| 1. |
- Lara, Sandra, et al.
(författare)
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Exploring complement-dependent cytotoxicity by rituximab isotypes in 2D and 3D-cultured B-cell lymphoma
- 2022
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Ingår i: BMC Cancer. - : BioMed Central. - 1471-2407. ; 22:1
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Tidskriftsartikel (refereegranskat)abstract
- Background The therapeutic IgG1 anti-CD20 antibody, rituximab (RTX), has greatly improved prognosis of many B-cell malignancies. Despite its success, resistance has been reported and detailed knowledge of RTX mechanisms are lacking. Complement-dependent cytotoxicity (CDC) is one important mode of action of RTX. The aim of this study was to systematically evaluate factors influencing complement-mediated tumor cell killing by RTX. Methods Different RTX isotypes, IgG1, IgG3, IgA1 and IgA2 were evaluated and administered on four human CD20(+) B-cell lymphoma cell lines, displaying diverse expression of CD20 and complement-regulatory protein CD59. Complement activation was assessed on lymphoma cells grown in 2 and 3-dimensional (3D) culture systems by trypan blue exclusion. CDC in 3D spheroids was additionally analyzed by Annexin V and propidium iodide staining by flow cytometry, and confocal imaging. Anti-CD59 antibody was used to evaluate influence of CD59 in RTX-mediated CDC responses. Statistical differences were determined by one-way ANOVA and Tukey post hoc test. Results We found that 3 out of 4 lymphomas were sensitive to RTX-mediated CDC when cultured in 2D, while 2 out of 4 when grown in 3D. RTX-IgG3 had the greatest CDC potential, followed by clinical standard RTX-IgG1 and RTX-IgA2, whereas RTX-IgA1 displayed no complement activation. Although the pattern of different RTX isotypes to induce CDC were similar in the sensitive lymphomas, the degree of cell killing differed. A greater CDC activity was seen in lymphoma cells with a higher CD20/CD59 expression ratio. These lymphomas were also sensitive to RTX when grown in 3D spheroids, although the CDC activity was substantially reduced compared to 2D cultures. Analysis of RTX-treated spheroids demonstrated apoptosis and necrosis essentially in the outer cell-layers. Neutralization of CD59 overcame resistance to RTX-mediated CDC in 2D-cultured lymphoma cells, but not in spheroids. Conclusions The results demonstrate that CDC outcome in CD20(+) B-cell lymphoma is synergistically influenced by choice of RTX isotype, antigen density, tumor structure, and degree of CD59 expression. Assessment of tumor signatures, such as CD20/CD59 ratio, can be advantageous to predict CDC efficiency of RTX in vivo and may help to develop rational mAbs to raise response rates in patients.
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| 2. |
- Lara, Sandra, et al.
(författare)
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Importance of antibody isotypes in antitumor immunity by monocytes and complement using human-immune tumor models
- 2021
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Ingår i: European Journal of Immunology. - : John Wiley & Sons. - 0014-2980 .- 1521-4141. ; 51:5, s. 1218-1233
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Tidskriftsartikel (refereegranskat)abstract
- Monoclonal antibodies (mAbs) have revolutionized clinical medicine, especially in the field of cancer immunotherapy. The challenge now is to improve the response rates, as immunotherapy still fails for many patients. Strategies to enhance tumor cell death is a fundamental aim, but relevant model systems for human tumor immunology are lacking. Herein, we have developed a preclinical human immune - three-dimensional (3D) tumor model (spheroids) to map the efficiency of tumor-specific isotypes for improved tumor cell killing. Different anti-CD20 Rituximab (RTX) isotypes alone or in combination, were evaluated for mediating complement-dependent cytotoxicity and antibody-dependent phagocytosis by human monocytic cells in 3D spheroids, in parallel with monolayer cultures, of human CD20(+) B-cell lymphomas. We demonstrate that the IgG3 variant of RTX has the greatest tumoricidal effect over other isotypes, and when combined with apoptosis-inducing RTX-IgG2 isotype the therapeutic effect can be substantially enhanced. The results show further that the treatment outcome by RTX isotypes is influenced by tumor morphology and expression of the complement inhibitor CD59. Hence, the human immune-3D tumor model is a clinical relevant and attractive ex vivo system to predict mAbs for best efficacy in cancer immunotherapy.
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| 3. |
- Lara, Sandra, et al.
