Lymphocyte Differentiation and Effector Functions

More than a decade has passed since the publication of the first report implicating T cells in the bone loss induced by ovariectomy (ovx).1 Since then there has been extraordinary progress in the understanding of the regulatory network that links the hemopoietic and the mesenchymal compartments of the bone marrow (BM), the interactions between the immune system and bone, and the role of lymphocytes as mediators of the effects of calciotrophic hormones in bone. Collectively this body of knowledge has led to the firm establishment of "osteoimmunology" as a novel discipline and a promising area of investigation. The objective of this perspective is to revisit the exciting hypothesis that T cells play a pivotal role in the mechanism of ovx-induced bone loss. The bone-sparing activity of estrogens is due to a repression of bone remodeling coupled with a balancing effect on bone formation and resorption.2 The dominant acute effect of estrogen is the blockade of new osteoclast formation, cells which arise by cytokine-driven proliferation and differentiation of monocyte precursors that circulate within the hematopoietic cell.3 This process is facilitated by BM stromal cells (SCs), which provide physical support for nascent osteoclasts and produce soluble and membrane-associated factors essential for the proliferation and differentiation of osteoclast precursors. This inhibitory effect on osteoclastogenesis is associated with a repressive effect on osteoblastogenesis.4 Another critical effect of estrogen is that of increasing osteoclast apoptosis5-7 while blocking the apoptosis of osteoblasts and osteocytes.2, 8 It has been proposed that the effects of estrogens on the generation of osteoblasts and the lifespan of osteoblasts and osteoclasts result from extranuclear actions of the endoplasmic reticulum (ER) and activation of cytoplasmic kinases.2, 7 The decline of ovarian function at menopause results in decreased production of estrogen and a parallel increase in pituitary follicle-stimulating hormone (FSH) levels. The combined effects of estrogen deprivation and elevated FSH production cause a marked stimulation of bone resorption9 and a period of rapid bone loss that is central to the pathogenesis of postmenopausal osteoporosis. In mice the acute effects of menopause are modeled by ovx, a procedure that stimulates bone resorption by increasing osteoclast formation3 and lifespan.5-7 This initial phase of bone loss is followed by a slower but more prolonged loss of mainly cortical bone due to incomplete refilling of the resorption cavities due to insufficient osteoblast (OB) activity and lifespan.10 An expansion of the osteoclastic pool is therefore the key mechanism responsible for the bone loss that occurs early after ovx. The net bone loss caused by ovx is limited, in part, by an increase in bone formation resulting from stimulated osteoblastogenesis.11 This compensation is fueled by an expansion of the pool of BM–SCs, increased commitment of such pluripotent precursors toward the osteoblastic lineage,11 and enhanced proliferation of early OB precursors.4 Subsequent escalations in OB apoptosis12, 13 extensions of osteoclast lifespan5, 6 and increased secretion of cytokines that suppress bone formation, such as interleukin 7 (IL-7) and tumor necrosis factor (TNF), are the likely reasons for why bone formation does not increase as much as resorption after ovx. The stimulatory effect of ovx on SCs is equally relevant for osteoclastogenesis as one of the consequences of estrogen deprivation is the formation of osteoblastic cells with an increased osteoclastogenic activity14; ie, the capacity to support osteoclast formation. Evidence now suggests that T cells play a pivotal role in the mechanism of ovx-induced bone loss. Core observations include reports showing that ovx fails to induce trabecular and cortical bone loss in: T-cell–deficient nude mice,1, 15-17, wild-type (WT) mice deleted of T cells by injection of anti–T cell antibodies,18 mice treated with Abatacept19 (an agent that blocks T cell costimulation and induces T cell anergy and apoptosis)20, 21 and mice lacking the T cell costimulatory molecule CD40 ligand (CD40L).18 The fact that nude mice are protected against ovx-induced bone loss has been confirmed independently.22 By contrast, Lee and colleagues23 showed that nude mice are protected against the loss of cortical, but not trabecular bone induced by ovx. In another study, T-cell–deficient and B-cell–deficient mice were found to lose bone after ovx.24 The discrepancy between our reports1, 15-19, 25 and those of others23, 24 is likely explained by differences in the experimental design, the lack of B cells in some models, and compensatory mechanisms such as an increase in natural killer (NK) cells producing the osteoclastogenic factor IL-17.26 Both CD4+ and CD8+ cells have been found to play a role in ovx-induced bone loss. CD4+ cells include the TH1, Th2, and Th17 subsets. Th17 are regarded as the most osteoclastogenic subset of T CD4+ cells because they produce high levels of IL-17, receptor activator of NF-κB ligand (RANKL), and TNF, and low levels of interferon gamma (IFNγ).27, 28 The differentiation of Th17 is inhibited by estrogen via a direct effect mediated by estrogen receptor alpha (ERα).29 The role of Th17 in ovx-induced bone loss remains to be determined because one study reported that IL-17R–null mice are more susceptible to ovx-induced bone loss than controls,30 while another group found these mice to be protected from ovx-induced bone loss.26 More abundant information is available about regulatory T cells (Tregs), a population capable of suppressing the effector function of TH1, Th2, and Th17 T cells. Tregs are defined by the expression of the transcription factor FoxP3. Tregs inhibit monocyte differentiation into osteoclasts in vitro and in vivo, and blunt bone resorption31, 32 through the secretion of IL-4, IL-10, and transforming growth factor beta 1 (TGFβ1).33 Attesting to the relevance of Tregs, studies have shown that estrogen increases the relative number of Tregs.27, 34 Moreover, transgenic mice overexpressing Tregs develop progressive high bone mass due to inhibition of bone resorption, and are protected against ovx-induced bone loss.35 Moreover, adoptive transfer of Tregs into T cell–deficient mice increases