Characterization of immune responses to anti-PD-1 mono and combination immunotherapy in hematopoietic humanized mice implanted with tumor xenografts.

BACKGROUND: The success of agents that reverse T-cell inhibitory signals, such as anti-PD-1/PD-L1 therapies, has reinvigorated cancer immunotherapy research. However, since only a minority of patients respond to single-agent therapies, methods to test the potential anti-tumor activity of rational combination therapies are still needed. Conventional murine xenograft models have been hampered by their immune-compromised status; thus, we developed a hematopoietic humanized mouse model, hu-CB-BRGS, and used it to study anti-tumor human immune responses to triple-negative breast cancer (TNBC) cell line and patient-derived colorectal cancer (CRC) xenografts (PDX). METHODS: BALB/c-Rag2nullIl2rγnullSIRPαNOD (BRGS) pups were humanized through transplantation of cord blood (CB)-derived CD34+ cells. Mice were evaluated for human chimerism in the blood and assigned into experimental untreated or nivolumab groups based on chimerism. TNBC cell lines or tumor tissue from established CRC PDX models were implanted into both flanks of humanized mice and treatments ensued once tumors reached a volume of ~150mm3. Tumors were measured twice weekly. At end of study, immune organs and tumors were collected for immunological assessment. RESULTS: Humanized PDX models were successfully established with a high frequency of tumor engraftment. Humanized mice treated with anti-PD-1 exhibited increased anti-tumor human T-cell responses coupled with decreased Treg and myeloid populations that correlated with tumor growth inhibition. Combination therapies with anti-PD-1 treatment in TNBC-bearing mice reduced tumor growth in multi-drug cohorts. Finally, as observed in human colorectal patients, anti-PD-1 therapy had a strong response to a microsatellite-high CRC PDX that correlated with a higher number of human CD8+ IFNγ+ T cells in the tumor. CONCLUSION: Hu-CB-BRGS mice represent an in vivo model to study immune checkpoint blockade to human tumors. The human immune system in the mice is inherently suppressed, similar to a tumor microenvironment, and thus allows growth of human tumors. However, the suppression can be released by anti-PD-1 therapies and inhibit tumor growth of some tumors. The model offers ample access to lymph and tumor cells for in-depth immunological analysis. The tumor growth inhibition correlates with increased CD8 IFNγ+ tumor infiltrating T cells. These hu-CB-BRGS mice provide a relevant preclinical animal model to facilitate prioritization of hypothesis-driven combination immunotherapies.

Testing gene function early in the B cell lineage in mb1-cre mice.

The mb1 gene encodes the Ig-alpha signaling subunit of the B cell antigen receptor and is expressed exclusively in B cells beginning at the very early pro-B cell stage in the bone marrow. We examine here the efficacy of the mb1 gene as a host locus for cre recombinase expression in B cells. We show that by integrating a humanized cre recombinase into the mb1 locus we obtain extraordinarily efficient recombination of loxP sites in the B cell lineage. The results from a variety of reporter genes including the splicing factor SRp20 and the DNA methylase Dnmt1 suggest that mb1-cre is probably the best model so far described for pan-B cell-specific cre expression. The availability of a mouse line with efficient cre-mediated recombination at an early developmental stage in the B lineage provides an opportunity to study the role of various genes specifically in B cell development and function.

Different sensitivity to receptor editing of B cells from mice hemizygous or homozygous for targeted Ig transgenes.

Ig knock-in mice have been used to study the relative contribution of receptor selection versus clonal selection in the control of autoreactive B cells. The anti-MHC class I 3-83Ig knock-in (3-83Igi) mice manifest extensive receptor editing in the presence of H-2(b). However, receptor editing is also observed on the H-2(d) background, although reactivity toward this antigen is below detection and its presence does not affect the generation of 3-83Ig(+) mature B cells in classical 3-83Ig transgenic mice. In this study we have analyzed the contribution of genetic background, B cell receptor signaling, and transgene copy number on the initiation and extent of receptor editing in the 3-83Igi;H-2(d) mice. Crossing the 3-83Ig insertion into either CD45-deficient H-2(d) mice or onto the BALB/c background reduces the extent of receptor editing and increases the fraction of 3-83Ig-expressing B cells, indicating that in the original line editing depends on B cell receptor signaling induced by cross-reacting antigen(s). However, receptor editing is still detectable in hemizygous 3-83Igi mice even on the BALB/c background, on which the 3-83 antibody was originally raised, whereas it is abrogated in homozygous 3-83Igi;H-2(d) animals. This latter observation indicates that immature B cells expressing immunoglobulin from single heavy and light chain loci, as they do physiologically, utilize receptor editing for an exquisite quality control of their antigen receptor that may only partly be based on self-reactivity.

