(18)F-FDG-PET/CT imaging in an IL-6- and MYC-driven mouse model of human multiple myeloma affords objective evaluation of plasma cell tumor progression and therapeutic response to the proteasome inhibitor ixazomib.

(18)F-fluorodeoxyglucose positron emission tomography (FDG-PET) and computed tomography (CT) are useful imaging modalities for evaluating tumor progression and treatment responses in genetically engineered mouse models of solid human cancers, but the potential of integrated FDG-PET/CT for assessing tumor development and new interventions in transgenic mouse models of human blood cancers such as multiple myeloma (MM) has not been demonstrated. Here we use BALB/c mice that contain the newly developed iMyc(ΔEµ) gene insertion and the widely expressed H2-L(d)-IL6 transgene to demonstrate that FDG-PET/CT affords an excellent research tool for assessing interleukin-6- and MYC-driven plasma cell tumor (PCT) development in a serial, reproducible and stage- and lesion-specific manner. We also show that FDG-PET/CT permits determination of objective drug responses in PCT-bearing mice treated with the investigational proteasome inhibitor ixazomib (MLN2238), the biologically active form of ixazomib citrate (MLN9708), that is currently in phase 3 clinical trials in MM. Overall survival of 5 of 6 ixazomib-treated mice doubled compared with mice left untreated. One outlier mouse presented with primary refractory disease. Our findings demonstrate the utility of FDG-PET/CT for preclinical MM research and suggest that this method will play an important role in the design and testing of new approaches to treat myeloma.

Delineation among eight major hematopoietic subsets in murine bone marrow using a two-color flow cytometric technique.

BACKGROUND: Many methods have been developed specifically for identifying hematopoietic progenitor cells in murine bone marrow, but few methods allow rapid identification of multiple bone marrow populations. We describe a new, simple method for identifying simultaneously eight populations in murine bone marrow with two-color flow cytometry and phenotypically define these populations. METHODS: Bone marrow was stained with anti-Ly-6C and anti-B220 (CD45R) in one fluorochrome and wheat germ agglutinin (WGA) in another fluorochrome. The eight populations identified in this way were defined further primarily by four-color flow cytometry. RESULTS: Six of the eight populations were characterized phenotypically as containing erythroid, granulocytic, mast, early B, mature B, and stem cell populations. Two additional populations with phenotypic characteristics of partially differentiated precursor cells also were identified. One population was Ly-6C/B220+ and WGA-. It also expressed markers associated with early B, T, and/or dendritic cell differentiation. The second population was Ly-6C(hi)WGA(hi)Mac-1+ and was negative for numerous other lineage-specific and precursor markers. Its morphology suggested monocytic differentiative potential. CONCLUSIONS: A two-color flow cytometric assay profiles six bone marrow populations with identifiable phenotypes and two additional unique, putative hematopoietic precursor populations.

The immune response modifiers imiquimod and R-848 are potent activators of B lymphocytes.

Imiquimod and R-848 are members of a family of immune response modifiers that stimulate cytokine production in monocyte/macrophages and dendritic cell cultures. This study evaluated the effects of the imidazoquinolines, imiquimod and R-848, on B lymphocyte activation. Both agents induced proliferation of murine T-cell-depleted and highly purified splenic B cell preparations as well as purified human B cells. Resting and activated B cells responded to these agents, with activated cells responding more efficiently. B cells from the LPS-hyporesponsive C3H/HeJ mice and guanosine-hyporesponsive SJL mice proliferated in response to imiquimod and R-848, indicating a different mechanism of action than lipopolysaccharide and guanine nucleosides. B cells were also stimulated by imiquimod and R-848 to produce increased immunoglobulin levels. Increased expression of a number of B cell activation markers were seen following imiquimod or R-848 stimulation. Finally, R-848 was shown to act as a vaccine adjuvant enhancing OVA-specific IgG2a levels while suppressing total IgE. These results indicate that R-848 and imiquimod are potent activators of B lymphocytes and are capable of augmenting antigen-specific immunoglobulin production.

SLP-76 expression is restricted to hemopoietic cells of monocyte, granulocyte, and T lymphocyte lineage and is regulated during T cell maturation and activation.

