Search for Effective Serum Tumor Markers for Early Diagnosis of Hepatocellular Carcinoma Associated with Hepatitis C.

The aim of the study was to identify the most effective serum tumor markers for early diagnosis of hepatocellular carcinoma based on the combination of diagnostic characteristics and correlations. Materials and Methods: There were observed 55 patients with chronic hepatitis C in the stage of liver cirrhosis with a verified diagnosis of hepatocellular carcinoma. The control group consisted of 55 patients with chronic hepatitis C at the stage of liver cirrhosis without hepatocellular carcinoma, comparable to the experimental group in terms of basic clinical profile. The following tumor markers were estimated in both groups: alpha-fetoprotein (AFP), alpha-fetoprotein-L3 (AFP-L3), annexin A2 (ANXA2), heparin-binding growth factor Midkine (MDK), glypican-3 (GPC3), des-gamma-carboxyprothrombin (DCP, PIVKA-II), dickkopf-related protein 1 (DKK-1), osteopontin (OPN), and Golgi protein 73 (GP73). There were also evaluated such indices as diagnostic sensitivity, specificity, positive predictive value, negative predictive value, likelihood ratio of a positive test, the possible correlation between alpha-fetoprotein and other tumor markers. The area under the ROC curve (AUC) was calculated at the 95% confidence interval. Results: The greatest sensitivity was revealed when using heparin-binding growth factor, annexin A2, osteopontin. Alpha-fetoprotein, alpha-fetoprotein-L3, glypican-3, des-gamma-carboxyprothrombin, dickkopf-related protein 1 had the best specificity. AUC>0.75 was found in annexin A2, heparin-binding growth factor, glypican-3, des-gamma-carboxyprothrombin, osteopontin, Golgi protein 73. The likelihood ratio of a positive test result was the highest for glypican-3. A significant correlation was found between alpha-fetoprotein and alpha-fetoprotein-L3, annexin A2, des-gamma-carboxyprothrombin. Conclusion: According to the aggregate indicators of diagnostic efficiency, heparin-binding growth factor, glypican-3, and osteopontin are the most promising tumor markers of those studied. When they are used, integral AUC values are above the average, the level of these tumor markers in the blood of patients with hepatocellular cancer does not correlate with alpha-fetoprotein. They are applicable for diagnosing liver cancer in AFP-negative patients. The combined use of AFP + GPC3, AFP + OPN has already shown their advantages. However, the efficacy of the combination of AFP + MDK, GPC3 + OPN has not been determined yet; therefore, significance of the combined use of these tumor markers in the diagnosis of liver cancer should be investigated in the near future.

Clinical and Experimental Evaluation of Diagnostic Significance of Alpha-Fetoprotein and Osteopontin at the Early Stage of Hepatocellular Cancer.

We evaluated the possibility of using an experimental model of hepatocellular carcinoma to study oncomarkers of primary liver cancer and compared the diagnostic efficacy of alpha-fetoprotein and osteopontin in the experiment and in clinical practice. Experimental studies were performed on a model of hepatocellular carcinoma induced by administration of diethyl nitrosamine to Fisher-344 rats. In addition, the levels of α-fetoprotein and osteopontin were determined in 35 patients with hepatocellular carcinoma detected at stages I-II according to TNM classification. The proposed model of liver cancer in rats reflects the sequence of stages characteristic of hepatocellular carcinoma in humans: liver fibrosis-cirrhosis-cancer. This model is applicable for the study of tumor markers at the early stage of tumor development. Osteopontin was found to have a more powerful diagnostic potential then alpha-fetoprotein.

[Application of alpha-fetoprotein and osteopontin combination for early diagnosis of hepatocellular carcinoma associated with hepatitis C.]