(författare)
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The Human Monocyte : A Circulating Sensor of Infection and a Potent and Rapid Inducer of Inflammation
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 23:7
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Tidskriftsartikel (refereegranskat)abstract
- Monocytes were previously thought to be the precursors of all tissue macrophages but have recently been found to represent a unique population of cells, distinct from the majority of tissue macrophages. Monocytes and intestinal macrophages seem now to be the only monocyte/macrophage populations that originate primarily from adult bone marrow. To obtain a better view of the biological function of monocytes and how they differ from tissue macrophages, we have performed a quantitative analysis of its transcriptome in vivo and after in vitro stimulation with E. coli LPS. The monocytes rapidly responded to LPS by producing extremely high amounts of mRNA for the classical inflammatory cytokines, IL-1α, IL-1β, IL-6 and TNF-α, but almost undetectable amounts of other cytokines. IL-6 was upregulated 58,000 times, from almost undetectable levels at baseline to become one of the major transcripts already after a few hours of cultivation. The cells also showed very strong upregulation of a number of chemokines, primarily IL-8, Ccl2, Ccl3, Ccl3L3, Ccl20, Cxcl2, Cxcl3 and Cxcl4. IL-8 became the most highly expressed transcript in the monocytes already after four hours of in vitro culture in the presence of LPS. A high baseline level of MHC class II chains and marked upregulation of super oxide dismutase (SOD2), complement factor B, complement factor C3 and coagulation factor 3 (F3; tissue factor) at four hours of in vitro culture were also observed. This indicates a rapid protective response to high production of oxygen radicals, to increase complement activation and possibly also be an inducer of local coagulation. Overall, these findings give strong support for monocytes acting primarily as potent mobile sensors of infection and rapid activators of a strong inflammatory response.
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| 4. |
- Paivandy, Aida, et al.
(författare)
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Quantitative In-Depth Transcriptome Analysis Implicates Peritoneal Macrophages as Important Players in the Complement and Coagulation Systems
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 23:3
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Tidskriftsartikel (refereegranskat)abstract
- To obtain a more detailed picture of macrophage (M phi) biology, in the current study, we analyzed the transcriptome of mouse peritoneal M phi s by RNA-seq and PCR-based transcriptomics. The results show that peritoneal M phi s, based on mRNA content, under non-inflammatory conditions produce large amounts of a number of antimicrobial proteins such as lysozyme and several complement components. They were also found to be potent producers of several chemokines, including platelet factor 4 (PF4), Ccl6, Ccl9, Cxcl13, and Ccl24, and to express high levels of both TGF-beta 1 and TGF-beta 2. The liver is considered to be the main producer of most complement and coagulation components. However, we can now show that M phi s are also important sources of such compounds including C1qA, C1qB, C1qC, properdin, C4a, factor H, ficolin, and coagulation factor FV. In addition, FX, FVII, and complement factor B were expressed by the M phi s, altogether indicating that M phi s are important local players in both the complement and coagulation systems. For comparison, we analyzed human peripheral blood monocytes. We show that the human monocytes shared many characteristics with the mouse peritoneal M phi s but that there were also many major differences. Similar to the mouse peritoneal M phi s, the most highly expressed transcript in the monocytes was lysozyme, and high levels of both properdin and ficolin were observed. However, with regard to connective tissue components, such as fibronectin, lubricin, syndecan 3, and extracellular matrix protein 1, which were highly expressed by the peritoneal M phi s, the monocytes almost totally lacked transcripts. In contrast, monocytes expressed high levels of MHC Class II, whereas the peritoneal M phi s showed very low levels of these antigen-presenting molecules. Altogether, the present study provides a novel view of the phenotype of the major M phi subpopulation in the mouse peritoneum and the large peritoneal M phi s and places the transcriptome profile of the peritoneal M phi s in a broader context, including a comparison of the peritoneal M phi transcriptome with that of human peripheral blood monocytes and the liver.
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| 5. |
- Akula, Srinivas
(författare)
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The mast cell transcriptome and the evolution of granule proteins and Fc receptors
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protection against disease-causing pathogens, known as immunity, involves numerous cells organs, tissues and their products. To able to understand the biology of immune cells (hematopoietic cells) and their role in an immune system, we have used several different methods, including transcriptome analyses, bioinformatics, production of recombinant proteins and analyses of some of them, focusing on the granule proteases by substrate phage display.Hematopoietic cells express surface receptors interacting with the constant region of immunoglobulins (Igs) known as Fc receptors (FcRs). These receptors play major roles in the immune system, including enhancing phagocytosis, activating antibody dependent cellular cytotoxicity and cell activation. A detailed bioinformatics analysis of FcRs reveals that the poly-Ig receptors (PIGR), FcR-like molecules and common signalling γ chain all appeared very early with the appearance of the bony fishes, and thereby represent the first major evolutionary step in FcR evolution. The FcμR, FcαμR, FcγR and FcεR receptors most likely appeared in reptiles or early mammals, representing the second major step in FcR evolution.Cells of several of the hematopoietic cell lineages contain large numbers of cytoplasmic granules, and serine proteases constitute the major protein content of these granules. In mammals, these proteases are encoded from four different loci: the chymase, the met-ase, the granzyme (A/K) and the mast cell tryptase loci. The granzyme (A/K) locus was the first to appear and came with the cartilaginous fishes. This locus is also the most conserved of the three. The second most conserved locus is the met-ase locus, which is found in bony fishes. The chymase locus appeared relatively late, and we find the first traces in frogs, indicating it appeared in early tetrapods.To study the early events in the diversification of these hematopoietic serine proteases we have analyzed key characteristics of a protease expressed by an NK-like cell in the channel catfish, catfish granzyme–like I. We have used phage display and further validated the results using a panel of recombinant substrates. This protease showed a strict preference for Met at the P1 (cleavage) position, which indicates met-ase specificity. From the screening of potential in vivo substrates, we found an interesting potential target caspase 6, which indicates that caspase-dependent apoptosis mechanisms have been conserved from fishes to mammals.A larger quantitative transcriptome analysis of purified mouse peritoneal mast cells, cultured mast cells (BMMCs), and mast cells isolated from mouse ear and lung tissue identified the major tissue specific transcripts in these mast cells as the granule proteases. Mast cell specific receptors and processing enzymes were expressed at approximately 2 orders of magnitude lower levels. The levels of a few proteases were quite different at various anatomical sites between in vivo and cultured BMMCs. These studies have given us a new insights into mast cells in different tissues, as well as key evolutionary aspects concerning the origins of a number of granule proteases and FcRs.
