Heinz Jacobs

Somatic Hypermutation in the Heavy Chain Locus Correlates with Transcription

Three mutant immunoglobulin heavy chain (IgH) insertion mice were generated in which a targeted nonfunctional IgH passenger transgene was either devoid of promoter (pdelta) or was placed under the transcriptional control of either its own RNA polymerase II-dependent IgH promoter (pII) or a RNA polymerase I-dependent promoter (pI). While the transgene mutation-frequency (0.85%) in memory B cells of pI mice was reduced compared to that in pII mice (1.4%), the distribution and the base exchange pattern of point mutations were comparable. In pdelta mice, the mutation frequency was drastically reduced (0.09%). The mutation frequencies correlated with the levels of transgene-specific pre-mRNA expressed in germinal center B cells isolated from the mutant mice.

DNA Double-Strand Breaks in Immunoglobulin Genes Undergoing Somatic Hypermutation

How rearranged immunoglobulin (Ig) genes are further diversified by somatic hypermutation is unknown. Using VDJ passenger Ig heavy chain (IgH) knockin mouse strains, we now demonstrate a high frequency of DNA double-strand breaks (DSBs) in the targeted VDJ passenger gene of germinal center (GC) B cells. These DSBs parallel the distribution of mutations in the targeted hypermutation domain and are found preferentially at RGYW motifs, the intrinsic hot spots of somatic hypermutation. The introduction of DSBs appears to depend on transcriptional activity. Thus, secondary diversification of rearranged V gene segments relates to an error-prone nonhomologous DSB repair system acting in B cells of the GC.

Heterogeneous Differentiation Patterns of Individual CD8+ T Cells

Dynamic Protection During an immune response, CD8+ T cells are recruited to provide protection. Most cells differentiate into short-lived effectors that help to clear the pathogen, whereas others form long-lived memory cells to protect against reinfection. Gerlach et al. (p. 635, published online 14 March) and Buchholz et al. (p. 630, published online 14 March) used in vivo fate mapping of mouse T cells with a defined specificity during a bacterial infection to show that the dynamics of the single-cell response are not uniform. The response of a particular T cell population is the average of a small number of clones that expand greatly (“large clones”) and many clones that only proliferate at low amounts (“small clones”). The memory pool arises largely from small clones whereas effectors are primarily made up of large clones. The single-cell dynamics as cytotoxic T cells respond to a bacterial infection are analyzed in mice. Upon infection, antigen-specific CD8+ T lymphocyte responses display a highly reproducible pattern of expansion and contraction that is thought to reflect a uniform behavior of individual cells. We tracked the progeny of individual mouse CD8+ T cells by in vivo lineage tracing and demonstrated that, even for T cells bearing identical T cell receptors, both clonal expansion and differentiation patterns are heterogeneous. As a consequence, individual naïve T lymphocytes contributed differentially to short- and long-term protection, as revealed by participation of their progeny during primary versus recall infections. The discordance in fate of individual naïve T cells argues against asymmetric division as a singular driver of CD8+ T cell heterogeneity and demonstrates that reproducibility of CD8+ T cell responses is achieved through population averaging.

