Donor Peripheral Blood Lymphocytes Research Articles

IMMU-07. HUMANIZED BI-SPECIFIC ANTIBODY SHOWS POTENT ACTIVITY IN PATIENT-DERIVED XENOGRAFT MODELS OF GLIOBLASTOMA

Abstract BACKGROUND Advancements in bioengineering have allowed bispecific Abs to develop with therapeutic applications in immuno-oncology. Here we describe a fully humanized bispecific t cell engager (BiTE) that binds the GBM-associated antigen interleukin 13 receptor alpha 2 (IL13Rα2) and the T Cell receptor CD3 epsilon (CD3ε) to elicit an anti-tumor immune response in patient-derived models of GBM. METHODS In vitro, BiTE-mediated activation of T Cells was assessed via flow cytometry as measurements of T cell activation markers CD25 and CD69 and cytokines IFNγ and TNFα. BiTE-directed T cell-mediated cytotoxicity was assessed in GBM 6 and GBM12patient-derived xenograft lines. In vivo, GBM6 patient-derived xenograft cells were intracranially implanted in NSG-MHC I/II DKO mice, reconstituted with HLA-matched donor peripheral blood lymphocytes (PBMC), and subsequently treated with 2.5 mg/kg BiTE via i.p. injection x 4 days/week of therapeutic BiTE for three weeks. Animals were followed for survival, and the brains of long-term survivors were analyzed for residual disease and T cell infiltration. RESULTS In vitro, BiTE potently activated T cells as determined by a high expression of CD25 and CD69 and the production of TNFα and IFNγ. BiTE also directed T cell-mediated cytotoxicity against GBM cells in a dose-dependent manner, with significant activity still seen in the low nanogram range. In vivo, BiTE significantly extended the survival of tumor-bearing animals in GBM6patient-derived xenograft models. Histological analysis of long-term surviving mice shows complete absence of tumor and robust presence of CD8+ T cells localized to the original site of tumor implantation. CONCLUSIONS Our data demonstrate the efficacy of humanized BiTE in patient-derived GBM models, and our results support the investigation of this novel therapeutic in a clinical trial setting.FUNDING: R01 NS122395-01 and R01 NS095642-01

Abstract 127: Single cell mapping of tumor infiltrating lymphocytes enables neoantigen-reactive T cell identification in metastatic human cancer

Abstract The adoptive transfer of tumor-infiltrating lymphocytes (TILs) expanded in vitro can mediate regressions of established tumors, yet only a minority of intratumoral T cells are reactive to cancer antigens while the majority represent bystander cells. This complexity underpins the need to phenotypically characterize T cells infiltrating the tumor microenvironment. In this study, we performed single-cell RNA and T cell receptor (TCR) sequencing (scRNA/TCR-seq) on over 45,000 T cells isolated from ten archival metastatic tumor samples whose primary histologies included colon, rectal, breast, and melanoma. By transcriptomic clustering of the T cells and mapping TCR identity data onto these blueprints, we observed a diversity of T cell phenotypes spanning from memory-like to dysfunctional effector-like, with clonal expansion occurring more widely in the effector-like cells. Through sequencing of peripheral blood lymphocytes (PBL) obtained at the time of tumor resection, we found T cell clusters that were either tumor enriched or distinctly PBL enriched, potentially representing recent tumor immigrants. Prior knowledge of 30 CD4 and CD8 tumor neoantigen-reactive TCRs (NeoTCRs) from eight patients allowed us to project these NeoTCRs onto the TIL transcriptomic map where we observed 380 total T cells bearing these NeoTCRs. The majority (>85%) of NeoTCRs were expressed by T cells within 2 clusters, one CD4+ and one CD8+, marked by expression of CXCL13, ENTPD1 (CD39), TOX, and PDCD1 (PD-1), indicating a dysfunctional state. We reasoned that other dominant T cell clonotypes within the NeoTCR clusters could contain additional novel NeoTCR clonotypes. We expressed these predicted NeoTCRs in donor PBL and screened them in vitro with antigen presenting cells (APCs) pulsed with peptides or tandem minigenes encoded by patient-specific tumor mutations, yielding 24 new NeoTCR clones (251 cells), which included neoantigen reactivities undetected in the same patients' fragment TIL screens. Using a NeoTCR signature score derived from the archival samples, three prospective patients' TIL were analyzed by scRNA/TCRseq and clusters scored according to their NeoTCR signature. TCRs reconstructed from these clusters and tested against patients' tumor-specific reagents yielded novel CD4 and CD8 NeoTCRs. Altogether, this study enabled successful enrichment and detection of tumor-specific NeoTCRs in the sequenced TIL of 12/12 patients for whom reactivity was either previously assessed or prospectively identified. Deconvolution of NeoTCRs from bystander TCRs within the tumor-immune microenvironment represents an important step in the development of personalized immunotherapy, as prospective NeoTCR isolation based on transcriptional phenotype of TIL will allow for rapid therapeutic development in the form of T cells expressing these tumor-specific TCRs. Citation Format: Frank J. Lowery, Sri Krishna, Rami Yossef, Praveen D. Chatani, Neilesh B. Parikh, Yong-Chen Lu, Nikolaos Zacharakis, Noam Levin, Ken-ichi Hanada, James C. Yang, Paul F. Robbins, Maria R. Parkhurst, Steven A. Rosenberg. Single cell mapping of tumor infiltrating lymphocytes enables neoantigen-reactive T cell identification in metastatic human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 127.

