T-cell Receptor Vβ Research Articles

Characterization of T cells specific for an epitope of human 60-kD heat shock protein (hsp) in patients with Behcet's disease (BD) in Japan.

BD is prevalent in the area of the Silk Route. It has been shown that hsp are involved in the T cell activation in patients with BD in the UK, where this disease has developed sporadically. We have thus examined whether the T cell response to the hsp-derived peptides may be induced in patients with BD in Japan, an east pole of the Silk Route. As with patients in the UK, the human 60-kD hsp peptide 336-351 also yielded vigorous proliferation of T cells in Japanese patients with BD, but neither in normal subjects nor in patients with rheumatoid arthritis (RA); there was significant association between proliferation by this peptide and the presence of ocular lesion, but not any other symptoms of BD. To clarify whether the peptide stimulates T cells as a polyclonal activator, a specific antigen or a superantigen-like substance, we analysed T cell receptor (TCR) usage of responding T cells by means of MoAbs specific for TCR Vbeta subfamily and polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP)-based technique. We found that T cells with certain TCR Vbeta subfamilies (including Vbeta5.2-3, 8, 13.6, 18, 21.3) were increased in circulation and responded to the hsp peptide in an antigen-specific fashion. In addition, TCR Vbeta gene-amplified products of freshly isolated T cells of patients with BD formed several bands in the PCR-SSCP analysis; some of them became prominent after stimulation with the peptide. This suggests that T cells in patients with this disease have already been expanded oligoclonally in vivo, which may be a result of stimulation by triggering antigens, including the hsp peptide. In addition, hsp peptide stimulation induced proinflammatory cytokine mRNA expression in peripheral blood mononuclear cells, including IL-8, tumour necrosis factor-alpha (TNF-alpha) and TNF-beta in eight out of eight patients studied. Taken together, the results suggest that hsp antigen may play a role in the pathogenesis of BD, not only in the area of the Silk Route, but also outside the Silk Route area.

T cell receptor (TCR) repertoire and function of human epidermal T cells: restricted TCR V alpha-V beta genes are utilized by T cells residing in the lesional epidermis in fixed drug eruption.

Human epidermis contains a phenotypically heterogeneous population of T cells. No information however, is available regarding the TCR repertoire of these T cells and their relevant physiologic and pathologic functions in vivo. To this end, T cells were prepared from the lesional epidermis in two patients with fixed drug eruption (FDE) and their phenotype, function and TCR repertoire were examined in parallel. Both epidermal T cells, termed FDE-1 and -2 cells, respectively, expressed alpha beta TCR, but displayed some phenotypic heterogeneity. These T cells were induced to display cytolytic activity by ligation of the CD3/TCR-alpha beta complex. Comparative analyses of TCR V alpha and V beta expression in the epidermal T cells and the paired peripheral blood lymphocytes (PBL) by quantitative polymerase chain reaction (PCR) demonstrated that the epidermal T cells, but not the paired PBL, utilized a very limited range of V alpha and V beta genes. These results indicate that some expansion or preferential migration of epidermal T cells that recognize a restricted set of antigens expressed within the epidermis could occur in situ following ingestion of the causative drug. The persistence of these epidermal T cells in FDE lesions suggests their pathologic role in a drug-induced flare.

Analysis of T-cell receptor Vβ gene from infiltrating T cells in insulitis and myocarditis in encephalomyocarditis virus-infected BALB/C mice

Encephalomyocarditis (EMC) virus induces insulin-dependent diabetes and myocarditis in several strains of mice. The T-cell receptor (TCR) Vβ genes of infiltrating T cells in the pancreas and myocardium of BALB/C mice infected with EMC virus D-variant (EMC-D virus) were analyzed. Using a nested two-step polymerase chain reaction (PCR), TCR Vβ cDNAs were cloned and sequenced. Two and four kinds of TCR Vβ clones were obtained from T cells infiltrating into the pancreas and myocardium of BALB/C mice infected with EMC-D virus, respectively. The infiltrating lymphocytes in the diabetic mice expressed Vβ 8.1, 8.2, and 8.3 genes predominantly. Previously, the use of Vβ 8.2 has been reported in autoimmune diseases such as murine experimental allergic encephalomyelitis (EAE) and non-obese diabetic (NOD) mouse. This study suggests that mice infected with EMC virus are a useful animal model for autoimmune diseases such as insulin-dependent diabetes.

