Depletion of αß+ T cells for a haploidentical hematopoietic stem cell transplantation in children.

A consistent and reproducible depletion technique is crucial for the successful transplantation of an ex vivo depleted graft. Our aim was to evaluate the efficacy of an ex vivo technique for depletion of αß+ T cells using a biotinylated anti-TCRαß monoclonal antibody, which was performed by one clinical nurse specialist. Between 2012 and 2017, 119 depletion procedures from 216 apheresis using the anti-TCRαß monoclonal antibody were performed on 105 pediatric patients. The median log depletion of αß+ T cells was 4.0 (range, 2.5-5.0). The median recovery rates of CD34+ , NK, and γδ+ T cells were 90.4%, 74.9%, and 75.9%, respectively. The efficacy of depletion of αß+ T cells significantly improved over time and the duration of the depletion procedure significantly decreased over time. Our study demonstrated that this procedure for depletion of αß+ T cells by skilled staff is highly effective at depleting target cells and obtaining CD34+ progenitor cells.

A mechanism for TCR sharing between T cell subsets and individuals revealed by pyrosequencing.

The human naive T cell repertoire is the repository of a vast array of TCRs. However, the factors that shape their hierarchical distribution and relationship with the memory repertoire remain poorly understood. In this study, we used polychromatic flow cytometry to isolate highly pure memory and naive CD8(+) T cells, stringently defined with multiple phenotypic markers, and used deep sequencing to characterize corresponding portions of their respective TCR repertoires from four individuals. The extent of interindividual TCR sharing and the overlap between the memory and naive compartments within individuals were determined by TCR clonotype frequencies, such that higher-frequency clonotypes were more commonly shared between compartments and individuals. TCR clonotype frequencies were, in turn, predicted by the efficiency of their production during V(D)J recombination. Thus, convergent recombination shapes the TCR repertoire of the memory and naive T cell pools, as well as their interrelationship within and between individuals.

Identification of a committed T cell precursor population in adult human peripheral blood.

Here, we report data concerning the discovery in adult human peripheral blood of a precursor cell population able to differentiate into CD4+CD3+ alpha beta + mature T cells. These cells, which represent 0.1-0.5% of total peripheral blood mononuclear cells (PBMC), express substantial levels of CD4, but lack CD3 surface expression. At a molecular level, they express the pre-T cell receptor alpha (pT alpha) gene, CD3-gamma, CD-delta and CD-epsilon, and RAG-1 recombination enzyme and have initiated rearrangements in the T cell receptor (TCR)-beta locus (D-J). Moreover, low levels of CD3 epsilon protein, but not of TCR-beta chain, can be detected in their cytoplasm. Our results suggest that CD4+CD3- cells identified in peripheral blood are different from CD3-CD4+CD8- thymocytes and may contain precursors of an extrathymic T cell differentiation pathway.

TCR-alpha/beta and CD19 depletion and treosulfan-based conditioning regimen in unrelated and haploidentical transplantation in children with acute myeloid leukemia.

We evaluated the depletion of TCR-alpha/beta cells from the graft of children with high-risk AML, who received transplantation from unrelated (n=20) and haploidentical donors (n=13). The preparative regimen included treosulfan, melphalan, fludarabine and anti-thymocyte globulin. Grafts were PBSC engineered by TCR-alpha/beta and CD19 depletion. The graft contained a median of 9 × 10(6)/kg of CD34+ and 20 × 10(3)/kg of αß-T cells. Post-transplant immune suppression included tacrolimus till day +30 and Mtx in 21 patients, tacrolimus in 5, Mtx in 2 and no prophylaxis in 5 patients. Sixteen patients received native or TCR-alpha/beta-depleted donor lymphocytes at a median of 47 (40-204) days. Median follow-up is 1.76 years. Primary engraftment was achieved in 33 patients (100%). Cumulative incidence of acute GvHD (aGvHD) grade 2-3 was 39 (26-60)%, half of them had skin-only aGvHD. Cumulative incidence of chronic GvHD was 30(18-50)%. Transplant-related mortality is 10(4-26)%. Event-free survival (EFS) is 60(43-76)% and overall survival (OS) is 67(50-84)% at 2 years. In a subgroup of patients, who received transplantation in CR, EFS is 66(48-84)% and OS-72(53-90)% at 2 years. Our data suggest that TCR-alpha/beta and CD19 depletion is a robust method of graft manipulation, which can be used to engineer grafts for children with AML.