IL-10 Restores MHC Class I Expression and Interferes With Immunity in Papillary Thyroid Cancer With Hashimoto Thyroiditis.

The incidence of papillary thyroid cancer (PTC) with concomitant Hashimoto thyroiditis (HT) is increasing. Interleukin (IL)-10 is a cytokine previously reported to be elevated in this condition. Evidence from multiple human malignancies showed IL-10 participated in tumor immunity and exhibited therapeutic potential. The aim of this study is to investigate whether IL-10 interferes with tumor immunity in PTC with concomitant HT. Expression of IL-10 and major histocompatibility complex (MHC) class Ⅰ were compared with PTC tissues with or without concomitant HT. PTC cell lines K1 and TPC-1 were stimulated with IL-10 and analyzed for MHC class Ⅰ expression afterward. T-cell activation, production of IL-2 and interferon (IFN)-γ and programmed death-1 (PD-1) expression were assessed by coculture of donor peripheral blood lymphocytes (PBLs) with IL-10-pretreated PTC cells. Programmed death-ligand 1 (PD-L1) expression was measured in PTC tissues and IL-10-pretreated cells of K1 and TPC-1. Increased levels of IL-10 and MHC class Ⅰ were observed in PTC with concomitant HT. IL-10 stimulation increased MHC class Ⅰ expression of PTC cells in vitro. Coculture of PBLs with IL-10-pretreated PTC cells enhanced T-cell activation (% cluster of differentiation [CD]25+ of CD3+T cells) and increased IL-2 production along with decreased IFN-γ secretion and PD-1 expression. Reduced PD-L1 expression was seen in PTC + HT tissue samples and IL-10-stimulated PTC cell lines. Elevated IL-10 expression in PTC with concomitant HT restores MHC class Ⅰ expression and interferes with tumor immunity. The potential mechanism of IL-10 in tumor immunity needs further investigation.

IL-17A increases MHC class I expression and promotes T cell activation in papillary thyroid cancer patients with coexistent Hashimoto\u2019s thyroiditis

BackgroundThe incidence of coexisting papillary thyroid cancer (PTC) and Hashimoto’s thyroiditis (HT) is increasing. The impact of HT on PTC prognosis and its possible mechanism remains controversial. Interleukin-17A (IL-17A) has been reported to participate in the pathogenesis of multiple autoimmune diseases and cancers. The aim of this study is to investigate the role of IL-17A in PTC with coexistent HT and evaluate the changes in tumor antigenicity.MethodsExpression of IL-17A and major histocompatibility complex (MHC) class I molecules were compared on PTC tissue samples with or without HT. PTC cell lines K1 and TPC-1 were stimulated with IL-17A and analyzed for MHC class I expression afterwards. Cluster of differentiation (CD) 8+T cell activation, production of Interleukin-2 (IL-2) and Interferon-gamma (IFN-γ) as well as the programmed death-1 (PD-1) expression on lymphocytes were assessed by coculture of donor peripheral blood lymphocytes (PBLs) with IL-17A pretreated PTC cells.ResultsElevated IL-17A and MHC class I expression were observed in PTC tissue samples with coexistent HT. Stimulation of PTC cells with IL-17A effectively increased MHC class I expression in vitro. Coculture of PBLs with IL-17A pretreated PTC cells resulted in enhanced T cell activation (%CD25+ of CD3+T cells) and increased IL-2 production along with decreased IFN-γ secretion and PD-1 expression of the lymphocytes.ConclusionsPapillary thyroid cancer with coexisting Hashimoto’s thyroiditis presents elevated MHC class I expression, which may be the result of IL-17A secretion. T cell activation is enhanced in vitro by IL-17A and may provide future utility in PTC immunotherapy.

