bluuebunny/crossref_metadata_2025 huggingface dataset:
A Search for Genes Specifying HSCs through the Development of a New Retroviral-Based System That Creates Nested Deletions in ES cells.
Authors: Bilodeau Melanie , Sauvageau Guy
Published: October 2019

With the intention of identifying novel DNA segments essential for hematopoietic stem cell (HSC) specification, we have taken advantage of retroviral-based delivery of loxP sequences to ES cells in order to create large genomic deletions. Cre-induced recombination between the integrated loxP-containing proviruses is selected based on the reconstitution of a functional neomycin resistance cassette. A unique set of complementary retroviruses (virus A and D) was finally identified after the generation and testing of more than 10 different cassettes. The nature of the rearrangements produced (deletions, inversions, etc.) was determined according to the sensitivity to selection marker genes incorporated inside the viruses and confirmed by Southern blot analysis of genomic DNA extracted from selected clones. The new technology was first tested on 11 different clones infected at a low MOI of virus D (to obtain one integrant per cell). This virus, selected under puromycin, contains also a neomycin resistance gene (neor), devoid of an initiation codon (ATG) and of a functional promoter. The proviral integration sites are now easily determined by inverse PCR using primers anchored at the neor gene. The second retrovirus (virus A), selected with hygromycin, integrates randomly another loxP site and a pgk promoter which precedes the ATG-loxP cassette. For the 11 primary clones tested to date, the number of secondary clones derived ranged between 7000 and 37 000. Recombination between the loxP sites, upon Cre addition, is selected by the functional reconstitution of the neomycin gene (i.e. pgk-ATG-loxP-neor). Out of 107cells electroporated with the cre plasmid, neomycin resistant clones were obtained for each population (ranging between 7 and 188). Rearranged tertiary clones (i.e., virus D+A+Cre) derived from a selected primary clone (no.9) have been studied extensively by Southern Blot analysis. Chromosomal rearrangements correlated with bands of expected sizes (3.0 kb compare to 3.4 kb for the unrearranged primary and secondary clones, using KpnI digests and a neomycin probe). Clonal analysis for integration of the second retrovirus (virus A) revealed at least 13 different deletions out of 34 tested. In one experiment, very preliminary results suggest that 3 out of these 13 clones bearing independent deletions failed to differentiate upon LIF removal. Among the other 10 primary clones saturated to date, 9 gave tertiary clones sensitive to puromycin (undergoing selection). Overall, these results document that our novel retroviral-based strategy is amenable to functional screening of genes that specify HSC in differentiating ES cells. Moreover, this technology is efficient (11 out of 11 clones tested), rapid and applicable to any cellular system.
HoxA10 Dramatically Alters the Immunophenotype of Early Hematopoietic Progenitors Derived In Vitro from ES Cells.
Authors: Iacovino Michelina , Osawa Mitsujiro , Kyba Michael K.
Published: October 2019

Hox genes are important regulators of normal hematopoiesis, where each step in differentiation from stem cell to differentiated progeny is associated with a particular Hox expression pattern. In addition, abnormal or disregulated Hox gene expression is often associated with leukaemia. In order to understand how Hox gene transcriptional networks govern normal and leukemic hematopoiesis, we have generated ES cell lines in which expression of representative Hox genes can be induced with doxycycline during in vitro differentiation. Previous work showed that the relatively 3′ Hox gene, HoxB4, promotes self-renewal of hematopoietic stem cells, as well as enabling a primitive to definitive hematopoietic transition. We report here the generation of an ES cell line with inducible expression of a relatively 5′ Hox gene, HoxA10. When expressed during the time at which the hematopoietic lineage first segregates from the endothelial lineage (day 4 of embryoid body (EB) differentiation), HoxA10 dramatically alters the immunophenotype of the EB hematopoietic compartment. Whereas 100% of hematopoietic colony forming cells (CFC) from a wild-type day 6 EB are double positive for c-kit and CD41, HoxA10 expression from day 4 to day 6 eliminates the CD41-positive population, while increasing the c-kit-positive population. CFCs were present at relatively normal numbers. In contrast, HoxB4 did not dramatically alter either the frequency or the immunophenotype of this early hematopoietic population, but significantly enhanced the frequency of mixed-lineage CFCs. We further compared the ability of these two Hox genes to promote the outgrowth of an undifferentiated hematopoietic population on OP9 from day 6 EBs. Whereas HoxB4 induced a rapid expansion of this population, concomitantly with ongoing myeloid commitment, HoxA10 induced a much slower outgrowth characterized by a prominent B-lymphoid component as well as myeloid and erythroid elements. We are currently assaying this HoxA10-induced hematopoietic population for adult repopulating activity.
Increased Non-Canonical Splicing of Autoantigen Transcripts Provides the Structural Basis for Expression of Untolerized Epitopes.
Authors: Yang Xiao-Feng , Ng Bernard , Yang Fan , Huston David P. , Peterson Leif E. , Wang Hong
Published: October 2019

Alternative splicing is important for increasing the complexity of the human proteome from a limited genome. Previous studies have shown that for some autoantigens, there is differential immunogenicity among alternatively spliced isoforms. In this report, we tested the hypothesis that alternative splicing is a common feature for transcripts of autologous proteins that are autoantigens. The corollary hypothesis tested was that non-autoantigen transcripts have a lower frequency of alternative splicing. We compared the extent of alternative splicing within 45 randomly selected self-proteins associated with autoimmune diseases to 9554 randomly selected proteins encoded in human genome using bioinformatics analysis. Isoform specific regions that result from alternative splicing were studied for their potential to be epitopes for antibodies or T cell epitopes. Our results demonstrate for the first time that alternative splicing occurred in 100% of the autoantigen transcripts. This is significantly higher that the approximately 42% rate of alternative splicing observed in 9554 randomly selected human gene transcripts. In addition, the majority of isoform-specific regions of these autoantigens encoded MHC restricted T cell antigen epitopes and autoantibody binding epitopes. Furthermore, 80% of the autoantigen transcripts underwent non-canonical alternative splicing, which is also significantly higher than the less than 1% rate in randomly selected gene transcripts. These studies suggest that non-canonical alternative splicing may be an important mechanism for the generation of untolerized epitopes that may lead to autoimmunity. Furthermore, the product of a transcript that does not undergo alternative splicing is unlikely to be a target antigen in autoimmunity.
Analysis of CD161+ T Cells in Human Peripheral Blood.
Authors: Takahashi Tsuyoshi , Dejbakhsh-Jones Sussan , Strober Samuel
Published: October 2019