Skin-resident memory cd8+ t cells trigger a state of tissue-wide pathogen alert

Resident memory T cells sound the alarm Immunological memory protects against reinfection. Resident memory T cells (TRM) are long-lived and remain in the tissues where they first encountered a pathogen (see the Perspective by Carbone and Gebhardt). Schenkel et al. and Ariotti et al. found that CD8+ TRM cells act like first responders in the female reproductive tissue or the skin of mice upon antigen reencounter. By secreting inflammatory proteins, TRM cells rapidly activated local immune cells to respond, so much so that they protected against infection with an unrelated pathogen. Iijima and Iwasaki found that CD4+ TRM cells protected mice against reinfection with intravaginal herpes simplex virus 2. Science, this issue p. 98, p. 101, p. 93; see also p. 40 Resident memory CD8+ T cells orchestrate a broad immune response in response to reinfection. [Also see Perspective by Carbone and Gebhardt] After an infection, pathogen-specific tissue-resident memory T cells (TRM cells) persist in nonlymphoid tissues to provide rapid control upon reinfection, and vaccination strategies that create TRM cell pools at sites of pathogen entry are therefore attractive. However, it is not well understood how TRM cells provide such pathogen protection. Here, we demonstrate that activated TRM cells in mouse skin profoundly alter the local tissue environment by inducing a number of broadly active antiviral and antibacterial genes. This “pathogen alert” allows skin TRM cells to protect against an antigenically unrelated virus. These data describe a mechanism by which tissue-resident memory CD8+ T cells protect previously infected sites that is rapid, amplifies the activation of a small number of cells into an organ-wide response, and has the capacity to control escape variants.

Strand-biased defect in C/G transversions in hypermutating immunoglobulin genes in Rev1-deficient mice

Somatic hypermutation of Ig genes enables B cells of the germinal center to generate high-affinity immunoglobulin variants. Key intermediates in somatic hypermutation are deoxyuridine lesions, introduced by activation-induced cytidine deaminase. These lesions can be processed further to abasic sites by uracil DNA glycosylase. Mutagenic replication of deoxyuridine, or of its abasic derivative, by translesion synthesis polymerases is hypothesized to underlie somatic hypermutation. Rev1 is a translesion synthesis polymerase that in vitro incorporates uniquely deoxycytidine opposite deoxyuridine and abasic residues. To investigate a role of Rev1 in mammalian somatic hypermutation we have generated mice deficient for Rev1. Although Rev1−/− mice display transient growth retardation, proliferation of Rev1−/− LPS-stimulated B cells is indistinguishable from wild-type cells. In mutated Ig genes from Rev1−/− mice, C to G transversions were virtually absent in the nontranscribed (coding) strand and reduced in the transcribed strand. This defect is associated with an increase of A to T, C to A, and T to C substitutions. These results indicate that Rev1 incorporates deoxycytidine residues, most likely opposite abasic nucleotides, during somatic hypermutation. In addition, loss of Rev1 causes compensatory increase in mutagenesis by other translesion synthesis polymerases.

DNA double-strand breaks: prior to but not sufficient in targeting hypermutation.

The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination (CSR) of immunoglobulin (Ig) genes, both of which are associated with DNA double-strand breaks (DSBs). As AID is capable of deaminating deoxy-cytidine (dC) to deoxy-uracil (dU), it might induce nicks (single strand DNA breaks) and also DNA DSBs via a U-DNA glycosylase-mediated base excision repair pathway (‘DNA-substrate model’). Alternatively, AID functions like its closest homologue Apobec1 as a catalytic subunit of a RNA editing holoenzyme (‘RNA-substrate model’). Although rearranged Vλ genes are preferred targets of SHM we found that germinal center (GC) B cells of AID-proficient and -deficient Vλ1-expressing GC B cells display a similar frequency, distribution, and sequence preference of DSBs in rearranged and also in germline Vλ1 genes. The possible roles of DSBs in relation to AID function and SHM are discussed.

Overlapping functions of Hdac1 and Hdac2 in cell cycle regulation and haematopoiesis.

Histone deacetylases (HDACs) counterbalance acetylation of lysine residues, a protein modification involved in numerous biological processes. Here, Hdac1 and Hdac2 conditional knock‐out alleles were used to study the function of class I Hdac1 and Hdac2 in cell cycle progression and haematopoietic differentiation. Combined deletion of Hdac1 and Hdac2, or inactivation of their deacetylase activity in primary or oncogenic‐transformed fibroblasts, results in a senescence‐like G1 cell cycle arrest, accompanied by up‐regulation of the cyclin‐dependent kinase inhibitor p21Cip. Notably, concomitant genetic inactivation of p53 or p21Cip indicates that Hdac1 and Hdac2 regulate p53–p21Cip‐independent pathways critical for maintaining cell cycle progression. In vivo, we show that Hdac1 and Hdac2 are not essential for liver homeostasis. In contrast, total levels of Hdac1 and Hdac2 in the haematopoietic system are critical for erythrocyte‐megakaryocyte differentiation. Dual inactivation of Hdac1 and Hdac2 results in apoptosis of megakaryocytes and thrombocytopenia. Together, these data indicate that Hdac1 and Hdac2 have overlapping functions in cell cycle regulation and haematopoiesis. In addition, this work provides insights into mechanism‐based toxicities observed in patients treated with HDAC inhibitors.