bone mass, indicating that Tregs directly affect bone homeostasis without the need to engage other T cell lineages.35 Two mechanisms have been described to explain how T cells contribute to ovx-induced bone loss (Fig. 1). The first involves an increase in T cell activation, leading to enhanced production of TNF by BM T cells. The second is a regulatory crosstalk between T cells and SCs, resulting in enhanced production of osteoclastogenic cytokines by SCs. Schematic representation of the role of T cells in the mechanism by which ovx promotes osteoclastogenesis, osteoblastogenesis, and hemopoiesis. Estrogen deficiency promotes T cell activation by increasing the interaction of antigen (Ag)-loaded MHC molecules with bone marrow macrophages (BMM) and dendritic cells (DC) with the T cell receptor (TCR). The Ags are likely to be non-self peptides derived from the intestinal macrobiota. T cell activation also requires at least two costimulatory signals provided by the binding of BMM and DC-expressed CD40 and CD80 to the T cell surface molecules CD40L and CD28, respectively. A critical upstream event is the increased production of reactive oxygen species (ROS) that activate DCs by increasing their expression of CD80. The expansion of T cells in the BM is partially driven by an ovx-induced increase in the thymic output of naïve T cells. Activated T cells secrete TNF that stimulates osteoclast formation primarily by potentiating the response to RANKL. In addition, T cell–expressed CD40L and DLK1/FA-1 increase the osteoclastogenic activity of SC by blunting their secretion of OPG and augmenting their production of RANKL, M-CSF, and other proinflammatory factors. The survival of naïve T cells and some memory T cells requires the low-affinity engagement of the T cell receptor (TCR) by a diverse repertoire of self-antigens (Ags) bound to major histocompatibility complex (MHC) molecules expressed on Ag-presenting cells.36, 37 By contrast, binding of foreign Ags such as bacterial Ags with MHC molecules is followed by high-affinity interactions with TCRs that drive T cell activation. Both low-affinity interactions with self-Ags and high-affinity foreign Ag/MHC/TCR interactions are referred to as "Ag presentation." Ovx induces T cell expression of activation markers and promotes T cell proliferation, expansion, and acquisition of effector functions. These are all features of T cells exposed to foreign Ags.36 The gastrointestinal tract is colonized for life with 100 trillion indigenous bacteria, creating a diverse ecosystem known as the microbiota, whose contributions to human health are profound.38 The macrobiota is likely to represent the source of foreign Ag that drives the expansion of T cells induced by ovx. Although direct verification of a role for macrobiota in T cell expansion is still lacking, compelling supportive data are available. For example, adoptive transfer of T cells into T cell–deficient mice is followed by rapid engraftment and expansion of donor T cells into the host. This process is driven by foreign Ags.36 Attesting to a role of the macrobiota, the expansion of transferred T cells into T cell–deficient mice is greatly reduced in host mice raised in a germ-free environment.36 Moreover, mice maintained in germ-free conditions display increased bone mass due to the lack of immune cell activation.39 T cells are key inducers of bone-wasting because ovx increases T cell TNF production to a level sufficient to augment RANKL-induced osteoclastogenesis.1 This effect is due to an increased number of TNF-producing T cells15 and enhanced production of TNF per cell.18, 40 Ovx also increases the population of premature senescent CD4 + CD28–T cells,40 a lineage that produces high levels of TNF. The presence of increased levels of T cell–produced TNF in the BM of ovx animals is well documented.15, 19, 40, 41 Studies have also shown that menopause increases T cell activation and T cell production of TNF and RANKL in humans.42, 43 The role of TNF in ovx-induced bone loss has been demonstrated in multiple models. For example, ovx fails to induce bone loss in TNF-null mice and in animals lacking the p55 TNF receptor.15 Likewise, transgenic mice insensitive to TNF due to the overexpression of a soluble TNF receptor,44 and mice treated with the TNF inhibitor TNF binding protein45 are protected from ovx-induced bone loss. The specific relevance of T cell TNF production in vivo was demonstrated by the finding that although reconstitution of nude recipient mice with T cells from WT mice restores the capacity of ovx to induce bone loss, reconstitution with T cells from TNF-deficient mice does not.15 The mechanism by which estrogen deficiency expands the pool of TNF-producing T cells is summarized in Figure 1 and involves reactivation of thymic function and induction of T cell activation in the BM. T cell activation is driven by enhanced Ag presentation by macrophages and dendritic cells (DCs).46, 47 The thymus undergoes progressive structural and functional decline with age, coinciding with increased circulating sex-steroid levels at puberty.48 By middle age most parenchymal tissue is replaced by fat, and in both mice and humans fewer T cells are produced and exported to secondary lymphoid organs. However, the thymus continues to generate new T cells even into old age.49, 50 In fact, active lymphocytic thymic tissue has been documented in adults up to 107 years of age.51 Under severe T cell depletion secondary to human immunodeficiency virus (HIV) infection, chemotherapy, or bone marrow transplant, an increase in thymic output (known as thymic rebound) becomes critical for long-term restoration of T cell homeostasis. For example, middle-aged women treated with autologous bone marrow transplants develop thymic hypertrophy and a resurgence of thymic T cell output, which contributes to the restoration of a wide T cell repertoire,52 although the intensity of thymic rebound declines with age. The mechanism driving thymic rebound is not completely understood, but one factor involved is IL-7.53 Both androgens and estrogen suppress thymic function.54, 55 Accordingly, castration reverses thymic atrophy and increases export of recent thymic emigrants to the periphery,56 whereas sex steroids inhibit thymic regeneration by promoting thymocyte apoptosis and arresting thymocyte/prelymphocyte differentiation.57 