Thymus transplantation in complete DiGeorge syndrome: immunologic and safety evaluations in 12 patients

AbstractComplete DiGeorge syndrome is a fatal condition in which infants have no detectable thymus function. The optimal treatment for the immune deficiency of complete DiGeorge syndrome has not been determined. Safety and efficacy of thymus transplantation were evaluated in 12 infants with complete DiGeorge syndrome who had less than 20-fold proliferative responses to phytohemagglutinin. All but one had fewer than 50 T cells/mm3. Allogeneic postnatal cultured thymus tissue was transplanted. T-cell development was followed by flow cytometry, lymphocyte proliferation assays, and T-cell receptor Vβ (TCRBV) repertoire evaluation. Of the 12 patients, 7 are at home 15 months to 8.5 years after transplantation. All 7 survivors developed T-cell proliferative responses to mitogens of more than 100 000 counts per minute (cpm). By one year after transplantation, 6 of 7 patients developed antigen-specific proliferative responses. The TCRBV repertoire showed initial oligoclonality that progressed to polyclonality within a year. B-cell function developed in all 3 patients tested after 2 years. Deaths were associated with underlying congenital problems. Risk factors for death included tracheostomy, long-term mechanical ventilation, and cytomegalovirus infection. Adverse events in the first 3 months after transplantation included eosinophilia, rash, lymphadenopathy, development of CD4-CD8- peripheral T cells, elevated serum immunoglobulin E (IgE), and possible pulmonary inflammation. Adverse events related to the immune system occurring more than 3 months after transplantation included thrombocytopenia in one patient and hypothyroidism and alopecia in one other patient. Thymic transplantation is efficacious, well tolerated, and should be considered as treatment for infants with complete DiGeorge syndrome.

Complete reconstitution of human lymphocytes from cord blood CD34+ cells using the NOD/SCID/γcnull mice model

AbstractEstablishment of an assay capable of generating all classes of human lymphocytes from hematopoietic stem cells (HSCs) will provide new insight into the mechanism of human lymphopoiesis. We report ontogenic, functional, and histologic examination results of reconstituted human lymphocytes in NOD/SCID/ γcnull mice after the transplantation of human cord blood (CB) CD34+ cells. After transplantation, human B, natural killer (NK), and T cells were invariably identified in these mice, even though no human tissues were cotransplanted. Immature B cells resided mainly in bone marrow (BM), whereas mature B cells with surface immunoglobulins were preferentially found in spleen. NK cells were identified in BM and spleen. T cells were observed in various lymphoid organs, but serial examinations after transplantation confirmed human T lymphopoiesis occurring in the thymus. These human lymphocytes were also functionally competent. Human immunoglobulin M (IgM), IgA, and IgG were detected in the sera of these mice. T cells showed a diverse repertoire of T-cell–receptor Vβ (TCR Vβ) chains, proliferated in response to phytohemagglutinin, and were cytotoxic against cell lines. NK activity was demonstrated using the K562 cell line. Immunohistochemical analysis revealed that human lymphocytes formed organized structures in spleen and thymus that were analogous to those seen in humans. In the thymus, CD4 and CD8 double-positive T cells were predominant and coexpressed CD1a and Ki-67, thereby supporting the notion that T lymphopoiesis was taking place. NOD/SCID/ γcnull mice provide a unique model to investigate human lymphopoiesis without the cotransplantation of human tissues.

Mycobacterium bovis BCG scar status and HLA class II alleles influence purified protein derivative-specific T-cell receptor V beta expression in pulmonary tuberculosis patients from southern India.