Liver cirrhosis in the outcome of hepatitis C is the leading cause of hepatocellular carcinoma (HCC) in the world. Early diagnosis and timely treatment of HCC are important for reducing mortality and increasing life expectancy of patients with hepatocellular carcinoma. To assess the risk of HCC, the definition of alpha-fetoprotein (AFP) in the blood is most widely used, but low sensitivity limits its diagnostic value. In 2012, a new HCC biomarker - osteopontin (OPN), which is a secreted phosphoprotein that has a high affinity for integrins was proposed. The level of acute renal failure begins to rise in the early stages of malignancy, before the period of HCC detection by imaging methods, and has significantly better sensitivity than AFP. The purpose of this study is to evaluate the diagnostic efficacy of the combined determination of alpha-fetoprotein and osteopontin in prospective monitoring of patients with chronic hepatitis C in the advanced phase of liver fibrosis. Monitoring of 588 patients with hepatitis C was carried out from February 2013 to February 2019. HCC was detected in 55 of them (2.6% per year). The combination of 2 biomarkers showed better diagnostic efficacy than alpha-fetoprotein and osteopontin separately: AUC 0.85 (95% CI 0.80-0.90) versus AUC 0.63 (95% CI 0.57-0, 70) and AUC 0.82 (95% CI 0.77-0.88), respectively. This combination showed a sensitivity of 85.5% and made it possible to diagnose HCC with a prognostic level of a positive result of 72.3% at 19,4±0,8 weeks before the diagnosis was confirmed by instrumental imaging methods (ultrasound, MRI, CT). In the combined variant, ARF made the greatest contribution to the increase in diagnostic efficacy (AUC). At an early and very early stage of HCC development, isolated HCC elevations were found in only 5.4% of patients. Conclusion: the combined use of alphafetoprotein and osteopontin as a diagnostic panel can be recommended for monitoring patients with liver cirrhosis in the outcome of hepatitis C and predicting HCC at an early stage of development.

Developmental regulation of p53-dependent radiation-induced thymocyte apoptosis in mice.

The production of T cell receptor αß(+) (TCRαß(+) ) T lymphocytes in the thymus is a tightly regulated process that can be monitored by the regulated expression of several surface molecules, including CD4, CD8, cKit, CD25 and the TCR itself, after TCR genes have been assembled from discrete V, D (for TCR-ß) and J gene segments by a site-directed genetic recombination. Thymocyte differentiation is the result of a delicate balance between cell death and survival: developing thymocytes die unless they receive a positive signal to proceed to the next stage. This equilibrium is altered in response to various physiological or physical stresses such as ionizing radiation, which induces a massive p53-dependent apoptosis of CD4(+) CD8(+) double-positive (DP) thymocytes. Interestingly, these cells are actively rearranging their TCR-α chain genes. To unravel an eventual link between V(D)J recombination activity and thymocyte radio-sensitivity, we analysed the dynamics of thymocyte apoptosis and regeneration following exposure of wild-type and p53-deficient mice to different doses of γ-radiation. p53-dependent radio-sensitivity was already found to be high in immature CD4(-) CD8(-) (double-negative, DN) cKit(+) CD25(+) thymocytes, where TCR-ß gene rearrangement is initiated. However, TCR-αß(-) CD8(+) immature single-positive thymocytes, an actively cycling intermediate population between the DN and DP stages, are the most radio-sensitive cells in the thymus, even though their apoptosis is only partially p53-dependent. Within the DP population, TCR-αß(+) thymocytes that completed TCR-α gene recombination are more radio-resistant than their TCR-αß(-) progenitors. Finally, we found no correlation between p53 activation and thymocyte sensitivity to radiation-induced apoptosis.

Multiple sclerosis-associated retrovirus particles cause T lymphocyte-dependent death with brain hemorrhage in humanized SCID mice model.

