slan-defined subsets of CD16-positive monocytes: impact of granulomatous inflammation and M-CSF receptor mutation.

Human monocytes are subdivided into classical, intermediate, and nonclassical subsets, but there is no unequivocal strategy to dissect the latter 2 cell types. We show herein that the cell surface marker 6-sulfo LacNAc (slan) can define slan-positive CD14(+)CD16(++) nonclassical monocytes and slan-negative CD14(++)CD16(+) intermediate monocytes. Gene expression profiling confirms that slan-negative intermediate monocytes show highest expression levels of major histocompatibility complex class II genes, whereas a differential ubiquitin signature is a novel feature of the slan approach. In unsupervised hierarchical clustering, the slan-positive nonclassical monocytes cluster with monocytes and are clearly distinct from CD1c(+) dendritic cells. In clinical studies, we show a selective increase of the slan-negative intermediate monocytes to >100 cells per microliter in patients with sarcoidosis and a fivefold depletion of the slan-positive monocytes in patients with hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS), which is caused by macrophage colony-stimulating factor (M-CSF) receptor mutations. These data demonstrate that the slan-based definition of CD16-positive monocyte subsets is informative in molecular studies and in clinical settings.

Human leukocyte antigen class II quantification by targeted mass spectrometry in dendritic-like cell lines and monocyte-derived dendritic cells.

The major histocompatibility complex II (HLA-II) facilitates the presentation of antigen-derived peptides to CD4+ T-cells. Antigen presentation is not only affected by peptide processing and intracellular trafficking, but also by mechanisms that govern HLA-II abundance such as gene expression, biosynthesis and degradation. Herein we describe a mass spectrometry (MS) based HLA-II-protein quantification method, applied to dendritic-like cells (KG-1 and MUTZ-3) and human monocyte-derived dendritic cells (DCs). This method monitors the proteotypic peptides VEHWGLDKPLLK, VEHWGLDQPLLK and VEHWGLDEPLLK, mapping to the α-chains HLA-DQA1, -DPA1 and -DRA1/DQA2, respectively. Total HLA-II was detected at 176 and 248 fmol per million unstimulated KG-1 and MUTZ-3 cells, respectively. In contrast, TNF- and LPS-induced MUTZ-3 cells showed a 50- and 200-fold increase, respectively, of total α-chain as measured by MS. HLA-II protein levels in unstimulated DCs varied significantly between donors ranging from ~ 4 to ~ 50 pmol per million DCs. Cell surface HLA-DR levels detected by flow cytometry increased 2- to 3-fold after DC activation with lipopolysaccharide (LPS), in contrast to a decrease or no change in total HLA α-chain as determined by MS. HLA-DRA1 was detected as the predominant variant, representing > 90% of total α-chain, followed by DPA1 and DQA1 at 3-7% and ≤ 1%, respectively.

Expression of HLA-DR molecules by keratinocytes, and presence of Langerhans cells in the dermal infiltrate of active psoriatic plaques.

Immunoperoxidase staining of skin sections and immunofluorescence analysis of keratinocyte suspensions obtained from suction blisters of psoriatic plaques were performed using an mAb, Josh 524.4.1, and Fab'2 fragments of a rabbit antiserum, both of which are directed against nonpolymorphic determinants of HLA-DR molecules. HLA-DR+ keratinocytes were present in plaques, but not normal-appearing skin, from a significant portion of patients with active psoriasis. Double-labelling immunofluorescence experiments with either the monoclonal or polyclonal anti-HLA-DR antibody, in conjunction with the mAb OKT6, which identifies DR+ Langerhans cells, demonstrated that HLA-DR molecules were present on OKT6- keratinocytes. The dermal infiltrate of psoriatic plaques contained T cells expressing the activation antigens, IL-2 receptor (Tac) and HLA-DR, as well as macrophages and OKT6+ cells. There was little difference in the characteristics of the dermal infiltrate between the lesions with or without HLA-DR+ keratinocytes. OKT6+ presumptive Langerhans cells were also found in the dermal infiltrates of patients with lichen planus, contact dermatitis, spongiotic dermatitis, erythema multiforme, basal and squamous cell carcinoma. Studies of keratinocyte suspensions showed that 7-84% of keratinocytes were HLA-DR+. Flow cytometry experiments showed that keratinocytes at all stages of differentiation were HLA-DR+. However, the stem cell-enriched population contained the highest proportion of HLA-DR+ cells. HLA-DR expression by keratinocytes correlated with disease activity. The expression was reversible with successful medical therapy. HLA-DR+ keratinocytes may activate T cells directly or may present an as yet unknown antigen to T cells. These studies provide further support for the hypothesis that immunological mechanisms play an important role in the pathogenesis of psoriasis.

