antitumor immunity ͉ type I interferon ͉ syngenic tumor ͉ implant model ͉ gene-disrupted mice

Mycobacterium tuberculosis (M. tb) is an intracellular pathogen that can replicate within infected macrophages. The ability of M. tb to arrest phagosome maturation is believed to facilitate its intracellular multiplication. Rab GTPases regulate membrane trafficking, but details of how Rab GTPases regulate phagosome maturation and how M. tb modulates their localization during inhibiting phagolysosome biogenesis remain elusive. We compared the localization of 42 distinct Rab GTPases to phagosomes containing either Staphylococcus aureus or M. tb. The phagosomes containing S. aureus were associated with 22 Rab GTPases, but only 5 of these showed similar localization kinetics as the phagosomes containing M. tb. The Rab GTPases responsible for phagosome maturation, phagosomal acidification and recruitment of cathepsin D were examined in macrophages expressing the dominant-negative form of each Rab GTPase. LysoTracker staining and immunofluorescence microscopy revealed that Rab7, Rab20 and Rab39 regulated phagosomal acidification and Rab7, Rab20, Rab22b, Rab32, Rab34, Rab38 and Rab43 controlled the recruitment of cathepsin D to the phagosome. These results suggest that phagosome maturation is achieved by a series of interactions between Rab GTPases and phagosomes and that differential recruitment of these Rab GTPases, except for Rab22b and Rab43, to M. tb-containing phagosomes is involved in arresting phagosome maturation and inhibiting phagolysosome biogenesis.

SummarySite-specific phosphorylation of intermediate filament (IF) proteins on serine and threonine residues leads to alteration of the filament structure, in vitro and in vivo. Protein kinases involved in cell signaling and those activated in mitosis dynamically control spatial and temporal organization of intracellular IF phosphorylation. Thus, IF phosphorylation appears to be one of the most predominant strategies in coordinating intracellular organization of the IF Accepted 6 March 1996 network.

We report the identification of two novel minor histocompatibility antigens (mHAgs), encoded by two separate single nucleotide polymorphisms on a single gene, BCL2A1, and restricted by human histocompatibility leukocyte antigen (HLA)-A*2402 (the most common HLA-A allele in Japanese) and B*4403, respectively. Two cytotoxic T lymphocyte (CTL) clones specific for these mHAgs were first isolated from two distinct recipients after hematopoietic cell transplantation. Both clones lyse only normal and malignant cells within the hematopoietic lineage. To localize the gene encoding the mHAgs, two-point linkage analysis was performed on the CTL lytic patterns of restricting HLA-transfected B lymphoblastoid cell lines obtained from Centre d'Etude du Polymorphisme Humain. Both CTL clones showed a completely identical lytic pattern for 4 pedigrees and the gene was localized within a 3.6-cM interval of 15q24.3–25.1 region that encodes at least 46 genes. Of those, only BCL2A1 has been reported to be expressed in hematopoietic cells and possess three nonsynonymous nucleotide changes. Minigene transfection and epitope reconstitution assays with synthetic peptides identified both HLA-A*2402– and B*4403-restricted mHAg epitopes to be encoded by distinct polymorphisms within BCL2A1.

The Mycobacterium bovis bacillus Calmette-Guérin cell-wall skeleton (BCG-CWS) activates Toll-like receptor (TLR) 2 and TLR4, but unlike the typical TLR4 agonist bacterial lipopolysaccharide barely induces type 1 IFN. BCG-CWS has been used for adjuvant immunotherapy for patients with cancer. We investigated the adjuvant potential of BCG-CWS for induction of CTLs subsequent to TLR-mediated dendritic cell (DC) maturation, using a syngeneic mouse tumor model (B16 melanoma in C57BL/ 6). We evaluated the retardation of tumor growth and cytotoxic response in wild-type and MyD88؊/؊ mice immunized with tumor debris and/or BCG-CWS. Delays in tumor growth and cytotoxic response were induced by immunization with a mixture of BCG-CWS emulsion and the tumor. BCG-CWS was capable of activating DCs ex vivo by the criteria of CD80/CD86 up-regulation and cytokine (interleukin-12, tumor necrosis factor-␣) induction. Efficient tumor suppression and ex vivo cytokine induction did not occur in MyD88-deficient mice and cells, suggesting that the MyD88 adapter is crucial for induction of tumor cytotoxicity. Because TLR4 is involved in both MyD88-dependent and -independent pathways and the latter affects DC maturation, our findings indicate that both pathways cooperate to induce CTL-based tumor immunity.

