IntroductionT regulatory (T reg ) cells play an important role in the maintenance of self-tolerance, control of auto-immunity, and regulation of T-cell homeostasis, and they modulate overall immune responses against infectious agents and tumor cells. 1 Natural T reg cells develop during normal T-cell maturation in the thymus and represent 5% to 10% of the CD4 ϩ cell compartment in the peripheral blood. 2 These cells express CD4 and CD25 surface antigens as well as CTLA-4, GITR, CD103, CD62L, CD69, CD134, CD71, CD54, and CD45RA. 3 The suppressive activity of T reg cells is associated with the overexpression of FOXP3, a member of the forkhead/winged helix family, which acts as a transcriptional repressor. 4 T reg cells suppress CD25 Ϫ CD4 ϩ T-cell proliferation on the basis of cell-cell contact and suppress immune responses by secreting immunosuppressive cytokines such as IL-10 and TGF-␤. 5 A significant impairment of T-cell function is observed in patients with multiple myeloma (MM) and patients with monoclonal gammopathy of undetermined significance (MGUS). Although phenotypic and functional aberrations in CD4 and CD8 cells have been described in MM and MGUS, 6-9 the biologic basis for these abnormalities remains unclear. Because T reg cells play an important role in modulating normal immune responses, the abnormal T reg -cell activity in myeloma patients could contribute to immune dysfunction in MM and could provide a new target to enhance immune responses. Therefore, in this study we evaluated natural T reg -cell number and function in patients with MGUS and MM and compared them with those of healthy donors. Study design Phenotypic characterizationCD4 ϩ CD25 ϩ T reg -cell numbers were analyzed by flow cytometric analyses in peripheral-blood mononuclear cells (PBMCs) collected from healthy donors, patients with MGUS, and patients with newly diagnosed MM. Approval for these studies was obtained from the institutional review board of the Dana-Farber Cancer Institute and the Veterans Administration (VA) Boston Healthcare System. Informed consent was provided according to the Declaration of Helsinki. Measurement of FOXP3 expressionAs FOXP3 is specifically expressed by T reg cells and is required for their suppressive activity, we analyzed the proportion of PBMCs expressing intracellular FOXP3 using anti-FOXP3 antibody (eBiosciences, San Diego, CA) using dual-color flow cytometry and multiphoton microscopy. Level of protein expression was quantitated by Western blotting and by real-time reverse transcription-polymerase chain reaction (RT-PCR) using previously described methods. 10 Suppressive activity of T regulatory cellsTo evaluate the function of T reg cells, PBMCs were first depleted of CD25 ϩ T cells (which contain T reg cells) by positive selection using anti-CD25-coated microbeads (Miltenyi Biotech, Auburn, CA), according to the manufacturer's instructions. 11 PBMCs depleted of CD25 ϩ cells and control PBMCs containing CD25 ϩ cells were stimulated with anti-CD3 antibody for 3 days, and proliferation was measu...

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Histone deacetylases (HDAC) are therapeutic targets in multiple cancers. ACY241, an HDAC6 selective inhibitor, has shown anti-multiple myeloma (MM) activity in combination with immunomodulatory drugs and proteasome inhibitors. Here we show ACY241 significantly reduces the frequency of CD138 MM cells, CD4CD25FoxP3 regulatory T cells, and HLA-DRCD11bCD33 myeloid-derived suppressor cells; and decreases expression of PD1/PD-L1 on CD8 T cells and of immune checkpoints in bone marrow cells from myeloma patients. ACY241 increased B7 (CD80, CD86) and MHC (Class I, Class II) expression on tumor and dendritic cells. We further evaluated the effect of ACY241 on antigen-specific cytotoxic T lymphocytes (CTL) generated with heteroclitic XBP1unspliced (YISPWILAV) and XBP1spliced (YLFPQLISV) peptides. ACY241 induces co-stimulatory (CD28, 41BB, CD40L, OX40) and activation (CD38) molecule expression in a dose- and time-dependent manner, and anti-tumor activities, evidenced by increased perforin/CD107a expression, IFN-γ/IL-2/TNF-α production, and antigen-specific central memory CTL. These effects of ACY241 on antigen-specific memory T cells were associated with activation of downstream AKT/mTOR/p65 pathways and upregulation of transcription regulators including Bcl-6, Eomes, HIF-1 and T-bet. These studies therefore demonstrate mechanisms whereby ACY241 augments immune response, providing the rationale for its use, alone and in combination, to restore host anti-tumor immunity and improve patient outcome.

Purpose The efficacy of peptide vaccines may be enhanced by stimulating immune cells with multiple peptides derived from distinct tumor-associated antigens. We have evaluated the heteroclitic XBP1 US184–192 (YISPWILAV), heteroclitic XBP1 SP367–375 (YLFPQLISV), native CD138260–268 (GLVGLIFAV), and native CS1239–247 (SLFVLGLFL) peptides, which have strong HLA-A2 affinity and immunogenicity in combination, for their ability to elicit multiple myeloma antigen-specific responses. Experimental Design Multipeptide-specific cytotoxic T lymphocytes (MP-CTL) were generated by the stimulation of CD3+ T lymphocytes from HLA-A2+ individuals with either autologous mature dendritic cells or T2 cells pulsed with a cocktail of these four peptides. Results The peptide cocktail did not compromise tumor antigen-specific activity of CTL. MP-CTL displayed increased total, effector memory (CCR7−CD45RO+), and activated (CD69+) CD3+CD8+ T lymphocytes. In addition, MP-CTL demonstrated IFN-γ production, cell proliferation, and cytotoxicity against HLA-A2+ multiple myeloma cells, including HLA-A2+ MM patients’ cells. Importantly, MP-CTL showed specific responses in functional assays to each relevant peptide, but not to an irrelevant HLA-A2 specific CMV pp65 (NLVPMVATV) peptide. Conclusions These results highlight the potential therapeutic application of vaccination with a cocktail of HLA-A2 specific peptides to induce CTL with a broad spectrum of immune responses against multiple myeloma antigens.