CD161 (NKR-P1) is a natural killer receptor of C-type lectin superfamily found on natural killer (NK) cells. A significant number of both human CD4+ and CD8+ T cells express CD161. However, little is known about the extended phenotype or function of CD161+ T cells. Here, we analyzed this population in normal human peripheral blood by immunofluorescent staining, and multi-color flow cytometric analysis (N=15). Amongst CD4+ T cells, a mean of 22% showed intermediate staining for CD161 (CD161int) and the remainder were CD161−. Almost all the CD4+CD161int TCRαβ+ T cells had the CD45RO+ memory phenotype, and did not express the CD16 or CD56 NK markers (<5%). CD4+CD161− T cells were a mixture of CD45RO+ and CD45RA+ cells. Invariant natural killer T (NKT) cells with the Vα24+/Vβ11+ TCR are known to express CD161, but CD4+CD161int T cells contained less than 1 % of these invariant NKT cells. After in vitro stimulation with αCD3 and αCD28 mAb, CD4+CD161int T cells produced larger amounts of both Th1 and Th2 type cytokines compared with CD4+CD161− T cells, especially IFN-γ, IL-4 and IL-10. However, there was no difference in the proliferative response between CD161− and CD161int CD4+ T cells, and CD4+CD161int T cells had no suppressor functions against autologous cell proliferation. In CD8+ T cells, there were two populations that were CD161 positive. One was CD161hi (mean; 11%) and another was CD161int (mean; 9%). Almost all CD161int and CD161hi CD8+ T cells had the CD45RO+ memory phenotype, and few expressed the Vα24+/Vβ11+ TCR NKT cell marker (<1%) or CD16 (<5%). CD8+CD161hi cells contained around 15% of CD56+ and CD8+CD161int cells contained around 5% of CD56+. CD8+CD161hi T cells had decreased expression of CD8β and 40% of CD8+CD161hi T cells expressed only CD8α. Conversely, most of the CD8α+CD8β− T cells in human peripheral blood expressed CD161. After in vitro stimulation with αCD3 and αCD28 mAb, CD8+CD161hi T cells secreted no IFN-γ, TNF-α or IL-2. Both CD8+CD161− and CD8+CD161int T cells produced all three cytokines. CD8+CD161hi T cells failed to proliferate after αCD3+αCD28 stimulation, though CD8+CD161− and CD8+CD161int T cells made vigorous proliferative responses. Thus, it appeared that CD8+CD161hi T cells were anergic. This anergic state could not be reversed by addition of IL-2. CD8+CD161+ T cells had no suppressor function against autologous cell proliferation nor cytotoxicity against the NK or NKT sensitive tumor cell lines, K562 and Jurkat. In conclusion, the CD161 marker can be used to identify subsets of CD4+ and CD8+ T cells that differ in their extended phenotypes and functions. In addition, we identified a unique subset of anergic CD8+CD161hi T cells that express a memory phenotype with a low level of CD8β.
The Spectrum of Human T Cell Receptor (TCR)-Vβ Frequencies Are Established Prior to Thymic Selection.
Authors: Melenhorst Jan J. , Follmann Dean , Fujiwara Hiroshi , Barrett Austin J.
Published: October 2019

Human T cell development is characterized by three phases of clonal selection and expansion: (1) β-selection which occurs independent of ligand recognition; (2) Positive thymic selection resulting in the outgrowth of T cell clones with an intermediate affinity for self-MHC/ self-peptide complexes; (3) Post-thymic antigen-driven expansion. The T cell receptor (TCR) Vβ frequencies in the peripheral blood could therefore be determined by T cell selection and expansion at one or all phases. To determine whether Vβselection patterns originate at the first phase of thymic proliferation we used flow cytometric analysis to quantitate 21 TCRVβ proteins in peripheral blood T cells of 23 HLA-disparate healthy adults. A common rank order of TCRVβ allele frequencies was found in both CD4 and CD8 T cell subsets (figure A). We found the same spectrum of HLA-independent TCR Vβ frequencies in three published studies of healthy adults. Furthermore, the percentages of CD4 cells expressing a particular Vβ were almost identical in a total of 151 data points, and was significantly correlated in CD8 cells (R2 = 0.79; slope 0.61; p < 0.00001). A significant correlation in TCRVβ allele frequencies in CD4 cells and CD8 cells was identified in all data sets (R2 = 0.73, slope 0.54, p < 0.00001)(figure B). Individuals over 50 years of age showed a 5-fold increase in variability of Vβ frequency in CD4 cells than younger subjects. The difference in the median values was statistically significant (p = 0.03) using a permutation test, indicating that post-thymic antigen recognition skewed the mature αβ T cell repertoire predominantly in elderly individuals. Our data therefore indicate that the patterns of TCRVβ protein frequencies are shaped during β-selection, conserved throughout life and are only weakly modified by antigen-driven thymic and post-thymic selection and expansion. A genetic basis for this underlying TCR Vβ frequency pattern remains undefined since there was no association of TCRVβ expression frequencies with chromosomal (5′-3′) organization. However, the reproducible pattern of TCR Vβ frequencies between individuals of diverse HLA types and distinct origins suggests that they are determined by a common genetic mechanism.

