Abnormalities of the Immune System and Inflammation in MDS Pathogenesis. Shahram Kordasti MSc(Med Imm), MD, PhD Department of Haematological Medicine

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1 Abnormalities of the Immune System and Inflammation in MDS Pathogenesis Shahram Kordasti MSc(Med Imm), MD, PhD Department of Haematological Medicine

2 What is the role of immune system in MDS? Oligoclonal expansion of CD8 + T-cells that resolve following IST in responsive patients. Persistent oligoclonality in relapsing and non responsive patients Cytokines Elaine M Sloand ( ) CD8+ T cell clonal expansion c-myc Surnivin CDK1

3 CD4 + T cells in BMFs 52 patients (30 men, 22 women) median age of 64.5 years Low risk n=18, Intermediate risk n=25, High risk n=9 Cytogenetic: Normal 49%, 5q- 17%, Complex 16%, Other 18% Kordasti, et al Carlsten, M et al, Leukemia 2010

4 Tregs and MDS prognosis Higher number of Tregs correlates with poorer prognosis in lower risk MDS Josephine D. Kahn, et al Haematologica 2015

5 Immunogenic cell death (ICD) Early events Late cellular response

6 Immunogenic mutations (IMs) Absence of Neoantigen: Smouldering inflammation which may contribute into genomic instability and promote malignant transformation. Combination of ICD and IMs is likely to form an effective antigen specific immune response Hacklet al. Nature Reviews (2016)

7 Effects of chronic inflammation Sallman et al Front. Onc 2016 Noemi A. Zambetti, et al. Cell Stem Cell, 2016

8 MDSCs in MDS 42 pre-treatment MDS patients (median age 69.5 y) was analysed. Twenty-three patients also had bone marrow samples available for analysis. Chen et al 2013 Kittang A, et al, Oncoimmunology 2016

9 Somatic mutation related neoantigens Can be predicted based on the type of mutation and HLA-type Daniel S. Chen1 & Ira Mellman Nature 541, , 2017)

10 Impact of neoantigens on survival in MDS Characteristics Patients with neoantigens Patients with no neoantigens P value Number Combined UK & German cohort Age in years (median) NS Sex (Male / Female) 86 /43 31 /20 NS Type of MDS NS Control + Neoag + WT1 RARS 10 6 RCUD 7 1 IPSS Categories RCMD Isolated 5q- 1 2 RAEB Other NS Percent survival 50 IFN-y Low Intermediate Intermediate High 6 4 Number of mutations (median) P< Follow up Progression to AML Vital Status Yes 26/129 (20.1%) 8/51 (15.7%) NS Neoantigen (n=129) No Neoantigen (n=51) Dead 28/129 15/51 NS Collaboration with Prof Uwe Platzbecker P<0.05. HR ( ) Tom Coats, et all, ASH 2017,

11 Multivariate factors influence tolerance and immunity Patient A Patient B Cancer immune set point Daniel S. Chen & Ira Mellman Nature 541, , 2017)

12 Summary (1) Immune response in MDS consists of two main components: o Immunogenic cell death and subsequent cellular response. There is an immune response switch from AA/LR MDS to HR MDS and AML. CD4 + T cells are regulated by the inflammatory environment. CD4 + T cells could orchestrate the overall immune response in MDS and Tregs play an important role in defining immune set point.

13 CD4 + T cells plasticity Manual gating (Expert gating) CD45-RA Tr I Tr III Tr II FOXP3

14 Trends in Immunology , DOI: ( /j.it ) Cytome (CyTOF; mass cytometry)

15 Identification of Treg subset by automated clustering. Kordasti et al. Blood 2016;128:

16 Function and ontogeny of Treg subpopulations. Kordasti et al. Blood 2016;128:

17 Tregs in MDS CD4 T cells CD8 T cells DCs B cells CD25 FOXP3 CD95 Tregs Coats et al, unpublished

18 TotalTreg Abundance of Tregs HD vs MDS TregA TregB **** NS % CD4+ unstim % CD4+ unstim % CD4+ unstim HD MDS Treg B Treg A M 0 HD MDS % CD4+ unstim HD ** Non-RAEB * RAEB CD4+ CD127lo CD25hi FOXP3hi Helios hi CTLA4hi % CD4+ unstim HD Non-RAEB TregA ** NS RAEB CD4+ CD127lo CD25hi FOXP3hi Helios hi, CTLA4hi, CD95 Lo, CCR4 lo, CD45RO lo, (Treg A) % CD4+ unstim Treg B Treg A H TregB Non-RA CD4+ CD127lo CD25hi FOXP3hi Helios hi CTLA4hi, CD95hi, CCR4hi, CD45ROhi (TregB) * RA

19 Why Treg B are low? Apoptotic + dead cells post-fas- L/Apoptotic + dead cells pre-fas-l After adding FAS-L (5 µg/ml) for 5 h, Treg B (and CD4 + ) have a higher rate of apoptotic and dead cells than Treg A. Benedetta Costantini, ASH 2017

20 Immune check point DCs Patient 1 Tregs CD4+ T cells B cells CD8+ T cells DCs Patient 2 Tregs CD4+ T cells B cells CD8+ T cells PD-1+ PD-1++ Coats et al, ASH 2017

21 PD-1 ++ cluster of immune cells D- CTLA-4 Coats et al, unpublished PD-1 PD-1 HR-MDS LR-MDS HD CD CD45RA CD11b CD16 CD19 CD123 CCR6 CD154 CD103 CD161 CD8a CD69 CD127 CXCR3 Tbet CD25 CCR4 CD38. CCR7 FOXP3 HLADR CD28 GATA3 CXCR4 CD3 CD62L CD7 CD27 CD4 CD45RO CTLA.4 Helios CD279 CD95 RAEB CD CD45RA CD11b CD16 CD19 CD123 CCR6 CD154 CD103 CD161 CD8a CD69 CD127 CXCR3 Tbet CD25 CCR4 CD38. CCR7 FOXP3 HLADR CD28 GATA3 CXCR4 CD3 CD62L CD7 CD27 CD4 CD45RO CTLA.4 Helios CD279 CD95 Non RAEB CD CD45RA CD11b CD16 CD19 CD123 CCR6 CD154 CD103 CD161 CD8a CD69 CD127 CXCR3 Tbet CD25 CCR4 CD38. CCR7 FOXP3 HLADR CD28 GATA3 CXCR4 CD3 CD62L CD7 CD27 CD4 CD45RO CTLA.4 Helios CD279 CD95 Expression HD Expression RAEB Expression Non RAEB

22 Treg signature in the presence of neoantigens Neoantigen - Neoantigen + Diggins et al., "At the Bench: Precision Medicine with Single Cell Mass Cytometry," under review Coats et al, unpublished

23 Treg subset identification in diagnostic lab Dr Robin Ireland Mr Alan Dunlop CD45, CD3, CD4, CD25, CD127, CD46RA, CCR4, CD95 Treg A Treg B

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26 DNMTi Induce Interferon Responses Aza-Upregulated Viral Defense Genes Are Significantly Correlated with ERVs in Primary Tumors and Correlate with Sensitivity to Immune Therapy Chiapinelli et al Cell 2015 Costantini B et al. Haematologica 2013;98:

27 Summary (2) Using a less biased and unsupervised method, we have identified 2 distinct subpopulation of Tregs. The frequency of these subpopulation could predict response to IST in AA and potentially disease progression in MDS. Inflammatory environment (particularly FAS mediated) affects the composition of Tregs. Predictor for response to immunotherapy (i.e. CPI)?

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