T-Cell receptor Vbeta repertoire CDR3 length diversity differs within CD45RA and CD45RO T-cell subsets in healthy and human immunodeficiency virus-infected children

The T-cell receptor (TCR) CDR3 length heterogeneity is formed during recombination of individual Vbeta gene families. We hypothesized that CDR3 length diversity could be used to assess the fundamental differences within the TCR repertoire of CD45RA and CD45RO T-cell subpopulations. By using PCR-based spectratyping, nested primers for all 24 human Vbeta families were developed to amplify CDR3 lengths in immunomagnetically selected CD45RA and CD45RO subsets within both CD4(+) and CD8(+) T-cell populations. Umbilical cord blood mononuclear cells or peripheral blood mononuclear cells obtained from healthy newborns, infants, and children, as well as human immunodeficiency virus (HIV)-infected children, were analyzed. All T-cell subsets from newborn and healthy children demonstrated a Gaussian distribution of CDR3 lengths in separated T-cell subsets. In contrast, HIV-infected children had a high proportion of predominant CDR3 lengths within both CD45RA and CD45RO T-cell subpopulations, most commonly in CD8(+) CD45RO T cells. Sharp differences in clonal dominance and size distributions were observed when cells were separated into CD45RA or CD45RO subpopulations. These differences were not apparent in unfractionated CD4(+) or CD8(+) T cells from HIV-infected subjects. Sequence analysis of predominant CDR3 lengths revealed oligoclonal expansion within individual Vbeta families. Analysis of the CDR3 length diversity within CD45RA and CD45RO T cells provides a more accurate measure of disturbances in the TCR repertoire than analysis of unfractionated CD4 and CD8 T cells.     

Vγ9Vδ2 T Cells in Human Legionellosis

In humans, expansion of circulating Vγ9Vδ2 T cells seems to be a pathophysiological denominator shared by protozoan and intracellular bacterial diseases. The assumption was tested here on legionellosis, a condition conforming to the category but not yet described with respect to γδ T cells. Levels of Vγ9Vδ2 T cells in peripheral blood were measured at various intervals in 14 subjects undergoing a Pontiac fever-like disease, shown by serological investigation to be caused by Legionella micdadei. In samples obtained 4 to 6 days after the onset of the disease, the mean percentage (± the standard deviation) of Vγ9Vδ2⁺ T cells among CD3⁺ cells was 1.0% ± 0.5%, compared to 5.0% ± 3.9% in healthy control subjects (P < 0.001). Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset, mean peak levels were as high as ≈15%. During the next 6 months, values slowly declined, although without reaching the normal range. Percentages of γδ⁺ T cells expressing tumor necrosis factor alpha or gamma interferon in response to phorbol myristate acetate were assayed in vitro. At 14 to 16 days after the onset of disease, the expression of both cytokines was increased (P < 0.01), whereas at 5 to 7 weeks, the expression of tumor necrosis factor alpha was decreased (P < 0.05), possibly reflecting modulation of an inflammatory response. In conclusion, Pontiac fever was found to be associated with a pronounced and long-lasting expansion of Vγ9Vδ2 T cells, implying that the subset may also be pathophysiologically important in a mild and transient form of intracellular bacterial diseases. Surprisingly, the expansion was preceded by a depletion of circulatory Vγ9Vδ2 T cells. Possibly, Vγ9Vδ2 T cells are initially recruited to a site of infection before they expand in response to antigen and occur in high numbers in blood.     

Angiocentric CD3+ T-Cell Infiltrates in Human Immunodeficiency Virus Type 1-Associated Central Nervous System Disease in Children

