Immune Ablation Research Articles

Dynamic changes of phenotype and function of natural killer cells in peripheral blood before and after thermal ablation of hepatitis B associated hepatocellular carcinoma and their correlation with tumor recurrence

BackgroundThermal therapy induces an immune response in patients with hepatocellular carcinoma (HCC), but the dynamic characteristics of the natural killer (NK) cell immune response post-thermal ablation remain unclear. We conducted a prospective longitudinal cohort study to observe the dynamic changes of phenotype and function of NK cells in peripheral blood before and after thermal ablation of hepatitis B-associated HCC and their correlation with tumor recurrence.MethodsFifty-six patients clinically and pathologically confirmed with hepatitis B-associated HCC were selected for thermal ablation. Peripheral blood was collected on day 0, day 7, and month 1. NK cell subsets, receptors, and killing function were detected by flow cytometry, and the LDH levels were examined. Overall recurrence and associated variables were estimated using Kaplan–Meier, log-rank, and Cox proportional-hazards analyses.ResultsThe frequency of CD3−CD56+ cells was increased on day 7 (P < 0.01) without significant differences between D0 and M1. NKG2D, NKp44, NKp30, CD159a, and CD158a expression was increased on M1 (all P < 0.05). The granzyme B and IFN-γ expression in NK cells were higher on M1 vs. D0 (P < 0.05). On day 7, the NK cell lysis activity of the target K562 cells was increased (P < 0.01) but decreased on M1 (P < 0.05). Survival analysis showed that CD158a expression and IFN-γ and perforin release on day 0 were associated with the risk of HCC recurrence. Cox regression analysis showed that the expression changes in CD56, NKp46, granzyme B, and perforin (D7-D0) induced by thermal ablation were associated with recurrence-free survival (RFS) of patients with HCC.ConclusionThermal ablation increased the frequency and function of CD3−CD56+ NK cells in the peripheral blood of patients with HCC. These cells tended to be more differentiated and activated. Notably, expression levels of NK cell receptors NKp46, perforin, and granzyme B were associated with RFS.

Limitations of Bu/Flu-based preparative regimen in CTLA-4 haploinsufficiency to achieving disease phenotype correction

BackgroundCTLA-4 haploinsufficiency (CTLA4-HI) is a broad immune dysregulation disorder characterized by multisystem autoimmunity and immune deficiency. In this report, we highlight the limitations of Busulfan-Fludarabine (Bu-Flu)-based conditioning regimens for HSCT in CTLA4-HI patients. MethodsThree patients with CTLA4-HI underwent HSCT at three centers between 2018–2019. Nine patients with nonmalignant disorders, without T cell (TC) dysregulation, who underwent allo-HSCT with Bu-Flu-based regimens were utilized as comparison. Further, we performed TC subset chimerism studies in CD4 recent thymic emigrants (RTE), CD4 and CD8 naïve TC cells, T regulatory cells (Tregs), circulating T follicular helper cells (cTfh), and CD4 and CD8 effector memory (EM). ResultsTwo patients (P1, P2) underwent MSD and 1 patient, MUD BM HSCT (P3). Cumulative Busulfan AUC was 66–95 mg*h/L and Fludarabine was 160 mg/m2. Only P1 and P2 had Alemtuzumab 0.3–0.48 mg/kg. Neutrophil (Day +18) and platelet (Day +24) engraftment was achieved in all patients with 100% donor myeloid chimerism. None had aGVHD. Mean TC chimerism at engraftment, 3 and 6 months, 1 year, 2 years, and last follow-up in the CTLA4-HI group was 20%, 26%, 25%, 45%, 46%, and 49%, respectively; lower than the comparison cohort – 59%, 52%, 70%, 87%, 93%, and 97%, respectively. With low TC chimerisms, all three patients had relapse of autoimmune manifestations, necessitating immune-modulatory therapy. No advantage of donor T cells in Treg, cTfh, CD4, or CD8 EM compartments was noted. P1 died unexpectedly at 25 months post-HSCT while still on sirolimus. P2 continues on medications for cytopenias. P3 came off sirolimus 3 years post-HSCT without autoimmune manifestations. CD4 RTE remains mixed donor (69%) >3 years post-HSCT (P2), suggesting incomplete thymic niche clearance, and chimerisms were only 54% in Tregs and 52% in cTfh. Correspondingly, P2 immunophenotype showed CD4 and CD8 activation with expansion of cTfh and decreased Treg frequency, suggesting ongoing immune dysregulation. ConclusionsIn our CTLA4-HI cohort, Bu-Flu-based conditioning resulted in inadequate immune ablation, low TC chimerisms, and incomplete or delayed disease phenotype correction. Moreover, no donor Treg benefit was noted in CTLA4-HI. Thus, in addition to augmenting immunoablation, strategies to enhance graft vs. immune dysregulation must be explored.

