Vβ Families Research Articles

Rapid analysis of the Vβ repertoire of CD4 and CD8 T lymphocytes in whole blood

Determination of the T cell receptor Vβ repertoire of human CD4 and CD8 populations is a useful immunological tool, particularly in the investigation of superantigen involvement in various disease states. We describe the optimisation of a rapid technique for the simultaneous evaluation of 24 Vβ families of the T cell receptor of CD4 and CD8 positive lymphocytes in whole blood by flow cytometry adapting a commercially available monoclonal antibody kit. The technique described is reliable and reproducible, and we describe its use as a potential diagnostic tool in patients with staphylococcal and streptococcal toxic shock syndromes.

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.

Transduction and selection of human T cells with novel CD34/thymidine kinase chimeric suicide genes for the treatment of graft-versus-host disease

Clinical trials evaluating the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) suicide gene therapy system for the control of graft-versus-host disease (GVHD) have been limited by low transduction efficiencies and inefficient selection procedures. In this study, we designed and evaluated a novel chimeric suicide gene consisting of the extracellular and transmembrane domains of human CD34 and full-length HSV-tk (ΔCD34-tk). High-efficiency transfer of ΔCD34-tk to primary human T cells was accomplished after a single exposure to VSV-G-pseudotyped, Moloney murine leukemia virus-based retrovirus 48 h after activation of human PBMCs with anti-CD3 and anti-CD28 antibodies immobilized on magnetic beads. Using an optimized 5-day transduction and selection procedure, transduction efficiencies averaged 71%, with isolation purities greater than 95% and yields exceeding 90%. The immunoselected T cells were selectively eliminated by GCV (IC50 ∼3 nM), maintained a normal subset composition, exhibited a polyclonal TCR Vβ family repertoire, and contained 5 or 6 vector copies per transduced cell when optimally transduced. No increase in GCV sensitivity was observed upon incorporation of highly active mutant HSV-tk enzymes into the ΔCD34-tk suicide gene. T cells modified with the ΔCD34-tk gene using the optimized protocol should improve the overall efficacy of the HSV-tk/GCV suicide gene therapy method of GVHD control.

Host stem cells can selectively reconstitute missing lymphoid lineages in irradiation bone marrow chimeras

AbstractThe regulatory elements governing the process of lymphopoiesis from pluripotential stem cells to mature lymphocytes are not well understood. In this study we found that in bone marrow chimeras made by reconstituting lethally irradiated normal mice with bone marrow taken from genetically B-cell–deficient animals (μMT.B6 → F1) the B-cell compartment is reconstituted with host-derived B cells. Similarly, in animals reconstituted with bone marrow taken from mice with genetic deficiencies in the development of T cells (TCR–/– → F1) or both B and T cells (RAG–/– → F1), the missing lymphocyte lineage(s) was specifically reconstituted from host-derived cells. In all chimeras, all other blood lineages were generated from donor-derived stem cells. Control chimeras (B6 → F1) had only donor-derived hematopoietic cells as expected. The reconstituted, host-derived lymphoid compartments contained normal functional cell populations as determined by the presence of T cells expressing all 16 common TCR Vβ families, and the presence of all antibody isotypes in the serum. Reconstituted TCR–/– → F1 chimeras were also able to mount T-cell proliferative responses to foreign antigens equal to those of control animals. This observation would seem to suggest that during lymphopoietic reconstitution, missing lymphoid lineages can dictate their own reconstitution.

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.

T cell Vbeta repertoires in childhood vasculitides.

