Multiple Myeloma and Colorectal Cancer

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1 Multiple Myeloma and Colorectal Cancer From Systems Immunology to Single Cells Leo Hansmann Mark M. Davis Lab Department of Microbiology&Immunology Stanford University

2 Multiple Myeloma Monoclonal disease of plasma cells in the bone marrow The myeloma cells produce monoclonal immunoglobulin and/or light chains Develops from pre cancer diseases called monoclonal gammopathy of undetermined significance (MGUS) or asymptomatic myeloma (AM) Malignant plasma cells are not present in the peripheral blood UT Houston Morning Report Swerdlow et al.

called monoclonal gammopathy of undetermined significance (MGUS) or asymptomatic myeloma (AM)

3 CyTOF phenotyping panel surface markers TCR αβ TCR γδ CD3 CD4 CD8 CD7 CD28 CD57 CD25 CD19 CD24 CD10 CD5 CD27 IgD IgM CD38 CD5 CD14 CD16 CD11c CD123 CD1c CD56 HLA DR CD66 CD45 CD45RA CD45RO CCR7 functional/ intracellular markers CD20 IL4 IFNγ IL17A IFNα TNFα unstimulated PMA/Ionomycin R848 CpG2216/Dotap Dota malemide live/dead stain In total 39 markers stimuli conditions

CD45RO CCR7 functional/ intracellular markers CD20 IL4 IFNγ IL17A IFNα TNFα unstimulated

4 PBMC phenotyping

5 Significant population frequency differences between study groups

6 Diversity of PBMC phenotypes in the peripheral blood MM (treatment naïve) MM (no treatment during the last 6M) pre cancer healthy

7 PCA separates MM from pre cancer and healthy controls MM (treatment naïve) MM (no treatment during the last 6M) pre cancer healthy

8 CD38 + CD24 lo CD27 + B cells contribute to PC1 in unstimulated cells and after PMA/Ionomycin stimulation

9 CD38 + CD24 lo CD27 + B cells in MM peripheral blood

10 CD38 + CD24 lo CD27 + B cells are uniquely expanded in MM peripheral blood

11 CD38 + CD24 lo CD27 + B cell expansion is associated with multiple myeloma activity

12 Citrus analysis of PBMC from MM patients and healthy controls

13 CD27 is significantly differentially expressed in PBMC subsets of MM patients when compared to healthy controls ** FDR<0.01

14 CD57 is significantly differentially expressed in PBMC subsets of MM patients when compared to healthy controls

15 Single cell sorting of CD24 lo CD38 + CD27 + B cells pre gated on CD19 + CD20 + B cells

16 Immunoglobulin light chain genes of CD24 lo CD38 + CD27 + B cells are polyclonal and show somatic mutations

17 Conclusions Multiple Myeloma CyTOF analysis of the immunological landscape in the states of cancer and pre cancer allows the detection of unique cancer associated phenotypes in human peripheral blood CD24 lo CD38 + CD27 + B cells are uniquely expanded in the peripheral blood of multiple myeloma patients The expansion of CD24 lo CD38 + CD27 + B cells is associated with multiple myeloma activity as measured by serum immunoglobulin light chain levels Single cell sequencing confirmed polyclonality and V gene mutations in these B cells most likely characterizing them as an expanded memory cell population with aberrant CD24 and CD38 expression Phosphorylation responses after B cell receptor or TLR 9 stimulation confirm the memory character of CD24 lo CD38 + CD27 + B cells

activity as measured by serum immunoglobulin light chain levels Single cell sequencing confirmed polyclonality and V gene mutations in these B cells most likely characterizing them as an

18 The single cell approach to TCR diversity and T cell plasticity a Single-Cell Sort Preamp Reaction: TCR primers + Cytokine primers + Transcription factor primers 1 TCR V (D) J C One-Step RT PCR with combined TCR +phenotype primers 1 Cytokine/ Transcription Factors 1 1 Reaction 1 CDR3 TCR sequencing, reactions 1 2 with barcoding Nested cytokine PCR + barcoding Nested PCR with combined TCR primers Nested PCR with combined phenotyping primers V (D) J C Reaction 2 Combine, Deep Sequence CDR3 Barcoding + Paired-End Addition for Deep Sequencing (TCR) Barcoding + Paired-End Addition for Deep Sequencing (phenotyping) V (D) J C Reaction 3 ~ 90% TCR sequencing efficiency CDR3 Combine, Purify, Sequence with Illumina MiSeq

combined TCR primers Nested PCR with combined phenotyping primers 2 1 2 1 V (D) J C 1 2 2 1 Reaction 2 Combine, Deep Sequence CDR3 Barcoding + Paired-End Addition for Deep Sequencing (TCR)

19 Single T cell phenotyping

20 TILs from colorectal cancer show clonal expansion and distinct immune phenotypes Han et al. Nat Biotechnol 2014

21 TILs are phenotypically different from normal colon or peripheral blood T cells Han et al. Nat Biotechnol 2014

22 Conclusions Next generation sequencing allows the detailed identification of immune phenotypes along with TCR sequences in single tumor infiltrating lymphocytes CD4 + T cells infiltrating colorectal cancer show clonal expansion and distinct phenotypes in contrast to CD4 + T cells from non cancerous colon tissue Mass Cytometry and next generation sequencing allow immune phenotyping in extraordinary depth leading to The Identification of cancer associated immune phenotypes in peripheral blood and The detection of uniquely expanded T cell clones in the cancer microenvironment

23 Acknowledgements Davis Lab Mark Davis and Yueh hsiu Chien Arnold Han William O Gorman Cesar Angel Lopez Jacob Glanville All other Davis Lab members Robinson Lab Lisa Blum Scalfone Chia Hsin Ju Stanford Cancer Center Michaela Liedtke Lawrence Okumoto Stanford Cytogenetics Dana Bangs Vanderbilt Jonathan Irish

specific B cells Humoral immunity lymphocytes antibodies B cells bone marrow Cell-mediated immunity: T cells antibodies proteins

specific B cells Humoral immunity lymphocytes antibodies B cells bone marrow Cell-mediated immunity: T cells antibodies proteins Adaptive Immunity Chapter 17: Adaptive (specific) Immunity Bio 139 Dr. Amy Rogers Host defenses that are specific to a particular infectious agent Can be innate or genetic for humans as a group: most microbes