275 Flow cytometry and nanoString provide a comprehensive cell- and gene-based tumor profile following checkpoint inhibition in a murine bladder cancer model

BackgroundBladder cancer (BC) is the thirteenth leading cause of cancer-related deaths.1 Five checkpoint immunotherapies that target the PD-1/PD-L1 axis are FDA approved, and gene- and protein-based approaches are helping to identify new combination treatment strategies for therapeutic intervention.2 Using the murine MB49 model for BC, we demonstrate how non-targeted immune gene expression profiling can combine with flow cytometry to provide a gene and cell-specific signature for the tumor microenvironment and help identify potential targets for novel treatment approaches.MethodsAnimals with established MB49 tumors were treated with anti-mPD-1 or isotype control antibodies. Tumors were collected 7 days after the last treatment. Flow cytometry examined anti-mPD-1 treatment-induced immunophenotypic modulation for eleven tumor-infiltrating immune subsets. The mouse PanCancer IO 360™ panel (NanoString) provided transcriptomic analysis of 770 immuno-oncology-related genes. The ROSALIND™ platform (OnRamp BioInformatics) was used to identify differentially regulated genes between treatment groups (±1.5 fold-change; p <0.05).ResultsAnti-mPD-1 had moderate anti-tumor activity, with a 58% tumor growth inhibition at day 18 post-implant in treated compared to control animals. Flow cytometry revealed anti-mPD-1 triggered an increase in tumor-infiltrating CD8+ T cells (45%) compared to control animals. Furthermore, the CD8+ T cell phenotype was altered by anti-mPD-1 treatment. The percentage of CD8+ T cells that expressed ICOS and LAG-3 was increased in tumors from anti-mPD-1 treated animals (22% and 35% respectively). A reduction in PD-1 expression was also observed (33%). In myeloid cells, iNOS expression increased in tumor-associated macrophages from treated animals compared to controls. NanoString analysis revealed 62 genes were differentially regulated in tumors from anti-mPD-1 treated animals compared to controls. ROSALIND analysis classified 30 of the genes as regulators of interferon, cytotoxicity, antigen presentation, and cytokine/chemokine signaling. Also, among the genes upregulated by anti-mPD-1 were IDO, HAVCR2 (TIM-3), and CSFR1, which can promote tumor growth and are clinical targets actively being investigated for new immunotherapies.ConclusionsNanoString analysis complemented flow cytometry to provide a comprehensive profile of the MB49 tumor. Together, these data demonstrate that anti-mPD1 increases T cell recruitment into the tumor and upregulates the expression of genes known to enhance T cell recruitment and anti-tumor activity. iNOS protein upregulation indicates that anti-mPD-1 treatment may also exert effects by reprogramming M2 macrophages towards an M1 phenotype. Upregulation of IDO, HAVCR2, and CSFR1 genes may effectively counteract anti-mPD-1 treatment. Further investigation may elucidate clinical implications for inhibitors of these gene products as combination treatment partners with anti-mPD-1.ReferencesSaginala K, Barsouk A, Aluru JS, Rawla P, Padala SA, Barsouk A. Epidemiology of bladder cancer. Med Sci 2020;1:15–26. Lopez-Beltran A, Cimadamore A, Blanca A, Massari F, Vau N, Scarpelli M, Cheng L, Montironi R. Immune checkpoint inhibitors for the treatment of bladder cancer. Cancers 2021;1:131–146.Ethics ApprovalN/A

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Enhance T Cell Immunotherapy By Targeting PIM-2 Kinase

Blood ◽

10.1182/blood.v128.22.815.815 ◽

2016 ◽

Vol 128 (22) ◽

pp. 815-815 ◽

Cited By ~ 1

Author(s):

Anusara Daenthanasanmak ◽

Yongxia Wu ◽

Supinya Iamsawat ◽

Hung D Nguyen ◽

Shikhar Mehrotra ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Cd8 T Cells ◽