Restoration of thymic function after castration occurs in young58 as well as in very old rodents.59, 60 In accordance with the notion that estrogen deficiency induces a rebound in thymic function, ovx increases the thymic export of naïve T cells.61 Indeed, stimulated thymic T cell output accounts for ∼50% of the increase in the number of T cells in the periphery. Moreover, thymectomy decreases the bone loss induced by ovx by ∼50%, thus demonstrating that the thymus plays a previously unrecognized role in the pathogenesis of ovx-induced bone loss in mice.61 The remaining bone loss is a consequence of the peripheral expansion of naïve and memory T cells. This finding suggests the tantalizing hypothesis that estrogen deficiency–induced thymic rebound may be responsible for the exaggerated bone loss in young women undergoing surgical menopause62 or for the rapid bone loss characteristic of women in their first 5 to 7 years after natural menopause.63 Indeed, an age-related decrease in estrogen deficiency–induced thymic rebound could mitigate the stimulatory effects of sex steroid deprivation and explain why the rate of bone loss in postmenopausal women diminishes as aging progresses.63 The most upstream effects of ovx in the BM are to stimulate the production of reactive oxygen species (ROS) and to impair the generation of antioxidants.10, 19, 64, 65 In response to ovx, ROS are produced by most BM cells, including T cells.41 ROS play an important role in postmenopausal bone loss by generating a more oxidized bone microenvironment.66, 67 Multiple enzymatic pathways regulate the intracellular redox state through modulation of ROS levels.68 Ovx blunts the BM levels of glutathione (GSH), a critical ROS scavenger, and reduces expression of APE1/Ref-1 and Prx-1 proteins, which collectively limit the production of intracellular ROS.69 ROS have important direct effects on osteoblasts and osteoclasts; these effects have been addressed elsewhere.7, 70, 71 However, additional pivotal effects of ROS include expanding the pool of mature DCs that express the costimulatory molecule CD80, and increasing DC-mediated Ag presentation.19 Antioxidants potently inhibit DC differentiation and their ability to activate T cells,72, 73 in part by suppressing expression of MHC class II and costimulatory molecules in response to Ag.74 N-acetyl-cysteine (NAC), which acts as an intracellular scavenger of ROS by restoring intracellular concentrations of GSM, can block DC maturation75 and DC-mediated T cell activation.76 In vivo support for a role of ROS is provided by experiments demonstrating that administration of antioxidants prevents ovx-induced bone loss,19, 64, 70 while depletion of glutathione by buthionine sulfoximine (BSO), which inhibits glutathione synthesis, enhances bone loss.64 Bone loss caused by BSO has significant similarities to bone loss induced by estrogen deficiency, as both processes are TNF-dependent.77 A second, direct upstream effect of estrogen deficiency is to blunt BM levels of TGFβ,78 a powerful repressor of T cell activation. TGFβ acts as an immunosuppressant by inhibiting T cell activation and T cell production of inflammatory cytokines, including IFNγ. Demonstrating the relevance of the repressive effects of TGFβ on T cell function, mice with T cell–specific blockade of TGFβ signaling were found to be completely resistant to the bone-sparing effects of estrogen.16 Gain of function experiments confirmed that elevation of the systemic levels of TGFβ prevents ovx-induced bone loss and bone turnover.16 The key downstream mechanism by which ovx increases Ag presentation by macrophages is a stimulatory effect on the expression of the gene encoding Class II Transactivator (CIITA). The product of CIITA is a non-DNA binding factor induced by IFNγ that functions as a transcriptional coactivator at the MHC II promoter.79 Increased CIITA expression in macrophages results from ovx-mediated increases in IFNγ production by CD4+ T cells and the responsiveness of CIITA to IFNγ.46 This cytokine was initially described as an anti-osteoclastogenic cytokine because it is a potent inhibitor of osteoclastogenesis in vitro.80 The notion that IFNγ is an inhibitor of bone resorption was reinforced by the finding that silencing of IFNγR−/− signaling leads to a more rapid onset of collagen-induced arthritis and bone resorption81 as compared to WT controls, and by the report that IFNγ decreases serum calcium and osteoclastic bone resorption in nude mice.82, 83 Several mechanisms have been proposed to explain the anti-osteoclastogenic activity of IFNγ, including inhibition of RANKL signaling through the degradation of TNF receptor–associated factor 6 (TRAF6),80 stimulation of apoptosis mediated by Fas/FasL signals,84 and inhibition of RANK and c-Fms gene expression.85 However, the finding that IFNγ is an effective treatment for osteopetrosis both in humans86 and rodents87 demonstrates that the net effect of IFNγ in vivo is to stimulate osteoclastic bone resorption. In keeping with a net pro-resorptive effect of IFNγ in vivo are reports demonstrating that IFNγ−/− and IFNγR−/− mice are protected against ovx-induced bone loss.17, 46 Mice lacking IFNγ production are also protected against infection-induced alveolar bone loss,88 whereas in erosive tuberculoid leprosy and psoriatic arthritis IFNγ production correlates positively with tissue destruction.89 In addition, randomized controlled trials have shown that IFNγ does not prevent bone loss in patients with rheumatoid arthritis (RA),90 nor the bone-wasting effect of cyclosporine A.91 Finally, disruption of IFNγ signaling in vivo results in a strong and sustained inhibition of markers of osteoclastic activity.92 These opposing in vitro and in vivo effects of IFNγ are explained by the fact that IFNγ influences osteoclast formation via both direct and indirect effects.17 IFNγ directly blocks osteoclast formation through targeting of maturing osteoclast.93 However, IFNγ is also a potent inducer of antigen presentation and thus of T cell activation. Therefore, when IFNγ levels are increased in vivo, activated T cells secrete pro-osteoclastogenic factors and this activity offsets the anti-osteoclastogenic effects of IFNγ.17 It should also be mentioned that it is now recognized that IFNγ affects bone turnover by promoting the commitment of BM–SCs into the osteoblastic lineage