RasGRP1 Transduces Low-Grade TCR Signals which Are Critical for T Cell Development, Homeostasis, and Differentiation

AbstractTwo important Ras-guanyl nucleotide exchange factors, Sos and RasGRP1, control Ras activation in thymocytes. However, the relative contribution of these two exchange factors to Ras/ERK activation and their resulting impact on positive and negative selection is unclear. We have produced two lines of RasGRP1−/− TCR transgenic mice to determine the effect of RasGRP1 in T cell development under conditions of defined TCR signaling. Our results demonstrate that RasGRP1 is crucial for thymocytes expressing weakly selecting TCRs whereas those that express stronger selecting TCRs are more effective at utilizing RasGRP1-independent mechanisms for ERK activation and positive selection. Analysis of RasGRP1−/− peripheral T cells also revealed hitherto unidentified functions of RasGRP1 in regulating T cell homeostasis and sustaining antigen-induced developmental programming

Familial Recurrence of Cerebral Palsy with Multiple Risk Factors

The recurrence of cerebral palsy in the same family is uncommon. We, however, report on two families with two or more affected siblings. In both families, numerous potential risk factors were identified including environmental, obstetric, and possible maternal effects. We hypothesize that multiple risk factors may lead to the increased risk of recurrence of cerebral palsy in families. Intrinsic and maternal risk factors should be investigated in all cases of cerebral palsy to properly counsel families on the risk of recurrence. Recent studies of genetic polymorphisms associated with cerebral palsy are considered with reference to our observations in these two families

RETRACTED: Autoimmunity as the Consequence of a Spontaneous Mutation in Rasgrp1

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).This article has been retracted at the request of the Authors.The authors have agreed to retract the paper because of the falsification of the Western blot in Figure 6A. The figure shows a defect in Ras activation, labeled as RasGTP, following TCR engagement, in thymocytes isolated from a RasGRP1 lag mutant mouse strain. This data set is one of several that show signaling and functional deficiencies identified for cells with lost of function of RasGRP. The authors stand by the validity of the other figures, results and interpretation in this paper. This matter was investigated by the Massachusetts Institute of Technology and the Office of Research Integrity at the United States Department of Health & Human Services, which found the figure was falsified by Luk Van Parijs, who is solely responsible. The authors deeply regret any inconvenience resulting from the publication of this data

RasGRP1, but not RasGRP3, is required for efficient thymic β-selection and ERK activation downstream of CXCR4.

T cell development is a highly dynamic process that is driven by interactions between developing thymocytes and the thymic microenvironment. Upon entering the thymus, the earliest thymic progenitors, called CD4(-)CD8(-) 'double negative' (DN) thymocytes, pass through a checkpoint termed "β-selection" before maturing into CD4(+)CD8(+) 'double positive' (DP) thymocytes. β-selection is an important developmental checkpoint during thymopoiesis where developing DN thymocytes that successfully express the pre-T cell receptor (TCR) undergo extensive proliferation and differentiation towards the DP stage. Signals transduced through the pre-TCR, chemokine receptor CXCR4 and Notch are thought to drive β-selection. Additionally, it has long been known that ERK is activated during β-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the β-selection events in mice lacking RasGRP1, RasGRP3 and RasGRP1 and 3. We report that RasGRP1 KO and RasGRP1/3 DKO deficient thymi show a partial developmental block at the early DN3 stage of development. Furthermore, DN3 thymocytes from RasGRP1 and RasGRP1/3 double knock-out thymi show significantly reduced proliferation, despite expression of the TCRβ chain. As a result of impaired β-selection, the pool of TCRβ(+) DN4 is significantly diminished, resulting in inefficient DN to DP development. Also, we report that RasGRP1 is required for ERK activation downstream of CXCR4 signaling, which we hypothesize represents a potential mechanism of RasGRP1 regulation of β-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the β-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway

RasGRP1 KO and RasGRP1/3 DKO thymocytes display impaired proliferation of DN3 and inefficient transition from DN3E to DN3L. A.

<p>Intracellular TCRβ (TCRβ_i.c.) by forward scatter (FSC) profiles of DN3 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁺) from B6 (n = 8), 1KO (n = 6), 3KO (n = 9) and DKO (n = 12) thymi. <b>B.</b> Frequencies of DN3 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁺) and DN4 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁻) expressing intracellular TCRβ (TCRβ_i.c.). <b>C.</b> Ratio of frequencies of TCRβ_i.c.⁺ DN3E/DN3L ((DN3E/DN3L)TCRβ_i.c.⁺). <b>D.</b> CD98 by CD25 profiles of Thy1.2⁺CD44⁻ cells from 1 and 2 day DN3E-OP9-DL1 co-cultures; data are representative of 3 independent experiments. <b>E.</b> Frequencies of BrdU⁺ DN3 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁺) and DN4 (CD4⁻CD8⁻Thy1.2⁺CD44^–CD25⁻) from B6 (n = 5), 1KO (n = 5), 3KO (n = 6) and DKO (n = 7) mice injected with BrdU i.p. 2h prior to euthanasia. *p<0.05, **p<0.01 and ***p<0.001.</p

RasGRP1 KO and RasGRP1/3 DKO DN4 thymocytes show an increased frequency of γδ T cells, despite normal numbers and frequencies of mature thymic γδ T cells. A.

<p>CD3 by γδTCR profiles of bulk thymocytes from B6 (n = 7), 1KO (n = 3), 3KO (n = 6) and DKO (n = 9) thymi. <b>B.</b> Frequencies and numbers of mature CD3⁺γδTCR⁺ γδ T cells. <b>C.</b> left, frequencies of surface TCRβ⁺ DN4 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁻) cells from B6 (n = 8), 1KO (n = 6), 3KO (n = 9) and DKO (n = 12) thymi; right, frequencies of surface γδTCR⁺ DN4 cells. ***p<0.001.</p

RasGRP1 KO, RasGRP3 KO and RasGRP1/3 DKO thymocytes show intact survival.

<p>Percentages of DN3 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁺), DN4 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁻) and DP (CD4⁺CD8⁺Thy1.2⁺) showing active caspase 3.</p

RasGRP1 KO and RasGRP 1/3 DKO show inefficient thymocyte development beyond DN3.

<p><b>A.</b> CD44 by CD25 profiles of DN thymocytes (CD4⁻CD8^–Thy1.2⁺CD3^lo) from B6 (n = 9), 1KO (n = 6), 3KO (n = 9) and DKO (n = 13) thymi. <b>B.</b> left, numbers of DN3 (CD4⁻CD8⁻CD3^loCD44⁻CD25⁺) thymocytes and DN4 (CD4⁻CD8⁻CD3^loCD44⁻CD25⁻) thymocytes; right, ratio of frequencies of DN3/DN4.</p