H-2Ld-alloreactive T cell hybridomas utilize diverse V alpha and V beta T cell receptor chains.

We have sequenced the TCRs from Ld-specific alloreactive T cell hybridomas, whose reactivities we have found to be quite representative of those of a primary dm2 anti-BALB/cJ mixed lymphocyte reaction. We find V beta 6, V beta 7, V beta 8 and V beta 10 gene segments. V alpha usage is diverse, although closely related to that from peptide-specific Ld-restricted CTLs. V alpha-V beta selection provides evidence of preferential pairing. Amino acid frequency analysis shows that the alpha CDR2 region is rich in charged amino acids, in contrast to the beta CDR2 region. Our data suggests the beta chain may be more immunoglobulin-like than the alpha chain, and that charge complementarity may be important in TCR-MHC interactions. We do not consider our results to be contradictory to those previously reported but rather they may represent an early, more diverse response.

Quantifying Distribution of Flow Cytometric TCR-Vß Usage with Economic Statistics.

Measuring changes of the T cell receptor (TCR) repertoire is important to many fields of medicine. Flow cytometry is a popular technique to study the TCR repertoire, as it quickly provides insight into the TCR-Vß usage among well-defined populations of T cells. However, the interpretation of the flow cytometric data remains difficult, and subtle TCR repertoire changes may go undetected. Here, we introduce a novel means for analyzing the flow cytometric data on TCR-Vß usage. By applying economic statistics, we calculated the Gini-TCR skewing index from the flow cytometric TCR-Vß analysis. The Gini-TCR skewing index, which is a direct measure of TCR-Vß distribution among T cells, allowed us to track subtle changes of the TCR repertoire among distinct populations of T cells. Application of the Gini-TCR skewing index to the flow cytometric TCR-Vß analysis will greatly help to gain better understanding of the TCR repertoire in health and disease.

Prokaryotic production of the human T cell receptor Vbeta2 chain and binding to toxic-shock syndrome toxin-1.

The human T-cell receptor Vbeta2-, D- and J-encoding domains were PCR-amplified from MOLT-4 total cDNA and subcloned in Escherichia coli. The V/D/J fragment was subsequently transferred to a prokaryotic expression vector in frame with a polyhistidine-encoding prosequence which enabled us to affinity-purify the fusion protein with IMAC (immobilized metal-ion affinity chromatography [correction of chromatorgraphy]). Since the recombinant (re-) human T-cell receptor Vbeta2 fusion protein (Vbeta2 sol) produced in E.coli was found to be insoluble, purification was carried out under denaturating conditions. The purified and renatured re-protein, Vbeta2 sol, was immunoreactive with an anti-Vbeta2 monoclonal antibody in an ELISA assay. The specificity of Vbeta2 sol was shown by its binding in vitro to the staphylococcal superantigen TSST-1, but not to the Staphylococcus aureus exotoxin-1 (SEA).

Direct assessment of junctional diversity in rearranged T cell receptor beta chain encoding genes by combined heteroduplex and single strand conformation polymorphism (SSCP) analysis.

In order to define the extent of T cell heterogeneity and clonality, unique DNA sequences in the junctional region in rearranged T cell receptor (TcR) genes can be studied. For this purpose we have adapted a non-denaturing nucleic acid gel electrophoresis procedure to detect TcR junctional diversity. Detection of junctional diversity is based upon electrophoretic separation of single stranded (ss) and double stranded (ds) DNA molecules via mobility shifts due to nucleotide sequence polymorphism. To examine the capacity of this nucleic acid gel electrophoresis procedure to detect nucleotide sequence polymorphism in the CDR 3 region within TcR V beta gene family sequences polymerase chain reaction (PCR) amplified TcR V beta 5.1/5.4 and V beta 14 cDNA sequences were analyzed. The results of this study showed that (1) the single strand conformation polymorphism (SSCP) procedure has a low capacity to discriminate between diverse TcR V beta cDNA sequences due to comigration of the ssDNA molecules, which results in an underestimation of the heterogeneity in a given T cell population; (2) comigrating ssDNA and/or dsDNA (homoduplex) molecules can be separated by the formation of heteroduplex molecules; these heteroduplex molecules provide essential additional information on the degree of nucleotide sequence polymorphism in the CDR 3 region within the TcR V beta cDNA sequences; (3) the double strand conformation polymorphism (DSCP) procedure provides a fast and reliable procedure to detect junctional diversity within the sequences tested. Using DSCP a more detailed assessment of amplified TcR V beta cDNA sequences can be obtained as compared with SSCP analysis only. Data obtained by gel analysis were very similar to those obtained by conventional bacterial cloning and DNA sequencing procedures on the corresponding cDNA clones. In conclusion, this new gel electrophoresis procedure allows a direct assessment of the extent of T cell heterogeneity and clonality by screening junctional diversity in TcR chain encoding sequences in clinical conditions with (oligo)clonal expansion of T lymphocytes.