A retroviral element (multiple sclerosis-associated retrovirus, MSRV) defining a family of genetically inherited endogenous retroviruses (human endogenous retrovirus type W, HERV-W) has been characterized in cell cultures from patients with multiple sclerosis. Recently, MSRV retroviral particles or the envelope recombinant protein were shown to display superantigen activity in vitro, but no animal model has yet been set up for studying the pathogenicity of this retrovirus. In the present study, the pathogenicity of different sources of MSRV retroviral particles has been evaluated in a hybrid animal model: severe combined immunodeficiency (SCID) mice grafted with human lymphocytes and injected intraperitoneally with MSRV virion or mock controls. MSRV-injected mice presented with acute neurological symptoms and died within 5 to 10 days post injection. Necropsy revealed disseminated and major brain hemorrhages, whereas control animals did not show abnormalities (P <.001). In ill animals, reverse transcriptase-polymerase chain reaction (RT-PCR) analyses showed circulating MSRV RNA in serum, whereas overexpression of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma was evidenced in spleen RNA. Neuropathological examination confirmed that hemorrhages occurred prior to death in multifocal areas of brain parenchyma and meninges. Further series addressed the question of immune-mediated pathogenicity, by inoculating virion to SCID mice grafted with total and T lymphocyte-depleted cells in parallel: dramatic and statistically significant reduction in the number of affected mice was observed in T-depleted series (P <.001). This in vivo study suggests that MSRV retroviral particles from MS cultures have potent immunopathogenic properties mediated by T cells compatible with the previously reported superantigen activity in vitro, which appear to be mediated by an overexpression of proinflammatory cytokines.

TCRA gene rearrangement in immature thymocytes in absence of CD3, pre-TCR, and TCR signaling.

During thymocyte differentiation, TCRA genes are massively rearranged only after productively rearranged TCRB genes are expressed in association with pTalpha and CD3 complex molecules within a pre-TCR. Signaling from the pre-TCR via the CD3 complex is thought to be required to promote TCRA gene accessibility and recombination. However, alphabeta(+) thymocytes do develop in pTalpha-deficient mice, showing that TCRalpha-chain genes are rearranged, either in CD4(-)CD8(-) or CD4(+)CD8(+) thymocytes, in the absence of pre-TCR expression. In this study, we analyzed the TCRA gene recombination status of early immature thymocytes in mutant mice with arrested thymocyte development, deficient for either CD3 or pTalpha and gammac expression. ADV genes belonging to different families were found rearranged to multiple AJ segments in both cases. Thus, TCRA gene rearrangement is independent of CD3 and gammac signaling. However, CD3 expression was found to play a role in transcription of rearranged TCRalpha-chain genes in CD4(-)CD8(-) thymocytes. Taken together, these results provide new insights into the molecular control of early T cell differentiation.

Multiple sclerosis retrovirus particles and recombinant envelope trigger an abnormal immune response in vitro, by inducing polyclonal Vbeta16 T-lymphocyte activation.

A retroviral element (MSRV) defining a family of genetically inherited endogenous retroviruses (HERV-W) has recently been characterized in cell cultures from patients with multiple sclerosis (MS). To address the possible relationship with MS, direct detection of circulating virion RNA was proposed but revealed technically difficult to perform in standardized conditions, in the face of multiple endogenous HERV-W copies. A parallel approach has evaluated MSRV potential pathogenicity in relation to characteristic features of multiple sclerosis, in particular, T-lymphocyte-mediated immunopathology. We report here that MSRV particles induce T-lymphocyte response with a bias in the Vbeta16 chain usage in surface receptor, whatever the HLA DR of the donor. A recombinant MSRV envelope-but not core-protein reproduced similar nonconventional activation. Molecular analysis of Vbeta CDR3 showed that Vbeta16 expansions are polyclonal. Our results thus provide evidence that MSRV envelope protein can trigger an abnormal immune response with similar characteristics to that of superantigens.

Murine dendritic cells derived from myeloid progenitors of the thymus are unable to produce bioactive IL-12p70.

