Human T cell differentiation : basic aspects and their clinical applications

Immune recognition plays a central role in our understanding of the function of the immune system. The ability to specifically recognize foreign antigens allows selective but efficient actions of the immune system against all kinds of pathogens. This is mediated by antigen-specific receptors on B and T lymphocytes. Immunoglobulin (lg) molecules represent the antigenspecific receptors of B lymphocytes, while the T cell receptor (TeA) has this function in T lymphocytes (1). Although these two types of antigen receptors have remarkable similarities in protein structure and their encoding genes, they differ significantly in their ability to interact with antigens (1). Via their surface membrane lg (Smlg) molecules, B lymphocytes are able to recognize antigens in their native configuration either free in solution, on surfaces or on cell membranes (1). TeA molecules ofT lymphocytes can only recognize processed or degraded antigens which are physically associated with major histocompatibility complex (MHC) molecules (2,3). This TeA-mediated recognition is therefore called MHC-restricted antigen recognition (2,3). Expression of Smlg or TcA molecules by lymphocytes is acquired during lymphoid differentiation via several rearrangement processes in the lg or TcR genes (3-7). B lymphopoiesis mainly occurs in the bone marrow (8), while the thymus is thought to represent the main tissue compartment for T lymphopoiesis (9-11 ). During T cell differentiation in the thymus the T lymphocytes are "educated" for their future functions, i.e. T cells which recognize self antigens are eliminated (negative selection), while positive selection occurs for T cells which recognize foreign (non-self) antigens in association with self-MHC molecules (12-14). Upon recognition of a TeA-compatible antigen, T lymphocytes are activated, start to proliferate and exhibit their regulatory or cytotoxic functions (2). These T cell functions play a central role in the regulation of the immune system. The T lymphocytes probably coordinate immune processes via cellular interactions and lymphokines and in this way adjust and harmonize the actions of the immune system. The TeA consists of two chains, which are closely associated with the CD3 protein complex (TcA-CD3). Th

Basic helix-loop-helix proteins E2A and HEB induce immature T-cell receptor rearrangements in nonlymphoid cells

T-cell receptor (TCR) gene rearrangements are mediated via V(D)J recombination, which is strictly regulated during lymphoid differentiation, most probably through the action of specific transcription factors. Investigated was whether cotransfection of RAG1 and RAG2 genes in combination with lymphoid transcription factors can induce TCR gene rearrangements in nonlymphoid human cells. Transfection experiments showed that basic helix-loop-helix transcription factors E2A and HEB induce rearrangements in the TCRD locus (Ddelta2-Ddelta3 and Vdelta2-Ddelta3) and TCRG locus (psi Vgamma7-Jgamma2.3 and Vgamma8-Jgamma2.3). Analysis of these rearrangements and their circular excision products revealed some peculiar characteristics. The Vdelta2-Ddelta3 rearrangements were formed by direct coupling without intermediate Ddelta2 gene segment usage, and most Ddelta2-Ddelta3 recombinations occurred via direct coupling of the respective upstream and downstream recombination signal sequences (RSSs) with deletion of the Ddelta2 and Ddelta3 coding sequences. Subsequently, the E2A/HEB-induced TCR gene recombination patterns were compared with those in early thymocytes and acute lymphoblastic leukemias of T- and B-lineage origin, and it was found that the TCR rearrangements in the transfectants were early (immature) and not necessarily T-lineage specific. Apparently, some parts

Comparative analysis of Ig and TCR gene rearrangements at diagnosis and at elapse of childhood precursor-B–ALL provides improved strategies for selection of stable PCR targets for monitoring of minimal residual disease

Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient-specific polymerase chain reaction (PCR) targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), but they might be unstable during the disease course. Therefore, we performed detailed molecula

Antigen receptor sequencing of paired bone marrow samples shows homogeneous distribution of acute lymphoblastic leukemia subclones

In B-cell precursor acute lymphoblastic leukemia, the initial leukemic cells share the same antigen receptor gene rearrangements. However, due to ongoing rearrangement processes, leukemic cells with different gene rearrangement patterns can develop, resulting in subclone formation. We studied leukemic subclones and their distribution in the bone marrow and peripheral blood at diagnosis

Molecular and flow cytometric analysis of the Vβ repertoire for clonality assessment in mature TCRαβ T-cell proliferations