Cell separation based on the reversible interaction between calmodulin and a calmodulin-binding peptide.

A cell separation system based on the calcium-dependent interaction of calmodulin (CM) with a calmodulin-binding peptide (CBP) has been developed. The prototype of this system utilizes an indirect method to label the target cell population. Cells are first labeled with a primary monoclonal antibody directed to a specific cell surface antigen, then with a secondary affinity reagent, consisting of a polyclonal goat anti-mouse IgG (GAM-IgG) that has been cross-linked to a CBP derived from the sequence of the rabbit skeletal muscle myosin light chain kinase. In the presence of Ca2+, the CBP on the cells labeled with GAM-IgG-CBP binds to biotinylated calmodulin (CM-Biotin) with high affinity. The target cells are then captured with a solid-phase streptavidin. The unbound non-target cells are washed away and the immobilized target cells are released by chelating Ca2+ with EGTA. The specificity of the GAM-IgG-CBP and CM-Biotin and the feasibility of using this system to separate cells was demonstrated using the KG-1 human acute myelogenous leukemia cell line. KG-1 cells were fractionated on the basis of cell surface expression of HLA-DR. The cell selection reagents and the cell separation process did not affect KG-1 cell viability while cells selected by this procedure were 90% pure with a yield of 75%. This cell separation system also was used for rare cell isolation from normal human peripheral blood mononuclear cells. T cells expressing the Vbeta5 T cell receptor, which represent < 5% of the unfractionated cells, were isolated with 89% viability, 72% purity, 80% yield, and retained the ability to respond to activation signals as measured by blast transformation. The results from this study show that a cell selection system based on the reversible interaction between CM and a CBP can be applied to gently and efficiently isolate cells from a heterogeneous starting population that are free of the solid matrix without exposure to the stresses of mechanical or enzymatic release.

Soluble T cell receptors: detection and quantitative assay in fluid phase via ELISA or immuno-PCR.

To establish the concentration range in which soluble murine T cell receptors (sTCR), derived from the Th2 clone D10, exhibited biological activity, and to follow production and purification of D10 sTCR, we devised four quantitative immunoassays: three ELISA systems, and an immuno-PCR assay. The direct ELISA, employed hamster anti-TCR beta monoclonal antibody (H57), which detects all types of alpha beta TCR, regardless of their variable regions, and had a detection limit of about 6 ng/ml sTCR. The indirect sandwich ELISA employed anti-V beta 8 as capture antibody, and had a detection limit of 600 pg/ml. With the direct sandwich ELISA, that also employed anti-V beta 8, TCR concentrations as low as 100 pg/ml could be detected. The ELISA assays were specific for soluble alpha beta TCR, and showed no cross-reactivity when employing two control hamster anti-gamma delta TCR mAbs (GL3 and UC7), or with anti-TCR beta and monoclonal hamster IgG as a control antigen. Further, we demonstrated that in some assays where use of passive binding ELISA plates resulted in a high background, replacement with covalent binding ELISA plates resulted in an acceptable low background value. With the immuno-PCR assay, concentrations of sTCR as little as 0.8 pg/ml could be detected. In summary, the assays described here may prove valuable in investigating the occurrence and amount of sTCR in vitro and in vivo.

Soluble HIV-specific T cell receptor: expression, purification and analysis of the specificity.