Superantigens (SAgs) are potent stimulators of T cells bearing specific Vbeta T cell receptors (TCR) and may play a role in the aetiopathogenesis of systemic vasculitis, although this remains contentious. To investigate the possible aetiological role of SAgs, this study examined peripheral blood T cell Vbeta repertoires in children with systemic vasculitis. FACS analysis of 17 different peripheral blood T cell Vbeta families was performed in 20 healthy control children, 27 disease control children with nonvasculitic inflammatory disease, 25 children with primary systemic vasculitis, six patients with Kawasaki disease (KD) and six patients with Henoch-Schönlein purpura (HSP). There was a significantly increased variance of CD4 Vbeta12 and Vbeta17, and CD8 Vbeta1 in the primary systemic vasculitis group compared to control and disease controls. Moreover, 80% of the primary systemic vasculitis children had one or more CD4 Vbeta expansions or deletions, compared with 30% of controls (P < 0.002), and 37% of the disease controls (P < 0.002). In the KD group, the mean percentage of CD4 Vbeta2 T cells was higher than in controls or disease controls. In the HSP group, there was no consistent skewing of the T cell Vbeta repertoire. We have observed changes in the T cell Vbeta repertoire in children with vasculitis over and above those observed in disease controls. While these data provide impetus for further research into this contentious field, they do not resolve unequivocally the question of the role of SAgs in childhood vasculitic syndromes.

Protein kinase C-dependent phosphorylation of the β-subunit of the voltage-sensitive potassium channels (K vβ2)

The K vβ proteins are members of the aldo-keto reductase (AKR) superfamily that interact with the cytoplasmic face of the pore-forming α-subunits of the voltage-sensitive K + channels. The physiological functions of K vβ are unclear, although some members of the K vβ family impart rapid inactivation to non-inactivating K + channels. Structural analyses reveal that the NADPH-binding site as well as the active site architecture and residues of other AKR proteins are conserved in the K vβ proteins. The K vβ2 displays high-affinity NADPH binding, although no catalytic activity for this protein has been reported. Recent studies show that K vβ2 is constitutively associated with protein kinase C (PKC) ζ via the zeta-interacting protein (ZIP). The primary structure of K vβ displays 25 PKC consensus phosphorylation sites. We report that incubation of recombinant K vβ2 with PKCα in vitro leads to rapid phosphorylation of the protein. Stimulation of PKC by phorbol-12-myristate-13-acetate (PMA) also induced the phosphorylation of K vβ2 expressed in COS-7 cells. Deletion of the first 35 amino acids of the variable N-terminus led to a substantial decrease in the overall extent of phosphorylation in vitro, indicating that the N-terminus peptide is the preferred site of PKC phosphorylation. Analysis of the phosphorylated protein by HPLC coupled with an ion-trap electrospray mass spectrometer revealed phosphorylation of Ser-266 located near the NADPH-binding site of the protein. We propose that phosphorylation could affect the N-terminus-mediated β–α interactions or the binding of NADP(H) to the conserved AKR domain of the K vβ proteins.

T Cell Receptor V-beta Repertoires in Kawasaki Disease and Primary Systemic Vasculitides of Childhood

Introduction: Despite conflicting evidence and much debate, superantigenic stimulation of the immune system in Kawasaki disease (KD) remains an attractive hypothesis since there is considerable overlap between the clinical and immunological phenotypes of KD and classical superantigen-mediated diseases such as the toxic shock syndrome. Moreover, although there are limited data in adults suggesting that SAgs may be involved in the initiation of other primary systemic vasculitides, no such data exists for children. Methods: To investigate the possible aetiological role of SAgs, this study examined peripheral blood TCR Vb repertoires in children with KD (n=6), polyarteritis nodosa (PAN, n=23), Wegener's granulomatosis (WG, n=1), and microscopic polyangiitis (MPA, n=1). 20 normal children and 30 children with non-vasculitic inflammatory disease, or recipients of renal allografts served as controls and disease controls respectively. 3 colour FACS analysis of peripheral blood mononuclear cells stained with conjugated monoclonal antibodies to CD3, CD4, CD8, and 17 different Vb families was performed. Results: The mean % of CD4+ T cells bearing Vb2 was significantly increased in the KD group versus controls and disease controls (p=0.03 and p=0.01 respectively). Individual KD patients were also noted to have CD4+ T cell Vb expansions other than Vb2 (Vb5.1 n=2; Vb12 n=1). 60% of the primary systemic vasculitis patients had one or more TCR Vb expansions in the CD4+ lymphocyte population, compared with 30% of the controls (p=0.02-0.05), and 36% of the disease controls (p=0.05-0.1). Unlike KD, however, the pattern of Vb families expanded in individual patients was more diverse, perhaps indicative of the involvement of several different SAgs. Follow-up of 7 primary systemic vasculitis patients demonstrated a normalization of the CD4+ T cell Vb repertoire following induction of remission. Conclusion: Our preliminary data provide indirect evidence for an aetio-pathogenetic role for SAgs in KD and primary systemic vasculitides affecting children.