Abstract A23: Neuropilin-1 expression on regulatory T cells in ovarian cancer

Abstract Introduction: Regulatory T cells (Treg) are a subpopulation of CD4+ T cells that suppress autoimmune responses, but also prevent clearance of tumors and chronic viral infections. In ovarian cancer, a higher frequency of tumor-infiltrating Treg is associated with poor prognosis, but insights into the mechanisms governing the survival and suppressive activity of Treg in this context are limited. Our group has recently described a signaling axis through neurophilin-1 (NRP1) on Treg in murine models of cancer that promotes the survival and suppressive function of Treg. Genetic knockout of NRP1 on murine Treg leads to reduced tumor growth and increased survival, underscoring the importance of NRP1+ Treg in suppressing antitumor immunity. Given the importance of NRP1+ Treg in murine cancer models, we sought to explore the expression pattern of NRP1 on tumor-infiltrating Treg from patients with ovarian cancer. Materials and Methods: Ovarian tumors were obtained through the health sciences tissue bank at the University of Pittsburgh, and ovarian ascites fluid was obtained through Magee Women’s Research Institute. Peripheral blood was obtained from healthy donors through the Red Cross. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation, and tumor-infiltrating lymphocytes were isolated by first mechanically disrupting the tumor, followed by treatment for 15 minutes at 37°C with 50 ug/mL Liberase DL. For flow cytometric analysis of Treg, cells from blood and tumors were first stained for surface markers, followed by live/dead discrimination, fixation and permeabilization, and staining for intracellular markers. Cells were analyzed on an LSR Fortessa at the Hillman Cancer Center Flow Core. For functional assays, Treg were selected from ascites fluid and assayed for their ability to suppressive naïve CD8+ T cells ex vivo. Wilcoxon rank sum tests were used to assess differences between groups, and a two-sided alpha less than 5% was considered significant. Results: A total of 12 healthy donors and 20 ovarian cancer patients were studied. Intracellular and surface NRP1 expression was assessed by flow cytometry on CD4+CD25+FOXP3+ Treg from healthy donor peripheral blood and tumor-infiltrating lymphocytes from either ovarian cancer tumors or ascites fluid from patients with ovarian cancer. Intracellular NRP1 was expressed on a median of 1.8% (interquartile range [IQR]: 0.69% to 4.8%) of Treg from healthy donors compared with a median of 66% (IQR: 28% to 90%; p<0.001) and 82% (IQR: 38% to 99%; p=0.004) of Treg from ovarian tumor-infiltrating lymphocytes (TIL) and ascites, respectively. Surface NRP1 was detected on 0.1% (IQR: 0% to 1%) of Treg in PBMC from healthy donors, compared with 11.1% (IQR: 1.1% to 60%; p=0.01) on ovarian TIL and 82% (IQR: 14% to 82%; p=0.0015) on ascites. Comparing expression of intracellular NRP1 on Treg from N=2 benign ovarian tissues versus N=14 malignant ovarian tumors, there was a trend towards increased NRP1 expression (median 9.3% versus 54.5%, respectively; p=0.044). Finally, Treg isolated from ascites fluid were capable of suppressing CD8+ T cells ex vivo proliferation in a dose-dependent manner. Conclusions and Future Directions: Both intracellular and surface NRP1 are expressed more frequently on tumor-infiltrating Treg and Treg from ascites fluid compared to Treg from healthy donor peripheral blood. Although the number of patients currently included in this study is small, there was also evidence that intracellular NRP1 expression on Treg was higher on malignant compared with benign tissue. Additionally, Treg from ascites fluid were capable of suppressing CD8 cells ex vivo. Future studies will evaluate whether blockade of NRP1 on human Treg limits either the survival or suppressive capacity of Treg. Note: This abstract was not presented at the conference. Citation Format: Anthony R. Cillo, Tullia Bruno, Francesmary Modugno, Robert Edwards, Dario Vignali. Neuropilin-1 expression on regulatory T cells in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A23.

Abstract B044: Patient-specific immunotherapy through TCR gene transfer

Abstract Adoptive cell transfer (ACT) of autologous ex vivo expanded tumor-infiltrating lymphocytes has shown to mediate significant tumor regression in metastatic melanoma. This finding has demonstrated the capacity of endogenous T cells to mount an immune response against and eliminate cancer cells. Two clinically relevant approaches to adoptive cell transfer are ACT of expanded TIL and ACT of gene-modified peripheral blood lymphocytes (PBLs). The former approach consists of isolation of tumor-infiltrating lymphocytes from tumor biopsies, ex vivo expansion and re-infusion after treatment with a lymphodepleting regimen. In the latter, PBLs are engineered to express T-cell receptors (TCRs) recognizing common melanoma tumor antigens. While both approaches have shown different extents of clinical benefit, several limitations hamper clinical efficacy. T cell specificity, phenotype and differentiation state of ex vivo expanded TIL are largely unknown, while ACT of gene-modified lymphocytes is limited by the use of TCRs recognizing common tumor antigens, which can be differentially expressed between patients. Furthermore, the use of these TCRs is often limited to a handful of HLA types. These factors can lead to reduced therapeutic efficacy and applicability. Here we aim to compare the tumor control of T cells expressing ‘personalized’ tumor-reactive TCRs and expanded tumor-infiltrating lymphocytes after adoptive cell transfer in a xenograft mouse model. To this end, highly tumor-reactive T cell receptors are isolated from TIL on a per-patient basis. These TCRs are introduced into donor peripheral blood lymphocytes, forming a population of tumor-reactive cells tailored to the patient tumor. These cells are subsequently tested for efficacy in a xenograft mouse model bearing autologous (patient-derived) tumor. Using the outlined approach, we aim to identify determinants of therapy efficacy. Preliminary results have shown dramatic tumor regression after ACT of patient-specific gene-modified T cells, leading to complete remission of tumors in all treated mice. In contrast, tumors of mice treated with TIL or control T cells did not show significant reduction of tumor burden, when compared to untreated controls. Citation Format: Lorenzo F. Fanchi, Laura Bies, Ji-Ying Song, Carsten Linnemann, Ton Schumacher. Patient-specific immunotherapy through TCR gene transfer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B044.

Spectrum of produced GAG gene viral proteins in lymphocytes infected HIV-1 NL4-3 strains in vitro