Purified protein derivative (PPD) RT23-recalled T-cell receptor (TCR) V beta expression was studied in the peripheral blood of 42 pulmonary tuberculosis patients and 44 healthy controls from southern India, a region where tuberculosis is endemic. Forty-eight-hour whole-blood cultures in the presence or absence of PPD-RT23 were set up, and at the end of the culture period total RNA was extracted and cDNA was synthesized. Expression of various TCR V beta families was assessed by using family-specific primers. PPD-specific expression (usage) of TCR V beta families 4, 6, 8 to 12, and 14 was found in more controls than patients. Among the responders (individuals who showed PPD-specific expression), endemic controls had significantly higher responses than the patients had for TCR V beta families 2, 3, 7, 13, and 17. The majority of the patients did not show usage of most of the TCR V beta families, and this was attributed to T-cell downregulation. A four-way nested classification analysis revealed that TCR V beta family 1, 5, 9, 12, and 13 usage in the context of HLA class II high-risk alleles (DRB1*1501, DRB1*08, and DQB1*0601) and Mycobacterium bovis BCG scar status were the determining factors in susceptibility and resistance to tuberculosis. The healthier status of controls was attributed to the wider usage of many TCR V beta families readily recalled by PPD, while the disease status of the patients was attributed to TCR V beta downregulation and the resultant T-cell (memory cell?) unresponsiveness. Host genetics (HLA status) and BCG vaccination (scar status) seem to play important roles in skewing the immune response in adult susceptibility to pulmonary tuberculosis through TCR V beta usage.

T-cell receptor repertoire in healthy Sardinian subjects

The T-cell receptor (TCR) Vβ gene usage of CD4 + and CD8 + T-cell subpopulations was evaluated by flow cytometric analysis and by CDR3 spectratyping in healthy subjects belonging to Sardinian population, which is ethnically homogeneous and genetically distant from all other Italian and Caucasoid groups. As described in healthy Caucasian subjects, we found a nonrandom Vβ gene usage and in some Vβ families a significant skewed reactivity toward CD4 + T cells. Moreover, different subjects showed expansions in some Vβ subfamilies, mainly in the CD8 + T cells. By CDR3 spectratyping analysis we found a significantly higher degree of skewness in the TCR Vβ repertoire of CD8 + than in that of CD4 + T cells. The similarity found in the TCR Vβ gene usage between the population examined and other Caucasoid groups suggest that the shape of the TCR repertoire is more influenced by rearrangement processes than ethnic background. However, genetic polymorphisms may condition the expression levels of some Vβs, determining the variability of the TCR repertoire between different populations. Finally, the profound perturbations evidenced in the CD8 + T cell subpopulation could be related to a different response to the antigenic stimulation between CD8 + and CD4 + T lymphocytes.

Application of the Molecular Analysis of the T-Cell Receptor Repertoire in the Study of Immune-Mediated Hematologic Diseases

The basis for the vast recognition spectrum of the T-cell receptor (TCR) can be determined by the rearrangement and recombination of the variable, diversity and joining regions of the variable portions of β (B) and α (A) chains as well as their recombination and modification. Analysis of the TCR rearrangement has been routinely used to detect clonality for the diagnosis of lymphoid malignancies. However, molecular analysis of the TCR repertoire can be a powerful tool in the study of T-cell responses to pathogens and in autoimmune diseases. The concept of the oligoclonality in the context of cellular immune responses is based on the presence of immunodominant T-cell clones within distinct T-cell subpopulations used for analysis. Under normal circumstances, a limited number of clones undergo periodic expansions in reaction to foreign antigens. Under pathologic conditions, though, the derailment of immune regulation allows expansions of specific and potentially pathogenic T-cell clones. For example, large granular lymphocyte (LGL) leukemia illustrates an extreme expansion of a single T-cell clone associated with a distinct autoimmune pathology, which suggests an exaggerated clonal response to a specific antigenic target. In immune-mediated bone marrow failure syndromes, clonal rearrangement of the TCR cannot be detected in unseparated blood or marrow. Nevertheless, individual T-cell clones can significantly expand and may allow for demonstration of oligoclonality in selected T-cell populations. These subpopulations are defined, for example, by a specific β (B)-chain usage or other phenotypic markers. Given the diversity of the TCR recognition spectrum, the task of identifying immunodominant clonotypes derived from unique complementarity determining region-3 (CDR3) sequences is very complex. However, expanded T-cell clones likely represent immunodominant responses which can be detected on the molecular level using analysis of the individual TCR VB-chain representation, CDR3 size fragment skewing, and determination of the frequency of individual clonotypic sequences. In the future, TCR VB clonotypes may be applied as a diagnostic tool, analogous to serologic markers. As an investigative tool in hematology, molecular analysis of the TCR utilization pattern and the detection of immunodominant clonotypes represents a novel approach in the study of immune-mediated hematologic diseases, such as aplastic anemia (AA), some forms of myelodysplasia (MDS), anti-leukemic immune surveillance, graft-versus-leukemia effects and graft-versus-host disease (GvHD).