Figure Figure
The Human Plasma Cell Gene Expression Program.
Authors: De Vos John , Hose Dirk , Reme Thierry , Goldschmidt Hartmut , Rossi Jean-Francois , Cremer Friedrich , Klein Bernard
Published: October 2019

Seven purified peripheral blood memory B-cells (BM), seven in-vitro-generated polyclonal plasmablastic cells (PPC) and seven purified bone marrow mature plasma cells (BMPC) were studied by oligonucleotide microarrays. All samples were obtained from healthy volunteers. The gene expression profiling of these samples was determined with Affymetrix pan genomic U133A + B arrays (44 928 oligonucleotide probesets). We determined that 2313 genes were differentially expressed between these three B cell categories (P 〈 0.01 by a Kruskal-Wallis test and a ratio between two categories 〉 3).

These 2313 genes were classified into six categories, according to the expression profile: early plasma cell genes (EPC), late plasma cell genes (LPC), genes lost early during plasma cell differentiation (LEPC), genes lost late during plasma cell differentiation (LLPC), genes upregulated only in plasmablasts (PBO) and genes lost only in plasmablasts (LPBO). As expected, Ig transcripts where essentially classified as EPC. As a corollary, genes involved in protein synthesis or degradation, transmembrane transporters and metabolism genes were overrepresented in EPC genes. Interestingly, genes involved in intercellular communication and extracellular matrix were enriched in LPC, highlighting the fact that mature plasma cells develop tight interactions with the bone marrow environment. Of note, genes involved in cell cycle are upregulated mainly in plasmablasts, whereas antiapoptotic genes are lost in plasmablasts only.

Mains genes known to be involved in plasma cell differentiation display an expression profile in agreement with published data, as illustrated for transcription factors in Figure 1, validating this DNA microarray dataset. However most of these 2313 genes have either never been described yet or have no yet been linked to plasma cell differentiation. The description of those genes among our genome whose expression vary most during plasma cell differentiation will be an essential step in understanding the biology of a cell type essential to immune defenses and involved in deadly diseases.

Figure 1: Transcription factors involved in plasma cell differentiation. Color indicates the expression profile category. For each gene is given the ratio of the mean expression value in plasma cell samples (PPC and BMPC) to the mean expression value in BM. UPR: Unfolded Protein Response. Figure 1:. Transcription factors involved in plasma cell differentiation. Color indicates the expression profile category. For each gene is given the ratio of the mean expression value in plasma cell samples (PPC and BMPC) to the mean expression value in BM. UPR: Unfolded Protein Response.
VCAM-1 Expression in B-Lymphopoiesis and Leukemia.
Authors: Young Faith M. , Felgar Raymond E. , Eyssallenne Antonia P. , Bottaro Andrea , Bushnell Timothy P.
Published: October 2019

Vascular Cell Adhesion Molecule-1 (VCAM; CD106), a member of the Ig Superfamily of molecules, binds to the β-1 integrin, Very Late Antigen-4 (VLA-4; CD49d); this interaction plays an integral role in leukocyte trafficking as well as lymphocyte-stromal cell interactions. VCAM can be shed from the surface of cells, and, in humans, serum levels of soluble VCAM (sVCAM) parallel activity and remission states in acute lymphocytic leukemia (ALL) and inflammation. Although widely investigated as a stromal-cell associated molecule, our lab and others have recently identified VCAM expression on normal bone-marrow derived B-lymphoid cells.

Using FACS technology, we found that surface expression of VCAM is closely modulated at specific stages of B cell development, with relatively high levels on the pro-B cell population, down-modulation in pre-B cells at the onset of immunoglobulin (Ig) gene rearrangement, and subsequent re-expression at variable levels in immature and mature peripheral B cell subsets. We have verified VCAM transcripts by cDNA PCR in highly purified populations of murine precursor B cells. Normal human bone marrow precursor B-lymphoid populations (hematogones) also demonstrate VCAM surface protein expression. Finally, in an animal model of BCR/ABL+ ALL, we found that VCAM expression is dramatically increased on lymphoblasts when compared to normal reference populations in bone marrow and spleen. VCAM expression in human lymphoid malignancies is currently under investigation.

Antibody-mediated VCAM cross-linking on primary B-cell precursors ex-vivo generates intracellular reactive oxygen species, demonstrating that signaling through this molecule has functional consequences. Intriguingly, in-vivo, VCAM expression is limited to B-lymphoid cells harvested from tissues such as bone marrow, spleen and lymph node; since, in the same animal, peripheral blood lymphocytes and peritoneal cells do not express readily detectable levels of the surface antigen. VCAM-expressing B-lymphoid cells cultured ex-vivo gradually lose surface expression over 24 hours. The tissue-associated modulation of VCAM expression is preserved in the murine Ph+ lymphoblasts; leukemia cells isolated from the peripheral blood express very low levels of surface VCAM compared to those harvested from bone marrow or spleen. Our data suggests that VCAM expression is dependent on tissue-specific microenvironmental signals in-vivo.

