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The recent progress in tumor immunology is a striking example of the successful application of modern biotechnology to understand the complex phenomenon of immune-mediated cancer rejection. Tumor antigens were identified and successfully utilized in active immunization trials to induce tumor antigen-specific T cells. This achievement has left, however, clinicians and researchers perplexed by the paradoxical observation that immunization-induced T cells can recognize tumor cells in standard assays but cannot induce tumor regression. A closer look at T cell physiology and tumor biology suggests that this observation is not so surprising. Here, we argue that successful immunization is one of several steps required for tumor clearance while more needs to be understood about how T cells localize and are effective within a tumor microenvironment impervious to the execution of their effector function.

Fetal/neonatal immune responses generally are considered to be immature and weaker than that of adults. We have studied the cord-blood T cells of newborns congenitally infected with Trypanosoma cruzi, the protozoan agent of Chagas disease. Our data demonstrate a predominant activation of CD8 T cells expressing activation markers and armed to mediate effector functions. The analysis of the T-cell receptor beta chain variable repertoire shows the oligoclonal expansion of these T lymphocytes, indicating that activation was driven by parasite antigens. Indeed, we have detected parasite-specific CD8 T cells secreting interferon-γ after coincubation with live T cruzi. This response is enhanced in the presence of recombinant interleukin-15, which limits the T-cell spontaneous apoptosis. These findings point out that the fetal immune system is more competent than previously appreciated, since fetuses exposed to live pathogens are able to develop an adultlike immune CD8 T-cell response.

Vaccine-based strategies are currently under investigation as a means of inducing tumour-specific immune responses and improving the clinical outcome of multiple myeloma (MM) patients in remission after high-dose chemotherapy and peripheral blood progenitor cell (PBPC) infusion. The immune competence of these patients was investigated by determining the overall diversity of the T-cell receptor (TCR) repertoire in the peripheral blood (PB) and bone marrow (BM). The average time after transplantation was 13 months. The clonality and reciprocal usage of BV gene segments (TCRBV repertoire) was estimated at the cDNA level and membrane protein expression. The TCRBV repertoire of MM was severely disrupted compared with age-matched normal donors. On average, one-third of the total repertoire in both the PB and the BM consisted of T cells expressing oligoclonal TCRbeta transcripts. Flow cytometry showed an increased frequency of abnormally expanded BV subfamilies at both sites. BV expansions were predominantly CD8+ and had the phenotype of antigen-experienced memory T cells as well as T cells with the naive phenotype. Oligoclonality was not restricted to phenotypically expanded BV subfamilies, but also involved normally represented BV subfamilies. The TCR repertoire of MM in remission was then compared with monoclonal gammopathy of undetermined significance (MGUS) and MM patients at diagnosis. The degree of TCR diversity was similar in age-matched normal donors and MGUS, but progressively decreased from MGUS to MM at diagnosis and then to MM in remission. These data indicate that: (1) there is a long-lasting and severe disruption of TCR diversity after high-dose chemotherapy and PBPC infusion, and (2) the extent of TCR disruption may affect the clinical outcome of vaccine-based strategies delivered at the stage of minimal residual disease.

Interaction of CD95 (Apo-1/Fas) and its ligand (CD95L) plays an important role in the regulation of the immune response, since CD95+ lymphocytes may be killed after engagement of the CD95 receptor. Studying the CD95/CD95L system in 40 cases of breast cancer, the malignant cells expressed CD95L, but lost CD95 expression, when compared with non-malignant mammary tissue. Jurkat T cells incubated on breast cancer sections underwent CD95L-specific apoptosis. The rate of apoptosis correlated with the CD95L mRNA levels of the tissue samples. In four breast cancer cell lines, CD95L expression was increased by interferon-gamma (IFN-gamma), which resulted in higher levels of CD95L-specific apoptosis in co-cultured Jurkat T cells. Since IFN-gamma is mainly secreted by activated T cells, up-regulation of CD95L in breast cancer cells in response to IFN-gamma may thus counterselect activated tumour-infiltrating T cells and favour the immune escape of breast cancer. As demonstrated by inhibition of matrix metalloproteinases, CD95L expressed on breast cancer cells can also be shed from the cell membrane into the culture supernatant. Supernatants derived from cultured breast cancer cells induced apoptosis in Jurkat T cells via CD95L. In breast cancer patients, depletion of CD4+ and CD8+ peripheral blood lymphocytes was significantly correlated with CD95L expression in the tumours. This might be suggestive for a relationship between CD95L expression by breast cancer and systemic immunosuppression.

CD95 ligand expression has been observed in various malignancies. Studying the CD95 ligand (CD95L) and receptor (CD95) system in eight non-malignant mammary tissues and 40 breast cancer tissues, mRNA and protein expression was determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence. mRNA levels of CD95L correlated positively ( r=0·90; p< 0·01) and transmembrane CD95 inversely ( r=−0·88; p< 0·01) with histopathological grading of the breast tumours: CD95L mRNA levels were low in adenomas, but increased by 20-fold in grade I, 120-fold in grade II, and 310-fold in grade III breast cancer. In contrast, CD95 mRNA levels were low in high-grade carcinomas, but high in benign mammary tissues. Since CD95L acts as an efficient inducer of apoptosis in CD95+ cells, apoptotic cells were identified on the tissue sections. Tumour-infiltrating lymphocytes and stromal cells in close proximity to CD95L-expressing breast cancer underwent apoptosis. As a functional test, CD95+ target cells were cultured on breast cancer tissue sections. The target cells underwent apoptosis when cultured on breast cancer sections, but could be rescued when CD95L was specifically blocked by a CD95–Fc fusion molecule. The data suggest an inverse regulation of CD95 ligand and receptor expression during dedifferentiation of breast cancer. Killing of bystander cells by the CD95L-expressing breast tumour could be involved in tissue invasion. Copyright © 1999 John Wiley & Sons, Ltd.

