RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci.

Coordinated recombination of homologous antigen receptor loci is thought to be important for allelic exclusion. Here we show that homologous immunoglobulin alleles pair in a stage-specific way that mirrors the recombination patterns of these loci. The frequency of homologous immunoglobulin pairing was much lower in the absence of the RAG-1-RAG-2 recombinase and was restored in Rag1-/- developing B cells with a transgene expressing a RAG-1 active-site mutant that supported DNA binding but not cleavage. The introduction of DNA breaks on one immunoglobulin allele induced ATM-dependent repositioning of the other allele to pericentromeric heterochromatin. ATM activated by the cleaved allele acts in trans on the uncleaved allele to prevent biallelic recombination and chromosome breaks or translocations.

Association between the Igk and Igh immunoglobulin loci mediated by the 3' Igk enhancer induces 'decontraction' of the Igh locus in pre-B cells.

Variable-(diversity)-joining (V(D)J) recombination at loci encoding the immunoglobulin heavy chain (Igh) and immunoglobulin light chain (Igk) takes place sequentially during successive stages in B cell development. Using three-dimensional DNA fluorescence in situ hybridization, here we identify a lineage-specific and stage-specific interchromosomal association between these two loci that marks the transition between Igh and Igk recombination. Colocalization occurred between pericentromerically located alleles in pre-B cells and was mediated by the 3' Igk enhancer. Deletion of this regulatory element prevented association of the Igh and Igk loci, inhibited pericentromeric recruitment and locus 'decontraction' of an Igh allele, and resulted in greater distal rearrangement of the gene encoding the variable heavy-chain region. Our data indicate involvement of the Igk locus and its 3' enhancer in directing the Igh locus to a repressive nuclear subcompartment and inducing the Igh locus to decontract.

RUNX transcription factor-mediated association of Cd4 and Cd8 enables coordinate gene regulation.

T cell fate is associated with mutually exclusive expression of CD4 or CD8 in helper and cytotoxic T cells, respectively. How expression of one locus is temporally coordinated with repression of the other has been a long-standing enigma, though we know RUNX transcription factors activate the Cd8 locus, silence the Cd4 locus, and repress the Zbtb7b locus (encoding the transcription factor ThPOK), which is required for CD4 expression. Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart. Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8. Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells.

Close proximity to Igh is a contributing factor to AID-mediated translocations.

Class switch recombination (CSR) has the potential to generate genomic instability in B cells as activation-induced cytidine deaminase (AID), which mediates this process, is known to target many sites outside Igh. Nonetheless we do not fully understand what factors influence AID targeting genome-wide. Given that errors in CSR can lead to dangerous, oncogenic chromosomal translocations it is important to identify the elements that determine which genes are at risk of being "hit" and could be involved in aberrant rearrangements. Here we have investigated the influence of nuclear organization in determining "off-target" activity and the choice of fusion partners. Our studies indicate that the vast majority of known AID-mediated Igh translocation partners are found in chromosomal domains that contact this locus during class switching. Further, these interaction domains can be used to identify other genes that are hit by AID.

VH replacement in primary immunoglobulin repertoire diversification.

The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. VH replacement represents a RAG-mediated secondary rearrangement in which an upstream VH element recombines with a rearranged VHDHJH joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original VH element and the conventional RSS of the invading VH gene, leaving behind a footprint of up to five base pairs (bps) of the original VH gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that VH replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive VHDHJH allele. Using this mouse model, we characterized the role of VH replacement in the diversification of the primary Ig repertoire through the modification of productive VHDHJH rearrangements. Our results indicate that VH replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies.

Chromosome dynamics and the regulation of V(D)J recombination.

Perhaps no process has provided more insight into the fine manipulation of locus accessibility than antigen receptor rearrangement. V(D)J recombination is carried out by the lymphoid-specific recombination-activating (RAG 1 and 2) proteins and the non-homologous end joining machinery; yet, it occurs only at specific loci (or portions of loci) during specific developmental stages. This spatiotemporal restriction of recombination is achieved through precise alterations in locus accessibility. In this article, we discuss the work of our laboratory in elucidating how nuclear sublocalization, chromosome conformation, and locus interactions contribute to regulating this complex process. We also discuss what is known about how key factors in B-cell development (such as the ubiquitously expressed helix loop helix protein E2A, the B-cell specific transcription factors EBF1 and Pax5, and the interleukin-7 cytokine signaling pathway) exert their effects through changes in nuclear dynamics.

Chronic viral infection alters PD-1 locus subnuclear localization in cytotoxic CD8+ T cells.