Antigen capture and major histocompatibility class II compartments of freshly isolated and cultured human blood dendritic cells.

Dendritic cells (DC) represent potent antigen-presenting cells for the induction of T cell-dependent immune responses. Previous work on antigen uptake and presentation by human DC is based largely on studies of blood DC that have been cultured for various periods of time before analysis. These cultured cells may therefore have undergone a maturation process from precursors that have different capacities for antigen capture and presentation. We have now used immunoelectron microscopy and antigen presentation assays to compare freshly isolated DC (f-DC) and cultured DC (c-DC). f-DC display a round appearance, whereas c-DC display characteristic long processes. c-DC express much more cell surface major histocompatibility complex (MHC) class II than f-DC. The uptake of colloidal gold-labeled bovine serum albumin (BSA), however, is greater in f-DC, as is the presentation of 65-kD heat shock protein to T cell clones. The most striking discovery is that the majority of MHC class II molecules in both f-DC and c-DC occur in intracellular vacuoles with a complex shape (multivesicular and multilaminar). These MHC class II enriched compartments (MIIC) represent the site to which BSA is transported within 30 min. Although MIIC appear as more dense structures with less MHC class II molecules in f-DC than c-DC, the marker characteristics are very similar. The MIIC in both types of DC are acidic, contain invariant chain, and express the recently described HLA-DM molecule that can contribute to antigen presentation. CD19+ peripheral blood B cells have fewer MIIC and surface MHC class II expression than DCs, while monocytes had low levels of MIIC and surface MHC class II. These results demonstrate in dendritic cells the elaborate development of MIIC expressing several of the components that are required for efficient antigen presentation.

Human Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes home preferentially to and induce selective regressions of autologous EBV-induced B cell lymphoproliferations in xenografted C.B-17 scid/scid mice.

C.B-17 scid/scid (severe combined immunodeficiency [SCID]) mice inoculated with peripheral blood lymphocytes from Epstein-Barr virus (EBV)-seropositive donors, or with EBV-transformed lymphoblastoid B cell lines (EBV-LCL), develop lethal human EBV+ B cell lymphoproliferative disorders (EBV-LPD) with characteristics similar to those arising in immunodeficient patients. Using this model, we examined the capacity of human effector cells to control human EBV-LPD. SCID mice received rabbit anti-asialo GM1 antiserum to abrogate endogenous natural killer-cell function. Preliminary experiments showed that adoptive transfer of peripheral blood mononuclear cells (PBMC), purified T cells, interleukin (IL) 2-activated PBMC or anti-CD3-activated T cells derived from EBV-seropositive donors did not result in improved survival of treated mice (in vivo effector/target ratio 2:1 to 1:1). In contrast, EBV-specific cytotoxic T lymphocytes (CTL), derived from EBV-seropositive donors and expanded in vitro, exhibited strong EBV-specific and HLA-restricted activity both in vitro and in vivo. SCID mice inoculated intraperitoneally with autologous but not with HLA-mismatched EBV-LCL had significantly improved survival relative to untreated mice after inoculation of EBV-specific CTL either intraperitoneally (P<0.001) or intravenously (P<0.001) (in vivo effector/target ratio 1:1). SCID mice bearing large subcutaneous EBV+ tumors and treated intravenously with 10(7) EBV-specific CTL achieved complete tumor regression. Both CTL- and CTL-plus-IL-2-treated mice survived significantly longer than untreated animals or animals treated with IL-2 alone (P = 0.0004 and P<0.02, respectively). SCID mice bearing two subcutaneous EBV+ tumors, one autologous and the other HLA mismatched to the EBV-specific CTL donor, had regression of only the autologous tumor after intravenous infusion of 10(7) EBV-specific CTL. Moreover, we could demonstrate preferential homing of PKH26-labeled EBV-specific CTL to autologous but not to HLA-mismatched EBV+ tumors as early as 24 h after intravenous adoptive transfer. Immunophenotypic analyses also demonstrated preferential infiltration of T cells into the autologous EBV+ tumor in SCID mice bearing both the autologous and either fully HLA-mismatched or genotypically related haplotype-sharing EBV+ tumors. The human T cells infiltrating EBV+ tumors were CD3+ and, predominantly, CD8+CD4-. Our results indicate that EBV-specific CTL preferentially localize to and infiltrate EBV+ tumors bearing the appropriate HLA antigens and thereafter induce targeted regressions of disease.

