Expression Of HLA Class Research Articles

141 POSTER Effect of population and gender on chemotherapeutic agent-induced cytotoxicity

Transgenic (Tg) mice expressing HLA class I alleles and lacking murine MHC class I represent a useful model for the pre-clinical evaluation of human vaccines, which focus on induction of CD8+ T-cell responses. We have developed a platform to be used in Tg mice for exploring the immunogenicity of T-cell targets, whose immunologic epitopes have yet to be defined. To test the attributes of the evaluation system in the context of an important human pathogen, we have explored multiple antigens from cytomegalovirus (CMV). A panel of recombinant modified vaccinia Ankara (MVA) vectors, expressing various CMV proteins (CMV–MVA) was used to immunize HLA-A*0201, B*0702 and A*1101 Tg mice. Immune splenocytes were in vitro stimulated (IVS) either using syngeneic lipo-polysaccharide activated lymphoblasts or Tg HLA-I matched human EBV-transformed B-lymphoblastoid cells (LCL), both loaded with peptide libraries, encompassing the CMV protein under investigation. IVS performed with peptide library loaded lymphoblasts failed to provide a reliable stimulation. In contrast, the usage of LCL as antigen presenting cells (APC) of CMV peptide libraries resulted in a consistent and specific amplification of the Tg T-cell response in animals immunized with CMV–MVAs. The LCL IVS method reliably allowed defining the immunogenicity and immunodominant CD8+ T-cell regions of uncharacterized CMV antigens. The combination of CMV–MVA vectors, unbiased pools of CMV-specific peptide libraries presented by Tg HLA-I matched LCL constitutes a valid tool for the pre-clinical evaluation of model candidate vaccines. This convenient method could find application to investigate the immunogenicity profile of cancer antigens or proteins from infectious human pathogens.

Prognostic significance of HLA class I expression in osteosarcoma defined by anti‐pan HLA class I monoclonal antibody, EMR8‐5

With the goal of establishing efficacious peptide-based immunotherapy for patients with bone and soft tissue sarcomas, we previously identified the cytotoxic T lymphocyte-defined osteosarcoma antigenic gene Papillomavirus binding factor. The present study was designed to determine the status of HLA class I expression in osteosarcoma and other bone and soft tissue sarcomas. Seventy-four formalin-fixed paraffin-embedded specimens of various bone and soft tissue sarcomas, including 33 osteosarcomas, were stained with the anti-HLA class I monoclonal antibody EMR8-5, which we recently generated. The expression of HLA class I was lost or downregulated in 46 of these specimens (62%). With respect to osteosarcoma, loss or downregulation of HLA class I expression was seen in 13 (52%) of 25 primary tumors and seven (88%) of eight metastatic tumors. In six of 11 HLA class I-negative osteosarcoma specimens, the expression of beta-2 microglobulin was also lost. Subsequently the prognostic significance of HLA class I expression was analyzed in 21 patients with osteosarcoma who had completed multidrug neoadjuvant chemotherapy and undergone adequate surgery. Patients with osteosarcoma highly expressing HLA class I showed significantly better overall and event-free survival than those with HLA class I-negative osteosarcoma. In contrast, such prognostic significance of HLA class I expression was not found in 15 patients with malignant fibrous histiocytoma of soft tissue. These findings suggest that the class I-restricted cytotoxic T lymphocyte pathway plays a major role in immune surveillance of patients with osteosarcoma.

Up-regulation of HLA class-I antigen expression and antigen-specific CTL response in cervical cancer cells by the demethylating agent hydralazine and the histone deacetylase inhibitor valproic acid