Immune Reconstitution and Tolerance after Allogeneic Hematopoietic Stem Cell Transplantation

We have evaluated recovery of CD56 positive and other cell types following allogeneic stem cell transplantation and have found that the recovery of CD56 positive cells was faster than other lymphoid cells after allogeneic stem cell transplantation, while the recovery of CD4 positive cells was markedly delayed. Chimerism analysis showed that mixed chimerism was often observed in younger (<30 years old) patients. Mixed chimerism in older (≥30 years old) patients was associated with rejection and relapse, while this was not found in younger patients. Among the chimerism of various cell populations, donor-derived CD56-positive cells are important in early engraftment when determined in allogeneic nonmyeloablative stem cell transplantation (allo-NST), regardless of the proportion of donor-derived CD3-positive cells. Complementarity-determining region three (CDR3) size spectratyping in T-cell receptor (TCR) chain subfamilies (Vβ) showed that high level of diversity in TCR Vβ repertoire is important for a late rejection and skewed TCR Vβ repertoire is correlated with the occurrence of graft-versus-host disease (GVHD) especially chronic GVHD. Expression of inhibitory natural killer (NK) cell receptors such as CD158b and CD94/NKG2A on peripheral CD3-negative and -positive cells were increased in parallel with GVHD. Interestingly, these cells appeared to control GVHD, while preserving graft-versus-leukemia (GVL) effect. Analysis of cytokine gene expression in peripheral blood mononuclear cells showed that type 1 helper T cells (Th1)-derived cytokines increased in severe GVHD, while Th2-derived cytokines such as IL-4, IL-10 and IL-13 increased in mild GVHD. These results indicate that Th2 cells suppress GVHD, although Th1 cells augment GVHD. Taken together, evaluation of immune reconstitution and tolerance in patients receiving allogeneic stem cell transplantation from the various viewpoints is essential and useful to obtain better clinical outcome.

Binding kinetics of superantigen with TCR and MHC class II.

There are several key differences between the nature of interactions of a T-cell receptor (TCR) with a conventional MHCpeptide complex and a bacterial superantigen (SAg). Apart from being powerful T-cell mitogens, these SAgs do not require antigen processing, they interact with the Vβ region of TCR and their binding sites lie outside the peptide-binding groove of the major histocompatibility complex (MHC) class II molecule (-). The plasticity of TCR binding to MHC-peptide complex has been appreciated only recently from crystal structures of TCR-MHC complex (,). However, the breadth of response to different MHCpeptide ligands is limited, whereas bacterial SAgs commonly interact with several Vβ elements. This results largely from the fact that much of the contact is with main-chain atoms instead of side-chain residues of TCR Vβ (,,). Taking into account these differences and particularly the strength of the SAg effect on T-cell activation, it was reasonable to predict that bacterial SAgs would interact with higher affinity and that they would also show different binding kinetics.

CD8αα memory effector T cells descend directly from clonally expanded CD8α+βhigh TCRαβ T cells in vivo

