Tumor Immunology and Tumor Evolution: Intertwined Histories

Background As consolidation therapy for acute myeloid leukemia (AML), allogeneic hematopoietic stem-cell transplantation provides a benefit in part by means of an immune-mediated graft-versus-leukemia effect. We hypothesized that the immune-mediated selective pressure imposed by allogeneic transplantation may cause distinct patterns of tumor evolution in relapsed disease. Methods We performed enhanced exome sequencing on paired samples obtained at initial presentation with AML and at relapse from 15 patients who had a relapse after hematopoietic stem-cell transplantation (with transplants from an HLA-matched sibling, HLA-matched unrelated donor, or HLA-mismatched unrelated donor) and from 20 patients who had a relapse after chemotherapy. We performed RNA sequencing and flow cytometry on a subgroup of these samples and on additional samples for validation. Results On exome sequencing, the spectrum of gained and lost mutations observed with relapse after transplantation was similar to the spectrum ...

https://www.nejm.org/doi/pdf/10.1056/NEJMoa1808777

Immune Escape of Relapsed AML Cells after Allogeneic Transplantation

Activation of NKT cells using the glycolipid α-galactosylceramide (α-GalCer) has availed many investigations into their immunoregulatory and therapeutic potential. However, it remains unclear how they respond to stimulation in vivo, which costimulatory pathways are important, and what factors (e.g., Ag availability and activation-induced cell death) limit their response. We have explored these questions in the context of an in vivo model of NKT cell dynamics spanning activation, population expansion, and subsequent contraction. Neither the B7/CD28 nor the CD40/CD40L costimulatory pathway was necessary for cytokine production by activated NKT cells, either early (2 h) or late (3 days) after initial stimulation, but both pathways were necessary for normal proliferative expansion of NKT cells in vivo. The proapoptotic Bcl-2 family member Bim was necessary for normal contraction of the NKT cell population between days 3–9 after stimulation, suggesting that the pool size is regulated by apoptotic death, similar to that of conventional T cells. Ag availability was not the limiting factor for NKT cell expansion in vivo, and a second α-GalCer injection induced a very blunted response, whereby cytokine production was reduced and further expansion did not occur. This appeared to be a form of anergy that was intrinsic to NKT cells and was not associated with inhibitory NK receptor signaling. Furthermore, NKT cells from mice prechallenged with α-GalCer in vivo showed little cytokine production and reduced proliferation in vitro. In summary, this study significantly enhances our understanding of how NKT cells respond to primary and secondary antigenic challenge in vivo.

NKT cell stimulation with glycolipid antigen in vivo

The 70-kDa heat shock proteins (HSP70s) are abundantly present in cancer, providing malignant cells selective advantage by suppressing multiple apoptotic pathways, regulating necrosis, bypassing cellular senescence program, interfering with tumor immunity, promoting angiogenesis and supporting metastasis. This direct involvement of HSP70 in most of the cancer hallmarks explains the phenomenon of cancer "addiction" to HSP70, tightly linking tumor survival and growth to the HSP70 expression. HSP70 operates in different states through its catalytic cycle, suggesting that it can multi-function in malignant cells in any of these states. Clinically, tumor cells intensively release HSP70 in extracellular microenvironment, resulting in diverse outcomes for patient survival. Given its clinical significance, small molecule inhibitors were developed to target different sites of the HSP70 machinery. Furthermore, several HSP70-based immunotherapy approaches were assessed in clinical trials. This review will explore different roles of HSP70 on cancer progression and emphasize the importance of understanding the flexibility of HSP70 nature for future development of anti-cancer therapies.

https://www.mdpi.com/2073-4409/9/3/587/pdf

HSP70 Multi-Functionality in Cancer.

Extracellular heat shock proteins (ex-HSPs) have been found in exosomes, oncosomes, membrane surfaces, as well as free HSP in cancer and various pathological conditions, also known as alarmins. Such ex-HSPs include HSP90 (α, β, Gp96, Trap1), HSP70, and large and small HSPs. Production of HSPs is coordinately induced by heat shock factor 1 (HSF1) and hypoxia-inducible factor 1 (HIF-1), while matrix metalloproteinase 3 (MMP-3) and heterochromatin protein 1 are novel inducers of HSPs. Oncosomes released by tumor cells are a major aspect of the resistance-associated secretory phenotype (RASP) by which immune evasion can be established. The concepts of RASP are: (i) releases of ex-HSP and HSP-rich oncosomes are essential in RASP, by which molecular co-transfer of HSPs with oncogenic factors to recipient cells can promote cancer progression and resistance against stresses such as hypoxia, radiation, drugs, and immune systems; (ii) RASP of tumor cells can eject anticancer drugs, targeted therapeutics, and immune checkpoint inhibitors with oncosomes; (iii) cytotoxic lipids can be also released from tumor cells as RASP. ex-HSP and membrane-surface HSP (mHSP) play immunostimulatory roles recognized by CD91+ scavenger receptor expressed by endothelial cells-1 (SREC-1)+ Toll-like receptors (TLRs)+ antigen-presenting cells, leading to antigen cross-presentation and T cell cross-priming, as well as by CD94+ natural killer cells, leading to tumor cytolysis. On the other hand, ex-HSP/CD91 signaling in cancer cells promotes cancer progression. HSPs in body fluids are potential biomarkers detectable by liquid biopsies in cancers and tissue-damaged diseases. HSP-based vaccines, inhibitors, and RNAi therapeutics are also reviewed.

