Combination antiretroviral therapy results in a rapid increase in T cell receptor variable region beta repertoire diversity within CD45RA CD8 T cells in human immunodeficiency virus-infected children

Human immunodeficiency virus (HIV) type 1 disrupts the T cell receptor (TCR) variable region (V) beta repertoire in CD8 T cells by impairing thymic capacity and skewing postthymic cellular maturation. The TCR repertoire was examined using spectratyping of CDR3 length diversity within CD45RA and CD45RO CD8 T cells in HIV-infected and healthy children. In healthy children, CDR3 lengths displayed Gaussian distribution in both CD45RA and CD45RO subsets. Vbeta families in HIV-infected children displayed a large proportion of perturbations in both subsets. High virus load and advanced immunosuppression correlated with increased perturbations within CD45RA but not CD45RO CD8 T cells. After therapy and virus suppression, there was rapid reestablishment of Gaussian distributions in CD45RA cells. HIV-1-induced disruption of TCR diversity within CD45RA CD8 T cells correlates with disease progression. Suppression of viral replication by treatment results in the rapid correction of TCR diversity in this CD8 subset because of emergence of new T cells from the thymus.     

Antigen-recognition sites of micromanipulated T cells in patients with acquired aplastic anemia

OBJECTIVE: Acquired aplastic anemia (AA) is a rare disorder characterized by pancytopenia and hypocellular bone marrow. Though experimental and clinical data suggest that AA represents a T cell-mediated disease, neither the immune response nor the nature of inciting antigen(s) have been characterized so far. The identification of a restricted T cell repertoire by PCR techniques in total lymphocyte populations supports an antigen-driven T cell response. In order to investigate the clonal composition, we analyzed the gene rearrangements of the T cell receptor (TCR) variable beta chain (Vbeta) at the single-cell level. PATIENTS AND METHODS: CD3(+) T lymphocytes were micromanipulated from peripheral blood and bone marrow samples of 8 AA patients and healthy controls. Subsequently amplified VDJ gene segments of the TCRVbeta chain were analyzed for functional rearrangements. More than 500 functionally rearranged TCR loci were studied for Vbeta/Jbeta gene segment usage and molecular composition of the complementary-determining region 3 (CDR3). RESULTS: In comparison to healthy controls, the Vbeta sequences confirmed a highly restricted T cell repertoire in AA patients at the single-cell level. Both in bone marrow and peripheral blood a predominance of Vbeta13 and Jbeta2S7 was observed. Furthermore, individual clonal T-cell expansion was identified in the majority of patients. However, deduced CDR3 amino acid sequences revealed a high variability without common motifs among the 8 patients. CONCLUSION: Individual clonal T-cell expansion with high diversity of the antigen-binding sites among the analyzed patients argues for the predominance of private inciting epitopes in AA.     

Structured antiretroviral treatment interruptions in chronically HIV-1-infected subjects

The risks and benefits of structured treatment interruption (STI) in HIV-1-infected subjects are not fully understood. A pilot study was performed to compare STI with continuous highly active antiretroviral therapy (HAART) in chronic HIV-1-infected subjects with HIV-1 plasma RNA levels (VL) <400 copies per ml and CD4(+) T cells >400 per microl. CD4(+) T cells, VL, HIV-1-specific neutralizing antibodies, and IFN-gamma-producing HIV-1-specific CD8(+) and CD4(+) T cells were measured in all subjects. STIs of 1-month duration separated by 1 month of HAART, before a final 3-month STI, resulted in augmented CD8(+) T cell responses in all eight STI subjects (P = 0.003), maintained while on HAART up to 22 weeks after STI, and augmented neutralization titers to autologous HIV-1 isolate in one of eight subjects. However, significant decline of CD4(+) T cell count from pre-STI level, and VL rebound to pre-HAART baseline, occurred during STI (P = 0.001 and 0.34, respectively). CD4(+) T cell counts were regained on return to HAART. Control subjects (n = 4) maintained VL <400 copies per ml and stable CD4(+) T cell counts, and showed no enhancement of antiviral CD8(+) T cell responses. Despite increases in antiviral immunity, no control of VL was observed. Future studies of STI should proceed with caution.     

T cell expansions in lymph nodes and peripheral blood in HIV-1-infected individuals: effect of antiretroviral therapy.

