In vivo splenic CD11c cells downregulate CD4 T-cell response thereby decreasing systemic immunity to gene-modified tumour cell vaccine

Abstract Objective: EBV-specific CD8+ T cells have been extensively studied in various settings, and appear to play a major role in the control of EBV-related malignancies. In contrast, it is still unclear whether EBV-specific CD4+ T cells play a role in vivo. To study this question, an assay was developed to measure the CD4+ T-cell response towards two EBV antigens, in both healthy (n=14) and HIV-infected subjects (n=23). In addition, both HAART-treated (n=12) and untreated HIV+ individuals (n=14) - including progressors to EBV-related lymphoma - were studied longitudinally. Methods: EBV-specific CD4+ T cells were stimulated with peptide pools from latent protein EBNA1 and lytic protein BZLF1, and detected by measurement of IFNg-production. Results: After direct ex vivo stimulation, EBNA1 or BZLF1-specific IFNg- (and/or IL2) producing CD4+ T cell numbers were low, and measurable in less than half of the subjects studied (either HIV- and HIV+). Therefore, PBMC were cultured for 12 days in the presence of peptides and IL2 (from day 3), and then restimulated with peptides, allowing specific and reproducible expansion of EBV-specific CD4+ T cells, independent of HLA type and ex vivo antigen processing. Interestingly, numbers of EBV-specific CD4+ T cells inversely correlated with EBV viral load, implying an important role for EBV-specific CD4+ T cells in the control of EBV in vivo. Untreated HIV-infected individuals had a lower CD4+ T cell response to EBNA1 and BZLF1 as compared to healthy EBV carriers and HAART-treated HIV+ subjects. In longitudinal samples, EBNA1-specific, but not BZLF1-specific T-cell numbers increased after HAART, while EBV load was not affected by treatment. In all the progressors to EBV-related lymphoma, EBV-specific CD4+ T cells were lost at least 24 months before lymphoma diagnosis. Conclusions: Both cross-sectional and longitudinal data suggest an important role for EBV-specific CD4+ T cells in the control of EBV-related malignancies. Furthermore, it seems that HAART treatment leads to recovery of EBNA1-specific, but not BZLF1-specific CD4+ T-cell responses, implying changes in the latency pattern of EBV, despite an unaltered cell-associated EBV DNA load. Thus, early HAART treatment might prevent loss of specific CD4+ T-cell help and progression to NHL.

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Targeting Myeloma-Associated Macrophage Inhibits Multiple Myeloma Progression By Enhancing Anti-Tumor Cytotoxic CD4+ T Cell Response

Blood ◽

10.1182/blood.v128.22.481.481 ◽

2016 ◽

Vol 128 (22) ◽

pp. 481-481

Author(s):

Wang Qiang ◽

Yong Lu ◽

Rong Li ◽

Zhen Cai ◽

Jian Hou ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Cell Response ◽

Myeloma Cell ◽

T Cell Response ◽

Cd4 T Cell ◽

Antibody Treatment ◽

Stimulating Factor

Abstract Background: Multiple myeloma (MM) is an incurable plasma cell cancer characterized by tumor cell accumulation and expansion in the bone marrow (BM). One of the major problems is that MM BM microenvironment is a tumor promoting and immune suppressive milieu. We previously discovered that macrophages (MФs), in particular myeloma-associated MФs (mMФs), heavily infiltrated into MM BM and mediated chemoresistance in MM. Colony-stimulating factor 1 receptor (CSF1R), also known as macrophage colony-stimulating factor receptor (M-CSFR), plays an important role as regulator of the development, morphology, survival, and functions of tissue macrophages as well as tumor-associated macrophages (TAMs). CSF1R blockade by inhibitors and antibodies has been shown promising to treat different tumors, such as glioma, pancreatic cancer, and diffuse-type giant cell tumor. We here assessed the impact of CSF1R blockade by CSF1R antibodies from Imclone (Lilly) on human and murine mMФs, MM growth in vivo, and anti-myeloma immune response in the BM microenvironment as well as chemotherapy in MM. Methods: MФ infiltration was determined in the bone marrow (BM) patients with MGUS (n=6), MM (n=6), and compared to healthy donors (HD, n=6). CSF1R signaling blockade was assessed in the monocytes differentiation with M-CSF in the presence of CSF1R antibody or IgG control. CSF1R antibody impact on MФs growth/viability/proliferation was measured by trypan blue exclusion analysis, MTS, and Ki67 flow cytometry analysis. Tumor burden of 5TGM-1-bearing mice with CSF1R blockade treatment was determined by in vivo bioluminescent imaging, ELISA of IgG2b in mouse serum, and flow cytometry analysis of CD138+ myeloma cell infiltration in mouse BM. The effect of CSF1R antibody treatment on mMФ depletion and M1/M2 polarization was determined by flow cytometry and real-time PCR. Impact of CSF1R antibody treatment on cytotoxic CD8+ and CD4+ T cell immune response was measured by intracellular granzyme B flow cytometry and granzyme B ELISPOT. Effector cell-mediated MM cytotoxicity in the presence of mMФs with or without CSF1R treatment was measured by CD138/Annexin V flow cytometry. Survival rate of MM-bearing mice with CSF1R antibody and chemotherapy was evaluated using Kaplan-Meier estimates and log-rank tests using GraphPad Prism 5 software. Results: MΦ accumulation in BM was associated with myeloma development. Blocking CSF1R by humanized and murine CSF1R monoclonal antibodies (CS4 and CS7) not only inhibited monocyte survival and differentiation but also suppressed human and mouse mMΦ survival and development in vitro. Further, Targeting of MФs by either CS7 antibody or DT-mediated MФ killing in LysmCre X Csf1rLsL-DTR C57BL/6 mouse had marked inhibitory effects on established myeloma progression. CSF1R blockade by CS7 treatment reprogrammed the tumor microenvironment toward to an anti-tumor phenotype. CSF1R antibody treatment reduced myeloma cell load in mouse BM, however the anti-MM activity by CSF1R antibody was abolished in immunodeficient Rag-/- mice. Strikingly we found CSF1R blockade mediated anti-MM effect mainly based on CD4+ T cell response by CD8 and CD4 neutralizing antibody. Correspondingly cytotoxic anti-MM CD4+ T cell response enhanced by CSF1R antibody treatment was confirmed by ex vivo ELISPOT and in vitro cytotoxicity assay. Additionally, CSF1R antibody treatment promoted MM drug sensitivity. Conclusion: Our data demonstrated thatMФs play an important role in MM growth and development. Targeting mMФs by CSF1R blockade achieves anti-MM activity by enhancing cytotoxic CD4+ T cell response and promotes chemotherapy on MM, therefore suggesting therapeutic strategies based on interfering with myeloma-macrophage interactions. Disclosures No relevant conflicts of interest to declare.

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