Adoptive Immunotherapy Research Articles

1955P Glycan-programmed T cell immunity: Effective adoptive T cell transfer in a CRC preclinical model

1955P Glycan-programmed T cell immunity: Effective adoptive T cell transfer in a CRC preclinical model

Abstract P183: Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus Induce Inflammation and Salt Sensitivity of Blood Pressure in Humanized Mice

Salt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular disease (CVD), even in normotensive people. Systemic lupus erythematosus (SLE) is also a risk factor for CVD and hypertension. SLE and CVD are on the rise, and it is not known if this is attributable to the increased dietary salt in the modern diet and SSBP. Isolevuglandins (IsoLGs) adduct self-proteins forming neoantigens in antigen-presenting cells (APC), activate T cells, and produce cytokines that promote sodium retention, kidney damage, SLE, and SSBP. We hypothesized the adoptive transfer of peripheral blood mononuclear cells (PBMCs) from patients with SLE into immunodeficient mice (NSG/MHCI/MHCII-/-) would increase inflammation and SSBP after high salt feeding. In vitro studies were performed using PBMCs from patients with SLE and treated with and without high salt, and myeloid cell activation and cytokine expression were measured with flow cytometry. We humanized mice with PBMCs from patients with SLE (n=7) and controls without autoimmune disease (n=7) in animal studies. The mice were fed a 4% high-salt diet for two weeks. Blood pressure was measured with radiotelemetry, immune phenotyping was performed with flow cytometry, and vascular reactivity was assessed in mesenteric arteries using wire myography. A high salt diet significantly increased systolic blood pressure in lupus mice compared to the controls (133.0 vs.116.4 mmHg, p=0.001). APC infiltration (macrophages, monocytes, dendritic cells), proinflammatory markers, and oxidative stress (Isolevuglandins, Nox2) were significantly higher in the kidneys of lupus mice. In lupus mice, classical monocyte and proinflammatory markers were significantly elevated in the spleen lymphocytes, and proinflammatory markers were increased in the kidney, spleen, and aorta in lupus mice. These mice also exhibited mesenteric artery vascular dysfunction (p<0.05). Lupus mice tended to excrete more urinary albumin than the controls (25.89 vs. 17.24 µg/ml p=0.142), suggesting kidney injury. High salt treatment in vitro increased intermediate and nonclassical monocytes with increased CD86, IsoLG, and TNF-alpha expression compared to normal salt-treated cells. In conclusion, our findings suggest that immune cells from patients with SLE, in response to high salt, increase SSBP and the proinflammatory milieu in the kidney, aorta, and spleen; impair vascular function; and worsen albuminuria in immunodeficient mice.

Role of N6-methyladenosine RNA modification in cancer.

N6-methyladenosine (m6A) is the most abundant modification of RNA in eukaryotic cells. Previous studies have shown that m6A is pivotal in diverse diseases especially cancer. m6A corelates with the initiation, progression, resistance, invasion, and metastasis of cancer. However, despite these insights, a comprehensive understanding of its specific roles and mechanisms within the complex landscape of cancer is still elusive. This review begins by outlining the key regulatory proteins of m6A modification and their posttranslational modifications (PTMs), as well as the role in chromatin accessibility and transcriptional activity within cancer cells. Additionally, it highlights that m6A modifications impact cancer progression by modulating programmed cell death mechanisms and affecting the tumor microenvironment through various cancer-associated immune cells. Furthermore, the review discusses how microorganisms can induce enduring epigenetic changes and oncogenic effect in microorganism-associated cancers by altering m6A modifications. Last, it delves into the role of m6A modification in cancer immunotherapy, encompassing RNA therapy, immune checkpoint blockade, cytokine therapy, adoptive cell transfer therapy, and direct targeting of m6A regulators. Overall, this review clarifies the multifaceted role of m6A modification in cancer and explores targeted therapies aimed at manipulating m6A modification, aiming to advance cancer research and improve patient outcomes.

1085P Adoptive cell therapy with TCR gene-engineered T cells directed against MAGE-C2-positive melanoma: An ongoing phase I trial

1085P Adoptive cell therapy with TCR gene-engineered T cells directed against MAGE-C2-positive melanoma: An ongoing phase I trial

Advancements in Immunotherapy for Breast Cancer: Mechanisms, Efficacy, and Future Directions.