BackgroundDNA hypermethylation and histone deacetylation are epigenetic events that contribute to the absence or downregulated expression of different components of the tumor recognition complex. These events affect the processing and presentation of antigenic peptides to CTLs by HLA class-I molecules. In this work evaluated the effect of the DNA hypomethylating agent hydralazine and the histone deacetylase inhibitor valproic acid, on the expression of HLA class-I molecules and on the antigen-specific immune recognition of cervical cancer cells.MethodsCell lines C33A (HPV-), CaSki (HPV-16+) and MS751 (HPV-18+) were treated with hydralazine and valproic acid to assess the expression of HLA class-I molecules by flow cytometry and RT-PCR. Promoter methylation of HLA class-I -A, -B and C, was also evaluated by Methylation-Specific PCR. Primary cervical tumors of four HLA-A*0201 allele patients were typed for HPV and their CTL's stimulated in vitro with the T2 cell line previously loaded with 50 μM of the HPV peptides. Cytotoxicity of stimulated CTL's was assayed against Caski and MS751 cells pre-treated with hydralazine and valproic acid.ResultsValproic acid and hydralazine/valproic acid up-regulated the constitutive HLA class-I expression as evaluated by flow cytometry and RT-PCR despite constitutive promoter demethylation at these loci. Hydralazine and valproic acid in combination but no IFN-gamma hyperacetylated histone H4 as evaluated by ChiP assay. The antigenic immune recognition of CaSki and MS751 cells by CTLs specific to HPV-16/18 E6 and E7-derived epitopes, was increased by VA and H/VA and the combination of H/VA/IFN-gamma.ConclusionThese results support the potential use of hydralazine and valproic acid as an adjuvant for immune intervention in cervical cancer patients whenever clinical protocols based on tumor antigen recognition is desirable, like in those cases where the application of E6 and E7 based therapeutic vaccines is used.

Expression of HLA class I and II on peripheral blood lymphocytes in HBV infection

坚持的肝炎 B 病毒(HBV ) 感染是为长期的肝炎 B，肝的肝硬化，和 hepatocellular 癌的最重要的原因。T 淋巴细胞， includingCD_4~+ 和 CD_8~+ T 房间，是主要作文招待细胞的免疫。而且， CD_8~+cells 玩在反肿瘤和反感染的主人免疫者反应的一个主要角色。HBV 感染在病人导致细胞的有免疫力的功能的混乱，这被证实了，特别在 T 淋巴细胞亚群和 cytokine 的管理功能的混乱。它在 HBV 和肿瘤房间，而是更少在过去在外部血淋巴细胞上感染到 HLA 类Ⅰ的表达式的 hepatocytes 上被付了更多的注意到 HLA 类Ⅰ的表达式。在这研究，我们与长期的肝炎 B，肝的肝硬化，和 hepatocellular 癌在病人在外部血淋巴细胞上计算了 HLA 班Ⅰ和Ⅱ的表达式并且试着为 celluar 免疫的混乱的机制提供新思维。

Expression of Endoplasmic Reticulum Aminopeptidases in EBV-B Cell Lines from Healthy Donors and in Leukemia/Lymphoma, Carcinoma, and Melanoma Cell Lines

Peptide trimming in the endoplasmic reticulum (ER), the final step required for the generation of most HLA class I-binding peptides, implicates the concerted action of two aminopeptidases, ERAP1 and ERAP2. Because defects in the expression of these peptidases could lead to aberrant surface HLA class I expression in tumor cells, we quantitatively assayed 14 EBV-B cell lines and 35 human tumor cell lines of various lineages for: 1) expression and enzymatic activities of ERAP1 and ERAP2; 2) ER peptide-trimming activity in microsomes; 3) expression of HLA class I H chains and TAP1; and 4) surface HLA class I expression. ERAP1 and ERAP2 expression was detectable in all of the EBV-B and tumor cell lines, but in the latter it was extremely variable, sometimes barely detectable, and not coordinated. The expression of the two aminopeptidases corresponded well to the respective enzymatic activities in most cell lines. A peptide-trimming assay in microsomes revealed additional enzymatic activities, presumably contributed by other unidentified aminopeptidases sharing substrate specificity with ERAP2. Interestingly, surface HLA class I expression showed significant correlation with ERAP1 activity, but not with the activity of either ERAP2 or other unidentified aminopeptidases. Transfection with ERAP1 or ERAP2 of two tumor cell lines selected for simultaneous low expression of the two aminopeptidases resulted in the expected, moderate increases of class I surface expression. Thus, low and/or imbalanced expression of ERAP1 and probably ERAP2 may cause improper Ag processing and favor tumor escape from the immune surveillance.

