Insulin-specific human T cells. Epitope specificity, major histocompatibility complex restriction, and alloreactivity to a diabetes-associated haplotype

T cells from an insulin-treated diabetic (ML, HLA DR1, w6) were stimulated in vitro with insulin, cloned at limiting dilution, and examined for their fine specificity and genetic restriction. T cell lines (TCL) derived from beef insulin stimulation were highly specific for epitopes on beef insulin, whereas pork insulin stimulation generated T cells that recognized determinants shared with beef insulin. Included among TCL reactive with pork insulin is one line (P2/9) that is autoreactive with human insulin. Antigen-presenting cells of known HLA type and monoclonal antibodies directed at class II major histocompatibility complex antigens were used to confirm the role of HLA-DR in restricting the response of insulin immune T cells. No preference or determinant selection within the donor's haplotypes was identified because either DR1 or DRw6 antigen-presenting cells could present the A loop of beef insulin. A TCL that recognized the A loop of beef insulin in association with DR1 was also alloreactive to HLA DR3, or a molecule closely linked to it, in the absence of insulin. A second T cell clone with insulin specificity and alloreactivity was also derived by allo stimulation of the donor's cells with DR3+ cells. When tested with a series of DR3+ stimulator cells, the alloreactivity was directed at diabetes-associated haplotypes. These data show that the T cell repertoire for insulin of a single diabetic donor encompasses that of multiple inbred animal strains and includes fine specificity for one to two amino acids, recognition of autologous insulin, and cross-reactivity with an allogeneic major histocompatibility complex antigen.     

Correlation between HbA1c, purified insulins, diabetic control, and insulin antibodies in diabetic children

Insulin antibodies were determined in sera from 38 children diagnosed as having juvenile diabetes for a duration of 0.7-15.2 years (median = 4.9 years). 8 children were treated with purified porcine insulins from the beginning of their disease, 16 children with bovine insulin NPH alone, and 14 children with non-purified, of whom 9 were later transferred to purified insulins. Serum insulin antibodies were measured by non-specific and specific methods using beef (B) and pork (P) antigens as described by Welborne and Sebriakova, respectively. 12/38 children had insulin binding levels similar to those of normal children, irrespective of the type of insulin used. The concentration of antibodies using radiolabelled B or P insulins as antigens were strongly correlated, by both the non-specific (p less than 0.01) and the specific (p less than 0.01) methods. Children with better score for diabetic control had significantly lower levels of insulin antibodies against B (p less than 0.05) and P (p less than 0.05) than those with poor diabetic control. There was also a significant positive correlation between mean HbA1c concentration and both B and P mean insulin antibody concentration (p less than 0.01). Finally, patients treated with purified porcine insulin had significantly lower levels of antibodies than patients with non-purified bovine insulin (p less than 0.05).     

T cell autoreactivity to insulin in diabetic and related non-diabetic individuals

T cell autoreactivity to insulin in type I diabetic and related non-diabetic individuals was analyzed. Peripheral T lymphocytes from both insulin-treated diabetic and untreated non-diabetic members of four families were found to proliferate in vitro in response to human insulin. T cell autoreactivity to insulin therefore does not appear to be diagnostic of the onset of type I diabetes. Highest T cell responses to human insulin were usually detected in insulin-dependent type I diabetes patients treated with a mixture of beef and pork insulin than with self insulin, the greater the dose of animal insulin the higher the T cell response. The T cell repertoires for self insulin appear to be similar in diabetics and non-diabetics based on their patterns of T cell reactivity to beef insulin, port insulin, human insulin, and various peptide of human insulin. The autoreactive T cells analyzed recognize two conformational epitopes of human insulin formed by interactions between A chain and B chain residues. One epitope is associated with the A chain loop and is present in the A1-A14/B1-B16 peptide, and the other epitope consists mainly of B chain residues located in the A16-A21/B10-B25 peptide. These two epitopes are present in amphipathic alpha-helical regions of insulin. HLA-DR (DR3, DR4, and DR5) and HLA-DQ (DQw2/DQw3) Ag can restrict these T cell responses to human insulin epitopes. The ability to detect insulin-specific autoreactive T cells in healthy non-diabetic individuals supports the hypothesis that autoreactive lymphocytes do not necessarily elicit autoimmune disease if present in an environment in which their activity is immunoregulated.     

