Long Term Humoral Immunity Is Dependent on CD28 Expression In Plasma Cells

Abstract Abstract 1737 Protective immunity against infection requires sustained antibody production by long-lived plasma cells (LLPC) that survive for years/decades within specialized niches. What regulates/supports this survival remains largely unknown. However, is has been shown normal and transformed (human multiple myeloma) LLPC are critically dependent on the bone marrow microenvironment including cell-to-cell interactions. Leading us to rationalize, modulating this interaction could either enhance antibody production for cancer vaccine development or conversely compromise the survival of transformed/normal LLPC in the bone marrow microenvironment. We have shown the T cell costimulatory receptor CD28 expressed on both normal and transformed LLPC plays an essential role. While LLPC and short-lived plasma cells (SLPC) both express CD28, its activation in vitro only significantly increases the survival and IgG production of LLPC. These observations led us to directly investigate the role of CD28 in LLPC survival as well as cell-cell interactions with CD80/CD86+ bone marrow derived dendritic cells (BMDC). Utilizing normal murine bone marrow and splenic PC as our model system we further investigated the role of CD28 in LLPC function and survival. We have previously shown, in vitro serum starvation experiments, direct activation of CD28 increased survival of LLPC by 12-fold (p<0.05), whereas CD28 activation of SLPC did not induce survival. Addition of BMDC improved the survival of LLPC 2-fold over that seen with media alone, and resulted in a significant increase in IgG production (p<0.001). In contrast, CD28-/- PC had no increase in survival when cocultured with BMDC, suggesting a direct role for CD28 in PC-DC interaction. Consistent with these findings we now show that in vivo, vaccinated bone marrow CD28-/-:μMT chimeras had significantly reduces long-term antibody titers and LLPC (but not SLPC) survival from t1/2 of 426 to 63 days. Additionally, LLPC CD28 modulates the microenvironment by inducing CD80/CD86+ stromal cell production of the supportive cytokine IL-6 (p<0.001 vs. BMDC/PC alone), which was abrogated by blocking CD80 and CD86 (p<0.05). From the above experiments we hypothesized IL-6 was playing a significant role in the survival of LLPC, however to our surprise LLPC cocultured with WT or IL-6-/- BMDC maintained equivalent LLPC numbers, interestingly however LLPC cocultures with BMDC showed a 3-fold increase of IgG compared to LLPC cocultured with IL-6-/- BMDC (p<0.001). These data suggest CD28 is a key molecular component in LLPC survival, whereas IL-6 contributes to Ig production. Our data demonstrates that signaling through CD28 directly supports the survival of LLPC, sustaining long term protective antibody titers. These findings suggest CD28 plays an important role in maintaining the quality of protective durable humoral immunity. Strategies to augment CD28 signaling may lead to greater LLPC survival and persistent antibody titers in cancer vaccine development. Conversely, blocking CD28 signaling could compromise the survival of transformed myeloma cells which are critically dependent on the bone marrow microenvironment. Disclosures: Boise: University of Chicago: Patents & Royalties.

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Sustained Antibody Responses Depend on CD28 Function in Bone Marrow Resident Plasma Cells

Blood ◽

10.1182/blood.v118.21.182.182 ◽

2011 ◽

Vol 118 (21) ◽

pp. 182-182

Author(s):

Cheryl H Rozanski ◽

Lindzy F Dodson ◽

Ramon Arens ◽

Louise M Carlson ◽

Lisa M Russell ◽

...

Keyword(s):

