Skewed T-cell receptor repertoire: more than a marker of malignancy, a tool to dissect the immunopathology of inflammatory diseases.

The highly diverse heterodimeric surface T cell receptor (TCR) gives the T lymphocyte its specificity for MHC-bound peptides needed to initiate antigen-recognition. In normal peripheral blood, spleen and lymph nodes, the TCR repertoire of the T lymphocytes is usually polyclonal. However, in malignancies such as leukemias, as well as in lymphoproliferative diseases of mature T cells, the TCR is a reflection of the clonality of the malignant cells and is therefore monoclonal. Several clinical conditions (mainly solid tumors and autoimmune diseases) have been described where the TCR repertoire is restricted. The ability to demonstrate clonal TCR usage provides a useful tool to dissect the immunopathology of inflammatory diseases. In this review we discuss these findings and propose to sub-divide diseases with restricted TCR repertoire into a group of conditions in which there is a known TCR ligand, as opposed to diseases in which the restricted TCR repertoire is the result of impaired T-cell development. This classification sheds light on the pathogenesis of several inflammatory diseases.

Strategies to overcome obstacles to successful immunotherapy of melanoma.

The immunogenicity of malignant melanomas has been recognized by the observed recruitment of tumor-specific cytotoxic T-cells (CTL), leading to the identification of several melanoma associated antigen (MAA). However, numerous strategies to treat melanoma with immunotherapy have resulted in only partial success. In this editorial, we discuss recent data related to the ability of tumors to elude immune responses. We therefore discuss different strategies to induce a clinically effective immune response. These approaches include 1) immunostimulation: including peptide/protein based vaccines, dendritic cell vaccines, and adoptive cell transfer; and 2) overcoming immunosuppression, including targeting of checkpoint molecules such as CTLA-4, circumventing the activity of Tregs, and assuring antigen expression by tumor cells (thwarting antigen silencing). Finally, we discuss recent advances in gene therapy, including adoptive therapy with engineered T cell receptors (TCRs). These issues lead to the conclusion that successful immunotherapy in malignant melanoma requires a combination of strategies aimed at both inducing immunostimulation and blocking immunosuppression.

A novel autocrine pathway of tumor escape from immune recognition: melanoma cell lines produce a soluble protein that diminishes expression of the gene encoding the melanocyte lineage melan-A/MART-1 antigen through down-modulation of its promoter.

We have observed that malignant melanoma cells produce a soluble protein factor(s), which down-regulates melanocyte lineage Melan-A/MART-1 Ag expression by melanoma cells with concomitant loss of recognition by Melan-A/MART-1-specific T cells. This down-modulation of Melan-A/MART-1 expression, which we refer to as "Ag silencing," is mediated via its minimal promoter, whereas the promoter for the restricting Ag-presenting HLA-A2 molecule is not affected. Significantly, this Ag silencing is reversible, as removal of factor-containing supernatants from Melan-A/MART-1-expressing cells results in up-regulation of the promoter for the gene encoding this Ag, and renewed expression of the protein. We have evaluated over 20 known factors, none of which accounts for the Ag-silencing activity of the melanoma cell culture supernatants. The existence of this autocrine pathway provides an additional novel explanation for melanoma tumor progression in vivo in the presence of CTL specific for this melanocyte lineage Ag. These observations may have important implications for Melan-A/MART-1-specific CTL-mediated immunotherapy of melanoma tumors.

Outer membrane protein A, peptidoglycan-associated lipoprotein, and murein lipoprotein are released by Escherichia coli bacteria into serum.