TCR rearrangement in lymphocytes infiltrating melanoma metastases after administration of autologous dinitrophenyl-modified vaccine.

Administration of a vaccine consisting of autologous melanoma cells modified with a hapten, dinitrophenyl (DNP), induces T cell infiltration of metastatic sites. We have reported an analysis of these infiltrating T cells, indicating that certain TCR-Vbeta gene segments are greatly overexpressed. In this study, we investigate the rearrangement of the TCR-Vbeta as well as the junctional diversity in T cells infiltrating melanoma metastases following treatment with DNP vaccine. In 19 of 26 control specimens, V-D-J length analysis showed the expected polyclonal patterns. In contrast, postvaccine tumors from 9 of 10 patients showed dominant peaks of V-D-J junction size in one or more Vbeta families. Dominant peaks were seen most frequently in six Vbeta families (Vbeta7, 12, 13, 14, 16, and 23) and were never seen in seven others. Further analysis of the oligoclonal Vbeta products showed dominant peaks in the J region as well. Of particular interest was the finding that Vbeta and Jbeta peaks were similar in inflamed metastases obtained at different times or from different sites from the same patient. Although 6 of 10 patients expressed HLA-A1, there was no common pattern of TCR rearrangements among them. Finally, the amplified PCR products from seven of these specimens were cloned and sequenced and the amino acid sequence of the complementarity-determining region 3 was deduced. In six of seven specimens, the same complementarity-determining region 3 sequence was repeated in at least two clones and in five of seven in at least three clones. Our study indicates that DNP vaccine induces the expansion of particular T cell clones that may be agents of its antitumor effects.

Longitudinal analysis of the T-cell receptor (TCR)-VA and -VB repertoire in CD8+ T cells from individuals immunized with recombinant hepatitis B surface antigen.

Recent studies have suggested that vaccination induces alterations in the T cell receptor (TCR) repertoire. We investigate the diversity of the TCR repertoire after immunization with a recombinant hepatitis B surface vaccine in seven healthy subjects in CD8+ T cells in peripheral blood lymphocytes. Cellular immune responses were monitored over time by sorting CD8 T cells followed by TCR-VA and -VB complementarity determining region 3 (CDR3) analysis. Frequency of individual VB families was determined by flow cytometry. TCR-VA/VB repertoires obtained from CD8+ T cells drawn after vaccination were compared to the TCR repertoire determined prior to vaccination. Monoclonal TCR transcripts could be detected exclusively in CD8+, but not in CD4+ T cells. Such monoclonal TCR transcripts were either stable in some individuals, or could only be detected at certain time points after vaccination. Sorting of monoclonal TCR-VB3+ T cells, which constituted up to 5% of the CD8+ T cell population from one individual, revealed that this T cell clone recognizes an epitope provided by the recombinant hepatitis B vaccine presented by MHC-class I on autologous antigen-presenting cells. Examination of the structural anatomy, defined by the TCR, and the frequency of T cells responding to the immunizing antigen may be helpful to provide surrogate markers to monitor cellular immune responses induced by protein antigens utilized for vaccination.

Direct ex vivo analysis of human CD4(+) memory T cell activation requirements at the single clonotype level.

