HLA alleles and drug hypersensitivity reactions.

The human leucocyte antigen (HLA) system is well known for its association with certain diseases such as ankylosing spondylitis, celiac disease and many others. More recently, severe and even fatal drug hypersensitivity reactions linked to particular HLA alleles have been discovered. The significance of these discoveries has led the European Medicines Agency (EMA) and its member state agencies to recommend HLA gene testing before initiation of drug treatment. To date, the following drugs have been identified as causing significant drug hypersensitivity reactions in patients who have the following HLA alleles: abacavir and HLA-B*57:01, carbamazepine and HLA-B*15:02/A*31:01 and finally allopurinol and HLA-B*58:01. This review will outline and discuss these three drugs and their associated HLA alleles as well as examine the pathogenesis of the drug hypersensitivity reactions.

Adenocarcinoma arising in a transplanted pancreas.

Tumor transmission or de novo tumor development in the transplanted organ is a rare event. Appreciation of the organ-specific risk factors for tumor development and careful inspection of the organ at procurement may reduce but not eliminate this complication. We report the first known combined kidney-pancreas recipient who developed adenocarcinoma in the transplanted pancreas. Molecular typing of the tumor by DNA sequencing supports donor derivation of the tumor. Despite cessation of immunosuppression and reconstitution of the recipient's immune response, the patient died from metastatic pancreatic adenocarcinoma. Comparison is made to the reported outcomes after diagnosis of renal cell carcinoma that appeared early after transplantation

Restoration of alloreactivity of melanoma by transduction with B7.1.

Melanoma cells are unusual because, unlike most epithelial tumors, constitutive expression of human leukocyte antigen (HLA) class II molecules is common. To elucidate the role of HLA class II expression in the immunopathogenesis of melanoma, the authors compared HLA class II+ melanoma cells to autologous B cells with respect to their ability to stimulate primary (naïve) histoincompatible lymphocytes and T-cell clones (antigen experienced). Using primary lymphocytes (peripheral blood lymphocytes [PBLs]), melanoma cells were nonstimulatory when compared to autologous B cells. To determine whether this was caused by defective antigen processing, the authors used alloreactive T-cell clones, which require alloantigen presentation by a histocompatible stimulator cell but not costimulation. Melanoma cells stimulated the alloreactive T-cell clones in two of three clones tested, indicating that they processed and presented alloantigen. To determine whether the failure of melanoma cells to stimulate primary lymphocytes was caused by their inability to costimulate the T cells, the authors transduced the melanoma cells with B7.1 and achieved stable expression in more than 95% of the cells. The transduced cells were highly stimulatory, eliciting a 17- to 25-fold increase in proliferation by the peripheral blood lymphocytes compared with controls. Indeed, B7-expressing melanoma cells were more stimulatory than autologous B cells, which elicited an 11- to 15-fold increase compared with controls. These data indicate that melanoma cells fail to stimulate primary lymphocytes because they do not deliver costimulatory signals. Engineering HLA class II+ melanoma cells to express high levels of B7.1 may provide a way to elicit primary T-cell responses to melanoma-associated antigens.

CD4(+) T cells kill HLA-class-II-antigen-positive melanoma cells presenting peptide in vitro.