Complexes containing lipopolysaccharide (LPS) and three outer membrane proteins (OMPs) are released by gram-negative bacteria incubated in human serum and into the circulation in an experimental model of sepsis. The same OMPs are bound by immunoglobulin G (IgG) in the cross-protective antiserum raised to Escherichia coli J5 (anti-J5 IgG). This study was performed to identify the three OMPs. The 35-kDa OMP was identified as outer membrane protein A (OmpA) by immunoblotting studies using OmpA-deficient bacteria and recombinant OmpA protein. The 18-kDa OMP was identified as peptidoglycan-associated lipoprotein (PAL) based on peptide sequences from the purified protein and immunoblotting studies using PAL-deficient bacteria. The 5- to 9-kDa OMP was identified as murein lipoprotein (MLP) based on immunoblotting studies using MLP-deficient bacteria. The studies identify the OMPs released into human serum and into the circulation in an experimental model of sepsis as OmpA, PAL, and MLP.

Release of gram-negative outer-membrane proteins into human serum and septic rat blood and their interactions with immunoglobulin in antiserum to Escherichia coli J5.

Prior studies indicate that 3 bacterial outer-membrane proteins (OMPs) are released into serum associated with lipopolysaccharide (LPS) and are bound by IgG in antiserum to Escherichia coli J5 (anti-J5 IgG). The present studies analyzed the interaction of the OMPs with anti-J5 IgG and evaluated their release in an infected burn model of gram-negative sepsis. Affinity purification studies were performed on filtrates of bacteria incubated in human serum and plasma from rats with sepsis by use of O chain-specific anti-LPS IgG and anti-J5 IgG. All 3 OMPs were captured from septic rat blood by anti-LPS IgG. Release of OMPs into serum was highest for immature bacterial cultures and was increased by antibiotics in vitro and in vivo. Anti-J5 IgG selectively captured an 18-kDa OMP released into serum and into plasma from septic rats. The results raise the possibility that anti-J5 IgG may, in part, protect via anti-OMP antibodies.

Melanoma antigen recognition by tumour-infiltrating T lymphocytes (TIL): effect of differential expression of melan-A/MART-1.

We have isolated, from an individual patient with metastatic melanoma, a series of eight TIL clones capable of lysing autologous melanoma cell targets. Six of the eight clones expressed TCRAV2S1 and lysed targets expressing HLA-A2 and the Melan-A/MART-1 peptide: AAGIGILTV. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis showed that the Melan-A/MART-1-specific clones were predominant in the bulk culture prior to cloning. However, the tumour progressed in vivo even in the presence of these tumour cell-lytic clones. Using the anti-Melan-A/MART-1 MoAb (A-103), we noted that Melan-A/MART-1 expression on three melanoma cell lines varied considerably during in vitro culture, in the absence of T cell immunoselection, relative to cell density. Tumour cells which spontaneously decreased Melan-A/MART-1 expression were less susceptible to specific TIL lysis. Melan-A/MART-1 expression and susceptibility to lysis increased in cells cultured at lower density. These data suggest that modulation of tumour antigen may account for tumour progression in the presence of tumour cell-lytic T lymphocytes. The observations suggest a possible explanation for the common finding of Melan-A/MART-1-specific lytic TIL in clinically progressing melanomas, as well as a possible pathway for therapeutic intervention.

Studies of the mechanism of cytolysis by tumour-infiltrating lymphocytes.

