Mimicry of a constitutively active pre–B cell receptor in acute lymphoblastic leukemia cells

Pre–B cells undergo apoptosis unless they are rescued by pre–B cell receptor–dependent survival signals. We previously showed that the BCR-ABL1 kinase that is expressed in pre–B lymphoblastic leukemia bypasses selection for pre–B cell receptor–dependent survival signals. Investigating possible interference of BCR-ABL1 with pre–B cell receptor signaling, we found that neither SYK nor SLP65 can be phosphorylated in response to pre–B cell receptor engagement. Instead, Bruton's tyrosine kinase (BTK) is constitutively phosphorylated by BCR-ABL1. Activated BTK is essential for survival signals that otherwise would arise from the pre–B cell receptor, including activation of PLCγ1, autonomous Ca(2+) signaling, STAT5-phosphorylation, and up-regulation of BCLX (L). Inhibition of BTK activity specifically induces apoptosis in BCR-ABL1 (+) leukemia cells to a similar extent as inhibition of BCR-ABL1 kinase activity itself. However, BCR-ABL1 cannot directly bind to full-length BTK. Instead, BCR-ABL1 induces the expression of a truncated splice variant of BTK that acts as a linker between the two kinases. As opposed to full-length BTK, truncated BTK lacks kinase activity yet can bind to BCR-ABL1 through its SRC-homology domain 3. Acting as a linker, truncated BTK enables BCR-ABL1–dependent activation of full-length BTK, which initiates downstream survival signals and mimics a constitutively active pre–B cell receptor

Defective lymphoid organogenesis underlies the immune deficiency caused by a heterozygous S32I mutation in IκBα.

Patients with ectodermal dysplasia with immunodeficiency (ED-ID) caused by mutations in the inhibitor of NF-κB α (IκBα) are susceptible to severe recurrent infections, despite normal T and B cell numbers and intact in vitro lymphocyte function. Moreover, the outcome of hematopoietic stem cell transplantation (HSCT) in these patients is poor despite good engraftment. Mice heterozygous for the IκBα S32I mutation found in patients exhibited typical features of ED-ID. Strikingly, the mice lacked lymph nodes, Peyer's patches, splenic marginal zones, and follicular dendritic cells and failed to develop contact hypersensitivity (CHS) or form germinal centers (GCs), all features not previously recognized in patients and typical of defective noncanonical NF-κB signaling. Lymphotoxin β receptor (LTβR)-driven induction of chemokines and adhesion molecules mediated by both canonical and noncanonical NF-κB pathways was impaired, and levels of p100 were markedly diminished in the mutant. IκBα mutant → Rag2(-/-), but not WT→IκBα mutant, bone marrow chimeras formed proper lymphoid organs and developed CHS and GCs. Defective architectural cell function explains the immunodeficiency and poor outcome of HSCT in patients with IκBα deficiency and suggests that correction of this niche is critical for reconstituting their immune function

Central memory CD8+ T cells become CD69+ tissue-residents during viral skin infection independent of CD62L-mediated lymph node surveillance.

Memory CD8+ T cells in the circulation rapidly infiltrate non-lymphoid tissues following infection and provide protective immunity in an antigen-specific manner. However, the subsequent fate of memory CD8+ T cells after entering non-lymphoid tissues such as the skin during a secondary infection is largely unknown. Furthermore, because expression of CD62L is often used to identify the central memory (TCM) CD8+ T cell subset, uncoupling the physical requirement for CD62L-mediated lymph node homing versus other functional attributes of TCM CD8+ T cells remains unresolved. Here, we show that in contrast to naïve CD8+ T cells, memory CD8+ T cells traffic into the skin independent of CD62L-mediated lymph node re-activation and provide robust protective immunity against Vaccinia virus (VacV) infection. TCM, but not effector memory (TEM), CD8+ T cells differentiated into functional CD69+/CD103- tissue residents following viral clearance, which was also dependent on local recognition of antigen in the skin microenvironment. Finally, we found that memory CD8+ T cells expressed granzyme B after trafficking into the skin and utilized cytolysis to provide protective immunity against VacV infection. Collectively, these findings demonstrate that TCM CD8+ T cells become cytolytic following rapid infiltration of the skin to protect against viral infection and subsequently differentiate into functional CD69+ tissue-residents

Compound heterozygous mutation of Rag1 leading to Omenn syndrome.