A significant proportion of brain tissue specimens from children with AIDS show evidence of vascular inflammation in the form of transmural and/or perivascular mononuclear-cell infiltrates at autopsy. Previous studies have shown that in contrast to inflammatory lesions observed in human immunodeficiency virus type 1 (HIV-1) encephalitis, in which monocytes/macrophages are the prevailing mononuclear cells, these infiltrates consist mostly of lymphocytes. Perivascular mononuclear-cell infiltrates were found in brain tissue specimens collected at autopsy from five of six children with AIDS and consisted of CD3⁺ T cells and equal or greater proportions of CD68⁺ monocytes/macrophages. Transmural (including endothelial) mononuclear-cell infiltrates were evident in one patient and comprised predominantly CD3⁺ T cells and small or, in certain vessels, approximately equal proportions of CD68⁺ monocytes/macrophages. There was a clear preponderance of CD3⁺ CD8⁺ T cells on the endothelial side of transmural infiltrates. In active lesions of transmural vasculitis, CD3⁺ T-cell infiltrates exhibited a distinctive zonal distribution. The majority of CD3⁺ cells were also CD8⁺ and CD45RO⁺. Scattered perivascular monocytes/macrophages in foci of florid vasculitis were immunoreactive for the p24 core protein. In contrast to the perivascular space, the intervening brain neuropil was dominated by monocytes/macrophages, microglia, and reactive astrocytes, containing only scant CD3⁺ CD8⁺ cells. Five of six patients showed evidence of calcific vasculopathy, but only two exhibited HIV-1 encephalitis. One patient had multiple subacute cerebral and brainstem infarcts associated with a widespread, fulminant mononuclear-cell vasculitis. A second patient had an old brain infarct associated with fibrointimal thickening of large leptomeningeal vessels. These infiltrating CD3⁺ T cells may be responsible for HIV-1-associated CNS vasculitis and vasculopathy and for endothelial-cell injury and the opening of the blood-brain barrier in children with AIDS.     

Strict Adherence to a Common Rank Order of T-Cell Receptor Vβ Usage in Human Leucocyte Antigen Disparate Individuals

In order to investigate the T-cell receptor (TCR) Vβ usage in different T-cell subsets, the authors performed flow cytometric analyses using a large panel of TCR Vβ-specific monoclonal antibodies on CD4⁺, CD8⁺ CD28⁺ and CD8⁺ CD28^? T cells from 15 random blood donors, six umbilical cords and seven human leucocyte antigen (HLA) identical non-twin sibling pairs. The authors found that the proportion of T cells expressing each Vβ gene product was similar within CD4⁺ and CD8⁺ CD28⁺ T cells from all samples studied. For these T-cell subsets a rank order of Vβ usage could be identified which was adhered to by all donors. In contrast, within CD8⁺ CD28^? T cells a wide variation of Vβ usage was found between individuals, and no rank order correlation could be detected. Members of HLA identical sibling pairs were found to be no more similar in their usage of Vβ gene products than pairs of HLA disparate random blood donors. Groups of individuals sharing HLA antigens were no different from the groups not sharing such antigens in their usage of Vβ segments. The results suggest that HLA polymorphisms play no more than a minor part in determining TCR Vβ usage.     

Self-activation of Vγ9Vδ2 T cells by exogenous phosphoantigens involves TCR and butyrophilins

The high cytotoxic activity of Vγ9Vδ2 T lymphocytes against tumor cells makes them useful candidates in anticancer therapies. However, the molecular mechanism of their activation by phosphoantigens (PAgs) is not completely known. Many studies have depicted the mechanism of Vγ9Vδ2 T-cell activation by PAg-sensed accessory cells, such as immune presenting cells or tumor cells. In this study, we demonstrated that pure resting Vγ9Vδ2 T lymphocytes can self-activate through exogenous PAgs, involving their TCR and the butyrophilins BTN3A1 and BTN2A1. This is the first time that these three molecules, concurrently expressed at the plasma membrane of Vγ9Vδ2 T cells, have been shown to be involved together on the same and unique T cell during PAg activation. Moreover, the use of probucol to stimulate the inhibition of this self-activation prompted us to propose that ABCA-1 could be implicated in the transfer of exogenous PAgs inside Vγ9Vδ2 T cells before activating them through membrane clusters formed by γ9TCR, BTN3A1 and BTN2A1. The self-activation of Vγ9Vδ2 T cells, which leads to self-killing, can therefore participate in the failure of γδ T cell-based therapies with exogenous PAgs and should be taken into account.     