In order to determine the mechanism of tumour destruction by tumour-infiltrating lymphocytes (TIL), we examined the ability of both CD4+ and CD8+ effector TIL, and TIL clones, to manifest granzyme-mediated and Fas-mediated destruction of tumour targets. In many in vitro studies TIL have been shown to manifest anti-tumour reactivity, yet many tumours escape immunological destruction. To investigate the role of Fas expression and the concomitant sensitivity to the inducibility of apoptotic death, we derived TIL from four melanomas and one glioma. The glioma, and all but one of the melanomas, expressed Fas, but Fas-mediated apoptosis could only be detected if the targets were treated with cyclohexamide. The melanomas and the glioma all expressed detectable cytoplasmic Bcl-2 protein, known to exert anti-apoptotic activity. Lysis of tumours by CD8-enriched cultures and CD8+ clones was Ca2+-dependent and could not be modified by an anti-Fas MoAb. In CD4-enriched cultures or CD4+ clones with cytotoxic potential against tumour cells, cytotoxicity was also Ca2+-dependent. As Ca2+-dependent cytotoxicity is usually the result of secretion of perforin/granzyme-B, we investigated the presence of perforin in cytotoxic CD4+ clones and demonstrated the presence of granular deposits of this enzyme in some of the CD4+ clones. Although an anti-Fas MoAb did not block the lysis of melanoma targets by CD4+ clones, the examination of Fas-dependent targets demonstrated that these clones also had the potential to kill by the Fas/Fas ligand system. These data suggest that the predominant mechanism in tumour killing by TIL appears to be perforin-granzyme-dependent, and that the solid tumour cell lines we studied are less susceptible to Fas-mediated apoptosis. As non-apoptotic pathways may enhance tumour immunogenicity, exploitation of the perforin-granzyme-dependent cytotoxic T lymphocyte (CTL) pathways may be important for achieving successful anti-tumour responses.

Oligoclonality of Vdelta1 and Vdelta2 cells in human peripheral blood mononuclear cells: TCR selection is not altered by stimulation with gram-negative bacteria.

Despite the enormous potential repertoire of gammadelta T cells, there are several observations which suggest that the expressed gammadelta repertoire in the periphery of normal individuals is often quite restricted. To assess selective expansions among gammadelta T cells from both adult and newborn blood samples, PBMC from 12 normal adults and cord blood from 15 normal newborns were analyzed for TCRDV1 and TCRDV2 junctional diversity by CDR3 size spectratyping and single-strand conformational polymorphism. Although TCRBV usage showed extensive heterogeneity in adults and newborns, both populations often showed CDR3 region restriction for TCRDV1 and TCRDV2. Analysis of the CDR3 spectratype patterns of newborn twins suggested that clonal selection for TCRDV is independent of genetic background. The possible role of Gram-negative bacteria in driving selective responsiveness of gammadelta T cells in PBMCs from adults was examined by in vitro stimulation with Escherichia coli and Pseudomonas aeruginosa. Donors whose TCRDV repertoire was highly clonal in the unstimulated blood cells showed the same predominant clones among the bacteria-stimulated cultures. In individuals whose gammadelta T cells were less restricted, in vitro stimulation did not select for clonality; rather, the TCRDV repertoires were similar before and after bacterial stimulation. Together, these data indicate that gammadelta T cells are often clonally restricted in adults as well as in newborns and suggest that the prominent stimulatory activity of Gram-negative bacteria does not by itself account for the restriction or diversity of the gammadelta T cell repertoire.

Antiserum against Escherichia coli J5 contains antibodies reactive with outer membrane proteins of heterologous gram-negative bacteria.

The binding of IgG in antiserum to Escherichia coli J5 to the surface of Enterobacteriaceae and to cell wall fragments released from serum-exposed bacteria was studied in a search for potentially protective epitopes other than lipopolysaccharide (LPS). IgG titers to multiple heterologous gram-negative smooth bacteria increased following incubation of the bacteria in serum and decreased following absorption with serum-exposed heterologous bacteria. IgG eluted from absorbing bacteria bound to at least three conserved bacterial outer membrane proteins (OMPs), but not LPS, as assessed by immunoblotting. The same OMPs were present in LPS-containing macromolecular cell wall fragments released by incubation of heterologous gram-negative bacteria in human serum. Part of the protection offered by J5 antiserum could be from binding of IgG to conserved OMPs at the bacterial surface or to OMPs in cell-wall fragments released from dying bacteria.

T cell receptor usage by HLA-DR3-specific T cell clones isolated from a renal allograft.

