A Novel TCR Transgenic Model Reveals That Negative Selection Involves an Immediate, Bim-Dependent Pathway and a Delayed, Bim-Independent Pathway

A complete understanding of negative selection has been elusive due to the rapid apoptosis and clearance of thymocytes in vivo. We report a TCR transgenic model in which expression of the TCR during differentiation occurs only after V(D)J-like recombination. TCR expression from this transgene closely mimics expression of the endogenous TCRα locus allowing for development that is similar to wild type thymocytes. This model allowed us to characterize the phenotypic changes that occurred after TCR-mediated signaling in self-reactive thymocytes prior to their deletion in a highly physiological setting. Self-reactive thymocytes were identified as being immature, activated and CD4loCD8lo. These cells had upregulated markers of negative selection and were apoptotic. Elimination of Bim reduced the apoptosis of self-reactive thymocytes, but it did not rescue their differentiation and the cells remained at the immature CD4loCD8lo stage of development. These cells upregulate Nur77 and do not contribute to the peripheral T cell repertoire in vivo. Remarkably, development past the CD4loCD8lo stage was possible once the cells were removed from the negatively selecting thymic environment. In vitro development of these cells occurred despite their maintenance of high intracellular levels of Nur77. Therefore, in vivo, negatively selected Bim-deficient thymocytes are eliminated after prolonged developmental arrest via a Bim-independent pathway that is dependent on the thymic microenvironment. These data newly reveal a layering of immediate, Bim-dependent, and delayed Bim-independent pathways that both contribute to elimination of self-reactive thymocytes in vivo

The Paf oncogene is essential for hematopoietic stem cell function and development

The Paf oncogene is highly expressed in cycling hematopoietic stem cells (HSCs) and is required for the development of long-term HSCs

Selective Depletion of Eosinophils or Neutrophils in Mice Impacts the Efficiency of Apoptotic Cell Clearance in the Thymus

Developing thymocytes undergo a rigorous selection process to ensure that the mature T cell population expresses a T cell receptor (TCR) repertoire that can functionally interact with major histocompatibility complexes (MHC). Over 90% of thymocytes fail this selection process and die. A small number of macrophages within the thymus are responsible for clearing the large number of dying thymocytes that must be continuously cleared. We studied the capacity of thymic macrophages to clear apoptotic cells under acute circumstances. This was done by synchronously inducing cell death in the thymus and then monitoring the clearance of apoptotic thymocytes. Interestingly, acute cell death was shown to recruit large numbers of CD11b+ cells into the thymus. In the absence of a minor CSF-1 dependent population of macrophages, the recruitment of these CD11b+ cells into the thymus was greatly reduced and the clearance of apoptotic cells was disrupted. To assess a possible role for the CD11b+ cells in the clearance of apoptotic cells, we analyzed mice deficient for eosinophils and mice with defective trafficking of neutrophils. Failure to attract either eosinophils or neutrophils to the thymus resulted in the impaired clearance of apoptotic cells. These results suggested that there is crosstalk between cells of the innate immune system that is necessary for maximizing the efficiency of apoptotic cell removal

Altered Development of NKT Cells, γδ T Cells, CD8 T Cells and NK Cells in a PLZF Deficient Patient

In mice, the transcription factor, PLZF, controls the development of effector functions in invariant NKT cells and a subset of NKT cell-like, γδ T cells. Here, we show that in human lymphocytes, in addition to invariant NKT cells, PLZF was also expressed in a large percentage of CD8+ and CD4+ T cells. Furthermore, PLZF was also found to be expressed in all γδ T cells and in all NK cells. Importantly, we show that in a donor lacking functional PLZF, all of these various lymphocyte populations were altered. Therefore, in contrast to mice, PLZF appears to control the development and/or function of a wide variety of human lymphocytes that represent more than 10% of the total PBMCs. Interestingly, the PLZF-expressing CD8+ T cell population was found to be expanded in the peripheral blood of patients with metastatic melanoma but was greatly diminished in patients with autoimmune disease

Ectopic T Cell Receptor-α Locus Control Region Activity in B Cells Is Suppressed by Direct Linkage to Two Flanking Genes at Once