B-lymphoid expression of both VCAM and its ligand VLA-4 is a surprising finding that has broad implications regarding leukemic cell interaction with endothelial cells, the bone marrow retention and trafficking of precursor- and leukemic-B cell populations, and the interpretation of an extensive experimental database predicated on the stromal-cell specificity of VCAM expression and function.
Direct Real Time Visualization of Platelet Calclium Signaling In Vivo: Role of Platelet Activation and Thrombus Formation in a Living Mouse.
Authors: Dubois Christophe , Panicot-Dubois Laurence , Furie Barbara C. , Furie Bruce
Published: October 2019

Intracellular calcium mobilization plays a critical role in platelet signaling. Upon platelet activation, an intracellular calcium mobilization leads to the activation of various intracellular and membrane proteins, including integrins involved in both platelet shape change and aggregation. The goal of the present study was to monitor platelet calcium mobilization in vivo in an intact animal and to determine which intracellular pathways are dominant in platelet accumulation into the developing thrombus. Platelets were isolated from mice, washed, loaded with a calcium-sensitive fluorochrome, Fura2-AM and then infused into a recipient mouse. We studied Fura2-AM loaded platelet incorporation during arterial thrombus development following laser injury of the vessel wall in the cremaster microcirculation of living mice using high speed intravital widefield digital microscopy. Fura-2 loaded platelets were monitored by excitation at 380 nm; this fluorescence reports the basal calcium levels in platelets. Calcium mobilization was monitored by excitation at 340 nm where the fluorescence intensity reflects Fura2-calcium complex formation. We observed that platelets bind to the growing thrombus independent of calcium mobilization. However, the stable incorporation of platelets into the thrombus correlated with a significant intracellular calcium increase. Once the thrombus reached maximal size at about 100 seconds, the calcium mobilization also reached maximal intensity. Subsequently, platelets that did not mobilize calcium dissociated from the thrombus. We confirmed these observations by using platelets treated with the calcium chelators, BAPTA-AM or EGTA-AM. We observed a significant inhibition of platelet accumulation into the thrombus, indicating that the intracellular calcium increase is necessary in vivo for the stable accumulation of the platelets into the thrombus. We also evaluated the involvement in vivo of two platelet agonists, ADP and thromboxane A2 (TxA2), on calcium mobilization and platelet incorporation into thrombi. When platelets were treated with aspirin or with the P2Y1 antagonist A3P5P (adenosine 3′-phosphate-5′-phosphate), we observed a partial decrease in both calcium mobilization and platelet accumulation into the thrombus. These results indicate that TxA2 and ADP via the P2Y1 receptor are involved in vivo in platelet activation upon vessel wall injury in this thrombosis model. When platelets were treated with both compounds, we completely inhibited the calcium increase and the incorporation of platelets into the thrombus. Altogether, our results directly show, for the first time in vivo, the importance of the calcium mobilization on platelet accumulation into the developing thrombus. The platelet agonists TxA2 and ADP both play an important and complementary role on platelet activation by acting on the mobilization of the intracellular calcium.
Immunoglobulin G Fc Receptor Polymorphisms and Clinical Course in Follicular Lymphoma Patients.
Authors: Weng Wen-Kai , Rosenberg Abby , Levy Ronald
Published: October 2019

Patients with low-grade B cell lymphoma, such as follicular lymphoma usually have an indolent clinical course. In most of the cases, patients do not require therapy at the time of diagnosis. Instead, patients can be monitored from months to years before the need for therapy. However, the time interval from diagnosis to the first therapy in these patients can vary significantly. In addition, the overall clinical course is heterogeneous in these patients with variable length of survival after diagnosis. The pathobiological basis of this hetergenisity is not known. We have recently found that IgG Fc receptor (FcγR) polymorphisms correlated with clinical outcome after immunotherapies in patients with follicular lymphoma. In one case, FcγRIIIa 158 V/F and FcγRIIa 131 H/R polymorphisms predicted the response to anti-CD20 antibody, rituximab therapy probably due to their role in the antibody-dependent cellular cytotoxicity. One question is whether the FcγR polymorphisms are correlated with the general clinical course of follicular lymphoma due toq their role in innate immunity. We therefore tested if FcγRIIIa 158 V/F, FcγRIIa 131 H/R and FcγRIIb 232 I/T polymorphisms predict clinical course of a group of 307 patients with follicular lymphoma. None of the three FcγR polymorphisms tested correlated with the time interval from diagnosis to first therapy, or with overall survival in this group of patients. We then tested whether FcγR polymorphisms have predictive value on clinical response to chemotherapy. Of 307 patients, only 158 patients received chemotherapy alone as first therapy, while the rest of the patients have received additional idiotype vaccination or rituximab. In the analysis of these 158 patients, there was no correlation between the FcγR polymorphisms and response rate to chemotherapy or time to progression after chemotherapy. Our results did not find any association between the three FcγR polymorphisms tested and clinical course of disease or response to chemotherapy in patients with follicular lymphoma, suggesting that the predictive value of FcγR polymorphisms on the clinical responses to rituximab is specific to the monoclonal antibody therapy.
Lymphocyte Count Persistence and Early Recovery Predicts Superior Survival and Is Independent of the International Prognostic Index in Patients Treated with CHOP Chemotherapy for Diffuse Large B Cell Lymphoma.
Authors: Porrata Luis F. , Ristow Kay M. , Markovic Svetomir N. , Persky Daniel , Habermann Thomas M.
Published: October 2019