Pim Kinases ◽

Target Organs ◽

Cell Immunotherapy ◽

Donor T Cells ◽

Anti Tumor Activity ◽

Deficient Mice

Abstract PIM kinases, a family of serine/threonine kinases, play an important role in regulating cell survival, cell proliferation, transcription activation and protein translation. Of the three isoforms, PIM-1, PIM-2 and PIM-3, some of them are overly expressed in several hematological malignancies as well as solid tumors. Thus, these kinases have been targeted clinically in several cancer studies. However, the roles of PIM kinases in T cells from previous studies are inconclusive and the functions of each isoform in these cells are yet to be investigated. In our study, we focused on the role of PIM-2 kinase in T cell responses to allo-antigen and tumor using pre-clinical models. Using allogeneic bone marrow transplantation (allo-BMT) models, we transferred allogeneic T cells isolated from mice deficient for Pim-2 (Pim-2-/-, FVB background) in comparison to WT and PIM1/3double deficient mice (PIM1/3-/-) into lethally irradiated Balb/C recipients. Our data revealed that upon allogeneic stimulation, PIM-2-/- T cells induced accelerated graft-versus-host-disease (GVHD) compared to WT or PIM1/3-/- T cells (Fig.1A). In addition, PIM-2-/- donor T cells were highly activated with significantly increased proliferation and IFN-γ production in recipient spleen and GVHD target organs. Donor PIM-2-/- T cells also expressed high levels of chemokine receptor, CXCR3 and a4b7 integrin (gut homing receptor) that correlated with increased capacity of donor T-cell migration to GVHD target organs such as liver and gut. In graft-versus-leukemia (GVL) study, PIM-2-/- donor T cells had roughly 4-fold higher ability to mediate GVL effect to WT and PIM1/3-/- T cells (Fig.1B). Negative regulation of T-cell responses to allo-antigens by PIM-2 was independently verified by restoring PIM-2 expression on PIM-2-/- T cells or inhibiting PIM kinases in PIM1/3-/- T cells. Altogether, the data from allo-BMT studies suggested that PIM-2 isoform functions as a negative regulator in T cell allo-response in contrast to PIM-1 and PIM-3 kinases. We then focused on evaluating T-cell immunity against syngeneic tumor. By administering tumor (TS-1 breast tumor in FVB background) into WT and PIM-2-/- mice either subcutaneously or intravenously, we observed that PIM-2-deficient mice efficiently control tumor growth in sharp contrast to WT mice (Fig. 1C). Mechanistic study revealed that tumor rejection in PIM-2-/- recipient relies on CD8+ tumor infiltrating lymphocytes (TILs) with a significant up-regulation of IFN-g, TNF-a and Fas-L expression. Lower infiltration of regulatory T cells and down regulation of PD-1 expression on T cells recovered from tumor site were also observed in PIM-2 deficient mice. Moreover, the tumor growth rate in PIM-2-/- mice after T-cell depletion with antibody was similar to that of WT mice. To further exclude the effect of other immune cells involved in tumor regression, we adoptively transferred WT and PIM-2-/- CD8+ T cells into WT mice with pre-established TS-1 tumor. CD8+ T cells deficient for PIM-2 was more efficient in controlling of tumor growth than WT or PIM1/3-/- CD8+ T cells. These data indicate that inhibition of PIM-2 in T cells display superior anti-tumor activity. In conclusion, PIM-2 kinase possesses a distinct role in anti-tumor immunity compared to the other two isoforms and it represents a potential target to enhance anti-tumor activity of adoptive T-cell immunotherapy. Disclosures No relevant conflicts of interest to declare.

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Skin dendritic cells in melanoma are key for successful checkpoint blockade therapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-000832 ◽

2021 ◽

Vol 9 (1) ◽

pp. e000832

Author(s):