Immune Ablation and Stem Cell Rescue in Two Pediatric Patients with Progressive Severe Chronic Graft-Versus-Host Disease

Transplantation of allogeneic hematopoietic stem cells represents an established treatment for children with high-risk leukemia. However, steroid-refractory chronic graft-versus-host disease (SR-cGvHD) represents a severe life-threatening complication, for which there is no standard therapy. After failing several lines of immunosuppressive and biological treatment, we applied an immunoablative therapy with re-transplantation of purified CD34+ donor stem cells to reset the aberrant immune system. Two pediatric patients, who had been transplanted for high-risk acute lymphoblastic leukemia, underwent the procedure. Interestingly, enough stem cells could be mobilized, harvested, and purified to be used as grafts more than one year after allogeneic transplantation under intensive immunosuppressive therapy and ongoing SR-cGvHD. With a follow-up of 8 and 22 months, respectively, both patients are without immunosuppressive therapy and do not show signs of active disease. Regeneration of skin manifestations started promptly, other damaged organs did not progress and continue to show recovery from severe fibrotic transformation. Bone marrow function is robust and T cell receptor repertoires showed polyclonal immune reconstitution. In conclusion, stem cell harvest and re-transplantation of human CD34+-selected allogeneic stem cells is possible and represents a new therapeutic option in SR-cGvHD by resetting a profoundly disturbed immune network.

Antibody based conditioning for allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapeutic option for many patients with hematological malignancies and nonmalignant hematopoietic disorders. To achieve stable engraftment of donor hematopoietic stem cells (HSCs), recipient HSC deletion is needed to create space for incoming donor HSCs and donor HSCs must escape immune rejection by the recipient. Conventional allo-HSCT requires high dose of irradiation and/or chemotherapy to produce sufficient host stem cell and immune system ablation to permit donor HSC engraftment. However, these procedures also result in nonspecific tissue injury that can cause short- and long-term adverse effects as well as incite and amplify graft-versus-host-disease (GVHD). The delivery of targeted radiotherapy to hematopoietic tissues with the use of a radioimmunoconjugate (ROIC) as a part of transplant preparative regimen has shown clinical benefits. ROIC clinical data provide evidence for decreased relapse without increased transplant-related mortality by delivering higher targeted radiation to sites of malignancy than when given in a nontargeted fashion. An alternative approach to allo-HSCT has been developed and tested in preclinical mouse models in which nonmyeloablative preconditioning with low dose of the alkylating agent (busulfan) or lower systemic dose of irradiation combined with co-stimulatory pathway blockade (CTLA4-Ig, anti-CD40L monoclonal antibody) and/or immunosuppressive drugs have been used. Under these conditions, mixed chimerism and transplantation tolerance to fully MHC mismatched donor marrow was observed. Recently, several novel proof-of-concept antibody-mediated preconditioning methods have been developed that can selectively target hematopoietic stem and immune cells with minimal overall toxicity. Antibody-drug-conjugate (ADC) combined with reduced intensity conditioning or high dose ADC as single dose monotherapy have shown promise for allo-HSCT in preclinical models. The purpose of the current review is to discuss the literature exploring antibody-based conditioning that includes native antibody, radiolabeled antibody conjugates, and ADC for allo-HSCT.

Retrospective Evaluation and Significance of Neutrophil-to-Lymphocyte Ratio Prior to and 1 month Following Microwave Ablation of Hepatocellular Carcinoma.

