ALBERT

Description: Global monitoring of tropical cyclones (TC) is enhanced by the unique capabilities provided by the day–night band (DNB), a sensor included on the Visible Infrared Imaging Radiometer Suite (VIIRS) flying on board the Suomi National Polar-Orbiting Partnership (SNPP) satellite. The DNB, a low-light visible–near-infrared-band passive radiometer, can leverage unconventional (i.e., nonsolar) sources of visible light illumination such as moonlight to infer storm structure at night. The DNB provides an unprecedented capability to resolve moonlit clouds at high resolution, offering numerous potential benefits to both operational TC analysts and researchers developing new methods of monitoring TCs occurring within the largely data-void tropical oceanic basins. DNB digital data provide significant enhancements over older nighttime visible data from the Defense Meteorological Satellite Program’s (DMSP) Operational Linescan System (OLS) by leveraging accurate calibration, high sensitivity, and sub-kilometer-scale imagery that covers 2–3 times the moon’s lunar cycle than the OLS. By leveraging these attributes, DNB data can enable the use of automated objective applications instead of subjective image interpretation. Here, the authors detail ways in which critical information about TC structure, location, intensity changes, shear environment, lightning, and other characteristics can be extracted when the DNB data are used in isolation or in a multichannel approach with coincident infrared (IR) channels.

Description: Rituximab (Rtx) has shown significant therapeutic activity in follicular lymphoma (FL) patients, yet it’s exact mechanism of action has not been fully defined. Although killing of FL cells through complement dependent cytolysis, antibody dependent cellular cytotoxicity or direct induction of apoptosis may contribute to its effectiveness, these mechanisms are unlikely to be the only ones as; the clinical and molecular responses to Rtx may continue for months after the last dose and; the median duration of the second response to Rtx is longer than that of the first, findings that cannot be explained solely by the above mechanisms. Rtx induced FL cell death likely results in the release of tumor antigens in a pro-inflammatory environment which we hypothesize may provoke a cell-mediated lymphoma specific immune response. Indeed, others have suggested that such a “vaccinal” effect may be an additional mechanism of action but to our knowledge there has been no direct evidence to support this in Rtx treated patients. Methods: To provide support for this hypothesis we examined lymphoma idiotype (Id) specific T-cell responses in peripheral blood mononuclear cells (PBMC) from three patients with relapsed FL. PBMC were obtained prior to and 4–6 weeks after the last of 4 weekly doses (375mg/m2 q week) of Rtx. A lymph node biopsy was obtained prior to Rtx to generate the patient’s Id protein. Dendritic cells (DC) were generated from pre-Rtx PBMC and pulsed with; no protein (control); the patient’s Id protein (Id); or an irrelevant (Irr) protein (Id from another patient). For patient 1, pre- and post-Rtx PBMC were stimulated with the DC for 1 week, while for patients 2 and 3, PBMC were stimulated for 1 week then re-stimulated for another week with fresh DC. Effectors were then assayed in triplicate for IFN-γ producing cells by Elispot. A second independent experiment was conducted in patients 2 and 3 (ie. this study describes data from 3 patients, 5 separate experiments). Results: Pre-Rtx: There was no consistent increase in the number of Id specific or Irr protein specific T-cells as compared to that of control T-cells. Post-Rtx: Whereas there was no consistent increase in the number of Irr protein specific T-cells as compared to that of control T-cells, in all three patients (including both replicates for patients 2 and 3) there was a consistent increase in the number of Id specific T-cells as compared to that of control T-cells. When composite data from all three patients were analyzed using a mixed ANOVA, the following p-values were obtained: Comparison Pre-Rtx Post-Rtx Control vs. Id 0.03 0.0003 Id vs. Irr 0.3 0.0005 Control vs. Irr 0.2 0.9 Comparison Pre-Rtx Post-Rtx Control vs. Id 0.03 0.0003 Id vs. Irr 0.3 0.0005 Control vs. Irr 0.2 0.9 Conclusions: These data provide, to our knowledge, the first support for the hypothesis that Rtx treatment results in an increase in Id specific T-cell responses in FL patients. If indeed this is a mechanism of Rtx activity, then clinical strategies to augment this postulated vaccinal effect, such as anti-CTLA-4 antibodies or Id vaccination post Rtx, may further increase the clinical potential of this agent and change the way we develop combination therapies. Further study of immune responses in a larger number of FL patients treated with Rtx is warranted and ongoing.

