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Introduction The HER2 + tumor immune microenvironment is composed of macrophages, natural killer cells, and tumor infiltrating lymphocytes, which produce pro-inflammatory cytokines. Determining the effect of T-cells on HER2 + cancer cells during therapy could guide immunogenic therapies that trigger antibody-dependent cellular cytotoxicity. This study utilized longitudinal in vitro time-resolved microscopy to measure T-cell influence on trastuzumab in HER2 + breast cancer. Methods Fluorescently-labeled breast cancer cells (BT474, SKBR3, MDA-MB-453, and MDA-MB-231) were co-cultured with CD4 + T-cells (Jurkat cell line) and longitudinally imaged to quantify cancer cell viability when treated with or without trastuzumab (10, 25, 50 and 100 μg/mL). The presence and timing of T-cell co-culturing was manipulated to determine immune stimulation of trastuzumab-treated HER2 + breast cancer. HER2 and TNF-α expression were evaluated with western blot and ELISA, respectively. Significance was calculated using a two-tailed parametric t-test. Results The viability of HER2 + cancer cells significantly decreased when exposed to 25 μg/mL trastuzumab and T-cells, compared to cancer cells exposed to trastuzumab without T-cells (p = 0.01). The presence of T-cells significantly increased TNF-α expression in trastuzumab-treated cancer cells (p = 0.02). Conversely, cancer cells treated with TNF-α and trastuzumab had a similar decrease in viability as trastuzumab-treated cancer cells co-cultured with T-cells (p = 0.32). Conclusions The presence of T-cells significantly increases the efficacy of targeted therapies and suggests trastuzumab may trigger immune mediated cytotoxicity. Increased TNF-α receptor expression suggest cytokines may interact with trastuzumab to create a state of enhanced response to therapy in HER2 + breast cancer, which has potential to reducing tumor burden.

Chemotherapy-induced immunosuppression poses an additional challenge to its limited efficacy in pancreatic cancer (PC). Here we investigated the effect of gemcitabine on macrophages, which are the first line of immune-defense mechanisms. We observed an increased presence of macrophages in orthotopic human pancreatic tumor xenografts from mice treated with gemcitabine as compared to those from vehicle only-treated mice. Conditioned media from gemcitabine-treated PC cells (Gem-CM) promoted growth, migration and invasion of RAW264.7 macrophage. In addition, Gem-CM also induced upregulation of M2-polarized macrophage markers, arginase-1 and TGF-β1. Cytokine profiling of gemcitabine-treated PC cells identified IL-8 as the most differentially-expressed cytokine. Incubation of Gem-CM with IL-8 neutralizing antibody diminished its ability to induce growth, migration and invasion of RAW264.7 macrophages, but did not abrogate their M2 polarization. Together, our findings identify IL-8 as an important mediator in the gemcitabine-induced infiltration of macrophages within the pancreatic tumor microenvironment and suggest the requirement of additional mechanism(s) for macrophage polarization.

Background The potency of T regulatory (TREG) cells to inhibit T helper (Th)-driven immune cell responses has been linked to increased intracellular cyclic-AMP (cAMP) levels of TREG cells. In an ovalbumin (OVA)-driven allergic asthma mouse model, moderate aerobic exercise increases TREG cell function in a contact-dependent manner that leads to a significant reduction in chronic inflammation and restoration of lung function. However, the mechanism, whereby exercise increases TREG function, remains unknown and was the focus of these investigations. Exercise can communicate with TREG cells by their expression of β2-adrenergic receptors (β2-AR). Activation of these receptors results in an increase in intracellular levels of cyclic-AMP, potentially creating a potent inhibitor of Th cell responses. Results For the allergic asthma model, female wildtype BALB/c mice were challenged with OVA, and exercised (13.5 m/min for 45 min) 3×/week for 4 weeks. TREG cells were isolated from all mouse asthma/exercise groups, including β2-AR−/− mice, to test suppressive function and intracellular cAMP levels. In these studies, cAMP levels were increased in TREG cells isolated from exercised mice. When β2-AR expression was absent on TREG cells, cAMP levels were significantly decreased. Correlatively, their suppressive function was compromised. Next, TREG cells from all mouse groups were tested for suppressive function after treatment with either a pharmaceutical β2-adrenergic agonist or an effector-specific cAMP analogue. These experiments showed TREG cell function was increased when treated with either a β2-adrenergic agonist or effector-specific cAMP analogue. Finally, female wildtype BALB/c mice were antibody-depleted of CD25+CD4+ TREG cells (anti-CD25). Twenty-four hours after TREG depletion, either β2-AR−/− or wildtype TREG cells were adoptively transferred. Recipient mice underwent the asthma/exercise protocols. β2-AR−/− TREG cells isolated from these mice showed no increase in TREG function in response to moderate aerobic exercise. Conclusion These studies offer a novel role for β2-AR in regulating cAMP intracellular levels that can modify suppressive function in TREG cells.