Psoriasis is a chronic inflammatory cutaneous disease of unknown etiology. Activation of T cells is thought to play a major role in the pathophysiology of psoriasis. In order to gain insight into the nature of the antigen (superantigen or nominal protein antigen) involved in psoriatic lesions, we have used a RT-PCR method to analyze the frequency of the 24 T cell receptor V beta chain (TCRBV) subfamilies and the size of the antigen-binding region (CDR3), using the immunoscope assay, in skin lesions of patients with chronic plaque-type psoriasis. Semi-quantitative analysis showed that no significant difference in V beta subfamily usage could be detected in T lymphocytes infiltrating lesional skin as compared to blood lymphocytes. Alternatively, determination of the size distribution of the CDR3 of all the V beta subfamilies revealed only in psoriatic skin a marked TCR oligoclonality defined by the presence in 3 to 5 V beta subfamilies of a single predominant CDR3 size which was associated with a unique V beta-J beta combination. Identical patterns of CDR3 length and V beta-J beta combination profiles were found in symetrical lesional sites from two psoriatic patients. This type of skewed CDR3 size profile is reminiscent of a local stimulation of T lymphocytes by nominal protein antigens. These data suggest that T lymphocytes infiltrating plaque-type psoriatic skin comprise expansions of oligoclonal T cells in response to stimulation by an antigen present in the skin.

Igs contain unique portions, collectively termed idiotypes (Id), that can be recognized by the immune system. Id expressed by tumor cells in B-cell malignancies can be regarded as tumor-specific antigens and a target for vaccine immunotherapy. We have started a vaccination trial in multiple myeloma (MM) using Id-specific proteins conjugated to keyhole limpet hemocyanin (KLH) as immunogens and low doses of subcutaneous granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-2 (IL-2) as immunoadjuvants. Twelve patients who had previously been treated with high-dose chemotherapy followed by peripheral blood progenitor cell (PBPC) transplantation entered this study from August 1995 to January 1998. All patients were in first remission at the time of vaccination. They received subcutaneous injections of Id vaccines and immunoadjuvants in an outpatient setting. The generation of Id-specific T-cell proliferative responses was documented in 2 patients, whereas a positive Id-specific delayed-type hypersensitivity (DTH) reaction was observed in 8 of the 10 patients studied. DTH specificity was confirmed in 1 patient by investigating the reactivity to synthetic peptides derived from the VDJ sequence of the tumor-specific Ig heavy chain. None of the patients generated soluble immune responses to Id, whereas the generation of soluble and cellular immune responses to KLH was observed in 100% and 80%, respectively. Eleven patients completed the treatment, whereas 1 patient failed to finish owing to progression of disease. Freedom from disease progression (FFDP), measured from the date of first Id/KLH injection to the date of first treatment after vaccination or last follow-up, ranged from 9 to 36 months. These data indicate that the immune competence status of MM patients is still susceptible to specific immunization after high-dose chemotherapy and PBPC transplantation. It remains to be determined whether generation of Id-specific immune responses can reduce the relapse rate of patients with minimal residual disease.

CD95 (Apo-1/Fas) ligand (CD95L) expression has been observed in various malignancies. In human primary cell lines from a squamous cell carcinoma (SCC) of the vulva, the effect of cisplatin (CDDP) and IFNγ on the expression of CD95L and its 2 receptor isoforms, CD95 transmembrane (CD95tm) and CD95 soluble receptor, was studied at the mRNA and protein levels. Addition of CDDP and IFNγ increased CD95L mRNA levels in the primary cell line 6-fold and 1.7-fold, respectively. In comparison, CD95tm mRNA levels were diminished by CDDP but increased 8-fold upon IFNγ challenge. CD95L expressed by SCC cells was functionally relevant since these cells were able to induce CD95-specific apoptosis in autologous lymphocytes from the SCC-bearing patient. Thus, CD95L expression in SCC may contribute to tumor-associated immunosuppression, which may be modulated by CDDP and IFNγ. In tumor samples of the primary SCC, CD95L expression was enhanced in the area of the border between invasive tumor tissue and surrounding stroma cells. The locally restricted over-expression of CD95L was congruent with the arrangement of apoptotic stroma cells in the direct vicinity of invading tumor tongues, suggesting a role as invasion factor for CD95L. Int. J. Cancer 80:564–572, 1999. © 1999 Wiley-Liss, Inc.