Polymorphisms and Lack of or Aberrant Expression of HLA Class I and II May Influence Antigen Presentation in Classical Hodgkin Lymphoma.

In a previously performed population-based genetic screening analysis we found that polymorphisms in the HLA class I region are specifically associated with EBV-positive classical Hodgkin lymphoma(cHL)(Diepstra et al., Lancet 2005; 365:2216). We now studied the expression of HLA class I and HLA class II in this population (n=450) by immunohistochemistry. HLA-B/C, HLA-G, beta-2-microglobulin and HLA-DP/DQ/DR staining was performed on formalin fixed paraffin embedded tissue sections, and HLA-DM and HLA-DO staining on frozen sections. Hodgkin-Reed Sternberg (HRS) cells showed cell surface expression of HLA class I in 37.5% (159/424) of cHL tumors and expression was observed predominantly in EBV-positive cases (73%; 103/141). Expression of HLA-G, a molecule known to enable evasion from natural killer cell responses, was found in 67% of the cases and was associated with absence of MHC class I and EBV. Only 58% of cHL cases showed HLA class II positive staining in more than 50% of the HRS cells (224/387). The non-classical HLA class II protein HLA-DM, essential for the loading of HLA class II with antigenic peptides was reduced or absent in HRS cells in 7 out of 10 HLA class II positive cases, whereas HLA-DO, the natural repressor of HLA-DM, was not or minimally expressed. Impaired expression of HLA-DM may prevent presentation of antigens at the level of individual HRS cells. Reanalysis of the genotyping data revealed an association of HLA class I positive cases with the known susceptibility polymorphisms in the HLA class I region. However, this association was stronger for the EBV-positive cases than for the HLA class I positive cases. Within the group of EBV-positive cHL patients, the same predisposing alleles were present in both HLA class I negative and positive cases, suggesting that the negative cases probably did express HLA class I during early pathogenesis. Finally, in cases with HLA class II expression on the HRS cells, a genetic association with the HLA class II region was found. These changes may influence (EBV) antigen presentation by HRS cells in cHL.

Defining MHC class II T helper epitopes for WT1 tumor antigen.

The product of Wilms' tumor gene 1 (WT1) is overexpressed in diverse human tumors, including leukemia, lung and breast cancer, and is often recognized by antibodies in the sera of patients with leukemia. Since WT1 encodes MHC class I-restricted peptides recognized by cytotoxic T lymphocytes (CTL), WT1 has been considered as a promising tumor-associated antigen (TAA) for developing anticancer immunotherapy. In order to carry out an effective peptide-based cancer immunotherapy, MHC class II-restricted epitope peptides that elicit anti-tumor CD4(+) helper T lymphocytes (HTL) will be needed. In this study, we analyzed HTL responses against WT1 antigen using HTL lines elicited by in vitro immunization of human lymphocytes with synthetic peptides predicted to serve as HTL epitopes derived from the sequence of WT1. Two peptides, WT1(124-138) and WT1(247-261), were shown to induce peptide-specific HTL, which were restricted by frequently expressed HLA class II alleles. Here, we also demonstrate that both peptides-reactive HTL lines were capable of recognizing naturally processed antigens presented by dendritic cells pulsed with tumor lysates or directly by WT1+ tumor cells that express MHC class II molecules. Interestingly, the two WT1 HTL epitopes described here are closely situated to known MHC class I-restricted CTL epitopes, raising the possibility of stimulating CTL and HTL responses using a relatively small synthetic peptide vaccine. Because HTL responses to TAA are known to be important for promoting long-lasting anti-tumor CTL responses, the newly described WT1 T-helper epitopes could provide a useful tool for designing powerful vaccines against WT1-expressing tumors.