The low-affinity receptor for IgE (CD23) on B lymphocytes is spatially associated with HLA-DR antigens.

Two hybridomas that produce the mAbs 135 and 449 B4 were obtained that inhibited the binding of IgE to the Fc epsilon RL/CD23 on the EBV-transformed B cell line RPMI 8866. mAb 135 was obtained from a mouse immunized with RPMI 8866 cells, whereas mAb 449B4 was obtained from a mouse immunized with a partially purified preparation of Fc epsilon RL/CD23 obtained as the eluate of an IgE immunoabsorbent loaded with a soluble extract of RPMI 8866 cells. These two mAbs bound to Fc epsilon RL/CD23- cell lines and precipitated two polypeptides with 36,000 Mr and 28,000 Mr, which were the HLA-DR alpha and beta chains, respectively. Immunoprecipitation with mAb 135 of NP-40 lysates from dithio-bis(succinimidyl propionate) (DSP) crosslinked 125I-labeled RPMI 8866 or normal B cells incubated with rIL-4 showed three polypeptides with 42,000, 36,000, and 28,000 Mr. The 42,000 Mr polypeptide is identical to the Fc epsilon RL/CD23 since it could be precipitated by the anti-Fc epsilon RL/CD23 mAb 25 after resolubilization from the SDS-PAGE gel. Immunoprecipitations of the crosslinked cell extracts carried out with the anti-Fc epsilon RL/CD23 mAb 25 yielded the same three polypeptides. Furthermore, when RPMI 8866 or rIL-4 preincubated normal B cells were solubilized with a digitonin buffer, which prevents the dissociation of noncovalently linked polypeptide complexes, mAb 135 and mAb 25 precipitated complexes composed of three molecules with 42,000, 36,000, and 28,000 Mr. The well-characterized anti-HLA-DR mAb L243 was unable to block the binding of either IgE or mAb 135 to RPMI 8866 cells, although it could immunoprecipitate the complex (HLA-DR-Fc epsilon RL/CD23) from crosslinked cell lysates. Since mAb 135 and L243 were able to both bind the RPMI 8866 cells, it demonstrates that they bind to different epitopes of the HLA-DR complex, the mAb 135 epitope of the HLA-DR molecule being close to the IgE binding site of the Fc epsilon RL/CD23. These data demonstrated that the Fc epsilon RL/CD23 and HLA-DR antigens are spatially associated on the B cell membrane.

Distinct intracellular compartments involved in invariant chain degradation and antigenic peptide loading of major histocompatibility complex (MHC) class II molecules.

Major histocompatibility complex (MHC) class II molecules are transported to intracellular MHC class II compartments via a transient association with the invariant chain (Ii). After removal of the invariant chain, peptides can be loaded onto class II molecules, a process catalyzed by human leukocyte antigen-DM (HLA-DM) molecules. Here we show that MHC class II compartments consist of two physically and functionally distinct organelles. Newly synthesized MHC class II/Ii complexes were targeted to endocytic organelles lacking HLA-DM molecules, where Ii degradation occurred. From these organelles, class II molecules were transported to a distinct organelle containing HLA-DM, in which peptides were loaded onto class II molecules. This latter organelle was not directly accessible via fluid phase endocytosis, suggesting that it is not part of the endosomal pathway. Uptake via antigen-specific membrane immunoglobulin resulted however in small amounts of antigen in the HLA-DM positive organelles. From this peptide-loading compartment, class II-peptide complexes were transported to the plasma membrane, in part after transit through endocytic organelles. The existence of two separate compartments, one involved in Ii removal and the other functioning in HLA-DM-dependent peptide loading of class II molecules, may contribute to the efficiency of antigen presentation by the selective recruitment of peptide-receptive MHC class II molecules and HLA-DM to the same subcellular location.