HLA-DR1 (DRB1*0101) and DR4 (DRB1*0401) use the same anchor residues for binding an immunodominant peptide derived from human type II collagen.

Rheumatoid arthritis is an autoimmune disease in which susceptibility is strongly associated with the expression of specific HLA-DR haplotypes, including DR1 (DRB1*0101) and DR4 (DRB1*0401). As transgenes, both of these class II molecules mediate susceptibility to an autoimmune arthritis induced by immunization with human type II collagen (hCII). The dominant T cell response of both the DR1 and DR4 transgenic mice to hCII is focused on the same determinant core, CII(263-270). Peptide binding studies revealed that the affinity of DR1 and DR4 for CII(263-270) was at least 10 times less than that of the model Ag HA(307-319), and that the affinity of DR4 for the CII peptide is 3-fold less than that of DR1. As predicted based on the crystal structures, the majority of the CII-peptide binding affinity for DR1 and DR4 is controlled by the Phe(263); however, unexpectedly the adjacent Lys(264) also contributed significantly to the binding affinity of the peptide. Only these two CII amino acids were found to provide binding anchors. Amino acid substitutions at the remaining positions had either no effect or significantly increased the affinity of the hCII peptide. Affinity-enhancing substitutions frequently involved replacement of a negative charge, or Gly or Pro, hallmark amino acids of CII structure. These data indicate that DR1 and DR4 bind this CII peptide in a nearly identical manner and that the primary structure of CII may dictate a different binding motif for DR1 and DR4 than has been described for other peptides that bind to these alleles.     

Heterogeneity and specificity of human anti-insulin antibodies determined by isoelectric focusing

Anti-insulin antibodies are present in the majority of insulin treated diabetics, and in some cases these antibodies have been found to be highly specific for limited epitopes on the molecule. To determine how the human response differs from that seen in inbred animals, we have examined the heterogeneity and specificity of human anti-insulin antibodies by isoelectric focusing (IEF). In addition, we have used human insulin to examine the extent of autoreactivity in the serum of subjects treated with animal insulins. The majority of diabetic sera exhibited complex IEF spectra that were composed of discrete bands and unresolved smears. Autoradiography using 125I-beef, pork, and human insulin revealed some affinity differences; however, the predominant antibodies were capable of binding all insulins, including human. These specificity studies were extended by comparing competitive inhibition with excess cold insulins, and sera with highly specific binding of the A chain loop of beef insulin were identified. The spectra by IEF of these highly specific sera were found to be variable. Our results indicate that the majority of insulin-treated diabetics develop a heterogeneous antibody response that is more complex than the response of inbred animals and includes reactivity with autologous insulin. Although infrequent, individuals having antibodies directed at limited regions of the molecule can be identified and will provide valuable tools for dissecting this complex response.     

An influenza A virus-specific and HLA-DRw8-restricted T cell clone cross-reacting with a transcomplementation product of the HLA-DR2 and DR4 haplotypes

The clone TA10 is a T3+ T4+ T8- proliferative and cytolytic human T cell clone. This clone has been shown to be specific for the hemagglutinin of influenza A Texas virus and restricted by an HLA class II molecule associated with the DRw8-Dw8.1 phenotype. Here we show that TA10 and all of its subclones can also react with eight HLA-DRw8 negative, Epstein-Barr virus (EBV)-transformed cell lines or phytohemagglutinin blasts in the absence of influenza antigens. All of these cell lines are HLA-DR2/DR4 with a classic DR2 long haplotype. The only nonreactive HLA-DR2/DR4 cell line observed bears a DR2 short haplotype. Only heterozygous HLA-DR2/DR4 but not parental DR2 or DR4 EBV-transformed cell lines can be recognized by TA10, indicating that the cross-reacting determinant is a transcomplementation product between HLA-DR2 and HLA-DR4 haplotypes. DR-specific, but not DQ- or DP-specific monoclonal antibodies, inhibit in the proliferation assay and in the chromium release test both the DRw8-Dw8.1-restricted and the anti-DR2/DR4 reactions. These results show that HLA-DR-restricted, anti-viral human T cell clone can evidence cross-reactivity for allospecific class II molecules of the major histocompatibility complex, and human CTL can recognize transcomplementation products of class II HLA genes. In addition, the results suggest that a beta-chain coded for by an HLA-DR gene and associated with an alpha-chain coded for by a still unidentified but possibly HLA-DQ gene constitute this functional transcomplementation product.     