In order to evaluate the T cell receptor (TCR) usage by clones of human allograft infiltrating lymphocytes, this study utilized polymerase chain reaction (PCR) amplification of TCR transcripts from five clones which were previously shown to react with a human leucocyte antigen (HLA)-DR3 mismatch between a living related kidney donor and recipient. The five CD4+ (CD8-) clones, which were selected for TCR analysis, proliferated in response to HLA-DR3 and three of the clones were also cytotoxic against the same target cells. After identification of the TCRAV and TCRBV usage of the clones, the sequence of the TCR alpha and beta were determined by direct sequencing of the PCR product. The results indicate that several different TCRAV and TCRBV gene segments are used among the different clones, but the two clones that were both cytotoxic and proliferative in response to HLA-DR3 shared identical TCRAV27-J42-C and TCRBV13-D1-J1S2-C1 transcripts. The additional three clones showed various TCRAV and TCRBV transcripts, but evaluation of the CDR3 region of the TCR beta chain, corresponding to the peptide antigen binding sites, demonstrated shared amino acid motifs which resulted both from germline sequences and combinations of n-region and germline-derived codons. These results suggest that the repertoire for anti-HLA-DR3-reactive clones can include a diverse expression of TCR, but there may be selection for some clones, as well as conserved motifs in the CDR3 region of anti-DR3 specific clones.

In vivo accumulation of the same anti-melanoma T cell clone in two different metastatic sites.

In a patient with progressing metastatic melanoma, we showed that the same autologous tumor-cytolytic CD8+ tumor infiltrating lymphocyte (TIL) clone accumulated in two separate metastatic sites. This clone, which represented three of eight independently derived clones from a tumor deposit on the skin of the abdomen, also represented two of eight clones derived from a skin lesion on the shoulder. This clone could be identified by its use of a unique TCRBV2-nD1n-J1S6 sequence, and could also be detected by single-stranded conformational polymorphism (SSCP) as the dominant TCRBV2-expressing clone among CD8+ TILs propagated from both shoulder and abdominal lesions. Using SSCP analysis, we also demonstrated that this clone was dominant in the fresh tumor tissue and in all TILs in which CD8+ were strongly represented, including several separate but parallel cultures. The SSCP pattern for this clone was not apparent among CD4+ TILs or CD8+ peripheral blood mononuclear cells. The SSCP analysis of the tumor tissue prior to in vitro culture is an indication that the selection for this anti-tumor cytotoxic T cell clone was a reflection of its in vivo accumulation. Thus, we provide evidence that melanomas are immunogenic and able to select for cytotoxic antitumor-specific TIL clones that are expanded in vivo and can circulate to accumulate in different tumor sites. However, because these clones were isolated from progressing tumor metastases, the accumulation of these specific cytotoxic T cells was not sufficient to contain tumor growth.

Escherichia coli and Pseudomonas aeruginosa induce expansion of V delta 2 cells in adult peripheral blood, but of V delta 1 cells in cord blood.

Human peripheral blood T cells proliferate in response to Escherichia coli and Pseudomonas aeruginosa. We observed that during the first few days after stimulation a large percentage of the responding PBMC were gamma delta T cells. In our study we characterized the early T cell responses of freshly isolated adult and newborn PBMC to soluble preparations of heat-killed E. coli and P. aeruginosa. Specimens from all healthy adults tested showed intense proliferation in response to both bacterial preparations; at 6 days, the responding cells were mainly T cell blasts, of which high percentages (up to 80%) were gamma delta T cells, most expressing V delta 2/V gamma 9. All newborn blood specimens tested also showed T cell proliferative responses, which included a marked expansion of gamma delta T cells, mainly of the V delta 1 subset. Populations of purified V delta 1 and V delta 2 T cells were obtained from adult PBMC following stimulation with E. coli; both subsets proliferated upon rechallenge with the bacterial preparations. Protease treatment of the bacterial preparations did not appreciably affect their ability to induce expansion of gamma delta T cells in either adult or cord blood, indicating that the stimulatory components were not proteins. The response of gamma delta T cells from newborns indicates that prior exposure to bacterial products is not necessary and suggests that gamma delta T cells are important elements in natural immunity to these extracellular organisms.

