Immuno-Hematology

Our group at the Dept of Experimental Immunology has studied apoptosis regulation in normal and pathological immune cells since 2002. Since 2014, we are expanding our research lines to include wider fundamental and translational aspects in Immuno-Hematology.

We study human and (genetically modified) murine tissues in two themes of research, centered around Chronic Lymphocytic Leukemia. Our projects are sponsored by NWO, DCF, the European Union, private funding agencies, the AMC foundation and we collaborate in sponsored research agreements with pharmaceutical companies.

In addition, three NWO Veni laureates started their projects in 2014, of which two have finished: Felix Wensveen studied selection of high affinity T and B cells, and Victor Peperzak investigated regulation of pro-survival Mcl-1 in malignant B cells, and has moved to the University of Utrecht end of 2015. Rianne van der Windt studies Immunometabolism (see dedicated sections below).

Theme 1. Hemato-oncological work on Chronic Lymphocytic Leukemia (CLL), in close collaboration with hematologist dr Arnon Kater. We study three interrelated areas:

a) CLL genetics and molecular diagnostics (PhDs Alexander Leeksma and Zhenghao Chen, technicians Ingrid Derks and Dieuwertje Luijks)

b) The leukemic microenvironment and novel drugs (Postdoc Erik Slinger, PhDs Fabio Brocco, Marco Haselager, Raquel Delgado, technican Hanneke ten Burg)

c) Interaction between the immune system and CLL (PhDs Iris de Weerdt, Tom Hofland, Anne Martens, technican Sanne Terpstra and MD Sanne Tonino)

Theme 2 Shaping the immune response. We study the adaptive immune response, in particular the function of B and T lymphocytes. The ultimate goal of this research is to apply the insight obtained in novel vaccination strategies and immunotherapies. We focus on two topics:

a) Clonal diversity and affinity (Veni laureate Felix Wensveen)

b) Cellular metabolism (PhD Armando van Bruggen, Veni laureate Rianne van der Windt and MD Arnon Kater)

Theme 1 Hemato-oncological work on CLL

a) CLL genetics and molecular diagnostics. Mutations or deletions of the tumor suppressor p53 or its upstream kinase ATM are prime determinants of poor prognosis in CLL. We have implemented an in-house RT-MLPA probe kit to detect functional defects in these pathways (1-3). These studies were performed in close collaboration with the European Research Initiative on CLL (ERIC). In addition, we are analysing the novel deleterious mutations that have been described in recent years in CLL in Notch, SF3B1 and MyD88 genes. We have reported our findings on the effects of SF3B1 mutation on the DNA damage response (4), and will continue this work by Alexander Leeksma. A novel project will investigate the effects of CLL cancer mutations on the metabolism of the leukemic cells (Zhenghao Chen).

b) The leukemic microenvironment and novel drugs. Most research on CLL is done on cells obtained from peripheral blood of patients. Yet, these cells do not represent the cycling CLL cells that reside in so-called proliferation centres in lymph nodes and bone marrow. There, they are protected from apoptosis and more resistant against cancer therapeutics. It is generally assumed that chemo-resistant clones from those sites cause the inevitable relapses that occur in this disease.

  • We study peripheral blood CLL cells in co-culture systems that mimick the chemoresistant and/or proliferative situation in lymph nodes using CD40 and cytokines which represent T cell signals (5).
  • A recently finished project has studied other components of the microenvironment, such as the effects of monocytic cells on CLL cells (6, 7).
  • Two novel projects will address important signaling pathways (BCR, CD40, TLR9) from the cell surface to NF-kB and CLL survival (Marco Haselager, Raquel Delgado)
  • The best studied model for CLL is the TCL-1 transgenic mouse. The BH3-only protein Noxa is overexpressed in CLL (7), and its expression is lower in lymph node than in peripheral blood (8), which suggests that is linked with survival and/or drug sensitivity. We found decreased lifespan of Tcl-1/NoxaKO mice (see figure 1), confirming a role for Noxa in CLL pathobiology (8). In addition, together with various pharmaceutical companies, we have started to investigate combined Ibrutinib and Venetoclax treatment in the TCL1 model.
  • Novel treatments. Pre-clinical studies to assess the efficacy of novel drugs and drug combinations that might circumvent chemoresistance. Compounds we are currently studying various BH3 mimetics (see figure 2), kinase inhibitors, novel CD20 and CD40 antibodies (9-11).
  • Side studies of Clinical trials– to be updated soon!

