Hematopoietic expression of a chimeric murine-human CALR oncoprotein allows the assessment of anti-CALR antibody immunotherapies in vivo.

Myeloproliferative neoplasms (MPNs) are characterized by a pathologic expansion of myeloid lineages. Mutations in JAK2, CALR and MPL genes are known to be three prominent MPN disease drivers. Mutant CALR (mutCALR) is an oncoprotein that interacts with and activates the thrombopoietin receptor (MPL) and represents an attractive target for targeted therapy of CALR mutated MPN. We generated a transgenic murine model with conditional expression of the human mutant exon 9 (del52) from the murine endogenous Calr locus. These mice develop essential thrombocythemia like phenotype with marked thrombocytosis and megakaryocytosis. The disease exacerbates with age showing prominent signs of splenomegaly and anemia. The disease is transplantable and mutCALR stem cells show proliferative advantage when compared to wild type stem cells. Transcriptome profiling of hematopoietic stem cells revealed oncogenic and inflammatory gene expression signatures. To demonstrate the applicability of the transgenic animals for immunotherapy, we treated mice with monoclonal antibody raised against the human mutCALR. The antibody treatment lowered platelet and stem cell counts in mutant mice. Secretion of mutCALR did not constitute a significant antibody sink. This animal model not only recapitulates human MPN but also serves as a relevant model for testing immunotherapeutic strategies targeting epitopes of the human mutCALR.

Assessment of lymphocyte development in radiation bone marrow chimeras.

Transplantation of marrow between mouse strains congenic for CD45 after lethal irradiation establishes hematopoiesis driven by genetically marked cells in recipient animals. After several weeks, peripheral blood or primary and secondary lymphoid organs of transplant recipients can be evaluated for the presence of donor-derived cells. Two- or three-color flow cytometry can be used to identify the progeny of transplanted cells, to document their cell-surface phenotypes, and to follow development of T, B, and myeloid lineages in vivo.

In vivo assessment of the relative contributions of deletion, anergy, and editing to B cell self-tolerance.

In normal B cell development, a large percentage of newly formed cells bear receptors with high levels of self-reactivity that must be tolerized before entry into the mature B cell pool. We followed the fate of self-reactive B cells expressing high affinity anti-hen egg lysozyme (HEL) Ag receptors exposed in vivo to membrane HEL in a setting in which the anti-HEL L chain was "knocked-in" at the endogenous L chain locus. These mice demonstrated extensive and efficient L chain receptor editing responses and had B cell numbers comparable to those found in animals lacking membrane Ag. BrdU labeling indicated that the time required for editing in response to membrane HEL was approximately 6 h. In mice transgenic for soluble HEL, anti-HEL B cells capable of editing showed evidence for both editing and anergy. These data identify receptor editing as a major physiologic mechanism by which highly self-reactive B cells are tolerized to membrane and soluble self-Ags.

Quantitative assessment of the stem cell self-renewal capacity.

Little is known about the manner in which hematopoietic stem cells (HSCs) self-renew. To address this issue, we used a serum-free single-cell culture, followed by transplantation of cultured cells into lethally irradiated mice. CD34-negative or low, c-Kit-positive, Sca-1-positive, lineage marker-negative (CD34-KSL) cells are highly enriched for murine bone marrow HSCs. Successful long-term reconstitution with a single CD34-KSL cell enabled us to study in vitro self-renewal of HSC at clonal level. Using this clonal cell transplantation system, we examined the effect of various cytokines on CD34-KSL cells. Among the cytokines examined, stem cell factor (SCF) and thrombopoietin (TPO) were minimum cytokines to induce cell division of CD34-KSL cells most efficiently. Similarly, multilineage repopulating activity was detected in the cells derived from a significant portion of single cells after culture in the presence of TPO and SCF. However, SCF + IL-3, SCF + IL-6, or SCF + IL-11 + FL appeared to be less effective for self-renewal of HSCs. The activity of HSCs as indicated by repopulation unit (RU) remaining after culture with SCF and TPO was not so different from that of freshly isolated HSCs. However, there was a substantial loss of HSC number in these cultured cells. Taken together, this study provides definitive proof that one HSC can generate at least one HSC in vitro.

Assessment of lymphocyte development in radiation bone marrow chimeras.

The Basic Protocol utilizes congenic strains of mice that differ at the CD45 (common leukocyte antigen) locus. Bone marrow-derived cells from these strains of mice can be identified by monoclonal antibodies specific for the two allelic variants of the CD45 molecule. Because this antigen is expressed on all nucleated blood cells, it provides a convenient genetic marker for following the progeny of transplanted bone marrow cells. Transplantation of marrow between mouse strains congenic for CD45 after lethal irradiation establishes hematopoiesis driven by genetically marked cells in recipient animals. The first support protocol can be used to establish appropriate radiation doses for use in the Basic Protocol. After several weeks, peripheral blood or primary and secondary lymphoid organs of transplant recipients can be evaluated for the presence of donor-derived cells using the second support protocol. Two- or three-color flow cytometry can be used to identify the progeny of transplanted cells, to document their cell-surface phenotypes, and to follow development of T, B, and myeloid lineages in vivo.