Anastasia Prokopi ◽

Christoph H Tripp ◽

Bart Tummers ◽

Florian Hornsteiner ◽

Sarah Spoeck ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Tumor Growth ◽

Tumor Progression ◽

Cd8 T Cells ◽

Checkpoint Blockade ◽

Tumor Stages ◽

Tumor Immunogenicity

BackgroundImmunotherapy with checkpoint inhibitors has shown impressive results in patients with melanoma, but still many do not benefit from this line of treatment. A lack of tumor-infiltrating T cells is a common reason for therapy failure but also a loss of intratumoral dendritic cells (DCs) has been described.MethodsWe used the transgenic tg(Grm1)EPv melanoma mouse strain that develops spontaneous, slow-growing tumors to perform immunological analysis during tumor progression. With flow cytometry, the frequencies of DCs and T cells at different tumor stages and the expression of the inhibitory molecules programmed cell death protein-1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) on T cells were analyzed. This was complemented with RNA-sequencing (RNA-seq) and real-time quantitative PCR (RT-qPCR) analysis to investigate the immune status of the tumors. To boost DC numbers and function, we administered Fms-related tyrosine 3 ligand (Flt3L) plus an adjuvant mix of polyI:C and anti-CD40. To enhance T cell function, we tested several checkpoint blockade antibodies. Immunological alterations were characterized in tumor and tumor-draining lymph nodes (LNs) by flow cytometry, CyTOF, microarray and RT-qPCR to understand how immune cells can control tumor growth. The specific role of migratory skin DCs was investigated by coculture of sorted DC subsets with melanoma-specific CD8+ T cells.ResultsOur study revealed that tumor progression is characterized by upregulation of checkpoint molecules and a gradual loss of the dermal conventional DC (cDC) 2 subset. Monotherapy with checkpoint blockade could not restore antitumor immunity, whereas boosting DC numbers and activation increased tumor immunogenicity. This was reflected by higher numbers of activated cDC1 and cDC2 as well as CD4+ and CD8+ T cells in treated tumors. At the same time, the DC boost approach reinforced migratory dermal DC subsets to prime gp100-specific CD8+ T cells in tumor-draining LNs that expressed PD-1/TIM-3 and produced interferon γ (IFNγ)/tumor necrosis factor α (TNFα). As a consequence, the combination of the DC boost with antibodies against PD-1 and TIM-3 released the brake from T cells, leading to improved function within the tumors and delayed tumor growth.ConclusionsOur results set forth the importance of skin DC in cancer immunotherapy, and demonstrates that restoring DC function is key to enhancing tumor immunogenicity and subsequently responsiveness to checkpoint blockade therapy.

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Pre-existing intratumoral CD8 T cells substantially contribute to control tumors following therapeutic anti-CD40 and polyI:C based vaccination

10.1101/2020.08.31.275420 ◽

2020 ◽

Author(s):

Aaron D. Stevens ◽

Timothy N.J. Bullock

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Tumor Growth ◽

Cd8 T Cells ◽

Tumor Control ◽

Cell Trafficking ◽

T Cell Trafficking ◽

Cell Numbers

ABSTRACTBackgroundDendritic cells are potently activated by the synergistic action of CD40 stimulation in conjunction with signaling through toll like receptors, subsequently activating antigen specific T cells. Cancer vaccines targeting the activation of dendritic cells in this manner show promise in murine models and are being developed for human cancer patients. While vaccine efficacy has been established, further investigation is needed to understand the mechanism of tumor control and how vaccination alters tumor infiltrating immune cells.MethodsMice bearing established murine melanoma tumors were vaccinated with agonist anti-CD40, polyI:C, and tumor antigen. Intratumoral T cell numbers, differentiation state, proliferation, and survival were assessed by flow cytometry. T cell effector function was measured both within the tumor and ex vivo by flow cytometry. T cell trafficking was blocked to examine changes to intratumoral T cells present at the time of vaccination.ResultsVaccination led to increased intratumoral T cell numbers and delayed tumor growth. Expansion of T cells and tumor control did not require trafficking of T cells from the periphery. The increase in intratumoral T cells was associated with an acute burst in proliferation but not changes in viability. Intratumoral T cells had lower PD-1 and Eomes expression but were less functional after vaccination on a per cell basis. However, the increased intratumoral T cell numbers yielded increased effector T cells per tumor.ConclusionsPre-infiltrated CD8 T cells are responsive to CD40/TLR-mediated vaccination and sufficient for vaccination to delay tumor growth when additional T cell trafficking is blocked. This indicates that the existing T cell response and intratumoral DC could be critical for vaccination efficacy. This also suggests that circulating T cells may not be an appropriate biomarker for vaccination efficacy.

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CD122-directed interleukin-2 treatment mechanisms in bladder cancer differ from αPD-L1 and include tissue-selective γδ T cell activation

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-002051 ◽

2021 ◽

Vol 9 (4) ◽

pp. e002051

Author(s):

Ryan Michael Reyes ◽

Yilun Deng ◽

Deyi Zhang ◽

Niannian Ji ◽

Neelam Mukherjee ◽

...

Keyword(s):

Bladder Cancer ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Combination Treatment ◽