Breast cancer is a major global health challenge characterized by its diverse biological behavior and varying treatment responses. Traditional therapies, including surgery, radiation, chemotherapy, hormonal therapy, and targeted therapy, have significantly advanced breast cancer treatment but are often limited by issues such as resistance, side effects, and variable efficacy. Immunotherapy has emerged as a transformative approach, leveraging the body's immune system to target and eliminate cancer cells. This review provides a comprehensive overview of recent advancements in immunotherapy for breast cancer, detailing the mechanisms of various therapeutic strategies, including checkpoint inhibitors, monoclonal antibodies, cancer vaccines, adoptive cell therapy, and oncolytic virus therapy. We evaluate the efficacy of these approaches in different stages of breast cancer, highlighting successes and challenges encountered in clinical settings. The review also addresses the current limitations of immunotherapy, such as treatment-related adverse effects, resistance mechanisms, and issues of cost and accessibility. We discuss promising future directions, including emerging targets, combination therapies, and personalized medicine approaches. By integrating recent research and clinical trial data, this review aims to elucidate the potential of immunotherapy to revolutionize breast cancer treatment, offering insights into its future role in improving patient outcomes and shaping the landscape of oncological care.

Pharmacologic HIF stabilization activates costimulatory receptor expression to increase antitumor efficacy of adoptive T cell therapy.

Adoptive cell transfer (ACT) is a therapeutic strategy to augment antitumor immunity. Here, we report that ex vivo treatment of mouse CD8+ T cells with dimethyloxalylglycine (DMOG), a stabilizer of hypoxia-inducible factors (HIFs), induced HIF binding to the genes encoding the costimulatory receptors CD81, GITR, OX40, and 4-1BB, leading to increased expression. DMOG treatment increased T cell killing of melanoma cells, which was further augmented by agonist antibodies targeting each costimulatory receptor. In tumor-bearing mice, ACT using T cells treated ex vivo with DMOG and agonist antibodies resulted in decreased tumor growth compared to ACT using control T cells and increased intratumoral markers of CD8+ T cells (CD7, CD8A, and CD8B1), natural killer cells (NCR1 and KLRK1), and cytolytic activity (perforin-1 and tumor necrosis factor-α). Costimulatory receptor gene expression was also induced when CD8+ T cells were treated with three highly selective HIF stabilizers that are currently in clinical use.

Harnessing the Power of NK Cell Receptor Engineering as a New Prospect in Cancer Immunotherapy.

Natural killer (NK) cells have recently gained popularity as an alternative for cancer immunotherapy. Adoptive cell transfer employing NK cells offers a safer therapeutic option compared to T-cell-based therapies, due to their significantly lower toxicity and the availability of diverse autologous and allogeneic NK cell sources. However, several challenges are associated with NK cell therapies, including limited in vivo persistence, the immunosuppressive and hostile tumor microenvironment (TME), and the lack of effective treatments for solid tumors. To address these limitations, the modification of NK cells to stably produce cytokines has been proposed as a strategy to enhance their persistence and proliferation. Additionally, the overexpression of activating receptors and the blockade of inhibitory receptors can restore the NK cell functions hindered by the TME. To further improve tumor infiltration and the elimination of solid tumors, innovative approaches focusing on the enhancement of NK cell chemotaxis through the overexpression of chemotactic receptors have been introduced. This review highlights the latest advancements in preclinical and clinical studies investigating the engineering of activating, inhibitory, and chemotactic NK cell receptors; discusses recent progress in cytokine manipulation; and explores the potential of combining the chimeric antigen receptor (CAR) technology with NK cell receptors engineering.

Unlocking the Potential: Epstein-Barr Virus (EBV) in Gastric Cancer and Future Treatment Prospects, a Literature Review.