Phenotypic analysis of alveolar macrophages and lymphocytes following allergen inhalation by atopic subjects with mild asthma

The airway inflammation associated with allergic asthma is initiated through a complex interaction of antigen-presenting cells (APC) and T lymphocytes resulting in the release of a cascade of cytokines regulating the progress of the allergic inflammatory response. In the present study the state of alveolar macrophage (AM) and T cell activation was investigated following induction of allergic airway inflammation in individuals with atopic asthma. Eleven individuals with mild, atopic asthma received cumulated allergen inhalations. Before and one day after challenge, bronchoalveolar lavage (BAL) was performed, and peripheral blood samples obtained. Ten healthy individuals served as controls. The expression of cell surface markers by BAL fluid AMs and T cells, and by blood T cells, was investigated by flow cytometry. All patients developed early asthmatic reactions (EAR) with increased numbers of eosinophils and mast cells in BAL fluid following allergen challenge. After allergen challenge, patients had relatively fewer pulmonary CD4+ T cells expressing CD69 and HLA class II and also relatively fewer pulmonary CD8+ T cells expressing HLA class II, compared to before challenge. An increased quantitative expression of CD14 and CD86 was seen within the AM population following allergen challenge. The results indicate a recruitment of non-activated, immature macrophages and CD4+ T cells to the airways as well as an altered phenotype pattern within the AM population following induction of allergic airway inflammation by allergen inhalation challenge in asthma.

HLA class II restricted T-cell receptor gene transfer generates CD4+ T cells with helper activity as well as cytotoxic capacity

Both cytotoxic T cells and helper T cells are important in immune responses against pathogens and malignant cells. In hematological malignancies which express HLA class II molecules, immunotherapy may be directed to HLA class II restricted antigens. We investigated whether it is possible to engineer HLA class II restricted T cells with both antigen-specific cytolytic activity and the capacity to produce high amounts of cytokines. CD4+ and CD8+ peripheral-blood-derived T cells were retrovirally transduced with the HLA class II restricted minor histocompatibility antigen dead box RNA helicase Y (DBY)-specific TCR. The TCR-transduced CD4+ T cells exerted DBY-specific cytolytic activity, produced Th0, Th1, or Th2 cytokines, and proliferated upon DBY-specific stimulation. TCR-transduced CD8+ T cells exerted cytolytic activity which equaled the level of cytolytic activity of the TCR-transferred CD4+ T cells. Cotransfer of CD4 enhanced the cytolytic activity of the TCR-transduced CD8+ T cells, but introduction of CD4 was not sufficient to generate DBY-specific CD8+ T cells with the capacity to produce high amounts of cytokines. In this study, we demonstrated the feasibility to engineer T cells with antigen-specific cytolytic activity, as well as the ability to produce significant amounts of cytokines, by TCR transfer to CD4+ T cells.

Human adipose derived stem cells suppress lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli

To study immunological properties of adipose derived stem cells (ADSC) and their in vitro immunomodulatory effects on lymphocytes. ADSC was isolated from fat tissue by liposuction and culture expanded. Cells at passage 2 were observed for the expression of HLAI, HLAII by FACs analysis. Peripheral blood lymphocytes (PBL) were cultured with allogeneic ADSCs at dose of 1 x 10(5). A third-party ADSCs at dose of 1 x 10(2), 1 x 10(3), 1 x 10(4), 1 x 10(5) were added to the ongoing two-way mixed lymphocyte reactions (MLR) for 6 days. Value of CPM (count per minute) was calculated with luminescence counter. Flow cytometry showed that undifferentiated ADSCs express HLA class I but not class II. Addition of interferon gamma (IFN-gamma) for 48 hours induced greater than 90% of cells to express HLA class II. No lymphocyte response was induced by allogeneic ADSCs as stimulators. Results were similar using ADSCs pretreated with IFN-gamma. ADSCs elicited inhibit function of mixed lymphocyte cultures in a dose dependent way. Even if ADSCs were pretreated with IFN-gamma, the suppression ability was maintained. Undifferentiated ADSCs do not elicit alloreactive lymphocyte proliferative responses and could modulate immune responses in vitro.