Treatment Efficacy ◽

Interleukin 2 ◽

Γδ T Cells ◽

T Cell Subsets ◽

Γδ T Cell

BackgroundAnti-programmed death-ligand 1 (αPD-L1) immunotherapy is approved to treat bladder cancer (BC) but is effective in <30% of patients. Interleukin (IL)-2/αIL-2 complexes (IL-2c) that preferentially target IL-2 receptor β (CD122) augment CD8+ antitumor T cells known to improve αPD-L1 efficacy. We hypothesized that the tumor microenvironment, including local immune cells in primary versus metastatic BC, differentially affects immunotherapy responses and that IL-2c effects could differ from, and thus complement αPD-L1.MethodsWe studied mechanisms of IL-2c and αPD-L1 efficacy using PD-L1+ mouse BC cell lines MB49 and MBT-2 in orthotopic (bladder) and metastatic (lung) sites.ResultsIL-2c reduced orthotopic tumor burden and extended survival in MB49 and MBT-2 BC models, similar to αPD-L1. Using antibody-mediated cell depletions and genetically T cell-deficient mice, we unexpectedly found that CD8+ T cells were not necessary for IL-2c efficacy against tumors in bladder, whereas γδ T cells, not reported to contribute to αPD-L1 efficacy, were indispensable for IL-2c efficacy there. αPD-L1 responsiveness in bladder required conventional T cells as expected, but not γδ T cells, altogether defining distinct mechanisms for IL-2c and αPD-L1 efficacy. γδ T cells did not improve IL-2c treatment of subcutaneously challenged BC or orthotopic (peritoneal) ovarian cancer, consistent with tissue-specific and/or tumor-specific γδ T cell contributions to IL-2c efficacy. IL-2c significantly altered bladder intratumoral γδ T cell content, activation status, and specific γδ T cell subsets with antitumor or protumor effector functions. Neither IL-2c nor αPD-L1 alone treated lung metastatic MB49 or MBT-2 BC, but their combination improved survival in both models. Combination treatment efficacy in lungs required CD8+ T cells but not γδ T cells.ConclusionsMechanistic insights into differential IL-2c and αPD-L1 treatment and tissue-dependent effects could help develop rational combination treatment strategies to improve treatment efficacy in distinct cancers. These studies also provide insights into γδ T cell contributions to immunotherapy in bladder and engagement of adaptive immunity by IL-2c plus αPD-L1 to treat refractory lung metastases.

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702 TJ-CD4B (ABL111), a Claudin18.2-targeted 4-1BB tumor engager induces potent tumor-dependent immune response without dose-limiting toxicity in preclinical studies

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.702 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A730-A730

Author(s):

Wenqing Jiang ◽

Zhengyi Wang ◽

Zhen Sheng ◽

Jaeho Jung ◽

Taylor Guo

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

Gene Expression Analysis ◽

Cell Activation ◽

Clinical Development ◽

Cynomolgus Monkeys ◽

Anti Tumor Activity

Background4-1BB (CD137) is a co-stimulatory receptor that stimulates the function of multiple immune cells. Its ability to induce potent anti-tumor activity makes 4-1BB an attractive target for immuno-oncology. However, clinical development of a monospecific 4-1BB agonistic antibody has been hampered by dose-limiting hepatic toxicities. To minimize systemic toxicities, we have developed a novel Claudin18.2 (CLDN18.2) x 4-1BB bispecific antibody, TJ-CD4B (ABL111) that stimulates 4-1BB pathway only when it engages with Claudin 18.2, a tumor-associated antigen specifically expressed in gastrointestinal cancers. TJ-CD4B (ABL111) is now being evaluated in patients with advanced solid tumors in a first-in-human trial (NCT04900818).MethodsTJ-CD4B (ABL111) was evaluated in vivo using the human 4-1BB knock-in mice bearing CLDN18.2 expressing MC38 tumor cells. Pharmacodynamic effects upon treatment were characterized in tumor tissue and blood. Immunophenotyping of the tumor microenvironment (TME) and peripheral blood was performed by flow cytometry. Soluble biomarkers were measured using Luminex-based multiplex assay. In-depth gene expression analysis was performed on primary human CD8+ T cells that were co-cultured with CLDN18.2 expressing cells in the presence of anti-CD3 using NanoString nCounter®. Pharmacokinetic (PK) and toxicity study were performed in cynomolgus monkeys.ResultsTJ-CD4B (ABL111) elicited complete tumor regression in 13 out of 18 MC38 tumor bearing mice given at a dose above 2 mg/kg. Dose-dependent anti-tumor activity was associated with enhanced T cell activation in TME and expansion of memory T cells in the peripheral blood. Increased CD8+ T cells number and proliferation were observed in both tumor nest and surrounding stroma while the level of soluble 4-1BB in the serum was also elevated in response to the treatment. In vitro gene expression analysis by Nanostring revealed TJ-CD4B(ABL111) effectively activated immune pathways characterized by IFN?-signaling and T cell inflammation. Preclinically, TJ-CD4B was well tolerated at the repeated doses up to 100 mg/kg/wk in cynomolgus monkeys without the adverse influence on the liver function which is generally affected by 4-1BB activation. Besides, no cytokine release or immune activation was observed in the periphery.ConclusionsTJ-CD4B (ABL111) is a novel CLDN18.2 dependent 4-1BB bispecific agonist antibody that induced T cell activation and memory response in tumor with CLDN18.2 expression, leading to a strong anti-tumor activity in vivo. TJ-CD4B did not induce systemic immune response nor hepatic toxicity due to the CLDN18.2 dependent 4-1BB stimulation. These data warrant the current clinical development in phase I trial to validate the safety properties and tumor specific responses.