Concentrations of Circulating β-Chemokines Do Not Correlate with Viral Load in Human Immunodeficiency Virus-Infected Individuals

The CC or β-chemokines MIP-1α, MIP-1β, and RANTES are the primary components of human immunodeficiency virus type 1 (HIV-1)-suppressive soluble factors in vitro. We studied the relationship between the concentrations of MIP-1α, MIP-1β, and RANTES in plasma and HIV viral load in HIV-infected subjects. The HIV-positive patient group (n = 140) had significantly lower concentrations of all three β-chemokines (MIP-1α, P < 0.0005; MIP-1β, P < 0.005; RANTES, P < 0.0005) than the control group (n = 58 for MIP-1α, n = 27 for MIP-1β, and n = 59 for RANTES). In addition, we divided the patient group into three subgroups (high, moderate, and low) based on the number of HIV-1 RNA copies in the plasma (as measured by quantitative HIV RNA PCR). Again, all three subgroups had significantly lower concentrations of the β-chemokines than the HIV-negative control group. However, there was no significant difference in plasma β-chemokine concentrations among the three subgroups within the patient group (P < 0.3). Although our results demonstrate that HIV-infected individuals had significantly lower concentrations of circulating β-chemokines than healthy uninfected control subjects, we found no correlation between the concentrations of β-chemokines in plasma and HIV-1 viral load in HIV-infected individuals.     

Inverted Vδ1/Vδ2 ratio within the T cell receptor (TCR)-γδ T cell population in peripheral blood of heart transplant recipients

We investigated the levels of TCR-γδ T cells and their subpopulations Vδ1 and Vδ2 in the peripheral blood lymphocytes (PBL) of 28 heart transplant (HTx) patients. Patients (n = 10) receiving cyclosporin A (CsA) for treatment of a nephrotic syndrome (NS) and 10 healthy individuals served as controls. There was no difference in levels of TCR-γδ T cells between the different groups. However, an elevated proportion of Vδ1⁺γδ T cells was found in the PBL of HTx patients, especially when these cells were present in their graft-infiltrating lymphocyte (GIL) cultures. Vδ1⁺γδ T cells of HTx patients showed normal expression of CD45RO and lacked the activation markers CD25 and HLA-DR. After expanding in IL-2-containing medium, PBL cultures of HTx patients more often were dominated by Vδ1 cells than PBL cultures of controls, in which Vδ2 cells were predominantly grown. The aberrant composition of the TCR-γδ population in HTx patients was not a result of immunosuppressive medication, since the proportion Vδ1⁺γδ T cells was normal in the PBL of the NS patients receiving a similar dose of CsA. It is postulated that long-term antigenic stimulation by the graft, at low level, might be responsible for the altered composition of the γδ pool in the HTx patients. Since no donor HLA-specific γδ T cells have been detected, other ligands, such as heat shock proteins, may be involved.     

Comparative Analysis of Human Cytomegalovirus-Specific CD4+ T-Cell Frequency and Lymphoproliferative Response in Human Immunodeficiency Virus-Positive Patients

Evaluation of human cytomegalovirus (HCMV)-specific T-helper immunity could contribute in optimizing anti-HCMV therapy in human immunodeficiency virus (HIV)-infected patients. Testin the lymphoproliferative response (LPR) is the standard technique used to evaluate T-helper response, but its use in the routine diagnostic laboratory setting can be problematic. The most promising new alternative technique is the determination of HCMV-specific CD4⁺ T-cell frequency by flow cytometry detection of intracellular cytokine production after short-term antigen-specific activation of peripheral blood mononuclear cells. HCMV-specific LPR and CD4⁺ T-cell frequency were compared in a group of 78 blood samples from 65 HIV-infected patients. The results showed concordance in 80.7% of samples. In addition, comparative analysis of sequential blood samples from 13 HIV-infected patients showed that while in about half of patients the T-helper HCMV-specific immune response remained stable during highly active antiretroviral therapy (HAART), in the other half declining levels of the HCMV-specific CD4⁺-mediated immune response were determined by either one or both assays. In conclusion, our data suggest that the determination of HCMV-specific CD4⁺ T-cell frequency can be considered a valuable alternative to the LPR test for the detection of HCMV-specific T-helper response in HIV-infected patients. It could facilitate wider screening of anti-HCMV T-helper activity in HIV-infected patients, with potential benefits for clinicians in deciding strategies of discontinuation or maintenance of anti-HCMV therapy.     