Umbilical cord blood (CB) constitutes a promising alternative to bone marrow for allogeneic transplantation and is increasingly used because of the reduced severity of graft-versus-host disease after CB transplantation. We have compared the T-cell receptor β chain (TCRB) diversity of CB lymphocytes with that of adult lymphocytes by analyzing the complementarity determining region 3 (CDR3) size heterogeneity. In marked contrast to adult samples, we observed bell-shaped profiles in all of the 22 functional β-chain variable (BV) subfamilies that reflect the lack of prior antigenic stimulation in CB samples. However, the mean CDR3 size and BV usage were comparable between CB and adult samples. BJ2 (65%) segments were used preferentially to BJ1 (35%), especially BJ2S7, BJ2S5, BJ2S3, and BJ2S1, in both CB and in adult lymphocytes. We therefore conclude that although naive as reflected by the heterogeneity of the CDR3 size, the TCRBV repertoire appears fully constituted at birth. The ability to expand TCRB subfamilies was confirmed by stimulation with staphylococcal superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxin A. This study provides the basis for future analysis of the T-cell repertoire reconstitution following umbilical CB transplantation.

Very large 'naïve' human antibody repertoires have been obtained from RT-PCR cloned VH and VL variable regions. They are used as starting material for the assembly of medium sized combinatorial libraries or so called multicombinatorial libraries. In nonimmunized individuals immunoglobulin messenger RNAs are poorly expressed, which can be a serious limitation for cloning efficiency. To overcome this problem two complementary strategies have been used: a nonspecific polyclonal activation of B cells, and a secondary PCR amplification technique to ensure recovery of Ig messengers in large amount and without introducing any bias.

Renal-cell carcinoma (RCC) is one of the human cancers which respond best to immunotherapy. To better characterize the mechanism of the immune response in RCC, we analyzed the T-cell receptor (TCR) beta-chain repertoire in primary RCC, metastases and paired peripheral blood lymphocytes (PBL) from 9 patients. For 3 of these, we analyzed T cells recovered from normal kidney, or from tumor-involved lymph nodes as well as tumor-infiltrating lymphocytes (TIL) expanded in vitro for adoptive immunotherapy. The initial semi-quantitative RT-PCR method for definition of the Vbeta gene usage was not informative enough to distinguish intratumoral clonal T-cell expansions. In contrast, the length pattern analysis of the complementary determining regions 3 (CDR3) allowed oligoclonal T-cell populations to be detected in fresh TIL form the 9 patients with RCC. Furthermore, these oligoclonal TIL populations were not present in normal renal tissue, autologous PBL or tumor-involved lymph nodes. Different clonal T-cell expansions were identified in the primary tumor and in a pulmonary metastasis from the same patient. The detection of clonal T-cell populations observed in RCC suggests an in situ expansion in response to potential tumor antigens. This report provides an overall and accurate description of the T-cell repertoire in a significant number of samples from patients with RCC.

The murine receptor for the Fc portion of IgG is a molecule expressed by cells of the immune system. This study suggests the hypothesis that Fc gamma receptor type II B I (Fc gamma RIIB I) functions as a progression-enhancing factor when expressed ectopically on non-lymphoid tumor cells. It has been shown previously that BALB/c 3T3 cells transformed in vitro with polyoma virus (PyV) do not express Fc gamma RII but acquire the expression of this receptor following an in vivo passage in syngeneic mice. The specific Fc gamma RII transcript present in tumor cells was identified in this report as Fc gamma RIIB I (BI). In order to determine whether or not the ectopically expressed Fc gamma RII plays a role in the progression of these transformed cells, PyV-transformed 3T3 cells were transfected with BI-cDNA. The BI transfected cells were tested for their ability to form local tumors in syngeneic mice, as compared to transfected cells which express the co-transfecting neomycine resistance (neores) DNA alone or together with the lacZ gene. Fc gamma RIIB I expressors exhibited a significantly higher tumorigenic phenotype than FcR-negative controls, though both types of cells exhibited the same growth curve in vitro. The ability of Fc gamma RIIB I to act as a potentially tumorgenicity-enhancing factor was also demonstrated as Fc gamma RII was expressed by tumor cells, originating from inoculated Fc gamma RIIB I-transfected cells, or from inoculation of a mixture of receptor-positive and -negative cells. B I-expressing cells dominated the tumor-cell population over non-expressors. This dominance strengthened the hypothesis that FcR plays a role in tumor progression in vivo.

Resume La reponse de lymphocytes T specifique d'antigene joue un role essentiel dans la physiopathologie des allergies cutanees. Les lymphocytes T de type alpha/beta reconnaissent les antigenes sous forme de peptides associes a des molecules du complexe majeur d'histocompatibilite. La specificite de cette reconnaissance est due a un grand nombre de regions variables des chaines alpha et beta qui, comme les immunoglobulines, sont produites a la suite de rearrangements genomiques permettant de juxtaposer deux ou trois segments codant respectivement pour des regions variables, de diversite et de jonction. Le repertoire des lymphocytes T alpha/beta correspond a l'ensemble des combinaisons de chaines alpha et beta differentes presentes dans une population de lymphocytes T. La complexite d'un tel repertoire peut etre etudiee a l'aide d'anticorps specifiques de familles de regions variables ou a l'aide de methodes de retrotranscription et amplification permettant d'etudier les ARNm codant pour les chaines du recepteur T. Dans cette revue nous presentons les bases moleculaires de la diversite de la chaine beta, une methode recente d'analyse de repertoires T, et nous discutons les informations disponibles sur le repertoire T dans le psoriasis, les eczemas de contact et la dermatite atopique.