CD4+ T Cell Responses to SSX-4 in Melanoma Patients

Genes of the synovial sarcoma X breakpoint (SSX) family are expressed in different human tumors, including melanomas, but not in adult somatic tissues. Because of their specific expression at the tumor site, SSX-encoded Ags are potential targets for anticancer immunotherapy. In this study, we have analyzed CD4+ T cell responses directed against the Ag encoded by SSX-4. Upon in vitro stimulation of PBMC from four melanoma patients bearing Ag-expressing tumors with a pool of long peptides spanning the protein sequence, we detected and isolated SSX-4-specific CD4+ T cells recognizing several distinct antigenic sequences, mostly restricted by frequently expressed HLA class II alleles. The majority of the identified sequences were located within the Krüppel-associated box domain in the N-terminal region of the protein, indicating a high potential immunogenicity of this region. Together our data document the existence of CD4+ T cells specific for multiple SSX-4 derived sequences in circulating lymphocytes from melanoma patients and encourage further studies to assess the impact of SSX-4-specific T cell responses on disease evolution in cancer patients.

The dilemma of screening for antibodies against low‐frequency human platelet antigens

The dilemma of screening for antibodies against low‐frequency human platelet antigens

Expression of HLA class I antigen and proteasome subunits LMP-2 and LMP-10 in primary vs. metastatic breast carcinoma lesions

Malignant transformation of breast epithelia is frequently associated with an altered expression of MHC products and of antigen processing molecular machinery. The consequent impairment of tumor immune recognition is thought to confer to tumor cells a selective advantage with respect to survival and metastatization. In order to understand if metastatic breast cancer lesions might be associated with a defective proteasome subunit expression that, in turn, might limit the peptide availability and prevent stable cell surface HLA class I-tumor antigen expression, we studied by immunostaining the expression of beta2-microglobulin, HLA class I antigens and proteasome subunits LMP-2 and LMP-10 in 35 matched primary and metastatic human breast carcinoma lesions. Overall, we found a downregulation of LMP-2 in 51.4% of the lesions, of LPM-10 in 45.7% of the lesions, of HLA class I heavy chain in 40.0% of the lesions, while beta2-microglobulin was downregulated in 25.7% of the lesions studied. In most primary and metastatic lesions the downmodulation of each antigen examined was coordinated. In the cases where a selective downmodulation of antigens was observed in the primary or in the metastatic lesion (with the exception of beta2-microglobulin), it was rather observed in the primary lesions. However, LMP-10 showed a significant selective downmodulation in the metastases as well. Antigen downmodulation does not appear therefore to represent a strategy for the primary tumor to metastasize successfully.

Expansion of Inhibitory NK Receptor (CD94/NKG2A)-Expressing CD8 T Cells from Various Blood Mononuclear Cells with Cytolytic Activity Against Primary Leukemic Cells.