Neutrophil-to-lymphocyte ratio (NLR) recently demonstrated predictive value for hepatocellular carcinoma (HCC) recurrence after thermal ablation. Microwave ablation (MWA) has been shown to induce changes in the immune landscape after HCC treatment. This study aims at identifying predictors of local tumor progression (LTP) and post-treatment NLR kinetics after MWA. Data from 108 consecutive patients who underwent percutaneous MWA of 119 HCCs with a 2450Hz/100W generator in two institutions from October 2014 to September 2021 were retrospectively reviewed. Forty-five HCCs (42 patients) met inclusion criteria for analysis (technique efficacy, pre- and post-treatment NLR availability, follow-up > 6months, absence of complications). NLR was analyzed prior to therapy and at 1-month follow-up; difference between the two time points was defined as ΔNLR1stFU. After a median follow-up of 25months, LTP occurred in 18 HCCs (40%) and 18 patients (42.9%). Multivariate competing risk regression comprising ΔNLR1stFU > 0, cirrhosis etiology and subcapsular location showed that the only independent predictor of LTP was ΔNLR1stFU > 0, on both a per-patient (HR = 2.7, p = 0.049) and per-tumor (HR = 2.8, p = 0.047) analysis. ΔNLR1stFU > 0 occurred in 24/42 patients (57.1%). In this subgroup, higher rates of female patients (p = 0.026), higher mean baseline NLR (p < 0.0001) and lower mean energy/size (p = 0.006) were observed. Upon ROC curve analysis, energy/size < 1414J/mm predicted ΔNLR1stFU > 0 with 76% sensitivity and 70% specificity (AUC = 0.74). NLR increase after ablation was the only independent predictor of LTP, supporting the role of balance between systemic inflammation and immunity in recurrence after MWA. Ablation energy/tumor size predicted NLR increase, reinforcing the concept of immune ablation. III.

Depletion of Lysozyme M+ Cells Offers Cardiovascular, but not Cerebral, Protection from Noise‐Induced Stress

Background and Hypothesis Increased leukocyte extravasation in the aortic endothelium leads to a pro-oxidant and pro-inflammatory environment which facilitates the development of endothelial dysfunction. Moderate hypertension, endothelial dysfunction, and increases in oxidative stress are associated with exposure to the novel cardiovascular risk, noise pollution. Since we have previously established a role for myelomonocytes (LysM+ cells) in the deleterious effects of noise exposure, we hypothesized that partial ablation of these cells would protect from cardiovascular consequences. Methods LysM+ immune cells were depleted with diphtheria toxin (DTX) in LysMCre+/-iDTR+/- mice for 6 days prior to noise exposure (mean SPL; 72 dBA) and 4 days concurrently. Blood pressure was determined by tail cuff method. Endothelial dysfunction was measured with isometric tension recordings. Protein and gene analysis were carried out using Western Blot, dot blot, and rtPCR. Oxidative stress measurements included dihydroethidium HPLC and staining. Inflammatory cell infiltration was assessed via FACS. Astrocyte and microglia activation were measured by GFAP+ and Iba1+ staining. Results Ablation of LysM+ cells successfully normalized the blood pressure as well as aortic and retinal vessel endothelial function of mice exposed to 4 days of noise. Noise caused a significant increase in superoxide production in aortic and cardiac tissue. Mice with ablated LysM+ cells were protected from increased oxidative stress and its effects. The DTX +Noise group was protected from expression of inflammatory genes in aortic tissue. These effects were not seen in brain or cortical tissue, where microglia and astrocyte activation were increased and there was a general failure of protection from noise in inflammatory gene (NFκB2, CD40L, TNFα) and protein (CD68, LysM, CD40, IL6) expression. Noise, DTX, and DTX +Noise groups were all found to have heightened levels of stress hormones in plasma. Conclusion Ablation of myelomonocytic cells was successful in rescuing mice from the cardiovascular damage associated with noise exposure, suggesting that these cells play a critical role in the damage incurred upon noise exposure. However, this protection was only found in the vasculature and a distinctly different outcome was discovered in the brain, where a neuroinflammatory phenotype persisted. Heightened levels of hormones indicate that the stress response following noise exposure remains intact in the brain following immune ablation, but is not carried forth to the vasculature, both implicating LysM+ cells in the oxidative damage caused by noise exposure and also in playing an important role the hormone signaling resultant from stress reactions.

ExoSTING, an extracellular vesicle loaded with STING agonists, promotes tumor immune surveillance

Cyclic dinucleotide (CDN) agonists of the STimulator of InterferoN Genes (STING) pathway have shown immune activation and tumor clearance in pre-clinical models. However, CDNs administered intratumorally also promote STING activation leading to direct cytotoxicity of many cell types in the tumor microenvironment (TME), systemic inflammation due to rapid tumor extravasation of the CDN, and immune ablation in the TME. These result in a failure to establish immunological memory. ExoSTING, an engineered extracellular vesicle (EV) exogenously loaded with CDN, enhances the potency of CDN and preferentially activates antigen presenting cells in the TME. Following intratumoral injection, exoSTING was retained within the tumor, enhanced local Th1 responses and recruitment of CD8+ T cells, and generated systemic anti-tumor immunity to the tumor. ExoSTING at therapeutically active doses did not induce systemic inflammatory cytokines, resulting in an enhanced therapeutic window. ExoSTING is a novel, differentiated therapeutic candidate that leverages the natural biology of EVs to enhance the activity of CDNs.