CD45RA expression on γδ and αβ T cells emigrating from the fetal and postnatal thymus

We have compared the expression of CD45RA on αβ and γδ T cells emigrating from the fetal and postnatal thymus. The fetal and postnatal thymus export both CD45RA⁺ and CD45RA^- T cells. The number of γδ⁺CD45RA⁺ T cells was remarkably constant regardless of stage of ontogeny or T cell maturity. Around 5--8% of γδ thymic emigrants, thymocytes and peripheral blood lymphocytes expressed CD45RA in both fetal and postnatal animals. In contrast to γδ T cells, up to one quarter of both fetal and postnatal αβ emigrants expressed CD45RA. Post-thymic maturation of CD45RA expression on αβ emigrants, which occurred both before and after birth, appeared to be antigen independent.     

Allogeneic Vγ9Vδ2 T-cell immunotherapy exhibits promising clinical safety and prolongs the survival of patients with late-stage lung or liver cancer

Vγ9Vδ2 T cells are promising candidates for cellular tumor immunotherapy. Due to their HLA-independent mode of action, allogeneic Vγ9Vδ2 T cells can be considered for clinical application. To apply allogeneic Vγ9Vδ2 T cells in adoptive immunotherapy, the methodology used to obtain adequate cell numbers with optimal effector function in vitro needs to be optimized, and clinical safety and efficacy also need to be proven. Therefore, we developed a novel formula to improve the expansion of peripheral γδ T cells from healthy donors. Then, we used a humanized mouse model to validate the therapeutic efficacy of expanded γδ T cells in vivo; furthermore, the expanded γδ T cells were adoptively transferred into late-stage liver and lung cancer patients. We found that the expanded cells possessed significantly improved immune effector functions, including proliferation, differentiation, and cancer cell killing, both in vitro and in the humanized mouse model. Furthermore, a phase I clinical trial in 132 late-stage cancer patients with a total of 414 cell infusions unequivocally validated the clinical safety of allogeneic Vγ9Vδ2 T cells. Among these 132 patients, 8 liver cancer patients and 10 lung cancer patients who received ≥5 cell infusions showed greatly prolonged survival, which preliminarily verified the efficacy of allogeneic Vγ9Vδ2 T-cell therapy. Our clinical studies underscore the safety and efficacy of allogeneic Vγ9Vδ2 T-cell immunotherapy, which will inspire further clinical investigations and eventually benefit cancer patients.     

The Effects of Mitogens, IL-2 and Anti-CD3 Antibody on the T-Cell Receptor Vβ Repertoire

Phytohaemagglutinin (PHA), Concanavalin A (Con A), interleukin-2 (IL-2), and monoclonal antibodies to CD3 (CD3MoAbs) are used for the assessment of the T-cell receptor (TCR) BV gene family expression in autoimmune disorders and multiple sclerosis, and to produce clones for assessment of cytokine profiles in progressive human immunodeficiency virus infection. The authors examined the effects of these stimulants on the TCR Vβ repertoire of resting and blastic CD4⁺ and CD8⁺ normal human peripheral blood lymphocytes, using three-colour cytofluorometry and a panel of anti-TCR Vβ monoclonal antibodies. IL-2 was associated with an increased percentage of blastic CD4⁺ cells expressing V_β5.1 (from median of 3.7% to 8.0%, P  = 0.0002) and blastic CD8⁺ cells expressing V_β5.3 (1.0 to 1.5%, P  = 0.0039). CD3MoAb caused a slight increase in V_β6.7 + blastic CD4⁺ cells (4.5 to 6.9%, P  = 0.0078). PHA did not alter the Vβ repertoire of blastic cells. Con A caused skewing in CD8⁺ blastic cells, toward expression of V_β5.2/5.3 (3.1 to 8.1%) and V_β5.3 (0.8 to 4.8%) ( P  = 0.0020). Thus, IL-2 stimulation causes slight alterations in the Vβ repertoire that should be taken into account in certain research settings. Con A produced skewing in CD8⁺ blastic cells suggesting that, in the presence of CD8, either Con A binds selectively to certain Vβ or the three-dimensional complex created by Con A's binding to other T-cell surface molecules induces preferential V_β5 stimulation.     

