Adoptive Transfer Of Immunity Research Articles

Adoptive immune transfer from donors offers Anti-HBV protection to HBsAb-negative patients after Allo-HSCT.

Adoptive transfer of hepatitis B virus (HBV) immunity may occur following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Here, we investigated the adoptive transfer of HBV immunity in 112 patients without HBV surface antibody (HBsAb) (HBsAb-) at the time of their first allo-HSCT. After allo-HSCT, HBV-DNA（87.5%） and HBsAg（11.1%%）cleared in HBsAg+ patients. All HBsAg- patients acquired HBsAb immediately. Nevertheless, HBsAb titers subsequently declined, and 39/67 (58.2%) patients lost HBsAb during follow-up. The 5-year overall survival (OS) was better in patients who lost HBsAb. Multivariate analysis showed that the independent risk factors for OS were lack of cytomegalovirus (CMV) clearance, acute graft-versus-host disease (aGVHD), and no HBsAb loss. Overall, adoptive immune transfer offers anti-HBV protection to patients without HBsAb, as they acquire HBsAb and clear HBV-DNA and HBsAg, while HBsAb loss after allo-HSCT predicts better survival.

Impact of Donor and Recipient SARS-CoV-2 Vaccination or Infection on Immunity after Hematopoietic Cell Transplantation

The role of donor and recipient Coronavirus disease 2019 (COVID-19) immunologic status pre-transplantation has not been fully investigated in allogeneic hematopoietic stem cell transplantation (HSCT) recipients. Given the poor immunogenicity to vaccines in this population and the serious outcomes of COVID-19, adoptive transfer of immunity may offer important insight into improving protection for this vulnerable population. In this study, we evaluated the role of adoptive transfer of immunity at 1 month post-transplantation and 6 months post-transplantation after vaccination of recipients, based on pre-transplantation severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination and infection exposures of both recipient and donor. Using banked specimens from related donor allogeneic HSCT recipients and clinical data from both donors and recipients, anti-Spike (S) IgG titers were analyzed at 1, 3, and 6 months post-transplantation according to prior SARS-CoV-2 immunologic exposures. Recipients were excluded if they had received SARS-CoV-2 monoclonal antibodies or had infection in the first 6 months post-transplantation. Of the 53 recipient-donor pairs, 29 donors and 24 recipients had prior SARS-CoV-2 immunologic exposure. Recipient-donor pairs with no prior SARS-CoV-2 exposure (D0R0) had significantly lower anti-S IgG titers at 1 month compared to those with prior exposures (D1R1) (D0R0: median, 2.43 [interquartile range (IQR), .41 to 3.77]; D1R1: median, 8.42; IQR, 5.58 to 12.20]; P=.008). At 6 months, anti-S IgG titers were higher in recipients who were vaccinated at 3 months post-transplantation in the D1R1 cohort (median IgG, 148.34; IQR, 92.36 to 204.33) compared with the D0R0 cohort (median IgG, 38.74; IQR, 8.93 to 119.71). Current strategies should be optimized to enhance SARS-CoV-2 protection for HSCT recipients, including augmentation of the immune response for both donors and recipients prior to transplantation.

Perspective: An International Fludarabine Shortage: Supply Chain Issues Impacting Transplantation and Immune Effector Cell Therapy Delivery

Host immune depletion has been recognized as a necessary step for successful adoptive immune cell transfer in both the autologous and allogeneic settings. The chemotherapy agent fludarabine as an immune suppressive agent has a central role in multiple conditioning regimens for both transplantation and immune effector cell therapies. With the recent and sudden recognition of an imminent worldwide fludarabine shortage, novel approaches to overcome supply chain disruption are needed, including exploration of alternative therapies. The fludarabine shortage has highlighted the need to prioritize the development of institutional algorithms for maintaining ongoing clinical trials and standard of care procedures in the setting of critical drug shortages.

Progress and Prospect of Immunotherapy for Triple-Negative Breast Cancer.