Immune Modulation Plus Tumor Ablation: Adjuvants and Antibodies to Prime and Boost Anti-Tumor Immunity In Situ.

In situ tumor ablation techniques, like radiotherapy, cryo- and heat-based thermal ablation are successfully applied in oncology for local destruction of tumor masses. Although diverse in technology and mechanism of inducing cell death, ablative techniques share one key feature: they generate tumor debris which remains in situ. This tumor debris functions as an unbiased source of tumor antigens available to the immune system and has led to the concept of in situ cancer vaccination. Most studies, however, report generally modest tumor-directed immune responses following local tumor ablation as stand-alone treatment. Tumors have evolved mechanisms to create an immunosuppressive tumor microenvironment (TME), parts of which may admix with the antigen depot. Provision of immune stimuli, as well as approaches that counteract the immunosuppressive TME, have shown to be key to boost ablation-induced anti-tumor immunity. Recent advances in protein engineering have yielded novel multifunctional antibody formats. These multifunctional antibodies can provide a combination of distinct effector functions or allow for delivery of immunomodulators specifically to the relevant locations, thereby mitigating potential toxic side effects. This review provides an update on immune activation strategies that have been tested to act in concert with tumor debris to achieve in situ cancer vaccination. We further provide a rationale for multifunctional antibody formats to be applied together with in situ ablation to boost anti-tumor immunity for local and systemic tumor control.

Infection of Immunocompetent Conventional Mice with Shiga Toxin-Producing E. coli: The DSS+STEC Model.

Previous methods of infecting mice with Shiga toxin-producing E. coli (STEC) required suppression of host immune function or ablation of the gut microbiota to induce susceptibility to gastrointestinal colonization. Consequently, many pathogen-host interactions occurring in immunocompetent hosts during STEC infection and Shiga toxicosis have remained unclear. The following protocol describes the use of dextran sulfate sodium (DSS) to induce a mild colitis in immunocompetent conventional C57BL/6 mice to facilitate susceptibility to STEC infection by oral gavage. STEC colonization in infected mice was confirmed by recovery of live STEC via fecal cultures and quantified via quantitative polymerase chain reaction of fecal DNA for the STEC-specific gene eae. DSS colitis is well established, broadly reproducible, and does not require specialized equipment or high levels of technical proficiency to be a useful method of inducing susceptibility to gastrointestinal STEC colonization. The DSS+STEC mouse model provides a novel and useful tool for the exploration of local STEC-host interactions in the gut environment and the pathogenesis of Shiga toxicosis.

Long-term survival of combined ablation therapy and tremelimumab with or without durvalumab in advanced hepatocellular carcinoma.

e16689 Background: Immune checkpoint inhibitor therapy has recently been approved for the treatment of patients with Hepatocellular Cancer (HCC). Data on long-term survival is lacking and the predictors of good outcomes are unknown. The combination of locoregional therapies (transarterial chemoembolization or radiofrequency ablation) plus tremelimumab (trem) with or without durvalumab (durva) was studied in patients with advanced HCC. We report the long-term survival and analyze predictors of good response in two trials of immune checkpoint and ablation treatment in advanced HCC. Methods: Adult patients (pts) with pathologically confirmed HCC with advanced or sorafenib refractory disease who satisfied the eligibility criteria were enrolled in the studies (NCT02821754, NCT01853618). They received treatment with trem, 10 mg per kilogram every 4 weeks for six doses followed by 3 monthly infusions (23 pts) or trem 75 mg flat dose every 28 days for four doses and then durva 1500 mg flat dose every 28 days (35 pts). Subtotal radiofrequency or chemoablation was done on day 36. Results: The average overall survival in the 58 pts was 10.1 months (range 0.9 to 74.2 months); 41.6% were alive at 12 months and 5.5 % were alive at 61 months. A select group of patients had exceptional overall survivals up to 67 months. Predictors of improved overall survival included the presence of higher grade immune related (irAEs) (OR 0.235, p = 0.075) and a partial or stable disease response per RECIST. No new late toxic effects were noted during this long term follow up. Conclusions: Impressive overall survival times of up to 68.8 months were observed with ICI and subtotal ablation in patients with advanced HCC; the presence of irAEs and the tumor response per RECIST may be predictive of better overall survivals. To our knowledge this has not been previously reported in the literature. Clinical trial information: NCT02821754,NCT01853618 .