We have produced soluble T cell receptor (TCR) derived from a human CD8(+) cytotoxic T lymphocyte (CTL) clone D3 that recognizes the immunodominant HIV Gag peptide SLYNTVATL (SL9) in association with major histocompatibility complex (MHC) class I protein HLA-A2. Drosophila Schneider cells (S2) were used to express genes coding the TCR alpha and beta chains under an inducible promoter. Both chains were labeled with two different tags: a (His)(6) was introduced at the C-terminal end of alpha chain, while beta chain was terminated by c-myc. Since an isolated alpha chain is unstable unless it is associated with a beta chain, this design permits rapid separation of alpha,beta-heterodimer from unpaired beta chain in a single step of Ni-NTA Agarose chromatography yielding 90% pure alpha,beta-TCR. Introduction of the c-myc epitope to the beta chain allows capture of soluble D3 from the culture supernatant by immobilized anti-c-myc antibody, without the need for receptor purification. The TCR specificity was then examined by analyzing the binding of peptide-HLA-A2/tetramer in an ELISA assay. Using this assay, we have also evaluated the binding of monomeric SL9-HLA-A2 complex to the immobilized D3 TCR and determined that the affinity measurement of the D3-SL9-HLA-A2 reaction is similar to that obtained by a biosensor instrument. We propose that the approach described here is generally useful for purification of other soluble TCRs and will allow rapid analysis of their specificity.

Identification of a novel human T-cell receptor V beta subfamily by genomic cloning.

Although a large number of human TCRBV gene segment sequences have been reported, the extent of the germline repertoire is still not precisely known. Most TCRBV gene segments have been identified in cDNA clones. However, genes expressed on only a small number of peripheral T cells may be more easily detectable by analysis of genomic DNA. In the present study, screening of cosmid clones containing the BV24S1 gene segment revealed the presence of a novel TCRBV gene segment defining a new subfamily, BV25S1. The nucleotide sequence of the gene contained a single open reading frame and encoded structurally important amino acids at correct positions. Southern blot analysis indicated that the BV25 subfamily contained only this single member. A single nucleotide polymorphism was identified by nucleotide sequencing of the gene from multiple individuals. Amplification of rearranged BV25S1 genes from cDNA derived from PBLs confirmed that the BV25S1 gene segment was capable of normal rearrangement and transcription.

Structure and diversity of the T-cell receptor alpha chain in rhesus macaque and chimpanzee.

We cloned and sequenced cDNA for the TCRAC1 of a healthy rhesus monkey and chimpanzee. TCRAC1 from both nonhuman primates show extensive conservation compared to the human sequence and to other mammals. A possible primate-specific insertion near the hinge region of the TCRAC1 region is described. Characterization of 18 rhesus macaque and eight chimpanzee TCRA chain cDNA clones derived from inverse PCR revealed 12 different TCRAV and 16 different TCRAJ regions which corresponded closely to known human counterparts. One functional rhesus macaque TCRDV-TCRAJ rearrangement was detected, suggesting a genomic organization of the macaque TCRD locus which is similar to humans. At the genomic level, a single TCRAC1 gene segment was detected in rhesus macaque and chimpanzee. The close phylogenetic relationship between primates shown here for TCRA chain components supports the use of these species as animal models of human immune-mediated disease.

Expression of gamma/delta T cell receptors in porcine thymus.

Swine possess an extraordinarily high number of T lymphocytes with the phenotype CD4-CD8- in peripheral blood as well as in lymphoid tissues. This subpopulation is subdivided into at least four subsets defined by the expression of CD2 and three biochemically distinct gamma/delta T cell receptors. The four subsets differ largely in their pattern of lymphoid homing in that CD2- subsets, historically referred to as null lymphocytes, predominate in the circulating pool, whereas CD2+ subsets are enriched in lymphoid tissues. Here we document the expression of all three types of gamma/delta T cell receptors by CD4-CD8- porcine thymocytes, which provides the first evidence for a thymic origination of all subsets of porcine gamma/delta T lymphocytes. The biochemical analysis shows that three distinct gamma-chains form disulfide-bonded cell surface heterodimers with a common delta-chain and that glycosylation of all chains is already completed within the thymus. Surprisingly, CD2- subsets, which are known to be enriched among thymic emigrants and which numerically predominate in peripheral blood, are underrepresented in the thymus, suggesting a high export rate.

Graves' disease thyroid tissue transplants in scid mice: persistent selectivity in hTcR Va gene family use.