OBJECTIVE: To evaluate dynamics in CD8 T cell expansions during highly active antiretroviral therapy (HAART). DESIGN: Various T cell subsets were isolated from blood and lymph nodes and analysed for T cell receptor (TCR) diversity. METHODS: TCR complementarity determining region 3 (CDR3) spectratyping and single-strand conformation polymorphism (SSCP) analyses were performed in combination with sequencing to assess clonality of the subsets. RESULTS: Strongly skewed CDR3 patterns in total CD8 cells and the CD8 subsets CD45RO+CD27+ and CD45RO-CD27+ showed substantial dynamics in dominant CDR3 sizes, resulting in relative improvement of CDR3 size diversity in the first months of therapy. During sustained treatment, TCR diversity changed only moderately. SSCP profiles confirmed oligoclonality of TCR CDR3 perturbations. Various dominant CDR3 sizes for CD4 and CD8 T cells present in lymph nodes, but not in peripheral blood mononuclear cells, before the start of therapy emerged in peripheral blood early during therapy. CONCLUSIONS: HAART induces substantial changes in CD8 TCR diversity, eventually resulting in improvement of the repertoire. Clonal expansions observed in lymph nodes before therapy were observed in peripheral blood after therapy, suggesting that recirculation of CD4 and CD8 T cells from lymph nodes contributes to the early T cell repopulation. Decreased immune activation and possibly naive T cell regeneration subsequently decreased clonal expansions and perturbations in the CD8 TCR repertoire.     

The TCR Vbeta repertoire usage of T-cells from cord blood induced by chronic myelogenous leukemia associated antigen

Understanding the clonality and restricted usage of the TCR Vbeta repertoire of expanded T-cells induced by the chronic myelogenous leukemia (CML) associated antigen may be useful in helping design new immunotherapeutic strategies specifically for CML. T-cells from cord blood that had been stimulated by different stimulators (IL-2, PHA, CML cells, K562 cells and bcr-abl peptide) were amplified in vitro by liquid T-cell culture and the mixed lymphocyte and tumor cell culture (MLTC) method. By using the RT-PCR, the CDR3 segments of 24 variable region genes of TCR beta was analyzed in T-cells from 22 cases of cord blood before and after T-cell culture, to observe the usage of TCR Vbeta repertoire. The PCR products were further labeled with fluorescence and analyzed by the Genescan technique for the CDR3 size, to evaluating clonality of the detectable TCR Vbeta T-cells. Only a part of 24 Vbeta subfamily T-cells (3-15 subfamilies) could be detected, however, all of the 24 TCR Vbeta subfamily of T-cells were detected after in vitro culture with PHA or IL-2+anti-CD3 antibody. The number of expressed TCR Vbeta subfamilies was gradually reduced by prolonging the time of culture with CML-associated antigens. The restricted expression of TCR Vbeta subfamilies and oligoclonal expansion of Vbeta21 T-cells were found in cultured T-cells induced by CML cells, K562 cells or bcr-abl peptide. In conclusion, T-cells from cord blood may have the potential capability of proliferation in different TCR Vbeta subfamily T-cells, and the ability for restricted expression and clonal expansion, when T-cells were induced by CML associated antigen.     

Contribution of T cell receptor affinity to overall avidity for virus-specific CD8+ T cell responses

Prior analysis has characterized the clonal characteristics of effector CD8(+) T cells specific for the prominent influenza A virus nucleoprotein (NP) and acid polymerase (PA) peptides presented by H2D(b). Using a single-cell approach and determination of CDR3beta profiles, a limited, predominantly "public" repertoire was found for CD8(+)D(b)NP(366)(+)Vbeta8.3+ cells, whereas diverse and "private" T cell antigen receptor (TCR)beta clonotypes were typical of the CD8(+)D(b)PA(224)(+)Vbeta7+ response. This single-cell approach has now been used to relate the contributions of particular clonotypes (or affinities) to high-avidity TCRs, as defined by binding under conditions of limiting tetramer availability. At least by the measure of CDR3beta usage, no difference could be found between total and high-avidity populations in the spectrum of TCR-pMHC affinities throughout the limited, and relatively public, CD8(+)D(b)NP(366)(+)Vbeta8.3+ populations. Conversely, the more even (by clone size), diverse, and private CD8(+)D(b)PA(224)(+)Vbeta7+ response was characterized by the clear partitioning of the largest T cell clones in the high-avidity compartment. These results suggest that the relatively constrained CD8(+)D(b)NP(366)(+)Vbeta8.3+ set utilizes a relatively narrow range of affinities, whereas the broader CD8(+)D(b)PA(224)(+)Vbeta7+ response is induced at a range of TCR-pMHC affinities. Thus, whereas TCR sequence (or affinity) appears to contribute substantially to the avidity profile of diverse virus-specific CD8+ populations, other mechanisms may be prominent where the TCR spectrum is more limited.     