Improved polymerase chain reaction (PCR)-based methods now permit a more in-depth analysis of the repertoire of T cells recovered in biological samples from mice and humans. At a certain level of resolution, the diversity of the T-cell repertoire can be readily estimated and clonal expansions become easily detectable. As discussed here by Christophe Pannetier, Jos Even and Philippe Kourilsky, these improvements allow a better appreciation of the degree of reproducibility of immune responses, both in mice and humans, and should have a significant impact on clinical investigations.

Cloned BALB/c 3T3 cells transformed in vitro with polyoma virus (PyV) acquired a higher tumorigenicity phenotype after a single in vivo passage. Some of the in vivo passaged cells (CTC cells) exhibited also a higher metastatic phenotype than cells from the same clones that were maintained only in culture (C cells). A phenotypic comparison between CTC and C cells was performed. It was found that most CTC lines exhibited a higher binding to laminin compared to their clonal C cell ancestors. Some CTC cells were less sensitive to the cytotoxic effects of TNF-alpha than the corresponding C cells. CTC cells originating from tumors which appeared after a long latency period (late tumors) tended to express Fc gamma RII while CTC cells originating from tumors which appeared after a short latency period (early tumors) as well as the corresponding C cells tended not to express Fc gamma RII. The expression of a membrane epitope recognized by a monoclonal antibody expressing specificity towards PyV transformed cells, was down-regulated on late tumor cells compared to early tumor cells. Transfection of cloned PyV-transformed BALB/c 3T3 cells with the beta 1Fc gamma RII gene augmented the tumorigenicity and metastatic phenotype of the transfectants compared to control transfectants.

The 5' and 3' flanking sequences of 14 members of the V kappa Ox (VK 4/5) gene family of BALB/c mice have been established. The family was unusual in the number of bases between the codon for Pro 95 and the heptamer sequence; most members contained four but there were also examples of none. A conserved leader sequence was used to amplify the genomic DNA of rearranged genes in order to analyze the spleen B cell repertoire of non-immunized animals. The library contained many members with virtually identical sequences to one or other of the already known members of the family. In addition, there were repeats of other sequences, allowing the definition of 12 hitherto undefined members of the family. Only 3 out of 96 could have originated by gene conversion, or as artefacts of the amplification procedure, and only 2 were putative somatic mutants. The frequency of expression of different members of the V kappa Ox gene family was not random, and some germ-line genes were unrepresented in the library. The high frequency of V kappa Ox1-J kappa 5 is in line with the dominance of this combination in the oxazolone response. An analysis of the junctional segment showed that although in most cases the diversity was due to trimming, there were exceptions indicating de novo additions (N or P bases). The average number of bases trimmed from the V kappa and the J kappa segments was not the same. There was no correlation in the number of bases trimmed from V kappa or J kappa in each recombination. The implications of asymmetric trimming in terms of the mechanism of recombination are discussed.

Purpose: Persistent infections by high-risk human papillomavirus (HPV) types are the main etiologic factor for cervical cancer. The objective of this study was to evaluate whether high-risk E7 oncoprotein is adequate as a marker for the detection of cervical cancer. Experimental Design: HPV typing was done in biopsies from 58 cervical carcinoma and 22 normal cervical squamous epithelia. The HPV-16 E7, HPV-18 E7, and HPV-45 E7 oncoprotein levels were monitored by immunohistochemistry and compared with those of p16INK4a and Ki67. Results: Fifty-five (94.8%) tumors were high-risk HPV-DNA–positive (46 HPV-16, 2 HPV-16 and HPV-18, 4 HPV-18, 1 HPV-33, and 2 HPV-45). HPV-DNA could not be detected in three tumors (5.2%). High HPV E7 oncoprotein levels were shown in 57 cervical cancers (98.3%), without correlation between expression levels and tumor stages. Conclusion: This is the first study which systematically analyzes the levels of the major HPV oncoproteins in cervical carcinomas demonstrating that the high-risk HPV E7 proteins are regularly expressed in these cancers. This suggests that high-risk E7 oncoproteins are necessary for cervical cancers and apparently essential as tumor marker.

Metastases are known to be more resistant to therapy than matching primary tumors, in particular they are less prone to apoptosis. In this study we investigated the functional interaction of a CTL clone (LT12) specific for a melanoma TA with the primary tumor (T1) versus its metastatic counterpart (G1). The CTL clone (LT12) was shown to lyse the primary T1 cells more efficiently in a classical cytotoxicity test. This differential susceptibility was not associated with MHC class I down-regulation and conjugate formation but correlated with a differential increase in Ca++ flux in the LT12 CTL when stimulated with the primary versus the metastatic tumor cells. Since LT12 uses perforin/granzyme B to kill its autologous target we analysed perforin and granzyme B mRNA expression in the CTL in the presence of either primary and metastatic melanoma cells. Quantitative PCR analysis showed an increased expression of granzyme B and perforin mRNA levels in LT12 when cocultured in the presence of the primary tumor. However, a similar level of (cytotoxic molecule) degranulation as revealed by CD107 expression was observed when LT12 was stimulated with T1 or G1 cells. These data suggest that the differential susceptibility of primary and metastatic melanoma cells involves at least in part their distinct potential to induce autologous CTL reactivity and the subsequent triggering of granzyme B and perforin in these cells.