Bone Marrow ◽

Signaling Pathway ◽

Humoral Immunity ◽

Antibody Production ◽

Plasma Cells ◽

Composite Element ◽

Antibody Titers

Abstract Abstract 182 Protective immunity against infection requires sustained antibody production by long-lived plasma cells (LLPC) that survive for years/decades within specialized niches. What regulates/supports this survival remains largely unknown. However, it has been shown that normal and transformed (human multiple myeloma) LLPC are critically dependent on the bone marrow microenvironment, including cell-to-cell interactions. This lead us to hypothesize that modulating these interactions could either enhance antibody production for vaccine development or, conversely, compromise the survival of transformed/normal LLPC in the bone marrow microenvironment. We have shown that the T cell costimulatory receptor CD28 expressed on both normal and transformed LLPC, plays an essential role in survival. While LLPC and short-lived plasma cells (SLPC) both express CD28, its activation in vitro only significantly increases survival and IgG production in LLPC. Consistent with these findings, we show in vivo, vaccinated bone marrow CD28−/−:μMT chimeras had significantly reduced long-term antibody titers and decreased LLPC (but not SLPC) t1/2 from 426 to 63 days. These findings demonstrate the existence of a distinct bone marrow (BM) LLPC subset necessary to sustain antibody titers, and establish a central role for CD28 function in the maintenance of plasma cells and humoral immunity. While CD28 signaling has been shown to play an important role in maintaining long-term humoral immune responses, the mechanism by which CD28 signaling affects PC function has not yet been determined. To further elucidate CD28 signaling in BM PC, we utilized CD28 conditional knock-in mice. In these mice, the CD28 cytoplasmic tail is mutated at either the YMNM or proline-rich motifs, resulting in an inhibition of PI3K or vav signaling, respectively. We found that CD28-vav signaling deficient BM PC were selectively depleted in vivo and could not be rescued by CD28 activation in in vitro serum starvation conditions. Furthermore, anti-CD28 mAb drove a 1.5 fold increase in Blimp-1 expression in BM PC, compared to control. This increase was regulated through the CD28-vav signaling pathway, as CD28 activation in CD28-vav signaling deficient BM PC did not increase Blimp-1 expression. To further determine if CD28 is acting directly on the Blimp-1 promoter, we examined in silico for a CD28RE composite element, previously reported to transcriptionally regulate IL-2 production in T cells and IL-8 production in myeloma cells. To our surprise, we found a CD28RE “like” site 4712bp upstream of the Blimp-1 start site. To confirm CD28 transcriptionally regulates Blimp-1 promoter activity, we transfected the CD28+ plasmacytoma cell line J558 with full-length or truncated Blimp-1 promoter constructs (i.e. 7000bp, 4500bp, 1500bp). We found CD28 activation enhances Blimp-1 activity in J558 cells transfected with full-length-Blimp-1, and this activity was lost when the promoter was truncated. Using site-directed mutagenesis, we confirmed the CD28RE is required for induction of Blimp-1 in PC. Furthermore, we show CD28 activation of Blimp-1 increases the BCMA receptor in BM PC. Taken together, our data suggests the CD28-vav signaling pathway in PC induces a CD28RE composite element, which is necessary for the induction of the key PC transcriptional regulator Blimp-1, required to maintain LLPC and humoral immunity. Disclosures: No relevant conflicts of interest to declare.

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Sustained antibody responses depend on CD28 function in bone marrow–resident plasma cells

Journal of Experimental Medicine ◽

10.1084/jem.20110040 ◽

2011 ◽

Vol 208 (7) ◽

pp. 1435-1446 ◽

Cited By ~ 111

Author(s):

Cheryl H. Rozanski ◽

Ramon Arens ◽

Louise M. Carlson ◽

Jayakumar Nair ◽

Lawrence H. Boise ◽

...

Keyword(s):

Bone Marrow ◽

Humoral Immunity ◽

Plasma Cells ◽

Significant Loss ◽

Population Reduction ◽

Antibody Titers ◽

T Cell Help ◽

Antibody Levels

Sustained long-term antibody levels are the cornerstone of protective immunity, yet it remains unclear how they are durably maintained. A predominant theory implicates antigen-independent antibody production by a subset of long-lived plasma cells (LLPCs) that survive within bone marrow (BM). Central tenets of this model—that BM LLPCs constitute a subset defined by intrinsic biology distinct from PCs in other tissues and contribute to long-term antibody titers—have not been definitively demonstrated. We now report that long-term humoral immunity depends on the PC-intrinsic function of CD28, which selectively supports the survival of BM LLPC but not splenic short-lived PC (SLPC). LLPC and SLPC both express CD28, but CD28-driven enhanced survival occurred only in the LLPC. In vivo, even in the presence of sufficient T cell help, loss of CD28 or its ligands CD80 and CD86 caused significant loss of the LLPC population, reduction of LLPC half-life from 426 to 63 d, and inability to maintain long-term antibody titers, but there was no effect on SLPC populations. These findings establish the existence of the distinct BM LLPC subset necessary to sustain antibody titers and uncover a central role for CD28 function in the longevity of PCs and humoral immunity.