The molecular mechanisms regulating the activity of the TCRα gene are required for the production of the circulating T cell repertoire. Elements of the mouse TCRα locus control region (LCR) play a role in these processes. We previously reported that TCRα LCR DNA supports a gene expression pattern that mimics proper thymus-stage, TCRα gene-like developmental regulation. It also produces transcription of linked reporter genes in peripheral T cells. However, TCRα LCR-driven transgenes display ectopic transcription in B cells in multiple reporter gene systems. The reasons for this important deviation from the normal TCRα gene regulation pattern are unclear. In its natural locus, two genes flank the TCRα LCR, TCRα (upstream) and Dad1 (downstream). We investigated the significance of this gene arrangement to TCRα LCR activity by examining transgenic mice bearing a construct where the LCR was flanked by two separate reporter genes. Surprisingly, the presence of a second, distinct, reporter gene downstream of the LCR virtually eliminated the ectopic B cell expression of the upstream reporter observed in earlier studies. Downstream reporter gene activity was unaffected by the presence of a second gene upstream of the LCR. Our findings indicate that a gene arrangement in which the TCRα LCR is flanked by two distinct transcription units helps to restrict its activity, selectively, on its 5′-flanking gene, the natural TCRα gene position with respect to the LCR. Consistent with these findings, a TCRα/Dad1 locus bacterial artificial chromosome dual-reporter construct did not display the ectopic upstream (TCRα) reporter expression in B cells previously reported for single TCRα transgenes

Development of spontaneous EAE in TCR transgenic mice.

<p>(<b>A</b>) RAG1 deficient mice were monitored for twelve months for overt signs of EAE (hind leg paraparesis). The day of the onset of EAE in individual mice is indicated on the chart. (<b>B</b>) More than 60% of MBPαβ mice and nearly all OVαβ TCR mice remained healthy throughout the course of the study, whereas 100% of the TCR transgenic RAG1 deficient became sick. Onset of disease was at an average of eleven weeks of age in all three susceptible mice.</p

The relative affinity of the OVαβ and MBPαβ TCRs.

<p>Antibody blocking studies show that the OVαβ TCR is of much lower affinity for the I-A^u:Ac1-11 peptide ligand as compared to the MBPαβ TCR. (<b>A</b>) Purified CD4⁺ T cells from MBPαβ TCR RAG1^−/− and OVαβ TCR RAG1^−/− mice were incubated with the indicated amount of antibody against Vβ8 (F23.1) and then challenged with titrated amounts of the Ac1-11 peptide presenting by B10.PL APCs. (<b>B</b>) Purified T cells were activated with 3uM Ac1-11, presented by B10.PL APCS, in the presence of titrated amounts of blocking antibody. Assays were pulsed after 48 hours and harvested at 72 hours. Each condition was done in triplicate and the data was averaged. One representative experiment of three is shown. (<b>C</b>) For clarity, the data from (B) are presented as the percent of the maximal response of the T cells without the blocking antibody.</p

Development of OVαβ TCR expressing thymocytes.

<p>The strong skewing of thymocytes expressing the high affinity MBPαβ TCR towards the CD4 lineage is not seen in OVαβTCR expressing mice. Expression of only the MBPβ chain does not markedly alter T cell development. (<b>A</b>) Single cell suspensions of thymocytes (top) and lymph node cells (bottom) from OVαβ, MBPαβ, MBPβ transgenic and transgene negative mice were stained with antibodies against CD4, CD8 and TCR Cβ. Only TCR Cβ⁺ cells are shown in the lymph node cell plots. The number indicates the percent of cells in the quadrant. Only subtle variations in the percent of cells within each quadrant were noted during repeat experiments. (<b>B</b>) CD4 SP thymocytes from OVαβ TCR transgenic mice express a high percentage of endogenous TCRα chains, whereas MBPαβ TCR transgenic mice predominantly express the transgene encoded Vα2 chain. Single cell suspensions of thymocytes from the indicated mouse strain were stained with antibodies against CD4, CD8 and individually with each of the four available TCR Vα specific antibodies. The percent of cells within the CD4 single positive population expressing each of the Vαs is shown. More than 1×10⁶ events were collected per sample. One of two experiments is shown. Greater than 98% of T cells in the OVαβ, MBPαβ and MBPβ mice expressed the transgene encoded Vβ8.2 TCR. (<b>C</b>) Increased levels of proliferation in response to the Ac1-11 peptide indicate a substantial number of antigen specific T cells develop in the OVαβ mice. CD4 T cells were enriched by negative selection. Double cultures were set up with 2.5×10⁴ CD4⁺ T cells and 1×10⁵ T cell depleted, irradiated B10.PL splenocytes plus the indicated amount of the MBP Ac1-11 peptide. Assays were pulsed with 3[H]thymidine after 48 hours and harvested after an additional 24 hours. Each condition was set up in triplicate and data was averaged. One example of three experiments is shown.</p