ABSTRACT
T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-) Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRß locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications.
Subject(s)
Cell Culture Techniques/methods , Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor/immunology , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor/immunology , Precursor Cells, T-Lymphoid/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocytes, Regulatory/immunology , Adaptor Proteins, Signal Transducing , Animals , Antigens, CD/genetics , Antigens, CD/immunology , CD3 Complex/genetics , CD3 Complex/immunology , Calcium-Binding Proteins , Cells, Cultured , Female , Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor/genetics , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor/genetics , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/immunology , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice , Mice, Inbred BALB C , Mice, Knockout , Precursor Cells, T-Lymphoid/cytology , Receptors, Antigen, T-Cell, alpha-beta/genetics , Stromal Cells , T-Lymphocytes, Regulatory/cytologyABSTRACT
The requirement for Notch signaling during T-cell development has been extensively studied. Nevertheless, the developmental stage at which it is required and whether additional signaling pathways are needed are still poorly understood. By using a stromal-cell-free culture system, we show that sorted double-negative 3 (DN3) thymocytes only require a Delta-like-4-induced Notch signal to differentiate into double-positive (DP) cells. This differentiation process is preTCR-α dependent. DN3 cells undergo 4-5 proliferation cycles, and the addition of the chemokine CXCL12 improves proliferation. IL-7 blocks the differentiation of DN3 cells to DP cells but not the Notch-induced proliferation of cultured DN3 cells. The impaired differentiation correlates with an inhibition of Rag-2 up-regulation. Overall, the in vitro stromal-cell-free culture system presented here also provides a powerful and unique tool for studying the mechanisms involved in the positive and negative selection of T cells.
Subject(s)
Cell Differentiation/immunology , Chemokine CXCL12/immunology , Interleukin-7/immunology , Intracellular Signaling Peptides and Proteins/immunology , Membrane Proteins/immunology , Signal Transduction/immunology , Thymocytes/cytology , Adaptor Proteins, Signal Transducing , Animals , Calcium-Binding Proteins , Cell Separation , Chemokine CXCL12/metabolism , Female , Flow Cytometry , Interleukin-7/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Real-Time Polymerase Chain Reaction , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Thymocytes/immunology , Thymocytes/metabolismABSTRACT
Rectal transmission is one of the main routes of infection by human immunodeficiency virus type 1 (HIV-1). To efficiently study transmission mechanisms and prevention strategies, a small animal model permissive for rectal transmission of HIV is mandatory. We tested the susceptibility of RAG2(-/-)gamma(c)(-/-) mice transplanted with human cord blood hematopoietic stem cells to rectal infection with HIV. We rectally exposed these humanized mice to cell-free and cell-associated HIV. All mice remained HIV negative as assessed by plasma viral load. The same mice infected intraperitoneally showed high levels of HIV replication. In the gut-associated lymphatic tissue, we found disproportionately smaller numbers of human cells than in other lymphoid organs. This finding may explain the observed resistance to rectal transmission of HIV. To increase the numbers of local HIV target cells and the likelihood of HIV transmission, we treated mice with different proinflammatory stimuli: local application of interleukin-1beta, addition of seminal plasma to the inoculum, or induction of colitis with dextran sodium sulfate. These procedures attracted some human leukocytes, but the transmission rate was still very low. The humanized mice showed low levels of human engraftment in the intestinal tract and seem to be resistant to rectal transmission of HIV, and thus they are an unsuitable model for this application.