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767 Activation of CD8+ T cells in the presence of multiple TLR agonists affects the expression of T-cell checkpoint receptors via IL-12 and type-1 interferon

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.767 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A802-A802

Author(s):

Donghwan Jeon ◽

Douglas McNeel

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cancer Immunology ◽

Receptor Expression ◽

Tlr Agonists ◽

Tumor Bearing ◽

Pathway Activation ◽

Checkpoint Receptors ◽

Anti Tumor Activity

BackgroundT-cell checkpoint receptors are expressed when T-cell are activated, and activation of these receptors can impair the function of T-cells and their anti-tumor efficacy.1 We previously found that T-cells activated with cognate antigen increase the expression of PD-1, while this can be attenuated by the presence of specific Toll-like receptor (TLR) agonists.2 3 This effect was mediated by IL-12 secretion from professional antigen presenting cells and resulted in CD8+ T cells with greater anti-tumor activity. In the current report, we sought to determine whether combination of TLR agonists can further affect the expression of T-cell checkpoint receptors and improve T-cell anti-tumor immunity.MethodsOT-1 CD8+ T cells were stimulated with peptide (SIINFEKL) and dendritic cells (DC) in the presence of two different TLR agonists. The cells were collected and evaluated for the expression of T-cell checkpoint receptors (PD-1, CTLA-4, CD160, CD244, LAG-3, TIM-3, TIGIT and VISTA) by flow cytometry, and for transcriptional changes by RNA-seq. Purified DC were stimulated with TLR combinations and evaluated for cytokine release by ELISA. The anti-tumor efficacy of vaccination using peptide and TLR agonist combinations was evaluated in EG7-OVA tumor-bearing mice.ResultsActivation of CD8+ T cells in the presence of specific TLR ligands resulted in decreases in expression of PD-1 and/or CD160. These changes in T-cell checkpoint receptor expression were modestly affected when TLR ligands were used in combination, and notably with combinations of TLR1/2, TLR3, and TLR9 agonists. Immunization of tumor-bearing mice, co-administered with combinations of these agonists, showed greater anti-tumor effects. However, while the effect of TLR1/2 and/or TLR9 was abrogated in IL12KO mice, TLR3 demonstrated anti-tumor activity when co-administered with peptide vaccine. RNA sequencing of TLR-conditioned CD8+ T-cells revealed IL-12 pathway activation, and IFNß pathway activation following TLR3 stimulation. Stimulation of DC with TLR3 agonist, alone or in combination with other TLR agonists, resulted in increased IL-12 and IFNß secretion. Co-incubation of OT-1 splenocytes with rIL12 and/or rIFNß during peptide activation led to reduced expression of PD-1, and this could be reversed with antibodies blocking IL12R or IFNAR-1.ConclusionsMultiple TLR agonists can modulate the expression of T-cell checkpoint receptors, notably PD-1, by upregulating the secretion of IL-12 and IFNß. These data provide the mechanistic rationale for choosing optimal combinations of TLR ligands to use as adjuvants to improve the efficacy of anti-tumor vaccines.ReferencesJin H-T, et al. Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proceedings of the National Academy of Sciences 2010;107(33):14733–14738.Zahm CD, Colluru VT, McNeel DG. Vaccination with high-affinity epitopes impairs antitumor efficacy by increasing PD-1 expression on CD8+ T cells. Cancer Immunology Research 2017;5(8):630–641.Zahm CD, et al. TLR stimulation during T-cell activation lowers PD-1 expression on CD8+ T Cells. Cancer Immunology Research 2018;6(11):1364–1374.

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A Detailed Phenotypic Analysis Using Six Color Flow Cytometry of Lymphocyte Subsets Mobilized with AMD3100 Compared to G-CSF.

