Kinetics of immune cell reconstitution predict survival in allogeneic bone marrow and G-CSF–mobilized stem cell transplantation

Abstract The clinical utility of monitoring immune reconstitution after allotransplant was evaluated using data from Blood and Marrow Transplant Clinical Trials Network BMT CTN 0201 (NCT00075816), a multicenter randomized study of unrelated donor bone marrow (BM) vs granulocyte colony-stimulating factor (G-CSF)–mobilized blood stem cell (G-PB) grafts. Among 410 patients with posttransplant flow cytometry measurements of immune cell subsets, recipients of G-PB grafts had faster T-cell reconstitution than BM recipients, including more naive CD4+ T cells and T-cell receptor excision circle–positive CD4+ and CD8+ T cells at 3 months, consistent with better thymic function. Faster reconstitution of CD4+ T cells and naive CD4+ T cells at 1 month and CD8+ T cells at 3 months predicted more chronic graft-versus-host disease (GVHD) but better survival in G-PB recipients, but consistent associations of T-cell amounts with GVHD or survival were not seen in BM recipients. In contrast, a higher number of classical dendritic cells (cDCs) in blood samples at 3 months predicted better survival in BM recipients. Functional T-cell immunity measured in vitro by cytokine secretion in response to stimulation with cytomegalovirus peptides was similar when comparing blood samples from BM and G-PB recipients, but the degree to which acute GVHD suppressed immune reconstitution varied according to graft source. BM, but not G-PB, recipients with a history of grades 2-4 acute GVHD had lower numbers of B cells, plasmacytoid dendritic cells, and cDCs at 3 months. Thus, early measurements of T-cell reconstitution are predictive cellular biomarkers for long-term survival and response to GVHD therapy in G-PB recipients, whereas more robust DC reconstitution predicted better survival in BM recipients.

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An Accelerated CD8+, but Not CD4+, T-Cell Reconstitution Associates with a More Favorable Outcome Following HLA-Haploidentical HSCT: Results from a Retrospective Study of the Cell Therapy and Immunobiology Working Party of the EBMT

Blood ◽

10.1182/blood.v126.23.1929.1929 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1929-1929

Author(s):

Attilio Bondanza ◽

Loredana Ruggeri ◽

Dimitris Ziagkos ◽

Chiara Bonini ◽

Christian Chabannon ◽

...

Keyword(s):

Overall Survival ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Reconstitution ◽