and their differentiation into mature osteoblasts.94 Accordingly, treatment with IFNγ reverses ovx-induced bone loss by promoting bone formation.92 Another mechanism by which estrogen regulates T cell TNF production is by repressing the production of IL-7, a potent lymphopoietic cytokine and an inducer of bone destruction in vivo.95 Attesting to the relevance of this factor, IL-7 levels are significantly elevated following ovx,61, 96 and in vivo IL-7 blockade is effective in preventing ovx-induced bone destruction.96 The elevated BM levels of IL-7 contribute to the expansion of the T cell population in peripheral lymphoid organs through several mechanisms. First, IL-7 directly stimulates T cell proliferation.61 Second, IL-7 increases antigen presentation by upregulating the production of IFNγ. Third, IL-7 and TGFβ regulate The in TGFβ signaling characteristic of estrogen deficiency may to stimulate IL-7 thus driving the of osteoclastogenic cytokine production and bone In estrogen deficiency, IL-7 bone loss by suppressing bone formation and thus bone formation from resorption. A that the crosstalk between T cells and SCs is relevant for ovx-induced bone loss was provided by the finding that activated T cells induce SC apoptosis via the ligand a which blunts the compensatory increase in bone formation that bone loss in ovx More abundant information is available about the T crosstalk driven by the also known as is a key surface ligand expressed on T CD40L to and several CD40 is expressed on cells, hemopoietic and cells of the osteoblastic CD40L has been to because T cells, through the production of the anti-osteoclastogenic factor by B mice a reduced bone due to exaggerated bone bone has also been found in by a in which CD40L production is due to a in the CD40L However, mice lacking T cell–expressed CD40L are protected against hormone bone the that CD40L may in while promoting bone resorption conditions of bone Studies with mice and with WT mice treated with the CD40L have that silencing of CD40L completely prevents ovx-induced bone A mechanism was shown to be First, silencing of CD40L blocks the activation of T cells and the resulting production of TNF. Second, CD40L is for ovx to increase the proliferation and the differentiation of SCs and their capacity to support osteoclast formation through enhanced production of factor and RANKL, and secretion of a critical additional mechanism by which T cells bone homeostasis in ovx mice is through a crosstalk between T cells and SCs that results in enhanced osteoclastogenesis to a enhanced In ovx increases the number of activated T cells that the expression of and RANKL by SCs, and ovx the SC production of The net result is a significant increase in the rate of The interaction of CD40L with CD40 on SCs in the of estrogen deficiency to the effects of costimulation on B cell OPG the of osteoclast formation in of bone loss. An additional mechanism of T crosstalk the novel of bone mass antigen has been for a known as This factor can be to generate a soluble known as In conditions DLK1/FA-1 are produced by SCs, B cells, and T cells. These factors stimulate osteoclastogenesis and block by the production of TNF, IL-7, and other inflammatory cytokines by SCs. Ovx increases the production of DLK1/FA-1 by activated CD4+ and CD8+ T cells, resulting in a stimulation of the production of osteoclastogenic cytokines by SCs. Attesting to the relevance of mice are significantly protected against ovx-induced bone The hypothesis that the immune system is pivotal for the bone loss about by menopause remains to be demonstrated in it remains to the of why the immune system in and is involved in the mechanism of ovx-induced bone loss. may by postmenopausal bone loss as an of an event critical for the need to stimulate bone resorption in the This process is essential to the increased for calcium about by The for this event is the acute in estrogen levels A second to the period is in associated with is by proliferation of the and formation of These are by and is now that regulates and to by the production of RANKL and a system that is for bone homeostasis is also a key of The in levels in the period results in reduced tissue levels of RANK and RANKL, with the effect of the for The increase in the and TNF levels in the BM induced by estrogen levels the of calcium from the to the and then to the a state of to the is induced by an increase in the number of A to the of is the loss of such immune and the restoration of a immune This is through a decrease in the resulting from the acute in steroid levels. However, a between immune to the and bone is provided by the that OPG is expressed by human it is to that of ovarian function induces bone loss through an immune response because natural has these key within the immune system to (Fig. Schematic representation of in the period and of the involved cells and The that estrogen and are followed by a stimulation of bone resorption, to and of immune to the Activated T cells drive bone resorption by TNF. A decrease in the number of Tregs induces the of the immune The loss of OPG may contribute to the and the increased bone resorption. A decrease in levels of RANKL and RANK induce the of the for with increased of calcium from the the in the contribute to an event critical for The data the hypothesis that of ovarian function induces bone loss through an immune response because natural has key within the immune system to The data support the hypothesis that the bone loss induced by estrogen deficiency is due to a complex of hormones and cytokines that to the process of bone Ovx T cell TNF production by increasing T cell activity in the and the peripheral lymphoid organs. T cell precursors the BM and to the T cell and expansion in the of from the these new T cells to peripheral lymphoid including the BM Ovx induces T cell activation in the BM in part by directly promoting antigen and in part via stimulation of IL-7 and IFNγ production and of TGFβ The net result of these actions is an increase in the number of TNF-producing T cells. The elevated levels of TNF increase RANKL-induced osteoclast formation. Estrogen deficiency also T cell activation and osteoclastogenesis by leading to an in The combined effect of IFNγ and ROS enhances Ag T cell activation. T cells stimulate RANKL and production by SCs, through CD40L and The that has of