Blood ◽

10.1182/blood.v104.11.408.408 ◽

2004 ◽

Vol 104 (11) ◽

pp. 408-408 ◽

Cited By ~ 1

Author(s):

Yoshiyuki Takahashi ◽

S. Chakrabarti ◽

R. Sriniivasan ◽

A. Lundqvist ◽

E.J. Read ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Lymphocyte Subsets ◽

Phenotypic Analysis ◽

Color Flow ◽

Cd34 Cells

Abstract AMD3100 (AMD) is a bicyclam compound that rapidly mobilizes hematopoietic progenitor cells into circulation by inhibiting stromal cell derived factor-1 binding to its cognate receptor CXCR4 present on CD34+ cells. Preliminary data in healthy donors and cancer patients show large numbers of CD34+ cells are mobilized following a single injection of AMD3100. To determine whether AMD3100 mobilized cells would be suitable for allografting, we performed a detailed phenotypic analysis using 6 color flow cytometry (CYAN Cytometer MLE) of lymphocyte subsets mobilized following the administration of AMD3100, given as a single 240mcg/kg injection either alone (n=4) or in combination with G-CSF (n=2: G-CSF 10 mcg/kg/day x 5: AMD3100 given on day 4). Baseline peripheral blood (PB) was obtained immediately prior to mobilization; in recipients who received both agents, blood was analyzed 4 days following G-CSF administration as well as 12 hours following administration of AMD3100 and a 5th dose of G-CSF. AMD3100 alone significantly increased from baseline the PB WBC count (2.8 fold), Absolute lymphocyte count (ALC: 2.5 fold), absolute monocyte count (AMC: 3.4 fold), and absolute neutrophil count (ANC: 2.8 fold). Subset analysis showed AMD3100 preferentially increased from baseline PB CD34+ progenitor counts (5.8 fold), followed by CD19+ B-cells (3.7 fold), CD14+ monocytes (3.4 fold), CD8+ T-cells (2.5 fold), CD4+ T-cells (1.8 fold), with a smaller increase in CD3−/CD16+ or CD56+ NK cell counts (1.6 fold). There was no change from baseline in the % of CD4+ or CD8+ T-cell expressing CD45RA, CD45RO, or CD56, CD57, CD27, CD71 or HLA-DR. In contrast, there was a decline compared to baseline in the mean percentage of CD3+/CD4+ T-cells expressing CD25 (5.5% vs 14.8%), CD62L (12.1% vs 41.1%), CCR7 (2.1% vs 10.5%) and CXCR4 (0.5% vs 40.9%) after AMD3100 administration; similar declines in expression of the same 4 surface markers were also observed in CD3+/CD8+ T-cells. A synergistic effect on the mobilization of CD34+ progenitors, CD19+ B cells, CD3+ T-cells and CD14+ monocytes occurred when AMD3100 was combined with G-CSF (Figure). In those receiving both AMD3100 and G-CSF, a fall in the % of T-cells expressing CCR7 and CXCR4 occurred 12 hours after the administration of AMD3100 compared to PB collected after 4 days of G-CSF; no other differences in the expression of a variety activation and/or adhesion molecules on T-cell subsets were observed. Whether differences in lymphocyte subsets mobilized with AMD3100 alone or in combination with G-CSF will impact immune reconstitution or other either immune sequela (i.e. GVHD, graft-vs-tumor) associated with allogeneic HCT is currently being assessed in an animal model of allogeneic transplantation.

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Therapeutic Anti-Tumor Immunity Mediated by Transiently Engrafting Allogeneic Lymphocytes: The “Allogeneic Effect” Revisited.

Blood ◽

10.1182/blood.v106.11.5255.5255 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5255-5255

Author(s):

Heather J. Symons ◽

M. Yair Levy ◽

Jie Wang ◽

Xiaotao Zhou ◽

Ephraim J. Fuchs

Keyword(s):

T Cells ◽

Immune System ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Host Immune System ◽