Responses of human T cells to dominant discrete protein antigens of Escherichia coli and Pseudomonas aeruginosa.

Normal human beings have circulating T lymphocytes that proliferate in response to Escherichia coli and Pseudomonas aeruginosa. We performed the present study to characterize the nature of the responding T cells and to determine whether distinct or shared conventional antigens, superantigens or polyclonal activators account for T cell proliferation. Long term antigen-specific T cell lines were generated by repeated stimulation of PBMC from four donors with soluble antigen preparations of E. coli or P. aeruginosa. This resulted in the emergence of distinct T cell populations, which responded to strains of either E. coli or P. aeruginosa, but not to both. Trypsin treatment of the bacterial preparations largely eliminated their ability to stimulate the T cells. The T cell lines were predominantly CD4+ and their proliferation to bacterial antigens was optimal using autologous APC. E. coli T cell lines proliferated not only in response to the E. coli strain with which they were initially selected, but also to four different strains of E. coli, as well as to several related Gram-negative species. P. aeruginosa selected T cells exhibited proliferative responses to six different P. aeruginosa strains, but not to the other Gram-negative species. The finding that repeated stimulation of PBMC with E. coli or P. aeruginosa leads to CD4+ T cells highly reactive with conventional protein antigens specific either for E. coli or P. aeruginosa indicates that these bacteria possess separate dominant protein antigens that drive the proliferation of peripheral blood T cells.

Longitudinal analysis of T cell receptor (TCR) gene usage by human immunodeficiency virus 1 envelope-specific cytotoxic T lymphocyte clones reveals a limited TCR repertoire.

Human immunodeficiency virus 1 (HIV-1) infection is associated with a vigorous cellular immune response that allows detection of cytotoxic T lymphocyte (CTL) activity using freshly isolated peripheral blood mononuclear cells (PBMC). Although restricting class I antigens and epitopes recognized by HIV-1-specific CTL have been defined, the effector cells mediating this vigorous response have been characterized less well. Specifically, no studies have addressed the breadth and duration of response to a defined epitope. In the present study, a longitudinal analysis of T cell receptor (TCR) gene usage by CTL clones was performed in a seropositive person using TCR gene sequences as a means of tracking responses to a well-defined epitope in the glycoprotein 41 transmembrane protein. 10 CTL clones specific for this human histocompatibility leukocyte antigen-B14-restricted epitope were isolated at multiple time points over a 31-mo period. All clones were derived from a single asymptomatic HIV-1-infected individual with a vigorous response to this epitope that was detectable using unstimulated PBMC. Polymerase chain reaction amplification using V alpha and V beta family-specific primers was performed on each clone, followed by DNA sequencing of the V-D-J regions. All 10 clones utilized V alpha 14 and V beta 4 genes. Sequence analysis of the TCR revealed the first nine clones isolated to also be identical at the nucleotide level. The TCR-alpha junctional region sequence of the tenth clone was identical to the junctional region sequences of the other nine, but this clone utilized distinct D beta and J beta gene segments. This study provides evidence that the observed high degree of HIV-1-specific CTL activity may be due to monoclonal or oligoclonal expansion of specific effector cells, and that progeny of a particular CTL clone may persist for prolonged periods in vivo in the presence of a chronic productive viral infection. The observed limited TCR diversity against an immunodominant epitope may limit recognition of virus variants with mutations in regions interacting with the TCR, thereby facilitating immune escape.

T cell receptor gene rearrangements and cytotoxic activities of clones isolated from tumour-infiltrating lymphocytes (TIL) from melanoma patients.

