Neuropilin-1 Is Expressed on Highly Activated CD4+ Effector T Cells and Dysfunctional CD4+ Conventional T Cells from Naive Mice.

Neuropilin-1 (Nrp-1) is a well described marker molecule for CD4+Foxp3+ thymus-derived regulatory T cells (Tregs). In addition, a small population of CD4+Foxp3- conventional (conv) T cells expresses Nrp-1 in naive mice, and Nrp-1 expression has been described to be upregulated on activated CD4+ T cells. However, the function of Nrp-1 expression on CD4+ non-Tregs still remains elusive. In this study, we demonstrate that Nrp-1 expression was induced upon stimulation of CD4+Foxp3- T cells in vitro and during an ongoing immune response in vivo. This activation-induced Nrp-1+CD4+ T cell subset (iNrp-1+) showed a highly activated phenotype in terms of elevated CD25 and CD44 expression, enhanced production of proinflammatory cytokines, and increased proliferation compared with the Nrp-1-CD4+ counterpart. In contrast, Nrp-1+CD4+Foxp3- conv T cells from naive mice (nNrp-1+) were dysfunctional. nNrp-1+CD4+ conv T cells upregulated activation-associated molecules to a lesser extent, exhibited impaired proliferation and produced fewer proinflammatory cytokines than Nrp-1-CD4+ conv T cells upon stimulation in vitro. Moreover, the expression of PD-1 and CTLA-4 was significantly higher on nNrp-1+CD4+Foxp3- T cells compared with iNrp-1+CD4+Foxp3- T cells and Nrp-1-CD4+Foxp3- T cells after stimulation and under homeostatic conditions. Strikingly, transfer of Ag-specific iNrp-1+CD4+ conv T cells aggravated diabetes development, whereas Ag-specific nNrp-1+CD4+ conv T cells failed to induce disease in a T cell transfer model of diabetes. Overall, our results indicate that Nrp-1 expression has opposite functions in recently activated CD4+ non-Tregs compared with CD4+ non-Tregs from naive mice.

Heroin-assisted treatment of heroin-addicted patients normalizes regulatory T cells but does not restore CD4+ T cell proliferation.

Heroin dependence may result in suppression of adaptive immune responses. Previously, we demonstrated an increase in relative numbers of inhibitory CD4+ regulatory T cells (Tregs) and impaired proliferative activity of CD4+ T cells from heroin-addicted patients in contrast to patients in opioid maintenance therapy and healthy controls. However, it remains elusive whether heroin has a direct impact on the CD4+ T cell compartment or whether observed effects result from stressful living conditions. Here, we analyzed the frequencies of Tregs and the proliferation as well as the cytokine production of stimulated CD4+ T cells from heroin-addicted patients with use of illicit heroin, patients in heroin-assisted treatment (HAT), and patients in methadone maintenance therapy (MMT). Relative numbers of CD4+ Tregs were significantly enhanced in patients with illicit heroin abuse compared with patients in HAT or MMT. Notably, CD4+ T cells from patients in HAT and from persons using illicit heroin showed significant reduced proliferation and secretion of the pro-inflammatory cytokines IFN-γ and IL-6 upon stimulation in vitro. From these results, we conclude that structured programs such as HAT and MMT dampen elevated frequencies of Tregs in heroin-addicted patients, whereas chronic heroin administration irrespective of using illicit heroin or receiving HAT has a direct impact on the proliferative activity and cytokine production of CD4+ T cells.

Meta-Analysis Reveals Significant Sex Differences in Chronic Lymphocytic Leukemia Progression in the Eµ-TCL1 Transgenic Mouse Model.

The Eµ-TCL1 transgenic mouse model represents the most widely and extensively used animal model for chronic lymphocytic leukemia (CLL). In this report, we performed a meta-analysis of leukemia progression in over 300 individual Eµ-TCL1 transgenic mice and discovered a significantly accelerated disease progression in females compared to males. This difference is also reflected in an aggressive CLL mouse model with additional deletion of Tp53 besides the TCL1 transgene. Moreover, after serial adoptive transplantation of murine CLL cells, female recipients also succumbed to CLL earlier than male recipients. This sex-related disparity in the murine models is markedly contradictory to the human CLL condition. Thus, due to our observation we urge both careful consideration in the experimental design and accurate description of the Eµ-TCL1 transgenic cohorts in future studies.

CD74 is dispensable for development of chronic lymphocytic leukemia in Eµ-TCL1 transgenic mice.

