AKT1 and AKT2 maintain hematopoietic stem cell function by regulating reactive oxygen species.

Although AKT is essential for multiple cellular functions, the role of this kinase family in hematopoietic stem cells (HSCs) is unknown. Thus, we analyzed HSC function in mice deficient in the 2 isoforms most highly expressed in the hematopoietic compartment, AKT1 and AKT2. Although loss of either isoform had only a minimal effect on HSC function, AKT1/2 double-deficient HSCs competed poorly against wild-type cells in the development of myeloid and lymphoid cells in in vivo reconstitution assays. Serial transplantations revealed an essential role for AKT1 and AKT2 in the maintenance of long-term HSCs (LT-HSCs). AKT1/2 double-deficient LT-HSCs were found to persist in the G(0) phase of the cell cycle, suggesting that the long-term functional defects are caused by increased quiescence. Furthermore, we found that the intracellular content of reactive oxygen species (ROS) is dependent on AKT because double-deficient HSCs demonstrate decreased ROS. The importance of maintaining ROS for HSC differentiation was shown by a rescue of the differentiation defect after pharmacologically increasing ROS levels in double-deficient HSCs. These data implicate AKT1 and AKT2 as critical regulators of LT-HSC function and suggest that defective ROS homeostasis may contribute to failed hematopoiesis.

Akt1 and Akt2 promote peripheral B-cell maturation and survival.

Although the 3 isoforms of Akt regulate cell growth, proliferation, and survival in a wide variety of cell types, their role in B-cell development is unknown. We assessed B-cell maturation in the bone marrow (BM) and periphery in chimeras established with fetal liver progenitors lacking Akt1 and/or Akt2. We found that the generation of marginal zone (MZ) and B1 B cells, 2 key sources of antibacterial antibodies, was highly dependent on the combined expression of Akt1 and Akt2. In contrast, Akt1/2 deficiency did not negatively affect the generation of transitional or mature follicular B cells in the periphery or their precursors in the BM. However, Akt1/2-deficient follicular B cells exhibited a profound survival defect when forced to compete against wild-type B cells in vivo. Altogether, these studies show that Akt signaling plays a key role in peripheral B-cell maturation and survival.

Histone H2AX stabilizes broken DNA strands to suppress chromosome breaks and translocations during V(D)J recombination.

The H2AX core histone variant is phosphorylated in chromatin around DNA double strand breaks (DSBs) and functions through unknown mechanisms to suppress antigen receptor locus translocations during V(D)J recombination. Formation of chromosomal coding joins and suppression of translocations involves the ataxia telangiectasia mutated and DNA-dependent protein kinase catalytic subunit serine/threonine kinases, each of which phosphorylates H2AX along cleaved antigen receptor loci. Using Abelson transformed pre-B cell lines, we find that H2AX is not required for coding join formation within chromosomal V(D)J recombination substrates. Yet we show that H2AX is phosphorylated along cleaved Igkappa DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation. We also show that H2AX prevents chromosome breaks emanating from unrepaired RAG endonuclease-generated TCR-alpha/delta locus coding ends in primary thymocytes. Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation. We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs.

Critical roles of the PI3K/Akt signaling pathway in T cell development.

Thymocyte development requires an integration of extracellular cues to enforce lineage commitment at multiple defined checkpoints in a stage-specific manner. Critical signals from the pre-TCR, Notch, and the receptor for interleukin-7 (IL-7) dictate cellular differentiation from the CD4(-)CD8(-) (double negative) stage to the CD4+CD8+ (double positive) stage. The PI3K/Akt signaling pathway is required to translate these extracellular signaling events into multiple functional outcomes including cellular survival, proliferation, differentiation, and allelic exclusion at the beta-selection checkpoint. However, a complete understanding of the contributions made by the PI3K/Akt pathway in thymocyte development has not been straightforward. This review highlights studies that support the model that the PI3K/Akt pathway is essential for thymocyte survival. We provide new evidence that Akt-mediated survival is not solely due to the increased expression of Bcl-xL but also is a consequence of the role played by Akt to support metabolism in proliferating thymocytes.

Akt1 and Akt2 are required for alphabeta thymocyte survival and differentiation.

The beta-selection checkpoint in alphabetaT lymphocyte development occurs at the double negative (DN) 3 (CD4(-)CD8(-)CD25(+)c-kit(-)) stage, when further differentiation requires a signal from the newly rearranged TCR beta chain. Thymocytes with mutations in key signaling molecules in the phosphatidylinositol 3-kinase-Akt pathway manifest defects in survival, proliferation, and differentiation past the beta-selection checkpoint. However, little information is available regarding the role of Akt itself in thymocyte development. In this study, we explore the role of the two Akt isoforms most highly expressed in the thymus, Akt1 and Akt2, in early T cell development. Using several complementary approaches, we find that deletion of Akt1 results in only minor defects in thymocyte development. The Akt1(-/-)Akt2(-/-) thymocytes manifest a severe developmental block at the DN3 stage and ultimately fail to repopulate the T cell compartment of an irradiated host. Further, we show that Akt1(-/-)Akt2(-/-) DN3 cells have decreased glucose uptake and die in response to TCR stimulation in vitro. Study of thymocytes from the genetically altered mice suggests that the cause of the developmental defect is due to apoptosis, partially caused by decreased cellular growth and metabolism at the DN3 stage. Our results show that Akt protects thymocytes from cell death during the beta-selection checkpoint.

Enfuvirtide resistance mutations: impact on human immunodeficiency virus envelope function, entry inhibitor sensitivity, and virus neutralization.

Enfuvirtide (ENF/T-20/Fuzeon), the first human immunodeficiency virus (HIV) entry inhibitor to be licensed, targets a structural intermediate of the entry process. ENF binds the HR1 domain in gp41 after Env has bound CD4, preventing conformational changes needed for membrane fusion. Mutations in HR1 that confer ENF resistance can arise following ENF therapy. ENF resistance mutations were introduced into an R5- and X4-tropic Env to examine their impact on fusion, infection, and sensitivity to different classes of entry inhibitors and neutralizing antibodies. HR1 mutations could reduce infection and fusion efficiency and also delay fusion kinetics, likely accounting for their negative impact on viral fitness. HR1 mutations had minimal effect on virus sensitivity to other classes of entry inhibitors, including those targeting CD4 binding (BMS-806 and a CD4-specific monoclonal antibody [MAb]), coreceptor binding (CXCR4 inhibitor AMD3100 and CCR5 inhibitor TAK-779), or fusion (T-1249), indicating that ENF-resistant viruses can remain sensitive to other entry inhibitors in vivo. Some HR1 mutations conferred increased sensitivity to a subset of neutralizing MAbs that likely target fusion intermediates or with epitopes preferentially exposed following receptor interactions (17b, 48D, 2F5, 4E10, and IgGb12), as well as sera from some HIV-positive individuals. Mechanistically, enhanced neutralization correlated with reduced fusion kinetics, indicating that, in addition to steric constraints, kinetics may also limit virus neutralization by some antibodies. Therefore, escape from ENF comes at a cost to viral fitness and may confer enhanced sensitivity to humoral immunity due to prolonged exposure of epitopes that are not readily accessible in the native Env trimer. Resistance to other entry inhibitors was not observed.