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Direct contact between human primitive hematopoietic progenitors and bone marrow stroma is not required for long-term in vitro hematopoiesis

Blood ◽

10.1182/blood.v79.11.2821.bloodjournal79112821 ◽

1992 ◽

Vol 79 (11) ◽

pp. 2821-2826 ◽

Cited By ~ 2

Author(s):

CM Verfaillie

Keyword(s):

Bone Marrow ◽

Direct Contact ◽

Hematopoietic Progenitors ◽

Proliferation And Differentiation ◽

Bone Marrow Cultures ◽

Marrow Stroma ◽

Blood Elements

Long-term bone marrow cultures support both differentiation and conservation of primitive human hematopoietic progenitors in the absence of exogenous cytokines. It is believed that hematopoiesis in such cultures requires direct contact between hematopoietic progenitors and stroma. In the present study, we demonstrate that primitive progenitors physically separated from the stromal layer by a 0.45- microns microporous membrane continue to generate differentiated progenitors for at least 8 weeks. Moreover, primitive progenitors are conserved to a greater extent under these conditions, as when cultured in direct contact with the stroma. However, excessive production of granulocyte-macrophage progenitors occurs when primitive progenitors are not allowed to interact directly with the stroma. Thus, direct contact between hematopoietic and stromal cells is not required for either differentiation or conservation of primitive hematopoietic progenitors but is essential for the regulated production of mature blood elements. These findings can now be used to define the role of diffusible factors and cell-cell or cell-extracellular matrix adhesion events in the regulation of conservation, proliferation, and differentiation of primitive human hematopoietic progenitors in vitro.

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The role of bone marrow microenvironment in the progression of multiple myeloma from monoclonal gammopathy of undetermined significance

Oncohematology ◽

10.17650/1818-8346-2021-16-3-26-32 ◽

2021 ◽

Vol 16 (3) ◽

pp. 26-32

Author(s):

A. S. Khudovekova ◽

Ya. A. Rudenko ◽

A. E. Dorosevich

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Monoclonal Gammopathy ◽

Plasma Cells ◽

Bone Marrow Microenvironment ◽

Genetic Mutations ◽

Microbiome Composition ◽

The Impact ◽

Undetermined Significance

Multiple myeloma is a tumor of plasma cells, one of the most common malignant blood diseases. It is preceded by a stage called monoclonal gammopathy of undetermined significance, from which true multiple myeloma develops in only a small percentage of cases. It was assumed that this process is associated with the accumulation of genetic mutations, but in recent years there is increasing evidence that the bone marrow microenvironment plays a key role in progression and that it can become a target for therapy that prevents the myeloma development. The review considers the role of mesenchymal stem cells, immune system cells, endotheliocytes, fibroblasts, adipocytes, osteoclasts and osteoblasts in multiple myeloma progression, as well as the impact of the sympathetic nervous system and microbiome composition.

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MCL1 Inhibitors in Multiple Myeloma

Blood ◽

10.1182/blood-2019-121104 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. SCI-12-SCI-12

Author(s):

Karin Vanderkerken ◽

Kim De Veirman ◽

Ken Maes ◽

Eline Menu ◽

Elke De Bruyne

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Therapeutic Option ◽

Therapy Resistance ◽

Bone Marrow Microenvironment ◽

Molecular Heterogeneity

Apoptosis plays a key role, not only in normal homeostasis but also in protection against genomic instability. Protection against apoptosis is a hallmark of cancer and is mainly regulated by the overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-Xl or Mcl-1. This results in increased survival of the tumor cells and resistance to therapy. This presentation will focus on MCL-1 (myeloid cell leukemia 1), its expression and its role as potential target in multiple myeloma (MM). MCL1 gene regions are one the most amplified gene regions in several human cancers and Mcl-1 activity is often associated with therapy resistance and relapse. Mcl-1 binds to and sequesters the pro-apoptotic BH3 proteins, thereby preventing apoptosis. Mcl-1 is overexpressed on MM cells from newly diagnosed patients compared to normal plasma cells and in MM cells at relapse. This overexpression is furthermore associated with a shorter survival of these patients. Increased Mcl-1 expression can result either from genetic lesions or by induction through interaction with the bone marrow microenvironment. Its expression is correlated with the molecular heterogeneity of the myeloma patients; while the CCDN1 group has high BCL2 and low MCL-1 expression; the MMSET and MAF group has high MCL-1 and low BCL2 expression. Unlike Bcl-2 and Bcl-Xl, Mcl-1 has a large unstructured aminoterminus and its activity is mainly dependent on posttranslational modifications. The bone marrow microenvironment, by producing high levels of interleukin 6, also induces the upregulation of Mcl-1. Furthermore, our group recently demonstrated that not only stromal cells in the bone marrow microenvironment, but also MDSC (myeloid derived suppressor cells) induce survival of MM cells by increasing Mcl-1 levels through the AMPK pathway. As such, these data suggest the potential therapeutic benefit of targeting Mcl-1 in MM patients. Developing the first-generation inhibitors appeared to be challenging, especially in view of the occurrence of unwanted off target effects. Recent preclinical data with new, selective Mcl-1 inhibitors show promising anti-tumor effects both in vitro and in in vivo myeloma models, either alone or in combination with the Bcl-2 selective inhibitor, venetoclax, especially as it was demonstrated that high levels of MCL-1 are associated with venetoclax resistance in MM. In addition, it was also shown that proteasome inhibition can trigger Mcl-1 accumulation, further pointing to the importance of Mcl-1 inhibition. Induction of NOXA, as an inhibitor of Mcl-1, is also suggested as a therapeutic option, especially in combinations with other drugs. Clinically, following preclinical results, several new Mcl-1 inhibitors have entered phase I trials. Most of them are still recruiting patients, and as such too early to have results. Disclosures No relevant conflicts of interest to declare.