Whereas most peripheral CD8(+) alphabeta T cells highly express CD8alphabeta heterodimer in healthy individuals, there is an increase of CD8alpha(+)beta(low) or CD8alphaalpha alphabeta T cells in HIV infection or Wiskott-Aldrich syndrome and after bone marrow transplantation. The significance of these uncommon cell populations is not well understood. There has been some question as to whether these subsets and CD8alpha(+)beta(high) cells belong to different ontogenic lineages or whether a fraction of CD8alpha(+)beta(high) cells have down-regulated CD8beta chain. Here we assessed clonality of CD8alphaalpha and CD8alpha(+)beta(low) alphabeta T cells as well as their phenotypic and functional characteristics. Deduced from surface antigens, cytotoxic granule constituents, and cytokine production, CD8alpha(+)beta(low) cells are exclusively composed of effector memory cells. CD8alphaalpha cells comprise effector memory cells and terminally differentiated CD45RO(-)CCR7(-) memory cells. T-cell receptor (TCR) Vbeta complementarity-determining region 3 (CDR3) spectratyping analysis and subsequent sequencing of CDR3 cDNA clones revealed polyclonality of CD8alpha(+)beta(high) cells and oligoclonality of CD8alpha(+)beta(low) and CD8alphaalpha cells. Importantly, some expanded clones within CD8alphaalpha cells were also identified within CD8alpha(+)beta(high) and CD8alpha(+)beta(low) subpopulations. Furthermore, signal-joint TCR rearrangement excision circles concentration was reduced with the loss of CD8beta expression. These results indicated that some specific CD8alpha(+)beta(high) alphabeta T cells expand clonally, differentiate, and simultaneously down-regulate CD8beta chain possibly by an antigen-driven mechanism. Provided that antigenic stimulation directly influences the emergence of CD8alphaalpha alphabeta T cells, these cells, which have been previously regarded as of extrathymic origin, may present new insights into the mechanisms of autoimmune diseases and immunodeficiencies, and also serve as a useful biomarker to evaluate the disease activities.

Analysis of the clonal expansion of TCR Vβ T cells in patients with CML after DLI

Objective To investigate the clonal expansion of T cell receptor (TCR) Vβ subfamily T cells which were considered as GVL effective cells after donor lymphocytes infusion (DLI) in patients with relapse chronic myelogenous leukemia (CML) after allogeneic bone marrow transplantation (allo-BMT).

Changes in T-cell receptor VB repertoire in aplastic anemia: effects of different immunosuppressive regimens

We studied the degree and the pattern of skewing of the variable region of beta-chain (VB) T-cell receptor (TCR) repertoire in aplastic anemia (AA) at initial presentation and after immunosuppression using a high-resolution analysis of the TCR VB complementarity-determining region 3 (CDR3). Age-matched healthy individuals and multitransfused patients with non-immune-mediated hematologic diseases were used as controls. In newly diagnosed AA, the average frequency of CDR3 size distribution deviation indicative of oligoclonal T-cell proliferation was increased (44% +/- 33% vs 9% +/- 9%; P =.0001); AA patients with human leukocyte antigen (HLA)-DR2 and those with expanded paroxysmal nocturnal hemoglobinuria clones showed more skewed VB repertoires. Nonrandom oligoclonal patterns were found for VB6, VB14-16, VB21, VB23, and VB24 subfamilies in more than 50%, and for VB15, VB21, and VB24 in more than 70% of AA patients with HLA-DR2. Patients received immunosuppression with antithymocyte globulin (ATG)/cyclosporine (CsA) or cyclophosphamide (CTX) with CsA in combination, and their VB repertoire was reanalyzed after treatment. Whereas no significant change in the degree of VB skewing in patients who had received ATG was seen, patients treated with CTX showed a much higher extent of oligoclonality within all VB families, consistent with a profound and long-lasting contraction of the T-cell repertoire. VB analysis did not correlate with the lymphocyte count prior to lymphocytotoxic therapy; however, after therapy the degree of VB skewing was highly reflective of the decrease in lymphocyte numbers, suggesting iatrogenic gaps in the VB repertoire rather than the emergence of clonal dominance. Our data indicate that multiple specific clones mediate the immune process in AA.

Mucosal tolerance to a bacterial superantigen indicates a novel pathway to prevent toxic shock.