CD27 is a member of the TNF-Receptor superfamily expressed on CD4+ and CD8+ T cells, on NK and NKT cells and on B cells. It promotes T cell co-activation, proliferation, clonal expansion and differentiation into antigen specific cytotoxic and memory T cells after stimulation with its ligand CD70. Its stimulatory signal is mediated via the NFkB pathway, but also via the phosphatidylinositol 3 kinase and the protein kinase B. Moreover, CD27 signaling influences the innate immune response via a direct activation of NK cells and a subsequent secretion of interferon-gamma (IFN-γ). CD27 plays a central role in immunological responses and by promoting T cell and NK cell activation it contributes to anti-tumor immunity. Previous studies have demonstrated tumor growth inhibition with anti-CD27 agonistic monoclonal antibodies in different mice models for solid and hematological tumors. This mechanism of action can be partly explained by the recruitment of IFN-γ producing CD8+ T cells within the tumor. CD27 is a promising target for antitumor therapy. Kineta has generated a library of 147 fully human anti-CD27 monoclonal antibodies after immunization of Trianni mice. From this library, a lead candidate with strong agonistic proprieties has been selected. This anti-CD27 antibody originates from a unique clade after alignment of the variable heavy chains. Kineta’s lead candidate demonstrates selectivity and cross-reactivity with Non-Human Primate (NHP)-CD27 but not with the mouse-CD27. It also induces strong NFkB signaling in a Jurkat T cell-reporter, either soluble or cross-linked. It also induces T cell proliferation and secretion of pro-inflammatory cytokines in vitro. This T cell activation occurs only in the presence of a TCR engagement preventing future risks of spontaneous activation of naïve T cells in vivo. Our lead antibody also induces direct activation of NK cells demonstrated by the expression of CD69 on their surface. We have evaluated the anti-tumor properties of our lead antibody as a single agent in vivo in human CD27 Knock-In (KI) mice. Our anti-CD27 candidate induces a significant anti-tumor activity in the EG7 thymoma model. We have also demonstrated the anti-tumor efficacy of this lead candidate in Raji cells implanted in Scid mice. Preliminary experiments performed in human CD27 KI mice have demonstrated a long half-life of our antibody at different concentrations. Epitope characterization, NHP pharmacokinetic analysis and additional in vivo studies of our lead anti-CD27 antibody in different tumor models use as a single agent and in combination with different check-point inhibitors are on-going. Citation Format: Shawn Iadonato, Jessica Cross, Nathan Eyde, Emily Frazier, Neda Kabi, Chen Katz, Remington Lance, Kurt Lustig, Yulia Ovechkina, David Peckham, Shaarwari Sridhar, Carla Talbaux, Isabelle Tihista, Mei Xu, Thierry Guillaudeux. CD27 an emerging immuno-oncology target at the cross-roads of innate and adaptive anti-tumor immune responses [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A60.