From systemic T cell self-reactivity to organ-specific autoimmune disease via immunoglobulins.

Rheumatoid arthritis is a common and debilitating autoimmune disease whose cause and mechanism remain a mystery. We recently described a T cell receptor transgenic mouse model that spontaneously develops a disease with most of the clinical, histological, and immunological features of rheumatoid arthritis in humans. Disease development in K/BxN mice is initiated by systemic T cell self-reactivity; it requires T cells, as expected, but B cells are also needed, more surprisingly. Here, we have identified the role of B cells as the secretion of arthritogenic immunoglobulins. We suggest that a similar scenario may unfold in some other arthritis models and in human patients, beginning with pervasive T cell autoreactivity and ending in immunoglobulin-provoked joint destruction.

Rearrangement and expression of immunoglobulin light chain genes can precede heavy chain expression during normal B cell development in mice.

In mouse mutants incapable of expressing mu chains, VkappaJkappa joints are detected in the CD43(+) B cell progenitors. In agreement with these earlier results, we show by a molecular single cell analysis that 4-7% of CD43(+) B cell progenitors in wild-type mice rearrange immunoglobulin (Ig)kappa genes before the assembly of a productive VHDHJH joint. Thus, mu chain expression is not a prerequisite to Igkappa light chain gene rearrangements in normal development. Overall, approximately 15% of the total CD43(+) B cell progenitor population carry Igkappa gene rearrangements in wild-type mice. Together with the results obtained in the mouse mutants, these data fit a model in which CD43(+) progenitors rearrange IgH and Igkappa loci independently, with a seven times higher frequency in the former. In addition, we show that in B cell progenitors VkappaJkappa joining rapidly initiates kappa chain expression, irrespective of the presence of a mu chain.

Kappa chain monoallelic demethylation and the establishment of allelic exclusion.

Allelic exclusion in kappa light-chain synthesis is thought to result from a feedback mechanism by which the expression of a functional kappa light chain on the surface of the B cell leads to an intracellular signal that down-regulates the V(D)J recombinase, thus precluding rearrangement of the other allele. Whereas such a feedback mechanism clearly plays a role in the maintenance of allelic exclusion, here we provide evidence suggesting that the initial establishment of allelic exclusion involves differential availability of the two kappa alleles for rearrangement. Analysis of kappa+ B-cell populations and of individual kappa+ B cells that have rearranged only one allele demonstrates that in these cells, critical sites on the rearranged allele are unmethylated, whereas the nonrearranged allele remains methylated. This pattern is apparently generated by demethylation that is initiated at the small pre-B cell stage, on a single allele, in a process that occurs prior to rearrangement and requires the presence in cis of both the intronic and 3' kappa enhancers. Taken together with data demonstrating that undermethylation is required for rearrangement, these results indicate that demethylation may actually underly the process of allelic exclusion by directing the initial choice of a single kappa allele for rearrangement.

Ku80 is required for immunoglobulin isotype switching.