Quantitative expression of epitopes defined by murine monoclonal antibodies on DR molecules from different HLA haplotypes

Monoclonal antibodies 50D6 and 21r5, reactive with human class II molecules, were analyzed quantitatively by flow cytometry and cellular radioimmunoassay for their binding to cells of different HLA-DR types. Monoclonal antibody 50D6 bound equally to cells of all DR types tested except DR7, where no reactivity was observed. Monoclonal antibody 21r5 was reactive with all cells. However, the percentage of DR molecules at the cell surface expressing 21r5 epitope varied with the DR type and increased as follows: DR3 = DR7 less than DR2 less than DR5 less than DR4 less than DR1. MAb 50D6 reacted with an epitope spatially related to but distinct from the 21w4 epitope present on all DR molecules. The 50D6 epitope was shown to be present on isolated DR1 molecules.     

Assessment of the role of determinant selection in genetic control of the immune response to insulin in H-2b mice.

The immune response to insulin is regulated by MHC class II genes. Immune response (Ir) gene-linked low responsiveness to protein Ags can be mediated by the low affinity of potential antigenic determinants for MHC molecules (determinant selection) or by the influence of MHC on the functional T cell repertoire. Strong evidence exists that determinant selection plays a key role in epitope immunodominance and Ir gene-linked unresponsiveness. However, the actual measurement of relative MHC-binding affinities of all potential peptides derived from well-characterized model Ags under Ir gene regulation has been very limited. We chose to take advantage of the simplicity of the structure of insulin to study the mechanism of Ir gene control in H-2b mice, which respond to beef insulin (BINS) but not pork insulin (PINS). Peptides from these proteins, including the immunodominant A(1-14) determinant, were observed to have similar affinities for purified IAb in binding experiments. Functional and biochemical experiments suggested that PINS and BINS are processed with similar efficiency. The T cell response to synthetic pork A(1-14) was considerably weaker than the response to the BINS peptide. We conclude that the poor immunogenicity of PINS in H-2b mice is a consequence of the T cell repertoire rather than differences in processing and presentation.     

DR antigen expression on ovarian carcinoma cells does not correlate with their capacity to elicit an autologous proliferative response

Summary Expression of HLA-DR antigens by purified preparations of human ovarian carcinoma cells freshly isolated from surgical specimens was examined in parallel with the capacity of tumor cells to elicit a blastogenic response from autologous lymphocytes in mixed lymphocyte-tumor culture (MLTC) assay. Of 21 tumor preparations, 11 (52%) reacted with monoclonal antibodies 279 and/or 949 specific for a monomorphic determinant of HLA-DR antigens, with heterogeneous positivity, ranging between 30% and 95%. In this series of patients positive MLTC occurred in 8/21 individual experiments. The HLA-DR expression was proportionally similar in tumors giving positive MLTC (4/8=50%) and negative MLTC (7/13=53%). The lack of correlation between DR expression on tumor cells and stimulatory activity in autologous MLTC and the fact that DR-negative tumors could induce lymphocyte stimulation, support the hypothesis that blastogenesis occurs upon recognition of tumor-associated antigens, different from DR molecules, possibly tumor-specific antigens.     

Analysis of families at risk for insulin-dependent diabetes mellitus reveals that HLA antigens influence progression to clinical disease.