Enterotoxins with superantigenic properties secreted during systemic Staphylococcus aureus infection are responsible for toxic shock. We show that intranasal administration of staphylococcal enterotoxin A (SEA), but not a recombinant SEA lacking superantigenic activity, protected mice against lethal systemic SEA challenge. Protection was superantigen specific since intranasal exposure to SEA would not protect against death caused by subsequent toxic shock syndrome toxin 1 systemic challenge. Protection was neither due to selective depletion of SEA-specific T-cell receptor Vbeta families nor due to production of neutralizing anti-SEA antibodies. Importantly, the production of interleukin 10 (IL-10) induced by "tolerization" (that is, by the induction of immunological tolerance) contributed to the observed protection against lethal superantigen-triggered disease. In support of this notion we found that (i) significantly increased levels of IL-10 in sera of "tolerized" animals (that is, animals rendered tolerant) and (ii) IL-10(-/-) mice could not be tolerized by mucosal SEA administration. Altogether, this is the first study to show that mucosal tolerance to a superantigen is readily triggered by means of immunodeviation.

T-cell receptor bias in patients with allergic bronchopulmonary aspergillosis

CD4+ Th2 helper cell mediated immune responses have been shown to play a crucial role in the pathogenesis of ABPA. HLA and TCR are the candidate genes, which can influence the specificity of these responses. We have previously established a strong association of HLA DR2/5 in ABPA susceptibility. The study was designed to determine whether allergen specific T cell express a limited usage of T cell receptor (TCR) Vβ gene repertoire in ABPA and to find an association of susceptible HLA-DR determinants with the identified TCR gene segments. TCR Vβ typing was performed on antigen specific T cell lines from 14 ABPA and 12 nonABPA patients. The majority of ABPA patients (86%) expressed allergen specific T cells with Vβ13 genes indicating its role in susceptibility, whereas in nonABPA controls, Vβ1 genes T cell repertoires were predominantly expressed. The unrestricted pattern of Vβ gene amplification seen before antigen stimulation suggests an oligoclonal expansion of a specific T cell population in response to the allergen Asp f 1 in ABPA and nonABPA patients. The increased usage of Vβ13 in ABPA and Vβ1 in nonABPA indicates their importance in susceptibility and resistance, respectively.

Improved assessment of T-cell receptor (TCR) VB repertoire in clinical specimens: combination of TCR-CDR3 spectratyping with flow cytometry-based TCR VB frequency analysis.

Antigen-specific T-cell responses may be described by combining three categories: (i) the specificity and effector functions of a T-cell population, (ii) the quantity of T-cell responses (i.e., the number of responding T cells within the CD4/CD8 population), and (iii) the "quality" of T cells (defined by the T-cell receptor [TCR] structure). Several methods to measure T-cell responses are now available including evaluation of T-cell precursors using limiting dilution, the enzyme-linked immunospot assay, ex vivo TCR variable (v)-segment analysis determined by flow cytometry, and TCR-CDR3 length analysis (spectratyping), as well as identification of peptide-specific T cells using major histocompatibility complex (MHC) class I tetramers containing appropriate peptides. Until now, only a limited set of MHC-peptide complexes have been available as tetramer complexes. We demonstrate that CD8(+) or CD4(+) T cells in patients with cancer can be molecularly defined using a combination of spectratyping (TCR structure and "molecular composition") plus the implementation of an antibody panel directed against 21 individual VB TCR chains ("quantity" of T-cell families). This approach is instrumental in defining and comparing the magnitudes of CD4(+) or CD8(+) T-cell responses over time in individual patients, in comparing the TCR VA and VB repertoire in different anatomic compartments, and in comparing the TCR VA-VB diversity with that in normal healthy controls. This method provides the means of objectively defining and comparing the TCR repertoire in patients undergoing vaccination protocols and underlines the necessity to calibrate the TCR-CDR3 analysis with a qualitative assessment of individual TCR VB families.

Antigen-driven T-cell selection in patients with cervical cancer as evidenced by T-cell receptor analysis and recognition of autologous tumor.