Leukemic cells and tumor cells can be escaped from allogeneic recognition by usual cytotoxic T cells because of the low expression level of HLA class I molecules. It has recently been shown that inhibitory natural killer cell receptors (NKRs) on not only NK cells but also on T cells negatively regulate NK cell and T cell functions through their binding to MHC class I molecules. The C-type lectin superfamily inhibitory NKR (CD94/NKG2A) heterodimer recognizes an HLA-E that preferably bound to a peptide derived from the signal sequences of most HLA class I. Therefore, CD94 can monitor the global status of HLA class I on the tumor and leukemic cells and induce cytolytic attack without inhibitory signal against HLA class I decreased target cells. In this study, we expanded CD94-expressing T cells from four different sources of blood mononuclear cells (BMCs) and then investigated their cytolytic characteristics against patients' primary leukemic cells in order to develop a potential strategy of cell therapy for hematological malignancy.We could get more than 100 fold expansion of CD94-expressing CD8 T cells from normal donor PBMC, apheresed PBMC without G-CSF mobilization from normal donor, apheresed PBMC with G-CSF mobilization from patients after chemotherapy and cord blood after 7 days culture with immobilized anti-CD3 monoclonal antibody (1μg/mL) and IL-15 (5 ng/mL). Cytolytic activities of purified CD94-expressing cells using magnetic cell sorting (MACS) (CD94 > 90%) detected by 4 hours 51 Cr release assay against HLA class I intermediate primary leukemic cells (AML M0, M2, M4, CML CP, BC, MDS overt) (50 < mean fluorescence intensity (MFI) < 150) were 35.6 ± 12.8 % (n=21). However, CTL activities against HLA class I high primary leukemic cells (ATL, ALL, LBL)(MFI>150) were lower than 10 % (6.5 ± 4.2, n=5). Also, CTL activities against HLA class I very high PHA autoblasts and alloblasts (MFI>200) were lower than 5 % (4.0 ± 3.6, n=11). Although the cytolytic activity of CD94-expressing cells roughly depends on the expression of HLA class I molecules in inverse proportion, adhesion molecules and also activating molecules such as NKG2D on effector cells might be important for the regulation of the killing activity. In fact, anti-NKG2D mAb (50 μg/mL) suppressed the cytolytic activity of CD94-expressing cells against patients' primary leukemic cells (% reduction of cytolytic activity, 22.5± 5.9, n=13). Furthermore, anti-LFA-1 mAb (20 μg/mL) suppressed the cytolytic activity of CD94-expressing cells much more effectively than did anti-NKG2D mAb(% reduction of cytolytic activity, 74.2±15.5, n=13, p<0.01). Our data indicated that the cytolytic activity of inhibitory NKR-expressing cells depends at least partially on NKG2D-activating NKR and also required adhesion through LFA-1. In this study, we were able to expand CD94-expressing CD8 T cells which have both inhibitory receptors (NKG2A) and stimulatoy receptors (NKG2D) as well as LFA-1 and ICAM-1 from four different sources of BMCs. Therefore, it may be possible to expand CD94-expressing cells from various sources of BMCs with cytolytic activity against both autologous and allogeneic primary leukemic cells for a new strategy of cell therapy.

Dendritic Cells Co-Developing from Human CD34+ Cells Following Incubation with Thrombopoietin and Tumor Necrosis Factor-α Phagocytose Self-Megakaryocytic Progenitors and Induce Autologous T-Cell Proliferation.

Background. Thrombopoietin (TPO) and tumor necrosis factor-α (TNF-α) sustain differentiation and proliferation of CD34+ cells toward dendritic cells (DC) in the presence of multi-acting cytokines. Therefore, we hypothesized that stimulation of human CD34+ cells with TPO and TNF-α might co-develop megakaryocytic progenitors and DC, which may relate to the induction of immune tolerance and autoimmunity in megakaryopoiesis. Materials and Methods. Highly purified human CD34+ cells were cultured in liquid phase with TPO, with or without TNF-α, and induced to undergo megakaryocytic differentiation. We enumerated megakaryocytic progenitor cells using the specific markers CD41, CD42b and CD61, and DC using CD4, CD11c, CD80, CD83, CD86 and CD123. The character and roles of co-developing non-megakaryocytic cells in the presence of TNF-α were analyzed using fluorescent activating cell sorter, enzyme immunohistochemistry, confocal microscopy and autologous mixed lymphocyte reaction (AMLR). Results. When CD34+ cells were cultured for 7 days in the presence of TPO at 100 ng/ml, the generated cells predominantly expressed CD41 (95±2%), CD42b (54±12%) and CD61 (96±2%), while rarely expressed CD11c (1.6±1.3%), CD80 (0.1±0.1%), CD83 (0.8±0.6%) or CD86 (3.3±1.9%). In contrast, addition of TNF-α at 100 ng/ml significantly decreased cells expressing CD41 (3.0±0.6%), CD42b (3.3±1.0%) or CD61 (3.2±0.9%), but did not affect the number of total cells. In the presence of TNF-α, the generated cells expressed HLA class I (100%) and HLA class II (100%), and a substantial number of cells became positive for CD11c (37±1%), even costimulatory molecules, such as CD80 (2.4±1.9%), D83 (8±4%) and CD86 (18±7%). TNF-α induced apoptosis of megakaryocytic cells. Immature CD11c+ DC was physically associated with apoptotic and CD61+ cells and was capable of endocytosing CD61+ cells. All of CD11c+ cells co-expressed c-mpl, CD4 and CD123, and about a half of CD11c+ cells co-expressed CD86. Cells generated by TNF-α and TPO (DC: TPO+TNF-α) induced autologous T cell proliferation in AMLR assay, however, cells generated by TNF-α alone (DC: TNF-α) did not (Figure 1A). Immunophenotypic analysis of both populations showed the higher expression of co-stimulatory molecules such as CD80, CD83 and CD86 in cells generated by TNF-α and TPO (Figure 1B). Conclusions. Non-megakaryocytic cells co-generated from human CD34+ cells during megakaryocytic differentiation in the presence of TPO and TNF-α express DC phenotypes. The CD4+/CD11c+/CD123+ DC subset physically and selectively associates with developing immature megakaryocytic cells and then obtains and captures self-substances and are functional in AMLR. These findings suggest that DC generated from human CD34+ cells under megakaryocytic and inflammatory co-stimuli obtain a functional role and possibly leading to the antigen presentation to induce immunity or tolerance against megakaryocytic cells and/or platelets. [Display omitted]

