T cell manipulation of the graft: Yes J.H. Frederik Falkenburg Department of Hematology L M U C
Allogeneic Hematopoietic Stem Cell Transplantation (SCT) for non-malignant disorders: no need for anti-tumor effect Chemotherapy and/or irradiation for reduction of endogenous hematopoiesis Immune suppression with chemotherapy, irradiation and/or anti-t cell therapy to prevent rejection Transplantation of stem cells to obtain recovery of donor hematopoiesis in the patient Restoration of immunity
Allogeneic Hematopoietic Stem Cell Transplantation (SCT) for Leukemia/Lymphoma Chemotherapy and/or irradiation for tumor reduction and to allow competitive repopulation of donor cells Immune suppression to prevent rejection Transplantation of stem cells to obtain recovery of (donor) hematopoiesis in the patient and obtain tolerance to donor Restoration of immunity Immune mediated elimination or suppression of the tumor (GVL/GVT) by donor (T) cells
Donor T cells: tolerance or GvHD and/or GVL Autologous T cells and T cells from a homozygous twin do not induce GVHD or GVL: alloreactivity is likely to be essential for both reactivities. Complete T cell depletion of the graft without T cell administration is likely to abolish the advantage of (partially) HLA matched allosct T cell treatment is an (the?) essential part of allosct
T cell depletion from the graft In general, T cell depletion Reduces or abolishes GVHD Reduces or abolishes GVL Impairs immune reconstitution Obviously, the relevance of the effects differs per disease, risk characteristics, patient group etc. In general, no improvement of survival, although less severe chronic GVHD
T cell manipulation of the graft? Yes To change the biology of transplantation!
Separation of GVHD from GVL reactivity possible? GVHD is a clinical disease with variable severity caused by T cells attacking (non-hematopoietic) tissues from the patient GVL is a beneficial reactivity caused by T cells attacking hematopoietic tissues from the patients GVHD is frequently associated with GVL reactivity GVL reactivity can occur in the absence of GVHD
GVHD is frequently associated with GVL reactivity GVL reactivity can occur in the absence of GVHD WHY? Donor Lymphocyte Infusion (DLI) for smoldering disease or mixed chimerism allows the characterization of alloreactive T cell responses in the absence of other therapeutic interventions Clonal analysis of T cells during clinical responses in the presence or absence of GVHD
Kinetics of mhag specific T cells after DLI for multiple myeloma M-protein LB-ADIR-1F LB-ECGF-1H HA-1 9 3 M-protein (g/l 6 3 2 1 mhag specific T ce (% of CD8+) 0-9 +39 +72 +78 0 +4 +7 +9 +12 +14 +40 0 SCT DLI. weeks after
Tetramer analysis of a patient responding to DLI for mixed chimerism (AML) 6000 LB-ERAP1-1R 15 LB-ARHGDIB-1R LB-WNK1-1I + celspermlblod Tetramer 4000 2000 LB-PDCD11-1F LB-SSR1-S LB-PRCP-1D % patient chimersism 10 5 patientchimerism(%) _ 0 donor -32-6 3 5 6 7 9 10 11 12 13 15 26 0 weeks after DLI 10
GVHD is frequently associated with GVL reactivity GVL reactivity can occur in the absence of GVHD GVHD is mediated by a (highly) polyclonal immune response against broadly expressed antigens (including the malignant cells) usually under inflammatory conditions Selective GVL reactivity is mediated by an oligoclonal immune response, especially under non-inflammatory conditions
Factors influencing the balance between GVHD and GVL reactivity Donor T cell repertoire Specificity Naive or memory repertoire (threshold) Dose Antigen presentation Microenvironment and tissue damage Inflammatory circumstances
GVHD after allogeneic non T cell depleted SCT High incidence of GVHD: Unselected donor repertoire High numbers of T cells Tissue damage due to conditioning High numbers of patient derived APC, activated in the GVHD target tissues Homeostatic proliferation due to lymphopenia Inflammatory circumstances (target susceptibility)
Possible solutions to shift the balance between GVHD and GVL and GVI reactivity Unselected donor repertoire Administration of T cell subsets (CD4 T cells?) Administration of T cell selected to selectively recognize recipient hematopoietic cells Administration of T cell selected to selectively recognize pathogens (CMV/EBV/aspergilus)
Possible solutions to shift the balance between GVHD and GVL and GVI reactivity High numbers of T cells Adapt the numbers of T cells to the circumstances and the match between donor and recipient
