Hergen Spits

Serum-free medium for generation and propagation of functional human cytotoxic and helper T cell clones

A serum-free lymphocyte culture medium is described in which serum is replaced by bovine serum albumin, transferrin, insulin, ethanolamine and a mixture of saturated and unsaturated fatty acids (linoleic acid, oleic acid and palmitic acid). In this serum-free medium proliferative and cytotoxic responses induced in mixed lymphocyte culture were comparable with those obtained in medium containing serum. Antigen-specific cytotoxic and helper T cells were isolated and could be propagated in serum-free medium without loss of function.

Phosphatidylinositol-3-OH kinase and nutrient-sensing mTOR pathways control T lymphocyte trafficking

Phosphatidylinositol-3-OH kinase (PI(3)K) and the nutrient sensor mTOR are evolutionarily conserved regulators of cell metabolism. Here we show that PI(3)K and mTOR determined the repertoire of adhesion and chemokine receptors expressed by T lymphocytes. The key lymph node–homing receptors CD62L (L-selectin) and CCR7 were highly expressed on naive T lymphocytes but were downregulated after immune activation. CD62L downregulation occurred through ectodomain proteolysis and suppression of gene transcription. The p110δ subunit of PI(3)K controlled CD62L proteolysis through mitogen-activated protein kinases, whereas control of CD62L transcription by p110δ was mediated by mTOR through regulation of the transcription factor KLF2. PI(3)K-mTOR nutrient-sensing pathways also determined expression of the chemokine receptor CCR7 and regulated lymphocyte trafficking in vivo. Hence, lymphocytes use PI(3)K and mTOR to match metabolism and trafficking.

Distinct roles of the phosphatidylinositol 3-kinase and STAT5 pathways in IL-7-mediated development of human thymocyte precursors.

Here, we define the IL-7R-activated signal that promotes survival and proliferation of T cell progenitors and demonstrate that it is distinct from the signals that induce differentiation. We show that IL-7 activates PKB and STAT5 in human thymocytes. Into T cell precursors we introduced chimeric receptors with a cytoplasmic domain of the IL-7R that is no longer able to activate PI-3K/PKB and STAT5 and tested the transduced cells in a fetal thymic organ culture. We also examined the T cell precursor activity of progenitors expressing dominant-negative forms of PI-3K or STAT5B. These experiments revealed that PI-3K/PKB activation is essential for the survival and proliferation of T cell precursors and suggest that STAT5 activated by IL-7 mediates T cell differentiation.

Immortalization of Human CD8+ T Cell Clones by Ectopic Expression of Telomerase Reverse Transcriptase

Replicative senescence of T cells is correlated with erosion of telomere ends. Telomerase plays a key role in maintaining telomere length. Therefore, it is thought that telomerase regulates the life span of T cells. To test this hypothesis, we have over-expressed human telomerase reverse transcriptase in human CD8+ T cells. Ectopic expression of human telomerase reverse transcriptase led to immortalization of these T cells, without altering the phenotype and without loss of specificity or functionality. As the T cells remained dependent on cytokines and Ag stimulation for their in vitro expansion, we conclude that immortalization was achieved without malignant transformation.

Early stages in the development of human T, natural killer and thymic dendritic cells

Summary: T‐cell development is initiated when CD34+ pluripotent stem cells or their immediate progeny leave the bone marrow Co migrate to the thymus. Upon arrival in the thymus the stem cell progeny is not yet committed to the T‐cell lineage as it has the capability to develop into T, natural killer (NK) and dendritic cells (DC). Primitive hematopoietic progenitor cells in the human thymus express CD34 and lack CD la. When these progenitor cells develop into T cells they traverse a number of checkpoints. One early checkpoint is the induction of T‐cell commitment, which correlates with appearance of CD la and involves the loss of capacity to develop into NK cells and DC and the initiation of T‐cell receptor (TCR) gene rearrangements, Basic helix‐loop‐helix transcription factors play a role in induction of T‐cell commitment. CDla+CD34+ cells develop into CD4+CD8α+β+ cells by upregulating first CD4, followed by CD8α and then CD8β. Selection for productive TCRβ gene rearrangements (β selection) likely occurs in the CD4+CD8α+β‐ and CD4+CD8α+β+ populations. Although the T and NK‐cell lineages arc closely related to each other, NK cells can develop independently of the thymus. The fetal thymus is most likely one site of NK‐cell development.

STAT5 regulates the self-renewal capacity and differentiation of human memory B cells and controls Bcl-6 expression

It is unknown how B cells that mature during a germinal center reaction decide between plasma or memory cell fate. Here we describe a previously unknown subpopulation of B cells in the human germinal center that is characterized by tyrosine phosphorylated transcriptional activator STAT5. These cells had an activated centrocyte phenotype and had abundant expression of BCL6 but low expression of PRDM1, both encoding transcriptional repression proteins. Using RNA interference and ectopic expression of constitutively activated forms of STAT5, we demonstrate here a function for STAT5 in the self-renewal of B cells in vitro. STAT5b isoform seemed to directly upregulate Bcl-6, and ectopic expression of Bcl-6 in B cells resulted in self-renewal and inhibition of plasma cell differentiation. These data indicate that activation of STAT5 is involved in regulation of memory B cell differentiation.