Chk1, one of the critical transducers in DNA damage/replication checkpoints, prevents entry into mitosis through inhibition of Cdk1 activity. However, it has remained unclear how this inhibition is cancelled at the G 2 /M transition. We reported recently that Chk1 is phosphorylated at Ser 286 and Ser 301 by Cdk1 during mitosis. Here, we show that mitotic Chk1 phosphorylation is accompanied by Chk1 translocation from the nucleus to the cytoplasm in prophase. This translocation advanced in accordance with prophase progression and was regulated by Crm-1-dependent nuclear export. Exogenous Chk1 mutated at Ser 286 and Ser 301 to Ala (S286A/S301A) was observed mainly in the nuclei of prophase cells, although such nuclear accumulation was hardly observed in wild-type Chk1. Induction of S286A/S301A resulted in the delay of mitotic entry. Biochemical analyses using immunoprecipitated cyclin B 1 -Cdk1 complexes revealed S286A/S301A expression to block the adequate activation of Cdk1. In support of this, S286A/S301A expression retained Wee1 at higher levels and Cdk1-induced phosphorylation of cyclin B 1 and vimentin at lower levels. A kinase-dead version of S286A/S301A also localized predominantly in the nucleus but lost the ability to delay mitotic entry. These results indicate that Chk1 phosphorylation by Cdk1 participates in cytoplasmic sequestration of Chk1 activity, which releases Cdk1 inhibition in the nucleus and promotes mitotic entry.

Cytomegalovirus (CMV)-specific T-cell immunity plays an important role in protection from CMV disease in immunocompromised patients. Identification of cytotoxic T-lymphocyte (CTL) epitopes is essential for monitoring T-cell immunity and also for immunotherapy. In this and previous studies, CMV-pp65-specific CTL lines were successfully generated from all of 11 CMV-seropositive healthy donors, using pp65-transduced CD40

Here we report the identification of a novel human leukocyte antigen (HLA)-B44-restricted minor histocompatibility antigen (mHA) with expression limited to hematopoietic cells. cDNA expression cloning studies demonstrated that the cytotoxic T lymphocyte (CTL) epitope of interest was encoded by a novel allelic splice variant of HMSD, hereafter designated as HMSD-v. The immunogenicity of the epitope was generated by differential protein expression due to alternative splicing, which was completely controlled by 1 intronic single-nucleotide polymorphism located in the consensus 5 splice site adjacent to an exon. Both HMSD-v and HMSD transcripts were selectively expressed at higher levels in mature dendritic cells and primary leukemia cells, especially those of myeloid lineage. Engraftment of mHA ؉ myeloid leukemia stem cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID)/␥c null mice was completely inhibited by in vitro preincubation with the mHA-specific CTL clone, suggesting that this mHA is expressed on leukemic stem cells. The patient from whom the CTL clone was isolated demonstrated a significant increase of the mHA-specific T cells in posttransplantation peripheral blood, whereas mHA-specific T cells were undetectable in pretransplantation peripheral blood and in peripheral blood from his donor. These findings suggest that the HMSD-v-encoded mHA (designated ACC - IntroductionMinor histocompatibility antigens (mHAs) are major histocompatibility complex (MHC)-bound peptides derived from cellular proteins encoded by polymorphic genes. Following human leukocyte antigen (HLA)-matched allogeneic hematopoietic cell transplantation (HCT), donor-recipient disparities in mHAs can induce a favorable graft-versus-leukemia (GVL) effect that is often associated with graft-versus-host disease (GVHD). 1-3 Significant efforts have been made to identify mHAs, particularly those specific for hematopoietic cells, since such mHAs are speculated to contribute to the GVL effect. The first report on the identification of a hematopoietic lineage-specific mHA, HA-1, was generated by the Goulmy group in 1998 (den Haan et al 4 ) as a result of biochemical analysis of peptides eluted from HLA-A*0201 molecules. The only other mHAs with selective expression in hematopoietic cells described to date are HA-2 5 ; ACC-1 and ACC-2 6 ; and DRN-7, 7 HB-1, 8,9 and PANE1, 10 the latter 2 of which are B-cell lineage-specific. Thus, identification of more mHAs should facilitate a better understanding of the biology of GVL and the development of effective immunotherapy to induce GVL reactions.Immunogenicity of most autosomal mHAs identified to date results from single-nucleotide polymorphisms (SNPs) that cause amino-acid substitutions within epitopes, leading to the differential display/recognition of peptides between HCT donor and recipient via several mechanisms: peptide binding to MHC observed in HA-1/A2-, 4 HA-2-, 5 and CTSH-encoded mHAs 11 ; proteasomal cleavage in HA-3 12 ; peptide transport in HA-8 13 ; and altered recognition o...