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| 9. |
- Andrén, Maria, 1976-
(författare)
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The Role of Fc Gamma Receptors in Experimental Arthritis
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Induction of collagen-induced arthritis (CIA), an animal model for human rheumatoid arthritis, is dependent on anti-collagen type II (CII) antibodies. The effector mechanism by which autoantibodies contribute to inflammatory reactions in autoimmune diseases is not well understood. In this thesis I have studied the effector pathways used by IgG anti-CII antibodies to initiate arthritis, namely the IgG Fc receptors (FcγRs) and the complement system. We have found that FcγRIII is crucial for development of CIA, as CII-immunized mice lacking this receptor do not develop arthritis and IgG1 and IgG2b anti-CII antibodies require FcγRIII to trigger arthritis when transferred to naïve mice. The antibody-mediated arthritis was further enhanced in mice deficient in the inhibitory FcγRIIB, indicating that FcγRIIB regulates the activation of FcγRIII. Furthermore, we demonstrate that FcγRIII exist as three distinct haplotypes in mice, FcγRIII:H, FcγRIII:V and FcγRIII:T. Mice expressing the FcγRIII:H haplotype are more susceptible to CIA than mice expressing the FcγRIII:V haplotype, indicating that certain FcγRIII haplotype predisposes for CIA. We also show that the most likely FcγRIII-expressing effector cell in CIA is the macrophage, since FcγRIII-expressing macrophages exclusively can induce arthritis in FcγRIII-deficient mice challenged for CIA.The complement system was also investigated in development of CIA. We found that this effector pathway is also necessary for onset of arthritis, as CIA was inhibited by treatment with anti-complement factor 5 (C5) antibodies. C5-deficient mice could neither develop CIA unless provided with C5-containing sera. Taken together, the work presented in this thesis indicates that FcγRs and the complement system are crucial for the induction of experimental arthritis. These findings are important for understanding the mechanisms behind rheumatoid arthritis and blocking of these effector pathways may in the future be used as treatment of rheumatoid arthritis.
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| 10. |
- Carnrot, Cecilia, et al.
(författare)
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Marginal zone B cells are naturally reactive to collagen type II and are involved in the initiation of the immune response in collagen-induced arthritis
- 2011
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Ingår i: Cellular & Molecular Immunology. - : Springer Science and Business Media LLC. - 1672-7681 .- 2042-0226. ; 8:4, s. 296-304
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies against type II collagen (CII) are essential for development of collagen-induced arthritis (CIA), but how and where the B-cell response to CII is initiated is not fully known. We show here that naive DBA/1 mice display naturally reactive IgM and IgG anti-CII producing B cells prior to immunization. The CII-reactive B cells were observed in the spleen and recognized as marginal zone (MZ) B cells. After CII immunization, CII-specific B cells expanded rapidly in the spleen, in contrast to the lymph nodes, with the initial response derived from MZ B cells and later by follicular (FO) B cells. This was evident despite that the MZ B cells were subject to stringent tolerance mechanisms by having a greater Fc gamma receptor IIb expression than the FO B cells. Further, the MZ B cells migrated to the FO areas upon immunization, possibly providing antigen and activating FO T cells and subsequently FO B cells. Thus, around CIA onset increased numbers of IgG anti-CII producing FO B cells was seen in the spleen, which was dominated by IgG2a- and IgG2b-positive cells. These data demonstrate that CII-reactive MZ B cells are present before and expand after CII immunization, suggesting an initiating role of MZ B cells in the development of CIA.
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