Detection of peripheral HIV-1-specific memory B cells in patients untreated or receiving highly active antiretroviral therapy

OBJECTIVES: To examine whether polyclonal activation of circulating B cells, in a process that involves CD40-CD40 ligand and cytokine interactions, could induced HIV-1-specific memory B cells to synthesize HIV-1-specific antibodies. METHODS: B cells from 26 HIV-1-infected patients were cultured with a CD40L-transfected cell line plus interleukins 2 and 10 and tested for their secretion of HIV-1- and Toxoplasma gondii-specific antibodies. RESULTS: In vitro activated B lymphocytes from HIV-1-infected patients secreted anti-HIV-1-specific antibodies. B cells from HIV-1-infected patients as well as those from controls chronically infected by T. gondii synthesized T. gondii-specific antibodies. HIV-1-specific IgG-, IgA- or IgM-secreting B cells represented approximately 1 x 10(-4) to 1 x 10(-5) of total circulating B cells and 1 x 10(-2) to 1 x 10(-3) of immunoglobulin-secreting cells. HIV-1-specific memory B cells were found in 9/9 untreated patients and in 8/17 patients receiving highly active antiretroviral therapy (HAART). The other nine patients showed a normal CD40-CD40L B cell response and six of them produced T. gondii-specific antibody B cells. The follow-up of seven patients indicated that HIV-1-specific memory B cells became undetectable after 8 to 46 months of HAART, whereas T. gondii-specific memory B cells persisted in parasite coinfected patients. CONCLUSIONS: Circulating memory HIV-1-specific B cells were detected in untreated patients and in about half of the patients taking HAART, suggesting that persistent low-level ongoing viral replication is not sufficient to maintain HIV-1-specific memory B cells.     

Analysis of the CDR3 region of alpha/beta T‐cell receptors (TCRs) and TCR BD gene double‐stranded recombination signal sequence breaks end in peripheral blood mononuclear cells of T‐lineage acute lymphoblastic leukemia

Recently, numerous reports have highlighted the restriction of the CDR3 length of T‐cell receptor (TCR) beta chain in T‐cells infiltrating solid tumors and hematological malignancies. However, these studies ignored the restriction of CDR3 length of TCR alpha chain and few of them attempted to reveal the mechanisms of the oligo‐clonal expansion of T cells in the tumors. The primary aims of this study were twofold to: (i) analyze the CDR3 length of TCR alpha and beta chain in peripheral blood mononuclear cells of T‐lineage acute lymphoblastic leukemia (T‐ALL); and (ii) discover the relationship between the clonality of T cells and the process of TCR rearrangement in peripheral T cells. To this end, we investigated the TCR BV and TCR AV family spectratypes of two T‐ALL patients and healthy controls using the immunoscope spectratyping technique. We found that the spectratypes exhibited a Gaussian distribution in healthy controls. However, the TCR repertoires of the two patients were highly restricted in the number of different TCR BV and TCR AV family members present. Furthermore, we found that the peripheral blood mononuclear cells (PBMC) of two T‐ALL patients had the recombination signal sequence (RSS) 5′‐ and 3′‐breaks end in the TCR BD2 gene using a specialized ligation‐mediated polymerase chain reaction, implying the ongoing recombination of the TCR beta gene. Analysis of the particular CDR3 length of TCR alpha/beta T cells might be helpful for further study of the individualized therapy of T‐ALL. This information will also be helpful in exploring new immunological pathogenesis and facilitating the design of a T‐ALL vaccine, as well as in improving our understanding of healthy human T‐cell development.     

DNA immunization with HIV early genes in HIV type 1-infected patients on highly active antiretroviral therapy

The aim of this study was to evaluate the immunological responses induced by DNA plasmids containing HIV regulatory genes administered in combination in HIV-1-infected patients with pretreatment with highly active antiretroviral treatment (HAART). The study is a double-blind, randomized, and placebo-controlled study, including 15 asymptomatic HIV-1-infected patients on stable HAART for at least 6 months and with plasma HIV RNA levels below 50 copies/ml. Ten patients received a combination of rev, tat, and nef intramuscularly (im) at weeks 0, 4, and 16 at increasing doses giving totals of 300 (100 x 3), 900 (300 x 3), and 1800 (600 x 3) micrograms DNA. Five patients received saline in the same amounts im. Antigen-specific cytotoxic T lymphocyte (CTL) levels were preserved or increased and new T lymphocyte proliferative responses were induced in the group immunized with the HIV DNA genes. No increase in antibody levels was noted. Despite a 10-fold higher vaccine dose, patients on HAART did not respond better to vaccination compared to non-HAART patients included in a previous study where the genes were administered separately. Combining the regulatory genes rev, tat, and nef in increasing doses may reduce the anticipated augmentation of HIV-specific T cell proliferative and CTL responses. Viral suppression did not seem to further improve the initial vaccine responses of patients with comparable CD4 levels.     