The peripheral blood absolute lymphocyte count (ALC) post-autologous stem cell transplantation is an independent predictor for survival in non-Hodgkin’s lymphoma. The role of ALC recovery during and after standard CHOP chemotherapy for newly diagnosed diffuse large B cell lymphoma (DLBCL) has not been reported. We hypothesized that ALC recovery during/after CHOP chemotherapy has a direct impact on survival. 135 consecutive newly diagnosed patients with DLBCL treated with CHOP from 1994 through 2000 were retrospectively analyzed. The primary end point was to assess the role of ALC recovery during and after CHOP on progression-free survival (PFS) and overall survival (OS). The ALC recovery before each of the 6 cycles and at 3 months follow-up after completion of therapy were analyzed. Of the 135 patients, 41 patients received concomitant radiation therapy. The median age was 64 years (range: 21–83) and median follow-up was 46 months (range: 1–124). Superior OS and PFS were identified in patients achieving the ALC cut-off value that discriminated clinical outcomes at a high level of significance for blood counts obtained before cycles 1 (ALC ≥ 1.5 x 109/L, OS = not reached vs 54 months, p< 0.0048; PFS = not reached vs 23 months, p <0.0005), 2 (ALC ≥1.5 x 109/L, OS = not reached vs 59 months, p <0.0255; PFS = not reached vs 30 months, p <0.0082), 3(ALC ≥1.2 x 109/L, OS = not reached vs 59 months, p<0.0074; not reached vs 30 months, p <0.0060), and at 3 months (ALC ≥ 1.2 x 109/L, OS = not reached vs 60 months, p< 0.0080; PFS = not reached vs 42 months, p < 0.0017). Similar cut-off points for cycles 4 through 6 could not be identified. The ALC recovery between each cycles 1–3 and at 3 months were not independent of each other. Multivariate analysis demonstrated ALC for cycles 1–3 and at 3 months post CHOP to be independent prognostic factor for OS and PFS when compared to other significant prognostic factors including International Prognostic Index and radiation therapy. Patients were stratified into three groups based on whether or not they achieved cut-off values of ALC in the first 3 cycles: group I= ALC achieved in at least 2 of 3 cycles; group II= ALC achieved in only 1/3 cycles; and group III= ALC cut-off not achieved. A trend towards worse OS (p< 0.0035) (Figure 1) and PFS (p< 0.0003) was identified if patients did not achieve any of the cut-off values of ALC in the first 3 cycles. These data further support the hypothesis that there is a critical role of lymphocyte (immune) recovery during and after CHOP chemotherapy in DLBCL.
FoxP3, a Key Molecule in CD4+CD25+ Regulatory T Cells, Express in Adult T Cell Leukemia/Lymphoma Cells and Relates to Clinicopathological Features.
Authors: Karube Kennosuke , Ohshima Koichi , Suzumiya Junji , Harada Mine , Kikuchi Masahiro
Published: October 2019

AIMES: Adult T cell leukemia/lymphoma (ATLL) is an aggressive neoplastic disease and opportunistic infections often occur in patients with ATLL. However, the underlying mechanisms of such infections remain unknown. Recently, regulatory T cells (Treg), characterized by coexpression of CD4 and CD25, are proposed as a new T cell group with definite function. Treg suppresses normal T cells proliferation in vitro and play an important role to suppress autoimmune disease in vivo. But the deregulated proliferation such immunosuppressive T cells may induce immunodeficient status. We analyzed the expression of forkhead/winged helix transcription factor (FoxP3), known to be important for the function and specific marker of Treg cells, on ATLL cells.

METHODS and RESULTS: FoxP3 expression was detected in both peripheral blood and lymph nodes in part of ATLL cases by real-time PCR and immunostaining (Figure). Next, we immunostained lymph node sections from 112 cases and 36 cases showed positivity for FoXP3. Morphologically, 112 ATLL cases were divided into three variants, namely pleomorphic cell type (61 cases), large cell type (45 cases), and anaplastic large cell type (16 cases). FoxP3 was expressed (more than 30% of the lymphoma cells) in a proportion of pleomorphic cell type (24 cases, 39.4%) and large cell type (12 cases, 26.7%) but no cases of anaplastic type showed positivity. Especially, strong expression (more than 50% of the lymphoma cells) was observed in 15 cases and 12 cases were pleomorphic cell type (Table). A proportion of FoxP3 positive ATLL cases showed intermingled EBV-infected transformed lymphocytes, suggesting local immunodeficient condition (Very few FoxP3 negative cases showed EBV-infected lymphocytes)(Table). Clinically, more proportion of FoxP3 positive ATLL cases showed opportunistic infection, for example Pneumocystis Carinii infections, Herpes zoster virus infection, antibiotics-refractory abscesses (Table).

CONCLUSION: A proliferation of FoxP3 positive lymphoma cells may contribute to the tumor invasion and opportunistic infection by inducing local and diffuse immunodeficient status respectively.

Figure Figure
PKC-beta 2 Protein Expression Predicts for Poor Response to Chemotherapy and Survival in Patients with Low Risk Diffuse Large B-Cell Lymphoma.
Authors: Espinosa Inigo , Briones Javier , Bordes Ramon , Brunet Salut , Martino Rodrigo , Sureda Ana , Prat Jaime , Sierra Jorge
Published: October 2019

The protein kinase C (PKC) superfamily includes conventional PKCs (alfa, beta 1, beta 2, and gamma) and PKC-mu. PKC plays an important role in the activation and survival of B cells. Recent studies using cDNA microarrays found that PKC-beta was overexpressed in refractary diffused large B-cell lymphoma (DLBCL). The purpose of this study is to analyze the clinical significance of PKC-beta 2 protein expression in a homogeneous series of patients with DLBCL. Seventy-six patients with primary DLBCL consecutively diagnosed between 1991 and 2002 were studied. The median age of patients was 58 years (range, 11–18 years), 30 women and 46 men. Advanced stage (III/IV) was observed in 36 cases (47%). Of 71 patients with available date, 36 (50%) presented with high serum lactic acid dehydrogenase (LDH) levels. The distribution according to the International Prognostic Index (IPI) (n = 71) was as follows: low risk, 32 cases (43%); low/intermediate risk, 17 cases (23%); high/intermediate risk, 8 cases (11%); and high risk, 14 cases (19%). All patients received an anthracycline-containing chemotherapy regimen. Formalin-fixed, paraffin-embedded tissue from 76 DLBCL tumors were immunostained with a monoclonal antibody against PKC-beta 2 protein. Tumors were considered positive for PKC-beta 2 if 5% o more of the tumor cells expressed the protein. The main clinical features (B symptoms, Ann Arbor stage, bulky disease, bone marrow disease, high LDH, IPI, and ECOG) of patients within the PKC-positive group were similar to those within the PKC-negative group. Twenty-six cases (34%) were positive for PKC-beta 2 protein. Within the patients whose tumors were PKC-positive, only 8 of 26 (31%) had a complete clinical remission, while that was achieved in the majority (31 of 50; 62%) of the PKC-negative patients (p = 0.015). Patients with PKC-positive tumors had a lower five-year disease free survival (DFS) (30% vs. 60%; p = 0.03) than the PKC-beta 2 negative group. Moreover, patients with low IPI whose tumors expressed PKC-beta 2 protein had an inferior five-year DFS (39% vs. 80%; p = 0.0046). Multivariate analysis confirmed the independent adverse prognostic value of PKC expression (OR = 3.7 [95%CI, 1.4–9.9]; p = 0.007) in patients with DLBCL and low IPI. These results indicate that PKC-beta 2 expression on DLBCL predicts for a low response rate to chemotherapy and unfavorable clinical outcome, specially in those patients with low IPI.
Histologic Subtypes Do Not Confer Unique Outcomes in Early-Stage Lymphoma: Long-Term Follow-Up of SWOG 8736.
Authors: Spier Catherine M. , LeBlanc Michael , Chase Ellen M. , Fisher Richard I. , Miller Thomas P.
Published: October 2019