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Role of cytokines in sustaining long-term human megakaryocytopoiesis in vitro

Blood ◽

10.1182/blood.v79.2.332.bloodjournal792332 ◽

1992 ◽

Vol 79 (2) ◽

pp. 332-337 ◽

Cited By ~ 1

Author(s):

RA Briddell ◽

JE Brandt ◽

TB Leemhuis ◽

R Hoffman

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Interleukin 1 ◽

Gm Csf ◽

Liquid Suspension ◽

Bone Marrow Cultures ◽

Macrophage Colony

An in vitro liquid suspension culture system was used to determine the role of cytokines in sustaining long-term human megakaryocytopoiesis. Bone marrow cells expressing CD34 but not HLA-DR (CD34+DR-) were used as the inoculum of cells to initiate long-term bone marrow cultures (LTBMC). CD34+DR- cells (5 x 10(3)/mL) initially contained 0.0 +/- 0.0 assayable colony-forming unit-megakaryocytes (CFU-MK), 6.2 +/- 0.4 assayable burst-forming unit-megakaryocytes (BFU-MK), and 0.0 +/- 0.0 megakaryocytes (MK). LTBMCs were recharged every 48 hours with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 1 alpha (IL-1 alpha), IL-3, and/or IL-6, alone or in combination. LTBMCs were demidepopulated weekly or biweekly, the number of cells and MK enumerated, and then assayed for CFU-MK and BFU-MK. LTBMCs receiving no cytokine(s) contained no assayable CFU-MK or BFU-MK and no observable MK. LTBMCs receiving GM-CSF, IL-1 alpha, and/or IL-3 contained assayable CFU-MK and MK but no BFU-MK for 10 weeks of culture. The effects of GM-CSF and IL-3, IL-1 alpha and IL-3, but not GM-CSF and IL-1 alpha were additive with regards to their ability to augment the numbers of assayable CFU-MK during LTBMC. LTBMCs supplemented with IL-6 contained modest numbers of assayable CFU-MK for only 4 weeks; this effect was not additive to that of GM-CSF, IL-1 alpha, or IL-3. The addition of GM-CSF, IL-1 alpha, and IL-3 alone or in combination each led to the appearance of significant numbers of MKs during LTBMC. By contrast, IL-6 supplemented cultures contained relatively few MK. These studies suggest that CD34+DR- cells are capable of initiating long-term megakaryocytopoiesis in vitro and that a hierarchy of cytokines exists capable of sustaining this process.

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Role of the bone marrow microenvironment in tumor transformation of plasma cell dyscrasias

Health and Ecology Issues ◽

10.51523/2708-6011.2021-18-1-2 ◽

2021 ◽

pp. 14-19

Author(s):

Zh. Kozich ◽

L. A. Smirnova ◽

V. N. Martinkov

Keyword(s):

Bone Marrow ◽

Clinical Characteristics ◽

Plasma Cells ◽

Bone Marrow Microenvironment ◽

Significant Progress ◽

Benign Diseases ◽

Genetic Mechanisms ◽

Tumor Transformation ◽

Plasma Cell Dyscrasias

Despite significant progress in the understanding of the pathogenesis of paraproteinemic hemoblastoses, these diseases remain incurable. In their development, they go through the stage of paraproteinemias, benign diseases, characterized by the detection of monoclonal paraprotein in the blood serum and / or urine, presence of clonal plasma cells in the bone marrow or in extramedullary tissues. It remains unclear why some paraproteinemias progress to multiple myeloma or other lymphoid tumors and how malignant progression occurs. An important role in the progression is played by molecular and genetic mechanisms, cytokines. Nevertheless, little is known about how the bone marrow microenvironment influences disease progression. In this review, we made an attempt to summarize the most significant biological, clinical characteristics on the course of paraproteinemias and the role of changes in the bone marrow microenvironment that contribute to malignant transformation.