Melanomas are most frequently infiltrated by actively proliferating T-lymphocytes (1). Some of these T-cells are cytolytic and recognize peptide antigens derived from melanoma-specific antigens (2). However, with the noteworthy exception of rare immune-mediated, sponaneous regressions of melanomas (3), or in the particular case of the halo nevus phenomenon in which normal melanocytes are killed by CD8(+)-specific T-cells (4), the ongoing melanocyte-specific T-cell responses are most frequently incapable of controlling the growth of the tumor, resulting in the malignant melanocytic tumors escaping an otherwise specific immune T-cell response. The understanding of the mechanisms that underlie the switch of efficient to inefficient (and vice versa) T-cell responses is thus of primary importance in conceiving specific immunotherapies of melanomas.

The generation of T lymphocytes with specific reactivity against tumor antigens is a prerequisite for effective adoptive transfer therapies. Melanoma-specific lymphocyte cultures can be established from tumor infiltrating lymphocytes (TILs) by in vitro culture in high levels of IL-2. We have optimized methods for generating melanoma-reactive TIL cultures from small resected tumor specimens. We report a retrospective analysis of 860 attempted TIL cultures from 90 sequential melanoma biopsy specimens from 62 HLA-A2+ patients. Multiple independent TIL derived from a single tumor often exhibited substantial functional and phenotypic variation. Tumor specific activity was detected in TIL from 29 (81%) of 36 patients screened. TIL cultures selected for high activity were generally capable of large numerical expansion using a single round of a rapid expansion protocol. Limited clonal T-cell populations in an oligoclonal TIL culture could confer specific tumor recognition in these highly selected, highly expanded TIL cultures. These methods were efficient at generating TILs suitable for adoptive transfer therapy.

The immune system has been implicated in the control of bladder tumor growth. To evaluate the clonality of bladder tumor-infiltrating T lymphocytes (TILs) in vivo, we studied the T-cell antigen receptor (TCR) repertoire in tumor biopsy specimens from 10 patients with transitional-cell carcinoma (TCC) of the bladder. Nine patients had a primary tumor, and one had a multifocal disease, consisting of two bladder tumors and three bilateral upper urinary tract sites of involvement. The following specimens from the nine patients with a primary tumor also were analyzed: a recurrent tumor from four patients, a metastatic lymph node from one patient, and peripheral blood from five patients. We used a high-resolution polymerase chain reaction (PCR) method to determine CDR3 (complementarity-determining region 3) size lengths of TCR beta-chain transcripts. Oligoclonal T-cell expansion was identified in all specimens, with a larger number of expanded clones in the tumors than in peripheral blood. Expanded clones were identified in several beta-chain variable region (BV) subfamilies and varied from one patient to the next and also in different specimens from the same patient. However, a number of clones with the same VJ combination and the same CDR3 size were identified in a given patient (in specimens collected either simultaneously or at different times), suggesting homogeneity in the immunogenic environment. Clonal T-cell expansion in patients with bladder cancer may reflect prolonged exposure of T lymphocytes to tumor antigens. Our findings provide a basis for functional studies to elucidate T lymphocyte-bladder tumor cell interactions.

The intestinal mucosal lesion in celiac disease is characterized by a predominant T-cell infiltration of both epithelium and lamina propria. However, a restricted use of T-cell receptors (TCR) in T lymphocytes infiltrating the jejunal mucosa of celiac patients has not been reported. Based on an immunohistochemical survey of jejunal biopsies from a cohort of untreated celiac patients, we demonstrated a small but significant increase of V beta 8.1/2+ T cells in the lamina propria, but not in the epithelium nor in the peripheral blood. Sequence analysis indicated the existence of a variable degree of clonality of V beta 8+ T cells in the celiac mucosa. More importantly, the recurrence of identical CDR3 regions in some patients was also observed. The altered distribution of V beta 8+ T cells and the presence of identical CDR3 regions in celiac patients, but not in controls was independently confirmed by CDR3 size analysis in a further cohort of patients. These findings suggest that disease-specific variations of the TCRBV8 repertoire are present in the small intestinal mucosa of untreated celiac patients.

Analysis of a large number of unrelated bone marrow transplantations (BMT) has shown that HLA-DP incompatibility did not detectably influence the risk for acute graft-versus-host disease (aGVHD). Accordingly, it was proposed that HLA-DP determinants did not function as transplantation antigens in the same way as HLA-A, -B, or -DR. We have previously shown that HLA-DP (as well as HLA-A, -B, -DQ, or -DR)-specific T cells could be isolated from skin biopsies of patients who developed an aGVHD after semiallogeneic BMT. Nevertheless, whether a single HLA-DP mismatched allele could induce a detectable allo-specific reaction in vivo after BMT remained to be established. To directly address this issue we studied one patient who presented aGVHD after receiving purified CD34+ bone marrow (BM) cells from an unrelated donor with a single HLA-DP mismatch in the GVHD direction. To characterize the immunological events associated with GVHD, we analyzed the peripheral T cell repertoire, the T cell receptor Vbeta diversity, and the specificity of T cells invading a skin biopsy at the onset of GVHD. Our results demonstrated that a large fraction of skin-infiltrating lymphocytes, which expressed diverse T cell receptors, were reactive against this single HLA-DPB1 *0501 mismatch and consequently that a single HLA-DP mismatch between BM donor and recipient can activate a strong T cell response in vivo.

