Magnetically localized and wash-free fluorescence immunoassay (MLFIA): proof of concept and clinical applications.

Immunoassays are used for many applications in various markets, from clinical diagnostics to the food industry, generally relying on gold-standard ELISAs that are sensitive, robust, and cheap but also time-consuming and labour intensive. As an alternative, we propose here the magnetically localized and wash-free fluorescence immunoassay (MLFIA): a no-wash assay to directly measure a biomolecule concentration, without mixing nor washing steps. To do so, a fluorescence no-wash measurement is performed to generate a detectable signal. It consists of a differential measurement between the fluorescence of fluorophores bound to magnetic nanoparticles specifically captured by micro-magnets against the residual background fluorescence of unbound fluorophores. Targeted biomolecules (antibodies or antigens) are locally concentrated on micro-magnet lines, with the number of captured biomolecules quantitatively measured without any washing step. The performance of the MLFIA platform is assessed and its use is demonstrated with several biological models as well as clinical blood samples for HIV, HCV and HBV detection, with benchmarking to standard analyzers of healthcare laboratories. Thus, we demonstrated for the first time the versatility of the innovative MLFIA platform. We highlighted promising performances with the successful quantitative detection of various targets (antigens and antibodies), in different biological samples (serum and plasma), for different clinical tests (HCV, HBV, HIV).

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.

Reassignment of the murine 3'TRDD1 recombination signal sequence.

T cell receptor genes are assembled in developing T lymphocytes from discrete V, D, and J genes by a site-specific somatic rearrangement mechanism. A flanking recombination signal, composed of a conserved heptamer and a semiconserved nonamer separated by 12 or 23 variable nucleotides, targets the activity of the rearrangement machinery to the adjoining V, D, and J genes. Following the rearrangement of V, D, or J genes, their respective recombination signals are ligated together. Although these signal joints are allegedly invariant, created by the head-to-head abuttal of the heptamers, some do exhibit junctional diversity. Recombination signals were initially identified by comparison and alignment of germ-line sequences with the sequence of rearranged genes. However, their overall low level of sequence conservation makes their characterization solely from sequence data difficult. Recently, computational analysis unraveled correlations between nucleotides at several positions scattered within the spacer and recombination activity, so that it is now possible to identify putative recombination signals and determine and predict their recombination efficiency. In this paper, we analyzed the variability introduced in signal joints generated after rearrangement of the TRDD1 and TRDD2 genes in murine thymocytes. The recurrent presence of identical nucleotides inserted in these signal joints led us to reconsider the location and sequence of the TRDD1 recombination signal. By combining molecular characterization and computational analysis, we show that the functional TRDD1 recombination signal is shifted inside the putative coding sequence of the TRDD1 gene and, consequently, that this gene is shorter than indicated in the databases.

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.

Simultaneous evaluation of lymphocyte subpopulations in the liver and in peripheral blood mononuclear cells of HCV-infected patients: relationship with histological lesions.

ABBREVIATIONS: Intrahepatic lymphocytes are believed to be involved in the immunopathogenesis of hepatitis C virus (HCV) infection and the evolution of HCV-induced hepatitis. In the present study, we examined the three main intrahepatic lymphocyte subsets, namely CD3+CD56- conventional T lymphocytes, CD3+CD56+ natural T (NT) lymphocytes and CD3-CD56+ natural killer (NK) lymphocytes in HCV-infected patients. The proportion of each lymphocyte subset was evaluated both in liver biopsies and in samples of peripheral blood lymphocytes (PBL) by flow cytometry in 21 patients with histologically proven chronic hepatitis C. Simultaneously, alanine aminotransferase (ALT) levels, viral load and histological lesions were assessed. Neither NT nor NK populations correlated with any biochemical, viral or histological parameters. Furthermore, Valpha24+ NT lymphocytes showed no preferential enrichment in the liver of HCV-infected patients. Regarding conventional T lymphocytes, a highly significant linear correlation was found between intrahepatic CD3+CD56- T lymphocytes and the Knodell score, a numerical score for assessing histological activity and fibrosis (r = 0.715, P < 0.0001) and more specifically with the periportal necrosis parameter, which is the main lesion of chronic hepatitis C. In addition, analysis of the peripheral compartment revealed a high correlation between values of CD3+CD56- lymphocytes and both Knodell score (r = 0.624, P = 0.003) and serum ALT levels and again with periportal necrosis. The strong correlation between the proportion of peripheral CD3+CD56- conventional T lymphocytes and the severity of hepatic lesions leads us to propose that evaluation of this accessible peripheral population could be used as an indicator test for the severity of histological lesions in chronic hepatitis C.