Disruption of αβ but not of γδ T cell development by overexpression of the helix-loop-helix protein Id3 in committed T cell progenitors

Enforced expression of Id3, which has the capacity to inhibit many basic helix–loop–helix (bHLH) transcription factors, in human CD34+ hematopoietic progenitor cells that have not undergone T cell receptor (TCR) gene rearrangements inhibits development of the transduced cells into TCRαβ and γδ cells in a fetal thymic organ culture (FTOC). Here we document that overexpression of Id3, in progenitors that have initiated TCR gene rearrangements (pre‐T cells), inhibits development into TCRαβ but not into TCRγδ T cells. Furthermore, Id3 impedes expression of recombination activating genes and downregulates pre‐Tα mRNA. These observations suggest possible mechanisms by which Id3 overexpression can differentially affect development of pre‐T cells into TCRαβ and γδ cells. We also observed that cell surface CD4−CD8−CD3− cells with rearranged TCR genes developed from Id3‐transduced but not from control‐transduced pre‐T cells in an FTOC. These cells had properties of both natural killer (NK) and pre‐T cells. These findings suggest that bHLH factors are required to control T cell development after the T/NK developmental checkpoint.

Immunogenicity, Including Vitiligo, and Feasibility of Vaccination With Autologous GM-CSF–Transduced Tumor Cells in Metastatic Melanoma Patients

PURPOSEnTo determine the feasibility, toxicity, and immunologic effects of vaccination with autologous tumor cells retrovirally transduced with the GM-CSF gene, we performed a phase I/II vaccination study in stage IV metastatic melanoma patients.nnnPATIENTS AND METHODSnSixty-four patients were randomly assigned to receive three vaccinations of high-dose or low-dose tumor cells at 3-week intervals. Tumor cell vaccine preparation succeeded for 56 patients (88%), but because of progressive disease, the well-tolerated vaccination was completed in only 28 patients. We analyzed the priming of T cells against melanoma antigens, MART-1, tyrosinase, gp100, MAGE-A1, and MAGE-A3 using human leukocyte antigen/peptide tetramers and functional assays.nnnRESULTSnThe high-dose vaccination induced the infiltration of T cells into the tumor tissue. Three of 14 patients receiving the high-dose vaccine showed an increase in MART-1- or gp100-specific T cells in the peripheral blood during vaccination. Six patients experienced disease-free survival for more than 5 years, and two of these patients developed vitiligo at multiple sites after vaccination. MART-1- and gp100-specific T cells were found infiltrating in vitiligo skin. Upon vaccination, the T cells acquired an effector phenotype and produced interferon-gamma on specific antigenic stimulation.nnnCONCLUSIONnWe conclude that vaccination with GM-CSF-transduced autologous tumor cells has limited toxicity and can enhance T-cell activation against melanocyte differentiation antigens, which can lead to vitiligo. Whether the induction of autoimmune vitiligo may prolong disease-free survival of metastatic melanoma patients who are surgically rendered as having no evidence of disease before vaccination is worthy of further investigation.

Early stages in human and mouse T-cell development.

One important question in lymphopoiesis is where stem cells commit to T-, B- and natural killer (NK)-cell lineages. Recent findings in human and mouse systems suggest that the thymus is seeded by a yet uncommitted progenitor cell. The earliest murine thymic progenitor cells have the capacity to develop into B, T and NK cells when introduced into the appropriate microenvironment. The mechanisms underlying T-cell commitment are unknown, but cytokines might be involved. The gamma-chain of the interleukin (IL)-2 receptor seems to play a role in development of T and NK cells, but the current data argue against a critical role for IL-2 in T- and NK-cell development. This suggests that the IL-2 receptor gamma-chain is part of a receptor for another cytokine, important for T- and NK-cell development. IL-7 might be involved in regulating T-cell receptor rearrangements and in proliferation of cells within the thymus.

In vitro-isolated human cytotoxic T-lymphocyte clones detect variations in serologically defined HLA antigens

T cells of two donors, JR (HLA-A23, 29; B7,7; G; DRw5) and HG (HLA-A2, 23; B40, w44; Cw4), were stimulated with cells from an HLA homozygous lymphoblastoid cell line JY (HLA-A2, 2; B7,7, C-, DRw4, 6) and cloned by limiting dilution after the third stimulation. Two cytotoxic T-cell (CTL) clones, JR-2-16 (from donor JR) and HG-31 (from donor HG), were used for detailed studies. The results of a panel study using lymphocytes from HLA-typed individuals and a study with two HLA recombinant families indicate that the antigens recognized by the CTL clones JR-2-16 and HG-31 were highly associated with HLA-A2 and HLA-B7, respectively. Blocking studies with a monoclonal antibody recognizing a framework determinant on HLA-A, -B and-C antigens and a monoclonal antibody reacting with HLA-A2 support the notion that JR-2-16 and HG-31 interact with the HLA-A2 and the HLA-B7 antigens per se. However, these clones did not recognize the HLA-A2 and HLA-B7 of all donors typed for these antigens, suggesting that the HLA-A2 and HLA-B7 antigens of these particular donors are variants of the serologically defined HLA antigens. These results indicate that in vitro-derived human CTL clones detect variations in the serologically defined allospecificities and can be used as reagents to elucidate the polymorphism of HLA antigens further.