Despite a large number of studies identifying expanded T-cell clones among infiltrating lymphocytes, little is known about their distribution in patients suffering from rheumatoid arthritis. To evaluate the clonality of alpha/beta T-cell populations in arthritic locations and PBL, we determined the CDR3 size lengths of TCR beta-chain transcripts using BV (Vbeta), BC (Cbeta), BJ (Jbeta), and clonotype-specific primers. Transcripts from PBL of healthy donors show gaussian profiles of approximately eight CDR3 size peaks in most BV subfamilies. Dominant peaks standing out above the normal background identify expansions of one or several T-cell clones within a given BV subfamily. The analysis of six patients suffering from rheumatoid arthritis showed clonal expansions in all samples including PBL. Synovial tissue infiltrates revealed less complex repertoires with a greater number of expanded clones than PBL. Expanded clones varied from one patient to another; no recurrences were observed. Most interestingly, identical clones were identified bilaterally in arthritic knee joints and PBL from the same patient. Our data show that given T-cell clones are not only locally expanded but can also be found in the periphery, and strongly suggest that many similar clones spread throughout the bodies of patients.

Many examples of oligoclonal T-cell expansion in infiltrated diseased tissues have been reported. However, it remains to be established whether such observations can be generalized and to what extent oligoclonal patterns obtained after in vitro culture of T-cell infiltrates reflect in vivo situations. Using new high resolution analysis which requires no in vitro cellular expansion, we detected such oligoclonal T-cell expansions in 7/7 melanoma tumour biopsies, 3/3 biopsies of inflammatory skin during acute graft versus host disease (aGVHD) after allogeneic bone marrow transplantation (alloBMT) and 7/7 synovial membranes from patients with rheumatoid arthritis. Thus, oligoclonal T-cell expansions are readily observed when a sufficiently sensitive detection method is used, suggesting that similar expansions are the rule among T-cell infiltrates in different diseases. This observation and the monitoring of the in vivo evolution of such expansion during the course of the disease and during in vitro culture should have important clinical implications.

Acute graft-versus-host disease after bone marrow transplantation with a single HLA-DPB1*1001 mismatch: involvement of different TCRBV subsets

Over the last decade, tumor immunology in general and tumor immunotherapy in particular have made important progress. Thanks to the discovery of tumor-associated antigens (TAA), and the consequent development of active-specific immunization protocols, TAA-specific T-cell responses can now be induced reproducibly in cancer patients. However, clinical responses directly ascribable to TAA-specific T cells occur only occasionally. It is not clear why TAA-specific T cells do not eliminate tumor cells in vivo. This paradoxical coexistence of antigen-bearing tumor cells and antigen-specific T cells constitutes a critical point of current investigation. In recent years, protein-ligand interaction-based methods aimed at the identification and characterization of T cells using antibodies (cell surface phenotyping, intracellular and secreted cytokine detection), or HLA-peptide multimers, have significantly improved the analysis of TAA-specific T cells. These methods, however, seem to have reached the limit of their usefulness. Transcriptional analysis may add sensitivity and resolution and may provide global pictures of the multifactorial requirements for an efficient immune response against tumors. In this review, we describe the use of molecular genetic methods, such as real time qRT-PCR, cDNA microarrays, and TCR repertoire analysis. In addition, we describe techniques for high-fidelity messenger RNA amplification that allow high-throughput analysis of samples obtained from minimal sources, such as HLA/peptide tetramer sorted antigen-specific T cells, laser capture dissection, or fine needle aspirates. Recent work discussed in this review summarizes the complementarity of transcriptional analysis as an essential tool that, in addition to conventional methods, may deepen and broaden the characterization of tumor-specific T cells.

The sequences required for transformation by the Gardner-Arnstein (GA) strain of feline sarcoma virus (GA-FeSV) were defined by site-directed, in vitro mutagenesis of molecularly cloned proviral DNA. Portions of the Ga-FeSV provirus, subcloned in the plasmid pBR322, were mutagenized by deletion or frameshift at Xho I restriction sites flanking the nucleotide sequences presumed to encode the GA-FeSV transforming polyprotein (P108 gag-fes ). The biological activity of subgenomic and reconstructed full-genome-length molecules was assayed by transfection and focus induction in NIH 3T3 cells. Both mutant and wild-type molecules containing the intact P108 gag-fes coding region induced foci of transformed cells at efficiencies between 10 4 and 10 5 focus-forming units per pmol of DNA; a deletion mutant lacking 3′-terminal v- fes sequences was completely nontransforming in parallel assays. Representative subcloned foci of transformed NIH 3T3 cells synthesized P108 gag-fes with associated in vitro protein kinase activity. Focus-forming viruses could be rescued from transformed subclones induced by full-length proviral DNA, but not from cells transformed by subgenomic DNA lacking a 3′ long terminal repeat (LTR). It was concluded that: (i) nucleotide sequences encoding P108 gag-fes and its associated kinase activity are responsible for transformation, (ii) the GA-FeSV 3′ env and LTR sequences are not required for focus induction, and (iii) the 3′ LTR is necessary for rescue of infectious FeSV RNA. A chimeric DNA containing the 5′ LTR and P108 gag-fes coding region of GA-FeSV joined to the 3′ LTR of Moloney murine sarcoma virus was both transforming and rescuable at high efficiency. Restriction analysis showed that passaged stocks of rescued transforming virus contained Moloney murine sarcoma virus U3 sequences at both proviral DNA termini, consistent with generally accepted models for LTR formation during reverse transcription. Wild-type GA-FeSV and the chimeric virus (here designated as GAHT), each rescued from NIH 3T3 cells with the same amphotropic murine leukemia virus, yielded approximately equal numbers of foci when titrated on CCL 64 mink cells. By contrast, on mouse NIH 3T3 cells, the focus-forming titer of GAHT was 1 to 2 log higher than that of FeSV. The foci induced on NIH 3T3 cells by GAHT appeared earlier and were reproducibly larger than those induced by GA-FeSV. Differences in transforming activity on NIH 3T3 cells were also found using colony formation in agar, showing that the more rapid appearance and larger size of foci formed in liquid media were not due to virus spread. These data suggest that transcriptional control signals within the viral LTR regulate the levels of the transforming gene product in a species-specific manner.