CD4(+) memory T cells continuously integrate signals transmitted through the TCR and costimulatory molecules, only responding when the intensity of such signals exceeds an intrinsic activation threshold. Recent data suggest that these activation thresholds can be regulated independently of TCR specificity, and that threshold tuning may constitute a major mechanism for controlling T cell effector activity. In this work we take advantage of the profound clonotypic hierarchies of the large human CD4(+) T cell response to CMV to study activation thresholds of fresh (unexpanded) memory T cells at the clonotypic level. We identified dominant responses to CMV matrix determinants mediated by single TCRB sequences within particular TCR-Vbeta families. The specific response characteristics of these single, Ag-specific, TCRB-defined clonotypes could be unequivocally determined in fresh PBMC preparations by cytokine flow cytometry with gating on the appropriate Vbeta family. These analyses revealed 1) optimal peptides capable of eliciting specific responses by themselves at doses as low as 2 pg/ml, with each log increase in dose eliciting ever-increasing frequencies of responding cells over a 4- to 5-log range; 2) significant augmentation of response frequencies at all submaximal peptide doses by CD28- and CD49d-mediated costimulation; 3) differential dose response and costimulatory characteristics for IFN-gamma and IL-2 responses; and 4) no association of activation requirements with the CD27-defined CD4(+) T cell memory differentiation pathway. Taken together these data confirm that triggering heterogeneity exists within individual CD4(+) memory T cell clonotypes in vivo and demonstrate that such single clonotypes can manifest qualitatively different functional responses depending on epitope dose and relative levels of costimulation.

Oligoclonal and polyclonal CD4 and CD8 lymphocytes in aplastic anemia and paroxysmal nocturnal hemoglobinuria measured by Vβ CDR3 spectratyping and flow cytometry

AbstractWe have hypothesized that in aplastic anemia (AA) the presence of antigen-specific T cells is reflected by their contribution to the expansion of a particular variable beta chain (Vβ) subfamily and also by clonal CDR3 skewing. To determine the role of disease-specific “signature” T-cell clones in AA, we studied preferential Vβ usage by flow cytometry and analyzed Vβ-CDR3 regions for the presence of oligoclonality. We first established the contribution of each Vβ family to the total CD4+ and CD8+ lymphocyte pool; in AA and paroxysmal nocturnal hemoglobinuria, a seemingly random overrepresentation of different Vβ families was observed. On average, we found expansion in 3 (of 22 examined) Vβ families per patient. When the contribution of individual Vβ families to the effector pool was examined, more striking Vβ skewing was found. Vβ-CDR3 size distribution was analyzed for the expanded Vβ families in isolated CD4+ and CD8+ populations; underrepresented Vβ families displayed more pronounced CDR3 skewing. Expanded CD4+Vβ subfamilies showed mostly a polyclonal CDR3 size distribution with only 38% of skewing in expanded Vβ families. In contrast, within overrepresented CD8+Vβ types, marked CDR3 skewing (82%) was seen, consistent with nonrandom expansion of specific CD8+ T-cell clones. No preferential expansion of particular Vβ families was observed, in relation to HLA-type. In patients examined after immunosuppressive therapy, an abnormal Vβ-distribution pattern was retained, but the degree of expansion of individual Vβ was lower. As Vβ skewing may correlate with relative Vβ size, oligoclonality in combination with numerical Vβ expansion can be applied to recognition of disease-specific T-cell receptors.

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.

A 320-kilobase artificial chromosome encoding the human HLA DR3-DQ2 MHC haplotype confers HLA restriction in transgenic mice.