Does Resetting the Immune System Fix Multiple Sclerosis?

Multiple sclerosis is the leading non-traumatic cause of disability in young adults, affecting up to 100,000 Canadians. This chronic inflammatory and neurodegenerative disease of the central nervous system leads to irreversible neurologic disability if inadequately controlled. Though many current medications are available that reduce inflammatory damage, most patients continue to show some evidence of disease activity and accrue disability. In this review, we discuss the role of immune ablation followed by autologous hematopoietic stem cell transplantation (AHSCT), a therapeutic option for select patients with a more aggressive disease course. By "resetting" the immune system with a variety of ablative conditioning regimens, followed by immune reconstitution, this therapy has shown a durable response in halting evidence of inflammatory activity in most patients, without the need for continued disease-modifying therapies (DMT). Since the introduction of this therapy, there have been advances in patient selection and supportive care, such that morbidity has significantly declined and treatment-related mortality is minimized. Recent phase-II trials have shown excellent results in efficacy and safety of AHSCT; however, challenges exist which require ongoing study. The future challenges include comparing the variety of AHSCT conditioning regimens with each other as well as with existing highly effective DMT; identifying patients with an aggressive disease course through novel biomarkers who may benefit the most from AHSCT; and surveillance of long-term outcomes of different treatment protocols. In select patients, replacing the immune system with AHSCT holds promise of fundamentally altering the trajectory of their aggressive disease course.

Immune Cell-Type Specific Ablation of Adapter Protein ADAP Differentially Modulates EAE

The cytosolic adhesion and degranulation-promoting adapter protein ADAP is expressed in various hematopoietic cells including T cells, NK cells, myeloid cells, and platelets but absent in mature B cells. The role of ADAP in T cell activation, proliferation and integrin activation is well-accepted. We previously demonstrated that conventional ADAP knockout mice show a significantly attenuated course of experimental autoimmune encephalomyelitis (EAE). To dissect the impact of different ADAP expressing cell populations on the reduced EAE severity, here, we generated lineage-specific conditional knockout mice. ADAP was deleted in T cells, myeloid cells, NK cells and platelets, respectively. Specific loss of ADAP was confirmed on the protein level. Detailed immunophenotyping was performed to assess the consequence of deletion of ADAP with regard to the maturation and distribution of immune cells in primary and secondary lymphoid organs. The analysis showed equivalent results as for conventional ADAP knockout mice: impaired thymocyte development in ADAPfl/fl Lck-Cre mice, normal NK cell and myeloid cell distribution in ADAPfl/fl NKp46-Cre mice and ADAPfl/fl LysM-Cre mice, respectively as well as thrombocytopenia in ADAPfl/fl PF4-Cre mice. Active EAE was induced in these animals by immunization with the myelin oligodendrocyte glycoprotein35−55 peptide. The clinical course of EAE was significantly milder in mice with loss of ADAP in T cells, myeloid cells and NK cells compared to ADAP-sufficient control littermates. Surprisingly, specific deletion of ADAP in platelets resulted in a more exacerbated disease. These data show that T cell-independent as well as T cell-dependent mechanisms are responsible for the complex phenotype observed in conventional ADAP knockout mice.

In situ thermal ablation of tumors in combination with nano-adjuvant and immune checkpoint blockade to inhibit cancer metastasis and recurrence

Tumor ablation therapies provide a minimally invasive approach to treat cancer. However, inhibition of cancer metastasis and recurrence after ablation is still a challenge in clinical trials. Here, we propose a strategy using combinatorial thermal ablation, adjuvants and immune checkpoint blockade (ICB) to inhibit metastatic tumor and recurrence via antitumor immune responses post tumor thermal ablation, which are frequently used in the clinic. Furthermore, a strong immune memory against cancer was observed 80 days after the primary tumor was ablated. Considering that all components in our design are approved by Food and Drug Administration (FDA), we provide a strategy based on clinically used cancer treatment technique that is promising in clinical translation.