To investigate the interaction between myeloid-derived suppressor cells (MDSCs) and nature killer cells during acute fulminate hepatitis. Acute fulminate hepatitis were induced by i.p. co-injection of LPS and D-GalN in mice, and the ratio of MDSCs,NK cells and the activation of NK cells in different tissues were analyzed by FACS at 0 h,1.5 h,3 h and 6 h. The percentage of MDSCs and NKG2D+NK cells in different tissues increased as acute fulminate hepatitis progressed, with the increased NK cells in liver tissue. The mean fluorescence intensity of NKG2D on NK cells in different tissues were also enhanced. MDSCs induce the proliferation and activation of NK cells in mice with acute fulminate hepatitis.

Identification of Biological and Pharmaceutical Mast Cell‐ and Basophil‐Related Targets

CD40 ligand (CD40L) is a well-known regulator of the adaptive immune response that induces and modulates the activation and differentiation of monocytes, dendritic cells, and lymphocytes. Less appreciated is its critical role in promoting the inflammatory response in CD40-expressing cells such as vascular endothelium. Expression of CD40L on TCR activated human CD4+ T cells is biphasic and upregulated by monocyte cell contact early and IL-2R signaling late. Here we demonstrate the existence of additional, distinct pathways that differentially regulate the expression of CD40L. We describe a novel link between vascular endothelial cell adhesion molecule 1 (VCAM-1), extracellular matrix (ECM) proteins, and CD40L expression on human CD4+T cells that constitutes an innate form of T cell costimulation modulated by the type and quantity of ECM protein present, with thrombospondin-1 inhibiting fibronectin-mediated costimulation of CD40L expression. Identification of this previously unrecognized β1 integrin pathway, which promotes CD40L expression on CD4+ T cells independently of monocytes and IL-2, provides new insights into our understanding of the potential role of CD40L in the establishment and progression of inflammatory disease.

Fibroblasts are ubiquitous cells that can adopt many functional states. As tissue-resident sentinels, they respond to acute damage signals and shape the earliest events in fibrotic and immune-mediated inflammatory diseases. Upon sensing an insult, fibroblasts produce chemokines and growth factors to organize and support the response. Depending on the size and composition of the resulting infiltrate, these activated fibroblasts may also begin to contract or relax thus changing local stiffness within the tissue. These early events likely contribute to the divergent clinical manifestations of fibrotic and immune-mediated inflammatory diseases. Further, distinct changes to the cellular composition and signaling dialogue in these diseases drive progressive fibroblasts specialization. In fibrotic diseases, fibroblasts support the survival, activation and differentiation of myeloid cells, granulocytes and innate lymphocytes, and produce most of the pathogenic extracellular matrix proteins. Whereas, in immune-mediated inflammatory diseases, sequential accumulation of dendritic cells, T cells and B cells programs fibroblasts to support local, destructive adaptive immune responses. Fibroblast specialization has clear implications for the development of effective induction and maintenance therapies for patients with these clinically distinct diseases.

Past studies with the live, double-mutant B. abortus (znBAZ) strain resulted in nearly complete protection of mice against pulmonary challenge with wild-type (wt) Brucella via a dominant CD8+ T cell response. To understand the contribution innate immune cells in priming CD8+ T cell responses, mice were nasally dosed with wt B. abortus, smooth vaccine strain 19 (S19), or znBAZ, and examined for innate immune cell activation. Flow cytometric analysis revealed that znBAZ, but not wt B. abortus nor S19 infection, induces up to a 5-fold increase in the frequency of IFN-γ-producing NK cells in mouse lungs. These NK cells express increased CXCR3 and Ki67, indicating their recruitment and proliferation subsequent to znBAZ infection. Their activation status was augmented noted by the increased NKp46 and granzyme B, but decreased NKG2A expression. Further analysis demonstrated that both lung caspase-1+ inflammatory monocytes and monocyte-derived macrophages secrete chemokines and cytokines responsible for NK cell recruitment and activation. Moreover, neutralizing IL-18, an NK cell-activating cytokine, reduced the znBAZ-induced early NK cell response. NK cell depletion also significantly impaired lung dendritic cell (DC) activation and migration to the lower respiratory lymph nodes (LRLNs). Both lung DC activation and migration to LRLNs were significantly impaired in NK cell-depleted or IFN-γ-/- mice, particularly the CD11b+ and monocytic DC subsets. Furthermore, znBAZ vaccination significantly induced CD8+ T cells, and upon in vivo NK cell depletion, CD8+ T cells were reduced 3-fold compared to isotype-treated mice. In summary, these data show that znBAZ induces lung IFN-γ+ NK cells, which plays a critical role in influencing lung DC activation, migration, and promoting protective CD8+ T cell development.

Targeting Mechanisms for Natural Killer Cell Dysfunction in Patients with Multiple Myeloma

Breast cancer is the most common cancer and the second leading cause of cancer death in women. Except for surgery, radiotherapy, chemotherapy and targeting therapy, breast cancer vaccine is now actively developed. However, the outcome of most therapeutic cancer vaccines was not satisfying probably due to the induction of immunosuppression in cancer patients. Myeloid-derived suppressor cells (MDSCs) are one of the major immunoregulatory cell populations, which play important roles not only in T-cell suppression but also in angiogenesis, lymphangiogenesis and metastasis during tumor progression. MDSCs are heterogeneous populations of immature myeloid cells, which expand and developed in responses to growth factors, cytokines and chemokines derived from cancer cells and then are released from bone marrow to peripheral tissues including tumor site. It is well known as tumor cells could influence the activation and proliferation of myeloid cells through release of cytokines during tumor progression to result in the accumulation of large number of MDSCs in cancer patients. However little is known about the relevance of metabolic status and accumulation of MDSCs in cancer patients during tumor progression. We therefore try to elucidate the metabolic changes during differentiation of myeloid cells into MDSCs. To intensively study the transcriptional program of MDSCs in comparison to their healthy counterparts, we perform DNA microarray analysis using mRNAs from monocytic MDSCs, granulocytic MDSCs from primary tumor sites of 4T1 tumor-bearing mice, monocytes and neutrophils from bone marrow of healthy mice. MDSCs up-regulated mRNA levels of genes related to angiogenesis and lymphangiogenesis e.g. VEGFa, angiopoietin 1, PROK2 (BV8), MMP9, MMP12, MMP13 and MMP14, genes of pro-inflammatory cytokines e.g. IL-1α, IL-6 and TNFα, genes of immunosuppressive molecules e.g. arginase 1, iNOS, PD-L1, PD-L2, CD83 and CD155. We are currently examining the metabolomics of these MDSCs and their healthy counterparts and expect to prove the association of certain metabolic changes with the differentiation of MDSCs. Citation Format: Li-Rung Huang, Shiou-Ling Jian, Yi-Jyun Jhou. The influence of breast cancer on the metabolic status of myeloid cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B78.