BACKGROUND: Individuals at risk for insulin-dependent diabetes mellitus (IDDM), with an affected first-degree relative, can be identified by the presence of islet cell antibodies (ICA). ICA-positive relatives progress at variable rates to IDDM and identification of those at highest risk is a prerequisite for possible preventative treatment. Those who develop IDDM may exhibit less genetic heterogeneity than their ICA-positive or ICA-negative relatives. Specific human leucocyte antigen (HLA) genes predispose to IDDM but could also influence the rate of progression of preclinical disease. Therefore, by comparing HLA antigen frequencies between first-degree relatives, we sought to identify HLA genes that influence progression to IDDM. MATERIALS AND METHODS: HLA antigen frequencies were compared in 68 IDDM, 53 ICA-positive, and 96 ICA-negative first-degree relatives from 40 Caucasoid families. Predictions were tested in a panel of 270 unrelated IDDM subjects. HLA typing was performed serologically (HLA class I and II) and by sequence-specific oligotyping (11th International Histocompatibility Workshop protocol) (HLA class II). ICA tests were measured by an internationally standardized indirect immunofluorescence assay. RESULTS: In general, known susceptibility class II HLA antigens increased in frequency and known protective class II HLA antigens decreased in frequency, from ICA-negative to ICA-positive to IDDM relatives. Thus, DR4 and DQ8 increased whereas DR2 and DQ6 decreased; DR3 and DQ2 increased from ICA-negative to ICA-positive relatives, but not further in IDDM relatives. The high-risk DR3, 4 phenotype increased across the three groups; DR4,X was unchanged, and DR3,X and DRX,X both decreased. The HLA class I antigen, A24, occurred more frequently in ICA-positive relatives who developed IDDM and, in 270 unrelated IDDM subjects, was associated with an earlier age at diagnosis of IDDM in those with the lower risk class II phenotypes DR4,4 and DR3,X. CONCLUSIONS: HLA-DR3 and DQ2 predispose to islet autoimmunity, but not development of clinical IDDM in the absence and DR4 and DQ8. DR4 and DQ8 predispose to the development of clinical IDDM in ICA-positive relatives, in combination with DR3-DQ2 and other haplotypes but not when homozygous. HLA-A24 is significantly associated with rapid progression to IDDM in ICA-positive relatives and with an earlier age at clinical diagnosis. Analysis of IDDM families reveals that HLA genes not only predispose to islet autoimmunity but influence progression to clinical disease. The findings have implications for identifying high-risk relatives as candidates for possible preventative therapy.     

MHC class II binding of peptides derived from HLA-DR 1.

The aim of these studies was to determine whether auto- and alloreactivity can arise from T cell recognition of MHC-peptides in context of syngeneic MHC. Four synthetic peptides derived from the first domain of the HLA-DR beta 1 * 0101 chain were used in limiting dilution analysis to prime T cells from HLA-DR1- and HLA-DR1+ responders. The frequency of T cells responding to these four peptides was similar in individuals with or without HLA-DR1. In both cases, the peptide corresponding to the nonpolymorphic sequence 43-62, was less immunogenic than peptides corresponding to the three hypervariable regions 1-20, 21-42, and 66-90, eliciting a lower number of reactive T cells. Experiments using a T cell line with specific reactivity to peptide 21-42 showed, however, that this response can be efficiently blocked by adding to the culture a nonpolymorphic sequence peptide. This suggests that alloreactivity can be blocked by use of monomorphic (self) peptides. The binding of both "monomorphic" and "polymorphic" synthetic DR1 peptides to affinity purified HLA-DR 1 and DR 11 molecules was measured using radiolabeled peptides and high performance size exclusion chromatography. The data showed that the polymorphic as well as monomorphic synthetic DR1 peptides bound to both DR1 and DR11 molecules. Competitive inhibition studies indicated that the monomorphic 43-62 peptide can block the binding of the polymorphic peptides, consistent with the results obtained in T cell cultures. Taken together these data suggest that anti-MHC autoreactive T cells are present in the periphery and that both auto and alloreactivity can be elicited by MHC peptides binding to MHC class II molecules.     

The clinical immunogenetics of viral hepatitis C in a Caucasoid population of western Siberia

The analysis of the immunogenetic studies on hepatitis C patients among the Caucasoid population of western Siberia has revealed a significant increase in the detection rate of antigens HLA-A10 and HLA-DR5, the combinations of DR2-DR5, DR5-DR7, DR1-B27 and the complete absence of antigen HLA-DR4, which is indicative of the fact that susceptibility and resistance to the development of the disease is associated with the genes of the main histocompatibility complex. In hepatitis of mixed etiology, B and C, a significant increase in the occurrence of HLA antigens: -A1, -B8, -DR1 and -DR3, as well as the combinations of A1-DR1, A1-DR3, A3-DR3, A9-A10, DR1-DR3, B8-DR3 is noted; at the same time a decrease in the occurrence of antigen DR4 and its combination with antigen HLA-A2 is observed.     