Breast cancer is the most commonly diagnosed cancer (estimated 2.3 million new cases in 2020) and the leading cause of cancer death (estimated 685,000 deaths in 2020) in women globally. Breast cancers have been categorized into four major molecular subtypes based on the immunohistochemistry (IHC) expression of classic hormone and growth factor receptors including the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), as well as a proliferation marker Ki-67 protein expression. Triple-negative breast cancer (TNBC), a breast cancer subtype lacking ER, PR, and HER2 expression, is associated with a high metastatic potential and poor prognosis. TNBC accounts for approximately only 15%–20% of new breast cancer diagnoses; it is responsible for most breast cancer–related deaths due to the lack of targeted treatment options for this patient population, and currently, systemic chemotherapy, radiation, and surgical excision remain the major treatment modalities for these patients with TNBC. Although breast cancer patients in general do not have a robust response to the immunotherapy, a subset of TNBC has been demonstrated to have high tumor mutation burden and high tumor-infiltrating lymphocytes, resembling the features observed on melanoma or lung cancers, which can benefit from the treatment of immune checkpoint inhibitors (ICIs). Therefore, the immunogenic nature of this aggressive disease has presented an opportunity for the development of TNBC-targeting immunotherapies. The recent US Food and Drug Administration approval of atezolizumab in combination with the chemotherapeutic agent nab-paclitaxel for the treatment of PD-L1-positive unresectable, locally advanced, or metastatic TNBC has led to a new era of immunotherapy in TNBC treatment. In addition, immunotherapy becomes an active research area, both in the cancer biology field and in the oncology field. In this review, we will extend our coverage on recent discoveries in preclinical research and early results in clinical trials from immune molecule-based therapy including cytokines, monoclonal antibodies, antibody–drug conjugates, bi-specific or tri-specific antibodies, ICIs, and neoantigen cancer vaccines; oncolytic virus-based therapies and adoptive immune cell transfer–based therapies including TIL, chimeric antigen receptor-T (CAR-T), CAR-NK, CAR-M, and T-cell receptor-T. In the end, we will list a series of the challenges and opportunities in immunotherapy prospectively and reveal novel technologies such as high-throughput single-cell sequencing and CRISPR gene editing-based screening to generate new knowledges of immunotherapy.

Adoptive Transfer of Immune Cells Into RAG2IL-2Rγ-Deficient Mice During Litomosoides sigmodontis Infection: A Novel Approach to Investigate Filarial-Specific Immune Responses.

Despite long-term mass drug administration programmes, approximately 220 million people are still infected with filariae in endemic regions. Several research studies have characterized host immune responses but a major obstacle for research on human filariae has been the inability to obtain adult worms which in turn has hindered analysis on infection kinetics and immune signalling. Although the Litomosoides sigmodontis filarial mouse model is well-established, the complex immunological mechanisms associated with filarial control and disease progression remain unclear and translation to human infections is difficult, especially since human filarial infections in rodents are limited. To overcome these obstacles, we performed adoptive immune cell transfer experiments into RAG2IL-2Rγ-deficient C57BL/6 mice. These mice lack T, B and natural killer cells and are susceptible to infection with the human filaria Loa loa. In this study, we revealed a long-term release of L. sigmodontis offspring (microfilariae) in RAG2IL-2Rγ-deficient C57BL/6 mice, which contrasts to C57BL/6 mice which normally eliminate the parasites before patency. We further showed that CD4+ T cells isolated from acute L. sigmodontis-infected C57BL/6 donor mice or mice that already cleared the infection were able to eliminate the parasite and prevent inflammation at the site of infection. In addition, the clearance of the parasites was associated with Th17 polarization of the CD4+ T cells. Consequently, adoptive transfer of immune cell subsets into RAG2IL-2Rγ-deficient C57BL/6 mice will provide an optimal platform to decipher characteristics of distinct immune cells that are crucial for the immunity against rodent and human filarial infections and moreover, might be useful for preclinical research, especially about the efficacy of macrofilaricidal drugs.

Hepatitis B Virus Infection in Patients Receiving Allogeneic Hematopoietic Stem Cell Transplantation.