Processing of Mycobacterium tuberculosis Bacilli by Human Monocytes for CD4+ αβ and γδ T Cells: Role of Particulate Antigen

Mycobacterium tuberculosis readily activates both CD4⁺ and Vδ2⁺ γδ T cells. Despite similarity in function, these T-cell subsets differ in the antigens they recognize and the manners in which these antigens are presented by M. tuberculosis-infected monocytes. We investigated mechanisms of antigen processing of M. tuberculosis antigens to human CD4 and γδ T cells by monocytes. Initial uptake of M. tuberculosis bacilli and subsequent processing were required for efficient presentation not only to CD4 T cells but also to Vδ2⁺ γδ T cells. For γδ T cells, recognition of M. tuberculosis-infected monocytes was dependent on Vδ2⁺ T-cell-receptor expression. Recognition of M. tuberculosis antigens by CD4⁺ T cells was restricted by the class II major histocompatibility complex molecule HLA-DR. Processing of M. tuberculosis bacilli for Vδ2⁺ γδ T cells was inhibitable by Brefeldin A, whereas processing of soluble mycobacterial antigens for γδ T cells was not sensitive to Brefeldin A. Processing of M. tuberculosis bacilli for CD4⁺ T cells was unaffected by Brefeldin A. Lysosomotropic agents such as chloroquine and ammonium chloride did not affect the processing of M. tuberculosis bacilli for CD4⁺ and γδ T cells. In contrast, both inhibitors blocked processing of soluble mycobacterial antigens for CD4⁺ T cells. Chloroquine and ammonium chloride insensitivity of processing of M. tuberculosis bacilli was not dependent on the viability of the bacteria, since processing of both formaldehyde-fixed dead bacteria and mycobacterial antigens covalently coupled to latex beads was chloroquine insensitive. Thus, the manner in which mycobacterial antigens were taken up by monocytes (particulate versus soluble) influenced the antigen processing pathway for CD4⁺ and γδ T cells.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is spread readily from person to person by inhalation of aerosolized mycobacteria (8). A hallmark of M. tuberculosis infection is the ability of most healthy individuals to control the infection by mounting an acquired immune response, in which antigen-specific T cells and mononuclear phagocytes arrest the growth of M. tuberculosis bacilli and maintain control over dormant bacilli within granulomas (reviewed in reference 25). This protective cellular immune response results in conversion of the tuberculin skin test from negative to positive and probably in increased resistance to reinfection with tubercle bacilli.CD4⁺ αβ-T-cell-receptor (αβ TCR)-bearing T cells (CD4⁺ T cells) are readily activated by mycobacterial antigens and have a dominant role in the protective immune response to M. tuberculosis in humans (2, 34). These CD4⁺ T cells not only secrete cytokines but also serve directly as cytotoxic effector cells against M. tuberculosis-infected macrophages (6). In addition to CD4⁺ T cells, M. tuberculosis antigens activate other human T-cell subsets such as γδ TCR⁺ T cells (γδ T cells) (15, 16, 18). Vδ2⁺ and Vγ9⁺ γδ T cells are particularly responsive to live M. tuberculosis (15). A role for both γδ and CD4⁺ T cells in protective immunity to acute M. tuberculosis infection has been demonstrated in murine models (20, 21, 26, 27). A recent study of humans suggests that Vγ9⁺ and Vδ2⁺ γδ T-cell numbers and function are reduced in tuberculosis patients (23).Functional comparisons of human CD4⁺ and γδ T-cell responses of healthy tuberculin-positive persons demonstrate that both T-cell subsets have similar cytotoxic effector functions for M. tuberculosis-infected monocytes and produce large amounts of gamma interferon (IFN-γ), with γδ T cells being slightly more efficient producers of IFN-γ than CD4⁺ T cells (37). Despite similarities in function, these two T-cell subsets differ in the mycobacterial antigens recognized by their TCRs and the manners in which antigens are presented to them by M. tuberculosis-infected mononuclear phagocytes. CD4⁺ T cells recognize a wide diversity of mycobacterial peptides in the context of class II major histocompatibility complex (MHC) molecules, which include secreted as well as somatic antigens (6, 13, 33, 37). In contrast, Vγ9⁺ and Vδ2⁺ γδ T cells, the dominant γδ TCR subsets activated by M. tuberculosis, recognize mycobacterial antigens in a non-MHC-restricted manner and the repertoire of antigens includes small phosphate-containing antigens such as TUBag’s (5, 9, 19, 22, 29, 36).Both blood monocytes and alveolar macrophages infected with M. tuberculosis are efficient antigen-presenting cells for mycobacterial antigen-specific CD4⁺ and γδ T cells (1, 5). However, little is known about how M. tuberculosis-infected mononuclear phagocytes process antigens for these two T-cell subsets. M. tuberculosis bacilli are taken up by mononuclear phagocytes through a variety of surface receptors, including complement receptor 4, mannose receptor, and complement receptor 3 (17, 31, 32). Within mononuclear phagocytes, the mycobacteria reside within phagosomes and modulate the phagosome by preventing fusion with acidic lysosomal compartments (7). Although the vacuolar membranes surrounding the phagosome acquire endosomal markers, the vesicular proton ATPase is actively excluded, resulting in an elevated pH of 6.3 to 6.5 compared to the normal lysosomal pH of 4.5 (7, 35). The elevated pH in the phagosome does not appear to inhibit the ability of mycobacterial antigens to be processed and presented to CD4⁺ and Vδ2⁺ γδ T cells. This study was undertaken to gain insight into the mechanisms used by monocytes infected with live M. tuberculosis bacilli to process mycobacterial antigens for presentation to both CD4⁺ and γδ T cells.     