Aim. To study spectrum of viral proteins secreted into the culture medium by the healthy donor peripheral blood lymphocytes infected by HIV-1 NL4-3 strain in vitro.Methods. We used a primary lymphocytes culture obtained from healthy donors. For synchronous infection of primary lymphocytes culture in vitro NL4-3 strain of human immunodeficiency virus (HIV) (NIH AIDS Research & Reference Reagents Program, USA) was used. The HIV-1 titer in the supernatants was determined by enzyme immunoassay (EIA p24gag Coulter). In addition, electrophoresis of clarified culture medium proteins in 12.5% polyacrylamide gel under denaturing conditions followed by electron transport onto a nitrocellulose membrane; immunoblotting for detection of virus-specific proteins using positive control serum to HIV-1 («Bio-Rad») were performed; protein markers kits LMW 94-14.4 kD, HMW 212-53 kD («Pharmacia Biotech») were used to determine the seroactive fractions molecular weight.Results.. HIV antigens spectrum in the culture medium was studied on the 4th, 9th and 13th days after lymphocyte culture infection with HIV. Within 13 days of virus reproduction both non-structural and structural proteins of HIV-1 gag gene are detected in the culture medium, spectrum and quantitative range of which varies with time. After 4 hours of HIV reproduction in primary lymphocytes culture in vitro large number of gag gene nonstructural proteins (p40, p55) are exported from cells into culture medium. Further, gag gene non-structural proteins may be cleaved into the structural proteins (p24/25, R18) under the cellular proteases action, which is caused by the proteolytic enzymes accumulation in the culture medium.Conclusion. In HIV reproduction in primary lymphocytes cultures soluble forms of the HIV-1 gag gene proteins are secreted into culture medium; in the initial stages of the infection HIV proteins spectrum in the culture medium of lymphocytes activated with phytohemagglutinin in vitro consists of only the HIV-1 precursor proteins (p55, p40) and at a later stages - also of the gag gene viral capsid proteins (p24/25, R18).

Adoptive T Cell Therapy for Polyomaviral Infections and Related Diseases

Introduction: Polyomaviruses (PyV) are a family of DNA-viruses responsible for serious morbidity and mortality in immunocompromised individuals including those with HIV or receiving immunosuppressive treatments for transplant or other conditions. BK polyomavirus (BKV) causes hemorrhagic cystitis and nephropathy in stem cell and organ transplant recipients. JC polyomavirus (JCV) causes progressive multifocal encephalopathy (PML) - an often fatal complication in immunosuppressed transplant recipients and in patients treated with natalizumab for multiple sclerosis. Merkel Cell Carcinoma (MCC) is an aggressive skin cancer of elderly or immunocompromised patients caused by Merkel Cell polyomavirus (MCV). There are no established antiviral drugs targeting PyVs and therefore current strategy is limited to reversing immunosuppression where possible. There is strong evidence that PyV can be controlled through T-cell based immunity and adoptive T cell transfer can cure or prevent BKV and JCV viral diseases after stem cell transplants (SCT). Similarly,evidence exists for immune susceptibility of MCC to T cell-mediated immune attack. JCV, BKV, and MCV are closely related and display a high degree of homology in the large T (LT) and VP1 antigens at the protein sequence level. Therefore, generation of a single T cell product containing a polyclonal cross-reactive donor T cell repertoire recognizing antigens from multiple PyV is an attractive strategy with potential clinical application for adoptive immunotherapy to treat patients suffering from HC, nephropathy, PML, MCC, and possibly other PyV-related diseases.Methods: T cells recognizing LT and VP1 antigens from BKV, MCV, and JCV were generated from healthy donor peripheral blood lymphocytes in two weekly stimulations against autologous, mature dendritic cells (DCs) pulsed with relevant peptide libraries or lentivirally transduced to express full length proteins. The cultures were supplemented with cytokines IL-7 and IL-15 to promote memory formation, and IL-2 was added after the second stimulation to promote expansion. These ex vivo generated T cells were tested against autologous DC targets pulsed with the relevant or irrelevant peptide libraries. In addition, to more accurately assess their potential activity in vivo, the T cells were tested against autologous DC targets lentivirally transduced to express naturally-processed epitopes of BKV and MCV antigens and analyzed for their ability to secrete polyfunctional cytokines by flow cytometry.Results: Flow cytometry revealed polyfunctional activity of BKV-primed cells against cognate BK peptide libraries and antigen-expressing transduced DCs as well as JC peptide libraries and MCV-expressing DCs in the production of TNFa, IFNg, and IL2 in both CD4 and CD8 subsets upon stimulation. The lentivirally engineered antigen-expressing DCs proved to be effective stimulators in generating BKV and MCV-recognizing T cells. While a high degree of cross-reactivity was observed among LT antigens of each virus, cross-reactivity among VP1 antigens was lower and largely donor dependent. T cells separately generated against VP1 antigens from Ia and IV serotypes of BKV showed no significant difference in activity between targets, indicating high overlap in TCR recognition of both serotypes. The antigen-primed T cultures could be highly enriched for antigen-specific T cells by CD137 (4-1BB) sorting 24 hours following simulation. T cells generated against JCV peptide libraries and MCV antigen expressing DCs displayed similar polyfunctional cross-reactivity among antigens from the three polyomaviruses, thus establishing the feasibility of generating a single human polyomavirus-targeted T cell product capable of recognizing multiple viruses within the polyomaviradae family.Conclusions: We have demonstrated successful generation of polyclonal cross reactive T cell repertoires targeted against the LT or VP1 antigens from BKV, JCV or MCV. The LT antigen-specific T cells were highly cross reactive among these PyV. There is a high degree of cross reactivity among the VP1-specific T cells from different serotypes of BKV. These universal PyV-specific T cells could have potential clinical application in the treatment and prevention of PyV infections and related diseases. DisclosuresNo relevant conflicts of interest to declare.