Considering a steady increase in the number of allogeneic hematopoietic stem cell transplantations (allo-HSCT) worldwide and the significant proportion of the world’s population that has been exposed to hepatitis B virus (HBV) infection, HBV reactivation following allo-HSCT remains an important issue for post-transplant morbidity and mortality. Antiviral prophylaxis can reduce HBV replication, severity of HBV-related hepatitis, and mortality; therefore, identification of patients at risk is crucial. It is recommended that all recipients and donors should be screened for active or prior HBV infection, including HBsAg, antiHBc, and antiHBs. Adoptive immunity transfer from the donor seems to have protective effects against HBV reactivation. Antiviral prophylaxis should be initiated in all HBsAg-positive patients. HBsAg-negative, antiHBc-positive patients remain at risk; therefore, antiviral prophylaxis should be considered if baseline serum HBV DNA is detectable. In HBsAg-negative, antiHBc-positive patients without detectable HBV DNA, close monitoring of viral load with an on-demand therapy is necessary. Entecavir or tenofovir rather than lamivudine are more appropriate for the emergence of lamivudine resistance. The treatment duration remains unclear, with 6- to 12-month therapy after cessation of immunosuppressive therapy commonly recommended. Here we review the updated evidence and recent recommendations regarding HBV reactivation in patients undergoing allo-HSCT for individualized therapy.

New Model of Splenocyte Transfer Exacerbates Salt‐Sensitive Renal Injury and Hypertension in Rats

Infiltration of immune cells into target organs, including the kidney, is a hallmark of hypertensive pathology, driving the use of various experimental manipulations to interrogate the influence of the immune system. Previous studies using RAG1−/− Dahl Salt‐Sensitive (SS) rats demonstrated that the adaptive immune system plays a vital role in potentiating the hypertensive phenotype. More specifically, we created a line of Dahl SS rats with a null mutation in the CD247 gene. CD3+ T cells are absent in these SSCD247−/− rats, but a B cell population is preserved. Compared to SS rats, we observe an attenuation in renal injury in SSCD247−/− rats when subjected to a high salt (HS) challenge (−109mg/day albuminuria), mirroring an attenuation in hypertension (−19mmHg). Though adoptive transfer studies are useful in mice models of hypertension, there has yet to be a study to demonstrate adoptive transfer of immune cells potentiating a genetic model of SS hypertension. Using the male T cell deficient Dahl SS rat, SSCD247−/−, we hypothesized that splenocyte transfer from SS wildtype rats into SSCD247−/− rats would populate the T cell compartment and exacerbate the SS phenotypes. Furthermore, we tested the hypothesis that splenocytes from high‐salt fed donors (HSS) would induce a more dramatic salt‐sensitive phenotype in the recipients than donors fed a low‐salt diet (LSS). The splenocyte transfer recipients (n=11/13) were compared to naïve SSCD247−/− rats (n=7). As engraftment of adoptively transferred cells is difficult to achieve reliably, we present a new approach demonstrating that transfer of splenocytes from adult rats into recipient pups at postnatal day 5 is feasible for robust population of the T cell compartment in the circulation and spleen. Using flow cytometry, greater than 80% of the constituted CD3+ T cells were CD4+ Helper T cells. In comparison to SSCD247−/− T cell deficient rats, by HS day 21, recipients receiving splenocyte transfer from both low‐salt and high‐salt fed donors showed an exacerbated rise in SS blood pressure (LSS, 150.3±19.0mHg; HSS, 155.2+/−20.8mmHg; vs SSCD24−/− 135.6±2.9mmHg; p<0.01) negating the effect originally observed in the knockout (+15–20mmHg). No difference was observed between LSS and HSS recipients. Mirroring the blood pressure, rats receiving the transfer developed greater renal damage assessed by albuminuria (+140mg/day, p<0.001) and outer medullary protein casting (p<0.05). To determine whether T cells contributed to the renal damage observed, we used flow cytometry to assess renal immune cell infiltration. Not only were the transferred cells able to infiltrate into the kidney, these T cells were more than 92% CD4+ cells. Not one phenotype showed any significant difference between those receiving splenocytes from low‐salt or high‐salt fed donors. These studies provide the first evidence that genetic salt‐sensitivity can be enhanced using adoptive immune cell transfer. Furthermore, we have documented that CD4+ cells infiltrate into the kidney which is accompanied by increased renal damage. Lastly, we note that the amount of salt in the diet of the donors does not affect the SS phenotypes of the recipients.Support or Funding InformationF31HL144084, HL116264, and HL137748

Adoptive donor immunity protects against resolved hepatitis B virus reactivation after allogeneic haematopoietic stem cell transplantation in the world's largest retrospective cohort study.