Description: The incorporation of rituximab, a chimeric anti-CD20 monoclonal antibody, into the therapeutic armamentarium for patients with follicular lymphoma (FL) has significantly improved treatment outcome for such patients. Despite the almost universal application of this therapy, however, its exact mechanism of action has not been completely defined. One proposed mechanism is that of a “vaccinal” effect, whereby FL cell kill by rituximab results in the elicitation of an FL-specific T-cell response. The demonstration that rituximab can even elicit such a response in patients has, to our knowledge, never been shown. We analyzed the response against the immunoglobulin expressed by the FL before and after rituximab monotherapy in 5 FL patients and found an increase in FL idiotype–specific T cells after rituximab in 4 of 5 patients. Our data thus provide “proof of principle” for the ability of passive immunotherapy with rituximab to elicit an active FL-specific cellular response.

Description: Regulatory T-cells (Tregs) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor reactive effector T-cells. We demonstrate that follicular lymphoma (FL) T-cells are hypo-responsive to CD3/CD28 costimulation, as assessed by proliferation of CFSE (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester) labeled cells, with only 3.11% ± 2.38 and 2.26% ± 1.76 of the CD8+ and CD4+ T-cells proliferating upon stimulation, respectively (n=7). In contrast, both normal lymph node (NLN), and reactive lymph node (RLN, lymphoid hyperplasia) T-cells proliferate significantly in response to costimulation. Specifically, NLN CD8+ and CD4+ T-cells demonstrate 35.2% ± 31.1 and 18.1% ± 15.9 cells proliferating upon stimulation, respectively (n=7). Similarly, upon stimulation, RLN CD8+ and CD4+ T-cells demonstrate 40.6% ± 22.6 and 40.3% ± 30.3 cells proliferating, respectively (n=5). We identify a population of FL infiltrating CD4+CD25+GITR+ T-cells that are significantly overrepresented within FL, 9.86% ± 6.70 (n=11) of the CD4+ T-cells, as compared to that seen in NLN, 0.70% ± 0.29 (n=13), or RLN, 1.40% ± 1.04 (n=5). These cells actively suppress the proliferation of autologous nodal CD8+ and CD4+ T-cells after costimulation, as CD25+ magnetic bead depletion of these cells in vitro restores proliferation of the remaining CD25− T-cells. Specifically, proliferation of FL CD8+CD25− and CD4+CD25− T-cells increases to 24.05% ± 11.46 and 10.53% ± 6.47, respectively, upon costimulation (n=4). The CD25+ enriched cell fraction contains functionally suppressive cells since add back of unlabelled CD25+ enriched cells to CFSE labeled CD25− cells results in a decrease in proliferation of the costimulated CD8+CD25− and CD4+CD25− T-cells, namely 7.59% ± 3.86 and 4.16% ± 1.79, respectively (n=4). These cells also suppress cytokine production (IFN-g, TNF-a and IL-2) from autologous nodal T-cells as assessed by multiplex analysis of culture supernatants. In addition to suppressing autologous nodal T-cells, the FL CD25+ enriched cells are also capable of suppressing proliferation of allogeneic CD8+CD25− and CD4+CD25− T-cells from NLN as well as normal donor peripheral blood lymphocytes (PBL), regardless of very robust stimulation of the target cells with plate bound anti-CD3 and anti-CD28 antibodies. The allogeneic suppression is not reciprocal, since CD25+ enriched cells derived from either NLN or normal donor PBL, used at the same ratio, are not capable of suppressing allogeneic CD8+CD25− and CD4+CD25− T-cells derived from FL and in fact, are less suppressive against autologous T-cells than are the FL derived CD4+CD25+ cells. Whether this is due to a higher proportion of functionally suppressive T cells within the FL derived CD25+ enriched cells, compared to that of NLN or normal donor PBL, or to an increased suppressive capacity of the FL derived CD25+ T cells is currently being investigated. These data show that FL infiltrating CD4+CD25+GITR+ T-cells have a phenotype and function consistent with Tregs and are very potent suppressors of lymphoma associated-CD8+ and CD4+ T-cells, and therefore may play an important role in lymphoma development, progression and response to treatment.