Dendritic cells (DC) are present at low density in the thymus where they mediate negative selection of self-reactive thymocytes. Previous reports suggest that thymic DC (TDC) are a single population of lymphoid-related DC. In this study, we documented the presence in the adult mouse thymus of an additional population of TDC exhibiting a myeloid phenotype (CD11c(+) CD8alpha(-) CD11b(+)). This population, which can be purified, represented approximately 20% of the total TDC and differs from the population of lymphoid TDC (CD11c(+) CD8(+) CD11b(-)) by its incapacity to produce IL-12p70 under double stimulation by LPS and anti-CD40. Furthermore, using an original culture system allowing expansion of DC from myeloid progenitors, we demonstrated that DC exhibiting a similar myeloid phenotype can be derived from a common DC/macrophage progenitor resident in the adult mouse thymus. We found that, in contrast with myeloid splenic DC expanded in the same conditions, these cultured TDC were unable to produce IL-12p70 under double stimulation by LPS and anti-CD40 or LPS and IFN-gamma. Thus, our results suggest that 1) adult mouse thymus contains at least two phenotypically and functionally distinct populations of DC; and 2) cultured myeloid DC derived from thymus and spleen differ by their ability to produce IL-12p70. The mechanisms underlying the differences in IL-12-secreting capacities of the cultured splenic and thymic DC are under current investigation.

Tetanus toxin L chain is processed by major histocompatibility complex class I and class II pathways and recognized by CD8+ or CD4+ T lymphocytes.

Tetanus toxin (TeNT) is a heterodimeric protein antigen, whose light chain (L) is translocated in the cytosol of neuronal target cells specifically to cleave its substrates, vesicle-associated membrane protein-2 (VAMP-2, or synaptobrevin) or cellubrevin. We report that the L chain behaves as a nominal antigen recognized by specific T-cell clones upon either class I- or II-restricted presentation. Three types of responses are observed: (i) a TeNT- and L-specific CD8+ T-cell response, that can be inhibited in a dose-dependent manner by the proteasome inhibitor clasto-Lactacystin beta-lactone; (ii) a CD4+ T-cell response specific for L but not TeNT, with recognition of a determinant processed in a chloroquine-sensitive and brefeldin A-resistant compartment; (iii) a CD4+ T-cell response against both L and TeNT, with processing in a brefeldin A-sensitive compartment. The L chain processing was investigated in U937 cells by internalization and localization of L chain by separation of the cell content by differential centrifugation experiments. After incubation with TeNT or L chain in the presence of H chain, the L chain was predominantly distributed in the cytosolic fraction, whereas incubation with L alone led to localization in a lysosome/membrane fraction. The distribution of the TeNT L chain in both cytosolic and endocytic compartments of the antigen-presenting cell accounted for its processing by both class I and class II pathways. Furthermore, an epitope overlapping with the zinc-binding region was recognized by CD4+ and CD8+ T cells.

A two-step culture method starting with early growth factors permits enhanced production of functional dendritic cells from murine splenocytes.

Dendritic cells (DC) are professional antigen presenting cells (APC) able to activate naive T cells and initiate the immune response. They are present in most tissues at very low concentrations and are difficult to isolate. DC can be obtained in larger numbers by their propagation from progenitors present in blood, bone marrow and spleen. However, biochemical studies and biological analysis of DC functions require very large numbers of these cells. In this paper, we described a two-step culture system using unfractionated splenocytes from BALB/c mice as a source of DC progenitors. The proliferative capacity of the progenitors is amplified in the first step of the culture (day 0-6) using different combinations of early acting cytokines combined or not with granulocyte-macrophage CSF (GM-CSF). The second step of the culture starts at day 6 with the removal of early growth factors in order to allow the differentiation and final maturation of DC during 2-3 weeks of culture with flt-3 ligand (flt-3L) and GM-CSF. The addition of Stem Cell Factor (SCF) or IL-6 to the standard combination of flt-3L+/-GM-CSF produces a large increase in the proliferation of GM and DC progenitors (28 times and 11 times respectively) in the first step of the culture. This proliferative wave of DC progenitors is followed by the production of a high percentage of immature and mature DC in flt-3L+GM-CSF stimulated cultures. The best combination of early cytokines in terms of proliferative activity and subsequent level of DC production was flt-3L+IL-6+GM-CSF, which permitted the generation of 1 to 2x10(9) DC from one single spleen. Using this growth factor cocktail, a mixture of immature (2/3) and mature (1/3) DC was produced until day 14 of culture, and levels of MHC class II and costimulatory molecules (CD40, B7.2) increased between 2 and 4 weeks of incubation, or within 2 days when stimulated by IL-4 or LPS. The splenic DC produced after 2 weeks of culture are fully functional, exhibiting a high capacity of endocytosis when immature, a strong stimulatory reactivity in mixed leukocyte reaction and consistently producing high levels of bioactive IL-12 p70 after CD 40 ligation in the presence of LPS between 13 and 43 days of culture.