Reactivation of hepatitis B virus (HBV) by reverse seroconversion (HBV-RS) after allogeneic haematopoietic stem cell transplantation (allo-HSCT) can occur in patients with resolved HBV infection (rHBV, defined as negative HBV surface antigen [HBsAg] and positive HBV core antibody), and may cause fatal hepatitis. To explore the risk factors, we retrospectively identified 817 consecutive patients who underwent allo-HSCT from 2005 to 2016 in this largest single centre cohort from National Taiwan Univerisity Hospital. Transplants using donors or recipients positive for HBsAg or HBV DNA were excluded, leaving 445 rHBV patients for analysis. The 3- and 5-year cumulative incidence of HBV-RS after allo-HSCT was 8·7% and 10·5%, respectively, at a median 16months after allo-HSCT. All had concurrent HBV reactivation. HBV flares developed in 19% of HBV-RS cases, but none experienced hepatic failure. Neither did it impact non-relapse mortality or overall survival. Multivariate analysis revealed that patients with donor lacking hepatitis B surface antibody and extensive chronic graft-versus-host disease (cGVHD) have the highest risk for HBV-RS, with 5-year incidence of 24·2%. In conclusion, adoptive immunity transfer from the donor seems to have protective effects against HBV-RS, which may alter future donor selection algorithms, and combined with extensive cGVHD provides a good target for risk-adaptive HBV prophylaxis.

Novel Approaches to Improve the Efficacy of Immuno-Radiotherapy.

Radiotherapy (RT) has been applied for decades as a treatment modality in the management of various types of cancer. Ionizing radiation induces tumor cell death, which in turn can either elicit protective anti-tumor immune responses or immunosuppression in the tumor micromilieu that contributes to local tumor recurrence. Immunosuppression is frequently accompanied by the attraction of immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs), M2 tumor-associated macrophages (TAMs), T regulatory cells (Tregs), N2 neutrophils, and by the release of immunosuppressive cytokines (TGF-β, IL-10) and chemokines. Immune checkpoint pathways, particularly of the PD-1/PD-L1 axis, have been determined as key regulators of cancer immune escape. While IFN-dependent upregulation of PD-L1 has been extensively investigated, up-to-date studies indicated the importance of DNA damage signaling in the regulation of PD-L1 expression following RT. DNA damage dependent PD-L1 expression is upregulated by ATM/ATR/Chk1 kinase activities and cGAS/STING-dependent pathway, proving the role of DNA damage signaling in PD-L1 induced expression. Checkpoint blockade immunotherapies (i.e., application of anti-PD-1 and anti-PD-L1 antibodies) combined with RT were shown to significantly improve the objective response rates in therapy of various primary and metastatic malignancies. Further improvements in the therapeutic potential of RT are based on combinations of RT with other immunotherapeutic approaches including vaccines, cytokines and cytokine inducers, and an adoptive immune cell transfer (DCs, NK cells, T cells). In the current review we provide immunological rationale for a combination of RT with various immunotherapies as well as analysis of the emerging preclinical evidences for these therapies.

Loss of HDAC11 Promotes Myeloid-Derived Suppressor Cells Inhibition of T Cell Function in a Murine Lymphoma Microenvironment