Graft-versus-lymphoma effects: clinical review, policy proposals, and immunobiology.

The indubitable existence of a graft-versus-lymphoma (GVL) effect is difficult to prove directly. This article reviews the difficulties in interpreting the current literature in this field and, with a number of caveats, argues for the existence of a clinically meaningful GVL effect in follicular, mantle cell, small lymphocytic, and Hodgkin lymphomas. The evidence, however, for a potent GVL effect in diffuse large-cell lymphoma and Burkitt lymphoma is not convincing. Policies for allografting in lymphoma are proposed on the basis of this evidence. The immunobiology of GVL effects is discussed--in particular, the expression of HLA class I and II and co-stimulatory molecules on lymphomas that influence the generation of alloreactive T cells--together with future directions in immunotherapy that may help to eradicate chemoresistant disease.

Superiority of thyroid peroxidase DNA over protein immunization in replicating human thyroid autoimmunity in HLA-DRB1*0301 (DR3) transgenic mice.

Murine experimental autoimmune thyroiditis (EAT), characterized by thyroid destruction after immunization with thyroglobulin (Tg), has long been a useful model of organ-specific autoimmune disease. More recently, porcine thyroid peroxidase (pTPO) has also been shown to induce thyroiditis, but these results have not been confirmed. When (C57BL/6 x CBA)F(1) mice, recently shown to be susceptible to mouse TPO-induced EAT, were immunized with plasmid DNA to human TPO (hTPO) and cytokines IL-12 or GM-CSF, significant antibody (Ab) titres were generated, but minimal thyroiditis was detected in one mouse only from the TPO + GM-CSF immunized group. However, after TPO DNA immunization of HLA-DR3 transgenic class II-deficient NOD mice, thyroiditis was present in 23% of mice injected with TPO + IL-12 or GM-CSF. We also used another marker for assessing the closeness of the model to human thyroid autoimmunity by examining the epitope profile of the anti-TPO Abs to immunodominant determinants on TPO. Remarkably, the majority of the anti-TPO Abs was directed to immunodominant regions A and B, demonstrating the close replication of the model to human autoimmunity. TPO protein immunizations of HLA-DR3 transgenic mice with recombinant hTPO did not result in thyroiditis, nor did immunization of other mice expressing HLA class II transgenes HLA-DR4 or HLA-DQ8, with differential susceptibility to Tg-induced EAT. Moreover, our efforts to duplicate exactly the experimental procedures used with pTPO also failed to induce thyroiditis. The success of hTPO plasmid DNA immunization of DR3(+) mice, similar to our reports on Tg-induced thyroiditis and thyrotropin receptor DNA-induced Graves' hyperthyroidism, underscores the importance of DR3 genes for all three major thyroid antigens, and provides another humanized model to study autoimmune thyroid disease.