PURPOSE: Most melanoma cell lines express HLA class II antigens constitutively or can be induced to do so with interferon gamma (IFNgamma). We have previously demonstrated that peptide-specific CD4(+) T cells proliferate in response to HLA-class-II-antigen-mediated peptide presentation by melanoma cells in vitro and produce interleukin-10 (IL-10) and (IFNgamma). We asked whether the responding T cells kill the tumor cells and, if so, whether direct cell contact was required. METHODS: Two HLA class II(+) melanoma cell lines derived from metastases were co-cultured with a human CD4(+) T cell clone specific for influenza hemagglutinin peptide (HA). T cells, melanoma, and HA were co-cultured for 48 h. Melanoma cells with and without HA and/or T cells served as controls. After 36 h, the medium was removed for cytokine analysis by enzyme-linked immunosorbent assay (ELISA). Twelve hours later non-adherent cells were washed away and the adherent melanoma cells were trypsinized and counted. Dual-chamber culture plates were used to determine whether cell contact and/or exposure to cytokine were required for tumor cell death. RESULTS: Melanoma cell counts were over 80% lower in wells containing T cells than in wells with melanoma and peptide alone (P < 0.05). ELISA of supernatants revealed production of IFNgamma and IL-10 by the responding T cells. Direct T cell contact with tumor cells was not required for tumor cell death, as melanoma cells were killed when they shared medium but had no contact with T cells responding to peptide presentation by HLA-class-II-antigen-positive melanoma cells in a separate chamber. Blocking antibody to IFNgamma but not IL-10 prevented melanoma cell death at levels of cytokine similar to that present in co-culture assays. CONCLUSIONS: Peptide-specific CD4(+) T cells kill melanoma cells in vitro when they recognize peptide presented by the tumor cell in the context of HLA class II antigen. Direct cell contact is not required, suggesting that it is a cytokine-mediated event. Immunotherapy, using primed CD4(+) T cells and peptide, may be beneficial in patients whose tumors express HLA class II antigens or can be induced to do so with IFNgamma.

Cytokine production by CD4+ T-cells responding to antigen presentation by melanoma cells.

Melanoma cells are unusual because, unlike most epithelial tumours, constitutive expression of HLA class II antigens is common. We have previously demonstrated that a peptide-specific CD4+ T-cell clone proliferates briskly in response to peptide and HLA class II expressing melanoma cell lines derived from metastases. Here we demonstrate that these CD4+ T-cells secrete large amounts of interferon-gamma (IFNgamma) and interleukin-10 (IL10), and insignificant quantities of IL2 or IL4, in response to peptide presentation by both melanoma and autologous B-cells. T-cells produced more IL10 when responding to peptide presentation by melanoma cells compared with B-cells, and less IFNgamma (P<0.01). Addition of IL12 did not alter the cytokines produced but increased the T-cell production of both, especially the production of IL10 in response to peptide presentation by melanoma cells. Our data suggest that differential cytokine production by CD4+ T-cells in response to peptide presentation by HLA class II expressing tumour cells may contribute to tolerance to tumour antigens.

Identification of antigenic escape variants in an immunodominant epitope of hepatitis C virus.

Numerous investigators have postulated that one mechanism by which hepatitis C virus (HCV) may evade the immune system is through the formation of escape mutants. This hypothesis is based largely on the observed mutability of the viral genome resulting in evolution of diverse quasispecies over the course of infection. That such diversification is a product of viral RNA polymerase infidelity, immune-driven selection or a combination of the two processes has not been addressed. We have examined sequence variability in a specific segment of HCV RNA encoding a known immunodominant region of the viral helicase, amino acids 358-375 of the non-structural 3 protein. Using sequence-specific oligonucleotide probe hybridization and automated DNA sequencing, we report a high frequency of mutations, essentially all of which result in amino acid replacements. To assess the biological impact of such mutations, corresponding chemically synthesized peptides were compared to wild-type peptide in T cell proliferation assays. We observed that a sizeable fraction of such peptides stimulated attenuated or negligible levels of proliferation by peripheral T cells from a chronically infected patient. This observation is consistent with expectations for immune-mediated selection of escape variants at the epitope level. We postulate that such a mechanism may be important in the immunopathogenesis of HCV infections.

Mutations in immunodominant T cell epitopes derived from the nonstructural 3 protein of hepatitis C virus have the potential for generating escape variants that may have important consequences for T cell recognition.