Myeloid-derived suppressor cells constitute a heterogeneous population of immature myeloid cells derived from bone marrow that negatively regulate both innate and adaptive immunity in the tumor microenvironment. Previous work in our lab had demonstrated that MDSCs lacking histone deacetylase 11 (HDAC11) displayed an increase in suppressive activity against IFN-γ producing CD8+ T cells. The upregulated suppressive activity of HDAC11KO MDSCs was associated with a more aggressive tumor growth pattern when compared with wild type control mice.Mechanistically we have found that tumor infiltrated HDAC11KO MDSCs isolated from lymphoma-bearing mice displayed up-regulation of expression and enzymatic activity of arginase 1 and Nos2, two enzymes that are crucial in regulating MDSCs suppressive function, when compare with wild type MDSCs. However, both arginase activity and NO production were at a similar level in the wild type and HDAC11KO MDSCs isolated from spleens of the same lymphoma-bearing mice. This finding suggests that HDAC11KO MDSCs are more suppressive within the tumor microenvironment. Moreover, the aberrant enzymatic activities of Arg1 and Nos2 in HDAC11KO MDSCs correlate with over-expression of the lineage-specific transcription factor C/EBPβ, which has been previously shown to be essential for the differentiation of functional MDSCs. Furthermore, ChIP analysis confirmed that HDAC11 is recruited to the C/EBPβ gene promoter where exerts a negative regulatory effect upon gene transcription.Unlike MDSC's in which absence of HDAC11 is associated with a suppressive phenotype, T-cell lacking HDAC11 are hyper-reactive and endowed with strong antitumor activity. To assess which phenotype will be the dominant one in vivo, we performed adoptive immune cell transfer experiments of both MDSC and/or T-cells from either wild type or HDAC11 KO mice into C57BL/6 lymphoma-bearing animals. The transfer of HDAC11KO MDSCs was able to eliminate, at least partially, the anti-tumor effect elicited by the HDAC11KO T cells in the lymphoma microenvironmentTaken together, we have uncovered a previously unknown role for HDAC11 as a transcriptional regulator of MDSCs phenotype and function in a murine lymphoma model. A better understanding of this novel role of HDAC11 in myeloid biology will lead to targeted epigenetic therapies to manipulate the suppressive effect of these immunoregulatory cells in vivo. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4967: HDAC11 function as a transcriptional regulator in immature myeloid cells to myeloid-derived suppressor cells transition

Abstract In normal myelopoiesis, immature myeloid cells (IMCs) differentiate into macrophages, neutrophils or dendritic cells, a process that is tightly controlled by transcription factors and epigenetic regulators. However, under tumor burden, IMCs differentiate into myeloid derived suppressor cells (MDSCs) and with subsequent up-regulation of immune suppressive factors and a pro-tumor effect. In prior studies, we found that MDSCs from HDAC11 KO mice displayed an increased T-cell suppressive activity that was associated with a more aggressive tumor growth as compared to MDSCs from wild type control mice. Unlike MDSC's in which absence of HDAC11 is associated with a suppressive phenotype, T-cell lacking HDAC11 are hyper-reactive and endowed with strong antitumor activity. To assess which phenotype will be the dominant one in vivo, we performed adoptive immune cell transfer experiments of MDSC and/or T-cells from HDAC11 KO mice into C57BL/6 tumor-bearing mice. The transfer of HDAC11KO MDSCs was able to eliminate, at least partially, the anti-tumor effect elicited by the adoptive transfer of HDAC11KO T cells. Mechanistically we have found that MDSCs lacking HDAC11 displayed up-regulation of expression and enzymatic activity of arginase 1 and Nos2, two enzymes that are crucial in regulating MDSCs suppressive function. The aberrant enzymatic activities of Arg1 and Nos2 in HDAC11KO MDSCs correlate with over-expression of the lineage-specific transcription factor C/EBPβ, which has been shown to be essential for the differentiation of functional MDSCs. Furthermore, ChIP analysis confirmed that HDAC11 is recruited to the C/EBPβ gene promoter where exerts a negative regulatory effect upon gene transcription. Taken together, we have uncovered a previously unknown role for HDAC11 as a transcriptional regulator of MDSCs function. A better understanding of this novel role of HDAC11 in myeloid biology will lead to targeted epigenetic therapies to manipulate the suppressive effect of these immunoregulatory cells. Citation Format: Jie Chen, Fengdong Cheng, Eva Sahakian, John Powers, Zi Wang, Alejandro Villagra, Javier Pinilla-Ibarz, Eduardo M. Sotomayor. HDAC11 function as a transcriptional regulator in immature myeloid cells to myeloid-derived suppressor cells transition [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 4967.

Cytomegalovirus retinitis in the post-cART era.

To review the epidemiology, diagnosis, and management of cytomegalovirus retinitis (CMVR) in the post-combined antiretroviral era (cART) era. Although cART has dramatically reduced CMVR incidence and morbidity in the HIV population, CMVR continues to cause significant vision loss in both HIV and non-HIV patients, especially amongst patients without immune reconstitution. Advances in imaging including ultra-widefield fundus and autofluorescence imaging, optical coherence tomography, and adaptive optics may reflect CMVR activity; however, the diagnosis remains a clinical one. There have been minimal advances in therapy, with several agents no longer available due to market concerns. Despite reduced incidence and morbidity in the post-cART HIV population, CMVR continues to cause vision loss amongst HIV and non-HIV patients. Diagnosis remains primarily clinical, and therapy centers upon immune reconstitution along with systemic and/or intravitreal antivirals. Further studies are necessary to determine whether advanced imaging can influence management, and whether novel antiviral agents or adoptive immune transfer have a role in treatment of drug-resistance CMVR.