Assessment of multi-organ system engraftment by genotypic typing using restriction fragment-length polymorphisms and by phenotypic typing using a microcytotoxicity assay.

We report the first application of Southern blotting techniques for the quantitative assessment of the donor or host origin of cell populations present in recipients of allogeneic or sex-mismatched syngeneic murine donor marrow grafts. The sensitivity of this assay system was noted to 1 to 5% for detection of a minor cell population by using cDNA probes that hybridize to single-copy sequences in the murine genome. The use of probes that generate distinguishing autoradiographic patterns due to strain-specific genomic sequence variations obviates the need for retroviral vector transfections (which potentially skew engraftment quantitation). Southern blotting analysis has provided definitive engraftment data in multiple cell populations isolated from both short-term and long-term allogeneic and syngeneic radiation chimeras. In contrast, H-2 typing in a microcytotoxicity assay, a standard typing technique for allogeneic murine donor cell engraftment, was noted to be less sensitive than Southern blotting. This occurred particularly in selected cell populations, in ill-appearing recipients, and in the early post-BMT period. Furthermore, because H-2 typing is a phenotypic assay, the results may be substantially influenced by the passive cell surface acquisition of host H-2 antigens, a process that is not evident with the use of genotyping techniques. Our results establish the superiority of Southern blotting techniques for the quantitation of donor cell engraftment and demonstrate the potential of this methodology when low-level detection of engrafted donor or residual host cells is of critical physiologic importance.

Characterization of antigen-specific, Ia-restricted, L3T4+ cytolytic T lymphocytes and assessment of thymic influence on their self specificity.

The goals of the present study were: (a) to generate antigen-specific L3T4+ cytolytic T lymphocytes (CTL), (b) to determine their major histocompatibility complex (MHC) restriction specificity, and (c) to assess the influence of thymic MHC determinants on their self specificity. We found that L3T4+ CTL specific for either trinitrophenyl (TNP)-modified self determinants or minor histocompatibility antigens could be generated from Lyt-2- responder T cells provided that the response cultures were supplemented with supernatants rich in helper factors. Such antigen-specific L3T4+ CTL were Ia-restricted by the criteria that they lysed only Ia+ target cells and that their lysis of Ia+ target cells was specifically inhibited by anti-Ia monoclonal antibodies. The relative frequency of L3T4+ pCTL was found to be only 5-10% of the total anti-TNP pCTL present in the spleens of normal mice. Finally, we utilized radiation bone marrow chimeras to assess the influence of the thymic haplotype on the self-Ia specificity of L3T4+ CTL. Both bulk culture and limiting dilution experiments revealed that the self-Ia specificity of L3T4+ anti-TNP CTL from F1----parent and A----B allogeneic chimeras was not markedly skewed toward the haplotype of the chimeric thymus. These results contrast with those obtained previously for L3T4+ anti-TNP Th cells and demonstrate that in the radiation bone marrow chimera model of T cell differentiation, the self specificity of Th cells but not pCTL is markedly influenced by the haplotype of the chimeric thymus.

Assessment of immunocompetence by limiting dilution analysis in long-term T cell depletion chimeras transplanted across the MHC barrier.

Chimeras were generated in a system in which donor C57BL/6 bone marrow plus spleen cells were T-cell-depleted prior to transplantation into lethally irradiated DBA/2 recipients. This protocol permits donor lymphohematopoietic engraftment and protects transplanted mice from development of lethal GVHD. The frequencies of alloantigen-specific cytotoxic T cells (CTL) and/or CTL precursors (CTL-P) in the chimera spleens were determined by limiting dilution analysis. This identified a small population of host-reactive CTL-P. The presence of host-reactive CTL-P in the absence of detectable anti-host immune response raises questions concerning the maintenance of the tolerant state in chimeras. Using mixtures of chimera and normal C57BL/6 splenocytes we found no evidence by limiting-dilution analysis for regulatory cells capable of dampening antihost immune reactivity in chimera spleens. We next measured the frequency of third-party-reactive CTL-P in chimeras. Chimeras displayed low CTL-P frequency by the 30th day posttransplant, which increased 15-21-fold over a five-month interval. Interestingly, both chimeric and irradiated syngeneic reconstituted control mice recovered anti-third-party CTL-P at a similar rate, but CTL-P levels never reached those measured in normal unirradiated control mice, suggesting that the radiation regimen has a long-lasting influence on host immunocompetence. In concomitant experiments we measured third-party CTL generation in MLC. Our findings suggest that measurement of CTL generation in MLC may be a less sensitive assessment of immunocompetence than LDA analysis. Our data also suggest that irradiated T-cell-depleted chimeras may suffer prolonged immunologic deficiencies based on reduced frequencies of alloreactive CTL-P.