One of the most disturbing features of hepatitis C virus (HCV) is its long-term persistence in the host. One hypothesis to explain this phenomenon is that HCV escapes immune recognition through its intrinsic hypermutability. To determine whether immunodominant T cell epitopes derived from HCV nonstructural 3 (NS3) protein might be subject to sequence variations leading to escape mutants, we examined sequence variations of one IL-2-producing epitope, NS3358-375, and one IL-10-producing epitope, NS3505-521. By PCR amplification, cloning, and sequencing, we observed significant sequence variations in the two epitopes, although the selection intensity for each epitope was different. For NS3358-375, more variants were observed, and for NS3505-521, fewer mutations were observed. Moreover, functional studies revealed that three NS3358-375 and one NS3505-521 variants failed to stimulate T cell proliferation, and two other NS3358-375 and NS3505-521 variants weakly stimulated T cell responses. Our results are consistent with immune selection of viral variants at the epitope level, which may enable HCV to evade host defenses over time.

Functionally distinct T-cell epitopes within the hepatitis C virus non-structural 3 protein.

Clearance of Hepatitis C Virus (HCV) infection is an uncommon phenomenon. To understand the mechanism of viral persistence despite active cellular and humoral responses, we examined the in vitro cytokine response of PBMC from an HCV sero-positive, asymptomatic individual to recombinant intact antigen and sixty-nine overlapping peptides of the HCV non-structural (NS) 3 protein. Whereas, intact antigen induced strong proliferation and significant levels of gammaIFN and IL-10, little or no IL-2 was produced. Only 7% of peptides induced IL-2, which also coincided with their ability to stimulate proliferation. In contrast, 38% of the peptides induced gammaIFN while 35% induced IL-10. All IL-2 stimulating peptides also induced significant levels of gammaIFN and among these, a peptide corresponding to residues 358-375 was the strongest. In addition, 16% of the peptides induced both gammaIFN and IL-10. Exogenous recombinant IL-10 inhibited proliferation and IL-2 induction in response to peptide 358-375. Furthermore, neutralization of IL-10 with an anti-IL-10 antibody resulted in enhanced IL-2 production in response to recombinant NS3 protein. We suggest that IL-10 inducing epitopes within HCV NS3 may thus down-regulate IL-2 dependent T-cell responses.

A site for CD4 binding in the beta 1 domain of the MHC class II protein HLA-DR1.

Using a lymphocyte binding assay, we have previously demonstrated that the CD4 protein can mediate cell adhesion by direct interaction with MHC class II molecules. In this report, we have used this assay to test whether synthetic peptides, corresponding to DR beta sequences, could inhibit CD4-class II adhesion. A peptide derived from sequences within the beta1 domain (DR beta 41-55), as well as two peptides derived from sequences within the beta 2 domain (DR beta 121-135 and DR beta 141-155), were shown to inhibit CD4-class II adhesion. Inasmuch as a site for CD4 binding in the beta 2 domain had been previously documented, these studies were designed to investigate the role of the beta 1 domain as an additional site of interaction with CD4. Sixteen site-specific mutations were engineered within the beta1 domain of DR beta 1*0101. Several mutations were shown to disrupt CD4-dependent T cell activation. Based on these results, we propose a model for the molecular interaction of CD4 with MHC class II proteins in which both the beta 1 and beta 2 domains of class II interact with the two amino-terminal Ig-like domains of CD4.

MHC class II-mediated antigen presentation by melanoma cells.

Constitutive expression of major histocompatibility complex (MHC) class II molecules is normally restricted to professional antigen-presenting cells (APCs) of the immune system, although it also occurs frequently in melanoma. Clinical evidence suggesting that MHC class II expression by melanoma is associated with tumor progression led us to postulate a role for MHC class II-mediated antigen presentation in this disease. First, we investigated whether melanoma cells derived from metastases can process antigen and/or present peptide vi MHC class II molecules to a peptide-specific CD4+ T-cell clone. In all cell lines tested, melanoma cells were able to process antigen and present peptide efficiently to CD4+ T cells, resulting in T-cell proliferation increased 5-26-fold over controls. Next, we found that CD28-mediated costimulation was not required, because blocking with CTLA-4Ig had no effect on the T-cell response to either melanoma or B cells as APCs. In contrast, blocking CD54 (ICAM-1) resulted in a decrease in proliferation in response to peptide presentation by melanoma but not B cells. These data demonstrate that MHC class II molecules on melanoma cells are functional and that antigen-processing pathways are intact. In addition, CD54 seems to play a significant role in peptide presentation by melanoma.