Clonality assessment through Southern blot (SB) analysis of TCRB genes or polymerase chain reaction (PCR) analysis of TCRG genes is important for diagnosing suspect mature T-cell proliferations. Clonality assessment through reverse transcription (RT)-PCR analysis of Vbeta-Cbeta transcripts and flow cytometry with a Vbeta antibody panel covering more than 65% of Vbeta domains was validated using 28 SB-defined clonal T-cell receptor (TCR)alphabeta(+) T-ALL samples and T-cell lines. Next, the diagnostic applicability of the V(beta) RT-PCR and flow cytometric clonality assays was studied in 47 mature T-cell proliferations. Clonal Vbeta-Cbeta RT-PCR products were detected in all 47 samples, whereas single Vbeta domain usage was found in 31 (66%) of 47 patients. The suspect leukemic cell populations in the other 16 patients showed a complete lack of Vbeta monoclonal antibody reactivity that was confirmed by molecular data showing the usage of Vbeta gene segments not covered by the applied Vbeta monoclonal antibodies. Nevertheless, this could be considered indirect evidence for the "clonal" character of these cells. Remarkably, RT-PCR revealed an oligoclonal pattern in addition to dominant Vbeta-Cbeta products and single Vbeta domain expression in many T-LGL proliferations, providing further evidence for the hypothesis raised earlier that T-LGL derive from polyclonal and oligoclonal proliferations of antigen-activated cytotoxic T cells. It is concluded that molecular Vbeta analysis serves to assess clonality in suspect T-cell proliferations. However, the faster and cheaper Vbeta antibody studies can be used as a powerful screening method for the detection of single Vbeta domain expression, followed by molecular studies in patients with more than 20% single Vbeta domain expression or large suspect T-cell populations (more than 50%-60%) without Vbeta reactivity

Wnt target genes identified by DNA microarrays in immature CD34+ thymocytes regulate proliferation and cell adhesion

The thymus is seeded by very small numbers of progenitor cells that undergo massive proliferation before differentiation and rearrangement of TCR genes occurs. Various signals mediate proliferation and differentiation of these cells, including Wnt signals. Wnt signals induce the interaction of the cytoplasmic cofactor beta-catenin with nuclear T cell factor (TCF) transcription factors. We identified target genes of the Wnt/beta-catenin/TCF pathway in the most immature (CD4-CD8-CD34+) thymocytes using Affymetrix DNA microarrays in combination with three different functional assays for in vitro induction of Wnt signaling. A relatively small number (approximately 30) of genes changed expression, including several proliferation-inducing transcription factors such as c-fos and c-jun, protein phosphatases, and adhesion molecules, but no genes involved in differentiation to mature T cell stages. The adhesion molecules likely confine the proliferating immature thymocytes to the appropriate anatomical sites in the thymus. For several of these target genes, we validated that they are true Wnt/beta-catenin/TCF target genes using real-time quantitative PCR and reporter gene assays. The same core set of genes was repressed in Tcf-1-null mice, explaining the block in early thymocyte development in these mice. In conclusion, Wnt signals mediate proliferation and cell adhesion, but not differentiation of the immature thymic progenitor pool

High CD33-antigen loads in peripheral blood limit the efficacy of gemtuzumab ozogamicin |(Mylotarg®) treatment in acute myeloid leukemia patients

Gemtuzumab ozogamicin (Mylotarg®) induces remission in approximately 30% of relapsed AML patients. We previously demonstrated that gemtuzumab infusion results in near-complete CD33 saturation in peripheral blood, and that saturating gemtuzumab levels result in continuous binding and internalization of gemtuzumab due to renewed CD33 expression. We now demonstrate that a high CD33-antigen load in peripheral blood is an independent adverse prognostic factor, likely due to peripheral consumption of gemtuzumab. Indeed, CD33 saturation in bone marrow is significantly reduced (40-90% saturation) as compared with CD33 saturation in corresponding peripheral blood samples (>90%). In vitro, such reduced CD33 saturation levels were strongly related with reduced cell kill. Apparently, high CD33-antigen loads in blood consume gemtuzumab and thereby limit its penetration into bone marrow. Consequently, CD33 saturation in bone marrow is reduced, which hampers efficient cell kill. Therefore, gemtuzumab should be administered at higher or repeated doses, or, preferably, after reduction of the leukemic cell burden by classical chemotherapy