Possible solutions to shift the balance between GVHD and GVL and GVI reactivity Tissue damage due to conditioning Postpone T cell infusion Homeostatic proliferation Administer T cells adapted to the time following chemotherapy/irradiation/antibody treatment
Induction of GVHD and GVL reactivity after HLA matched transplantation GVHD and GVL are mostly mediated by alloreactive donor T cells from the naive repertoire induced by patient derived antigen presenting cells (APC) These APC may be normal or malignant APC GVHD target organs contain high numbers of APC
Possible solutions to shift the balance between GVHD and GVL and GVI reactivity High numbers of patient derived APC, activated in the GVHD target tissues Postpone T cell administration: gradual replacement of patient APC by donor APC
Possible solutions to shift the balance between GVHD and GVL and GVI reactivity Inflammatory circumstances (target susceptibility) adapt the moment of T cell infusion increase (interferon) of decrease (MSC? Tregs?) inflammatory circumstances
Conclusion: T cell manipulation of the graft followed by adapted T cell infusion did not yet lead to better overall survival, but results in less severe (chronic) GVHD (quality of life). To improve allosct changes are necessary: Manipulate the graft Manipulate the T cell populations Manipulate the target
CD4 DLI: Preclinical mouse model NOD/scid mouse model of human Acute Lymphoblastic Leukemia (ALL) i.v. inoculation with primary human leukemic cells DLI (URD) CD3+ DLI CD4+ DLI day 0 day 35-42 Monitoring leukemic engraftment Monitoring post DLI leukemic progression emergence of T cells Stevanović S, Griffioen M, Nijmeijer BA, van Schie ML, Stumpf AN, Rutten CE, Willemze R, Falkenburg JH. Leukemia. 2011, in press
Phenotype of T cells following CD4 + DLI for ALL in NOD/scid Mice After CD4 + DLI only CD4 + T cells and no CD8 + T cells emerged CD3 + DLI CD4 + DLI only CD4 + T cells emerge CD8 PE CD4 FITC n=3 mice
Clinical response of a patient with refractory B cell leukemia responding to DLI DLI Minimal Skin GVHD % donor chimeris 100 90 80 70 60 50 40 30 20 10 PB BM 7 6 5 4 3 2 1 # malignant cells (*10 e 6/ml) 0 5 6 7 8 9 10 11 12 13 months following allo-sct 0 Patient and donor mismatched for both HLA-DP alleles
Ex vivo anti-leukemic response mediated by CD4+ HLA-DP specific T cells 100 80 60 # IFN-γ ELISPOTs 40 20 0 leukemic cells + HLA-DP moab + HLA-DQ moab ELISPOT analysis of circulating T cells after stimulation with leukemic cells in the presence or absence of blocking antibodies
Determination of an anti-leukemic CD4+ T cell response starting 6 weeks after DLI CD 4+ T cells CD 8+ T cells % malignant cells # IFN-γ ELISPOTs 70 60 50 40 30 20 10 DLI 50 40 30 20 10 malignant cells 0 0-8 0 6 10 14 21 pre-dli weeks after DLI PB BM
Identification of HLA-DPB1* 0201 and HLA-DPB1* 0301 specific T cell clones using an EBV-LCL panel study HLA-DPB1*0201 specific clone HLA-DPB1*0301 specific clone HLA-DPB1* 04 HLA-DPB1* 03 HLA-DPB1* 02 450 350 250 150 50 50 150 250 350 450 IFNγ (pg/ml) Donor: HLA-DPB1* 04 / 05 Patient: HLA-DPB1* 02 / 03 3 HLA-DPB1* 0201 18 HLA-DPB1* 0301 specific clones specific clones Rutten CE, et al. Leukemia. 2008;22:1387.
New approaches HLA class II specific CD4 T cells can mediate graftversus-leukemia reactivity CD4 T cells support immune reconstitution Since under non-inflammatory conditions HLA class II expression is mainly restricted to normal and malignant hematopoietic cells, HLA class II specific CD4 T cells may mediate GVL with no or limited GVHD
A randomized clinical study to study the effect of infusion of 10e6 CD4 T cells/ kg 3 months after T cell depleted SCT Conditioning with Cyclo/TBI or Busulfan/Fludarabine and alemtuzumab/atg T cell depleted transplant (alemtuzumab in the bag), and no post transplant immune suppression 6-8 weeks after transplantation, antibodies no longer present High risk patients receive low dose CD3 DLI at 3 months If not high risk: Randomized between CD4 T cells at 3 months or not Unmanipulated CD3 T cells (3x10e6/kg) at 6 months if no GVHD and mixed chimerism
Future applications Removal of the (naive) T cells from the graft Isolation and infusion of donor T cells specifically recognizing minor histocompatibility antigens or tumor associated antigens Isolation and infusion pathogen specific T cells Isolation and infusion virus specific T cells engineered to express an antigen specific T cell receptor
HLA class II expression on non-hematopoietic cells + + IFN-γ + DQ DR DP counts + + IFN-γ γ 10 0 10 1 10 2 10 3 10 4 MFI 10 0 10 1 10 2 10 3 10 4