Adoptive transfer of immunity against red mouth disease in rainbow trout (Oncorhynchus mykiss)

Enteric Red Mouth Disease (ERM) caused by Yersinia ruckeri is among the bacterial diseases in rainbow trout aquaculture that can be controlled by registered vaccines and part of these vaccines can be delivered by immersion which is the most convenient way of application inducing good antibody responses and ensuring decreased handling costs. However, the mechanisms by which fish are protected against Yersinia ruckeri are unclear. To shed light on this question we have bath vaccinated clonal rainbow trout donors by using a commercially available vaccine, and leukocytes have been isolated from vaccinated donors followed by adoptive cell transfer to naive fish recipients of the same trout clone. Control recipients were injected with same numbers of leukocytes from non-vaccinated clonal donors. Transfer between genetically identical donors and recipients ensured that immune cells were not rejected by the recipients. After transfer, both groups of recipients were bath infected with a virulent field strain of Yersinia ruckeri. While rainbow trout that have received leukocytes from vaccinated donors showed no mortality, less than 40% of control recipients survived the challenge. Flow cytometry using leukocytes from recipient survivors revealed that the percentage of IgM B cells was considerably higher in recipients that were injected with leukocytes from vaccinated donors when compared with control recipients, while no differences in the percentages of CD8 cells among groups were recorded. Being the first report on adoptive cellular transfer of immunity in a bacterial infection in fish these data suggest that immunity was rather transferred by IgM B cells, but not by CD8 cytotoxic T cells. . It can be further suggested that bath vaccination has caused an effective systemic immune response since protection could be transferred by non-mucosal leukocytes.

New Strategies in Sarcoma: Linking Genomic and Immunotherapy Approaches to Molecular Subtype.

There are more than 100 sarcoma subtypes, each uncommon and diagnostically challenging. Conventional chemotherapy has little benefit for most soft-tissue sarcomas; new treatment strategies are needed. Multiple recent genomic studies have provided detailed insights into sarcoma biology, including more accurate classification by molecular subtype, identification of recurrent mutations in oncogenic pathways, and evidence of epigenetic dysregulation. Advances in immunotherapy (adoptive immune cell transfer, tumor vaccine strategies, and immune checkpoint inhibition) have also provided a better understanding of how immuno-oncology might best be applied to sarcoma treatment, including connections to oncogenic pathways that may support combination strategies with conventional and targeted therapies. In this article, we review the latest sarcoma genomic studies and immuno-oncology developments and discuss how the findings suggest potential strategies to improve diagnosis and treatment across multiple sarcoma subtypes.

Abstract B49: Metabolic stress within tumors lead to exhaustion of melanoma antigen-specific CD8+ T cells