/pdf/roles-of-extracellular-hsps-as-biomarkers-in-immune-yxqkthh18l.pdf

Roles of Extracellular HSPs as Biomarkers in Immune Surveillance and Immune Evasion

The success of haematopoietic stem cell transplantation (HSCT) lies with the ability of the engrafting immune system to remove residual leukaemia cells via a graft versus leukaemia effect, caused either spontaneously post HSCT or via donor lymphocyte infusion. Graft versus leukaemia effects can also be initiated by allogeneic mismatched NK cells, antigen specific T cells and activated dendritic cells of leukemic origin. The history and further application of this graft versus leukaemia effect and the main mechanisms will be discussed and reviewed in this chapter.

/pdf/graft-versus-leukemia-effect-following-hematopoietic-stem-1uzl1lb95v.pdf

Graft-versus-Leukemia Effect Following Hematopoietic Stem Cell Transplantation for Leukemia.

Previous studies from Multhoff and colleagues reported that plasma membrane Hsp70 acts as a tumour-specific recognition structure for activated NK cells, and that the incubation of NK cells with Hsp70 and/or a 14-mer peptide derived from the N-terminal sequence of Hsp70 (TKDNNLLGRFELSG, TKD, aa 450-463) plus a low dose of IL-2 triggers NK cell proliferation and migration, and their capacity to kill cancer cells expressing membrane Hsp70. Herein, we have used flow cytometry to determine the influence of in vitro stimulation of peripheral blood mononuclear cells from healthy individuals with IL-2 or IL-15, either alone or in combination with TKD peptide on the cell surface expression of CD94, NK cell activatory receptors (CD16, NK2D, NKG2C, NKp30, NKp44, NKp46, NKp80, KIR2DL4, DNAM-1 and LAMP1) and NK cell inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2 and NKR-P1A) by CD3+CD56+ (NKT), CD3+CD4+, CD3+CD8+ and CD19+ populations. NKG2D, DNAM-1, LAMP1 and NKR-P1A expression was upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD in NKT, CD8+ T cells and B cells. CD94 was upregulated in NKT and CD8+ T cells. Concurrently, an increase in a number of CD8+ T cells expressing LIR1/ILT-2 and CD4+ T cells positive for NKR-P1A was observed. The proportion of CD8+ T cells that expressed NKG2D was higher after IL-2/TKD treatment, when compared with IL-2 treatment alone. In comparison with IL-15 alone, IL-15/TKD treatment increased the proportion of NKT cells that were positive for CD94, LAMP1 and NKRP-1A. The more potent effect of IL-15/TKD on cell surface expression of NKG2D, LIR1/ILT-2 and NKRP-1A was observed in B cells compared with IL-15 alone. However, this increase was not of statistical significance. IL-2/TKD induced significant upregulation of LAMP1 in CD8+ T cells compared with IL-2 alone. Besides NK cells, other immunocompetent cells present within the fraction of peripheral blood mononuclear cells were influenced by the treatment with low-dose interleukins themselves or in combination with hsp70 derived (TKD) peptide.

/pdf/influence-of-in-vitro-il-2-or-il-15-alone-or-in-combination-scs5y1a8ym.pdf

Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp 70 Derived 14-Mer Peptide (TKD) on the Expression of NK Cell Activatory and Inhibitory Receptors on Peripheral Blood T Cells, B Cells and NKT Cells.