During chronic infection, virus-specific CD8+ cytotoxic T lymphocytes (CTLs) progressively lose their ability to mount effective antiviral responses. This "exhaustion" is coupled to persistent upregulation of inhibitory receptor programmed death-1 (PD-1) (Pdcd1)-key in suppressing antiviral CTL responses. Here, we investigate allelic Pdcd1 subnuclear localization and transcription during acute and chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. Pdcd1 alleles dissociate from transcriptionally repressive chromatin domains (lamin B) in virus-specific exhausted CTLs but not in naive or effector CTLs. Relative to naive CTLs, nuclear positioning and Pdcd1-lamina dissociation in exhausted CTLs reflect loss of Pdcd1 promoter methylation and greater PD-1 upregulation, although a direct correlation is not observed in effector cells, 8 days post-infection. Genetic deletion of B lymphocyte-induced maturation protein 1 (Blimp-1) enhances Pdcd1-lamina dissociation in effector CTLs, suggesting that Blimp-1 contributes to maintaining Pdcd1 localization to repressive lamina. Our results identify mechanisms governing Pdcd1 subnuclear localization and the broader role of chromatin dynamics in T cell exhaustion.

A multifunctional element in the mouse Igκ locus that specifies repertoire and Ig loci subnuclear location.

Nonbiased V gene usage for V(D)J joining is essential for providing an optimal immune system, but no cis-acting sequence with this function has been uncovered. We previously identified a recombination silencer and heterochromatin targeting element in the Vκ-Jκ intervening sequence of germline Igκ transgenes, which we termed Sis. We now have generated Sis knockout mice in the endogenous locus. Intriguingly, Sis(-/-) mice exhibit a skewed Igκ repertoire with markedly decreased distal and enhanced proximal Vκ gene usage for primary rearrangement, which is associated with reduced occupancy of Ikaros and CCCTC-binding factor in the Vκ-Jκ intervening sequence in pre-B cells, proteins believed to be responsible for dampening the recombination of nearby Vκ genes and altering higher-order chromatin looping. Furthermore, monoallelic heterochromatin localization is significantly reduced in Sis(-/-) mice for Igκ in cis and IgH loci in trans in pre-B cells. Because Sis(-/-) mice still allelically excluded Igκ and IgH loci and still exhibited IgL isotype exclusion, we concluded that stable localization at pericentromeric heterochromatin is neither necessary nor sufficient for the establishment or maintenance of allelic exclusion. Hence, Sis is a novel multifunctional element that specifies repertoire and heterochromatin localization to Ig genes.

Intratumoral IL12 mRNA Therapy Promotes TH1 Transformation of the Tumor Microenvironment.

PURPOSE: While immune checkpoint inhibitors such as anti-PD-L1 are rapidly becoming the standard of care in the treatment of many cancers, only a subset of treated patients have long-term responses. IL12 promotes antitumor immunity in mouse models; however, systemic recombinant IL12 had significant toxicity and limited efficacy in early clinical trials. EXPERIMENTAL DESIGN: We therefore designed a novel intratumoral IL12 mRNA therapy to promote local IL12 tumor production while mitigating systemic effects. RESULTS: A single intratumoral dose of mouse (m)IL12 mRNA induced IFNγ and CD8+ T-cell-dependent tumor regression in multiple syngeneic mouse models, and animals with a complete response demonstrated immunity to rechallenge. Antitumor activity of mIL12 mRNA did not require NK and NKT cells. mIL12 mRNA antitumor activity correlated with TH1 tumor microenvironment (TME) transformation. In a PD-L1 blockade monotherapy-resistant model, antitumor immunity induced by mIL12 mRNA was enhanced by anti-PD-L1. mIL12 mRNA also drove regression of uninjected distal lesions, and anti-PD-L1 potentiated this response. Importantly, intratumoral delivery of mRNA encoding membrane-tethered mIL12 also drove rejection of uninjected lesions with very limited circulating IL12p70, supporting the hypothesis that local IL12 could induce a systemic antitumor immune response against distal lesions. Furthermore, in ex vivo patient tumor slice cultures, human IL12 mRNA (MEDI1191) induced dose-dependent IL12 production, downstream IFNγ expression and TH1 gene expression. CONCLUSIONS: These data demonstrate the potential for intratumorally delivered IL12 mRNA to promote TH1 TME transformation and robust antitumor immunity.See related commentary by Cirella et al., p. 6080.

B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage.

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.

Durable anticancer immunity from intratumoral administration of IL-23, IL-36γ, and OX40L mRNAs.