Abstract The 5-year survival rate of late-stage melanoma patients is less than 5%. Adoptive immune transfer has demonstrated the potency of melanoma-associated antigen (MAA)-specific T cells in inducing clinical responses. Nevertheless, cancer vaccines against melanoma that aim to induce such T cells have largely been ineffective in patients with advanced disease. One major barrier of cancer vaccine efficacy is immunoinhibition within the tumor microenvironment (TME). Vaccine-induced MAA-specific tumor infiltrating T cells (TILs) commonly show signs of T cell exhaustion within TME by upregulating co-inhibitory molecules combined with loss of functions. This has traditionally been viewed as the result of chronic antigen stimulation. However, our data show that TILs directed to an antigen that is not expressed by the tumor also become exhausted, which led us to hypothesize that exhaustion of TILs is linked to metabolic stress within the TME. Upon activation the energy production of T cells switches from oxidative phosphorylation (OXPHOS) to glycolysis, which requires enhanced uptake of glucose for ATP production. Glycolysis is also used by tumor cells, which may lead to glucose deprivation within the TME, forcing MAA-specific TILs to rely on OXPHOS. OXPHOS requires oxygen, which due to lack of blood supply can also be limiting within TME. TILs therefore face dual metabolic jeopardy, i.e., lack of glucose and oxygen, which may affect their functions and thereby impair the efficacy of cancer vaccines. Using a B16 mouse melanoma model and an adenoviral vector based polyepitope melanoma vaccine, we tested if vaccine-induced TILs experience metabolic stress within the TME and whether this contributes to T cell exhaustion. Our results indicate that tumors recruit vaccine-induced tyrosinase-related protein (Trp)-1-specific CD8+ T cells, which proliferate within TME, upregulate co-inhibitory markers and become functionally impaired. Activated CD8+ T cells directed to an unrelated antigen expressed by another vaccine were also recruited to the tumors; although they fail to proliferation they also increased co-inhibitors programmed death (PD)-1 and lymphocyte activation gene (LAG)-3 expression with downregulation of T-bet, a critical transcription factor regulating T cell effector functions. Comparing markers of mitochondrial functions and oxidative deficiencies between TILs and CD8+ T cells stimulated in vitro under conditions producing energy through OXPHOS or glycolysis and under normal or reduced oxygen levels, we showed that activated CD8+ TILs undergo oxidative stress evidenced by loss of mitochondrial membrane potential (MMP), increases of mitochondrial reactive oxygen species (MROS) and enhanced levels of hypoxia inducible factor 1 subunit α (HIF-1α) and its downstream target glucose transporter (Glut)1. This metabolic profile was largely mirrored by T cells activated in vitro under hypoxia. Furthermore, our data demonstrated links between the T cells metabolic stress and their exhaustion status. Enhanced expression of PD-1 was linked to lack of glucose as cells cultured in galactose significantly increased PD-1 compared to cells with access to glucose. Increased expression of LAG-3 was most pronounced on cells cultured under hypoxia or in medium with drug that stabilizes HIF-1α. Knocking down HIF-1α in activated CD8+ T cells cultured under hypoxia significantly reduced their LAG-3 expression and increased their T-bet levels, implying a direct impact of hypoxia-induced HIF-1α on the expression of co-inhibitor LAG-3 and overall exhaustion status of T cells. In summary, our data indicate that within the TME metabolic stress due to lack of glucose and oxygen rather than sustained activation causes T cell exhaustion. These findings suggest that lifting the metabolic stress of MAA-specific TILs may improve the therapeutic efficacy of cancer vaccines. Citation Format: Ying Zhang, Xiangyang Zhou, Raj Kurupati, Flavia Filisio, Abeer Hudaihed, Wynetta Giles-Davis, Hildegund CJ Ertl. Metabolic stress within tumors lead to exhaustion of melanoma antigen-specific CD8+ T cells. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B49.

Analysis of adverse events following the treatment of autologous cytokine-induced killer cells for adoptive immunotherapy in malignant tumour sufferers

Background: Adoptive immune cell transfer such as cytokine-induced killer (CIK) cells has become an important adjuvant approach in patients with tumours.Objectives: The aim of this study was to analyse the adverse events (AEs) that occur during the transfusion of autologous CIK cells and to identify the risk factors associated with these AEs.Methods: Cell infusion-associated AEs were evaluated according to National Cancer Institute Common Terminology Criteria. Analysis was performed from a single-centre data on 893 malignant tumour patients who received a total of 4088 transfusions from March 2008 to October 2013.Results: A total of 215/4088 (5.26%) transfusion cases from 893 patients presented with AEs (Grade 1 – 4); 204/215 (94.88%) were Grade 1 – 2, and 156/215 (72.56%) occurred within 24 h. The most common AEs were fever (0.88%), chills (0.56%) and fatigue (0.49%). The rare but severe AEs included anaphylactoid purpura, tumour lysis syndrome, anaphylactic shock, arthralgia. No transfusion-associated death was noticed. The mainly relative risk factors for AEs included transfer cycles and clinical stages.Conclusion: This study is a large-sample AEs research, to our knowledge, relative to immune cell transfusion from a single centre data analysis, revealing that autologous CIK cell therapy represents a fairly safe and well-tolerated treatment modality for malignant tumour patients, even rare severe, but not lethal AEs were observed in few patients.

Ex vivo assessment of cellular immune function-applications in patient care and clinical studies.