Human insulin autoantibody fine specificity and H and L chain use

Fine specificity and H and L chain isotypes of insulin autoantibodies in sera from 11 subjects were examined. None of these 11 subjects was treated with exogenous insulin. Two patterns of fine specificity were found. In one, the autoantibodies were specific for human insulin, with a requirement for threonine at B30. The conservative substitution in pork insulin (threonine to alanine) abrogated IgG binding by these sera. Insulin autoantibodies in other sera cross-reacted with beef, pork, and human insulin; not requiring threonine at B30. Reciprocal competitive inhibition experiments showed that epitopes recognized by the human specific insulin autoantibodies were exclusively on the B chain, whereas the cross-reactive sera contain autoantibodies that recognize both the B chain and combinatorial (A and B chain) epitopes. The fine specificity of cross-reactive insulin autoantibodies are thus similar to insulin antibodies from insulin-treated subjects. When IgG subclasses and L chains of insulin autoantibodies were examined, however, restricted C region usage was found. The hierarchy was IgG3 greater than G1 greater than G2 greater than G4; with one subclass dominant in each serum, although others were used. L chain use was similarly restricted. There was no correlation between isotype and fine specificity or between H and L chain type. It is concluded that heterogeneity of insulin autoantibodies is restricted. The response is probably more oligo- or pauciclonal than insulin antibody from insulin-treated subjects.     

Characterization of adult human marrow hematopoietic progenitors highly enriched by two-color cell sorting with My10 and major histocompatibility class II monoclonal antibodies

Monoclonal antibodies, My10 (HPCA-1) and major histocompatibility class II (HLA-DR), were used to enrich and phenotype normal human marrow colony-forming unit: granulocyte-macrophage (CFU-GM), burst-forming unit: erythroid (BFU-E), and multipotential colony-forming unit: granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM) progenitor cells. Nonadherent low density T lymphocyte-depleted marrow cells were sorted on a Coulter Epics 753 dye laser flow cytometry system with the use of Texas Red-labeled anti-My10 and phycoerythrin conjugated anti-HLA-DR. Cells were separated into populations with nondetectable expression of antigens (DR-My10-) or with constant expression of one antigen and increasing densities of the other antigen. More than 98% of the CFU-GM, BFU-E, and CFU-GEMM were found in fractions containing cells expressing both HLA-DR and My10 antigens. The cloning efficiency (CE) of cells in the DR-My10- cell fraction was 0.01%. In the antigen-positive sorted fractions, the CE was highest (up to 47%) in the fractions of cells expressing high My10 and low DR (My10 DR+) antigens and was lowest (2.5%) in the fraction of cells expressing low My10 and low DR (My10+DR+) antigens. Populations of cells varying in the density of HLA-DR, but not My10, antigens varied in the proportion and types of progenitor cells present. When My10-positive cells were sorted for HLA-DR density expression, the CE for CFU-GM was similar in the DR+ and DR++ fractions, but most of the BFU-E and CFU-GEMM were found in the DR+ fraction. Within the CFU-GM compartment, most of the eosinophil progenitors were found in the DR+ fraction, whereas a greater proportion of macrophage progenitors were detected in the DR++ fraction. CFU-GM and BFU-E in the fractions of cells positive for DR and My10 were assessed for responsiveness to the effects of recombinant human tumor necrosis factor-alpha, recombinant human interferon-gamma, and prostaglandin E1. Colony formation from CFU-GM was suppressed by the three molecules, and colony formation by BFU-E was suppressed by recombinant human tumor necrosis factor-alpha and interferon-gamma and enhanced, in the presence of T lymphocyte-conditioned medium, by prostaglandin E1 in all antigen-positive fractions.(ABSTRACT TRUNCATED AT 400 WORDS)     

HLA-DR typing in identical twins with insulin-dependent diabetes: difference between concordant and discordant pairs.

A total of 106 pairs of identical twins, of whom 56 were concordant and 50 discordant for insulin-dependent diabetes, were typed for HLA-DR. In both the concordant and discordant groups there was a high prevalence of the antigens DR3 and DR4, a low prevalence of DR5 and DR7, and a virtual absence of DR2. The heterozygous phenotype DR3,DR4 was more prevalent in concordant than discordant pairs. This was therefore the first demonstration of a genetic difference between concordant and discordant identical twin pairs. These findings suggest that possession of both DR3 and DR4 antigens confers a greater genetic predisposition to insulin-dependent diabetes than does the possession of either antigen alone.     