Gastric cancer (GC) is a complex disease with various etiologies. While Helicobacter pylori infection is still one of the leading risk factors for GC, increasing evidence suggests a link between GC and other infective agents such as Epstein Bar Virus (EBV). EBV-associated gastric cancer (EBVaGC) is now recognized as a distinct subgroup of GC, and the complex interactions between the virus and gastric mucosa may influence its development. A recent integrative analysis of the genome and proteome of GC tissues by The Cancer Genome Atlas project has identified EBVaGC as a specific subtype characterized by PIK3CA and ARID1A mutations, extensive DNA hyper-methylation, and activation of immune signaling pathways. These molecular characteristics are markers of the unique molecular profile of this subset of GC and are potential targets for therapy. This review aims to provide an overview of the current knowledge on EBVaGC. It will focus on the epidemiology, clinic-pathological features, and genetic characteristics of EBVaGC. Additionally, it will discuss recent data indicating the potential use of EBV infection as a predictive biomarker of response to chemotherapy and immune checkpoint inhibitors. The review also delves into potential therapeutic approaches for EBVaGC, including targeted therapies and adoptive immunotherapy, highlighting the promising potential of EBV as a therapeutic target.

Alginate-based artificial antigen presenting cells expand functional CD8+ T cells with memory characteristics for adoptive cell therapy

The development of artificial Antigen Presenting Cells (aAPCs) has led to improvements in adoptive T cell therapy (ACT), an immunotherapy, for cancer treatment. aAPCs help to streamline the consistent production and expansion of T cells, thus reducing the time and costs associated with ACT. However, several issues still exist with ACT, such as insufficient T cell potency, which diminishes the translational potential for ACT. While aAPCs have been used primarily to increase production efficiency of T cells for ACT, the intrinsic properties of a biomaterial-based aAPC may affect T cell phenotype and function. In CD8+ T cells, reactive oxygen species (ROS) and oxidative stress accumulation can activate Forkhead box protein O1 (FOXO1) to transcribe antioxidants which reduce ROS and improve memory formation. Alginate, a biocompatible and antioxidant rich biomaterial, is promising for incorporation into an aAPC formulation to modulate T cell phenotype. To investigate its utility, a novel alginate-based aAPC platform was developed that preferentially expanded CD8+ T cells with memory related features. Alginate-based aAPCs allowed for greater control of CD8+ T cell qualities, including, significantly improved in vivo persistence and augmented in vivo anti-tumor T cell responses.

Cbl-b inhibition promotes less differentiated phenotypes of T cells with enhanced cytokine production

For adoptive therapy with T cell receptor engineered T (TCR-T) cells, the quantity and quality of the final cell product directly affect their anti-tumor efficacy. The post-transfer efficacy window of TCR-T cells is keen to optimizing attempts during the manufacturing process. Cbl-b is a E3 ubiquitin ligase previously shown with critical negative impact in T cell functions. This study investigated whether strategic inclusion of a commercially available small inhibitor targeting Cbl-b (Cbl-b-IN-1) prior to T cell activation could enhance the quality of the final TCR-T cell product. Examination with both PBMCs and TCR-T cells revealed that Cbl-b-IN-1 treatment promoted TCR expression efficiency, T cell proliferation potential and, specifically, cell survival capability post antigenic stimulation. Cbl-b-IN-1 exposure facilitated T cells in maintaining less differentiated states with enhanced cytokine production. Further, we found that Cbl-b-IN-1 effectively augmented the activation of TCR signaling, shown by increased phosphorylation levels of Zeta-chain-associated protein kinase 70 (ZAP70) and phospholipase c-γ1 (PLCγ1). In conclusion, our results evidence that the inclusion of Cbl-b inhibitor immediately prior to TCR-T cell activation may enhance their proliferation, survival, and function potentials, presenting an applicable optimization strategy for immunotherapy with adoptive cell transfer.

Delivery Systems Developed for Treatment Combinations to Improve Adoptive Cell Therapy.

Adoptive cell therapy (ACT) has shown great success in the clinic for treating hematologic malignancies. However, solid tumor treatment with ACT monotherapy is still challenging, owing to insufficient expansion and rapid exhaustion of adoptive cells, tumor antigen downregulation/loss, and dense tumor extracellular matrix. Delivery strategies for combination cell therapy have great potential to overcome these hurdles. The delivery of vaccines, immune checkpoint inhibitors, cytokines, chemotherapeutics, and photothermal reagents in combination with adoptive cells, have been shown to improve the expansion/activation, decrease exhaustion, and promote the penetration of adoptive cells in solid tumors. Moreover, the delivery of nucleic acids to engineer immune cells directly in vivo holds promise to overcome many of the hurdles associated with the complex ex vivo cell engineering strategies. Here, these research advance, as well as the opportunities and challenges for integrating delivery technologies into cell therapy s are discussed, and the outlook for these emerging areas are criticlly analyzed.