Many solid cancers contain dysfunctional immune microenvironments. Immune system modulators that initiate responses to foreign pathogens could be promising candidates for reigniting productive responses toward tumors. Interleukin-1 (IL-1) and IL-12 cytokine family members cooperate at barrier tissues after microbial invasion, in human inflammatory diseases, and in antitumoral immunity. IL-36γ, in classic alarmin fashion, acts in damaged tissues, whereas IL-23 centrally coordinates immune responses to danger signals. In this study, direct intratumoral delivery of messenger RNAs (mRNAs) encoding these cytokines produced robust anticancer responses in a broad range of tumor microenvironments. The addition of mRNA encoding the T cell costimulator OX40L increased complete response rates in treated and untreated distal tumors compared to the cytokine mRNAs alone. Mice exhibiting complete responses were subsequently protected from tumor rechallenge. Treatments with these mRNA mixtures induced downstream cytokine and chemokine expression, and also activated multiple dendritic cell (DC) and T cell types. Consistent with this, efficacy was dependent on Batf3-dependent cross-presenting DCs and cytotoxic CD8+ T cells. IL-23/IL-36γ/OX40L triplet mRNA mixture triggered substantial immune cell recruitment into tumors, enabling effective tumor destruction irrespective of previous tumoral immune infiltrates. Last, combining triplet mRNA with checkpoint blockade led to efficacy in models otherwise resistant to systemic immune checkpoint inhibition. Human cell studies showed similar cytokine responses to the individual components of this mRNA mixture, suggesting translatability of immunomodulatory activity to human patients.

The Conserved ATM Kinase RAG2-S365 Phosphorylation Site Limits Cleavage Events in Individual Cells Independent of Any Repair Defect.

Many DNA lesions associated with lymphoid malignancies are linked to off-target cleavage by the RAG1/2 recombinase. However, off-target cleavage has mostly been analyzed in the context of DNA repair defects, confounding any mechanistic understanding of cleavage deregulation. We identified a conserved SQ phosphorylation site on RAG2 365 to 366 that is involved in feedback control of RAG cleavage. Mutation of serine 365 to a non-phosphorylatable alanine permits bi-allelic and bi-locus RAG-mediated breaks in the same cell, leading to reciprocal translocations. This phenomenon is analogous to the phenotype we described for ATM kinase inactivation. Here, we establish deregulated cleavage itself as a driver of chromosomal instability without the associated repair defect. Intriguingly, a RAG2-S365E phosphomimetic rescues the deregulated cleavage of ATM inactivation, reducing the incidence of reciprocal translocations. These data support a model in which feedback control of cleavage and maintenance of genome stability involves ATM-mediated phosphorylation of RAG2.

It takes two: communication between homologous alleles preserves genomic stability during V(D)J recombination.

Chromosome pairing is involved in X chromosome inactivation, a classic instance of monoallelic gene expression. Antigen receptor genes are also monoallelically expressed ("allelically excluded") by B and T lymphocytes, and we asked whether pairing contributed to the regulation of V(D)J recombination at these loci. We found that homologous immunoglobulin (Ig) alleles pair up during recombination. Homologous Ig pairing is substantially reduced in the absence of the RAG1/RAG2 recombinase, but a transgene expressing an active site RAG1 mutant (which binds but does not cleave DNA) rescues pairing in Rag1(-/-) developing B cells. RAG-mediated cleavage on one allele induces the other allele to relocate to pericentromeric heterochromatin (PCH), likely to ensure that only one allele is cut at a time. This relocation to PCH requires the DNA damage sensor ATM (ataxia telengiectasia mutated). In the absence of ATM, repositioning at PCH is diminished and the incidence of cleavage on both alleles is significantly increased. ATM appears to be activated by the introduction of a double-strand break on one allele to act in trans on the uncleaved allele, repositioning or maintaining it at PCH, to prevent bi-allelic recombination and chromosomal translocations.

Nuclear repositioning marks the selective exclusion of lineage-inappropriate transcription factor loci during T helper cell differentiation.

To address how heritable patterns of gene expression are acquired during the differentiation of Th1 and Th2 cells, we analyzed the nuclear position of lineage-restricted cytokine genes and their upstream regulators by 3-dimensional fluorescence in situ hybridization. During Th1 differentiation, GATA-3 and c-maf loci, which encode upstream regulators of Th2 cytokines, were progressively repositioned to centromeric heterochromatin as defined by a gamma-satellite repeat probe and/or the nuclear periphery, compartments that have been associated with transcriptional repression. A third transcription factor locus, T-bet, which controls Th1-specific programs, was subject to de novo CpG methylation in a Th2 cell clone. In contrast, we did not find repositioning of the cytokine gene loci IL-2, IL-3, IL-4 or IFN-gamma during T helper cell differentiation. Instead, IFN-gamma was constitutively associated with the nuclear periphery, even when primed for expression in Th1 cells. Our results suggest that Th1/Th2 lineage commitment and differentiation involve repositioning of the regulators of cytokine expression, rather than the cytokine genes themselves.