The lymphocytes which infiltrate tumours and are grown in vitro to be used in adoptive immunotherapy are often characterized by dominant rearrangement of their T cell receptor (TCR) genes. To investigate the frequency and function of cells contributing to the 'dominant' rearrangement, we have cloned two bulk cell lines of TIL derived from melanoma patients (TIL-1 and TIL-5). These IL-2-propagated TIL cell lines had a CD8+ phenotype and exerted strong cytotoxic activity against autologous melanoma cells, but not against the natural killer (NK)-sensitive K-562 cell line or LAK targets such as Daudi cells. We derived 40 clones from TIL-1 and 23 from TIL-5. All tested clones were CD3+, CD4-, CD8+ and expressed the alpha/beta TCR. From TIL-1, 27 of 40 clones, and 13/19 of the TIL-5 clones lysed autologous tumour cells. In contrast to the NK-negative bulk cultures, K-562 killing was detected in 21 of the TIL-1 clones and 17 of the TIL-5 clones. TIL-1 contained eight clones and TIL-5 two clones with lytic capacity against neither autologous tumour cells nor the K562 cell line, although these clones possessed lytic potential as evidenced in a lectin-mediated lysis assay. LAK activity was not detected in most clones. Cytotoxic activity against autologous tumour could be inhibited by preincubation with anti-CD3 or anti-HLA class I MoAbs. Of the 34 TIL-1 clones analysed, 15 shared a rearranged TCR beta EcoRI restriction fragment of approximately 9.5 kb with the bulk culture. Clones sharing the EcoRI 10.5-kb dominant band present in TIL-5 bulk culture were also isolated. When the pattern of TCR beta rearrangement was compared with the cytotoxic functions, the following conclusions could be drawn: (i) clones contributing to the dominant band had heterogeneous functions. Most killed autologous tumour cells, but clones with no cytotoxic activity or even with no proliferative capacity in response to autologous tumour cells were also detected among those contributing rearrangement; (ii) some clones that share an apparently identical rearranged band different from the 'dominant' rearrangement, may demonstrate the same cytotoxic function. In addition, our data suggest that many of the clones that share the dominant rearrangement originated from diverse progenitors. The high frequency of clonally diverse anti-tumour reactive TIL is likely to be a reflection of the in vivo selection of the TCR repertoire at the site of tumour.(ABSTRACT TRUNCATED AT 400 WORDS)

T lymphocyte responses to antigens of gram-negative bacteria in pyelonephritis.

We showed previously that large numbers of T lymphocytes accumulate within a few days in the kidneys of rats with ascending pyelonephritis induced with Escherichia coli or Pseudomonas aeruginosa. CD4+ T cells propagated from the lesions exhibited MHC-restricted proliferative responses to formalin-fixed bacteria of the species used to induce infection. In the present study we investigated further the nature of the antigens responsible for the T cell proliferation and studied the ability of different bacterial strains and species to produce proliferative responses. We found that heat-killed bacteria were more stimulatory than formalin-fixed bacteria, and that soluble supernatants of heat-killed organism were also effective. The stimulatory effects of supernatants were destroyed by trypsin and the responses were MHC-restricted. Twelve different E. coli strains, with or without characteristics of uropathogenicity in humans, were all highly stimulatory to T cells derived from a kidney infected with a single E. coli strain. Strains of Klebsiella pneumoniae, Enterobacter aerogenes, and Serratia marcescens--species of Enterobacteriaceae closely related to E. coli--were also stimulatory, whereas more distantly related bacteria--Proteus, Morganella, and P. aeruginosa--were not. T cells propagated from kidneys infected with P. aeruginosa responded to supernatants of this organism, but not to E. coli supernatants. We conclude that a protein antigen (or antigens) shared by strains of E. coli and related Enterobacteriaceae, but not by other gram-negative bacteria, produce MHC-restricted proliferative responses of CD4+ T cells that infiltrate rat kidneys infected with E. coli.

Phenotypic characteristics of lymphocyte populations isolated from middle gestation human placenta.