Major histocompatibility complex class II molecules induce the formation of endocytic MIIC-like structures.

During biosynthesis, major histochompatibility complex class II molecules are transported to the cell surface through a late endocytic multilaminar structure with lysosomal characteristics. This structure did not resemble any of the previously described endosomal compartments and was termed MIIC. We show here that continuous protein synthesis is required for the maintenance of MIIC in B cells. Transfection of class II molecules in human embryonal kidney cells induces the formation of multilaminar endocytic structures that are morphologically analogous to MIIC in B cells. Two lysosomal proteins (CD63 and lamp-1), which are expressed in MIIC of B cells, are also present in the structures induced by expression of major histocompatibility complex class II molecules. Moreover, endocytosed HRP enters the induced structures defining them as endocytic compartments. Exchanging the transmembrane and cytoplasmic tail of the class II alpha and beta chains for that of HLA-B27 does not result in the induction of multilaminar structures, and the chimeric class II molecules are now located in multivesicular structures. This suggests that expression of class II molecules is sufficient to induce the formation of characteristic MIIC-like multilaminar structures.

Evidence for the effects of a superantigen in rheumatoid arthritis.

While studying the alpha beta T cell receptor repertoire in rheumatoid arthritis (RA) patients, we found that the frequency of V beta 14+ T cells was significantly higher in the synovial fluid of affected joints than in the peripheral blood. In fact, V beta 14+ T cells were virtually undetectable in the peripheral blood of a majority of these RA patients. beta-chain sequences indicated that one or a few clones dominated the V beta 14+ population in the synovial fluid of individual RA patients, whereas oligoclonality was less marked for other V beta's and for V beta 14 in other types of inflammatory arthritis. These results implicate V beta 14-bearing T cells in the pathology of RA. They also suggest that the etiology of RA may involve initial activation of V beta 14+ T cells by a V beta 14-specific superantigen with subsequent recruitment of a few activated autoreactive v beta 14+ T cell clones to the joints while the majority of other V beta 14+ T cells disappear.

Cytoplasmic tail-dependent localization of CD1b antigen-presenting molecules to MIICs.

CD1 proteins have been implicated as antigen-presenting molecules for T cell-mediated immune responses, but their intracellular localization and trafficking remain uncharacterized. CD1b, a member of this family that presents microbial lipid antigens of exogenous origin, was found to localize to endocytic compartments that included the same specialized subset of endosomes in which major histocompatibility complex (MHC) class II molecules are proposed to bind endocytosed antigens. Unlike MHC class II molecules, which traffic to antigen-loading endosomal compartments [MHC class II compartments (MIICs)] primarily as a consequence of their association with the invariant chain, localization of CD1b to these compartments was dependent on a tyrosine-based motif in its own cytoplasmic tail.

Accumulation of HLA-DM, a regulator of antigen presentation, in MHC class II compartments.

The HLA-DM genes encode an unconventional HLA (human leukocyte antigen) class II molecule that is required for appropriate binding of peptide to classical HLA class II products. In the absence of DM, other class II molecules are unstable upon electrophoresis in sodium dodecyl sulfate and are largely associated with a nested set of peptides derived from the invariant chain called CLIP, for class II-associated invariant chain peptides. DMA and DMB associated and accumulated in multilaminar, intracellular compartments with classical class II molecules, but were found infrequently, if at all, at the cell surface. Thus, DM may facilitate peptide binding to class II molecules within these intracellular compartments.

[U-HO1. A new cell line derived from a primary refractory classical Hodgkin lymphoma]. / U-HO1. Eine neue Zelllinie, abstammend von einem primär therapierefraktären klassischen Hodgkin-Lymphom.

