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 Igκ 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-α/δ 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

Akt signaling maintains survival and proliferation in immature T cells and hematopoietic stem cells

Hematopoietic development and cell fate choices are regulated by signals from the extracellular environment. This study focuses on two developmental checkpoints in the hematopoietic system that are characterized by unique proliferative stages: (1) The DN3 (double negative 3) to DP (double positive) transition, otherwise known as β-selection, in T cell development and (2) The regulation of self-renewal in long-term hematopoietic stem cells. This thesis tests the hypothesis that Akt, a serine/threonine kinase, is required for both of these proliferative stages of hematopoietic development. The first set of experiments test whether a constitutively active Akt molecule is sufficient to rescue survival, proliferation, and differentiation in a mouse model that fails to generate mature T cells due to a block at the DN3 stage. In the second set of experiments, we ask if Akt is necessary for T cell development, specifically at the DN3 to DP transition. After defining the biological role for Akt signaling in thymocyte development, we next determine whether Akt also has a similar role in the development of other hematopoietic lineages. The hematopoietic stem cell pool is another exquisitely regulated developmental stage in hematopoiesis that is dependent on cell survival and proliferation. In the last set of experiments, we test the hypothesis that Akt would also be required for maintenance of the most primitive hematopoietic stem cell. In summary, we found that Akt is sufficient to promote differentiation to the DP stage in the absence of signals from the pre-TCR, and that Akt1 and Akt2 are also required for differentiation at the same pre-TCR mediated checkpoint. The biological role of Akt in T cell development is similar to its function in long-term hematopoietic stem cells (LT-HSCs). LT-HSCs require Akt signals to maintain self-renewal and survival of more mature progenitor cells. In conclusion, these studies provide evidence that Akt is required in proliferating hematopoietic cells during development, but is not required at the more quiescent stage of hematopoiesis

Akt signaling maintains survival and proliferation in immature T cells and hematopoietic stem cells

Hematopoietic development and cell fate choices are regulated by signals from the extracellular environment. This study focuses on two developmental checkpoints in the hematopoietic system that are characterized by unique proliferative stages: (1) The DN3 (double negative 3) to DP (double positive) transition, otherwise known as β-selection, in T cell development and (2) The regulation of self-renewal in long-term hematopoietic stem cells. This thesis tests the hypothesis that Akt, a serine/threonine kinase, is required for both of these proliferative stages of hematopoietic development. The first set of experiments test whether a constitutively active Akt molecule is sufficient to rescue survival, proliferation, and differentiation in a mouse model that fails to generate mature T cells due to a block at the DN3 stage. In the second set of experiments, we ask if Akt is necessary for T cell development, specifically at the DN3 to DP transition. After defining the biological role for Akt signaling in thymocyte development, we next determine whether Akt also has a similar role in the development of other hematopoietic lineages. The hematopoietic stem cell pool is another exquisitely regulated developmental stage in hematopoiesis that is dependent on cell survival and proliferation. In the last set of experiments, we test the hypothesis that Akt would also be required for maintenance of the most primitive hematopoietic stem cell. In summary, we found that Akt is sufficient to promote differentiation to the DP stage in the absence of signals from the pre-TCR, and that Akt1 and Akt2 are also required for differentiation at the same pre-TCR mediated checkpoint. The biological role of Akt in T cell development is similar to its function in long-term hematopoietic stem cells (LT-HSCs). LT-HSCs require Akt signals to maintain self-renewal and survival of more mature progenitor cells. In conclusion, these studies provide evidence that Akt is required in proliferating hematopoietic cells during development, but is not required at the more quiescent stage of hematopoiesis

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

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

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 G0 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