Abstract 3938: Acoustic immune priming of radiation therapy eradicates primary tumor and reduces distant metastases in triple-negative murine breast cancer

Abstract Introduction: Recently, we have shown that low energy focused ultrasound (FUS) treatment reversed tumor-induced T cell tolerance in the B16F10 melanoma model. FUS also induced protein unfolding and translocation of cellular chaperones on the cell surface, thereby providing danger signals for immune activation. To further study immune priming using FUS, we designed a novel immune priming ablation therapy, whereby non-ablative low energy FUS was delivered before ablative radiation therapy in murine triple-negative breast cancer for generating an in situ vaccine. Methods: Orthotopic, palpable (&amp;gt;5mm) 4T1 tumors, a poorly immunogenic, aggressive, triple-negative murine metastatic mammary carcinoma in Balb/c mice, were treated with FUS (15W (100 W/cm2) and 75W (500W/cm2); 100% duty cycle for 3 seconds using custom transducer in multi-frequency mode with or without RT (20Gy) for three consecutive days. Tumor growth, survival, and metastases were monitored. The tumor immune-infiltrate population was analyzed at day 7 post-treatment using flow cytometry. 18F-FDG PET was used to image distant metastases. For the abscopal study, mice were implanted with a primary (treated) and secondary (non-treated) contralateral tumor and monitored for tumor growth on both sites. Results: FUS treatment (75W) in combination with RT eradicated 86% (6/7) of tumors. Tumor growth was also considerably reduced in RT alone and 15W+RT groups compared to non-treated, with a relapse of 43% (3 of 7) in both the groups. There was significant reduction in regional metastatic spread in draining lymph nodes (DLN) (RT, 50% versus FUS (75W)+RT, 22%) and pulmonary metastases (RT, 60% versus FUS (75W)+RT, 22%). Tumors relapsed in DLN and lungs after FUS+RT treatment in only 2 out of 9 mice. At 7 weeks, FDG-PET scans showed no uptake in FUS+RT treated groups, compared to metastatic uptakes in lungs, lymph nodes, and liver in 3 out of 7 mice, treated with RT alone. There was a significant increase in tumor-infiltrating CD3+ lymphocytes, IFNg+CD8 T cells, Granzyme B+ CD8+ T cells and NKT cells, upon treatment with FUS+RT compared to the RT alone group. In contrast to RT alone, FUS+RT promoted suppression of untreated abscopal tumors in contralateral flank. Summary: In this study, we found that local treatment with FUS+RT resulted in primary tumor control, a significant reduction in distant metastasis with improved long-term survival and suppression of unirradiated, abscopal tumors, indicating that focal therapy with a combination of acoustic immune priming with ablative RT induced systemic immunity. Our acoustic immune priming ablation therapy provides a foundation for using physical energy-based focal therapies in combination with immunotherapies for in situ tumor vaccines. Citation Format: Saurabh Singh, Claudia Gutierrez-Chavez, Karin Skalina, Wade Koba, Indranil Basu, Stephen Barry, Chandan Guha. Acoustic immune priming of radiation therapy eradicates primary tumor and reduces distant metastases in triple-negative murine breast cancer [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 3938.

Regenerative medicine: the red planet for clinicians.

Regenerative medicine represents the forefront of health sciences and holds promises for the treatment and, possibly, the cure of a number of challenging conditions. It relies on the use of stem cells, tissue engineering, and gene therapy alone or in different combinations. The goal is to deliver cells, tissues, or organs to repair, regenerate, or replace the damaged ones. Among stem-cell populations, both haematopoietic and mesenchymal stem cells have been employed in the treatment of refractory chronic inflammatory diseases with promising results. However, only mesenchymal stem cells seem advantageous as both systemic and local injections may be performed without the need for immune ablation. Recently, also induced pluripotent stem cells have been exploited for therapeutic purposes given their tremendous potential to be an unlimited source of any tissue-specific cells. Moreover, through the development of technologies that make organ fabrication possible using cells and supporting scaffolding materials, regenerative medicine promises to enable organ-on-demand, whereby patients will receive organs in a timely fashion without the risk of rejection. Finally, gene therapy is emerging as a successful strategy not only in monogenic diseases, but also in multifactorial conditions. Several of these approaches have recently received approval for commercialization, thus opening a new therapeutic era. This is why both General Practitioners and Internists should be aware of these great advancements.