Melanocortin 1 Receptor-derived peptides are efficiently recognized by cytotoxic T lymphocytes from melanoma patients

BackgroundMelanocortin 1 Receptor (MC1R) is expressed in a majority of melanoma biopsies and cell lines. We previously demonstrated that three hydrophobic low-affinity HLA-A2-restricted MC1R-derived peptides: MC1R291–298, MC1R244–252 and MC1R283–291 can elicit cytotoxic T-lymphocytes (CTL) responses from normal donor peripheral blood lymphocytes (PBL). Moreover, peptide-specific CTL recognized a panel of MHC-matched melanomas, demonstrating that human melanoma cell lines naturally present MC1R epitopes. However, the natural presence of MC1R-specific T cells in melanoma patient's tumour and blood remains unknown. MethodsThe presence of anti-MC1R specific CD8+ T cells was established in a population of melanoma-specific T cells derived from peripheral blood mononuclear cells (PBMC) and tumour-infiltrating lymphocytes (TIL) from HLA-A2+ melanoma patients. ResultsCTLs specific for the three MC1R-derived peptides that lysed allogeneic HLA-A2+MC1R+ melanomas were elicited from PBMC, demonstrating the existence of an anti-MC1R T cell repertoire in melanoma patients. Moreover, TILs also recognized MC1R epitopes and HLA-A2+ melanoma cell lines. Finally, HLA-A2/MC1R244-specific CD8+ T cell clones derived from TILs and a subset of MC1R291 specific TILs were identified using HLA-A2/MC1R tetramers. ConclusionOur results demonstrate that MC1R-derived peptides are common immunogenic epitopes for melanoma-specific CTLs and TILs, and may thus be useful for the development of anti-melanoma immunotherapy.

AIM1 and LINE-1 Epigenetic Aberrations in Tumor and Serum Relate to Melanoma Progression and Disease Outcome

Aberrations in the methylation status of non-coding genomic repeat DNA sequences and specific gene promoter region are important epigenetic events in melanoma progression. Promoter methylation status in LINE-1 and Absent in melanoma-1(AIM1;6q21) associated with melanoma progression and disease outcome was assessed. LINE-1 and AIM1 methylation status was assessed in paraffin-embedded archival tissues(PEAT)(n=133) and melanoma patients’ serum(n=56). LINE-1 U-Index(hypomethylation) and AIM1 were analyzed in microdissected melanoma PEAT sections. The LINE-1 U-Index of melanoma(n=100) was significantly higher than that of normal skin(n=14) and nevi(n=12)(P=0.0004). LINE-1 U-Index level was elevated with increasing AJCC stage(P<0.0001). AIM1 promoter hypermethylation was found in higher frequency(P=0.005) in metastatic melanoma(65%) than in primary melanomas(38%). When analyzed, high LINE-1 U-Index and/or AIM1 methylation in melanomas were associated with disease-free survival(DFS) and overall survival(OS) in Stage I/II patients (P=0.017, 0.027; respectively). In multivariate analysis, melanoma AIM1 methylation status was a significant prognostic factor of OS(P=0.032). Furthermore, serum unmethylated LINE-1 was at higher levels in both stage III(n=20) and stage IV(n=36) patients compared to healthy donors(n=14)(P=0.022). Circulating methylated AIM1 was detected in patients’ serum and was predictive of OS in Stage IV patients (P=0.009). LINE-1 hypomethylation and AIM1 hypermethylation have prognostic utility in both melanoma patients’ tumors and serum.

Human Tumor-Derived Exosomes Selectively Impair Lymphocyte Responses to Interleukin-2

Exosomes are nanometer-sized vesicles, secreted by normal and neoplastic cells. The outcome following interaction between the cellular immune system and cancer-derived exosomes is not well understood. Interleukin-2 (IL-2) is a key factor supporting expansion and differentiation of CTL and natural killer (NK) cells but can also support regulatory T cells and their suppressive functions. Our study examined whether tumor-derived exosomes could modify lymphocyte IL-2 responses. Proliferation of healthy donor peripheral blood lymphocytes in response to IL-2 was inhibited by tumor exosomes. In unfractionated lymphocytes, this effect was seen in all cell subsets. Separating CD4(+) T cells, CD8(+) T cells, and NK cells revealed that CD8(+) T-cell proliferation was not inhibited in the absence of CD4(+) T cells and that NK cell proliferation was only slightly impaired. Other exosome effects included selective impairment of IL-2-mediated CD25 up-regulation, affecting all but the CD3(+)CD8(-) T-cell subset. IL-2-induced Foxp3 expression by CD4(+)CD25(+) cells was not inhibited by tumor exosomes, and the suppressive function of CD4(+)CD25(+) T cells was enhanced by exosomes. In contrast, exosomes directly inhibited NK cell killing function in a T-cell-independent manner. Analysis of tumor exosomes revealed membrane-associated transforming growth factor beta(1) (TGFbeta(1)), which contributed to the antiproliferative effects, shown by using neutralizing TGFbeta(1)-specific antibody. The data show an exosome-mediated mechanism of skewing IL-2 responsiveness in favor of regulatory T cells and away from cytotoxic cells. This coordinated "double hit" to cellular immunity strongly implicates the role of exosomes in tumor immune evasion.

Marking of peripheral T-lymphocytes by retroviral transduction and transplantation of CD34 + cells in a canine X-linked severe combined immunodeficiency model

A retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) was used to mark and dynamically follow vector-expressing cells in the peripheral blood of bone marrow transplanted X-linked severe combined immunodeficient dogs. CD34 + cells isolated from young normal dogs were transduced, using a 2 day protocol, with an amphotropic retroviral vector that expressed enhanced green fluorescent protein (EGFP) and the canine common gamma chain (γc) cDNAs. Following transplantation of the transduced cells, normal donor peripheral blood lymphocytes (PBL) appeared by 1 month post-bone marrow transplant (BMT) and rescued three of five treated dogs from their lethal immunodeficiency. PCR and flow cytometric analysis of post-BMT PBL documented the peripheral EGFP expressing cells as CD3 + T cells, which varied from 0% to 28%. Sorting of EGFP + and EGFP − peripheral blood T cells from two dogs, followed by vector PCR analysis, showed no evidence of vector shutdown. EGFP expression in B cells or monocytes was not detected. These marking experiments demonstrate that the transduction protocol did not abolish the lymphoid engraftment capability of ex vivo transduced canine CD34 + cells and supports the potential utility of the MSCV retroviral vector for gene transfer to XSCID affected canine hematopoietic progenitor cells (HPC).