Integrins αvβ3 and α5β1 Mediate Attachment of Lyme Disease Spirochetes to Human Cells

Borrelia burgdorferi (sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin α_IIbβ₃ was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferi sensu stricto, B. garinii, and B. afzelii). Here we show that B. burgdorferi also recognizes the widely expressed integrins α_vβ₃ and α₅β₁, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified α_vβ₃ and α₅β₁. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectious B. burgdorferi strain N40 to purified α_vβ₃ and α₅β₁ was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to α_vβ₃ was also shown by using a transfected cell line that expresses this receptor but not α_IIbβ₃. Attachment of B. burgdorferi N40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both α₅β₁ and α_vβ₃. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.     

Immunophenotypic Analysis of the TCR-Vβ Repertoire in 98 Persistent Expansions of CD3+/TCR-αβ+ Large Granular Lymphocytes : Utility in Assessing Clonality and Insights into the Pathogenesis of the Disease

At present, a major challenge in the initial diagnosis of leukemia of large granular lymphocytes (LGLs) is to establish the clonal nature of the expanded population. In the present study we have analyzed by flow cytometry immunophenotyping the TCR-Vbeta repertoire of 98 consecutive cases of persistent expansions of CD4(+) or CD8(+bright) CD3(+)/TCR-alphabeta(+) LGLs and compared the results with those obtained in molecular studies of TCR-beta gene rearrangements. Fifty-eight cases were considered to be monoclonal in molecular studies whereas in the remaining 40 cases there was no evidence for monoclonality (11 cases were considered oligoclonal and 29 polyclonal). The TCR-Vbeta repertoire was biased to the preferential use of one or more TCR-Vbeta families in 96% of cases, a total of 124 TCR-Vbeta expansions being diagnosed: one TCR-Vbeta expansion in 71 cases and two or more TCR-Vbeta expansions in 23 cases. The highest TCR-Vbeta expansion observed in each case was higher among monoclonal (74 +/- 19%) as compared to nonmonoclonal cases (24 +/- 14%) (P = 0.001), as did the fraction of LGLs that exhibited a TCR-Vbeta-restricted pattern (86 +/- 16% and 42 +/- 23%, respectively; P = 0.0001); by contrast, the proportion of cases displaying more than one TCR-Vbeta expansion was higher in the latter group: 7% versus 48%, respectively (P = 0.001). Results obtained in oligoclonal cases were intermediate between those obtained in polyclonal and monoclonal cases and similar results were observed for CD4(+) as for CD8(+bright) T-cell expansions. TCR-Vbeta families expressed in CD8(+bright) T-cell-LGL proliferations showed a pattern of distribution that mimics the frequency at which the individual TCR-Vbeta families are represented in normal peripheral blood T cells. Assuming that a given proliferation of LGLs is monoclonal whenever there is an expansion of a given TCR-Vbeta family of at least 40% of the total CD4(+) or CD8(+bright) T-cell compartment, we were able to predict clonality with a sensitivity of 93% and a specificity of 80%. By increasing the cut-off value to 60%, sensitivity and specificity were of 81% and 100%. In summary, our results suggest that flow cytometry immunophenotypic analysis of the TCR-Vbeta repertoire is a powerful screening tool for the assessment of T-cell clonality in persistent expansions of TCR-alphabeta(+) LGLs.