The leukocyte-specific adapter protein SLP-76 is known to augment the transcriptional activity of nuclear factor of activated T cells and AP-1 following TCR ligation. A role for SLP-76 in additional receptor-mediated signaling events is less clear. To define the pattern of SLP-76 expression during murine hemopoiesis, we stained cells isolated from various tissues with a combination of surface markers followed by intracellular staining with a fluorochrome-labeled SLP-76-specific Ab. In the bone marrow, SLP-76 expression is largely restricted to cells of granulocyte and monocyte lineage. Heterogeneous SLP-76 expression is first detected in the CD44+ CD25- subset within the CD3- CD4- CD8- thymocyte population. Interestingly, SLP-76 expression increases as thymocyte maturation progresses within the CD4- CD8- compartment but decreases as cells mature to a CD4+ CD8+ phenotype. SLP-76 expression is then up-regulated following selection and concomitant with maturation to a CD4+ or CD8+ phenotype. In the periphery, SLP-76 is expressed in T lymphocytes with no detectable expression in the B cell compartment. Exposure to the superantigen staphylococcal enterotoxin B augments SLP-76 expression in the reactive T cell subset. Furthermore, in vitro stimulation with TCR-specific Abs augments the existing levels of SLP-76. These data reveal that SLP-76 expression is coordinately regulated with surface expression of a pre-TCR or mature TCR complex during thymocyte development and that TCR ligation elicits signals that result in increased expression of SLP-76.

CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen.

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODN) cause B cell proliferation and Ig secretion, monocyte cytokine secretion, and activation of NK cell lytic activity and IFN-gamma secretion in vivo and in vitro. The potent immune activation by CpG ODN suggests possible utility for enhancing immune responses to vaccines. Mice immunized with recombinant hepatitis B virus surface Ag and a CpG ODN as an immune enhancer have titers of Abs against HBsAg (anti-HBs) that are five times higher than those of mice immunized with HBsAg and the standard adjuvant, aluminum hydroxide (alum). Ab titers in mice immunized with HBsAg and both CpG ODN plus alum were 35 times higher than the titers in mice immunized with alum alone, indicating a strong synergistic interaction between the CpG ODN and alum. ODN without CpG motifs had little or no immune-enhancing activity at the doses used herein. Alum induces a Th2 humoral response (mostly IgG1) and no CTL. In contrast, CpG ODN gives a strong Thl response with predominantly IgG2a Abs and CTL, even when mixed with alum. In vitro studies to determine possible mechanisms of CpG immune-enhancing effects show that CpG ODN induce expression of costimulatory molecules on Ag-presenting cells and drive B cell isotype switching in the appropriate cytokine milieu. These studies demonstrate that CpG ODN are promising new immune enhancers for vaccination applications.

Differential expression of CD22 (Lyb8) on murine B cells.

Previous studies have established the distribution, biochemistry and functional attributes of human CD22, a B cell-restricted glycoprotein. Recently, molecular cloning of the murine CD22 equivalent revealed this molecule to be the same as the previously described Lyb8 alloantigen. Using the anti-Lyb8 mAb Cy34.1.2, the present report documents the expression patterns of CD22 within the murine B cell compartment. The results demonstrate that in the bone marrow, murine CD22 is absent on the surface of pro-B cells, pre-B cells and newly emerging IgM+ B cells. CD22 is present at a low density on immature IgMhi B cells and fully expressed on mature recirculating B cells. In the periphery, murine CD22 is expressed at mature levels on all B cell subsets including follicular, marginal zone, B1 and switched B cells. Further studies showed CD22 to be retained on activated murine B cells for extended periods. Finally, in combination with CD23 and heat stable antigen, CD22 can be used to delineate the immature splenic B cells, and distinguish them from follicular and marginal zone cells. Together, the results demonstrate murine CD22 to be a useful pan marker for all mature B cell subsets.

Effect of pregnancy on thymic T cell development.