Follicular Lymphoma Infiltrating CD4+CD25+GITR+ Regulatory T-Cells Are Potent Suppressors of CD3/CD28 Co-Stimulated Autologous and Allogeneic CD8+CD25− and CD4+CD25− T-Cells.

Regulatory T-cells (Tregs) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor reactive effector T-cells. We demonstrate that follicular lymphoma (FL) T-cells are hypo-responsive to CD3/CD28 costimulation, as assessed by proliferation of CFSE (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester) labeled cells, with only 3.11% ± 2.38 and 2.26% ± 1.76 of the CD8+ and CD4+ T-cells proliferating upon stimulation, respectively (n=7). In contrast, both normal lymph node (NLN), and reactive lymph node (RLN, lymphoid hyperplasia) T-cells proliferate significantly in response to costimulation. Specifically, NLN CD8+ and CD4+ T-cells demonstrate 35.2% ± 31.1 and 18.1% ± 15.9 cells proliferating upon stimulation, respectively (n=7). Similarly, upon stimulation, RLN CD8+ and CD4+ T-cells demonstrate 40.6% ± 22.6 and 40.3% ± 30.3 cells proliferating, respectively (n=5). We identify a population of FL infiltrating CD4+CD25+GITR+ T-cells that are significantly overrepresented within FL, 9.86% ± 6.70 (n=11) of the CD4+ T-cells, as compared to that seen in NLN, 0.70% ± 0.29 (n=13), or RLN, 1.40% ± 1.04 (n=5). These cells actively suppress the proliferation of autologous nodal CD8+ and CD4+ T-cells after costimulation, as CD25+ magnetic bead depletion of these cells in vitro restores proliferation of the remaining CD25− T-cells. Specifically, proliferation of FL CD8+CD25− and CD4+CD25− T-cells increases to 24.05% ± 11.46 and 10.53% ± 6.47, respectively, upon costimulation (n=4). The CD25+ enriched cell fraction contains functionally suppressive cells since add back of unlabelled CD25+ enriched cells to CFSE labeled CD25− cells results in a decrease in proliferation of the costimulated CD8+CD25− and CD4+CD25− T-cells, namely 7.59% ± 3.86 and 4.16% ± 1.79, respectively (n=4). These cells also suppress cytokine production (IFN-g, TNF-a and IL-2) from autologous nodal T-cells as assessed by multiplex analysis of culture supernatants. In addition to suppressing autologous nodal T-cells, the FL CD25+ enriched cells are also capable of suppressing proliferation of allogeneic CD8+CD25− and CD4+CD25− T-cells from NLN as well as normal donor peripheral blood lymphocytes (PBL), regardless of very robust stimulation of the target cells with plate bound anti-CD3 and anti-CD28 antibodies. The allogeneic suppression is not reciprocal, since CD25+ enriched cells derived from either NLN or normal donor PBL, used at the same ratio, are not capable of suppressing allogeneic CD8+CD25− and CD4+CD25− T-cells derived from FL and in fact, are less suppressive against autologous T-cells than are the FL derived CD4+CD25+ cells. Whether this is due to a higher proportion of functionally suppressive T cells within the FL derived CD25+ enriched cells, compared to that of NLN or normal donor PBL, or to an increased suppressive capacity of the FL derived CD25+ T cells is currently being investigated. These data show that FL infiltrating CD4+CD25+GITR+ T-cells have a phenotype and function consistent with Tregs and are very potent suppressors of lymphoma associated-CD8+ and CD4+ T-cells, and therefore may play an important role in lymphoma development, progression and response to treatment.

A Phase I/II Trial Testing Immunization of Hepatocellular Carcinoma Patients with Dendritic Cells Pulsed with Four α-Fetoprotein Peptides

Alpha-fetoprotein (AFP) is a self protein expressed by fetal liver at high levels, but is transcriptionally repressed at birth. AFP is up-regulated in hepatocellular carcinomas, and patients with active disease could have plasma levels as high as 1 mg/mL. We previously identified four immunodominant HLA-A*0201-restricted peptides [hAFP(137-145) (PLFQVPEPV), hAFP(158-166) (FMNKFIYEI), hAFP(325-334) (GLSPNLNRFL), and hAFP(542-550) (GVALQTMKQ)] derived from human AFP that could stimulate specific T cell responses in healthy donor peripheral blood lymphocytes in vitro. We conducted a phase I/II clinical trial in which HLA-A*0201 patients with AFP-positive hepatocellular carcinoma were immunized with three biweekly intradermal vaccinations of the four AFP peptides pulsed onto autologous dendritic cells (DC). DCs were prepared from adherent peripheral blood mononuclear cells cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 7 days. Sixteen subjects were enrolled and 10 were treated. Peripheral blood lymphocytes were isolated from these patients before, during, and after AFP peptide/DC immunization and were tested ex vivo with MHC tetramer and IFNgamma ELISPOT analysis. Six of 10 subjects expanded statistically significant levels of AFP-specific T cells postvaccine to at least one peptide by MHC tetramer. Also, 6 of 10 subjects increased IFNgamma producing AFP-specific T cell responses to at least one of the peptides postvaccination, by ELISPOT. We conclude that the human T cell repertoire is capable of responding to the AFP self antigen after the administration of AFP peptide-pulsed DC even in an environment of high circulating levels of this oncofetal antigen.

