Michelle L. Joachims

CD73-Generated Adenosine Restricts Lymphocyte Migration into Draining Lymph Nodes

After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5′-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73−/− mice have normal sized lymphoid organs in the steady state, but ∼1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73+/+ and cd73−/− lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A2B receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-α. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73−/− mice is largely normalized by pretreatment with the selective A2B receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.

Human αβ and γδ Thymocyte Development: TCR Gene Rearrangements, Intracellular TCRβ Expression, and γδ Developmental Potential—Differences between Men and Mice

To evaluate the role of the TCR in the αβ/γδ lineage choice during human thymocyte development, molecular analyses of the TCRβ locus in γδ cells and the TCRγ and δ loci in αβ cells were undertaken. TCRβ variable gene segments remained largely in germline configuration in γδ cells, indicating that commitment to the γδ lineage occurred before complete TCRβ rearrangements in most cases. The few TCRβ rearrangements detected were primarily out-of-frame, suggesting that productive TCRβ rearrangements diverted cells away from the γδ lineage. In contrast, in αβ cells, the TCRγ locus was almost completely rearranged with a random productivity profile; the TCRδ locus contained primarily nonproductive rearrangements. Productive γ rearrangements were, however, depleted compared with preselected cells. Productive TCRγ and δ rearrangements rarely occurred in the same cell, suggesting that αβ cells developed from cells unable to produce a functional γδ TCR. Intracellular TCRβ expression correlated with the up-regulation of CD4 and concomitant down-regulation of CD34, and plateaued at the early double positive stage. Surprisingly, however, some early double positive thymocytes retained γδ potential in culture. We present a model for human thymopoiesis which includes γδ development as a default pathway, an instructional role for the TCR in the αβ/γδ lineage choice, and a prolonged developmental window for β selection and γδ lineage commitment. Aspects that differ from the mouse are the status of TCR gene rearrangements at the nonexpressed loci, the timing of β selection, and maintenance of γδ potential through the early double positive stage of development.

The TCR ligand-inducible expression of CD73 marks γδ lineage commitment and a metastable intermediate in effector specification

CD73 expression is induced in response to TCR ligation and identifies a population of thymocytes that are committed to the γδ T cell fate.

High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination.

Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus.

Restoration of Adenosine Deaminase-Deficient Human Thymocyte Development In Vitro by Inhibition of Deoxynucleoside Kinases

Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture to study ADA-deficient human thymocyte development in an “in vivo-like” environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular dATP. Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients.

Further Differentiation of Murine Double-Positive Thymocytes Is Inhibited in Adenosine Deaminase-Deficient Murine Fetal Thymic Organ Culture

Murine fetal thymic organ culture (FTOC) was used to investigate the mechanism by which a lack of adenosine deaminase (ADA) leads to a failure of T cell production in the thymus. We previously showed that T cell development was inhibited beginning at the CD4−CD8−CD25+CD44low stage in ADA-deficient FTOC initiated at day 15 of gestation when essentially all thymocytes are CD4−CD8−. In the present study, we asked whether thymocytes at later stages of differentiation would also be sensitive to ADA inhibition by initiating FTOC when substantial numbers of CD4+CD8+ thymocytes were already present. dATP was highly elevated in ADA-deficient cultures, and the recovery of αβ TCR+ thymocytes was inhibited by 94%, indicating that the later stages of thymocyte differentiation are also dependent upon ADA. ADA-deficient cultures were partially rescued by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or by the use of apoptotic protease-activating factor-1-deficient mice. Rescue was even more dramatic, with 60- to >200-fold increases in the numbers of CD4+CD8+ cells, when FTOC were performed with an inhibitor of adenosine kinase, the major thymic deoxyadenosine phosphorylating enzyme, or with bcl-2 transgenic mice. dATP levels were normalized by treatment with either carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or an adenosine kinase inhibitor, but not in cultures with fetal thymuses from bcl-2 transgenic mice. These data suggest that ADA deficiency leads to the induction of mitochondria-dependent apoptosis as a consequence of the accumulation of dATP derived from thymocytes failing the positive/negative selection checkpoint.

Inhibition of deoxynucleoside kinases in human thymocytes prevents dATP accumulation and induction of apoptosis

Thymocytes lacking adenosine deaminase (ADA) activity, a purine metabolism enzyme, accumulate intracellular dATP and consequently undergo apoptosis during development. We have analyzed the effect of ADA enzyme inhibition in human thymocyte suspension cultures with regard to accumulation of intracellular dATP and induction of apoptosis. We demonstrate that while inhibition of deoxycytidine kinase will prevent the accumulation of dATP and induction of apoptosis to a large degree, inhibition of both deoxycytidine kinase and adenosine kinase completely abrogates the accumulation of dATP and significantly reduces the induction of apoptosis. Thus, both deoxynucleoside kinases are involved in this model of ADA deficiency.