Analysis of patients with advanced non-Hodgkin’s lymphoma (NHL) suggests unique clinical presentations and outcome according to histologic subtype. Little is known of outcome as it relates to histologic subytpe in patients with limited disease. In a previous report (NEJM1998;339:21–6), 401 patients with limited disease (Stage I or non-bulky II) intermediate or high grade NHL, enrolled in SWOG study 8736, were randomized to chemotherapy or chemotherapy plus radiotherapy. Results at 5 years showed superior survival and less toxicity for those treated with 3 cycles of CHOP chemotherapy plus radiotherapy. These 401 patients, with additional followup and reclassification of histology according to World Health Organization (WHO) criteria, form the basis of the current report. Of the 401 pts, 2 were excluded because of clinical findings and 34 patients were excluded for insufficient pathology material. 365 pts were evaluable. On the basis of hematoxylin-eosin and immunohistochemistry results, pt cases were classified as diffuse large B cell (174pts), follicular, Grade 3 (36), Burkitt-like (27), peripheral T cell, unspecified (9), anaplastic large T cell (4), morphologically consistent with mantle cell (4), morphologically consistent with MALToma (3), morphologically diffuse small cleaved cell (2), unclassifiable (1), suggesting anaplastic large cell CD30+ (1), diffuse large cell without immunohistochemical results (104). At 10 years, there was no significant difference in overall survival (OS) or failure free survival (FFS) between the 2 treatments (OS, p=0.48; FFS, p=0.77) (Blood2001;98:724a). When analyzed by histology, including T lineage, neither OS nor FFS were different for any histologic subytpe (OS, p=0.94; FFS, p=0.99). In addition, the estimated shapes of the survival curves were similar for all histologic subtypes among these 365 evaluable pts. On the basis of these findings it does not appear that either the WHO or the Working Formulation (WF) classification system is able to predict outcome for limited stage disease pts treated with CHOP +/− radiotherapy. Therefore, histologic type should not be a criterion for exclusion for limited stage studies of lymphoma. Further, it does not appear that any histologic subtype presenting as early stage disease needs CNS prophylaxis or a unique chemotherapy treatment strategy. There was only one central nervous system (CNS) relapse (in a patient with testis involvement), and none among the 27 Burkitt-like pts. The lack of an observed survival plateau in the first 10 years of followup for these pts is in contrast to advanced stage pts who achieve a plateau within 6 years. The biology of limited stage lymphoma may be fundamentally different from that of advanced stage disease and more significanct in determining outcome than histologic type.
Clinical Relevance of Soluble HLA-I as Compared to b2-MicroglobulinLevels in Non-Hodgkin’s Lymphoma and Hodgkin’s Disease.
Authors: Albitar Maher , Vose Julie M. , Johnson Marcella M. , Do Kim-Ann , Day Amanda , Jilani Iman , Kantarjian Hagop , Keating Michael , O’Brien Susan M. , Verstovsek Srdan , Armitage James O. , Giles Francis J.
Published: October 2019

Higher levels of b-2 microglobulin (b2-M) have been reported to correlate with poor outcome in non-Hodgkin’s lymphoma (NHL) and Hodgkin’s disease (HD). However, b2-M is a subunit of the human leukocyte antigen-class I (HLA-I) molecule, which is composed of b2-M and the alpha heavy chain. Although soluble HLA-I (sHLA-I) protein has been detected in circulation and is believed to play a role in immunomodulation by inducing apoptosis in cytotoxic T-lymphocytes, the clinical relevance of sHLA-I levels in HD and NHL is not known. Here we report that serum sHLA-I levels are increased in NHL and HD. More importantly, levels of sHLA-I correlate with clinical behavior in NHL in a fashion similar to that of b2-M. In a multivariate model incorporating b2-M, sHLA-I, and IPI (International Prognostic Index), NHL patients with sHLA-I levels >312.60 mg/mL had significantly shorter survival than those with lower levels, independent of IPI score. Moreover, a model incorporating both sHLA-I (cut-point = 312.60 mg/mL) and b2-M (cut-point = 4.16 mg/L) levels were highly predictive of survival (Figure) in NHL patients. In contrast, in HD, b2-M levels correlated with clinical behavior whereas HLA-1 levels did not. These data not only establish the role of sHLA-I as an independent tumor marker in NHL that can be used to stratify patients, but also suggest that, in HD, b2-M and sHLA-I may reflect different processes. The role of sHLA-I as an immunomodulator may be responsible for its divergence from b2-M as an indicator of clinical behavior in HD. Further studies exploring the importance of this role in the biology of HD are needed.