Although increased awareness leading to early detection and prevention, as well as advancements in treatment strategies, have resulted in superior clinical outcomes, African American women with breast cancer continue to have greater mortality rates, compared to Caucasian American counterparts. Moreover, African American women are more likely to have breast cancer at a younger age and be diagnosed with aggressive tumor sub-types. Such racial disparities can be attributed to socioeconomic differences, but it is increasingly being recognized that these disparities may indeed be due to certain genetic and other non-genetic biological differences. Tumor microenvironment, which provides a favorable niche for the growth of tumor cells, is comprised of several types of stromal cells and the various proteins secreted as a consequence of bi-directional tumor-stromal cross-talk. Emerging evidence suggests inherent biological differences in the tumor microenvironment of breast cancer patients from different racial backgrounds. Tumor microenvironment components, affected by the genetic make-up of the tumor cells as well as other non-tumor-associated factors, may also render patients more susceptible to the development of aggressive tumors and faster progression of disease resulting in early onset, thus adversely affecting patients' survival. This review provides an overview of breast cancer racial disparity and discusses the existence of race-associated differential tumor microenvironment and its underlying genetic and non-genetic causal factors. A better understanding of these aspects would help further research on effective cancer management and improved approaches for reducing the racial disparities gaps in breast cancer patients.

Resistance to chemotherapeutic drugs remains a major cause of therapeutic failure in pancreatic cancer (PC) patients. Efficacy of chemotherapy is further limited by its debilitating effects on immune system. Apart from genetic abnormalities in tumor cells, the tumor immune- microenvironment plays a crucial role in PC chemoresistance. However, it is unclear how chemotherapy affects the cancer immunity or how it affects the tumor microenvironment (TME). In the present study, we demonstrate gemcitabine induces an increased infiltration of CD68+ M2 macrophages in pancreatic tumors isolated from an orthotopic xenograft murine model. Moreover, the conditioned media from PC cell lines cultured with gemcitabine (Gem-CM) promotes migration, invasion, and growth of RAW264.7 macrophage. Interestingly, Gem-CM induces an upregulation of tumor-associated or M2-polarized macrophage markers, arginase-1 and TGF-β1. Additionally, gemcitabine treatment of PC cell lines induces the expression of M2-polarization associated cytokines, growth factors and chemokines, including IL-8. IL-8 exhibits the greatest upregulation after culture with Gemcitabine. Further, IL-8 neutralization in Gem-CM diminished its ability to induce growth, migration, and invasion of RAW264.7 macrophages. Together, these findings suggest an indirect effect of gemcitabine on increasing macrophage trafficking and M2-polarization that potentially supports a pro-tumor microenvironment. These actions may contribute to the chemoresistance of PC. Prevention of gemcitabine-induced macrophage infiltration, growth and M2-polarization will offer a better strategy to counter chemotherapy-induced immunosuppression in PC. Citation Format: Sachin Kumar Deshmukh, Arun Bhardwaj, Nikhil Tyagi, Mohammad Aslam Khan, Sanjeev K. Srivastava, Ahmed AL-Ghadhban, Kari Dugger, James E. Carter, Ajay P. Singh, Seema Singh. Gemcitabine treatment induces immunosuppressive microenvironment in pancreatic cancer by promoting the infiltration, growth, and polarization of macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 113.

