presenting class II HLA antigens.

Human spleen cells were fractionated by percoll density gradient centrifugation and by sorting in the FACS with mixtures of fluorescent antibodies against T cells, B cells, monocytes, and Sig-bearing cells. Cells responsible for powerful MLR stimulation were class II HLA antigen-positive and were concentrated in preparations depleted of all the markers listed above. These cells represented 1-2% of the initial spleen cells. They were remarkably more active than other HLA class II antigen-positive cells. The procedure described allows rapid enrichment for the responsible cells. It should be useful for further characterization of these cells and for performing studies on their function.

Antigen presentation by adherent cells from human peripheral blood. Correlation between T-cell activation and expression of HLA-DQ and -DR antigens.

The ability of cells with different amounts of HLA-DQ or -DR to support T-cell proliferation in response to foreign antigens was investigated. Adherent cells were stained with monoclonal antibodies and sorted in the fluorescence-activated cell sorter (FACS) into (a) DQ-positive and DQ-negative subsets, with monoclonal anti-DQ; or (b) subsets expressing different density of DR determinants. Expression of HLA-DQ correlated with increased density of DR. The subset of cells expressing detectable DQ and increased density of DR was found to be more efficient in presenting mumps or tetanus toxoid antigen to T cells than were the DQ-negative, low-DR density adherent cells. Similar results were obtained with primary cultures of T cells from blood and with cloned antigen-specific T-cell lines, restricted by a single DR-subregion specificity. Our results suggest that quantitative variation in DR/DQ molecules expressed on monocytes correlates with their ability to support T-cell responses to nominal antigens. It is not clear whether this is due to only class II antigen density on the surface of the accessory cells or whether other factors are involved.

Different specificities of an HLA-DRw6 haplotype detected by alloreactive T lymphocytes.

DRw6 has been difficult to define serologically. In the present experiments we have developed T cell lines in order to characterize the components of a DRw6 haplotype. This was accomplished by priming T cells with allogeneic mononuclear cells mismatched for DRw6, Dw6, and MT2. Subsequently, three sublines with distinct reactivity patterns were derived by limiting dilution. The specificities detected by these sublines included: (a) a specificity found on a subset of cells positive for DRw6 which was inhibited by monoclonal antibodies against DS(DC), the human homologue of the murine IA-encoded molecules, (b) another DRw6-associated specificity blocked by an MT2-like antibody, and (c) an MT2-like specificity blocked by monoclonal antibodies reactive with a different MT2-associated determinant. These results show that more than one IE-like, as well as the DS/DC (IA-like) molecules, carry distinctive antigenic epitopes that can be recognized by allogeneic T cells. Primed T cell lines may be useful for a better definition of certain haplotypes which are at present difficult to characterize with serological reagents alone.

Cell mediated cytotoxicity against HLA-D region products expressed in monocytes and B lymphocytes. III. Inhibition by monoclonal antibodies and study of an HLA-B/DR recombinant.

Attempts to further define the antigens recognized by HLA-D-region specific cytotoxic lymphocytes were undertaken using monocytes and transformed B cell lines as target cells. Monoclonal antibody against framework determinants of HLA-DR antigens partially blocked cell mediated lysis, suggesting that at least a portion of the D-region specific cytotoxic cells recognized the HLA-DR determinants. The study of a family with an HLA-B/DR recombinant showed that the determinants recognized by allogeneic anti-HLA-D-region cytotoxic lymphocytes are encoded outside of HLA-B. In addition, cytotoxic lymphocytes specific for the HLA-D region could be generated with cells identical throughout the interval from HLA-A to B and disparate only to the left of HLA-B.

HLA-B27 polymorphism.