Effector Functions of Dendritic Cells in Cancer: Role of Cytotoxicity and Growth Inhibition.

The tumor microenvironment plays a critical role in modulating immune responses associated with tumorigenesis, tumor progression, and metastasis. Dendritic cells (DC) play a key role in preventing and progression of metastatic neoplasia by driving and restoring dysfunctional immune systems and obliterating immunosuppression, thus obstructing tumor evasion. In this review, we will discuss the functions of tumor-infiltrating DC in anti-tumor resistance, prevention of tumor recurrence, and immunosuppression. We will also describe DC metabolism, differentiation, and plasticity, which are essential for its function. Cancers like Lymphomas may be able to corrupt immune surveillance by reducing natural killer cell numbers. Thus, interactions between lymphoma and DC with reference to cytotoxicity may be an important event, likely to be mediated via activation with interferon-γ (IFN-γ) and Toll like receptors (TLR) ligands. Mechanisms of DC-mediated cytotoxicity and the role of apoptosis and death receptors, including the role played by nitric oxide, etc., are of immense significance. We will also look into the molecular mechanisms in the tumor microenvironment, reduced drug sensitivity, and tumor relapse, as well as methods for combating drug resistance and focusing on immunosuppressive tumor networks. We will address how DC mediated cytotoxicity in combination with drugs affects tumor growth and expansion in relation to checkpoint inhibitors and regulatory T cells. Innovative approaches for therapeutic modulation of this immunosuppressive adoptive DC immunotherapy will be highlighted, which is necessary for future personalized therapeutic applications.

The Freshwater Cyanobacterium Synechococcus elongatus PCC 7942 Does Not Require an Active External Carbonic Anhydrase.

Under standard laboratory conditions, Synechococcus elongatus PCC 7942 lacks EcaASyn, a periplasmic carbonic anhydrase (CA). In this study, a S. elongatus transformant was created that expressed the homologous EcaACya from Cyanothece sp. ATCC 51142. This additional external CA had no discernible effect on the adaptive responses and physiology of cells exposed to changes similar to those found in S. elongatus natural habitats, such as fluctuating CO2 and HCO3- concentrations and ratios, oxidative or light stress, and high CO2. The transformant had a disadvantage over wild-type cells under certain conditions (Na+ depletion, a reduction in CO2). S. elongatus cells lacked their own EcaASyn in all experimental conditions. The results suggest the presence in S. elongatus of mechanisms that limit the appearance of EcaASyn in the periplasm. For the first time, we offer data on the expression pattern of CCM-associated genes during S. elongatus adaptation to CO2 replacement with HCO3-, as well as cell transfer to high CO2 levels (up to 100%). An increase in CO2 concentration coincides with the suppression of the NDH-14 system, which was previously thought to function constitutively.

Nucleic Acid Armor: Fortifying RNA Therapeutics through Delivery and Targeting Innovations for Immunotherapy.

RNA is a promising nucleic acid-based biomolecule for various treatments because of its high efficacy, low toxicity, and the tremendous availability of targeting sequences. Nevertheless, RNA shows instability and has a short half-life in physiological environments such as the bloodstream in the presence of RNAase. Therefore, developing reliable delivery strategies is important for targeting disease sites and maximizing the therapeutic effect of RNA drugs, particularly in the field of immunotherapy. In this mini-review, we highlight two major approaches: (1) delivery vehicles and (2) chemical modifications. Recent advances in delivery vehicles employ nanotechnologies such as lipid-based nanoparticles, viral vectors, and inorganic nanocarriers to precisely target specific cell types to facilitate RNA cellular entry. On the other hand, chemical modification utilizes the alteration of RNA structures via the addition of covalent bonds such as N-acetylgalactosamine or antibodies (antibody-oligonucleotide conjugates) to target specific receptors of cells. The pros and cons of these technologies are enlisted in this review. We aim to review nucleic acid drugs, their delivery systems, targeting strategies, and related chemical modifications. Finally, we express our perspective on the potential combination of RNA-based click chemistry with adoptive cell therapy (e.g., B cells or T cells) to address the issues of short duration and short half-life associated with antibody-oligonucleotide conjugate drugs.