Immune Cell ◽

Cd4 T Cell ◽

Cd4 Counts ◽

Cell Counts ◽

T Cell Reconstitution

Abstract Introduction and Aim: HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is increasingly offered to patients with high-risk acute myeloid (AML) or lymphoid leukemia (ALL). Unfortunately, graft manipulation employed to overcome the HLA barrier significantly delays immune reconstitution, posing the patients at risk of infections. Accordingly, non-relapse mortality after haplo-HSCT clearly extends beyond day 100 post-transplant. Over the years, different approaches have been investigated to speed-up immune reconstitution. In the absence of validated immune biomarkers, it is however difficult to evaluate the clinical impact of accelerated immune reconstitution. The aim of this EBMT retrospective study is to explore immune-cell counts early after haplo-HSCT as predictive of its overall outcome. Methods and Patients: Among AML and ALL patients in the EBMT database who underwent haplo-HSCT in the period 2001-2012, criteria for study entry were survival beyond day 100 and availability of differential immune-cell counts (CD3+, CD4+, CD8+ T cells, CD19+ B cells, CD16+/CD56+ NK cells) within this period. Accordingly, statistical analysis was landmarked at day 100. Of 259 patients meeting these criteria (age 2-70, median 33), 67 (26%) were children. The underlying disease was AML in 162 cases (63%), while ALL in the remaining (including 5 cases of bi-phenotypic leukemia). Fifty-two percent of patients were transplanted in CR1. The stem-cell source was G-CSF mobilized peripheral blood in all but one patient (>99%) and 171 received TBI (66%). The graft was manipulated in 199 patients (78%), including CD34-selection (50%), ex vivo T-cell depletion (15%) or both (13%). Female-to-male transplants were 68 (26%), while 204 (79%) recipients were CMV seropositive. Sustained hematopoietic engraftment was reached in 246 patients (95%) Results: The estimated overall survival at 2yrs was43%. The estimated cumulative incidence of death due to relapse was 33%, while that of death due to other causes was 35% (51% of those were infections) The occurrence of grade III-IV GVHD and of chronic GVHD was 9% and 18% (7% extensive), respectively. As expected, overall survival was better in children (62% vs 36%, P=0.002 by Log-rank), who clearly had a lower incidence of death due to causes other than relapse compared with adults (10% vs 37%, P=0.0001). Negative prognostic factors for overall survival were any disease state other than CR1 at time of transplantation (P=0.002) and CMV seropositivity (P=0.009). Type of leukemia, TBI or graft manipulation had no effect on the outcome. By day 100 post-transplant, patients reached the following median immune-cell counts: 100 CD3+ T cells (range 0-2576), 30 CD4+ T cells (0-1714), 48 CD8+ T cells (0-1880), 276 CD16+/CD56+ NK cells (18-3581), 21 CD19+ B cells (0-790). Importantly, CD3+ counts above the first quartile (1Q) of the entire data set (29 cells per microL) were significantly associated with a better overall survival (P=0.0005 by Log rank) and a lower incidence of death due to causes other than relapse (P=0.002 by Gray test). The same held true for CD8+ counts (1Q: 15 cells per microL; P=0.003 on overall survival; P=0.0004 on death due to other causes). CD4+ counts also showed similar correlations, but at higher values (above the median). None of the other immune-cell counts analyzed correlated with clinical outcome. Strikingly, when challenged in multivariate analysis taking into account age category, CMV seropositivity, graft manipulation and CR1 status at transplant, CD3+ and CD8+ counts above the 1Q adjusted to fit optimal cut-off points were still significantly associated with a better overall survival (P=0.006 and P=0.015, respectively), but only CD8+ values associated with a lesser risk of death due to causes other than relapse (P=0.026). Conversely, similarly adjusted median CD4+ counts failed to show any association. Conclusions: Contrary to what is generally accepted, these results indicate that an accelerated CD8+, but not CD4+, T cell reconstitution associates with a more favorable clinical outcome after haplo-HSCT, likely due to its protective role against opportunistic viral infections. Moreover, they suggest that yet to be validated CD8+ cut-off points, rather than the commonly used arbitrary value of 200 CD4+ T cells per microL, should be considered as surrogate biomarkers in clinical trials. Disclosures Bonini: MolMed S.p.A: Consultancy.

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Promoting Regulation Via the Inhibition of DNAM-1 After Transplantation

Blood ◽

10.1182/blood.v120.21.338.338 ◽

2012 ◽

Vol 120 (21) ◽

pp. 338-338

Author(s):