Tumoricidal Activity ◽

Tumor Bearing ◽

Anti Tumor Activity ◽

Allogeneic Lymphocytes

Abstract The “allogeneic effect” refers to the induction of host B cell antibody synthesis or host T cell cytotoxicity, including tumoricidal activity, by an infusion of allogeneic lymphocytes. We have previously shown that treatment of mice with cyclophosphamide (Cy) followed by infusion of CD8+ T cell-depleted allogeneic spleen cells (Cy + CD8− DLI) induces anti-tumor activity in a model of minimal residual leukemia, even though the donor cells are eventually rejected by the host immune system. The purpose of the current investigation was to test the activity of Cy + CD8− DLI in the treatment of well-established cancer, and to characterize the mechanisms of the anti-tumor effect. BALB/c mice were inoculated intravenously (IV) with the syngeneic A20 lymphoma/leukemia or the RENCA renal cell carcinoma on day 0 and were then treated with nothing, Cy alone on day 14, or Cy + CD8− DLI from MHC-mismatched C57BL/6 donors on day 15. In both tumor models, the combination of Cy + CD8− DLI significantly prolonged survival compared to mice treated with nothing or with Cy alone. While depletion of CD4+ T cells from the DLI significantly diminished the beneficial effect of CD8− DLI, purified CD4+ T cells alone were inactive, demonstrating that donor CD4+ T cells and another population of cells were required for optimal anti-tumor activity. Several observations pointed to an active role for the host immune system in the anti-tumor activity of Cy + CD8− DLI. First, host T cells participated in the anti-tumor effect of treatment with Cy alone, since the drug’s activity was diminished in tumor-bearing scid mice or in normal BALB/c mice depleted of T cells. Second, while Cy + CD8− DLI caused no GVHD in tumor-bearing but immunocompetent BALB/c recipients, it caused fatal acute GVHD in either tumor-bearing scid or T-cell depleted BALB/c mice. Finally, the anti-tumor effect of Cy + CD8- DLI was also significantly inhibited in BALB/c mice that were depleted of CD8+ T cells. These results demonstrate that transiently engrafting T cells administered after Cy can induce significant anti-tumor effects against both solid and liquid tumors. We propose that upon recognition of alloantigen on host antigen-presenting cells (APCs), allogeneic donor CD4+ T cells deliver activating ligands to the APCs, thereby generating effective “help” to break tolerance in tumor-specific host CD8+ T cells. This mechanism may correspond to the “allogeneic effect” in the anti-tumor response described over three decades ago.

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The Absence of Vasoactive Intestinal Peptide Augments Allo-Reactivity and the Anti-Viral Response in Bone Marrow Transplantation.

Blood ◽

10.1182/blood.v110.11.3267.3267 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3267-3267

Author(s):

Lauren T. Southerland ◽

Jian-Ming Li ◽

Sohrab Hossain ◽

Cynthia Giver ◽

Wayne Harris ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Immune Responses ◽

Cd8 T Cells ◽

Cell Response ◽

Low Dose ◽

T Cell Response ◽

High Dose ◽

Mcmv Infection

Abstract Background: The severe morbidity and mortality associated with bone marrow transplantation (BMT) is caused by uninhibited immune responses to alloantigen and suppressed immune responses to pathogens. Vasoactive Intestinal Peptide (VIP) is an immunomodulatory neuropeptide produced by T-cells and nerve fibers in peripheral lymphoid organs that suppresses immune responses by induction of tolerogenic dendritic cells. In order to determine the immunoregulatory effects of VIP, we examined T-cell immune responses to allo- and viral-antigens in VIP knockout (KO) mice and mouse BMT recipients of hematopoietic cells from VIP KO donors. Methods: VIP KO mice and VIP WT littermates were infected with lethal or sub-lethal doses (5 × 104− 5 × 105 PFU) of murine cytomegalovirus (mCMV) and the T-cell response to viral antigen was measured by flow cytometry for mCMV peptide-MHC class 1-tetramer+ CD8+ T-cells. We transplanted 5 × 106 BM plus 1 × 106 splenocytes (SP) either from VIP KO or VIP WT donors in an C57BL/6 to F1(BL/6 × Balb/c) allo-BMT model and assessed survival, GvHD, donor T-cell expansion, chimerism, and response to mCMV vaccination and mCMV infection. Results: B-cell, αβ and γδ T-cell, CD8+ T-cell, CD11b+ myeloid cell, and dendritic cell numbers were equivalent between VIP KO and WT mice, while VIP KO mice had higher number of CD4+ and CD4+CD62L+CD25+ T-cells. Non-transplanted VIP KO mice survived mCMV infection better compared to VIP WT, with a brisker anti-viral T-cell response in the blood. In the allogeneic BMT setting, recipients of VIP KO BM plus VIP KO SP had more weight loss and lower (40%) 100 day post-transplant survival compared to the recipients of VIP KO BM plus WT SP (80% survival), recipients of WT BM plus KO SP (100% survival), and recipients of WT BM plus WT SP (80% survival). Recipients of VIP KO grafts had a significantly greater anti-mCMV response that peaked four days earlier than the tetramer response of mice transplanted with WT cells. This increased anti-viral response to vaccination correlated with a greater and more rapid T-cell response to secondary viral challenge. Conclusions: These experiments suggest that the absence of all VIP in the body, or the absence of VIP in a transplanted immune system, enhances anti-viral immunity and allo-immune responses. Modulation of the VIP pathway is a novel method to regulate post-transplant immunity. Figure 1: VIP knockout(KO) mice have an increased CMV tetramer response. VIP KO and VIP WT mice were infected (day 0) with either a sub-lethal low dose (5 × 10^4 PFU) or a lethal high dose (5 × 10^5 PFU) of CMV. Peripheral blood was stained for T cell markers and tetramer and analyzed by flow cytometry. On day 3, high dosed VIP KO mice had a higher number of tetramer positive CD8 T cells and better survival than WT mice (all high dose VIP WT died prior to day 10). VIP KO mice had a significant increase in tetramer positive CD8 T cells between days 3 and 10. *** p<0.01, difference between VIP KO and VIP WT littermate at designated dose level and day. Figure 1:. VIP knockout(KO) mice have an increased CMV tetramer response. VIP KO and VIP WT mice were infected (day 0) with either a sub-lethal low dose (5 × 10^4 PFU) or a lethal high dose (5 × 10^5 PFU) of CMV. Peripheral blood was stained for T cell markers and tetramer and analyzed by flow cytometry. On day 3, high dosed VIP KO mice had a higher number of tetramer positive CD8 T cells and better survival than WT mice (all high dose VIP WT died prior to day 10). VIP KO mice had a significant increase in tetramer positive CD8 T cells between days 3 and 10. *** p<0.01, difference between VIP KO and VIP WT littermate at designated dose level and day.

