12-Month Full Donor Chimerism Is Associated with Low-Grade Acute Graft Versus Host Disease (aGVHD) and Predicts Superior Outcomes in Reduced Intensity (RIC) Alemtuzumab-Based Transplants for MDS and AML

Introduction Reduced intensity (RIC) Alemtuzumab conditioned allogeneic stem cell transplants have enabled a successful graft versus leukaemia effect while GvHD rates were low. The impact of chimerism on outcome has scarcely been studied in RIC, Alemtuzumab-based transplants. Patient characteristics Retrospective analysis of 50 patients with high risk MDS (n=14) and AML (n=36) undergoing RIC-Alemtuzumab transplant in our center since 2006. Median age was 62 years (range 51-72) and there were 33 male and 17 female recipients. All donors were fully (10/10) matched in HLA-loci. 11 patients had a sibling donor while 39 had a matched unrelated donor. 29 patients were conditioned with fludarabine, melphalan and alemtuzumab while 21 patients received fludarabine, busulphan and alemtuzumab. Results The median follow-up of patients was 18.2 months (range 15-90). Median overall survival was 27.4 months and median progression free survival 33 months. At 3 months post-HSCT 72% (n=36) and 18% (n=9) of patients were full and mixed chimeras respectively. At 12 months post-HSCT n=19 and n=10 of patients were full and mixed donor respectively. 14 patients (28%) developed grade 1-2 GvHD while only 1 (2%) grade 3-4 GvHD. Patients who attained 12-month full donor chimerism in whole blood (WB), had a mean PFS of 85 months as compared to 81.5 months for mixed chimeras (p= 0.04). In 3-months, those who were full donor in WB had a mean PFS 95 months compared to 33 for mixed chimeras in WB (p= 0.016). 3, 12 -month T-cell chimerism did not influence OS and PFS. The presence of grade I-II GVHD was significantly associated with 12-month full donor chimerism in both WB and T-cells (p<0.05). Conclusion In our series of AML/MDS patients who received RIC Campath-based allografts, full donor chimerism is a surrogate marker for progression free and overall survival. All grade I/II aGVHD patients achieved full donor chimerism in whole blood. Disclosures Paneesha: Abvie: Honoraria. Kishore:celgene: Other: travel grant.

Natural Killer Cells Adapt to Major Histocompatibility Complex: Implications for Hematopoietic Cell Transplantation

Natural Killer (NK) cells play several important roles in hematopoietic cell transplantation. Host NK cells can reject allogeneic transplants that lack major histocompatibility complex (MHC) molecules of the host (so-called “missing self” reactivity), whereas donor NK cells in the marrow can promote engraftment, inhibit graft versus host disease, and mediate rejection of host leukemic cells (graft versus tumor (GVT) response). Like T cells, NK cells can distinguish allogeneic cells from self-cells, and “learn” self vs. non-self so as to establish self-tolerance. This presentation will summarize current understanding of some of these processes based primarily on studies in mice, and present new data that may be relevant for the success of human bone marrow transplantation. The self-tolerance of T cells and B cells is established in large part by developmental processes that either remove or suppress self-reactive clones as the cells differentiate from hematopoietic stem cells. Studies will be presented that show that NK cell self-tolerance is regulated quite differently, by an adaptation process that occurs quite rapidly and acts on mature NK cells. NK cell reactivity against susceptible untransformed cells (e.g., cells from animals lacking MHC of the NK cell donor) is rapidly lost after transfer of such NK cells to susceptible hosts, indicating that self-tolerance is an adaptation that occurs rapidly under non-inflammatory conditions. Irradiation chimeras were employed to identify the cell types that induce tolerance of NK cells to cells from MHC-deficient mice, as one model of missing self-recognition. Stable tolerance of wild type NK cells to MHC-deficient cells was established when NK cells were exposed in chimeras to either MHC-deficient hematopoietic cells or MHC-deficient non-hematopoietic cells. Interestingly, however, tolerance induced by exposure solely to MHC-deficient hematopoietic cells was unstable in the face of inflammatory conditions arising from infections or exposures to inflammatory cytokines in vivo. Specifically, mixed chimeras consisting of WT and MHC-deficient hematopoietic cells in WT hosts were stably chimeric until the mice were exposed to viral or bacterial infections, at which time the MHC-deficient cells were rapidly eliminated. In contrast, tolerance induced by non-hematopoietic cells was much more stable. Specifically, mixed chimeras consisting of WT and MHC-deficient hematopoietic cells in MHC-deficient hosts were stably chimeric whether or not they underwent infections. These findings suggest that tolerance may be imposed by multiple mechanisms depending on the cell types that induce tolerance, with distinct properties as a result. Most importantly from a clinical perspective, the results suggest that depending on the host/donor combination, intense infections may jeopardize the stability of bone marrow transplants. Previous studies have shown that tolerance of NK cells to MHC-deficient cells is accompanied by a state of hyporesponsiveness of the NK cells, characterized by low functional responses to stimulation via crosslinking of activating receptors ex vivo. Accordingly, it has been widely accepted that hyporesponsiveness to stimulation is the mechanism of NK cell self-tolerance when NK cells lack inhibitory receptors specific for host MHC molecules. However, our recent analysis of chimeric NK cells showed that this cannot be the sole explanation. When NK cells developed in MHC+ hosts in the presence of MHC-deficient hematopoietic cells, the cells became responsive to activating receptor stimulation despite being tolerant of MHC-deficient cells. Hence, tolerance can occur even when NK cells are responsive to stimulation. In contrast, when NK cells developed in MHC-deficient hosts, they became hyporesponsive. These data suggest that non-hematopoietic cells in the host impart hyporesponsiveness. Furthermore, exposure to MHC-deficient hematopoietic cells induces tolerance by a mechanism distinct from hyporesponsiveness. The latter mechanism is relatively unstable as it can be broken as a result of infections. Disclosures Raulet: Innate Pharma: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy.