Motoko Koyama ◽

Rachel D Kuns ◽

Stuart D Olver ◽

Katie E Lineburg ◽

Mary Lor ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Steady State ◽

T Cell ◽

Median Survival ◽

Cd4 T Cells ◽

Cell Expansion ◽

Acute Gvhd ◽

Cd4 T Cell

Abstract Abstract 338 Graft-versus-host disease (GVHD) is the major limitation of allogeneic hematopoietic bone marrow transplantation (BMT). Donor T cells play pivotal roles in GVHD and graft-versus-leukemia (GVL) effects and following BMT all T cell fractions, including regulatory T cells (Treg) express the DNAX accessory molecule-1 (DNAM-1, CD226) and T cell Immunoglobulin and ITIM domain (TIGIT) molecule. DNAM-1 is a co-stimulatory and adhesion molecule, expressed mainly by NK cells and CD8+ T cells at steady state to promote adhesion to ligand (CD155, CD112)–expressing targets and enhance cytolysis. TIGIT is a regulatory ligand expressed predominantly by Treg as steady state which competes for CD155 binding, We have analyzed the role of this pathway in GVHD and GVL. Lethally irradiated C3H/Hej (H-2k) mice were injected with bone marrow cells and T cells from MHC disparate wild-type (wt) or DNAM-1–/– C57Bl6 (H-2b) mice. Recipients of DNAM-1–/– grafts were protected from GVHD (survival 67% vs. 7%, P < .0001). We also confirmed the role of DNAM-1 in GVHD in a MHC-matched BMT model (B6 → BALB/B (H-2b)) where GVHD is directed to multiple minor histocompatibility antigens. Next we examined the donor populations expressing DNAM-1 which mediate this effect. DNAM-1 had little impact on acute GVHD severity in the B6 → bm1 BMT model where GVHD is directed against an isolated MHC class I mismatch and is CD8-dependent. In contrast, recipients of wt bone marrow and DNAM-1–/– CD4 T cells survived long-term (compared to recipients of wt CD4 T cells, survival 81% vs. 25%, P = .003) in the B6 → B6C3F1 BMT model, confirming the protection from GVHD is CD4-dependent. Donor CD4 T cell expansion and effector function (Th1 and Th17), and CD8 T cell expansion and cytotoxic function were equivalent in recipients of wt and DNAM-1–/– grafts. However the percentage and number of Treg were significantly increased in recipients of DNAM-1–/– grafts compared to those of wt grafts. The depletion of Treg from donor grafts eliminated the protection from GVHD seen in the absence of DNAM-1 signalling (median survival 16 days vs. 15.5 days, P = 0.53). Adoptive transfer experiments using FACS-sorted Treg were undertaken to compare the relative ability of B6.WT and B6.DNAM-1–/– Treg to suppress GVHD. The majority of recipients of DNAM-1–/– Treg survived beyond day 50 (median survival; day 56), demonstrating a superior ability to suppress acute GVHD relative to wt Treg where the median survival was day 36 (survival 47% vs. 0%, P = .001). These data demonstrate that donor DNAM-1 expression promotes GVHD in a CD4+ T cell-dependent manner via the inhibition of donor Foxp3+ Treg. Finally, the absence of donor DNAM-1 did not influence leukemia-specific mortality in multiple GVL models, regardless of whether the tumor expressed CD155 or not. Thus we demonstrate that the DNAM-1 pathway promotes GVHD, putatively due to competition with TIGIT on Treg, thereby inhibiting regulatory function. This provides support for therapeutic DNAM-1 inhibition to promote tolerance not only after transplant but also in relevant inflammatory based diseases characterized by T cell activation. Disclosures: No relevant conflicts of interest to declare.

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Peri-Transplant Steroids Enhance GVL and Attenuate GVHD By Redistributing Alloreactive Donor T Cells from the Gut into the Bone Marrow

Blood ◽

10.1182/blood-2019-129341 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3210-3210

Author(s):

Takayuki Inouye ◽

Motoko Koyama ◽

Ensbey Kathleen ◽

Nicholas Greene ◽

Luke Samson ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Cell Expansion ◽