We have reported previously that moderate intensity aerobic exercise training attenuates airway inflammation in a murine asthma model. Recent studies implicate regulatory T (Treg) cells in decreasing asthma-related airway inflammation; as such, the current study examined the effect of exercise on Treg cell function in a murine asthma model. Mice were sensitized with ovalbumin (OVA) prior to the start of exercise training at a moderate intensity 3x/week for 4weeks; exercise was performed as treadmill running (13.5m/min, 0% grade). Mice were OVA challenged repeatedly throughout the exercise protocol. At protocol completion, mice were analyzed for changes in the number and suppressive function of CD4(+)CD25(+)Foxp3(+) cells isolated from lungs, mediastinal lymph nodes, and spleens. Results show that exercise increased significantly the number of Foxp3(+) cells within the lungs and mediastinal lymph nodes, but not the spleens, of OVA-treated mice as compared with sedentary controls. Exercise also enhanced the suppression function of CD4(+)CD25(+)Foxp3(+) Treg cells derived from OVA-treated mice as compared with sedentary controls. Specifically, Treg cells from exercised, OVA-treated mice more effectively suppressed CD4(+)CD25(-) cell proliferation and Th2 cytokine production in vitro. Enhanced suppression was associated with increased protein levels of TGF-beta and lesser amounts of IL-10 and IL-17; however, blocking TGF-beta had no effect on suppressive functions. These data demonstrate that exercise-mediated increases in Treg cell function may play a role in the attenuation of airway inflammation. Further, these results indicate that moderate intensity aerobic exercise training may alter the Treg cell function within the asthmatic airway.

LFA-1 (CD11a/CD18) is a member of the β 2 -integrin family of adhesion molecules important in leukocyte trafficking and activation. Although LFA-1 is thought to contribute to the development of experimental autoimmune encephalomyelitis (EAE) primarily through its functions on effector T cells, its importance on other leukocyte populations remains unexplored. To address this question, we performed both adoptive transfer EAE experiments involving CD11a −/− mice and trafficking studies using bioluminescent T cells expressing luciferase under the control of a CD2 promoter (T-lux cells). Transfer of encephalitogenic CD11a −/− T cells to wild type mice resulted in a significant reduction in overall EAE severity compared to control transfers. We also observed, using in vivo imaging techniques, that CD11a −/− T-lux cells readily infiltrated lymph nodes and the CNS of wild type recipients with kinetics comparable to CD11a +/+ transfers, although their overall numbers in these organs were reduced. Surprisingly, transfer of encephalitogenic wild type T cells to CD11a −/− mice induced a severe and sometimes fatal EAE disease course, associated with massive T cell infiltration and proliferation in the CNS. These data indicate that LFA-1 expression on leukocytes in recipient mice plays an important immunomodulatory role in EAE. Thus, LFA-1 acts as a key regulatory adhesion molecule during the development of EAE, serving both pro- and anti-inflammatory roles in disease pathogenesis.

There has been a rapid growth of bioluminescence imaging applications in small animal models in recent years, propelled by the availability of instruments, analysis software, reagents, and creative approaches to apply the technology in molecular imaging. Advantages include the sensitivity of the technique as well as its efficiency, relatively low cost, and versatility. Bioluminescence imaging is accomplished by sensitive detection of light emitted following chemical reaction of the luciferase enzyme with its substrate. Most imaging systems provide 2-dimensional (2D) information in rodents, showing the locations and intensity of light emitted from the animal in pseudo-color scaling. A 3-dimensional (3D) capability for bioluminescence imaging is now available, but is more expensive and less efficient; other disadvantages include the requirement for genetically encoded luciferase, the injection of the substrate to enable light emission, and the dependence of light signal on tissue depth. All of these problems make it unlikely that the method will be extended to human studies. However, in small animal models, bioluminescence imaging is now routinely applied to serially detect the location and burden of xenografted tumors, or identify and measure the number of immune or stem cells after an adoptive transfer. Bioluminescence imaging also makes it possible to track the relative amounts and locations of bacteria, viruses, and other pathogens over time. Specialized applications of bioluminescence also follow tissue-specific luciferase expression in transgenic mice, and monitor biological processes such as signaling or protein interactions in real time. In summary, bioluminescence imaging has become an important component of biomedical research that will continue in the future.