Proviral HIV-DNA predicts viral rebound and viral setpoint after structured treatment interruptions

In HIV-1-infected patients with long-term undetectable viraemia on highly active antiretroviral treatment (HAART), we found that pre-HAART plasma viraemia and the baseline proviral DNA level were significantly associated with the viraemia setpoint during scheduled treatment interruptions. In long-term treated patients, pre-HAART viraemia may not be available, and in these circumstances proviral DNA, measured at the time of scheduled treatment interruption, can help to identify patients likely to reach a low viraemia setpoint after treatment interruption.     

Evaluation of modified vaccinia virus Ankara as an alternative vaccine against smallpox in chronically HIV type 1-infected individuals undergoing HAART

The fear of malevolent use of variola virus by terrorists has led to the implementation of a health care worker vaccination program and to the consideration of vaccination for the general public. However, due to concerns about side effects of the classical smallpox vaccine, especially for immunocompromised individuals, a safer vaccine is urgently needed. We characterized the immunogenicity of modified vaccinia virus Ankara (MVA), one of the more promising alternative smallpox vaccines, in a cohort of 10 chronically HIV-1-infected individuals undergoing highly active antiretroviral therapy (HAART). Nine subjects received smallpox vaccination as children while one subject was never vaccinated against smallpox. All the subjects had CD4 counts >400 cells/mm(3) and 8 out of 10 had undetectable viral loads. MVA was able to elicit humoral and cellular immune responses in the majority of individuals. Vaccinia-specific antibodies were mainly of the IgG class while T cells specific to vaccinia were predominantly CD8(+). The immune responses were maintained over 1 year. Similar vaccinia specific humoral immune responses were observed when our cohort of HIV-1-infected individuals was compared to smallpox-vaccinated healthy subjects. The observed immune responses suggest that the highly attenuated MVA could be used as a substitute vaccine against smallpox in chronically HIV-1-infected individuals undergoing HAART.     

Analysis of the CDR3 region of alpha/beta T-cell receptors (TCRs) and TCR BD gene double-stranded recombination signal sequence breaks end in peripheral blood mononuclear cells of T-lineage acute lymphoblastic leukemia

Recently, numerous reports have highlighted the restriction of the CDR3 length of T-cell receptor (TCR) beta chain in T-cells infiltrating solid tumors and hematological malignancies. However, these studies ignored the restriction of CDR3 length of TCR alpha chain and few of them attempted to reveal the mechanisms of the oligo-clonal expansion of T cells in the tumors. The primary aims of this study were twofold to: (i) analyze the CDR3 length of TCR alpha and beta chain in peripheral blood mononuclear cells of T-lineage acute lymphoblastic leukemia (T-ALL); and (ii) discover the relationship between the clonality of T cells and the process of TCR rearrangement in peripheral T cells. To this end, we investigated the TCR BV and TCR AV family spectratypes of two T-ALL patients and healthy controls using the immunoscope spectratyping technique. We found that the spectratypes exhibited a Gaussian distribution in healthy controls. However, the TCR repertoires of the two patients were highly restricted in the number of different TCR BV and TCR AV family members present. Furthermore, we found that the peripheral blood mononuclear cells (PBMC) of two T-ALL patients had the recombination signal sequence (RSS) 5'- and 3'-breaks end in the TCR BD2 gene using a specialized ligation-mediated polymerase chain reaction, implying the ongoing recombination of the TCR beta gene. Analysis of the particular CDR3 length of TCR alpha/beta T cells might be helpful for further study of the individualized therapy of T-ALL. This information will also be helpful in exploring new immunological pathogenesis and facilitating the design of a T-ALL vaccine, as well as in improving our understanding of healthy human T-cell development.     