Isotype switching is the DNA recombination mechanism by which antibody genes diversify immunoglobulin effector functions. In contrast to V(D)J recombination, which is mediated by RAG1, RAG2 and DNA double-stranded break (DSB) repair proteins, little is known about the mechanism of switching. We have investigated the role of DNA DSB repair in switch recombination in mice that are unable to repair DSBs due to a deficiency in Ku80 (Ku80(-/-)). B-cell development is arrested at the pro-B cell stage in Ku80(-/-) mice because of abnormalities in V(D)J recombination, and there are no mature B cells. To reconstitute the B-cell compartment in Ku80(-/-) mice, pre-rearranged VB1-8 DJH2 (mu i) and V3-83JK2 (kappa i) genes were introduced into the Ku80(-/-) background (Ku80(-/-)mu i/+kappa i/+). Ku80(-/-)mu i/+ kappai/+ mice develop mature mIgM+ B cells that respond normally to lipopolysaccharide (LPS) or LPS plus interleukin-4 (IL-4) by producing specific germline Ig constant region transcripts and by forming switch region-specific DSBs. However, Ku80(-/-)mu i/+kappa i/+ B cells are unable to produce immunoglobulins of secondary isotypes, and fail to complete switch recombination. Thus, Ku80 is essential for switch recombination in vivo, suggesting a significant overlap between the molecular machinery that mediates DNA DSB repair, V(D)J recombination and isotype switching.

V(D)J recombination in mature B cells: a mechanism for altering antibody responses.

The clonal selection theory states that B lymphocytes producing high-affinity immunoglobulins are selected from a pool of cells undergoing antibody gene mutation. Somatic hypermutation is a well-documented mechanism for achieving diversification of immune responses in mature B cells. Antibody genes were also found to be modified in such cells in germinal centers by recombination of the variable (V), diversity (D), and joining (J) segments. The ability to alter immunoglobulin expression by V(D)J recombination in the selective environment of the germinal center may be an additional mechanism for inactivation or diversification of immune responses.

Receptor editing in a transgenic mouse model: site, efficiency, and role in B cell tolerance and antibody diversification.

Mice carrying transgenic rearranged V region genes in their IgH and Igkappa loci to encode an autoreactive specificity direct the emerging autoreactive progenitors into a pre-B cell compartment, in which their receptors are edited by secondary Vkappa-Jkappa rearrangements and RS recombination. Editing is an efficient process, because the mutant mice generate normal numbers of B cells. In a similar nonautoreactive transgenic strain, neither a pre-B cell compartment nor receptor editing was seen. Thus, the pre-B cell compartment may have evolved to edit the receptors of autoreactive cells and later been generally exploited for efficient antibody diversification through the invention of the pre-B cell receptor, mimicking an autoreactive antibody to direct the bulk of the progenitors into that compartment.

A prematurely expressed Ig(kappa) transgene, but not V(kappa)J(kappa) gene segment targeted into the Ig(kappa) locus, can rescue B cell development in lambda5-deficient mice.

We generated surrogate light chain (SLC)-deficient mice carrying either a V(kappa)J(kappa)-C(kappa) transgene under the control of the kappa promoter and intron enhancer or a V(kappa)J(kappa) gene segment targeted into its physiological position. Efficient rescue of B cell development was seen in the former and partial rescue in the latter. This difference corresponded to a developmentally earlier onset of kappa chain expression from the conventional than from the targeted transgene. Thus, a kappa chain can substitute for SLC in development. However, mechanisms controlling gene expression in addition to gene rearrangements appear to restrict kappa chain expression largely to a cellular compartment into which mu chain-expressing B cell progenitors are selected with the help of the SLC.

Glutamate synthase genes of the diazotroph Azospirillum brasilense. Cloning, sequencing, and analysis of functional domains.

A 10-kilobase EcoRI fragment of Azospirillum brasilense genomic DNA was cloned in Escherichia coli. Two open reading frames of 4548 and 1446 base pairs (bp) were identified within the fragment as the structural genes for the alpha and beta subunits (gltB and gltD, respectively) of A. brasilense GltS. The organization of the gltBD region of A. brasilense differs from that of the corresponding region in E. coli: in A. brasilense, gltD is upstream relative to gltB, and its stop codon is separated by 141 bp from the first ATG of gltB. The deduced amino acid sequences reveal a high similarity with GltS from E. coli and with the ferredoxin-dependent GltS from maize. Binding domains for flavin cofactors and NADPH, a domain for glutamine binding and activation, and cysteine clusters for iron-sulfur centers formation were tentatively identified on the basis of sequence comparison with flavoproteins, pyridine nucleotide-dependent enzymes, amidotransferases, and iron-sulfur proteins.