Effects of Vitamin Supply on Spontaneous and Chemically Induced Mutagenesis in Human Cells

Chromosome aberrations in donor peripheral blood lymphocyte culture were evaluated before, 14 and 30 days after treatment with a vitamin/mineral complex. Treatment with the complex had no effect on spontaneous level of aberrant cells. The number of chromosome aberrations induced by dioxidine or cadmium chloride in vitro at the G2 stage decreased on days 14 and 30 of treatment with the complex.

Treatment of Resistant Leukemia by rIL-2 Activated NK Cells in Recipients of HLA Matched and Haploidentically Mismatched Stem Cell Allografts while Avoiding GVHD.

Although allogenenic stem cell transplantation may provide a cure for a growing number of patients with hematologic malignancies and several metastatic solid tumors, several problems remain to be solved. In routine medical practice transplant can be offered for patients with a matched donor available whereas the large majority of patients in need have no matched donor available. Although alloreactive lymphocytes may eliminate residual malignant cells, such an effect is accompanied by acute and chronic GVHD which may be hazardous even in recipients with perfectly matched allografts, and prohibitive in recipients treated with haploidentically mismatched allografts. On the other hand immunotherapy with intentionally mismatched allografts could provide a much more effective tool for eradication of tumor cells resistant to chemotherapy. We have pioneered a new approach for treatment of patients with resistant hematological malignancies (AML/MDS 5; ALL 1; Biphenotype 2; NHL 3; HD 1) using matched siblings (n=4), matched unrelated donor (n=1) or haploidentically mismatched donors (n=7). Prevention of rejection of mismatched allografts was accomplished by combination of fludarabine and deletion of donor reactive host lymphocytes by infusion of donor mononuclear blood cells and elimination of alloreactive lymphocytes susceptible to high-dose cyclosphosphamide (60mg/kgx3) one day later. Prevention of GVHD following infusion of G-CSF mobilized, haploidentically mismatched blood stem cells was accomplished using Miltenyi's immunomagentic beads coupled with anti-AC133 (n=6) or using anti-CD3 (n=1). No other anti-GVHD prophylaxis was used. Following transplantation, patients were treated with rIL-2 activated donor peripheral blood lymphocytes activated for 4 days at 37°C in 5% C02 in air incubator with rIL-2 6,000 IU/ml. T cell depletion was accomplished either by positive selection of CD56+ (n=10) or negative selection of CD3 (n=2) for optimal induction of graft vs leukemia (GVL) effects by mismatched and fully activated NK cells. One patient with resistant leukemia became disease free for 8 months but died of resistant aspergilosis which was evident prior to transplantation. Five out of 12 patients with intractable and fully resistant leukemia are alive with no GVHD and no evidence of disease 1–18 (median 13) months post transplantation. Based on our ongoing preliminary study we conclude that patients with resistant hematological malignancies may benefit from cell therapy mediated by rIL-2 activated donor lymphocytes, and most likely from intentionally mismatched haploidentical allografts following elimination of host anti-donor alloreactive lymphocytes and prevention of GVHD by positively or negatively selected stem cells, followed by immunotherapy with rIL-2 activated CD3 depleted NK cells. Intentionally mismatched rIL-2 activated NK cells represents a safe approach for elimination of residual tumor cells, aiming for induction of GVL while avoiding GVHD.

Covalent linking of proteins and cytokines to suture: enhancing the immune response of head and neck cancer patients.

The immune system of advanced stage head and neck cancer patients is frequently suppressed. Poor immune function has been correlated with poor clinical outcome. Immunotherapeutic strategies have been previously attempted in an effort to enhance immune function and improve survival. Previous studies have shown surgical suture can be transformed into an immune stimulant capable of activating the T lymphocytes of cancer patients. The development of a process for covalently linking proteins and cytokines to suture could have enormous potential for the in vivo manipulation of the immune system. We hypothesize proteins and cytokines can be covalently linked to surgical suture while preserving their functional properties. Prospective study testing normal donor and head and neck squamous cell carcinoma (HNSCC) patient lymphocytes. Polyester suture was acid hydrolyzed followed by reacting with 1-ethyl-3(-3-dimethylamino propyl carbodiimide) (EDAC) to create a suture-EDAC intermediate. Next, selected proteins (horseradish peroxidase [HRP] or bovine serum albumin [BSA]) or cytokines (interleukin [IL]-2 or interferon [IFN]-gamma) were reacted with the suture-EDAC intermediate to test the covalent linkage of the selected protein or cytokine to suture. Functional activity of the linked proteins was measured spectrophotometrically. The linking of cytokines to suture was tested by stimulating normal donor peripheral blood lymphocytes (PBL) or HNSCC patients' lymphocytes. The functional activity was confirmed by proliferation, enzyme linked immunoadsorbent assay (ELISA), and phenotype expression of T cells. RESULTS The conditions for optimally linking a protein to polyester suture were defined using HRP as a model protein. HRP retained its enzymatic activity. The optimal conditions for linking IL-2 or IFN-gamma were defined. The covalently linked cytokines retained their immune enhancing properties for stimulating PBL and lymph node lymphocytes (LNL) from HNSCC patients to proliferate, generate a TH1 immunologic profile of cytokines (IL-2, IL-12, IFN-gamma), and stimulate T lymphocytes. This is the first report to demonstrate that cytokines can be covalently linked to surgical sutures and retain their immune-stimulating properties. Proteins linked to suture also retained their enzymatic activity. The clinical implications of functionally active cytokines or proteins linked to surgical suture may be very significant in the future for manipulating the immune system in vivo or enhancing wound healing.