The Hodgkin cell line U-HO1 was established from a malignant pleural effusion of a 23-yr-old male patient during the end stage of refractory nodular sclerosing classical Hodgkin lymphoma (cHL). Since its establishment in 2005, U-HO1 has maintained stable characteristics in vitro and has a doubling time of about 4 days under standard culture conditions. U-HO1 forms typical Reed/Sternberg cells in suspension, is EBV negative, lacks HLA-ABC- but expresses HLA-D- proteins/CD74 and surface exposes CD15 together with CD30 in the absence of CD19 and CD20. Karyotype analysis of U-HO1 revealed a hyperdiploid karyotype with multiple clonal aberrations. Most significant is an elongated chromosome 2, der(2)t(2;10)(q35;q16.1)add(2)(p13). CGH analysis revealed the following imbalances: ish cgh dim(1)(p13p31)(p12q21), enh(2)(p13p23), dim(4)(q31.3qter), enh(6)(q22q27), enh(12), enh(18),enh(20)(q13.1pter). FISH analysis showed about six-fold amplification of REL and BCL-11A, thus, U-HO1 is prototypical for cHL in every aspect tested so far. Compared to other HL cell lines, U-HO1 proved far less genetically aberrant suggesting that U-HO1's imbalances suffice to cause the full-blown phenotype of primary refractory cHL.

[Adult celiac disease and intraepithelial lymphocytes. New options for diagnosis?]. / Enfermedad celíaca del adulto y linfocitos intraepiteliales. Nuevas opciones para el diagnóstico?

BACKGROUND: Permanent changes have been found in intraepithelial lymphocyte (IEL) subsets in patients with celiac disease. OBJECTIVE: The main aim of this study was to demonstrate the utility of determining CD3()/CD7(+) and T cell receptor (TCR) gamma-delta IEL subsets by flow cytometry as a diagnostic marker of adult celiac disease. PATIENTS AND METHODS: We performed a prospective study in a sample of 128 adult patients (70 with celiac disease and 58 controls). In all patients, distal duodenal biopsy was performed and IEL subsets were determined by flow cytometry. RESULTS: Patients with celiac disease showed an increase in gamma-delta IEL subsets and a decrease in CD3(-)/CD7(+) IEL subsets in comparison with the control group, independently of diet. CONCLUSIONS: The results indicate that IEL subset determination by flow cytometry could be useful to confirm diagnosis of celiac disease. IEL subsets should be investigated in diseases other than celiac disease, as well as in patients with potential or latent celiac disease.

Defective antigen presentation and novel structural properties of DR1 from an HLA haplotype associated with 21-hydroxylase deficiency.

We have segregated DR1+ individuals into two categories according to whether or not their class II+ cells stimulated T lymphocyte clones specific for or restricted to DR1. In a majority of cases (87%), failure to stimulate was a property of cells having the B14;DR1 haplotype and/or nonclassical 21-hydroxylase deficiency. Absence of clonal proliferation could not be explained by release of an intercellular suppressor factor or by stimulator cell absorption of interleukin 2. Homozygous cells inheriting both stimulatory (DR1n) and nonstimulatory (DR1x) haplotypes did not successfully mediate clonal expansion, implying that a trans acting factor operates intracellularly to modify both DR1 alleles or their products. Other DR alleles did not appear to be affected as evidence by normal proliferative responses of T lymphocyte clones restricted to DR2 or DR7 and stimulated by DR1x,2 and DR1x,7 cells, respectively. By two-dimensional gel analysis, we have further identified a 50-kD surface glycoprotein contained in anti-DR immunoprecipitates of DR1x, but not DR1n or non-DR1 cellular lysates. This 50-kD structure had antigenic and peptide identity to DR alpha and beta chains but was resistant to dissociation under conditions that normally separate DR alpha and beta (8 M urea plus 5% 2-mercaptoethanol); boiling in sodium dodecyl sulfate was required to segregate the component polypeptides of the 50-kD heterodimer. We postulate that a product of a novel combinatorial association between constitutive chains of DR may interfere with or compete for normal T cell receptor recognition of DR1 as both an alloantigen and a restricting element. We further propose that gene abnormalities within the class III region of a haplotype associated with nonclassical 21-hydroxylase deficiency may extend into the DR subregion of the major histocompatibility complex with consequent aberrations in DR1 presentation.