Figure Figure
CpG Combined with Chemotherapy in the Immunotherapy of Large Tumor.
Authors: Li Jiali , Levy Ronald
Published: October 2019

Unmethylated cytosine-phosphorothioate-guanine nucleotides (CpG) are immuostimulators that can activate both innate and adaptive immunity. By binding to its receptor, TLR9, CpG can activate dendritic cells and B cells by inducing the secretion of cytokines, and increasing the expression of costimulatory molecules, such as CD80 and CD86. In this study, a B type CpG, CpG1826, was given intratumorally in an A20 lymphoma animal model. CpG alone can delay the growth of tumor in this model, however, the majority of the treated tumor-bearing mice still die, and it has very little effect on systemic tumor cells. Here we propose a new therapeutic strategy, a combination of cyclophosphomide (CTX), followed by subsequent intratumoral injection of CpG. BALB/c mice were inoculated s.c. with 107 A20 cells in the lower back, and treatment started on day 17 when tumor reached approximately 1.0 cm in diameter. CTX (100mg/Kg) was given i.p. on day 17 and 18, and for the groups that received CpG, CpG was injected intratumorally on day 19,20,21,24, and 26 with 100μg/dose. As shown in Fig.1, the combination of CTX with CpG could cure well-established large size (>1.2cm in diameter) tumor, and mice remained tumor-free 5–6 months after the initial treatment. Conversely, the majority of mice that received CTX or CpG alone died of lymphoma. Tumor-free mice from this combined maneuver are protected from a lethal dose of A20 challenge even 5 months after the initial treatment. Our studies also showed that this combination is site dependent, as CTX combined with intratumoral injection of CpG had a much better therapeutic effect (100% survival), as compared to injection of CpG into a distant normal site (20% survival). Our studies also indicated that this combined maneuver is CD8+T cell dependent, as no therapeutic effect was observed when we repeated this experiment in a CD8+ T cell knockout model. We also demonstrated the systemic effect of this combination strategy in mice bearing tumors at two different sites. In combination with CTX, injection of one site with CpG controlled the growth of the second site tumor as opposed to a transient effect from chemotherapy or CpG alone. This led to significant prolongation of overall survival (Fig.2). In summary, we have shown that this combined chemoimmunotherapy strategy can cure both local and systemic tumor, which may provide a powerful antitumor therapy in the clinic.

Figure Figure
Development of Effective Immunotherapy for B-Cell Non-Hodgkin’s Lymphoma with CD19-Specific Cytotoxic T Cells.
Authors: Mihara Keichiro , Yanagihara Kazuyoshi , Imai Chihaya , Kimura Akiro , Campana Dario
Published: October 2019

Less than 60% of patients with B-cell non-Hodgkin’s lymphoma (B-NHL) can be cured with contemporary therapy. Using artificial receptors it is possible to redirect the specificity of immune cells to tumor-associated antigens, a strategy that holds great potential as a novel cancer therapy. Since B-NHL cells invariably express CD19, we transduced human peripheral blood T lymphocytes with a recently developed receptor (anti-CD19-BB-ζ), which consists of the single-chain variable domain (scFv) of an anti-CD19 monoclonal antibody, the hinge and transmembrane domains of CD8α, and the signaling domains of CD3ζ and 4-1BB. CD3ζ delivers the primary stimulus upon receptor engagement, while 4-1BB delivers co-stimulatory signals that are crucial for T-cell cytotoxicity. It has been shown that elicitation of 4-1BB signaling enhances the immune response to tumors in vivo, even when an immune response cannot be induced by CD28 stimulation. Retroviral transduction led to anti-CD19-BB-ζ expression in T cells with high efficiency: median percent of transduced cells was 60.3% (range, 25.7%–83.4%; n = 9). T lymphocytes expressing anti-CD19-BB-ζ expanded more vigorously that T cells transduced with receptors lacking 4-1BB and exerted powerful cytotoxicity against the CD19+ B-NHL cell lines Raji, Daudi, RL, and HT in vitro: at a 0.5: 1 effector: target ratio, mean (± SD) cell specific lymphoma cell killing was 96.6% ± 4.6% after 5–7 days of culture (4 experiments in each cell line). Transduced T cells were also effective against freshly isolated cells from patients with diffuse large, follicular large, Burkitt, and mantle cell lymphoma cultured on bone marrow-derived mesenchymal cells: in 10 samples, cell killing was 93.6% ± 5.7% at a 0.5: 1 ratio after 5–7 days of culture. Sensitivity to anti-CD19-BB-ζ-mediated killing was observed regardless of high Bcl-2 expression. T cells expressing anti-CD19-BB-ζ were also effective in a xenograft model of NHL, in which NOD/SCID mice were inoculated subcutaneously with lymphoma cells (1 x 107). Subsequent inoculation of T cells (2 x 106) transduced with anti-CD19-BB-ζ receptors significantly suppressed tumor growth, whereas inoculation of T cells transduced with empty control vector had no effect (3 mice for each treatment). These results provide a rationale for clinical testing of autologous T cells modified with anti-CD19-BB-ζ receptors in patients with aggressive or relapsed B-NHLs refractory to conventional therapy.
GCS-100, a Galectin 3 Antagonist, Is a Novel Caspase-9 Apoptosis Activating Agent for the Treatment of Indolent B-Cell Malignancies.
Authors: Cotter Finbarr E. , Su Bo , Corbo Margherita , Chang Y. , Schindler Joanne , Carver Brad , Fegan Christopher D. , Newland Adrian C. , Auer Rebecca L.
Published: October 2019

Introduction: GCS-100 is a novel polysaccharide agent derived from natural citrus pectin with the ability to inhibit Galectin 3 (Gal-3). It has demonstrated anti-cancer activity and has been administered to patients with solid tumors in a Phase I study demonstrating a good safety profile and a lack of myelosuppression. It has been reported to have three major pharmacological effects: anti-metastatic, anti-angiogenic and pro-apoptotic. This study has investigated the mechanism of induction of apoptosis by GCS-100 for therapeutic benefit in indolent B-cell lymphomas.