Pairing of Vbeta6 with certain Valpha2 family members prevents T cell deletion by Mtv-7 superantigen.

Superantigens (SAg) are proteins of bacterial or viral origin able to activate T cells by forming a trimolecular complex with both MHC class II molecules and the T cell receptor (TCR), leading to clonal deletion of reactive T cells in the thymus. SAg interact with the TCR through the beta chain variable region (Vbeta), but the TCR alpha chain has been shown to have an influence on the T cell reactivity. We have investigated here the role of the TCR alpha chain in the modulation of T cell reactivity to Mtv-7 SAg by comparing the peripheral usage of Valpha2 in Vbeta6(+) (SAg-reactive) and Vbeta8.2(+) (SAg non-reactive) T cells, in either BALB/D2 (Mtv-7(+)) or BALB/c (Mtv-7(-)) mice. The results show, first, that pairing of Vbeta6 with certain Valpha2 family members prevents T cell deletion by Mtv-7 SAg. Second, there is a strikingly different distribution of the Valpha2 family members in CD4 and CD8 populations of Vbeta6 but not of Vbeta8.2 T cells, irrespective of the presence of Mtv-7 SAg. Third, the alpha chain may play a role in the overall stability of the TCR/SAg/MHC complex. Taken together, these results suggest that the Valpha domain contributes to the selective process by its role in the TCR reactivity to SAg/MHC class II complexes, most likely by influencing the orientation of the Vbeta domain in the TCR alphabeta heterodimer.

Immunotherapy by non-myeloablative stem cell transplantation: study of the immune reconstitution. Arguments for distinct cell subsets in skin and blood.

INTRODUCTION: Non-myeloablative peripheral stem cell transplantation has been shown to induce tumour rejection in patients with acute leukaemia. However, the immunological mechanisms involved and the immune reconstitution achieved have not been investigated. MATERIALS AND METHODS: We describe the cases of two patients for whom we have studied the lymphocyte reconstitution achieved, using both phenotypic and genetic analyses of the T-cell repertoire, after peripheral stem cell transplantation. RESULTS: : In both cases we observed immune reconstitution with T-cell repertoire evolution and presence of activated CD8(+) T cells. In one of the patients an activated clone expressing Vbeta8 represents 46% of the CD8(+) cells. Expansion of this clone occurred in the absence of graft vs host disease symptoms. In the second case a skin lesion typical of graft vs host disease appeared after complete remission had been achieved. The T-cell repertoire in a biopsy of the lesion was distinct from that observed in the blood. CONCLUSION: Our study indicates that peripheral donor cells can effectively reconstitute a grafted patient while inducing an immune response against antigens expressed by the leukaemic/myeloma cells. Our data provide arguments for different populations of T cells associated with graft vs leukaemia/lymphoma and GVH effects.

Importance of thioredoxin in the proteolysis of an immunoglobulin G as antigen by lysosomal Cys-proteases.

For disulphide-bonded antigens, reduction has been postulated to be a prerequisite for proteolytic antigen processing, with subsequent production of major histocompatibility complex (MHC) class II binding fragments. The murine monoclonal immunoglobulin G (IgG) CE25/B7 was used as a multimeric antigen in a human model. Native IgG is highly resistant to proteolysis and has been previously found to be partially reduced at early steps of cell processing to become a suitable substrate for endopeptidases. The role of the oxidoreductase thioredoxin (Trx) was assessed in the reduction of the IgG by cleavage of H-L and H-H disulphide bonds. Recombinant human Trx (rTrx) has been assayed in a proteolytic in vitro system on IgG using endosomal and lysosomal subcellular fractions from B lymphoblastoid cells. rTrx is required in a dose-dependent manner for development of efficient proteolysis, catalysed by thiol-dependent Cys-proteases, such as cathepsin B. We demonstrated that cathepsin B activity was stimulated by the addition of rTrx. Thus, we propose that Trx-dependent IgG proteolysis occurred, on the one hand by means of the unfolding of the IgG after disulphide reduction, becoming a substrate of lysosomal proteases, and on the other hand by Cys-proteases such as cathepsin B that are fully active upon the regeneration of their activity by hydrogen donors.