GPR18 is required for a normal CD8αα intestinal intraepithelial lymphocyte compartment

Intraepithelial lymphocytes (IELs) play an important role in maintaining the physiology of the small intestine. The majority of mouse IELs express CD8αα and are either γδ or αβ T cells. Although the development and homing of CD8αα IELs have been studied in some detail, the factors controlling their homeostasis and positioning are incompletely understood. Here we demonstrate that G protein–coupled receptor 18 (GPR18) is abundantly expressed in CD8αα IELs and that mice lacking this orphan receptor have reduced numbers of γδT IELs. Mixed bone marrow chimera experiments reveal a markedly reduced contribution of GPR18-deficient cells to the CD8αα IEL compartment and a reduction in the CD8αβ T cell subset. These defects could be rescued by transduction with a GPR18-expressing retrovirus. The GPR18-deficient γδT IELs that remained in mixed chimeras had elevated Thy1, and there were less granzyme B+ and Vγ7+ cells, indicating a greater reduction in effector-type cells. Flow cytometric analysis indicated GPR18 deficiency more strongly affected the CD8αα cells in the intraepithelial compared with the adjacent lamina propria compartment. These findings establish a requirement for GPR18 in CD8αα and CD8αβ IELs, and we suggest the receptor has a role in augmenting the accumulation of CD8 T cells in the intraepithelial versus lamina propria compartment.

CXCR4 promotes B cell egress from Peyer’s patches

Peyer’s patches (PPs) play a central role in supporting B cell responses against intestinal antigens, yet the factors controlling B cell passage through these mucosal lymphoid tissues are incompletely understood. We report that, in mixed chimeras, CXCR4-deficient B cells accumulate in PPs compared with their representation in other lymphoid tissues. CXCR4-deficient B cells egress from PPs more slowly than wild-type cells, whereas CXCR5-deficient cells egress more rapidly. The CXCR4 ligand, CXCL12, is expressed by cells adjacent to lymphatic endothelial cells in a zone that abuts but minimally overlaps with the CXCL13+ follicle. CXCR4-deficient B cells show reduced localization to these CXCL12+ perilymphatic zones, whereas CXCR5-deficient B cells preferentially localize in these regions. By photoconverting KikGR-expressing cells within surgically exposed PPs, we provide evidence that naive B cells transit PPs with an approximate residency half-life of 10 h. When CXCR4 is lacking, KikGR+ B cells show a delay in PP egress. In summary, we identify a CXCL12hi perilymphatic zone in PPs that plays a role in overcoming CXCL13-mediated retention to promote B cell egress from these gut-associated lymphoid tissues.