Regulatory mechanism of peripheral tolerance: in vitro evidence for dominant suppression of host responses during the maintenance phase of tolerance to renal allografts in miniature swine

Background: Studies from our laboratory have demonstrated that a short course of cyclosporine leads to indefinite survival of renal allografts across an MHC class-I barrier in miniature swine. We have recently reported that a peripheral regulatory mechanism appears to be involved in the maintenance of this tolerance since peripheral blood lymphocytes (PBL), exposed to donor antigen in vitro specifically suppressed the generation of anti-donor cytotoxic activity by recipient-matched naive PBL. We have now further investigated the mechanism of this phenomenon to determine the level at which such regulation occurs, and investigated the phenotypes of the cells involved in maintaining dominant suppression. Materials and methods: PBL from long-term tolerant animals (>6 months after renal transplantation) were pre-stimulated in vitro with donor-type PBL. These cells were then incubated with recipient-matched naive responders and donor-type PBL stimulators in MLR assays. The proliferative activity of the cells was measured by [ 3H]-thymidine incorporation. Suppression was measured by inhibition of proliferation of naive cells in response to donor PBL when co-cultured with tolerant cells. Flow cytometry was used to study the phenotypes of cells that were present in cell cultures. Results: Primed PBL from tolerant animals markedly suppressed the proliferative response of recipient-matched naive cells to donor-matched stimulators in vitro. No suppression of proliferation was observed in response to third party stimulators, indicating that the suppression was donor-specific. Primed PBL from naive animals stimulated with donor antigen and co-cultured with unprimed recipient-matched naive cells also demonstrated reduced proliferative responses. However, this decrease in proliferation appeared to be due to a ‘burn-out’ phenomenon, as assessed by kinetic studies, rather than due to true suppression. Expression of CD25 increased on a sub-population of T cells from tolerant animals following priming with donor antigen. These cell then markedly inhibited further activation of CD25 positive cells in co-cultures with naive responder cells, suggesting a possible mechanism of suppression. Conclusion: These results suggest that the mechanism of tolerance to class-I-mismatched renal allografts, involves a population of regulatory cells that are capable of suppressing proliferative anti-donor responses.

Evolution of the Enzyme-Linked Immunosorbent Spot Assay for Post-Transplant Alloreactivity as a Potentially Useful Immune Monitoring Tool

Post-transplant monitoring of cellular immunity has the potential to guide alterations in medical therapy. To this end, our laboratory has developed an enzyme-linked immunosorbent spot (ELISPOT) assay for detection of peripheral blood alloimmunity. Peripheral blood lymphocytes (PBLs) from normal volunteers and from renal allograft recipients were tested against donor stimulator cells for their ability to respond in 'one-way' cytokine ELISPOT assays. T cell depletion of donor spleen or PBLs eliminated donor cell cytokine secretion while preserving the ability of these cells to present allo-antigen to responding T cells. Alloreactive IFN-gamma-producing PBLs derive from the memory T cell pool and are readily detectable in recipients of renal allografts taking immunosuppressant medications. A significant expansion of IFN-gamma-producing donor-reactive memory PBLs was detectable at 4-6 months post-transplant in those who had experienced an acute rejection episode compared with those with a stable post-transplant course. The data demonstrate the feasibility of repeated post-transplant monitoring of allograft recipients, and provide the foundation for improving the care of human transplant recipients through rational clinical decision-making based on measures of immune function.

Tolerance to vascularized kidney grafts in canine mixed hematopoietic chimeras.

Recent progress in allogeneic hematopoietic stem cell transplantation provides new methods for reliable engraftment with nonlethal conditioning regimens. These techniques have been successfully applied in the treatment of both malignant and nonmalignant diseases, but have not been fully exploited for their potential to tolerize recipients for organ transplantation. These studies were undertaken to test whether the tolerance of host immune cells toward donor hematopoietic cells in mixed hematopoietic chimeras extends to include a vascularized organ, the kidney. Using nonmyeloablative doses of total body irradiation, a short course of immunosuppression, and hematopoietic stem cells from marrow or peripheral blood sources, five dog lymphocyte antigen-identical canines were made to become stable mixed hematopoietic chimeras with no development of graft-versus-host disease or posttransplant lymphoproliferative disorder. Subsequently, renal transplantations were performed between stem cell donor and recipient littermates, and no additional immunosuppressive therapy was given after stem cell transplantation. All mixed chimeric dogs demonstrate different, but stable, levels of donor peripheral blood lymphocyte and granulocyte chimerism. With follow-up of longer than 1 year, all of the mixed chimeric dogs (five/five) have excellent renal function with normal serum creatinines (<1.5 mg/dl) and no pathological evidence of rejection on biopsies. In a major histocompatibility-matched model, minor antigen differences between donor and recipient are not sufficient to induce a host immune response to a vascularized kidney transplant in mixed hematopoietic chimeras.