Effector Memory ◽

Gi Tract ◽

Donor T Cells ◽

T Cell Expansion

Leukemia relapse represents a failure of graft-versus-leukemia (GVL) and remains the major limitation of allogeneic stem cell/bone marrow transplantation (BMT). Graft-versus-host disease (GVHD) within the gastrointestinal (GI) tract is the principal determinant of transplant-related mortality and is initiated by a network of alloantigen presentation by professional and non-professional APC that prime donor T cells in the GI tract and related lymphoid structures. Since GVL and lethal GVHD are mediated by donor T cells at spatially distinct sites; bone marrow (BM) and the GI tract respectively, we sought tractable approaches to spatially separate alloreactive responses at these two locations. The administration of high dose steroids in the peri-transplant period is permissive of T cell replete HLA-haploidentical BMT and significant GVL effects (Ogawa H, et al. BBMT. 2006). We utilized murine haploidentical BMT models (B6D2F1 → B6C3F1, B6 → B6D2F1) with recipient background MLL/AF9 primary acute myeloid leukemia (AML), with or without dexamethasone (Dex) administration (5 mg/kg/day i.p., days -1 to +5). Dex-treatment improved transplant survival (from 25% to 68% at day 100, P=0.0012) with significant reductions in GVHD histopathology specifically in the colon (histopathology scores 8.7±1.0 vs 4.6±0.8, P< 0.05), despite excellent leukemia control. To understand this paradox, we analyzed the kinetics of donor T cell expansion after BMT. In the mesenteric lymph node (mLN), Dex treatment significantly suppressed the expansion of both CD4 and CD8 T cells (3.3±0.3 x 105 vs 1.4±0.3 x 105, P< 0.001 and 4.2±0.4 x 105 vs 2.1±0.4 x 105, P< 0.01 respectively) and the activation of CD4 T cells (CD25 MFI: 2021±146 vs 1056±102, P< 0.01). In contrast, donor effector/memory CD44+ CD8 T cells were expanded in the BM of Dex treated recipients (1.9±0.3 x 105 vs 3.1±0.4 x 105, P< 0.05) that demonstrated high per cell cytolytic activity against leukemia (specific lysis: 65±2.4 % vs 62±2.6 % in untreated vs Dex-treated, P> 0.05). Surprisingly, there was no difference in proliferation (cell tracking dye dilution: 63±5.5 % vs 57±5.5 % in untreated vs Dex-treated, P> 0.05) or apoptosis (caspase-3: 6.6±0.4 % vs 6.1±0.6 %, caspase-8: 20±1.6 % vs 17±3.3 % in untreated vs Dex-treated, respectively, P> 0.05) of CD4 T cells in the mLN between the two groups. We undertook experiments with luciferase expressing T cells and noted that Dex-treatment preferentially inhibited T cell accumulation in the GI tract, but not marrow after BMT. Thus, it appeared that Dex treatment preferentially re-distributed donor T cells from the GI tract to the bone marrow. We next determined if Dex exerted effects via direct signaling to the donor T cell. We thus transplanted glucocorticoid receptor (GR)-deficient or intact T cells (GRfl/fl lck-Cre mice). Dex-treatment reduced donor CD4 T cell expansion in the mLN independent of their expression of the GR (untreated vs Dex-treated: 2.8±0.6 x 105 vs 1.2±0.3 x 105, lckCREGRfl/fl and 2.4±0.3 x 105 vs 1.4±0.4 x 105, GRfl/fl littermates, P< 0.05 both groups). Thus steroid effects were mediated indirectly, putatively via effects on recipient alloantigen presentation. There was a marked reduction in recipient dendritic cells (DC) and macrophages expressing the Ea peptide within MHC class II in the GI tract of Dex-treated recipients (terminal Ileum YAe+ DC number 896±93 vs 356±40, P< 0.01, YAe+ macrophage number 1035±136 vs 355±97, P< 0.01). In conjunction with this, expression of the gut homing integrin a4b7 expression was reduced in CD4 T cells from Dex treated recipient mLN (25±1.6 % vs 17±1.7 %, P< 0.01), while the marrow homing integrin VLA-4 (a4b1) was increased (a4: 62±2.2 % vs 75±1.6 %, P< 0.001, b1: 52±2.5 % vs 61±1.6 %, P< 0.05) in donor CD8 T cells from Dex treated recipient BM. Finally, Dex treatment enhanced GVL against a second primary AML (BCR/ABL-NUP98/HOXA9) relative to untreated recipients and those receiving post-transplant cyclophosphamide (PT-Cy) (relapse rate: 0% vs 40% vs 100% at day 35 in Dex vs untreated vs PT-Cy, PT-Cy vs Dex-treated, P< 0.0001; untreated vs Dex-treated, P=0.029). These data suggest a potential therapeutic strategy to modulate antigen presentation in the GI tract and consequent integrin imprinting that minimizes GVHD lethality whilst enhancing GVL within BM. Disclosures No relevant conflicts of interest to declare.

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Donor-Derived T Cells Can Be Rendered Hyporesponsive to Alloantigen without Loss of Pathogen or Tumor Immune Responses.