Renal denervation and CD161a immune ablation prevent cholinergic hypertension and renal sodium retention

Cholinergic receptor activation leads to premature development of hypertension and infiltration of proinflammatory CD161a+/CD68+ M1 macrophages into the renal medulla. Renal inflammation is implicated in renal sodium retention and the development of hypertension. Renal denervation is known to decrease renal inflammation. The objective of this study was to determine the role of CD161a+/CD68+ macrophages and renal sympathetic nerves in cholinergic-hypertension and renal sodium retention. Bilateral renal nerve denervation (RND) and immune ablation of CD161a+ immune cells were performed in young prehypertensive spontaneously hypertensive rat (SHR) followed by infusion of either saline or nicotine (15 mg·kg-1·day-1) for 2 wk. Immune ablation was conducted by injection of unconjugated azide-free antibody targeting rat CD161a+. Blood pressure was monitored by tail cuff plethysmography. Tissues were harvested at the end of infusion. Nicotine induced premature hypertension, renal expression of the sodium-potassium chloride cotransporter (NKCC2), increases in renal sodium retention, and infiltration of CD161a+/CD68+ macrophages into the renal medulla. All of these effects were abrogated by RND and ablation of CD161a+ immune cells. Cholinergic activation of CD161a+ immune cells with nicotine leads to the premature development of hypertension in SHR. The effects of renal sympathetic nerves on chemotaxis of CD161a+ macrophages to the renal medulla, increased renal expression of NKCC2, and renal sodium retention contribute to cholinergic hypertension. The CD161a+ immune cells are necessary and essential for this prohypertensive nicotine-mediated inflammatory response.NEW & NOTEWORTHY This is the first study that describes a novel integrative physiological interaction between the adrenergic, cholinergic, and renal systems in the development of hypertension, describing data for the role of each in a genetic model of essential hypertension. Noteworthy findings include the prevention of nicotine-mediated hypertension following successful immune ablation of CD161a+ immune cells and the necessary role these cells play in the overexpression of the sodium-potassium-chloride cotransporter (NKCC2) in the renal medulla and renal sodium retention. Renal infiltration of these cells is demonstrated to be dependent on the presence of renal adrenergic innervation. These data offer a fertile ground of therapeutic potential for the treatment of hypertension as well as open the door for further investigation into the mechanism involved in inflammation-mediated renal sodium transporter expression. Taken together, these findings suggest immune therapy, renal denervation, and, possibly, other new molecular targets as having a potential role in the development and maintenance of essential hypertension.

Differences in Peripheral and Tissue Immune Cell Populations Following Haematopoietic Stem Cell Transplantation in Crohn's Disease Patients.

Background and AimsRecent studies have shown the efficacy of autologous haematopoietic stem cell transplantation [HSCT] in severely refractory Crohn’s disease [CD] patients. HSCT is thought to eliminate auto-reactive cells; however, no specific studies of immune reconstitution in CD patients are available.MethodsWe followed a group of CD patients [n = 18] receiving autologous HSCT, with 50% of them achieving endoscopic drug-free remission. To elucidate the mechanisms driving efficacy, we monitored changes after HSCT in blood and intestine immune-cell composition. CD patients [n = 22] receiving anti-tumour necrosis factor [TNF]-α were included for comparison.ResultsSevere immune ablation followed by HSCT induced dramatic changes in both peripheral blood T and B cells in all patients regardless of the efficacy of the treatment. Endoscopic remission at week 52 following HSCT was associated with significant intestinal transcriptional changes. A comparison of the remission signature with that of anti-TNFα identified both common and unique genes in the HSCT-induced response. Based on deconvolution analysis of intestinal biopsy transcriptome data, we show that response to HSCT, but not to anti-TNFα, is associated with an expansion of naïve B-cells, as seen in blood, and a decrease in the memory resting T-cell content. As expected, endoscopic remission, in response to both HSCT and anti-TNFα, led to a significant reduction in intestinal neutrophil and M1 macrophage content.ConclusionsPeripheral blood immune remodelling after HSCT does not predict efficacy. In contrast, a profound intestinal T-cell depletion that is maintained long after transplant is associated with mucosal healing following HSCT, but not anti-TNFα.

Embryonic macrophages and microglia ablation alter the development of dorsal root ganglion sensory neurons in mouse embryos.

