Summary Microglia play critical roles in brain development, homeostasis, and neurological disorders. Here, we report that human microglial-like cells (iMGL) can be differentiated from iPSCs to study their function in neurological diseases, like Alzheimer’s disease (AD). We find that iMGLs develop in vitro similarly to microglia in vivo and whole transcriptome analysis demonstrates that they are highly similar to cultured adult and fetal human microglia. Functional assessment of iMGLs reveals that they secrete cytokines in response to inflammatory stimuli, migrate and undergo calcium transients, and robustly phagocytose CNS substrates. iMGLs were used to examine the effects of Aβ fibrils and brain-derived tau oligomers on AD-related gene expression and to interrogate mechanisms involved in synaptic pruning. Furthermore, iMGLs transplanted into transgenic mice and human brain organoids resemble microglia in vivo. Together, these findings demonstrate that iMGLs can be used to study microglial function, providing important new insight into human neurological disease.

Evidence from C57BL/6 mice suggests that CD8+ T cells, specific to the immunodominant HSV-1 glycoprotein B (gB) H-2b–restricted epitope (gB498–505), protect against ocular herpes infection and disease. However, the possible role of CD8+ T cells, specific to HLA-restricted gB epitopes, in protective immunity seen in HSV-1–seropositive asymptomatic (ASYMP) healthy individuals (who have never had clinical herpes) remains to be determined. In this study, we used multiple prediction algorithms to identify 10 potential HLA-A*02:01–restricted CD8+ T cell epitopes from the HSV-1 gB amino acid sequence. Six of these epitopes exhibited high-affinity binding to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01–positive, HSV-1–seropositive ASYMP individuals, the most frequent, robust, and polyfunctional CD8+ T cell responses, as assessed by a combination of tetramer, IFN-γ-ELISPOT, CFSE proliferation, CD107a/b cytotoxic degranulation, and multiplex cytokine assays, were directed mainly against epitopes gB342–350 and gB561–569. In contrast, in 10 HLA-A*02:01–positive, HSV-1–seropositive symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent clinical herpes disease) frequent, but less robust, CD8+ T cell responses were directed mainly against nonoverlapping epitopes (gB183–191 and gB441–449). ASYMP individuals had a significantly higher proportion of HSV-gB–specific CD8+ T cells expressing CD107a/b degranulation marker and producing effector cytokines IL-2, IFN-γ, and TNF-α than did SYMP individuals. Moreover, immunization of a novel herpes-susceptible HLA-A*02:01 transgenic mouse model with ASYMP epitopes, but not with SYMP epitopes, induced strong CD8+ T cell–dependent protective immunity against ocular herpes infection and disease. These findings should guide the development of a safe and effective T cell–based herpes vaccine.

Some cases of pre-B cell acute lymphoblastic leukemia (pre-B-ALL) are caused by the Philadelphia (Ph) chromosome-encoded BCR-ABL oncogene, and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL-mediated transformation in vitro; however, the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph + pre-B-ALL, we found that deletion of both Pik3r1 and Pik3r2, genes encoding class IA PI3K regulatory isoforms, severely impaired transformation. BCR-ABL-dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes, but the cells were smaller, proliferated more slowly, and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However, they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR, PI-103, was more effective than rapamycin at suppressing proliferation of mouse pre-B-ALL and human CD19 + CD34 + Ph + ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K, alone or together with mTOR, in the treatment of Ph + ALL.

Summary In the CNS, perivascular cells (“pericytes”) associate with endothelial cells to mediate the formation of tight junctions essential to the function of the blood-brain barrier (BBB). The BBB protects the CNS by regulating the flow of nutrients and toxins into and out of the brain. BBB dysfunction has been implicated in the progression of Alzheimer's disease (AD), but the role of pericytes in BBB dysfunction in AD is not well understood. In the developing embryo, CNS pericytes originate from two sources: mesoderm and neural crest. In this study, we report two protocols using mesoderm or neural crest intermediates, to generate brain-specific pericyte-like cells from induced pluripotent stem cell (iPSC) lines created from healthy and AD patients. iPSC-derived pericytes display stable expression of pericyte surface markers and brain-specific genes and are functionally capable of increasing vascular tube formation and endothelial barrier properties.

IntroductionThe Krüppel-like factor (KLF) family of transcription factors is related to the protein Krüppel in Drosophila melanogaster. 1,2 KLFs regulate cell-fate decisions, including development and differentiation of specific tissues. Each contains 3 conserved zinc finger DNA-binding motifs in the c-terminal domain and bind GC-rich regions with a consensus sequence of "CACCC." The functional specificity of distinct KLF family members is maintained through the transactivation and/or repression domains in the N-terminus of the protein and through differential tissue expression. KLF4 is highly expressed in epithelial tissues, and mouse genetic models have established a role for Klf4 in terminal epidermal differentiation. 3,4 KLF4 is also highly expressed in resting lymphocytes but strongly down-regulated in activated cells. 5,6 During human B-cell development, KLF4 is differentially expressed, with greater levels of mRNA in the small pre B-I and pre B-II stages compared with the large pre-B-cell stage. 7 Thus, KLF4 expression in the B lineage correlates with stages with low proliferation, implying a role in B-cell development similar to its function in terminal skin differentiation.Many KLFs have been implicated in suppression of proliferation. For example, KLF6 is a tumor suppressor in human prostate cancer, and disruption of KLF3 (BKLF) function induces a myeloproliferative disorder in mice. 8,9 KLF2, the isoform most closely related to KLF4, has been established as a quiescence factor in T lymphocytes. 10,11 KLF4 itself has been identified as a tumor suppressor gene in colon and gastric cancer based on studies that show loss of heterozygosity, and is also down-regulated in bladder cancer. 12-14 KLF4 promotes cellcycle arrest as shown by serum withdrawal and overexpression studies in multiple cell lines including in NIH 3T3 fibroblasts and RKO cells (a colonic epithelial cancer cell line). 15 KLF4 is up-regulated upon DNA damage in a p53-dependent fashion and can prevent both G 1 -to-S transition and G 2 -to-M transitions in colon cancer cell lines. 15-17 KLF4 promotes expression of the cell-cycle inhibitor protein p21 CIP but in situations where p21 CIP is inactivated, KLF4 can act as a tumor promoter by suppressing p53. 18,19 In a fraction of patients with B-cell acute lymphoblastic leukemia (B-ALL), tumor cells carry the Philadelphia (Ph) chromosome (9;22). The translocation encodes a fusion protein combining the breakpoint cluster region (BCR) protein with the Abelson tyrosine kinase (ABL). 20 Although the ABL kinase inhibitor imatinib has been effective with patients with Ph ϩ chronic myelogenous leukemia, appearance of resistance to this compound has mitigated the prospect of a cure for this leukemia. Furthermore, acute lymphoblastic leukemia caused by BCR-ABL is more refractory to treatment. 21 BCR-ABL, and the related murine oncoprotein v-Abl, activate many mitogenic signaling enzymes and transcription factors that have been extensively studied. 22,23 Identifying factors that must be silenced or inacti...