T cell recognition of MHC class II-associated peptides is independent of peptide affinity for MHC and sodium dodecyl sulfate stability of the peptide/MHC complex. Effects of conservative amino acid substitutions at anchor position 1 of influenza matrix protein19-31.

T cells recognize peptide fragments of Ags bound to MHC-encoded molecules. Pockets in the MHC peptide-binding groove accommodate a limited set of amino acid side chains present at anchor positions in peptide; however, the functional significance of accommodation of different side chains at an anchor position in peptide is not clear. A panel of T cell clones was evaluated to test the effect of conservative amino acid substitution at a primary peptide anchor position. Results of T cell stimulation studies were correlated with two well studied characteristics of the peptide/MHC complex, which are the affinity of peptide binding to MHC and the stability of the resulting complex upon PAGE in the presence of SDS. We found that formation of a functional complex required neither high affinity peptide binding nor SDS stability. Furthermore, T cell clones differed in their ability to recognize individual peptide variants, suggesting that some structural aspect of the peptide/MHC complex is influenced by interactions between peptide anchor residues and MHC pockets.

The predictive value of HLA-DR oligotyping for MLC responses.

Comparison of HLA proteins between a patient and potential unrelated marrow donors is difficult because many similar, but not identical, HLA proteins are expressed in the human population. A reliable and practical method to detect these subtle differences is provided by oligotyping, a new technique that identifies polymorphic sequences in the genes encoding the HLA proteins. Oligotyping was used to compare polymorphic HLA-DR sequences in 286 pairs of samples from patients and potential unrelated donors who were serologically matched for HLA-DR specificities. Oligotyping detected HLA-DR differences in 53% of these pairs and all mismatched pairs were reactive in primary mixed lymphocyte cultures. Where HLA-DR disparity was not detected by oligotyping, 37% of the pairs were nonreactive in MLC. The remaining 63% often contained an allele associated with the HLA-DRw11 serological specificity. In the absence of HLA-DRB1*11, oligotyping was predictive of MLC reactivity for samples with HLA-DR2, -DR4, and DRw52. In clinical settings, the ability to predict MLC reactivity on the basis of precise HLA typing provides an alternative to MLC. Further, the relationship between specific polymorphic sequences and reactivity in MLC may lead to more fundamental insights into the mechanisms involved in alloreactive responses.

Clonal analysis of HLA-DPw1 (SB1) associated allodeterminants: recognition of novel epitopes and evidence for quantitative variation in class II antigen expression.

Alloreactive human T-lymphocyte clones were derived from the SB1 HLA-DPw1)-specific, primed lymphocyte typing cell line SB1A used in the Ninth International Histocompatibility Workshop. The clones from two separate subclonings were analyzed for their proliferative patterns in panel experiments with cells from unrelated individuals and in family segregation analyses. While only one clone gave a perfect correlation with the DPw1 specificity, the maturity of clones recognized multiple specificities apparently associated with but not identical to HLA-DPw1. Most clones defined "splits" or subsets of DPw1 and some also displayed "extra" reactions with DPw1-negative stimulator cells. Further evidence was also found that, of the molecules bearing epitopic subsets associated with DPw1, some may be selectively expressed on the cell surface whereas the surface density of other DPw1-associated antigens may be varied. Thus, the HLA-DP region appears to encode a complex array of alloantigens and is in this regard similar to the HLA-DR region.

Inhibition of T cell proliferation by antibodies to synthetic peptides.