Structure and specificity of T cell receptors expressed by potentially pathogenic anti-DNA autoantibody-inducing T cells in human lupus.

The production of potentially pathogenic anti-DNA autoantibodies in SLE is driven by special, autoimmune T helper (Th) cells. Herein, we sequenced the T cell receptor (TCR) alpha and beta chain genes expressed by 42 autoimmune Th lines from lupus patients that were mostly CD4+ and represented the strongest inducers of such autoantibodies. These autoimmune TCRs displayed a recurrent motif of highly charged residues in their CDR3 loops that were contributed by N-nucleotide additions and also positioned there by the recombination process. Furthermore, Th lines from four of the five patients showed a marked increase in the usage of the V alpha 8 gene family. Several independent Th lines expressed identical TCR alpha and/or beta chain sequences indicating again antigenic selection. 10 of these Th lines could be tested further for antigenic specificity. 4 of the 10 pathogenic anti-DNA autoantibody-inducing Th lines responded to the non-histone chromosomal protein HMG and two responded to nucleosomal histone proteins; all presented by HLA-DR molecules. Another Th line responded to purified DNA more than nucleosomes. Thus, these autoimmune Th cells of lupus patients respond to charged epitopes in various DNA-binding nucleoproteins that are probably processed and presented by the anti-DNA B cells they selectively help.

Perforin-secreting killer cell infiltration and expression of a 65-kD heat-shock protein in aortic tissue of patients with Takayasu's arteritis.

Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of vascular cell injury in Takayasu's arteritis. To clarify the immunological mechanisms involved, we examined the expression of a cytolytic factor, perforin in infiltrating cells of aortic tissue samples from seven patients with Takayasu's arteritis. We also examined the expression of a 65-kD heat-shock protein (HSP-65), human leukocyte antigen classes I and II, and intercellular adhesion molecule-1 in the aortic tissue. Immunohistochemical studies showed that the infiltrating cells mainly consisted of gamma delta T lymphocytes, natural killer cells, macrophages, cytotoxic T lymphocytes and T helper cells, and that perforin was expressed in gamma delta T lymphocytes, natural killer cells, and cytotoxic T lymphocytes. In situ hybridization analysis also revealed expression of perforin mRNA in the infiltrating cells. Immunoelectron microscopic studies demonstrated that the infiltrating cells released massive amounts of perforin directly onto the surface of arterial vascular cells. We also found that expression of HSP-65, human leukocyte antigen classes I and II, and intercellular adhesion molecule-1 was strongly induced in the aortic tissue and might facilitate the recognition, adhesion and cytotoxicity of the infiltrating killer lymphocytes. These findings provide the first direct evidence that the infiltrating cells in the aortic tissue mainly consist of killer cells, and strongly suggest that these killer cells, especially gamma delta T lymphocytes, may recognize HSP-65 and play a critical role in the vascular cell injury of Takayasu's arteritis by releasing perforin.

Assessing the potential of immunohistochemistry for systematic gene expression profiling.

Immunohistochemistry (IHC) is a powerful technique for identifying sites of protein expression in tissues at the cellular and sub-cellular level. Here we have investigated the potential of using IHC for genome-wide expression screening by measuring the success rate and specificity of a panel of 35 monoclonal antibodies recognizing 5 well characterised CD antigens. Antibodies were pre-screened on acetone fixed frozen sections of spleen, tonsil and colon tissues. 19/35 antibodies gave staining with a success rate of 0/7 for JAM-2, 1/4 for CD99, 3/6 for CD138, 5/8 for CD45 and 10/10 for MHC-class II. 16/19 of these antibodies also gave staining on formalin fixed paraffin embedded tissue sections of tonsil and colon. All antibodies that had given staining were then profiled on tissues presented in human tissue microarrays. In the frozen microarrays 216 cores from 29 normal tissue types were present and in the formalin fixed paraffin array 344 cores from 35 normal and 4 cancers were represented. Where multiple antibodies were positive, there was evidence of consistent staining of the same tissues with several antibodies. In some cases differences in staining were observed potentially due to differential splice variants, polymorphisms or protein modification. With some antibodies there was evidence of cross-reactivity to inappropriate cells or structures. In addition the staining intensity with formalin fixation was changed quantitatively for some antibodies and in a few cases qualitatively, representing differential sensitivity of specific and non-specific epitopes to fixation. Accordingly, whilst IHC has potential for describing protein expression of unknown genes, these results emphasise a need to systematically address issues of specificity and sensitivity if appropriate profiles are to be described.