After activation, populations of antigen-specific T cells flow between sites of antigen expression, local lymphoid structures and other lymphoid and non-lymphoid organs. In this study, we documented the in vivo dynamics of a CD8(+) T cell response to antigen delivered using herpes simplex virus amplicon vectors and revealed several unexpected features. First, the T cells localized to the site of vector injection, as well as the draining lymph node within 24-48 h. Second, the major site to which T cells later redistributed were intra-abdominal lymphoid organs, including milky spots, mesenteric and lumbar lymph nodes. We determined the relationship between bioluminescent signal and antigen-specific T cell numbers in various lymphoid organs, and concluded that bioluminescent signal is a valid surrogate measure of T cell abundance in superficial lymph nodes, but not in deeper structures such as the spleen.

Lymphocyte activation gene (LAG)-3 (CD223) is a CD4-related activation-induced cell surface molecule that binds to MHC class II molecules with high affinity and negatively regulates T cell expansion and homeostasis. In this study, we show that LAG-3 inhibits CD4-dependent, but not CD4-independent, T cell function via its cytoplasmic domain. Although high affinity interaction with MHC class II molecules is essential for LAG-3 function, tailless LAG-3 does not compete with CD4 for ligand binding. A single lysine residue (K468) within a conserved “KIEELE” motif is essential for interaction with downstream signaling molecules. These data provide insight into the mechanism of action of this important T cell regulatory molecule.

Studies have indicated increased incidence and severity of allergic asthma due to western lifestyle and increased sedentary activity. Investigations also indicate that exercise reduces the severity of asthma; however, a mechanism of action has not been elucidated. Additional work implicates re-distribution of T helper (Th) cells in mediating alterations of the immune system as a result of moderate aerobic exercise in vivo. We have previously reported that exercise decreases T helper 2 (Th2) responses within the lungs of an ovalbumin (OVA)-sensitized murine allergic asthma model. Therefore, we hypothesized that exercise alters the migration of OVA-specific Th cells in an OVA-challenged lung. To test this hypothesis, wildtype mice received OVA-specific Th cells expressing a luciferase-reporter construct and were OVA-sensitized and exercised. OVA-specific Th cell migration was decreased in OVA-challenged lungs of exercised mice when compared to their sedentary controls. Surface expression levels of lung-homing chemokine receptors, CCR4 and CCR8, on Th cells and their cognate lung-homing chemokine gradients revealed no difference between exercised and sedentary OVA-sensitized mice. However, transwell migration experiments demonstrated that lung-derived Th cells from exercised OVA-sensitized mice exhibited decreased migratory function versus controls. These data suggest that Th cells from exercised mice are less responsive to lung-homing chemokine. Together, these studies demonstrate that moderate aerobic exercise training can reduce the accumulation of antigen-specific Th cell migration into an asthmatic lung by decreasing chemokine receptor function.

Experimental autoimmune encephalomyelitis as a model for demyelinating disease challenges the mindset that β2-integrins are redundant in function and potential therapeutic targets for multiple sclerosis. The β2-integrins are a subfamily of integrins expressed on leukocytes that play an essential role in leukocyte trafficking, activation, and many other functions. Studies in EAE, the animal model for multiple sclerosis, show differential requirements for β2-integrins in this disease model, ranging from critical in the case of LFA-1 (CD11a/CD18) to unimportant in the case of CD11d/CD18. Importantly, expression of β2-integrins on T cell subsets provides some clues as to the function(s) these adhesion molecules play in disease development. For example, transferred EAE studies have shown that Mac-1 (CD11b/CD18) expression on αβ T cells is critical for disease development, and the absence of LFA-1 on Tregs in recipient mice results in exacerbated disease. In this review, we summarize recent findings regarding the role of β2-integrins in demyelinating disease and new information about the role of β2-integrins with respect to alterations in Treg numbers and function. In addition, we discuss the potential for targeting β2-integrins in human demyelinating disease in light of the recent animal model studies.