PROBLEM: The thymus gland decreases in size during pregnancy. The significance of this alteration is not known. METHOD: In this report, we examined thymic function by evaluating the development of T lymphocytes in the thymus of pregnant Balb/c mice at 15 and 20 days gestation using multi-color flow cytometry. Comparative analysis was made with non-pregnant mice, post-partum lactating mice, and postpartum non-lactating mice. RESULTS: Progressive reduction of thymic size and cellularity during pregnancy was observed. All of the CD4 and CD8 defined subsets were reduced, with a disproportionate loss of CD4+, CD8+ double positive cells. Examination of the CD4-, CD8- double negative compartment revealed a predominance of TCR alpha, beta+ double negative cells, and a striking loss of precursor cells. The CD3-, CD4-, CD8- triple negative thymic subset was composed almost entirely of the earliest population (CD44+, CD25-), with the remaining maturational stages (CD44+, CD25+; CD44-, CD25+; and CD44-, CD25-) depleted. At 2 weeks postpartum, the subset ratios normalized, and the total cell count showed recovery. CONCLUSION: T cell development is blocked at the precursor level during the mouse pregnancy. These effects are transient, and gradual recovery is observed in the postpartum period.

An essential role for gp39, the ligand for CD40, in thymic selection.

The interactions between CD40 on B cells and its ligand gp39 on activated T helper cells are known to be essential for the development of thymus-dependent humoral immunity. However, CD40 is also functionally expressed on thymic epithelial cells and dendritic cells, suggesting that gp39-CD40 interactions may also play a role in thymic education, the process by which self-reactive cells are deleted from the T cell repertoire. Six systems of negative selection were studied for their reliance on gp39-CD40 interactions to mediate negative selection. In all cases, when the antigen/superantigen was endogenously expressed (in contrast to exogenously administered), negative selection was blocked by loss of gp39 function. Specifically, blockade of gp39-CD40 interactions prevented the deletion of thymocytes expressing V beta 3, V beta 11, and V beta 12, specificities normally deleted in BALB/c mice because of the endogenous expression of minor lymphocyte-stimulating determinants. Independent verification of a role of gp39 in negative selection was provided by studies in gp39-deficient mice where alterations in T cell receptor (TCR) V beta expression were also observed. Studies were also performed in the AND TCR transgenic (Tg) mice, which bear the V alpha 11, V beta 3 TCR and recognize both pigeon cytochrome c (PCC)/IEk and H-2As. Neonatal administration of anti-gp39 to AND TCR Tg mice that endogenously express H-2As or endogenously produce PCC prevented the deletion of TCR Tg T cells. In contrast, deletion mediated by high-dose PCC peptide antigen (administered exogenously) in AND TCR mice was unaltered by administration of anti-gp39. In addition, deletion by Staphylococcus enterotoxin B in conventional mice was also unaffected by anti-gp39 administration. gp39 expression was induced on thymocytes by mitogens or by antigen on TCR Tg thymocytes. Immunohistochemical analysis of B7-2 expression in the thymus indicated that, in the absence of gp39, B7-2 expression was substantially reduced. Taken together, these data suggest that gp39 may influence negative selection through the regulation of costimulatory molecule expression. Moreover, the data support the hypothesis that, for negative selection to some endogenously produced antigens, negative selection may be dependent on TCR engagement and costimulation.

The effect of in vivo IL-7 deprivation on T cell maturation.

A number of previous studies have suggested a key role for interleukin 7 (IL-7) in the maturation of T lymphocytes. To better assess the function of IL-7 in lymphopoiesis, we have deprived mice of IL-7 in vivo by long-term administration of a neutralizing anti-IL-7 antibody. In a previous report (Grabstein, K. H., T. J. Waldschmidt, F. D. Finkelman, B. W. Hess, A. R. Alpert, N. E. Boiani, A. E. Namen, and P. J. Morrissey. 1993. J. Exp. Med. 178:257-264), we used this system to demonstrate the critical role of IL-7 in B cell maturation. After a brief period of anti-IL-7 treatment, most of the pro-B cells and all of the pre-B and immature B cells were depleted from the bone marrow. In the present report, we have injected anti-IL-7 antibody for periods of up to 12 wk to determine the effect of in vivo IL-7 deprivation on the thymus. The results demonstrate a > 99% reduction in thymic cellularity after extended periods of antibody administration. Examination of thymic CD4- and CD8- defined subsets revealed that, on a proportional basis, the CD4+, CD8+ subset was most depleted, the CD4 and CD8 single positive cells remained essentially unchanged, and the CD4-, CD8- compartment actually increased to approximately 50% of the thymus. Further examination of the double negative thymocytes demonstrated that IL-7 deprivation did, indeed, deplete the CD3-, CD4-, CD8- precursors, with expansion of this subset being interupted at the CD44+, CD25+ stage. The proportional increase in the CD4-, CD8- compartment was found to be due to an accumulation of CD3+, T cell receptor alpha, beta + double negative T cells. Additional analysis revealed that anti-IL-7 treatment suppressed the audition/selection process of T cells, as shown by a significant reduction of single positive cells expressing CD69 and heat stable antigen. Finally, the effects of IL-7 deprivation on the thymus were found to be reversible, with a normal pattern of thymic subsets returning 4 wk after cessation of treatment. The present results thus indicate a central role for IL-7 in the maturation of thymic-derived T cells.