Omenn syndrome is a primary immunodeficiency disorder, featuring susceptibility to infections and autoreactive T cells and resulting from defective genomic rearrangement of genes for the T cell and B cell receptors. The most frequent etiologies are hypomorphic mutations in "non-core" regions of the Rag1 or Rag2 genes, the protein products of which are critical members of the cellular apparatus for V(D)J recombination. In this report, we describe an infant with Omenn syndrome with a previously unreported termination mutation (p.R142*) in Rag1 on one allele and a partially characterized substitution mutation (p.V779M) in a "core" region of the other Rag1 allele. Using a cellular recombination assay, we found that while the p.R142* mutation completely abolished V(D)J recombination activity, the p.V779M mutation conferred a severe, but not total, loss of V(D)J recombination activity. The recombination defect of the V779 mutant was not due to overall misfolding of Rag1, however, as this mutant supported wild-type levels of V(D)J cleavage. These findings provide insight into the role of this poorly understood region of Rag1 and support the role of Rag1 in a post-cleavage stage of recombination

Compound Heterozygous Mutation of Rag1 Leading to Omenn Syndrome

Omenn syndrome is a primary immunodeficiency disorder, featuring susceptibility to infections and autoreactive T cells and resulting from defective genomic rearrangement of genes for the T cell and B cell receptors. The most frequent etiologies are hypomorphic mutations in “non-core” regions of the Rag1 or Rag2 genes, the protein products of which are critical members of the cellular apparatus for V(D)J recombination. In this report, we describe an infant with Omenn syndrome with a previously unreported termination mutation (p.R142*) in Rag1 on one allele and a partially characterized substitution mutation (p.V779M) in a “core” region of the other Rag1 allele. Using a cellular recombination assay, we found that while the p.R142* mutation completely abolished V(D)J recombination activity, the p.V779M mutation conferred a severe, but not total, loss of V(D)J recombination activity. The recombination defect of the V779 mutant was not due to overall misfolding of Rag1, however, as this mutant supported wild-type levels of V(D)J cleavage. These findings provide insight into the role of this poorly understood region of Rag1 and support the role of Rag1 in a post-cleavage stage of recombination

p.R142* maternal and p.V779M paternal mutations.

<p>P1 harbors a maternally inherited c.424C>T mutation, resulting in a premature stop codon. <b>A.</b> Sequencing chromatogram demonstrating the presence of a heterozygous c.424C>T mutation. <b>B.</b> Alignment of the wildtype and mutant Rag1 cDNA and protein sequences. c.424C>T creates a premature stop codon at position 142 of the protein. <b>C.</b> P1 harbors a paternally inherited c.2335G>A mutation, resulting in the non-synonymous coding mutation p.V779M. Sequencing chromatogram demonstrating the presence of a heterozygous c.2335G>A mutation. <b>D.</b> Alignment of the wildtype and mutant Rag1 cDNA and protein sequences. c.2335G>A creates a missense p.V779M mutation in the Rag1 protein.</p

Rag1_R142* is a null mutant and Rag1_V779M is a hypomorphic mutant.

<p>A. Western analysis of Flag-tagged full-length Rag1 proteins expressed in Br3neo human fibroblast cells confirms that the wild-type (Rag1) and mutant (Rag1_V779M) proteins are expressed at comparable levels <i>in vivo</i>. B. Representative recombination data from using the indicated constructs for transient V(D)J recombination assays in Br3neo cells. C. Absolute recombination activity using wild-type Rag1 (hatched) or the p.V779M mutant (shaded) with signal-joint substrates (left) or coding-joint substrates (right). Results represent the mean ±s.d. of six independent experiments. D. Normalized recombination activity of the p.V779M mutant. Recombination activity of the p.V779M mutant on each substrate was normalized to the activity of wild-type Rag1. Results represent the mean ± s.d. of six independent experiments.</p