Purification of intracellular compartments involved in antigen processing: a new method based on magnetic sorting.

In the present study, we describe a method to specifically isolate intracellular compartments containing endocytosed antigen. We have demonstrated that isolated compartments represent a small proportion of the intracellular material, highly enriched in antigen. Antigen-containing vesicles are specifically sorted from other intracellular compartments, such as endoplasmic reticulum or Golgi apparatus, and from the plasma membrane. They remain functional in vitro since they can be acidified, and the antigen inside has been found to be partially proteolysed. In macrophages, kinetic analysis has revealed that the antigen is first found in compartments of endosomal density, carrying Rab 5 and Rab 7, then in late compartments of lysosomal density, which are rich in proteases. The global protein content of the compartments was mapped by two-dimensional electrophoresis. In B lymphocytes, this method has allowed the isolation of endocytic compartments emerging from receptor-mediated endocytosis of the antigen. After 2 h of chase, the antigen reached vesicles containing large amounts of MHC-class II molecules, invariant chain and human leucocyte antigen-DM, where peptide loading can occur.

Heat shock increases antigenic peptide generation but decreases antigen presentation.

The heat shock response is a universal and highly conserved cellular response to stress. We describe here the effect of elevated temperature on the capacity of B cells to present antigen. Heat shock markedly affects the ability of these cells to process and present tetanus toxin to class II-restricted T cell clones. Inhibition of antigen presentation is due neither to a modification of antigen capture nor to a variation of major histocompatibility complex (MHC) class II molecule synthesis and cell surface expression. Stressed and nonstressed B cells are able to present peptides loaded at the cell surface with the same efficiency. Nevertheless, heat shock leads to an increase of antigen peptide generation in subcellular compartments; an enhancement of cathepsin B activity is also observed. These data suggest that such a stress induces a failure in the intracellular peptide loading onto MHC class II molecules.

T cell receptor V beta repertoire in mice lacking endogenous mouse mammary tumor provirus.

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) V beta segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, -7, -8 and -9) and 38CH (Mtv-) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, -7, -8 or -9. The TcR V beta chain (TcR V beta) usage in these mice was analyzed using monoclonal antibodies specific for TcR V beta 2, V beta 3, V beta 4, V beta 5, V beta 6, V beta 7, V beta 8, V beta 11, V beta 12 and V beta 14 segments. Both Mtv-8+ mice and Mtv-9+ mice deleted TcR V beta 5+ and V beta 11+ T cells. Moreover, we also observed the deletion of TcR V beta 12+ cells by Mtv-8 and Mtv-9 products. Mtv-6+ and Mtv-7+ animals deleted TcR V beta 3+ and V beta 5+ cells, and TcR V beta 6+, V beta 7+ and V beta 8.1+ cells, respectively. Unexpectedly, TcR V beta 8.2+ cells were also deleted in some backcross mice expressing Mtv-7. TcR V beta 8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn-->Asp) in position 19 on the TcR V beta 8.2 fragment. Reactivities of BALB.D2 TcR V beta 8.2 and 38CH TcR V beta 8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR V beta 2+, V beta 4+ and V beta 8+ CD4+ T cell subsets in Mtv-8+ and Mtv-9+ mice, and TcR V beta 4+ CD4+ T cells in Mtv-6+ and Mtv-7+ mice, when compared with the T cell repertoire of Mtv- mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.

T cell regulation of collagen-induced arthritis in mice. III. Is T cell vaccination a valuable therapy?