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Generation of CD8 T Cell-Mediated Protective Immunity against Tumor Escapees

Blood ◽

10.1182/blood.v112.11.2623.2623 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2623-2623 ◽

Cited By ~ 1

Author(s):

Bindu Varghese ◽

Behnaz Taidi ◽

Adam Widman ◽

James Do ◽

R. Levy

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Tumor Growth ◽

Tumor Cells ◽

Cd8 T Cells ◽

Cell Lymphoma ◽

Ex Vivo ◽

B Cell Lymphoma ◽

Cd8 T Cell

Abstract Introduction: Anti-idiotype antibodies against B cell lymphoma have shown remarkable success in causing tumor regression in the clinic. In addition to their known ability to mediate ADCC, anti-idiotype antibodies have also been shown to directly inhibit the proliferation of tumor cells by sending negative growth signals via the target idiotype. However, further studies to investigate this mechanism have been hindered by the failure of patient tumor cells to grow ex vivo. Methods and Results: In order to study this phenomenon further, we developed an antibody against the idiotype on an A20 mouse B lymphoma cell line. A radioactive thymidine incorporation assay showed decreased A20 cell proliferation in the presence of the anti-id antibody ex vivo. In vivo, when mice were treated intraperitoneally (i.p.) with 100 μg of antibody 3 hours post-tumor inoculation (1×106 A20 subcutaneously (s.c.)), tumor growth was delayed for greater than 40 days after which the tumor began to grow once again. Further analysis of these escaping tumor cells by flow cytometry showed that that the tumor cells escaped the antibody-mediated immune response by down-regulating expression of idiotype and IgG on their surfaces although the cells retained idiotype expression intracellularly. This down-regulation of surface idiotype rendered the tumor cells resistant to both ADCC and signaling-induced cell death. The addition of an immunostimulatory bacterial mimic (CpG-DNA; 100 μg × 5 intratumoral (i.t.) injections; Days 2, 3 4, 6 & 8) to antibody therapy (Day 0; 100 μg i.p.) cured large established tumors (Day 0 = 1 cm2) and prevented the occurrence of tumor escapees (p<0.0001). Antibody plus CpG combination therapy in tumor-bearing mice deficient for CD8+ T cells demonstrated the critical role of CD8+ T cells in A20 tumor eradication (p<0.005). Depletion of CD4+ T cells was found to have no significant impact on the therapy. We also found that when mice were inoculated with two tumors and treated with anti-idiotype antibody (i.p.) followed by intratumoral CpG in just one tumor (Day 0=1 cm2; anti-idiotype antibody 100 μg Day 0; 100 μg CpG Days 2, 3, 4, 6 & 8), untreated tumors regressed just as well as CpG-treated tumors indicating a systemic anti-tumor immune response was generated. Conclusion: Anti-idiotype therapy, although effective in delaying tumor growth, frequently generates antigen-loss variants. However, we found that when anti-idiotype antibodies were combined with CpG, even large established tumors were cured due to systemic CD8+ T cell-dependent tumor immunity. Rather than simply mediating ADCC against a single tumor antigen, which requires the constant infusion of antibody to hamper tumor growth, we hypothesize a cytotoxic T-cell response against many tumor antigens was also generated. Such a diverse T-cell repertoire can prevent the emergence of tumor escapees and collectively provide long-lasting tumor protection. These pre-clinical results suggest that anti-tumor antibodies combined with CpG warrant further study in patients with B cell lymphoma.

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