BackgroundRapid clonal expansion of T cells occurs in response to antigenic challenges. The kinetics of the T cell response has previously been described using tissue-based studies performed at defined time points. Luciferase bioluminescence has recently been utilized for non-invasive analysis ofin vivobiologic processes in real-time.ResultsWe have created a novel transgenic mouse model (T-Lux) using a human CD2 mini-gene to direct luciferase expression specifically to the T cell compartment. T-Lux T cells demonstrated normal homing patterns within the intact mouse and following adoptive transfer. Bioluminescent signal correlated with T cell numbers in the whole body images as well as within specific organ regions of interest. Following transfer into lymphopenic (RAG2-/-) recipients, homeostatic proliferation of T-Lux T cells was visualized using bioluminescent imaging. Real-time bioluminescent analysis of CD4+T cell antigen-specific responses enabled real-time comparison of the kinetics and magnitude of clonal expansion and contraction in the inductive lymph node and tissue site of antigen injection. T cell expansion was dose-dependent despite the presence of supraphysiologic numbers of OVA-specific OT-II transgenic TCR T-Lux T cells. CD4+T cells subsequently underwent a rapid (3–4 day) contraction phase in the draining lymph node, with a delayed contraction in the antigen delivery site, with bioluminescent signal diminished below initial levels, representing TCR clonal frequency control.ConclusionThe T-Lux mouse provides a novel, efficient model for trackingin vivoaspects of the CD4+T cell response to antigen, providing an attractive approach for studies directed at immunotherapy or vaccine design.

Through the expression of inflammatory mediators and immune-related molecules, epithelial cells function as immune effector cells in a wide variety of tissues; the expression of the CD40 receptor on these cells contributes this role. Engagement of CD40 activates epithelial cells and results in their release of pro- and anti-inflammatory mediators as well as pro-fibrotic molecules. As such, epithelial CD40 has been implicated in the pathogenesis of inflammatory disorders, generation of self-tolerance, and rejection of allografts.

Moderate aerobic exercise has been documented to modify the immune system, specifically, by mediating a re-distribution of Th cells in vivo. In addition, it is well-established that exercise generates the soluble Beta 2-adrenergic receptor (B2-AR) agonist, epinephrine, in which Th cells can respond by their expression of B2-AR. Studies using a luciferase reporter for OVA-specific Th cells in an OVA-sensitized/challenged allergic asthma mouse model have shown a reduction in OVA-specific Th cell migration into the lungs when exercised, subsequently reducing asthma pathogenesis. In addition, investigations looking at lung-homing chemokine receptor presence on the surface of Th cells (CCR4 and CCR8) by flow cytometry revealed no difference between exercised and sedentary asthmatic mice. However, transmigration experiments demonstrated that exercised Th cell chemokine receptors, CCR4 and CCR8, are less functional than their sedentary counterparts. These data suggest exercised Th cells are less responsive to lung-homing chemokine gradients created by an asthmatic lung. Furthermore, studies using B2-AR−/− Th cells transferred to OVA sensitized/challenged recipient mice, showed a loss in exercise-mediated chemokine receptor desensitization in transmigration assays comparing exercised and sedentary B2-AR−/− Th cells. These studies demonstrate that moderate aerobic exercise training can alter Th cell migration into the asthmatic lung by modifying chemokine receptor function. Additionally, the data suggests a role for B2-AR signaling in mediating this exercise-induced chemokine receptor loss of function.