Pertubation of B and T cell development and predisposition to lymphomagenesis in Emu Bmi1 transgenic mice require the Bmi1 RING finger

Proviral activation of the Bmi1 gene has implicated Bmi1 as a collaborator of c-Myc in lymphomagenesis. To determine the effect of Bmi1 overexpression on hema- topoiesis and lymphomagenesis transgenic mice were generated that overexpress different forms of the Bmi1 protein in their lymphoid compartment. EμBmi1 transgenic mice, overexpressing the wild type Bmi1 protein showed a perturbed lymphoid development and were highly susceptible to B and T cell lymphomagenesis. Mutational analysis of the Bmi1 protein demonstrated that the conserved N-terminal RING finger and central part of Bmi1 are essential for its oncogenic potential whereas the C-terminal Pro-Ser rich region is not required. We have used provirus tagging in the EμBmi1 mice to identify genes that cooperate with Bmi1 in lymphomagenesis. MoMLV infection in EμBmi1 transgenic mice accelerated lymphoma development. Proviral activation of the Pim and Myc genes but not the Gfi1 gene were frequently observed in these tumors. These results demonstrate that Bmi1 is a potent oncogene and suggest that it plays an important role in early lymphoid development.

CD27 Is Acquired by Primed B Cells at the Centroblast Stage and Promotes Germinal Center Formation

Studies on human B cells have featured CD27 as a marker and mediator of the B cell response. We have studied CD27 expression and function on B cells in the mouse. We find that B cells acquire CD27 at the centroblast stage and lose it progressively upon further differentiation. It is not a marker for somatically mutated B cells and is present at very low frequency on memory B cells. Enrichment of CD27 among centroblasts and the presence of its ligand CD70 on occasional T and B cells in or near germinal centers (GCs) suggested a role for CD27/CD70 interactions in clonal B cell expansion. Accordingly, GC formation in response to influenza virus infection was delayed in CD27 knockout mice. CD27 deficiency did not affect somatic hypermutation or serum levels of virus-specific IgM, IgG, and IgA attained in primary and recall responses. Adoptive transfer of T and B cells into CD27/CD28−/− mice revealed that CD27 promotes GC formation and consequent IgG production by two distinct mechanisms. Stimulation of CD27 on B cells by CD28+ Th cells accelerates GC formation, most likely by promoting centroblast expansion. In addition, CD27 on T cells can partially substitute for CD28 in supporting GC formation.

A/T mutagenesis in hypermutated immunoglobulin genes strongly depends on PCNAK164 modification.

B cells use translesion DNA synthesis (TLS) to introduce somatic mutations around genetic lesions caused by activation-induced cytidine deaminase. Monoubiquitination at lysine164 of proliferating cell nuclear antigen (PCNAK164) stimulates TLS. To determine the role of PCNAK164 modifications in somatic hypermutation, PCNAK164R knock-in mice were generated. PCNAK164R/K164R mutants are born at a sub-Mendelian frequency. Although PCNAK164R/K164R B cells proliferate and class switch normally, the mutation spectrum of hypermutated immunoglobulin (Ig) genes alters dramatically. A strong reduction of mutations at template A/T is associated with a compensatory increase at G/C, which is a phenotype similar to polymerase η (Polη) and mismatch repair–deficient B cells. Mismatch recognition, monoubiquitinated PCNA, and Polη likely cooperate in establishing mutations at template A/T during replication of Ig genes.