Immunotherapies designed to activate or replace T-cell effector immunity, such as immune checkpoint blockade or adoptive transfer of T cells, are limited by the ability of subpopulations of tumor cells to escape T-cell immunity. This can occur through immunologic selection of tumor cells that present weak T-cell antigens or harbor genomic or epigenetic alterations that alter antigen processing or presentation or responses to interferon. Immunotherapy designed to activate or replace NK cell immunity may overcome these limitations through antigen and MHC class I-independent tumor cell recognition and killing. However, similar to suppression of T-cell function, local immunosuppression within the tumor microenvironment may limit the function of NK cells. We hypothesized that immature, immunosuppressive myeloid cells (myeloid derived suppressor cells, or MDSCs) may limit optimal function of NK cells and that NK cell control of tumors can be enhanced through abrogation of MDSC trafficking and tumor infiltration. To model new NK cell therapy products that are available for clinical investigation, we studied how MDSCs alter the ability of culturable murine NK (KIL) cell line to kill oral cancer cells. We used an oral cancer model, MOC2, that has few genomic alterations and forms highly aggressive, non-T cell-inflamed tumors in wild-type C57BL/6 mice. CXCR2+ neutrophilic-MDSCs (PMN-MDSCs) sorted from MOC2 tumors significantly suppressed the ability of KIL to kill MOC2 tumor cells in vitro as assessed by real-time impedance analysis. NK suppression mediated by PMN-MDSC appeared to be mediated by both cell surface TGFβ and arginase. In vivo, we aimed to block chemotaxis and MOC2 tumor infiltration of CXCR2+ PMN-MDSC with the use of SX-682, a clinical-stage small-molecule inhibitor of CXCR1/2. After validating that SX-682 blocks tumor PMN-MDSC trafficking into tumors, we treated mice bearing established MOC2 tumors with SX-682 and adoptive transfer of KIL, alone or in combination. While SX-682 alone produced no tumor growth inhibition (TGI) and KIL adoptive transfer alone produced modest TGI, the combination resulted in significant TGI and prolonged survival of mice. Immune correlative analysis revealed increased tumor infiltration of adoptively transferred KIL with inhibition of PMN-MDSC tumor infiltration. These data suggest that the efficacy of NK cell therapies can be enhanced with PMN-MDSC inhibition and support the clinical investigation of chemokine receptor inhibitors such as SX-682 in combination with NK cell therapy products. Citation Format: Sarah Greene, Yvette Robbins, Paul E. Clavijo, Dean Maeda, John Zebala, Jeffrey Schlom, Claudia Palena, Clint Allen. Natural killer cell therapy efficacy is enhanced through inhibition of myeloid-derived suppressor cell trafficking [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A16.

Central Memory T-Cell Differentiation Correlates with Depth of Response in Relapsed/Refractory Multiple Myeloma Patients Receiving Elotuzumab in Combination with Carfilzomib, Lenalidomide and Dexamethasone (Elo-KRd)

Targeting the crosstalk between cytokine-induced killer cells and myeloid-derived suppressor cells in hepatocellular carcinoma

The three-signal paradigm tries to capture how the innate immune system instructs adaptive immune responses in three well-defined actions: (1) presentation of antigenic peptides in the context of MHC molecules, which allows for a specific T cell response; (2) T cell co-stimulation, which breaks T cell tolerance; and (3) secretion of polarizing cytokines in the priming environment, thereby specializing T cell immunity. The three-signal model provides an empirical framework for innate instruction of adaptive immunity, but mainly discusses STAT-dependent cytokines in T cell activation and differentiation, while the multi-faceted roles of type I IFNs and IL-1 cytokine superfamily members are often neglected. IL-1α and IL-1β are pro-inflammatory cytokines, produced following damage to the host (release of DAMPs) or upon innate recognition of PAMPs. IL-1 activity on both DCs and T cells can further shape the adaptive immune response with variable outcomes. IL-1 signaling in DCs promotes their ability to induce T cell activation, but also direct action of IL-1 on both CD4+ and CD8+ T cells, either alone or in synergy with prototypical polarizing cytokines, influences T cell differentiation under different conditions. The activities of IL-1 form a direct bridge between innate and adaptive immunity and could therefore be clinically translatable in the context of prophylactic and therapeutic strategies to empower the formation of T cell immunity. Understanding the modalities of IL-1 activity during T cell activation thus could hold major implications for rational development of the next generation of vaccine adjuvants.