We have shown moderate aerobic exercise can increase both the number and function of T regulatory (TREG) cells that suppress Th2 responses of an ovalbumin (OVA)-driven murine asthma model by an unknown mechanism. Exercise can communicate with TREG cells with the systemic release of catecholamines that bind to Beta-2-adrenergic receptors (β2-AR) and generate cAMP signals that can affect cell function. In these studies, we investigated a role for β2-AR in the exercise-mediated modulation of TREG cell function. For these studies, TREG cells (CD4+CD25+) were depleted from female BALB/cJ mice by i.v. injection of anti-CD25. Either 500,000 wt or β2-AR-/- TREG cells were adoptively transferred into the TREG-depleted BALB/cJ mice. Mice were sensitized/challenged with OVA to mimic allergic asthma pathogenesis. Mice were moderately exercised 45 min post-OVA challenge on a treadmill for 1hr 3x/wk for 4wks. Upon protocol completion, mice were examined for changes in TREG cell suppressive function and their resulting TREG cAMP levels. Our results show that exercise increases TREG function in vitro as compared with sedentary controls. However, the increase in TREG function of exercised TREG cells was abrogated in the absence of β2-ARs. We conclude that moderate aerobic exercise attenuates asthma-related airway inflammation via a mechanism that involves increased TREG cell function modulated by their expression of β2-ARs and subsequent production of cAMP.

Exercise greatly influences the immune system; this includes an exercise-mediated redistribution of T cells in vivo. Increasing evidence demonstrates that Th cells play a central role in the pathogenesis of asthma. We reported previously that repeated bouts of aerobic exercise at a moderate intensity ameliorate asthma-related responses, e.g., airway inflammation and hyperresponsiveness. Studies using OVA-sensitized/challenged mice with luciferase expressing T cells provide a novel method to track T cell trafficking patterns in vivo in real-time. We found a significant reduction in proliferation and presence within the asthmatic lung of exercised mice in comparison with sedentary controls. Further, flow cytometry analysis demonstrated a difference in chemokine receptor profiles on the surface of T cells. The presence of CCR7, which mediates the exit out of lungs, was increased on exercised T cells. In addition, transmigration studies show exercised T cells have a decrease in response to chemokine gradients created by the asthmatic lung that arbitrate entry into the site of inflammation, CCL1 and CCL17. These data demonstrates that aerobic exercise training at a moderate intensity decreases Th cell migration into and increases Th cell exit out of the asthmatic lung; thereby, reducing the subsequent inflammation within the asthmatic lung. Future studies will investigate the underlying mechanism of this exercise-induced shift in homing receptor expression patterns on Th cells.

Exercise training has been shown to significantly reduce Th2 cytokines, airway hyper-responsiveness (AHR), and lung pathology in asthmatic mice. Some of these changes have been postulated to be due to regulatory T cells (Tregs). In these experiments, mice were induced into an asthmatic state (OVA) and exercised (45min, 3d/wk, at 60-70% of VO2max) for a total of 10 bouts. Tregs isolated from the spleens, lungs, and mediastinal lymph nodes of exercise-trained mice and stimulated in vitro were able to suppress effector cells at a significantly greater rate than Tregs from sedentary mice irrespective of whether or not they were induced to an asthmatic state (p < 0.05). Blocking TGF-β did not reduce Treg-mediated suppression in the exercise-trained group, but suppression was decreased in control mice following anti-TGF-β administration. In vitro Treg:effector co-cultures demonstrated significant reductions in Th2 and Th17 cytokine production in exercise-trained mice compared to their sedentary counterparts. Surface expression of Tregs, as assessed by Foxp3EGFP, was significantly higher in exercise-trained asthmatic mice compared to sedentary controls. We show for the first time that exercise training increases Treg expression and function in asthmatic and non-asthmatic mice.

CD223 (LAG-3) is an activation-induced cell surface molecule, structurally similar to the T cell coreceptor CD4, that binds MHC class II molecules with high affinity. Little is known about theexpression and function of murine CD223. Here, we show that mRNA expression is restricted to the thymic medulla, splenic red pulp and sparse cells in the adult brain cortex. In contrast, surprisingly high expression was seen in defined tracts at the base of the cerebellum and in the choroid plexus of day 7 postnatal brain. mCD223:Ig, but not CD4:Ig, fusion proteins stained cells expressing MHC class II molecules. Analysis of mCD223 cell surface expression was performed with a new monoclonal antibody (mAb) that recognizes an epitope in the D2 domain. Although it blocked mCD223 function in vitro, it did not block binding of mCD223 to MHC class II molecules. While very few TCRα β T cells in the spleen and thymus of naive mice express surface mCD223 (