Differences in the expressed HLA class I alleles effect the differential clustering of HIV type 1-specific T cell responses in infected Chinese and caucasians.

China is a region of the world with a rapidly spreading HIV-1 epidemic. Studies providing insights into HIV-1 pathogenesis in infected Chinese are urgently needed to support the design and testing of an effective HIV-1 vaccine for this population. HIV-1-specific T cell responses were characterized in 32 HIV-1-infected individuals of Chinese origin and compared to 34 infected caucasians using 410 overlapping peptides spanning the entire HIV-1 clade B consensus sequence in an IFN-gamma ELISpot assay. All HIV-1 proteins were targeted with similar frequency in both populations and all study subjects recognized at least one overlapping peptide. HIV-1-specific T cell responses clustered in seven different regions of the HIV-1 genome in the Chinese cohort and in nine different regions in the caucasian cohort. The dominant HLA class I alleles expressed in the two populations differed significantly, and differences in epitope clustering pattern were shown to be influenced by differences in class I alleles that restrict immunodominant epitopes. These studies demonstrate that the clustering of HIV-1-specific T cell responses is influenced by the genetic HLA class I background in the study populations. The design and testing of candidate vaccines to fight the rapidly growing HIV-1 epidemic must therefore take the HLA genetics of the population into account as specific regions of the virus can be expected to be differentially targeted in ethnically diverse populations.

P53 and HLA class-I expression are not down-regulated in colorectal cancer liver metastases.

p53 overexpression occurs in more than 50% of colorectal carcinomas, which makes it an interesting target for immunotherapy. HLA class I expression on tumor cells is required for the presentation of p53 peptides and an effective T-cell mediated-immune response to ensue. To analyze to which extent p53 and HLA-I expression in a primary tumor reflects expression in liver metastases, we investigated p53, HLA-A and HLA-B/C expression in 82 colorectal carcinomas and 143 associated liver metastases of 82 patients. We used the monoclonal antibodies DO-7 (p53), HCA2 (HLA-A) and HC-10 (HLA-B/C) on formalin-fixed, paraffin embedded tissue. The percentage of expressing cells was estimated. P53 was overexpressed in 73% of the colorectal carcinomas and 66% of liver metastases. HLA-A was expressed in 98% and 96% and HLA-B/C in 100% and 94% of colorectal cancers and liver metastases respectively. There were no significant differences between the primary tumors and the liver metastases for each marker. The concordance was also very high in those cases in which more than one metastasis was available. Discordant cases consisted of tumors in which expression of p53 or HLA-A was lost in the liver metastases, whereas it was present in only a few tumor cells in the primary tumor. The combined analysis of p53 and HLA-I expression in liver metastases demonstrated that both molecules were expressed in 63% of the cases. P53 and HLA-I were expressed in the majority of primary tumors and their associated liver metastases. This allows to select patients for p53-immunotherapy on the basis of p53 and HLA-I expression in the primary tumor.

Immunocytochemical demonstration of down regulation of HLA class-I molecule expression in human metastatic breast carcinoma.

Deficient expression of HLA class-I molecules observed in many cancers is suggested to influence both disease progression and potential efficacy of T cell-mediated immune therapy. Previous studies have attempted to correlate either primary breast cancer tumor grade with HLA class-I levels or the presence of HLA class-I-deficient cells in metastatic lesions with survival. In this study we evaluated the HLA class-I status of matched primary and secondary breast cancer lesions in order to ask the question: is metastasis of breast cancer associated with down-regulation of HLA class-I expression? Immunocytochemistry analysis shows a definitive correlation between diminished HLA class-I expression and dissemination of breast cancer to tumor-draining lymph nodes: both the total number of HLA class-I+ cells per sample and the levels of expression are dramatically decreased in secondary versus primary tumor lesions. These findings are consistent with the contention that the ability of breast cancer cells to escape the confines of the original tumor lesion requires down-regulation of HLA class-I expression and implies that enhancing HLA class-I expression in secondary breast cancer may have a beneficial effect on T-cell-mediated immunotherapy.