The 90-kDa heat shock protein (Hsp90) has become an important therapeutic target with ongoing evaluation in a number of malignancies. Although Hsp90 inhibitors have a high therapeutic index with limited effects on normal cells, they have been described to inhibit dendritic cell function. However, its effect on human immune effector cells may have significant clinical implications, but remains unexplored. In this study, we have evaluated the effects of Hsp90 inhibition on human T lymphocyte and NK cells, including their Ag expression, activation, proliferation, and functional activities. These studies demonstrate that Hsp90 inhibition irreversibly downregulates cell surface expression of critical Ags (CD3, CD4, CD8), the costimulatory molecule (CD28, CD40L), and αβ receptors on T lymphocytes, as well as activating receptors (CD2, CD11a, CD94, NKp30, NKp44, NKp46, KARp50.3) on NK cells. Hsp90 inhibition significantly reduced CD4 protein expression on T lymphocytes at both the cell surface and intracellular level, which was shown to be associated with aberrant regulation of Src-kinase p56Lck. Downregulation of the Ags triggered by Hsp90 inhibition on CD3+ T lymphocytes, both in CD4+ and CD8+ T cell subsets, was associated with a disruption in their cellular activation, proliferation, and/or IFN-γ production, when the inhibition occurred either in activated or inactivated cells. In addition, downregulation of key activating receptors on NK cells following Hsp90 inhibition resulted in decreased cytotoxicity against tumor cells. Therefore, these observations demonstrate the need to closely monitor immune function in patients being treated with a Hsp90 inhibitor and may provide a potential therapeutic application in autoimmune diseases.

ClinicalTrials.gov identifier: NCT01718899.

X-box binding protein 1 (XBP-1) is a transcription factor is essential for the differentiation of plasma cells and the unfolded protein response. XBP-1 is significantly up-regulated in myeloma cells compared to both normal plasma cells and B-cells. Selective and specific requirement of XBP-1 for differentiation of B cells to plasma cells and its further up-regulation in multiple myeloma (MM) cells makes it a promising target for immunotherapy directed at MM. We have evaluated XBP-1 as a target antigen to develop MM-specific immunotherapy. In order to generate XBP-1 antigen-specific cytotoxic T lymphocytes (CTLs), we have identified HLA-A2-specific peptides derived from non-spliced (short form) and spliced (long form) XBP-1 proteins. We have further modified the peptide by altering one amino acid, which is critical for HLA-A2 affinity, to obtain epitope, which has higher stability to HLA-A2 clefts. The modified XBP-1 peptides were able to evoke higher levels of IFN-g release compared to native peptides and were able to induce CTLs highly specific to MM, demonstrated by Calcein-released cytotoxicity assay. Cytotoxic activity of these XBP1 peptide-specific-CTLs against U266, a HLA-A2+/XBP-1+ MM cell line was 60% and 79% by CTLs stimulated by non-spliced peptide and 69% and 88% by CTLs stimulated by spliced peptide at Effector:Target ratios of 20:1 and 60:1, respectively. The CTLs did not lyse HLA-A2− or XBP-1− cells. These results were further confirmed by the novel CD107 cytotoxicity assay, which detects MM-specific cytolytic CD8+ T cells by flow-cytometric analyses. The CTLs generated with XBP-1 peptide displayed distinct phenotypes, showing high CD69+CD45RO+ cell population (62% in non-spliced peptide-CTLs, 67% in spliced peptide-CTLs, 4% in unstimulated control-CTLs) and CD45RA+CCR7+ cell population (1% in non-spliced peptide-CTLs, 1% in spliced peptide-CTLs, 24% in unstimulated control-CTLs). In conclusion, we report the identification of highly immunogenic heteroclitic XBP-1 epitopes that have ability to generate MM-specific CTLs. Confirmation of in vivo activity of these CTLs in SCID mouse model is currently underway prior to its evaluation in clinical studies.

We evaluated a cocktail of HLA-A2-specific peptides including heteroclitic XBP1 US184-192 (YISPWILAV), heteroclitic XBP1 SP367-375 (YLFPQLISV), native CD138260-268 (GLVGLIFAV) and native CS1239-247 (SLFVLGLFL), for their ability to elicit multipeptide specific cytotoxic T lymphocytes (MP-CTL) using T cells from smoldering multiple myeloma (SMM) patients. Our results demonstrate that MP-CTL generated from SMM patients’ T cells show effective anti-MM responses including CD137 (4-1BB) upregulation, CTL proliferation, IFN-γ production, and degranulation (CD107a) in an HLA-A2-restricted and peptide-specific manner. Phenotypically, we observed increased total CD3+CD8+ T cells (>80%) and cellular activation (CD69+) within the memory SMM MP-CTL (CD45RO+/CD3+CD8+) subset after repeated multipeptide stimulation. Importantly, SMM patients could be categorized into distinct groups by their level of MP-CTL expansion and anti-tumor activity. In high responders, the effector memory (CCR7-CD45RO+/CD3+CD8+) T cell subset was enriched, while the remaining responders’ CTL contained a higher frequency of the terminal effector (CCR7-CD45RO-/CD3+CD8+) subset. These results suggest that this multipeptide cocktail has the potential to induce effective and durable memory MP-CTL in SMM patients. Therefore, our findings provide the rationale for clinical evaluation of a therapeutic vaccine to prevent or delay progression of SMM to active disease.