Microglia are known to regulate several aspects of the development of the central nervous system. When microglia colonize the spinal cord, from E11.5 in the mouse embryo, they interact with growing central axons of dorsal root ganglion sensory neurons (SNs), which suggests that they may have some functions in SN development. To address this issue, we analyzed the effects of embryonic macrophage ablation on the early development of SNs using mouse embryo lacking embryonic macrophages (PU.1 knock-out mice) and immune cell ablation. We discovered that, in addition to microglia, embryonic macrophages contact tropomyosin receptor kinase (Trk) C+ SN, TrkB+ SN, and TrkA+ SN peripheral neurites from E11.5. Deprivation of immune cells resulted in an initial reduction of TrkC+ SN and TrkB+ SN populations at E11.5 that was unlikely to be related to an alteration in their developmental cell death (DCD), followed by a transitory increase in their number at E12.5. It also resulted in a reduction of TrkA+ SN number during the developmental period analyzed (E11.5-E15.5), although we did not observe any change in their DCD. Proliferation of cells negative for brain fatty acid-binding protein (BFABP- ), which likely correspond to neuronal progenitors, was increased at E11.5, while their proliferation was decreased at E12.5, which could partly explain the alterations of SN subtype production observed from E11.5. In addition, we observed alterations in the proliferation of glial cell progenitors (BFABP+ cells) in the absence of embryonic macrophages. Our data indicate that embryonic macrophages and microglia ablation alter the development of SNs.

Comparative utility of NRG and NRGS mice for the study of normal hematopoiesis, leukemogenesis, and therapeutic response

Cell-line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) in immune-deficient mice have revolutionized our understanding of normal and malignant human hematopoiesis. Transgenic approaches further improved in vivo hematological research, allowing the development of human-cytokine-producing mice, which show superior human cell engraftment. The most popular mouse strains used in research, the NOG (NOD.Cg-PrkdcscidIl2rγtm1Sug/Jic) and the NSG (NOD/SCID-IL2Rγ-/-, NOD.Cg-PrkdcscidIl2rγtm1Wjl/SzJ) mouse, and their human-cytokine-producing (interleukin-3, granulocyte-macrophage colony-stimulating factor, and stem cell factor) counterparts (huNOG and NSGS), rely partly on a mutation in the DNA repair protein PRKDC, causing a severe combined immune deficiency (SCID) phenotype and rendering the mice less tolerant to DNA-damaging therapeutics, thereby limiting their usefulness in the investigation of novel acute myeloid leukemia (AML) therapeutics. NRG (NOD/RAG1/2-/-IL2Rγ-/-) mice show equivalent immune ablation through a defective recombination activation gene (RAG), leaving DNA damage repair intact, and human-cytokine-producing NRGS (NRG-SGM3) mice were generated, improving myeloid engraftment. Our findings indicate that unconditioned NRG and NRGS mice can harbor established AML CDXs and can tolerate aggressive induction chemotherapy at higher doses than NSG mice without overt toxicity. However, unconditioned NRGS mice developed less clinically relevant disease, with CDXs forming solid tumors throughout the body, whereas unconditioned NRG mice were incapable of efficiently supporting PDX or human hematopoietic stem cell engraftment. These findings emphasize the contextually dependent utility of each of these powerful new strains in the study of normal and malignant human hematopoiesis. Therefore, the choice of mouse strain cannot be random, but must be based on the experimental outcomes and questions to be addressed.

FcγRI expression on macrophages is required for antibody-mediated tumor protection by cytomegalovirus-based vaccines.

Cytomegalovirus (CMV)-based vaccine vectors are promising vaccine platforms because they induce strong and long-lasting immune responses. Recently it has been shown that vaccination with a mouse CMV (MCMV) vector expressing the melanoma-specific antigen TRP2 (MCMV-TRP2) protects mice against outgrowth of TRP2-positive B16 melanoma tumors, and this protection was dependent on the induction of IgG antibodies. Here we demonstrate that, although mice lacking all receptors for the Fc part of IgG (FcγRs) develop normal IgG responses after MCMV-TRP2 vaccination, the protection against B16 melanoma was completely abrogated, indicating that FcγRs are indispensable in the downstream effector pathway of the polyclonal anti-TRP2 antibody response. By investigating compound FcγR-deficient mouse strains and by using immune cell type-specific cell ablation we show that the IgG antibody-mediated tumor protection elicited by MCMV-TRP2 mainly depends on FcγRI expression on macrophages, whereas FcγRIV plays only a modest role. Thus, tumor-specific antibody therapy might benefit from combination therapy that recruits FcγRI-expressing pro-inflammatory macrophages to the tumor micro-environment.