Material and Methods: GCS-100 was tested in varying doses with B-cell lymphoma and leukaemia cell lines including the DoHH2 (t(14;18)), Ramos (high and low Bcl-2 expression), BV173 (t(9;22)) and K562 cell lines, primary patient CD19 selected CLL cells and normal B-cells in short term cultures. The addition of chemotherapy post GCS-100 was then undertaken to investigate enhanced anti-tumor activity. Apoptosis was assessed by a number of parameters including mitochondrial transition pore permeability (MTPP) by DiOC6, caspase-8 and -9 activation by western analysis and membrane blebbing by MC540. Gal-3 expression was measured by western analysis and flow cytometry.

Results: The ability of GCS-100 to induce significant apoptosis in both malignant cell lines and primary patient CLL cells in vitro is clearly demonstrated with a minimal effect against normal B-cells (and stem cell cultures). The induced apoptotic pathway is the intrinsic mitochondrial and caspase-9 pathway. In addition, GCS-100 at low dosage will greatly enhance the apoptotic effect of chemotherapy, even in the presence of high levels of anti-apoptotic proteins such as Bcl-2. Immunoprecipitation has shown Bcl-2 binds to Gal-3. Gal-3 expression was increased in the malignant cells compared to the normal B-cells and could explain the selective activity.

Discussion: Gal-3 is a member of the b-galactoside-binding protein family that play significant roles in cellular interactions, cell growth, differentiation, resistance to apoptosis and loss of cell anchorage (anoikis). It shares the highly conserved BH1 functional receptor domain (NWGR) of the Bcl-2 gene family within its carbohydrate binding domain. The potential binding of GCS-100 to this region suggests this as its pro-apoptotic mechanism in B-cell malignancies. As shown in this study, normal B-cells with lower Gal-3 expression do not show significant apoptosis indicating GCS-100 is a selective anti-tumor agent. The possibility of using GCS-100 as an anti cancer agent, either alone or in combination with chemotherapy is suggested by this study. Its activity against indolent B-cell malignancies, such as follicular lymphoma (DoHH2 cell line) and CLL is particularly marked; a Phase 1 study is underway.
In Vitro and In Vivo Targeting and Therapy of an Antibody-Drug Conjugate (IMMU-110) in B-Cell Malignancies.
Authors: Sapra Puja , Stein Rhona , Pickett Jennifer , Govindan Serengulam V. , Cardillo Thomas M. , Damoci Christopher , Sheerin Agatha , Hansen Hans J. , Horak Ivan D. , Griffiths Gary L. , Goldenberg David M.
Published: October 2019

IMMU-110 is a drug immunoconjugate comprised of doxorubicin (DOX) conjugated to the humanized anti-CD74 monoclonal antibody (mAb), hLL1, at a DOX:mAb (mol/mol) ratio of 8:1. CD74 is a rapidly internalizing type-II transmembrane chaperone molecule associated with HLA-DR, and has high expression on human non-Hodgkin’s lymphoma (NHL) and multiple myeloma (MM) clinical specimens and cell lines. Here, we investigated the in vitro and in vivo efficacy of IMMU-110 in xenograft models of human NHL (Raji, Daudi) and MM (MC/CAR). In vitro cell binding of IMMU-110 with the CD74-positive cells was significantly higher than that of a non-specific isotype-matched mAb-DOX conjugate (DOX conjugated to a mAb against epithelial glycoprotein-1; DOX-hRS7), and was similar to that of naked hLL1. Both IMMU-110 and naked hLL1 bound CD74 with subnanomolar affinity. The in vitro cytotoxicity of IMMU-110 was significantly higher than non-specific antibody-DOX conjugate, DOX-hRS7, and was similar to free DOX in MC/CAR, Raji or Daudi human Burkitt’s lymphoma cells. In CD74-negative cell lines, IMMU-110 was significantly less toxic than free DOX, having similar cytotoxicity to DOX-hRS7. In vivo, IMMU-110 displayed a pharmacokinetic and biodistribution profile almost identical to that of hLL1 mAb. Both hLL1 mAb and IMMU-110 had a biphasic clearance from the circulation; the α and β half-life (t1/2) of IMMU-110 were 4.6 h and 157.9 h, respectively, and those of hLL1 were 5.4 h and 151.5 h, respectively. In biodistribution studies, no significant difference was observed between IMMU-110 and naked hLL1 with regards to normal tissue uptake. Neither IMMU-110 nor naked hLL1 mAb had a significant association with any normal body tissue. In therapy experiments, a single i.v. protein dose of 350 μg IMMU-110, injected 5 days after implantation of MC/CAR cells in SCID mice, resulted in curing 70% of the animals. Similar cure rates were observed when treatment with IMMU-110 was given 10 days after transplantation of MC/CAR cells. In the Raji xenograft model, 100% of animals were cured with a single protein dose of 120 μg IMMU-110, injected 5 days after implantation of cells. In survival studies, the efficacy of IMMU-110 was significantly better than naked hLL1, the combination of naked hLL1 and free DOX, or of a non-specific antibody-DOX conjugate, DOX-hRS7. In a tolerability study in SCID mice, no toxic effect of IMMU-110 was observed even at the highest dose tested (2.5 mg /mouse). In conclusion, treatment of B-cell lymphoma and myeloma xenograft models with single injections of IMMU-110 resulted in high levels of response and long-term survivors. IMMU-110 is being further developed as a potential therapeutic for the treatment of CD74-positive tumors.