CD74 is a surface protein expressed on immune cells, which acts as receptor for the chemokine macrophage migration inhibitory factor (MIF). Signaling via the MIF/CD74-axis has been reported to be important for the pathogenesis of chronic lymphocytic leukemia (CLL). We wanted to clarify the role of CD74 in MIF-induced signaling/leukemic development. In Eµ-TCL1 transgenic mice, occurrence of the leukemic phenotype was associated with increased surface CD74 expression. Eµ-TCL1+/+Cd74-/- mice showed similar kinetics and clinical features of CLL development as Eµ-TCL1+/+ mice. MIF stimulation of leukemic splenocytes led to AKT activation in a CD74-dependent manner. AKT activation was reduced in Cd74-deficient splenocytes in the presence of the oncogenic TCL1-transgene. Tumor cell apoptosis/proliferation were unaffected in Eµ-TCL1+/+Cd74-/- mice. Our data suggest that the need for active CD74 signaling is overcome in the leukemic context of TCL1-driven CLL, and that CD74 may have a dispensable role for CLL pathogenesis in this model.

LYN Kinase in the Tumor Microenvironment Is Essential for the Progression of Chronic Lymphocytic Leukemia.

Survival of chronic lymphocytic leukemia (CLL) cells strictly depends on the support of an appropriate tumor microenvironment. Here, we demonstrate that LYN kinase is essential for CLL progression. Lyn deficiency results in a significantly reduced CLL burden in vivo. Loss of Lyn within leukemic cells reduces B cell receptor (BCR) signaling including BTK phosphorylation, but surprisingly does not affect leukemic cell expansion. Instead, syngeneic CLL transplantation of CLL cells into Lyn- or Btk-deficient recipients results in a strongly delayed leukemic progression and prolonged survival. Moreover, Lyn deficiency in macrophages hinders nursing functions for CLL cells, which is mediated by direct contact rather than secretion of soluble factors. Taken together, LYN and BTK seem essential for the formation of a microenvironment supporting leukemic growth.

CD44 regulates the apoptotic response and promotes disease development in chronic lymphocytic leukemia.

The cell-surface glycoprotein CD44 is expressed in chronic lymphocytic leukemia (CLL), but its functional role in this disease is poorly characterized. We therefore investigated the contribution of CD44 to CLL in a murine disease model, the Eµ-TCL1 transgenic mouse, and in CLL patients. Surface CD44 increased during murine CLL development. CD44 expression in human CLL was induced by stimulation with interleukin 4/soluble CD40 ligand and by stroma cell contact. Engagement of CD44 by its natural ligands, hyaluronic acid or chondroitin sulfate, protected CLL cells from apoptosis, while anti-CD44 small interfering RNAs impaired tumor cell viability. Deletion of CD44 during TCL1-driven murine leukemogenesis reduced the tumor burden in peripheral blood and spleen and led to a prolonged overall survival. The leukemic cells from these CD44 knockout animals revealed lower levels of antiapoptotic MCL1, a higher propensity to apoptosis, and a diminished B-cell receptor kinase response. The inhibitory anti-CD44 antibodies IM7 and A3D8 impaired the viability of CLL cells in suspension cultures, in stroma contact models, and in vivo via MCL1 reduction and by effector caspase activation. Taken together, CD44 expression in CLL is mediated by the tumor microenvironment. As a coreceptor, CD44 promotes leukemogenesis by regulating stimuli of MCL1 expression. Moreover, CD44 can be addressed therapeutically in CLL by specific antibodies.

Relevance of individual Mo-box nucleotides to DNA binding by the related molybdenum-responsive regulators MopA and MopB in Rhodobacter capsulatus.

Either of two related molybdenum-responsive regulators, MopA and MopB, of Rhodobacter capsulatus is sufficient to repress the nitrogen-fixation gene anfA. In contrast, MopA (but not MopB) activates mop, which codes for a molybdate (Mo)-binding molbindin. Both regulators bind to conserved cis-regulatory elements called Mo-boxes. Single-base substitution of two highly conserved nucleotides within the anfA-Mo-box (T21C and C24T) had little effect on regulator binding and anfA expression as shown by DNA mobility shift assays and reporter gene fusions, respectively. In contrast to C24T, mutation C24A strongly diminished binding and repression by MopA and MopB, showing that different nucleotide substitutions at the same position may have very different effects. A triple mutation destroying the left half-site of the mop-Mo-box completely abolished mop expression by MopA, demonstrating the importance of the mop-Mo-box for mop activation. Two point mutations (T23A and T24C) still allowed binding by MopA, but abolished mop activation, most likely because these nucleotides overlap with the RNA polymerase-binding site. A mutant mop promoter, in which the mop-Mo-box was exchanged against the anfA-Mo-box, allowed activation by MopA, showing that a former repressor-binding site may act as an activator-binding site depending on its location relative to the other promoter elements.