Since T cells play a critical role in collagen-induced arthritis (CIA), CD4+ T cell hybridomas were derived from DBA/1 mice immunized with bovine type II collagen (CII). The hybrid clones selected were Thy-1-2+, CD4+, CD8-, T cell receptor (TcR) alpha beta + and produced interleukin-2 in response to CII peptides presented by I-Aq molecules. The clones were collagen type-specific and recognized CII from many species except the mouse. More precisely, the reactivity was directed against the immunodominant cyanogen bromide-cleaved fragment CB11(II). Analysis of the TcR carried by the T cell hybridomas showed that they used identical V alpha and J alpha (V alpha BMB, J alpha 20) gene segments and two distinct V beta (V beta 1 and V beta 4) associated with the J beta 2.5 gene segment. Interestingly, the junctional regions were highly conserved in structure and length. These findings may indicate a strong in vivo selection by the antigen for a particular combination of both alpha and beta chains of the TcR. Inoculation of irradiated anti-CII T cell hybrids into DBA/1 mice, before priming with CII, altered the course of the disease resulting in either a long-lasting suppression or an exacerbation of CIA whereas a control CD4+ hybridoma with an unrelated specificity did not influence the development of arthritis. However, the regulatory effect of anti-CII T cell clones was unpredictable, suggesting that the TcR structure may not solely account for the modulation of CIA and that T cell vaccination is not a reliable method for inducing suppression of CIA.

Analysis of the nucleotide sequence variation of the antigen-binding domain of DR alpha and DQ alpha molecules as related to the evolution of papillomavirus-induced warts in rabbits.

We previously found that regression of skin warts induced by the Shope cottontail rabbit papillomavirus in New Zealand White rabbits, as well as malignant conversion of persistent warts, are linked to a restriction fragment length polymorphism of the major histocompatibility complex class II DR alpha and DQ alpha genes. To find out whether this immunogenetic control could be connected with the antigen binding and presentation function of the alpha 1 domain of class II molecules, we have sequenced the exon 2 of the four DR alpha EcoRI and six of the seven DQ alpha PvuII restriction fragment length polymorphism alleles identified, and deduced the encoded amino acid sequences. We found no amino acid polymorphism among DR alpha alleles, indicating that the alpha 1 domain of the DR alpha chain does not condition wart regression or cancer development. In contrast, 27 of the 82 amino acids of the DQ alpha 1 domain were found variable, defining five amino acid sequence alleles. The restriction fragment length polymorphism allele linked to regression and another allele not linked to regression share the same alpha 1 domain, indicating that wart regression is rather conditioned by a closely linked gene. The most divergent DQ alpha 1 allele, however, was that associated with a higher risk of cancer. Alignment of rabbit and human DQ alpha exon 2 alleles disclosed that amino acid charge variations occur at positions assumed to be important for peptide binding in humans. By modulating the affinity for tumor-specific antigenic peptides, such transitions could affect immune surveillance and, thus, condition the risk for progression to carcinoma of papillomavirus-associated lesions.

Identification of an endogenous mammary tumor virus involved in the clonal deletion of V beta 2 T cells.

Expression of V beta (beta-chain variable region) gene segments was investigated in the Mus m. domesticus DDO strain, which possesses a large genomic deletion encompassing 20 of the 29 V beta gene segments known in BALB/c. Stainings using V beta-specific monoclonal antibodies revealed that up to 60% of the peripheral T cells use 3 V beta gene segments. Variable frequencies of V beta 2 T cells were observed among DDO individuals. Segregation analyses of F2 crosses between V beta 2-deletor mice and mammary tumor virus (Mtv)-free mice led to the identification of a new endogenous Mtv, named Mtv-DDO, mediating V beta 2 T cell clonal deletion. Mtv-DDO structure is conserved with the exception of the carboxy-terminal region as compared to other Mtv. Comparison between Mtv sharing the same V beta specificity and isolated from laboratory or wild mice confirms that a stretch of 11 amino acids, defined as the V beta-specific region, is required for the V beta-specific interaction. Limited substitutions in this region account for the shift of the Mtv specificity towards different V beta.