Characterization of HLA class II/peptide-TCR interactions of the immunodominant T cell epitope in Art v 1, the major mugwort pollen allergen

More than 95% of mugwort pollen-allergic individuals are sensitized to Art v 1, the major allergen in mugwort pollen. Interestingly, the CD4 T cell response to Art v 1 involves only one single immunodominant peptide, Art v 1(25-36) (KCIEWEKAQHGA), and is highly associated with the expression of HLA-DR1. Therefore, we investigated the molecular basis of this unusual immunodominance among allergens. Using artificial APC expressing exclusively HLA-DRB1*0101 and HLA-DRA*0101, we formally showed that DR1 acts as restriction element for Art v 1(25-36)-specific T cell responses. Further assessment of binding of Art v 1(25-36) to artificial HLA-DR molecules revealed that its affinity was high for HLA-DR1. Amino acid I27 was identified as anchor residue interacting with DR molecules in pocket P1. Additionally, Art v 1(25-36) bound with high affinity to HLA-DRB1*0301 and *0401, moderately to HLA-DRB1*1301 and HLA-DRB5*0101, and weakly to HLA-DRB1*1101 and *1501. T cell activation was also inducible by Art v 1(25-36)-loaded, APC-expressing HLA molecules other than DR1, indicating degeneracy of peptide binding and promiscuity of TCR recognition. Specific binding of HLA-DRB1*0101 tetramers containing Art v 1(19-36) allowed the identification of Art v 1(25-36)-specific T cells by flow cytometry. In summary, the immunodominance of Art v 1(25-36) relies on its affinity to DR1, but is not dictated by it. Future investigations at the molecular HLA/peptide/TCR and cellular level using mugwort pollen allergy as a disease model may allow new insights into tolerance and pathomechanisms operative in type I allergy, which may instigate new, T cell-directed strategies in specific immunotherapy.     

Ir gene control of the immune response to insulins. I. Pork insulin stimulates T cell activity in nonresponder mice

Immune responses by mice to heterologous insulins are controlled by H-2-linked Ir genes. In studies to determine the mechanism(s) of nonresponsiveness, we found that although pork insulin fails to stimulate antibody or proliferative responses in H-2b mice, it does stimulate enhanced responses to subsequent challenge with an immunogenic species of insulin, such as beef insulin. Experiments described in this communication analyze the cell type primed in H-2b mice by pork insulin using an adoptive transfer protocol. The results demonstrate that pork insulin primes T cells that can express helper activity when recipient mice are challenged with beef but not pork insulin. This helper T cell activity is insulin specific in both elicitation and effect but is dependent upon stimulation by beef insulin for expression. Our interpretation of these results is that 2 antigen-specific T cell subpopulations are required for the generation of insulin-specific antibody responses and that the Ir gene defect in this case is expressed as a failure of specific interaction of these 2 T cell populations.     

Tailored amino acid diversity for the evolution of antibody affinity

Antibodies are a unique class of proteins with the ability to adapt their binding sites for high affinity and high specificity to a multitude of antigens. Many analyses have been performed on antibody sequences and structures to elucidate which amino acids have a predominant role in antibody interactions with antigens. These studies have generally not distinguished between amino acids selected for broad antigen specificity in the primary immune response and those selected for high affinity in the secondary immune response. By studying a large data set of affinity matured antibodies derived from in vitro directed evolution experiments, we were able to specifically highlight a subset of amino acids associated with affinity improvements. In a comparison of affinity maturations using either tailored or full amino acid diversification, the tailored approach was found to be at least as effective at improving affinity while requiring fewer mutagenesis libraries than the traditional method. The resulting sequence data also highlight the potential for further reducing amino acid diversity for high affinity binding interactions.     

Effect of internalization and degradation of insulin on rat adipocyte insulin receptor binding kinetics

We have assessed the influence of nondisplaceable (internalized) insulin and insulin degradation during binding reactions at 37 degrees C on the numbers and affinities of insulin binding sites on isolated rat adipocytes. Corrections for nondisplaceable insulin caused a 33% reduction in the number of the high affinity sites (p less than 0.01) and a 24% reduction (p less than 0.01) in the number of the low affinity sites which was associated with a 20% increase (p less than 0.01) in affinity when a two-site model was applied. With a one-site model, the number of insulin receptors decreased by approximately 33% (p less than 0.01), but the affinity did not change. These results indicate that the internalization and degradation of insulin that occurs during the binding reaction can significantly affect the estimation of insulin binding kinetics. Potential variations in internalization and degradation of insulin by cells obtained under various physiological or pathologic conditions should, therefore, be taken into consideration in the interpretation of insulin binding data.