MHC class II haplotypes control the specificity of Th immune responses and susceptibility to many autoimmune diseases. Understanding the role of HLA class II haplotypes in immunity is hampered by the lack of animal models expressing these genes as authentic cis-haplotypes. In this study we describe transgenic expression of the autoimmune prone HLA DR3-DQ2 haplotype from a yeast artificial chromosome (YAC) containing an intact similar320-kb region from HLA DRA to DQB2. In YAC-transgenic mice HLA DR and DQ gene products were expressed on B cells, macrophages, and dendritic cells, but not on T cells indicating cell-specific regulation. Positive selection of the CD4 compartment by human class II molecules was 67% efficient in YAC-homozygous mice lacking endogenous class II molecules (Abeta(null/null)) and expressing only murine CD4. A broad range of TCR Vbeta families was used in the peripheral T cell repertoire, which was also purged of Vbeta5-, Vbeta11-, and Vbeta12-bearing T cells by endogenous mouse mammary tumor virus-encoded superantigens. Expression of the HLA DR3-DQ2 haplotype on the Abeta(null/null) background was associated with normal CD8-dependent clearance of virus from influenza-infected mice and development of CD4-dependent protection from otherwise lethal infection with Salmonella typhimurium. HLA DR- and DQ-restricted T cell responses were also elicited following immunization with known T cell determinants presented by these molecules. These findings demonstrate the potential for human MHC class II haplotypes to function efficiently in transgenic mice and should provide valuable tools for developing humanized models of MHC-associated autoimmune diseases.

Discordance between T-cell receptor expression and effector function in mice infected with Histoplasma capsulatum.

V beta 10(+) and V beta 14(+) T cells were selectively increased 7 to 14 days following infection in the lungs of naive mice infected with Histoplasma capsulatum. Following secondary challenge of immune mice, V beta 1(+) and V beta 8.1(+) cells were sporadically increased. Elimination of V beta 10(+) and V beta 14(+) cells from naive mice did not alter the course of infection over a period of 21 days. Thus, overexpression of V beta families does not necessarily signify a key role in host defense.

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.

Overlap of direct and indirect alloreactive T-cell repertoires when MHC polymorphism is limited to the peptide binding groove

The immune response to allogeneic-MHC molecules can be divided into two pathways based on the nature of the antigen. In the direct pathway, T cells respond to intact allogeneic MHC molecules, while in the indirect pathway T cells respond to allo MHC-derived peptides presented by self-MHC. The T-cell repertoire used in the direct and indirect alloresponse have not been compared in the same alloantigen system. Here, HLA-DR transgenic mice are used to compare the repertoires of T cells that respond to the same alloantigen through either the direct or the indirect pathway. Separate direct and indirect DR1 anti-DR4 T-cell lines were generated and the T cell repertoire was analyzed by molecular methods. The same six Vβ families were involved in both direct and indirect cultures indicating a complete overlap in the Vβ gene usage. A partial overlap at the clonotype level was observed as two identical clonotypic TCRs were observed in both direct and indirect cultures. Interestingly, the T cells observed in both cultures were public as the same TCRs were identified in cultures developed from independent mice. These results raise the prospect that immune suppression of selected T cells during allo-transplantation can simultaneously modulate direct and indirect alloreactivities.

Immune pathophysiology of aplastic anemia.