We characterized the T-cell receptor (TCR) repertoire in freshly harvested tumor lesions, in short-term-expanded CD4(+) tumor infiltrating lymphocytes (TIL) as well as in CD4(+) and CD8(+) peripheral blood lymphocytes (PBL) from three patients with cervical cancer. Skewing of the T-cell repertoire as defined by measuring the length of the complementarity-determining region 3 (CDR3) of the TCR VA and VB chains was observed in CD8(+) PBL, in freshly harvested tumor tissue, as well as in CD4(+) TIL. Comparative analysis of the TCR repertoire revealed unique monoclonal TCR transcripts within the tumor lesion which were not present in PBL, suggesting selection of TCR clonotypes due to antigenic stimulation. TCR repertoire analysis of the short-term (7-day) CD4(+) TIL lines revealed that the TCR composition is markedly different from that in CD4(+) PBL or in the freshly harvested tumor tissue. Only one-third of CD4(+) TIL lines showed HLA-DR-restricted recognition of autologous tumor cells as defined by cytolysis. These data provide support for the antigen-driven selection of T cells within cervical cancer lesions and suggest that analysis of the TCR repertoire may aid in obtaining an objective description of the immune response in patients with cervical cancer who are undergoing epitope-based immunotherapy.

Importance, mechanisms and limitations of the distant bystander effect in cancer gene therapy of experimental liver tumors

GCV-ablation of transplanted TK-positive liver tumors or the application of syngenic and allogenic HSV-TK/GCV oncolysates significantly reduced the size of synchronously growing untreated sister tumors in the liver. These TK-negative liver tumors constantly showed an increased infiltration by mononuclears (×4). The relative abundance of CD 4/8, NK and monocyte subtypes remained constant. The distant bystander effect was associated with a strong induction of GMCSF and IL-12 expression in the untreated TK-negative liver tumors. Analysis of the vβ T-cell receptor profiles from TK-negative tumors did not point to clonal lymphocyte expansions. These results support the view of the ‘distant bystander effect’ as a predominantly local phenomenon, which is mediated by resident immune effectors rather than by MHC I restricted CD 3 positive lymphocytes.

T-cell receptor Vbeta repertoire in nodal non-anaplastic peripheral T-cell lymphomas.

The aim of this study was to evaluate the T-cell receptor (TCR) Vbeta repertoire in the two main histological subtypes of nodal non-anaplastic peripheral T-cell lymphoma: Not Otherwise Specified (NOS) and angioimmunoblastic lymphoma (AIL). Frozen lymph node tissues of eight NOS and six AIL were analyzed. A reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to assess the expression of the 24 Vbeta gene families. Our study showed a broad TCR Vbeta repertoire in AIL and NOS, with a slight increase in the number of Vbeta families in AIL (16 vs 10 on agarose gels). Nevertheless, there was a clear difference in four cases. A predominant Vbeta family was observed in two NOS, whereas no predominant Vbeta family was observed in the AIL. Two AIL showed the whole Vbeta repertoire, whereas it was never observed in NOS. This pattern may help to categorize these histopathological entities and further suggests a differential T-cell response. These results show that numerous reactive T-cells are present both in AIL and NOS. Possibly, they play a role in the growth of these lymphomas.

Selective CD8+ T cells accumulate in the lungs of patients with allergic asthma after allergen bronchoprovocation.

Our objective was to study whether CD4+ or CD8+ T cells expressing particular T cell receptors (TCR) would accumulate in the lungs of patients with allergic asthma following allergen exposure. We thus analysed the TCR Valpha and Vbeta gene usage of CD4+ and CD8+ lung and peripheral blood lymphocytes (PBL) of eight patients with allergic asthma before and 4 days after inhalation challenge with the relevant allergen. Lung cells obtained by bronchoalveolar lavage (BAL) and paired PBL samples were analysed by flow cytometry using a panel of anti-TCR V-specific monoclonal antibodies that encompass = 50% of the T cell repertoire. Lung-limited T cell expansions were recorded in both the CD4+ and the CD8+ subsets. In BAL CD8+, out of a total of 126 analyses, the number of T cell expansions increased from two to 11 after challenge, some of them dramatic. In BAL CD4+ the frequency of expansions was moderately increased already before challenge, but remained unchanged. A few expansions that tended to persist were noted in PBL CD8+. When analysing the overall change in TCR V gene usage the largest changes were also recorded in the BAL CD8+ subset. Specific interactions between T cells and antigens may lead to an increased frequency of T cells using selected TCR V gene segments. In this study we demonstrate that following allergen bronchoprovocation in allergic asthmatic subjects, T cell expansions preferentially emerge in the lung CD8+ T cell subset.