We have analyzed the human T-cell receptor (hTcR) V alpha gene repertoire in thyroid tissue transplants of a patient with hyperthyroid Graves' disease. Blocks of thyroid tissue were transplanted subcutaneously into 10 mice with severe immunodeficiency (scid) and 4 weeks later 5 of the mice were injected intraperitoneally with autologous peripheral blood mononuclear cells (PBMC) (10(7) cells per mouse). After a further 3 weeks, mice were sacrificed and total cellular RNA and cDNA prepared from each of the explants. We used specific olingonucleotides in polymerase chain reactions (PCR) to amplify 18 different human hTcR V alpha gene families and the identity of the PCR fragments was confirmed by Southern blot analysis. Different samples of the donor thyroid tissue consistently expressed 9-10 of the 18 hTcR V alpha gene families screened (V alpha 1-7, 11, 12 & 15). A more marked bias in hTcR V gene family use was seen in each of the explants with a mean of only 2.8 V alpha gene families detected. After 7 weeks of transplantation, the thyroid explants largely reflected some of the same genes seen in the hTcR V gene repertoire of the donor tissue with particularly pronounced expression of V alpha 2 and V alpha 3 gene families. The transplantation of PBMC into the scid mice showed evidence for their accumulation within the transplanted thyroid tissues as judged by the appearance of additional hTcR V gene families expressed in these samples although the specificity of such accumulation remains unclear.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective usage of TCR V beta in tumor-specific CTL lines isolated from ovarian tumor-associated lymphocytes.

We have developed a series of six CTL lines exhibiting preferential killing of autologous tumour cells from tumour-associated lymphocytes (TALs) infiltrating malignant ovarian ascites. Five out of six CTL lines showed increased percentages of V beta 8.1+ cells, four showed increased percentages of V beta 5.3+ cells and one showed increased percentages of V beta 6.7+ cells. On the contrary, fresh isolated TALs or autologous PBMCs, when stimulated by OKT3 mAb or other TAL cultures with nonspecific cytolytic function, did not show preferential usage of any of these families. Most important, V beta 8.1+ and V beta 6.7+ T cells within the CTL-TALs mediated cytoxicity against autologous targets. This represents the first demonstration of preferential usage of TCR V beta in ovarian tumor-specific CTLs and suggests possible association between tumor surveillance and TCR V beta genes.

A new twist in TCR diversity revealed by a forbidden alphabeta TCR.

We report crystal structures of a negatively selected T cell receptor (TCR) that recognizes two I-A(u)-restricted myelin basic protein peptides and one of its peptide/major histocompatibility complex (pMHC) ligands. Unusual complementarity-determining region (CDR) structural features revealed by our analyses identify a previously unrecognized mechanism by which the highly variable CDR3 regions define ligand specificity. In addition to the pMHC contact residues contributed by CDR3, the CDR3 residues buried deep within the V alpha/V beta interface exert indirect effects on recognition by influencing the V alpha/V beta interdomain angle. This phenomenon represents an additional mechanism for increasing the potential diversity of the TCR repertoire. Both the direct and indirect effects exerted by CDR residues can impact global TCR/MHC docking. Analysis of the available TCR structures in light of these results highlights the significance of the V alpha/V beta interdomain angle in determining specificity and indicates that TCR/pMHC interface features do not distinguish autoimmune from non-autoimmune class II-restricted TCRs.

Identical TCR beta-chain rearrangements in streptococcal angina and skin lesions of patients with psoriasis vulgaris.