Aplastic anemia (AA) remains an elusive disease. Its pathophysiology is not only fascinating by the seemingly simple findings of cytopenia and marrow hypoplasia, but may also contain key information to the understanding of other fundamental processes such as stem cell regeneration, evolution, and immune control of clonal diseases. Although measurements of blood counts provide an objective tool to assess the disease activity and response to the therapy, immune pathophysiology of AA, as inferred from the successes of immunosuppression, provides only few other clinical clues. Similarly, the current laboratory evidence remains mostly indirect. In spite of the recognition of immune pathways of hematopoietic inhibition and apoptosis in AA, the fundamental question about the nature of the antigen(s) inciting or maintaining the pathologic immune response that ultimately leads to bone marrow failure, remains open. However, recognition of the immune targets may aid in understanding not only the pathogenesis but also many of clinical associations and the late squelae of AA. For example, abnormal cells in AA and myelodysplastic syndrome (MDS) MDS may harbor inciting antigens but the immune response lacks selectivity. Clonal selection pressure may be a result of this process or alternatively, emergence of tolerance could lead to the establishment of abnormal hematopoiesis. Clonal proliferation of large granular lymphocytosis could represent an example of an exaggerated response to an immunodominant hematopoietic antigen. In addition to the traditional functional or phenotypic analysis, pathologic immune response in AA can be studied on molecular level by identifying and quantitating T cell clones based on the presence of unique variable B-chain CDR3 sequences. Detection of clonal expansion is based on the observation that in infections and autoimmune conditions, the presence of antigenic drive will lead to the expansion and overrepresentation of T cell clones recognizing this antigen. However, simple analysis of clonal representation is not sufficient to resolve the complex nature of the immune repertoire in the context of genetic and clinical heterogeneity. Therefore, we analyzed VB and CDR3 repertoire in CD4 and CD8 cells, activated or effector cell subsets. To distinguish truly expanded and likely immunodominant clones, we first studied VB distribution and cloned CDR3 sequences from expanded VB families. Identified clonotypic sequences can be used to design molecular tests to quantitate the strength of pathologic immune response. Clonotype sharing has been confirmed in patients with similar clinical features indicating presence of common antigens. In addition, quantitative analysis showed correlation with the therapy response. Persistence and patterns of clonotypes may be helpful in the classification of immune-mediated marrow failure based on the immune characteristics and will allow inferences into the inciting pathways.

T cell receptor (TCR) Vbeta gene usage in bronchoalveolar lavage and peripheral blood T cells from asthmatic and normal subjects.

T cells are thought to play an important regulatory role in asthma, but little is known about the T cell repertoire of the human lung or whether asthma is associated with any specific repertoire changes. Flow cytometry and MoAbs to TCR VB (TCRBV) families were used to quantify bronchoalveolar lavage (BAL) and blood T cells from normal and atopic individuals. Clonality was then assessed by polymerase chain reaction (PCR) amplification of cDNA and gene scanning using consensus and family-specific TCRBV primers and confirmed by sequence analysis. In addition, blood and BAL T cell populations were studied pre- and post-allergen challenge in four patients with allergic asthma. The majority of TCRBV families detected in blood by MoAb staining were also represented in BAL. While differences between BAL and blood populations were evident in each individual studied, these differences were not consistent between individuals or between CD4+ and CD8+ T cell subpopulations. These results are in broad agreement with other published studies, but in contrast to previous work we found a consistent difference between TCRBV7 family usage in blood and BAL in all individuals studied, and a consistently increased proportion of CD4+ BAL T cells bearing BV5S2/3 in asthmatics only. After allergen challenge, the pattern of TCRBV gene usage was largely unchanged as judged by flow cytometry. Gene scanning of PCR products generated from consensus VB primers revealed polyclonal lymphocyte populations in blood and BAL from all seven atopic individuals: in one normal tested polyclonal populations were found in blood and oligoclonal populations in BAL. Selected families amplified with family-specific primers BV5S2/3, BV6 and BV7 (chosen because of their predominance in BAL compared with blood) were more variable and revealed predominant polyclonal populations in blood and polyclonal or oligoclonal populations in BAL. In one asthmatic patient a clonal BV5S2 family was found in BAL. Following allergen challenge there were no significant changes in polyclonality/oligoclonality/clonality in three cases, but in one case a clonal BV5S2 population was found after challenge, that had not been evident beforehand. The lung T cell repertoire is thus broadly representative of blood T cells, but shows population differences that may result from response to persistent exposure to airborne antigens common to normal and atopic individuals. Oligoclonal TCRBV family expansion appears to be primarily lung-specific but independent of atopic asthma, although our challenge data in one case support the concept that clonal populations may follow local allergen challenge. These data are consistent with selection and amplification of specific T cell families in the lung in response to local antigenic exposure.