Description: As we previously described, intra-peritoneal injection of single cell suspensions (including non-neoplastic cells) from 〉90% of primary B cell non-Hodgkin's lymphoma specimens results in lymphoma cell growth in the omentum of the NSG mouse strain (NOD-scid IL2rg null) (ASH Meeting on Lymphoma Biology 2014; abstract #144). To date, we have shown reproducible engraftment of 17/20 specimens (including 9 follicular, 5 marginal zone, 4 diffuse large B cell, 2 mantle cell). Molecular markers specific for each patient's tumor (including clone specific features of the IGK, IGH, BCL2 and BCL1 loci) provided verification that all 17 of the engrafted tumors contained the same clone identified in the original specimen. Each tumor was implanted and engrafted in at least 3 mice (range 3-41) and each had a characteristic and often distinctive time course of engraftment, propensity to disseminate, and histologic features, indicating that the behaviors of the xenografts reflected intrinsic properties of the original tumors. B cell engraftment required CD4+ T cells. B cells were purified by negative selection from three specimens with particularly robust engraftment, a FL (J1) and two MZL (M1 and W1). Depletion of non-B cells entirely ablated engraftment for all 3 tumors; mock depletion had no effect. Selective depletion of CD4+ but not CD8+ T cells ablated engraftment of both tumors assessed (J1 and M1). Finally, purified CD4+ cells (but not CD8+) fully reconstituted engraftment of purified B lymphoma cells from tumor M1. In addition, the xenograft model highlighted two unexpected features of these tumors: 1) The neoplastic B cells of some tumors were relatively resistant to engraftment compared to non-neoplastic B cells present in the same specimen. For 5 tumors (2 MZL and 3 FL), the number of human cells and the ratio of neoplastic to non-neoplastic B cells in the xenografts at two weeks were determined by performing bar-coded, ultra-deep sequencing on the entire mesentery including the omentum. For the 2 MZL, the fraction of B cells that was clonal was 〉 90% at 2 weeks ; in contrast, for 3 FL specimens, the fraction of B cells that were neoplastic dropped from 〉60% at time of implantation to less than 5%. These results indicate that neoplastic B cells do not necessarily have an engraftment advantage over non-neoplastic B cells. Where non-neoplastic cells can respond to proliferation/survival cues, neoplastic cells in the same FL specimens apparently resist the same signals. 2) We observed that B cells, including neoplastic B cells, have a striking propensity to differentiate into plasma cells when engrafted in the mice. In all specimens at the time of injection, neoplastic B cells comprised 〉60% of the cells and plasma cells were negligible. In contrast, at 2-4 weeks the number of CD20+ B cells fell to 20% of the cells in all xenografts. 4/17 xenografts (including 2 FL and 2 MZL tumors) assayed showed monoclonality of the plasma cells at two weeks (based on kappa:lambda light chain ratio). These results indicate that the neoplastic B cells of some tumors have a developmental plasticity in the xenograft that is not typically apparent in patients. In summary, a xenograft model reveals several features of low grade B cell lymphomas that are not apparent from static observations: 1) CD4+ T cells are essential to engraftment, suggesting that CD4+ T cells may be critical to lymphomagenesis; 2) neoplastic B cells of some tumors do not respond to the proliferation/survival cues to which non-neoplastic B cells are sensitive; 3) neoplastic B cells of some tumors differentiate into post-mitotic plasma cells, potentially eliminating these cells from the proliferative pool. Disclosures No relevant conflicts of interest to declare.

Description: Previous studies have suggested that murine T cells are tolerant to epitopes derived from germ line variable regions of immunoglobulin (Ig) heavy (VH) or light chains. This has lead to the prediction that germ line VH-region epitopes found in neoplastic B cells cannot be used to provoke an antitumor immune response. To test these assumptions and address the question of how such a vaccine may alter the normal B-cell response, an antibody-forming B-cell hybridoma (1H6) expressing a conserved germ line VH gene with specificity for dextran was generated and used as a tumor model. Using algorithms for predicting major histocompatibility complex (MHC) binding, potential MHC class I and II binding peptides were identified within the 1H6 VH region, synthesized, and tested for MHC binding and immunogenicity. We show that germ line VH peptides, when presented by dendritic cells, are immunogenic in vitro and provoke a tumor-specific protective immune response in vivo. We conclude that (1) it is possible to induce a T-cell response to germ line VH peptides; (2) such peptides can be used to generate a B-cell tumor-specific vaccine; and (3) a vaccine targeting VH peptides expressed by the dominant dextran-specific B-cell clonotype had no effect upon the magnitude of the normal B-cell response to dextran.