Regulated expression of a murine class I gene in transgenic mice.

The major histocompatibility complex class I genes play an essential role in the immune presentation of aberrant cells. To gain further insight into the regulation of the expression of these class I genes and to better define the functions of their protein products, we made use of the technique of gene transfer into the germ line of inbred mice. With the use of locus-specific DNA probes, we observed that a transgenic class I gene was expressed in a tissue-dependent fashion analogous to that of an endogenous class I gene. In addition, the level of expression of the transgenic gene was substantially higher that that of the endogenous gene. The availability of transgenic mice properly expressing a foreign murine class I gene provides a unique system to further define the role of the class I antigens in the maturation of the immune response and in determining the malignant and metastatic phenotypes of tumor cells.

Specific immune recognition of pancreatic carcinoma by patient-derived CD4 and CD8 T cells and its improvement by interferon-gamma.

Pancreatic carcinoma is a very aggressive disease and little is known about its immunobiology. We here describe the presence in pancreatic cancer patients of spontaneously induced functional CD4 and CD8 memory/effector T cells reactive to autologous tumor cells or to the pancreatic cancer associated antigen, MUC-1. Such specific cells were present in the bone marrow or peripheral blood of most of the 23 tested patients. Low dose stimulation of primary cultures of pancreatic cancer cells with 500 IU/ml IFN-gamma for 72 h enhanced HLA-I expression and induced the de novo expression of HLA-II molecules. This led to a much better immune recognition by autologous HLA-I restricted and purified CD8 T cells and allowed tumor cell recognition by HLA-II restricted purified CD4 T-helper cells. Thus, interferon-gamma appears to be a useful adjuvant cytokine to enhance the immunogenicity of a patients' tumor cells and their recognition by tumor reactive immune cells.

Expression and function of class II antigens on gastric carcinoma cells and gastric epithelia: differential expression of DR, DQ, and DP antigens.

The expression of human histocompatibility leukocyte antigen class II antigens on gastric epithelia and gastric carcinoma (GaCa) was studied with the use of murine monoclonal antibodies to DR, DQ, and DP antigens. DR and DP antigens but not DQ antigens were demonstrated in fundic glands of normal gastric epithelia, and DP+ cells were located in part of the DR+ epithelia. Of 15 GaCas examined, 11 expressed DR antigen, and the degree of the expression varied considerably among the specimens. DP antigen was found in 3 of the 11 DR+ carcinomas, and the DQ antigen was found in one of the 3 DR+ DP+ specimens. Thus the expression of class II antigens in normal gastric epithelia and GaCas appears to be in the order of DR, DP, and DQ. Studies on 3 GaCa cell lines (Kato III, MKN28, and MKN45) demonstrated that 1 line (Kato III) expressed DR antigen only, and the remaining lines were negative. Interferon (IFN)-gamma treatment enhanced the expression of DR antigen on Kato III cells and induced expression of DQ and DP antigens. The IFN-gamma treatment also induced expression of DR antigen but not DQ or DP antigens in 1 of the 2 negative cell lines. The induction of the class II antigens by IFN-gamma was shown to be dose dependent. However, maximal induction of DQ and DP antigens on the Kato III cells and DR antigens on MKN45 cells required 10 times more IFN-gamma than that needed for the maximal expression of DR antigen on Kato III. Northern blot analyses of cytoplasmic RNA from these cells were in agreement with and affirmed the above-described expression of the class II antigens on the cell lines. The DR antigen on the Kato III cells was capable of stimulating allogeneic lymphocytes in MLR, and its stimulatory activity was significantly enhanced by IFN-gamma. These results demonstrated a differential expression of class II antigens in the "DR, DP, and DQ order" in normal gastric epithelia, GaCa cells, and GaCa cell lines, suggesting different mechanisms acting discordantly on the expression of each of these antigens and that the DR antigen on the GaCa cell lines possesses MLR-stimulatory ability.