Regulatory T cells administration reduces anxiety-like behavior in mice submitted to chronic restraint stress.

Major depression disorder (MDD) and anxiety are common mental disorders that significantly affect the quality of life of those who suffer from them, altering the person's normal functioning. From the biological perspective, the most classical hypothesis explaining their occurrence relies on neurotransmission and hippocampal excitability alterations. However, around 30% of MDD patients do not respond to medication targeting these processes. Over the last decade, the involvement of inflammatory responses in depression and anxiety pathogenesis has been strongly acknowledged, opening the possibility of tackling these disorders from an immunological point of view. In this context, regulatory T cells (Treg cells), which naturally maintain immune homeostasis by suppressing inflammation could be promising candidates for their therapeutic use in mental disorders. To test this hypothesis, C57BL/6 adult male mice were submitted to classical stress protocols to induce depressive and anxiety-like behavior; chronic restriction stress (CRS), and chronic unpredictable stress (CUS). Some of the stressed mice received a single adoptive transfer of Treg cells during stress protocols. Mouse behavior was analyzed through the open field (OFT) and forced swim test (FST). Blood and spleen samples were collected for T cell analysis using cell cytometry, while brains were collected to study changes in microglia by immunohistochemistry. Mice submitted to CRS and CUS develop anxiety and depressive-like behavior, and only CRS mice exhibit lower frequencies of circulating Treg cells. Adoptive transfer of Treg cells decreased anxiety-like behavior in the OFT only in CRS model, but not depressive behavior in FST in neither of the two models. In CRS mice, Treg cells administration lowered the number of microglia in the hippocampus, which increased due this stress paradigm, and restored its arborization. However, in CUS mice, Treg cells administration increased microglia number with no significant effect on their arborization. Our results for effector CD4+ T cells in the spleen and microglia number and morphology in the hippocampus add new evidence in favor of the participation of inflammatory responses in the development of depressive and anxiety-like behavior and suggest that the modulation of key immune cells such as Treg cells, could have beneficial effects on these disorders.

Application and development of TCR-T in tumor therapy

As cancer has gradually become the world's deadliest long-term disease, the impact on the public health has been devastating. Especially the Melanoma which is a cancer with a very high malignant and a high fatality rate. With the emergence of adoptive cell therapy(ACT) based on T Cell Receptor-Gene Engineered T Cells (TCR-T), good results have been obtained in scientific research and clinical fields for a variety of malignant tumors. As TCR-T continues to enter the clinic, the FDA also has approved the first case of TCR-T in 2022, and more and more basic researches and clinical trial projects based on TCR-T are gradually being carried out. However, TCR-T therapy still has some defects, such as non-tumor tissue damage caused by off-target effect and insufficient affinity with tumor tissue. Therefore, the affinity and anti-tumor effectiveness of TCR-T should be improved in multiple aspects, and multi-field cooperation should be carried out to promote the development of basic research, a number of products should be introduced into the clinic, and the intervention of economic means to improve the accessibility of TCR-T. Not only brings the Gospel of cure to patients in the field of solid tumors, but also produces innovation in the field of non-solid tumors.

High-Risk Neuroblastoma Challenges and Opportunities for Antibody-Based Cellular Immunotherapy.

Immunotherapy has emerged as an attractive option for patients with relapsed or refractory high-risk neuroblastoma (HRNB). Neuroblastoma (NB), a sympathetic nervous system cancer arising from an embryonic neural crest cell, is heterogeneous clinically, with outcomes ranging from an isolated abdominal mass that spontaneously regresses to a widely metastatic disease with cure rates of about 50% despite intensive multimodal treatment. Risk group stratification and stage-adapted therapy to achieve cure with minimal toxicities have accomplished major milestones. Targeted immunotherapeutic approaches including monoclonal antibodies, vaccines, adoptive cellular therapies, their combinations, and their integration into standard of care are attractive therapeutic options, although curative challenges and toxicity concerns remain. In this review, we provide an overview of immune approaches to NB and the tumor microenvironment (TME) within the clinical translational framework. We propose a novel T cell-based therapeutic approach that leverages the unique properties of tumor surface antigens such as ganglioside GD2, incorporating specific monoclonal antibodies and recent advancements in adoptive cell therapy.