While T cell proliferation to antigen in the presence of antigen-presenting cells is well known to be readily inhibited by antibodies directed against Class II major histocompatibility complex (MHC) (Ia/HLA-DR) products, it has not been possible to inhibit proliferation by antibodies directed against the antigen. Because of the implications of these observations for targets of T cell recognition, this phenomenon was reinvestigated using human T cell clones, recognizing a small (24 amino acid) synthetic peptide (termed p20) derived from the influenza hemagglutinin-1 molecule. It was found that proliferation of clones to p20 was inhibited efficiently (less than 90%), using p20 as antigen, and rabbit anti-p20. Inhibition was possible either by coculturing p20 antigen and antibody to p20 with cloned T cells and antigen-presenting (E-) cells, or by pulsing antigen-presenting cells with antigen prior to a brief incubation with antibody before washing the E- cells and using them to stimulate cloned T cells. These results do not indicate why previous attempts had failed, but in view of the different techniques available now (cloned T cells, small synthetic polypeptides, and antibody raised against polypeptide) we investigated the influence of these parameters. It was found that, using cloned T cells, the form of the antigen was of importance, as antibody inhibition of the response to hemagglutinin or whole influenza A was much less apparent. These differences were interpreted as being due to greater access of anti-p20 to p20 than to hemagglutinin or influenza. If uncloned T cell lines were used, inhibition was also much harder to detect. This was interpreted as masking of inhibition of the response of some clones in the line by interleukin 2-induced recruitment.

Pharmacologic and biochemical modulation of human T-lymphocyte colony formation: hormonal influences.

The generation of T-cell colonies from human peripheral blood lymphocytes is a sensitive in vitro measure of cell-mediated immunity, considered to be under different and/or additional regulatory controls than short-term liquid cultures. The influences of steroids (aldosterone, estradiol, diethylstilbestrol, hydrocortisone, prednisolone, progesterone, testosterone), prostaglandins (PGA1, PGA2, PGB1, PGB2, PGE1, PGE2, PGF1 alpha), bradykinin, cyclic adenosine monophosphate (AMP), cyclic guanosine monophosphate (GMP), epinephrine, glucagon, histamine, insulin, luteinizing hormone, luteotropic hormone, serotonin, and thyroxin on the generation of both T-cell colonies in semisolid phase and induction of transformation in liquid culture was assessed in parallel assays. Steroids uniformly suppressed both types of culture systems, although colony formation appeared more sensitive by several hours of magnitude. In contrast, significant differences in the response of lymphocytes in colony formation assay, compared to liquid transformation, was noted for the other agents. Prostaglandins significantly inhibited colony formation even in the presence of as little as 10(-12) M PGE2; however, liquid culture responses were suppressed only by higher concentrations (10(-5) M) and enhanced transformation was found at lower concentrations (10(-9) M). Bradykinin, glucagon, and luteinizing hormone did not significantly influence either colony formation or liquid transformation. In contrast, cyclic AMP inhibited and cyclic GMP stimulated colony formation and liquid transformation. Histamine, insulin, epinephrine, and serotonin all had significant positive or negative influences on colony formation in concentrations that produced no detectable effects using conventional liquid transformation assays. Finally, correlation analysis of drug effects for each system extends the thesis that these assays quantitate different parameters of T-cell function. T-lymphocyte colony formation is a promising diagnostic tool for rapid screening of immune modulating agents.

Therapeutic manipulation of Nb rat prostatic adenocarcinomas: chemotherapeutic evaluation of autonomous tumors.

Nb rat autonomous prostatic adenocarcinomas were transplanted into several groups of synergeneic Nb rats. Chemotherapy using 5-fluorouracil, methotrexate, Adriamycin, and cyclophosphamide was evaluated. Significance was seen with the use of these agents with varying doses. Cyclophosphamide and 5-fluorouracil treated animals had complete tumor regression whereas Adriamycin and methotrexate treated animals had cessation of tumor volume increase in some groups after chemotherapy; however, neither agent causes complete tumor regression.