Figure 1:

Crossing NoxaKO mice with CLL-prone Tcl-1 transgenic mice shows accelerated mortality in Noxa/KO/Tcl-1 mice. (Erik Slinger unpublished)   Crossing NoxaKO mice with CLL-prone Tcl-1 transgenic mice shows accelerated mortality in Noxa/KO/Tcl-1 mice. (Erik Slinger unpublished)

Figure 2:

CLL cells cultured under conditions intended to mimic the lymph node environment become insensitive for ABT-737, a so-called BH3 mimetic drug that targets Bcl-2 and Bcl-XL. (Rachel Thijssen unpublished)   CLL cells cultured under conditions intended to mimic the lymph node environment become insensitive for ABT-737, a so-called BH3 mimetic drug that targets Bcl-2 and Bcl-XL. (Rachel Thijssen unpublished)

c) Interaction between the immune system and CLL. CLL is characterized by an immune dysfunction, which is presumed to result from aberrant functioning of both T cells as well as (leukemic) B cells. These conclusions are based on non-specific activation experiments of lymphocytes in vitro. In contrast, we recently demonstrated that the function of virus-specific T cells was still intact in CLL samples (see figure 3) (12). This demonstrates that the changes in T cell characteristics in CLL are more heterogeneous than presently assumed.

Several new immunotherapeutic strategies for CLL (e.g. immune checkpoint blockade and adoptive transfer of chimeric antigen receptor (CAR) T-cells) rely on T-cell mediated cell death. Although immune checkpoint blockade and CAR T-cell therapy have shown promising results in Non-Hodgkin lymphoma’s, first trials in CLL have shown only moderate responses thus far. The acquired T-cell dysfunction is generally considered to be responsible for the hampered effectivity of T-cell therapies in CLL. Therefore, understanding the biology of this acquired immune dysfunction in CLL, finding means to restore T-cell function, and identifying populations with retained functionality may provide solutions to improve the efficacy of these novel therapies in CLL patients. We have several ongoing projects on this theme:

  • We aim to thoroughly analyze the bidirectional interactions between CLL and T cells in the context of chronic viral infections, by studying how CLL affects the composition and function of (virus-specific) T cells and why various virus-specific T cells may be differentially affected by CLL.
  • We study subsets of cytotoxic immune cells (including gd T cells and NK cells) with specific characteristics which may make them excellent mediators in immune therapy.
  • In collaboration with the VUmc we explore novel forms of antibodies (nanobodies) for use in immunetherapy for CLL

Figure 3:

Interferon-γ production by CMV-specific T cells from healthy controls (HC) or CLL patients is comparable. CLL B cells are poor antigen presenters. (From te Raa et al Blood 2013)   Interferon-γ production by CMV-specific T cells from healthy controls (HC) or CLL patients is comparable. CLL B cells are poor antigen presenters. (From te Raa et al Blood 2013)

Theme 2 Shaping the immune response – ongoing work

a) Clonal diversity and affinity – Felix Wensveen. A key feature of the adaptive immune system is that it consists of millions of clones, each unique in its ability to bind antigen through its dedicated receptor. This system ensures an ability to recognize many different molecular structures of pathogens. However, its biological implication is that any given antigen activates many different clones which differ in their specificity and efficiency towards the invading pathogen. Systems must therefore be in place to select cells based on their antigen-affinity, in order to prevent wasting resources on cells of suboptimal efficiency. In B cells, we investigated how antigen-affinity selects for high-affinity clones in the first days after B cell activation. We found that B cell receptor affinity correlates with induction of the receptor for BAFF, an important cytokine that promotes B cell survival. BAFF stabilizes the pro-survival protein Mcl-1 through the PI3K signalling pathway. High-affinity B cells are therefore positively selected due to an increased ability to sustain Mcl-1 levels in response to BAFF. For memory T cells, increased diversity may in fact be beneficial in a recall response. T cells with sub-optimal specificity for the original infectious agent have an enhanced probability of recognizing re-infecting pathogens that have acquired mutations in their immune-dominant epitopes. Indeed, the memory cell pool is much more clonally diverse than the effector pool directed towards a given antigen. We investigated how this diversity is established and what control its boundaries. We find that low-affinity memory precursors proliferate less, but express higher levels of the transcription factor Eomes than cells of high affinity. In CD8 T cells, Eomes directly regulates the pro-survival protein Bcl-2, thus providing them with a survival advantage. This ensures that, despite their lower proliferation rate, low-affinity cells make a significant contribution to the memory cell pool.

b) Cellular metabolism. Recent studies have revealed the importance of metabolic processes for immune cell function (13, 14). In the tumor microenvironment tumor and T cells can interact, and may compete for nutrients, which can restrain T cell function. We perform several parallel studies:

  • We study how components of the tumor microenvironment impact CLL cell metabolism, and will use this knowledge for the rational design of novel therapies.
  • Since T cell function is impaired in CLL, we aim to elucidate alterations in T cell metabolism in CLL and determine their impact on T cell function. Initial findings show that CD8 T cells from CLL patients use more mitochondrial metabolism (oxidative phosphorylation) compared to T cells from age-matched healthy donors (see figure 4). In response to T cell receptor stimulation, CLL T cells show impaired switching to glycolysis. We currently further address how these metabolic changes develop, and how we can reverse those to improve T cell therapies.
  • We have previously established the importance of mitochondrial biogenesis and fatty acid oxidation during memory T cell formation. We currently aim to determine and target the underlying mechanisms regulating these processes in T cells in order to identify pathways for immune-therapeutics.