Chimeric antigen receptor (CAR) T cells have shown great promise in treating hematopoietic malignancies, such as leukemia and non-Hodgkin’s lymphoma. The efficacy and durability of CAR T cell therapy have been correlated with higher levels of T stem cell memory (TSCM) and T central memory (TCM) populations. Accordingly, various strategies to enrich these CAR T cell populations are of intense interest. It is well-established that the PI3K/mTOR pathway plays an important role in T cell activation and differentiation, and perturbations in this pathway can enhance T cell memory populations. Work from our group has demonstrated that MNK modifies mTOR signaling and T cell differentiation. Here, we demonstrate that the MNK1/2 inhibitor tomivosertib can substantially increase TCM and TSCM populations in both primary murine and human T cells. Tomivosertib treatment of murine OT-I T cells biases T cell differentiation towards a TCM (CD8+ CD44+ CD62L+) population upon SIINFEKL peptide stimulation in vitro without adverse effects on T cell proliferation, interferon-γ production or cytotoxic function. Similar effects are seen in vivo, where tomivosertib treatment also enriches the TCM cell pool in a SIINFEKL vaccine-induced OT-I adoptive T cell transfer model, which results in increased persistence as demonstrated by a higher memory-recall T cell response upon re-challenge. Furthermore, addition of tomivosertib during production of human CAR (anti-CD19 scFv-4-1BB-CD3ζ) T cells leads to a significantly increased population of TSCM (CD8+ CD45RO CD45RA+ CD27+ CD95+) cells. Based on these data, the combination of tomivosertib with CD19-directed CAR T cells was assessed in the CD19+ Pfeiffer DLBCL model in vivo. Daily oral administration of tomivosertib in combination with CD19-directed CAR T cells results in improved efficacy in comparison to either monotherapy alone, consistent with tomivosertib promoting and maintaining TSCM populations in vivo. Tomivosertib is currently in multiple Phase II clinical trials as a monotherapy or in combination with checkpoint inhibitors. Citation Format: Vikas K. Goel, Rajesh K. Sharma, Jocelyn Staunton, Craig R. Stumpf, Nathan P. Young, Peggy A. Thompson, Gary G. Chiang, Kevin R. Webster. Tomivosertib (eFT508), a potent and highly selective inhibitor of MNK1 and MNK2, enhances CAR T cell activity through modulating T cell differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-068.

Background: Women age 20-40 have a higher ten year risk for developing breast cancer than the five other leading cancers in this gender and age group combined. Women currently in their 30s have a 1/203 risk for breast cancer in the ensuing ten years. Cancers diagnosed in this age population of breast cancer have a higher risk for death for reasons that are not fully identified. Regulatory T cell (Tregs) and myeloid derived suppressor cells (MDSC) have a role in suppressing anti-tumor immunity. MDSC function through Jak/Stat pathway activation by generation of reactive oxygen species and/or arginase-1, and have been correlated pre-clinically with cancer progression and poorer prognostic features and outcomes. Our Young Women's Breast Cancer Translational Program seeks to identify immunologic differences potentially contributing to the poorer prognosis and potential targets for development of immunomodulatory treatment. Hypothesis: We hypothesized that newly diagnosed, treatment-naive young breast cancer cases would have higher percentage of circulating Tregs and MDSC with T cell suppressive function that similar age women without breast cancer. Methods: We conducted an IRB-approved, prospective translational study of women age 40 and under both affected and unaffected by breast cancer. Exclusion criteria included pregnancy, known autoimmunity or immunosuppressive medications, or other cancers. Tregs and MDSC were isolated from peripheral blood and phenotyped through standard protocols. The ability of MDSC to suppress T cell function were tested in co-culture assays for expression of activation marker CD25, production of gamma-interferon and production of arginase-1 and generation of reactive oxygen species. Results: As expected, Tregs were in higher number in young women with breast cancer than unaffecteds. Contrary to prior publications no difference was detected in number of MDSC identified in breast cancer (n=61) versus unaffected subjects (n=18). However, statistically significant increase in the MDSC ability to suppress T cell activation was identified in the breast cancer cohort with decreased CD25 expression and gamma-interferon production. MDSC from young women with breast cancer also secreted higher arginase-1 but not ROS from their MDSC. Level of MDSC suppression was independent of stage or biologic subtype of the breast cancer and did not correlate with the level of Tregs in the patient. Conclusions: The presence of equal rather than higher numbers of MDSC circulating in young onset breast cancer versus unaffected subjects was unexpected in comparison to prior publications. Our results may differ due to our larger sample size or the alignment by gender and age between the cohorts. Also, these are the first data on Tregs and MDSC to specifically focus on a young female population. Despite the equal numbers, MDSC from the breast cancer cohort demonstrated increased ability to suppress T cell function which was independent of cancer stage, biologic subtype or number of Tregs in the subject. These data suggest a functional difference to MDSC in the young breast cancer-bearing woman that could be exploited in even early stage breast cancer and that is not directly dependent on Tregs numbers. Moreover, the data indicate that unaffected young women have a higher than expected number of MDSC available for cancer to usurp and induce immune suppression. Identification of secretion of arginase-1 as a potential mechanism of immune suppression in young women's breast cancer warrants further investigation. Citation Format: Virginia F. Borges, Oscar Ramirez, Michelle Borakove, Elizabeth Manthey, Christina Finlayson, Anthony Elias, Martin McCarter, Kimberley Jordan, Jennifer Diamond, Nicole Kounalakis. Young women's breast cancer demonstrates increased immune suppression through ciculating regulatory T cells and myeloid-derived supressor cells independent of stage or subtype. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A98.