Tonsillar infection with Streptococcus pyogenes may induce several nonsuppurative autoimmune sequelae. The precise pathogenetic mechanisms behind this clinically well-established association are still unresolved. Using TCR analysis, we sought to identify a link between streptococcal tonsillitis and the T cell-mediated autoimmune response in psoriasis. Three patients with streptococcal-induced psoriasis underwent tonsillectomy. Using size spectratyping and sequencing of TCR beta-chain variable region gene (TCRBV) rearrangements, we compared the TCR usage of psoriatic skin lesions, blood, tonsils, and tonsillar T cells fractionated according to the expression of the skin address in "cutaneous lymphocyte-associated Ag" (CLA). TCRBV-size spectratype analysis of the blood lymphocytes, tonsils, and the CLA-negative tonsillar T cells revealed largely unselected T cell populations. Instead, TCRBV gene families of the psoriatic lesions and skin-homing CLA-positive tonsillar T cells displayed highly restricted spectratypes. Sequencing of TCRBV cDNA identified various clonal TCRBV rearrangements within the psoriatic lesions that indicated Ag-driven T cell expansion. Several of these clonotypes were also detected within the tonsils and, in one of the patients, within the small subset of CLA-positive tonsillar T cells, suggesting that T cells from the same T cell clones were simultaneously present within skin and tonsillar tissue. Because after tonsillectomy psoriasis cleared in all three patients our observations indicate that T cells may connect psoriatic inflammation to streptococcal angina. They suggest that the chronic streptococcal immune stimulus within the tonsils could act as a source for pathogenic T cells in poststreptococcal disorders, and they may help to explain why eliminating this source with tonsillectomy may improve streptococcal-induced sequelae.

Local anticandidal immune responses in a rat model of vaginal infection by and protection against Candida albicans.

Humoral (antibody [Ab]) and cellular Candida-specific immune responses in the vaginas of pseudoestrus rats were investigated during three successive infections by Candida albicans. After the first, protective infection, Abs against mannan and aspartyl proteinase antigens were present in the vaginal fluid, and their titers clearly increased during the two subsequent, rapidly healing infections. In all animals, about 65 and 10% of vaginal lymphocytes (VL) were CD3(+) (T cells) and CD3(-) CD5(+) (B cells), respectively. Two-thirds of the CD3(+) T cells expressed the alpha/beta and one-third expressed the gamma/delta T-cell receptor (TCR). This proportion slightly fluctuated during the three rounds of C. albicans infection, but no significant differences between infected and noninfected rats were found. More relevant were the changes in the CD4(+)/CD8(+) T-cell ratio, particularly for cells bearing the CD25 (interleukin-2 receptor alpha) marker. In fact, a progressively increased number of both CD4(+) alpha/beta TCR and CD4(+) CD25(+) VL was observed after the second and third Candida challenges, reversing the high initial CD8(+) cell number of controls (estrogenized but uninfected rats). The CD3(-) CD5(+) cells also almost doubled from the first to the third infection. Analysis of the cytokines secreted in the vaginal fluid of Candida-infected rats showed high levels of interleukin 12 (IL-12) during the first infection, followed by progressively increasing amounts of IL-2 and gamma interferon during the subsequent infections. No IL-4 or IL-5 was ever detected. During the third infection, VL with in vitro proliferative activity in response to an immunodominant mannoprotein antigen of C. albicans were present in the vaginal tissue. No response to this antigen by mitogen-responsive blood, lymph node, and spleen cells was found. In summary, the presence of protective Ab and T helper type 1 cytokines in the vaginal fluids, the in vitro proliferation of vaginal lymphocytes in response to Candida antigenic stimulation, and the increased number of activated CD4(+) cells and some special B lymphocytes after C. albicans challenge constitute good evidence for induction of locally expressed Candida-specific Ab and cellular responses which are potentially involved in anticandidal protection at the vaginal level.

T cell clonality in synovial fluid of a patient with rheumatoid arthritis: persistent but fluctuant oligoclonal T cell expansions.

Recently, oligoclonal T cell accumulation has been reported in affected joints of patients with rheumatoid arthritis. To characterize such clonally accumulating T cells, we quantitatively monitored their frequency by analyzing TCR B chains. As a result, despite a similar TCR BV usage between synovial fluid (SF) and PBL, obviously skewed TCR BJ usage was detected in SF. Subsequent DNA sequencing demonstrated that the skewed BJ gene usage in SF resulted from clonal T cell accumulation. The complementarity-determining region 3 of TCR B chains of the detected clones appeared to have homologous amino acid sequences. Further, one of the predominant TCR B chains of the clones was identical with that reported previously. In the follow-up study, most of the accumulated clones persisted; however, in some, proportions were drastically decreased or increased during the time course. In particular, one of the clones expanded sixfold, from 11 to 67%, in the BV8-BJ2S3 TCR population of SF, even though the clone was not detected in PBL. In summary, oligoclonal T cell accumulation in SF was persistent, but it appeared to fluctuate independently of the clonality in PBL. The expansion of the clones in SF may occur by antigenic stimuli within the joint.