Alloreactive CD4+ T cells play a central role in allograft rejection. However, the post‐transcriptional regulation of the effector program in alloreactive CD4+ T cells remains unclear. N6‐methyladenosine (m6A) RNA modification is involved in various physiological and pathological processes. Herein, we investigated whether m6A methylation plays a role in the allogeneic T‐cell effector program. m6A levels of CD4+ T cells from spleens, draining lymph nodes and skin allografts were determined in a skin transplantation model. The effects of a METTL3 inhibitor (STM2457) on CD4+ T‐cell characteristics including proliferation, cell cycle, cell apoptosis and effector differentiation were determined after stimulation of polyclonal and alloantigen‐specific (TEa; CD4+ T cells specific for I‐Eα52‐68) CD4+ T cells with α‐CD3/α‐CD28 monoclonal antibodies and cognate CB6F1 alloantigen, respectively. We found that graft‐infiltrating CD4+ T cells expressed high m6A levels. Administration of STM2457 reduced m6A levels, inhibited T‐cell proliferation and suppressed effector differentiation of polyclonal CD4+ T cells. Alloreactive TEa cells challenged with 40 μm STM2457 exhibited deficits in T‐cell proliferation and T helper type 1 cell differentiation, a cell cycle arrest in the G0 phase and elevated cell apoptosis. Moreover, these impaired T‐cell responses were associated with the diminished expression levels of transcription factors Ki‐67, c‐Myc and T‐bet. Therefore, METTL3 inhibition reduces the expression of several key transcriptional factors for the T‐cell effector program and suppresses alloreactive CD4+ T‐cell effector function and differentiation. Targeting m6A‐related enzymes and molecular machinery in CD4+ T cells represents an attractive therapeutic approach to prevent allograft rejection.

METTL3 inhibition reduces N6‐methyladenosine levels and prevents allogeneic CD4+ T‐cell responses

The long non-coding RNA (lncRNA) PVT1 was first found to activate variant translocations in the plasmacytoma of mice. Human lncPVT1 is located on chromosome 8q24.21, at the same locus as the well-known MYC oncogene. LncPVT1 has been found to promote the progression of various malignancies. Chemoresistance and radioresistance seriously affect tumor treatment efficacy and are associated with the dysregulation of physiological processes in cancer cells, including apoptosis, autophagy, stemness (for cancer stem cells, CSC), hypoxia, epithelial–mesenchymal transition (EMT), and DNA damage repair. Previous studies have also implicated lncPVT1 in the regulation of these physiological mechanisms. In recent years, lncPVT1 was found to modulate chemoresistance and radioresistance in some cancers. In this review, we discuss the mechanisms of lncPVT1-mediated regulation of cellular chemoresistance and radioresistance. Due to its high expression in malignant tumors and sensitization effect in chemotherapy and radiotherapy, lncPVT1 is expected to become an effective antitumor target and chemotherapy and radiotherapy sensitizer, which requires further study.

/pdf/long-non-coding-rna-pvt1-a-promising-chemotherapy-and-1z1ncpiy.pdf

Long non-coding RNA PVT1: A promising chemotherapy and radiotherapy sensitizer

Allogeneic CD8+ T cells are prominently involved in allograft rejection, but how their effector differentiation and function are regulated at a transcriptional level is not fully understood. Herein, we identified the basic leucine zipper ATF-like transcription factor (BATF) as a key transcription factor that drives the effector program of allogeneic CD8+ T cells. We found that BATF is highly expressed in graft-infiltrating CD8+ T cells, and its ablation in CD8+ T cells significantly prolonged skin allograft survival in a fully MHC-mismatched transplantation model. To investigate how BATF dictates allogeneic CD8+ T cell response, BATF–/– and wild-type (WT) CD8+ T cells were mixed in a 1:1 ratio and adoptively transferred into B6.Rag1 –/– mice 1 day prior to skin transplantation. Compared with WT CD8+ T cells at the peak of rejection response, BATF–/– CD8+ T cells displayed a dysfunctional phenotype, evident by their failure to differentiate into CD127–KLRG1+ terminal effectors, impaired proliferative capacity and production of pro-inflammatory cytokines/cytotoxic molecules, and diminished capacity to infiltrate allografts. In association with the failure of effector differentiation, BATF–/– CD8+ T cells largely retained TCF1 expression and expressed significantly low levels of T-bet, TOX, and Ki67. At the memory phase, BATF-deficient CD8+ T cells displayed impaired effector differentiation upon allogeneic antigen re-stimulation. Therefore, BATF is a critical transcriptional determinant that governs the terminal differentiation and memory responses of allogeneic CD8+ T cells in the transplantation setting. Targeting BATF in CD8+ T cells may be an attractive therapeutic approach to promote transplant acceptance.

Shuang Li

Papers

Graft-versus-Leukemia Effect Following Hematopoietic Stem Cell Transplantation for Leukemia.

Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp 70 Derived 14-Mer Peptide (TKD) on the Expression of NK Cell Activatory and Inhibitory Receptors on Peripheral Blood T Cells, B Cells and NKT Cells.

METTL3 inhibition reduces N6‐methyladenosine levels and prevents allogeneic CD4+ T‐cell responses

Long non-coding RNA PVT1: A promising chemotherapy and radiotherapy sensitizer

Ablation of BATF Alleviates Transplant Rejection via Abrogating the Effector Differentiation and Memory Responses of CD8+ T Cells