Effect of HIV on thymic function before and after antiretroviral therapy in children

Studies were undertaken to investigate the role of the thymus in T cell reconstitution in human immunodeficiency virus (HIV)-infected children treated with antiretroviral therapy. Nine pediatric patients who acquired HIV perinatally were treated with multidrug combinations of antiretroviral agents. Plasma virus load and CD4+ and CD8+ T cell subsets were measured, and thymus function was measured by quantifying T cell receptor rearrangement excision circles in peripheral blood. Patients with virus loads remaining >400 RNA copies/mL plasma were classified as virologic nonresponders. Thymus function was initially decreased in all subjects. After antiretrovirus therapy, peripheral CD4+ T cells increased in all subjects. Thymus function was restored in 4 of 5 virologic responders but in only 1 of 4 virologic nonresponders. This suggests that HIV has an adverse effect upon thymic function in pediatric HIV infection. Potent antiretroviral therapy restores thymic function but is affected by the degree to which virus suppression is achieved.     

Thymus transplantation restores the repertoires of forkhead box protein 3 (FoxP3)+ and FoxP3− T cells in complete DiGeorge anomaly

The development of T cells with a regulatory phenotype after thymus transplantation has not been examined previously in complete DiGeorge anomaly (cDGA). Seven athymic infants with cDGA and non-maternal pretransplantation T cell clones were assessed. Pretransplantation forkhead box protein 3 (Foxp3)⁺ T cells were detected in five of the subjects. Two subjects were studied in greater depth. T cell receptor variable β chain (TCR-Vβ) expression was assessed by flow cytometry. In both subjects, pretransplantation FoxP3⁺ and total CD4⁺ T cells showed restricted TCR-Vβ expression. The development of naive T cells and diverse CD4⁺ TCR-Vβ repertoires following thymic transplantation indicated successful thymopoiesis from the thymic tissue grafts. Infants with atypical cDGA develop rashes and autoimmune phenomena before transplantation, requiring treatment with immunosuppression, which was discontinued successfully subsequent to the observed thymopoiesis. Post-transplantation, diverse TCR-Vβ family expression was also observed in FoxP3⁺ CD4⁺ T cells. Interestingly, the percentages of each of the TCR-Vβ families expressed on FoxP3⁺ and total CD4⁺ T cells differed significantly between these T lymphocyte subpopulations before transplantation. By 16 months post-transplantation, however, the percentages of expression of each TCR-Vβ family became significantly similar between FoxP3⁺ and total CD4⁺ T cells. Sequencing of TCRBV DNA confirmed the presence of clonally amplified pretransplantation FoxP3⁺ and FoxP3⁻ T cells. After thymus transplantation, increased polyclonality was observed for both FoxP3⁺ and FoxP3⁻ cells, and pretransplantation FoxP3⁺ and FoxP3⁻ clonotypes essentially disappeared. Thus, post-transplantation thymic function was associated with the development of a diverse repertoire of FoxP3⁺ T cells in cDGA, corresponding with immunological and clinical recovery.     

Pre-operative sentinel lymph node localization in breast cancer with superparamagnetic iron oxide MRI: the SentiMAG Multicentre Trial imaging subprotocol

Objective:

Sentinel lymph node biopsy (SLNB) with a superparamagnetic iron oxide (SPIO) tracer was shown to be non-inferior to the standard combined technique in the SentiMAG Multicentre Trial. The MRI subprotocol of this trial aimed to develop a magnetic alternative for pre-operative lymphoscintigraphy (LS). We evaluated the feasibility of using MRI following the administration of magnetic tracer for pre-operative localization of sentinel lymph nodes (SLNs) and its potential for non-invasive identification of lymph node (LN) metastases.

Methods:

Patients with breast cancer scheduled to undergo SLNB were recruited for pre-operative LS, single photon emission CT (SPECT)-CT and SPIO MRI. T₁ weighted turbo spin echo and T₂ weighted gradient echo sequences were used before and after interstitial injection of magnetic tracer into the breast. SLNs on MRI were defined as LNs with signal drop and direct lymphatic drainage from the injection site. LNs showing inhomogeneous SPIO uptake were classified as metastatic. During surgery, a handheld magnetometer was used for SLNB. Blue or radioactive nodes were also excised. The number of SLNs and MR assessment of metastatic involvement were compared with surgical and histological outcomes.

Results:

11 patients were recruited. SPIO MRI successfully identified SLNs in 10 of 11 patients vs 11 of 11 patients with LS/SPECT-CT. One patient had metastatic involvement of four LNs, and this was identified in one node on pre-operative MRI.

Conclusion:

SPIO MRI is a feasible technique for pre-operative localization of SLNs and, in combination with intraoperative use of a handheld magnetometer, provides an entirely radioisotope-free technique for SLNB. Further research is needed for the evaluation of MRI characterization of LN involvement using subcutaneous injection of magnetic tracer.

Advances in knowledge:

This study is the first to demonstrate that an interstitially administered magnetic tracer can be used both for pre-operative imaging and intraoperative SLNB, with equal performance to imaging and localization with radioisotopes.     

The clonal composition of human CD4+CD25+Foxp3+ cells determined by a comprehensive DNA-based multiplex PCR for TCRB gene rearrangements

The characterization of the T-cell receptor (TCR) repertoire of CD4+ regulatory T cells (T(R)) has been limited due to the RNA degradation that results following permeabilization and fixation as routinely used for intracellular staining of Foxp3. In the present study the clonal composition of human umbilical cord blood (UCB) and adult peripheral blood mononuclear cell (PBMC) CD4+ T(R) and non-T(R) was characterized by a DNA-based multiplex PCR which allowed for the consistent clonotypic characterization of cells that have undergone fixation and permeabilization. To validate this method, CD8+ T cells from two HLA A()0201 individuals were sorted and compared clonotypically based upon their ability either to secrete interferon-gamma in response to a CMV pp65 epitope or to bind to the corresponding pMHC I tetramer. Clonotypes shared between the CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- subsets were observed in all 3 UCB and in one adult PBMCs, suggesting that na?ve and memory CD4+ T(R) can share the same clonotypes as CD4+ non-T(R) in humans.     

Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation

Flow cytometric detection of antigen-specific CD8+ T cells has previously been limited to MHC-class I tetramer staining or intracellular cytokine production, neither of which measure the cytolytic potential of these cells. Here we present a novel technique to enumerate antigen-specific CD8+ T cells using a marker expressed on the cell surface following activation induced degranulation, a necessary precursor of cytolysis. This assay measures the exposure of CD107a and b, present in the membrane of cytotoxic granules, onto the cell surface as a result of degranulation. Acquisition of cell surface CD107a and b is associated with loss of intracellular perforin and is inhibited by colchicine, indicating that exposure of CD107a and b to the cell surface is dependent on degranulation. CD107a and b are expressed on the cell surface of CD8+ T cells following activation with cognate peptide, concordant with production of intracellular IFNgamma. Finally, CD107-expressing CD8+ T cells are shown to mediate cytolytic activity in an antigen-specific manner. Measurement of CD107a and b expression can also be combined with MHC-class I tetramer labeling and intracellular cytokine staining to provide a more complete assessment of the functionality of CD8+T cells expressing cognate T cell receptors (TCR).