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.

Both TCR alpha and TCR delta chain diversity are regulated during thymic ontogeny.

TCRalpha and TCRdelta chains are coded by a common genetic locus using a single set of V gene segments (ADV segments). This article addresses the question of regulation of the use of the ADV segments by the TCRalpha and TCRdelta chains. Using both qualitative and quantitative analyses we have studied the use of 23 ADV gene families as part of TCRalpha and TCRdelta transcripts. A number of previously undetected rearrangement and transcription events are described, indicating that the intrathymic TCRdelta repertoire is much more diverse than previously supposed. Repertoire analysis at several developmental time points allowed the description of regulated waves of ADV gene use, not only for TCRdelta chains, but also for TCRalpha chains, during thymic ontogeny. Control of these waves appears to be linked directly to the ADV segments and their local chromatin environment, which may change over the course of T cell differentiation.

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.

Preferential ADV-AJ association during recombination in the mouse T-cell receptor alpha/delta locus.

The gene coding for a T-cell receptor (TCR) alpha chain is assembled from variable (ADV) and joining (AJ) genes located on Chromosome 14. Each of the 90 ADV genes can rearrange with any one of the 61 AJ genes. We have previously demonstrated that ADV and AJ gene segment use evolves with time, with a progressive opening of ADV and AJ regions of the locus. To define the rules governing the use of AJ genes by ADV genes belonging to one family, we carried out a detailed analysis of 268 combinations of ADV2 BALB/c transcripts. We found that the different ADV2 members use different sets of AJ genes depending on their location within the ADV locus: ADV2S7 (the most AJ proximal ADV2 member) rearranges mainly with the AJ genes located close to the TEA element, whereas 50% of the sequences for ADV2S8, which is distal to the AJ locus, use the most distal AJ genes. ADV2S5, an ADV2 member located in the middle of the ADV locus, is associated with a wider set of AJ genes, located in the center of the AJ locus. Taken together, our results indicate that, in addition to the progressive opening of the ADV and AJ loci, the chromosomal location of ADV and AJ genes is a factor affecting AJ use in BALB/c mice.

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.

TCR alpha-chain repertoire in pTalpha-deficient mice is diverse and developmentally regulated: implications for pre-TCR functions and TCRA gene rearrangement.

Pre-TCR expression on developing thymocytes allows cells with productive TCRB gene rearrangements to further differentiate. In wild-type mice, most TCRA gene rearrangements are initiated after pre-TCR expression. However, in pTalpha-deficient mice, a substantial number of alphabeta+ thymocytes are still produced, in part because early TCR alpha-chain expression can rescue immature thymocytes from cell death. In this study, the nature of these TCR alpha-chains, produced and expressed in the absence of pre-TCR expression, have been analyzed. We show, by FACS analysis and sequencing of rearranged transcripts, that the TCRA repertoire is diverse in pTalpha-/- mice and that the developmental regulation of AJ segment use is maintained, yet slightly delayed around birth when compared with wild-type mice. We also found that T cell differentiation is more affected by pTalpha inactivation during late gestation than later in life. These data suggest that the pre-TCR is not functionally required for the initiation and regulation of TCRA gene rearrangement and that fetal thymocytes are more dependent than adult cells on pTalpha-derived signals for their differentiation.

Co-operation between human CR1 (CD35) and CR2 (CD21) in internalization of their C3b and iC3b ligands by murine-transfected fibroblasts.

CR1 and CR2 are expressed as associated proteins on the B-lymphocyte surface. To investigate their respective contributions to the internalization of C3 fragments, transfected murine fibroblasts expressing human CR1, CR2, or both CR1 and CR2 were produced. CR1- and CR1-CR2-expressing cells bound C3b and C3b-dimer whereas CR2- and CR1-CR2-expressing cells bound iC3b and C3de. In all cases, maximum binding was achieved at low ionic strength. CR1-CR2-positive cells internalized two- to threefold more C3b and 1.5-fold more iC3b than CR1- and CR2-single-positive cells, respectively. Internalization of the anti-CR1 antibody J3D3, or C3de was at the same level, in both double-transfected and single-transfected cells. Furthermore, the internalization of C3b dimer by CR1-CR2 cells was impaired in the presence of OKB7, an anti-CR2-blocking antibody, but it was not altered in CR1 cells. Taken together, these findings suggest that CR1 and CR2 collaborate to internalize C3b and iC3b proteins. We suggest that the induction of conformational changes of the ligands enhances their binding to both receptors.