Putting a cap on the glycome: Dissecting human sialyltransferase functions

Human glycans are capped with sialic acids and these nine-carbon sugars mediate many of the biological functions and interactions of glycans. Structurally diverse sialic acid caps mark human cells as self and they form the ligands for the Siglec immune receptors and other glycan-binding proteins. Sialic acids enable host interactions with the human microbiome and many human pathogens utilize sialic acids to infect host cells. Alterations in sialic acid-carrying glycans, sialoglycans, can be found in every major human disease including inflammatory conditions and cancer. Twenty sialyltransferase family members in the Golgi apparatus of human cells transfer sialic acids to distinct glycans and glycoconjugates. Sialyltransferases catalyze specific reactions to form unique sialoglycans or they have shared functions where multiple family members generate the same sialoglycan product. Moreover, some sialyltransferases compete for the same glycan substrate, but create different sialic acid caps. The redundant and competing functions make it difficult to understand the individual roles of the human sialyltransferases in biology and to reveal the specific contributions to pathobiological processes. Recent insights hint towards the existence of biosynthetic rules formed by the individual functions of sialyltransferases, their interactions, and cues from the local Golgi environment that coordinate sialoglycan biosynthesis. In this review, we discuss the current structural and functional understanding of the human sialyltransferase family and we review recent technological advances that enable the dissection of individual sialyltransferase activities.

Sialidase-Chimeric Bioengineered Bacteria for Tumor-Sialoglycan-Triggered Solid Tumor Therapy.

Adoptive cell therapies for solid tumors are usually limited by off-target antigens, incapable tissue infiltration, and cell function exhaustion. In contrast, bacterial cells possess the inherent competencies of preferential tumor targeting, deep tissue penetration, and high intratumoral bioactivity and represent promising alternatives to overcome these challenges. Here, a sialic-acid-responsive regulatory gene circuit is engineered into Escherichia coli MG1655 to express cytolysin of hemolysin E (HlyE). Furthermore, sialidases are bioorthogonally decorated onto the surface of azido-functionalized bioengineered bacteria for recognizing tumor sialoglycans and cleaving their sialosides into free sialic acids. As chemical inducers, sialic acids feedbackingly activate the bacterial gene circuit to produce HlyE and lyse tumor cells. This study mimics the tumor antigen-induced cytotoxin production and cell lysis that occurs in chimeric antigen receptor T (CAR-T) cells yet surmounts the intrinsic limitations of adoptive cell therapies. Moreover, sialidase-mediated tumor cell desialylation also reverses the immunosuppressive effect of glycoimmune checkpoints and further improves the therapeutic effect of solid tumors.

Improving light availability and creating high-frequency light–dark cycles in raceway ponds through vortex-induced vibrations for microalgae cultivation: a fluid dynamic study

Limited light availability due to insufficient vertical mixing strongly reduces the applicability of raceway ponds (RWPs). To overcome this and create light–dark (L/D) cycles for enhanced biomass production through improved vertical mixing, vortex-induced vibration (VIV) system was implemented by the authors in a previous study to an existing pilot-scale RWP. In this study, experimental characterization of fluid dynamics for VIV-implemented RWP is carried out. Particle image velocimetry (PIV) technique is applied to visualize the flow. The extents of the vertical mixing due to VIV and the characteristics of L/D cycles were examined by tracking selected particles. Pond depth was hypothetically divided into three zones, namely dark, light Iimited and light saturated for detailed analysis of cell trajectories. It has been observed that VIV cylinder oscillation can efficiently facilitate the transfer of cells from light-limited to light-saturated zones. Among the cells that were tracked, 44% initially at dark zone entered the light-limited zone and 100% of initially at light-limited zone entered the light-saturated zone. 33% of all tracked cells experienced high-frequency L/D cycles with an average frequency of 35.69 s−1 and 0.49 light fraction. The impact of VIV was not discernible in the deeper sections of the pond, due to constrained oscillation amplitudes. Our findings suggest that the approximately 20% increase in biomass production reported in our previous study can be attributed to the synergistic effects of enhanced L/D cycle frequencies and improved light availability resulting from the transfer of cells from dark to light-limited zones. To further enhance the effectiveness of VIV, design improvements were developed. It was concluded that light availability could be significantly improved with the presented method for more effective use of RWPs.