Figure 4:

Increased oxygen consumption rates in CD8 T cells derived from a CLL patient compared to T cells from an age-matched healthy donor (van Bruggen unpublished)   Increased oxygen consumption rates in CD8 T cells derived from a CLL patient compared to T cells from an age-matched healthy donor (van Bruggen unpublished)

Group members

Faculty 

Prof. dr. Eric Eldering PhD

Prof. dr. Arnon P.Kater MD PhD

Dr. Sanne Tonino MD PhD

Postdocs

Dr Erik Slinger 
Dr Felix Wensveen (Veni laureate– finished February 2017, transferred to Croatia) 
Dr Rianne van der Windt (Veni laureate, expected finish end 2017)

PhD Students

Martijn H.A. van Attekum - obtained PhD in January 2017

Rachel Thijssen MSc – obtained PhD in November 2016

Iris de Weerdt

Tom Hofland

Alexander Leeksma

Armando van Bruggen

Fabio Brocco

Raquel Delgado

Anne Martens

Marco Haselager

Zhenghao Chen

Technicians

Ing. Ingrid A.M. Derks

Sanne Terpstra

Hanneke ter Burg MSc

Dieuwertje M.P. Luijks

References

1. E E, C.A. S, H.L. A, A G, I.A. D, A.F. dV, McElgunn CJ, and J.P. S. Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signaling pathways. Nucleic Acid Research. 2003;31(23):e153.2. Te Raa GD, Malcikova J, Mraz M, Trbusek M, Le Garff-Tavernier M, Merle-Beral H, Greil R, Merkel O, Pospisilova S, Lin K, et al. Assessment of TP53 functionality in chronic lymphocytic leukaemia by different assays; an ERIC-wide approach. Br J Haematol.2014;167(4):565-9.3. Te Raa GD, Malcikova J, Pospisilova S, Trbusek M, Mraz M, Garff-Tavernier ML, Merle-Beral H, Lin K, Pettitt AR, Merkel O, et al. Overview of available p53 function tests in relation to TP53 and ATM gene alterations and chemoresistance in chronic lymphocytic leukemia. Leuk Lymphoma. 2013;54(8):1849-53.4. Te Raa GD, Derks IA, Navrkalova V, Skowronska A, Moerland PD, van LJ, Oldreive C, Monsuur H, Trbusek M, Malcikova J, et al. The impact of SF3B1 mutations in CLL on the DNA-damage response. Leukemia. 2014.5. Pascutti MF, Jak M, Tromp JM, Derks IA, Remmerswaal EB, Thijssen R, van Attekum MH, van Bochove GG, Luijks DM, Pals ST, et al. IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells. Blood.2013;122(17):3010-9.6. van Attekum M, Terpstra S, Reinen E, Kater AP, and Eldering E. Macrophage-mediated chronic lymphocytic leukemia cell survival is independent of APRIL signaling. Cell death discovery. 2016;2(16020.7. van Attekum MH, Terpstra S, Slinger E, von Lindern M, Moerland PD, Jongejan A, Kater AP, and Eldering E. Macrophages confer survival signals via CCR1-dependent translational MCL-1 induction in chronic lymphocytic leukemia. Oncogene. 2017.8. Slinger E, Wensveen FM, Guikema JE, Kater AP, and Eldering E. Chronic lymphocytic leukemia development is accelerated in mice with deficiency of the pro-apoptotic regulator NOXA. Haematologica. 2016;101(9):e374-7.9. Peperzak V, Slinger E, Ter Burg J, and Eldering E. Functional disparities among BCL-2 members in tonsillar and leukemic B-cell subsets assessed by BH3-mimetic profiling. Cell death and differentiation. 2016.10. Thijssen R, Slinger E, Weller K, Geest CR, Beaumont T, van Oers MH, Kater AP, and Eldering E. Resistance to ABT-199 induced by microenvironmental signals in chronic lymphocytic leukemia can be counteracted by CD20 antibodies or kinase inhibitors. Haematologica. 2015.11. Thijssen R, Ter Burg J, Garrick B, van Bochove GG, Brown JR, Fernandes SM, Rodriguez MS, Michot JM, Hallek M, Eichhorst B, et al. Dual TORK/DNA-PK inhibition blocks critical signaling pathways in chronic lymphocytic leukemia. Blood. 2016;128(4):574-83.12. Te Raa GD, Pascutti MF, Garcia-Vallejo JJ, Reinen E, Remmerswaal EB, Ten Berge IJ, van Lier RA, Eldering E, van Oers MH, Tonino SH, et al. CMV-specific CD8+ T cell function is not impaired in chronic lymphocytic leukemia. Blood. 2013.13. van der Windt GJ, Everts B, Chang CH, Curtis JD, Freitas TC, Amiel E, Pearce EJ, and Pearce EL. Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development. Immunity. 2012;36(1):68-78.14. van der Windt GJ, and Pearce EL. Metabolic switching and fuel choice during T-cell differentiation and memory development. Immunol Rev. 2012;249(1):27-42.

Contact

For further information about our research and opportunities for work or collaborations, you can contact Prof. dr. Eric F. Eldering

+31 (0)20 566 7018