HLA-B27 is a serologic specificity that encompasses 25 different alleles that encode 23 different products (proteins): HLA-B*2701 to B*2723. These alleles are also called subtypes of HLA-B27, and they may have evolved from the most widespread subtype, B*2705. These subtypes are distinguished from changes mostly in exons 2 and 3, which encode the alpha 1 and alpha 2 domains of the B27 molecule, respectively. Occurrence of ankylosing spondylitis (AS) or related spondyloarthropathy (SpA) has thus far been documented in subjects possessing any one of the first ten (B*2701 to B*2710) subtypes studied. However, B*2706 in Southeast Asian and B*2709 in the Italian island population of Sardinia seem not to be associated with AS. The 13 most recent subtypes have not yet been studied for disease association. It is important to investigate which of them are and are not associated with AS and related SpA, and whether certain subtypes show any preferential association with some of the clinical features or forms of these diseases among the various ethnic/racial populations and geographic regions of the world. This is expected to provide clues as to the mechanism of disease association, and one of the strongest reasons to study the B27 subtypes is to learn the effects of the sequence variations on the peptide-binding specificity of the molecule. Among these peptides may be the putative arthritogenic peptide(s).

Antigen-specific HLA-restricted human T-cell lines. II. A GAT-specific T-cell line restricted by a determinant carried by an HLA-DQ molecule.

Human T-cell lines responsive to the polypeptide antigens GAT and (T,G)-A--L were developed. They were specific for the priming antigens and required the participation of accessory cells matched for HLA-linked determinants, as shown in family studies. In panel studies, the ability of accessory cells to present antigen was shown to be associated with HLA-D-region antigens. However, the specificity of these determinants did not fully correspond to any HLA antigens as currently defined. One GAT-specific subline, derived by limiting dilution, utilized a restriction determinant associated with, but distinct from, the DQw3 (MB3) allospecificity. Blocking studies with mouse monoclonal antibodies indicated that this restriction determinant was carried by HLA-DQ molecules. The epitopes recognized in these molecules appear to be distinct from the alloantigenic determinants currently defined by serology.

Antigen-specific HLA-restricted human T-cell lines. I. An MT3-like restriction determinant distinct from HLA-DR.

The results presented provide evidence that the HLA specificity known as MT3, BR4, or Hon7 can serve as a restriction epitope for the proliferation of certain T cells responding to mumps viral antigen. This restriction determinant was found to be HLA-linked in family studies, and to segregate centromeric to a crossover between HLA-B and DR in one family. In the population studied, the specificity was found to be associated with the DR antigens DR4, DR7, and DRw9, which are known to be associated with MT3. The ability of accessory cells to present mumps antigen in the context of this supertypic restriction determinant was blocked by a monoclonal antibody specific for MT3. Since MT3 (BR4, Hon7) has been shown to be expressed on molecules distinct from DR, our experiments suggest that such molecules are functionally important in antigen presentation to T cells.

The use of cellular reagents as an adjunct to typing for HLA-DRw10.

Two human T cell lines recognizing DRw10 were generated. One line was directly alloreactive, while a second cloned line recognized mumps virus in the context of a DRw10-like determinant. In panel studies, the lines responded only to DRw10 cells, and not to DR1 (DRw10-) cells. When compared to 5 anti-DRw10 sera, the cellular reagents gave virtually identical results. The generation and use of such cellular reagents may be of particular value for the identification of HLA-D-region specificities that are rare, poorly defined, or for which serologic typing reagents are limited.

Cell-mediated cytotoxicity against HLA-D-region products expressed in monocytes and B lymphocytes. IV. Characterization of effector cells using monoclonal antibodies against human T-cell subsets.

The cytotoxic effector cells that recognize HLA-D-region determinants and their precursors were characterized using monoclonal antibodies against human T lymphocytes and T-cell subsets. These studies were performed using MLC combinations giving rise to cytotoxic cells specific for both class I (HLA-A, B, C) and class II (HLA-D-region) antigens, and then tested against target cells displaying relevant antigens of only one class. Both class I and class II specific CTL (cytotoxic T lymphocytes) were inhibited by treatment with the OKT3 monoclonal antibody and complement, indicating that the effector cells were T lymphocytes. A major portion of class II specific CTL, and their precursors, were inhibited by OKT4 and complement, while class I specific CTL from the same cultures were not. The T4+T8- cell subset has previously been associated with helper or inducer functions, but not with cytotoxicity. The present findings indicate that class I and class II specific CTL, and their precursors, are different on the basis of the class of target antigen recognized and on the basis of surface phenotype detected by monoclonal antibodies.