The problem of the structural relationship between suppressive IgG-binding factor and low-affinity receptors for the Fc portion of IgG (Fc gamma RII) has not yet been solved. In the present work we have isolated a recombinant soluble Fc gamma RII containing only the two external domains of Fc gamma RII, and analyzed its biochemical characteristics and biological activity. A cDNA encoding Fc gamma RII was mutated by the creation of a stop codon at the Lys175 codon. L cells have been transfected with this cDNA inserted into an expression vector. A cell line was obtained that secretes recombinant soluble Fc gamma RII which reacts with a monoclonal anti-Fc gamma RII antibody and binds to IgG1, IgG2a and IgG2b murine isotypes but not to IgG3 or F(ab')2 fragments of IgG2a. The secreted molecule contains two molecular species of relative molecular mass (Mr) 44,000 and 34,000-38,000 and of pI 4.5 and 6.3. They correspond to different glycosylations of a single polypeptide of Mr 19,000. After purification to homogeneity, soluble Fc gamma RII has found to suppress secondary and primary in vitro antibody responses in a dose-dependent way. The present work shows that recombinant soluble Fc gamma RII has biochemical characteristics, immunoreactivity and biological activity similar to those of suppressive IgG-binding factor.

The nucleotide sequences of the Gardner-Arnstein feline sarcoma virus (FeSV) long terminal repeat and the adjacent leader sequences 5' to the viral gag gene were determined. These were compared with homologous portions of Synder-Theilen FeSV and with previously published sequences for Moloney murine sarcoma virus and simian sarcoma virus proviral DNA. More than 75% of the residues in the FeSV R and U5 regions were homologous to sequences within the same regions of the other viral long terminal repeats. Unexpectedly, alignment of the FeSV sequences with those of the Moloney murine sarcoma and simian sarcoma viruses showed similar extents of homology within U3. The homologous U3 regions included the inverted repeats, a single set of putative enhancer sequences, corresponding to a "72-base-pair" repeat, and sequences, including the CAT and TATA boxes, characteristic of eucaryotic promotors. The 5' leader sequences of both FeSV strains included a binding site for prolyl tRNA and a putative splice donor sequence. In addition, the FeSV leader contained a long open reading frame which was adjacent to and in phase with the ATG codon at the 5' end of the FeSV gag gene. The open reading frame could code for a signal peptide of about 7.4 kilodaltons. Our results support the concept that the virogenic portions of both FeSV and simian sarcoma virus were ancestrally derived from viruses of rodent origin, with conservation of regulatory sequences as well as the viral structural genes.

The mRNA of ten monoclonal phenyloxazolone (phOx) antibodies originating from the primary (day 7) response of C57BL/10 mice were partially sequenced. The sequences were analyzed together with those of two previously published antibodies. The C57BL response does not have a predominant subset of antibodies like the BALB/c response has (VH-Ox1/V kappa-Ox1 JK5). Probably, C57BL mice lack the VH-Ox1 gene and, as a consequence, their V kappa-Ox1 gene does not have a main role in the anti-phOx response. Five V kappa and six VH genes were found to participate. All five V kappa genes or their "alleles" had previously been found from the BALB/c response to 2-phenyloxazolone (phOx). On the other hand, the two strains use different VH genes for the anti-phOx response. Most C57BL antibodies were coded by VH genes of group 1 which has only minor role in the BALB/c response. The remaining VH genes were from group 7. Our data show that one V kappa segment (e.g. V kappa-Ox1) can code for anti-phOx antibodies with several, even widely different, VH genes. On the other hand, they emphasize the role of certain VH/VL gene combinations for the anti-phOx specificity. Thus, VH genes of group 7 were found to code for anti-phOx antibodies only together with the V kappa 45.1 gene.

E7 is the major oncoprotein of high-risk human papillomaviruses (HPV) which causes cervical cancer. To date E7 oncoproteins have not been investigated in cervical adenocarcinoma. In this study we generated a rabbit monoclonal anti-HPV-16 E7 antibody, RabMab42-3, which recognizes a conformational epitope in the E7 carboxy-terminal zinc-finger resulting in a strong increase in the sensitivity for the detection of cell-associated HPV-16 E7 protein relative to conventional polyclonal anti-HPV-16 E7 antibodies. Using RabMab42-3, we show that the subcellular localization of endogenous HPV-16 E7 oncoprotein varies during the cell cycle in cervical cancer cells. Moreover, we demonstrate for the first time that the HPV-16 E7 oncoprotein is abundantly expressed in cervical adenocarcinoma in situ and adenocarcinoma, suggesting an important role of HPV-16 E7 for the development of these tumors. Our findings suggest that the HPV-16 E7 oncoprotein could be a useful marker for the detection of cervical adenocarcinoma and their precursors.