Natural killer T cells reactive to a single glycolipid exhibit a highly diverse T cell receptor beta repertoire and small clone size

CD1d-restricted natural killer (NK) T cells reactive with the glycolipid alpha-galactosylceramide (alpha-GalCer) are a distinct lymphocyte sublineage. They express an invariant Valpha14-Jalpha18 T cell receptor (TcR), but the role of the beta chain has been controversial. Here, we have used CD1d tetramers to identify and isolate NK T cells based on their antigen specificity. In mice lacking germline Vbeta8, most of the alpha-GalCer-reactive T cells express either Vbeta2 or Vbeta7, strong Vbeta selection being revealed by the lack of an increase in other Vbeta regions. By contrast to the selection for complementarity determining region (CDR) 3beta sequences in some anti-peptide responses, alpha-GalCer-reactive T cells have polyclonal CDR3beta sequences. There is little CDR3beta sequence redundancy between organs or individual mice, and, surprisingly, there also is no evidence for organ-specific CDR3beta sequence motifs. These data argue against a T cell receptor-mediated self-reactivity for tissue-specific CD1d-bound ligands. Each NKT clone is represented by only 5-10 cells. This clone size is similar to naive conventional T cells, and much lower than that reported for memory T cells, although NK T cells have an activated/memory phenotype.     

Changes in T cell receptor repertoire associated with graft-versus-tumor effect and graft-versus-host disease in patients with relapsed multiple myeloma after donor lymphocyte infusion

Recent reports of clinical responses following donor lymphocyte infusions (DLI) in patients with relapsed multiple myeloma (MM) after allogeneic BMT have demonstrated the ability of allogeneic cells to mediate a graft-versus-myeloma (GVM) effect, but the mechanisms involved have not been determined. To identify changes in the T cell compartment associated with DLI, we performed a molecular analysis of the T cell receptor (TCR) repertoire in four patients with relapsed MM who received infusions of CD4+ lymphocytes from HLA-identical sibling donors. Three of the four patients demonstrated a clinical anti-myeloma response following DLI but also developed graft-versus-host disease (GVHD). The TCR repertoire was examined after PCR amplification of 24 Vbeta gene subfamilies. This method determines the relative utilization of each Vbeta gene subfamily and also allows the identification of clonal and oligoclonal T cell populations through analysis of CDR3 regions for each TCR Vbeta gene subfamily. Serial blood samples were obtained over at least a 1 year period before and after DLI and results compared to 10 normal donors. Serial analysis of CDR3 size profiles demonstrated the appearance of clonal T cell populations after DLI in each of the three responding patients. The appearance of some clones was noted within the first 3 months after DLI and coincided with decreasing levels of monoclonal paraprotein indicating an ongoing GVM response. Other T cell clones appeared at later time points and coincided with the development of GVHD. These findings demonstrate that T cell clones with different patterns of onset can be identified in the peripheral blood of MM patients following DLI. Further functional characterization of these distinct clonal expansions will be required to determine whether these T cell clones are mediators of either anti-myeloma or anti-host activity.     

Restricted T-cell receptor beta-chain usage by T cells autoreactive to beta(2)-glycoprotein I in patients with antiphospholipid syndrome

We recently identified CD4(+) T cells that are autoreactive to beta(2)-glycoprotein I (beta(2)GPI) and that promote antiphospholipid antibody production in patients with antiphospholipid syndrome (APS). In this study, T-cell receptor (TCR) beta chains of beta(2)GPI-reactive T cells were examined in 8 beta(2)GPI-responders, including 5 patients with APS and 3 healthy subjects, using polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis combined with in vitro stimulation of peripheral blood T cells with recombinant beta(2)GPI. The TCR Vbeta segments that expanded oligoclonally after stimulation with beta(2)GPI varied among responders, but the Vbeta7 and Vbeta8 segments were commonly detected in 6 and 4 beta(2)GPI-responders, respectively. Analysis of the complementarity-determining region 3 sequence of beta(2)GPI-reactive T cells revealed limited diversity, and all Vbeta7(+) TCRs had an amino acid motif of TGxxN/Q or minor variations. The Vbeta8(+) TCRs had another motif, PxAxxD/E. Surprisingly, an identical Vbeta7(+) TCRbeta chain was used by beta(2)GPI-reactive T cells in 3 patients with APS. There was no apparent difference in the TCRbeta usage between APS patients and healthy responders. Some of the Vbeta7(+) TCRs with the TGxxN/Q motif detected by PCR-SSCP analysis were also used by beta(2)GPI-specific CD4(+) T-cell clones responsive to an immunodominant epitope containing the major phospholipid-binding site. Depletion of Vbeta7(+) or Vbeta8(+) T cells from the peripheral blood mononuclear cell cultures significantly inhibited in vitro anti-beta(2)GPI antibody production in response to beta(2)GPI. Our results indicate preferential usage of TCRbeta chains by beta(2)GPI-reactive T cells. These TCRbeta chains can be reasonable targets for TCR-based immunotherapy for patients with APS.