In order to characterize the phenotypic composition of populations of lymphoid cells in maternal and fetal tissues during the period of middle gestation, mononuclear cells were isolated from maternal peripheral blood, fetal spleen, fetal thymus and placenta of 18-24 week pregnancies. Peripheral blood and placental isolates were stained for a number of lymphoid cell markers by indirect immunofluorescence and analyzed by flow cytometry. Studies were performed on both freshly isolated mononuclear cell preparations and in vitro cultured cells after selective expansion in interleukin 2 (IL2). Fresh placental mononuclear cell isolates were an average 20% CD3+; their CD4/CD8 ratios varied among individuals. An average of 68% of the lymphocytes isolated from maternal peripheral blood were CD3+. Placental and maternal peripheral blood isolates had comparable percentages of CD16+ and CD20+ cells, while CD56+ cells were present at significantly greater numbers in the lymphocyte compartment of placenta (17%) than in peripheral blood (3%; P < 0.01). Lymphocyte isolates were expanded by culture with IL2 and PHA and stained to determine if propagated lymphocyte populations are representative of initial isolates. Expansion of all lymphocyte isolates favored CD3 phenotypes and CD8 phenotypes. Compared to expanded placenta-derived populations, expanded peripheral blood lymphocytes were similar with regard to percentages of all phenotypes except gamma/delta T cells which represented more of placental lymphocytes (10%) than peripheral lymphocytes (5%; P < 0.01). Surface HLA typing determined propagated placenta-derived lymphocytes to be of maternal and not fetal origin. In vitro propagation of placental mononuclear cell isolates may therefore provide populations of maternal CD3+ lymphocytes for assessment of function and specificity.

Phenotypic characteristics of lymphoid populations of middle gestation human fetal liver, spleen and thymus.

Mononuclear cells isolated from liver, spleen and thymus of fetuses between 18 and 24 weeks gestational age were stained for a number of lymphoid cell markers by indirect immunofluorescence and analyzed by flow cytometry. Studies were carried out on freshly isolated mononuclear cell preparations and on cultured cells after selective expansion in interleukin 2 (IL2). Many mononuclear cells in fresh isolates of liver and spleen could not be identified with antibodies to mature T- and B-cell markers. An average of 3% of isolated liver cells and 34% of isolated spleen cells stained positively for CD3, and 19% of liver cells and 37% of spleen cells stained positively for CD20. Lymphoid cells of the fetal thymus were an average 67% CD3+, 76% CD4+, 84% CD8+, and showed greater CD45RO staining (93%) than mononuclear cells of other tissues. Propagation of liver and spleen cell populations in culture favored CD3 phenotypes and CD8 phenotypes. Propagated T cell populations of liver and spleen were primarily TCR alpha/beta+ (81% in liver, 85% in spleen), suggesting a selective advantage in IL2 expansion of alpha/beta T cells over gamma/delta T cells. Propagated gamma/delta T cells of liver and spleen were predominantly TCR gamma/delta 2+. Whereas propagated cells of liver and spleen consisted of approximately 10% gamma/delta+ cells, thymus-derived cells expanded in culture were only an average of 2% TCR gamma/delta+, demonstrating a rarity of IL2-responsive gamma/delta T cells in middle gestation fetal thymus.

T-cell receptor V-gene usage in synovial fluid lymphocytes of patients with chronic arthritis.

In this study we analyzed the usage frequencies of the TCR V-gene segments by alpha beta+ T cells present in synovial fluid of 17 patients with chronic arthritis, including rheumatoid arthritis. The results of this study, obtained from semiquantitative PCR analyses, showed that in all patients most of the TCR V alpha- and V beta-gene segments could be detected both in fresh PBMCs and in fresh SFMCs. The relative frequencies of use of these V-region genes were variable between the different patients. Although there was some skewing of increased usage frequencies of particular TCR V alpha and V beta genes among SFMC-derived TCRs when compared with PBMCs, we could not correlate such increased TCR V-gene usage with the inflammation in the joints as a disease-specific marker.