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Repurposing tofacitinib as an anti-myeloma therapeutic to reverse growth-promoting effects of the bone marrow microenvironment

10.1101/143206 ◽

2017 ◽

Author(s):

Christine Lam ◽

Megan Murnane ◽

Hui Liu ◽

Geoffrey A. Smith ◽

Sandy Wong ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Plasma Cells ◽

Single Agent ◽

Drug Repurposing ◽

Myeloma Cell ◽

Bone Marrow Microenvironment ◽

Growth Promoting

AbstractThe myeloma bone marrow microenvironment promotes proliferation of malignant plasma cells and resistance to therapy. Interleukin-6 (IL-6) and downstream JAK/STAT signaling are thought to be central components of these microenvironment-induced phenotypes. In a prior drug repurposing screen, we identified tofacitinib, a pan-JAK inhibitor FDA-approved for rheumatoid arthritis, as an agent that may reverse the tumor-stimulating effects of bone marrow mesenchymal stromal cells. Here, we validated bothin vitro, in stromal-responsive human myeloma cell lines, andin vivo, in orthotopic disseminated murine xenograft models of myeloma, that tofacitinib showed both single-agent and combination therapeutic efficacy in myeloma models. Surprisingly, we found that ruxolitinib, an FDA-approved agent targeting JAK1 and JAK2, did not lead to the same anti-myeloma effects. Combination with a novel irreversible JAK3-selective inhibitor also did not enhance ruxolitinib effects. RNA-seq and unbiased phosphoproteomics revealed that marrow stromal cells stimulate a JAK/STAT-mediated proliferative program in myeloma plasma cells, and tofacitinib reversed the large majority of these pro-growth signals. Taken together, our results suggest that tofacitinib specifically reverses the growth-promoting effects of the tumor microenvironment through blocking an IL-6-mediated signaling axis. As tofacitinib is already FDA-approved, these results can be rapidly translated into potential clinical benefits for myeloma patients.

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Immunologic Rebound After Cessation of Long-term Chemotherapy in Acute Leukemia

Blood ◽

10.1182/blood.v40.1.42.42 ◽

1972 ◽

Vol 40 (1) ◽

pp. 42-51 ◽

Cited By ~ 72

Author(s):

Luis Borella ◽

Alexander A. Green ◽

Robert G. Webster

Keyword(s):

Bone Marrow ◽

Antibody Production ◽

Immunosuppressive Drugs ◽

Lymphocytic Leukemia ◽

Cell Compartment ◽

Age Dependent ◽

Antibody Titers ◽

Kinetics Of ◽

Practical Implications

Abstract The aim of this study was to determine the kinetics of repopulation of blood and bone marrow lymphocytes, immunoglobulin synthesis, and antibody production after stopping long-term combination chemotherapy in children with acute lymphocytic leukemia (ALL) in continuous remission for 2½-3 yr. The phase of recovery after immunosuppressive drugs were discontinued was characterized by a rise of lymphocytes and increased immunoglobulin and antibody production, and it was dependent on the age of the patients at the beginning of treatment. Thus, after the drugs were stopped, blood and bone marrow lymphocytes and serum IgG and IgM increased significantly in the group of patients younger than 5 yr but not in the older group. Lymphocytosis was more pronounced and earlier in bone marrow, suggesting an initial expansion of this cell compartment. After the drugs were discontinued one-fourth of these patients had a rise in antibody to the Hong Kong influenza virus without evidence of reexposure to the same antigen. Although the mechanisms of this age-dependent immunologic rebound are uncertain, we postulate that the number of long-lived, drug-resistant, memory lymphocytes increases in peripheral blood as a function of age and antigenic stimulation. Practical implications derived from this study are: an increase of bone marrow lymphocytes above 40% in children with ALL in whom chemotherapy has been stopped does not indicate relapse but may be a manifestation of immunologic recovery; and a rise in antibody titers after cessation of immunosuppressive drugs may reflect immunologic rebound to an "old" antigen and not necessarily be secondary to an active infectious process.

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