Accessory cell function of human endothelial cells. I. A subpopulation of Ia positive cells is required for antigen presentation.

The expression of class I and class II HLA antigens on preparations of human endothelial cells, isolated from umbilical cord veins, was investigated by immunofluorescence. While virtually all endothelial cells expressed class I antigens, less than 1% were positive for class II antigens, as detected with a panel of 10 different monoclonal antibodies. Antigen specific T cell lines proliferated in response to mumps antigen in the presence of endothelial cells or blood monocytes from HLA-DR matched donors. However, these T cell lines failed to respond in the absence of accessory cells or when accessory cells from HLA-D-region mismatched cord donors were used. The ability of both monocytes and endothelial cells to present antigen was abolished by treatment of the cells with monoclonal antibodies specific for either class I or class II HLA antigens plus complement. Similar treatment with monoclonal antibodies specific for monocytes greatly reduced antigen presentation by endothelial cells. These results indicate that preparations of endothelial cells contain a subpopulation of Ia positive cells, distinct from monocytes, which are required for antigen presentation.

HLA-B27 and genetic predisposing factors in spondyloarthropathies.

With the mapping of the human genome having been completed, our ability to investigate and ideally better understand the genetic basis of rheumatic diseases is advancing at a rapid pace. Substantial evidence strongly favors a direct role for HLA-B27 in genetic susceptibility to ankylosing spondylitis and related spondyloarthropathies, although the underlying molecular basis has yet to be identified. HLA-B27 contributes only 16 to 50% of the total genetic risk for the disease, clearly indicating that other genes must be involved. However, no other putative disease genes have yet been absolutely proven. Potential genes include MHC (HLA class II, low molecular weight proteasome [LMP], transporter associated with antigen processing (TAP), tumor necrosis factor [TNF]-alpha, and major histocompatibility complex class I chain-related gene A (MICA), as well as non-MHC genes (IL-1RA, IL-6, IL-10, and CYP2D6). Genome-wide screens have identified other chromosomal areas of interest: 1p, 2q, 6p, 9q, 10q, 16q, and 19q. However, different studies have given conflicting results. HLA-B27 itself is a serologic specificity, which encompasses 25 different alleles that encode 23 different products (proteins): HLA-B*2701 to B*2723. These alleles may have evolved from the most widespread subtype, B*2705, and two of them, B*2706 in Southeast Asia and B*2709 in Sardinia, seem not to be associated with ankylosing spondylitis. The distinction between the disease associated and nonassociated subtypes may provide clues to the actual role of B27 in disease pathogenesis.

Restriction fragment length polymorphism of the human T cell receptor alpha gene. I. Two polymorphic restriction sites localized to different regions of the gene.

Previous studies have demonstrated restriction fragment length polymorphisms (RFLP) in the vicinity of the alpha and beta genes of the human T-cell receptor. In the course of experiments designed to discover additional polymorphic restriction sites, we found a new RFLP of the T-cell alpha gene recognized by the restriction enzyme Taq I. The site was localized to the interval between the most 3' joining (J) exon and the most 5' constant (C) region exon, about 7 kb distant from the previously described Bgl II polymorphic site which mapped to the vicinity of the 3' untranslated exon. With the use of these two polymorphic markers, four Ti-alpha alleles could be identified, allowing unambiguous assignment of all Ti-alpha genes in some families. These markers may be useful in identifying possible immune response genes or disease predisposition genes associated with the genes of the T-cell receptor for antigen.