Cellular ex vivo assays have a broad range of applications in patient care and clinical studies, especially when they are standardized and highly sensitive. As compared to analyses by molecular genetics such as the single nucleotide polymorphism (SNP) testing, they are usually more global. These assays partly mimic the in vivo situation, relying on a complex interaction of various immune cells. For example, they can be used to determine modulation of alloresponses by treatment or underlying disease, diagnose and quantify primary and secondary cellular immunodeficiency, follow-up vaccination responses, measure adoptive transfer of virus-specific immunity via hematopoietic stem cell or liver transplantation, assess allergy, antimicrobial immunity and also rare effector/memory cells directed against tumor antigens. This review will first shortly describe various cellular in vitro methods and then present applications, summarizing some own studies performed within the last 18 years.

Immunosuppressive Drugs Modulate the Replication of Hepatitis B Virus (HBV) in a Hydrodynamic Injection Mouse Model

Hepatitis B virus (HBV) reactivation and recurrence are common in patients under immunosuppression and can be controlled by hepatitis B immunoglobulin, antivirals, and hepatitis B vaccine. However, the detailed analysis of HBV infection under immunosuppression is essential for the prophylaxis and therapy for HBV reactivation and recurrence. In this study, HBV replication and T cell responses were analyzed in a HBV-transfected mouse model under immunosuppressive therapy. During the treatment, HBV replication was at a high level in mice treated with dexamethasone, cyclosporine, and cyclophosphamide, whereas was terminated in mice treated with mycophenolate mofetil. After the withdrawal, HBV replication was at low or high levels in the dexamethasone-treated mice or in both cyclosporine- and cyclophosphamide-treated mice. The early withdrawal of cyclosporine allowed the recovery of suppressed T cell responses and led to subsequent HBV clearance, while the adoptive immune transfer to the mice with HBV persistence led to HBV suppression. Taken together, long-term HBV persistence under immunosuppression depends on the immunosuppressive drugs used and on the treatment duration and is mediated by the suppressed intrahepatic CD8 T cell response. These data may be helpful for individualized immunosuppressive therapy in patients with high risk of HBV reactivation and recurrence, and the mouse system is suitable for studying HBV reactivation and recurrence under immunosuppression.

Immune therapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide. Currently, there are no treatment options available for a large number of these patients. One of the mechanisms that may contribute to tumor growth is the lack of an effective immune response toward viral antigens or other tumor-associated antigens (TAAs). Immunotherapy has been tested as a potential therapeutic option for these patients. Several methods of immune modulation for augmenting antitumor immunity are being explored and have been shown to be effective in suppressing HCC growth in animal models. Activation of HCC-specific response can be accomplished by targeting hepatitis B or C viral antigens, alpha-fetoprotein, or other TAAs. This review summarizes part of the recent data on the use of adoptive transfer of immunity against viral antigens, oral immune modulation against TAAs, and the use of pulsed innate immune cells and gut adjuvants for the suppression of HCC; it reviews some additional new immunotherapeutic approaches.

ANTI-TUMOR RESPONSES INDUCED BY LASER IRRADIATION AND IMMUNOLOGICAL STIMULATION USING A MOUSE MAMMARY TUMOR MODEL

Anti-tumor immunological response induced by local intervention is ideal for treatment of metastatic tumors. Laser immunotherapy was developed to synergize photothermal interaction with immunological stimulation for cancer treatment. Using an infrared laser, indocyanine green (ICG, as a light absorbing agent), and glycated chitosan (GC, as an immunostimulant), laser immunotherapy has resulted in tumor suppression and anti-tumor responses in pre-clinical as well as clinical studies. To further understand the mechanism of laser immunotherapy, the effects of laser and GC treatment without specific enhancement of laser absorption were studied. Passive adoptive immunity transfer was performed using splenocytes as immune cells. Spleen cells harvested from tumor-bearing mice treated by laser + GC provided 60% immunity in naive recipients. Furthermore, cytotoxicity and TNF-α secretion by splenocytes from treated mice also indicated that laser + G induced immunity was tumor-specific. The high level of infiltrating T cells in tumors after laser + GC treatment further confirmed a specific anti-tumor immune response. Therefore, laser + GC could prove to be a promising selective local treatment modality that induces a systemic anti-tumor response, with appropriate laser parameters and GC doses.