B-cell subsets defined by the Fc epsilon R.

The data summarized herein demonstrate the utility of the low-affinity Fc epsilon R in delineating murine B-cell subsets. In the peritoneal cavity, the Fc epsilon R appears to be a reliable marker in distinguishing between conventional (Fc epsilon R+) and Ly-1/sister (Fc epsilon R-) B cells. In the spleens of normal animals, flow cytometric and histologic studies established that a distinct population of Fc epsilon R- B cells is also present and comprises the marginal zones. Thus in the spleen, the Fc epsilon R may be the first murine marker to allow for selective purification and analysis of marginal zone B cells. Although it is unlikely that splenic Fc epsilon R- B cells are directly related to peritoneal Fc epsilon R- Ly-1/sister B cells, further studies will be required to address this question. Analysis of autoimmune mice revealed that the splenic Fc epsilon R- subset is greatly expanded in these animals and indicates that the Fc epsilon R may be a sensitive indicator of abnormalities within the B-cell compartment. Additional studies compared the functional capacity of Fc epsilon R+ and Fc epsilon R- B cells and tested the ability of these populations to isotype-switch and respond to polyclonal stimuli. The results showed that Fc epsilon R+ and Fc epsilon R- B cells from both the peritoneum and spleen can switch to produce IgG, and all but the peritoneal Fc epsilon R- B cells can switch to the IgE class. This latter result is certainly interesting and demonstrates an important functional difference between peritoneal and splenic Fc epsilon R- B cells. Finally, experiments with B-cell mitogens showed further differences between the Fc epsilon R+ and Fc epsilon R- subsets. Whereas Fc epsilon R- B cells appeared to be more sensitive to LPS stimulation, Fc epsilon R+ B cells were clearly more responsive to an anti-IgM signal. Taken together, the results show that the Fc epsilon R is likely to be useful in separating B cells with different phenotypic, histologic, and functional characteristics.

The expression of B cell surface receptors. III. The murine low-affinity IgE Fc receptor is not expressed on Ly 1 or 'Ly 1-like' B cells.

The distribution of IgE FcR (Fc epsilon R)-positive and -negative B cells was examined in normal adult mice. Using three-color flow cytometry, the expression of the Fc epsilon R was analyzed on various B-cell subsets present in the peritoneum and spleen. The results demonstrate that in the peritoneal cavity, the Fc epsilon R is not expressed on the large majority of Ly 1+ B cells and Ly 1-, Mac 1+ sister B cells. The receptor is present, however, on the small number of conventional B cells residing in the peritoneum. Although interleukin 4 (IL-4) can increase the levels of the Fc epsilon R on conventional B cells, incubation of Ly 1 and sister B cells with IL-4 did not result in the expression of the Fc epsilon R. When examining B cells present in the spleen, a small subset of B cells was consistently found to be Fc epsilon R-. These Fc epsilon R- cells were IgM-bright, IgD-dull and largely Ly 1- and Mac 1-negative. Staining of splenic tissue sections revealed that the Fc epsilon R- B cells were primarily localized to the marginal zones, whereas the Fc epsilon R+ B cells were found in the follicles. Taken together, the results indicate that the Fc epsilon R may be a useful marker in delineating the various B-cell subsets. In the peritoneum, the Fc epsilon R appears to discriminate conventional B cells from those of the Ly 1/sister lineage, and in the spleen it is likely to distinguish resting follicular B cells from Ly 1/sister and marginal zone B cells.