L'utilisation d'anticorps monoclonaux comme sonde pour les proteines et le cDNA comme sonde pour les acides nucleiques a permis l'etude de la regulation de l'expression genique de la chaine peptidique β du recepteur Fcγ II de la regulation de l'expression du recepteur a la surface membranaire et l'analyse moleculaire de la specificite isotypique du recepteur. L'activite biologique du recepteur et de facteurs solubles capable de lier l'IgG a ete etudiee

The integrated DNA provirus of the Gardner-Arnstein (GA) strain of feline sarcoma virus (FeSV) was molecularly cloned in a bacteriophage lambda vector. The cloned DNA fragment is 14.4 kilobase pairs long and contains a 6.7-kilobase provirus flanked by cellular sequences derived from nonproductively transformed mink cells. Transfection of mouse NIH/3T3 cells with the cloned DNA fragment induced foci of transformation at efficiencies of 10(4) focus-forming units/pmol of sarcoma virus DNA. Restriction endonuclease mapping and heteroduplex analyses were used to compare the GA-FeSV provirus with that of Snyder-Theilen (ST)-FeSV, a second strain that contains homologous transformation-specific sequences (v-fes). Both viruses have the general structure 5'-gag-fes-env-c region-3', each having retained portions of the feline leukemia virus (FeLV) gag and env genes. In addition to segments shared by the two sarcoma viruses, GA-FeSV contains 1.7 kilobases of extra sequences not found in ST-FeSV. Of these, at least 400-500 base pairs located near the 5' end of v-fes encode a portion of the GA-FeSV polyprotein; the remaining 1.2 kilobases are derived from the FeLV env gene but do not appear to encode any detectable product related to the FeLV envelope glycoprotein. The close homology of the v-fes sequences shows that GA- and ST-FeSV were formed by recombination of FeLV with similar portions of a cat cellular gene (c-fes).

Igs contain unique portions, collectively termed idiotypes (Id), that can be recognized by the immune system. Id expressed by tumor cells in B-cell malignancies can be regarded as tumor-specific antigens and a target for vaccine immunotherapy. We have started a vaccination trial in multiple myeloma (MM) using Id-specific proteins conjugated to keyhole limpet hemocyanin (KLH) as immunogens and low doses of subcutaneous granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-2 (IL-2) as immunoadjuvants. Twelve patients who had previously been treated with high-dose chemotherapy followed by peripheral blood progenitor cell (PBPC) transplantation entered this study from August 1995 to January 1998. All patients were in first remission at the time of vaccination. They received subcutaneous injections of Id vaccines and immunoadjuvants in an outpatient setting. The generation of Id-specific T-cell proliferative responses was documented in 2 patients, whereas a positive Id-specific delayed-type hypersensitivity (DTH) reaction was observed in 8 of the 10 patients studied. DTH specificity was confirmed in 1 patient by investigating the reactivity to synthetic peptides derived from the VDJ sequence of the tumor-specific Ig heavy chain. None of the patients generated soluble immune responses to Id, whereas the generation of soluble and cellular immune responses to KLH was observed in 100% and 80%, respectively. Eleven patients completed the treatment, whereas 1 patient failed to finish owing to progression of disease. Freedom from disease progression (FFDP), measured from the date of first Id/KLH injection to the date of first treatment after vaccination or last follow-up, ranged from 9 to 36 months. These data indicate that the immune competence status of MM patients is still susceptible to specific immunization after high-dose chemotherapy and PBPC transplantation. It remains to be determined whether generation of Id-specific immune responses can reduce the relapse rate of patients with minimal residual disease.

Although our knowledge and understanding of tumor-specific cytotoxic T lymphocytes (CTL) have expanded considerably, the long-term work needed to assay CTL has precluded their analysis in large numbers of patients. Moreover, in vitro culture steps may introduce major biases. New approaches to identify tumor-specific CTL clones would be helpful. As a means to describe the in situ immune status by T-cell repertoire analysis, we developed the Immunoscope approach, a PCR-based method that allows us to determine the spectra of CDR3 lengths of the TCR chains displayed by complex populations of T cells. We review here some of our data about melanoma. Tumor-infiltrating lymphocytes of a melanoma patient were analyzed by different means and melanoma-specific T-cell clones were derived. Two categories of tumor-specific CD8+ CTL clones were derived from the infiltrate of a tumor-proximal invaded lymph node. The majority of T-cell clones specifically lyse the autologous tumor cell lines and predominantly recognize the HLA-A2/MART-1(27-35) peptide complex. The in vivo representativity of such CTL was assessed by the immunoscope technology. Among three MART-1-specific clones, none was detectable in situ. The other kind of tumor-specific CD8+ CTL did not lyse autologous melanoma cell lines but lysed the "fresh" autologous tumor cells in a MHC class I dependent manner. The immunoscope approach revealed that one of the latter was detectable in situ among tumor-infiltrating lymphocytes although not among PBMC. These data indicate that melanoma-specific lymphocytes that could not have been selected through conventional screening procedures may be important in tumor rejection. Our results suggest that a better characterization of tumor-specific immune responses will be important for the optimization of specific immunotherapy strategies and the long-term follow-up of patients.