Blood ◽

10.1182/blood.v110.11.771.771 ◽

2007 ◽

Vol 110 (11) ◽

pp. 771-771 ◽

Cited By ~ 2

Author(s):

Jeff Davies ◽

Dongin Yuk ◽

Lee Nadler ◽

Eva Guinan

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

Cd8 T Cells ◽

Immune Reconstitution ◽

Acute Gvhd ◽

Alloreactive T Cells ◽

Cell Pool ◽

Donor T Cells ◽

Leukemia Antigen

Abstract The prevention of severe acute Graft-versus-Host Disease (GvHD) without impairment of immune reconstitution is the major challenge in HLA-mismatched hematopoietic stem cell transplantation (HSCT). One alternative to experimental strategies to selectively destroy or remove alloreactive T cells from the donor T cell pool is to induce hyporesponsiveness (anergy) in alloreactive T cells within the donor T cell pool and thus preserve the vast majority of T cell repertoire. We previously reported early clinical data of HLA-mismatched HSCT after alloanergization of donor bone marrow via ex vivo allostimulation in the presence of co-stimulatory blockade (CSB) with Cytotoxic T Lymphocyte Antigen-4 Immunoglobulin (CTLA4-Ig). Analysis of a larger cohort of such patients revealed a low rate of severe acute GvHD and very few clinically significant viral infections, with over 30% of patients (pts) surviving long-term without disease relapse. This suggested that CSB might indeed be controlling alloreactivity with preservation of pathogen-specific immunity and a graft-versus-leukemia (GvL) effect. We therefore sought to directly determine the effect of alloanergization of human donor T cells on alloreactivity, pathogen- and leukemia-antigen-specific immunity. After alloanergization via blockade of CD28-mediated co-stimulation with clinical-grade humanized anti-B7.1 and anti B7.2 antibodies, HLA-mismatched alloproliferative responses were reduced by 2 logs, a more efficient reduction in alloreactivity than previously reported with the use of CTLA4 Ig. Using CFSE-based labeling of human responder T cells we have demonstrated directly for the first time that alloanergization efficiently abrogates stimulator-specific alloproliferation in both CD4 and CD8 donor T cells, whereas third party responses are retained (Figure 1). Importantly, the strategy does not diminish the capacity of donor CD4 and CD8 T cells to mount a range of functional immune responses, including proliferation, cytokine production and cytotoxic responses, in response to stimulation with several human herpes viruses. We have also demonstrated that frequencies of WT1-specific IFN-g+ CD4 and CD8 T cells are not diminished after the process of alloanergization, showing that a T cell mediated GvL effect may be retained. Importantly we demonstrated retention of pathogen and leukemia antigen-specific responses to both MHC Class I- and II-restricted antigens and in both HLA-A2+ and non-HLA-A2+ responders. These data confirm that the technique of alloanergization can be used to provide non-alloreactive donor T cells without loss of beneficial CD4 and CD8 donor immunity. The optimal dose of HLA-mismatched alloanergized donor T cells that will improve immune reconstitution whilst controlling acute GvHD after HLA-mismatched HSCT remains to be defined. To answer this question, we have embarked on a dose-escalating clinical study of delayed alloanergized donor T cell infusion to improve immune reconstitution after haploidentical HSCT. Figure Figure

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Tigit, CD226 and PD-L1/PD-1 Are Highly Expressed By Marrow-Infiltrating T Cells in Patients with Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.2102.2102 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2102-2102 ◽

Cited By ~ 5

Author(s):

Mahesh Yadav ◽

Cherie Green ◽

Connie Ma ◽

Alberto Robert ◽

Andrew Glibicky ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Plasma Cells ◽

Color Flow

Abstract Introduction:TIGIT (T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif [ITIM] domain) is an inhibitory immunoreceptor expressed by T and natural killer (NK) cells that is an important regulator of anti-tumor and anti-viral immunity. TIGIT shares its high-affinity ligand PVR (CD155) with the activating receptor CD226 (DNAM-1). We have recently shown that TIGIT blockade, together with PD-L1/PD-1 blockade, provides robust efficacy in syngeneic tumor and chronic viral infection models. Importantly, CD226 blockade abrogates the benefit of TIGIT blockade, suggesting additional benefit of TIGIT blockade through elaboration of CD226-mediated anti-tumor immunity, analogous to CTLA-4/CD28 regulation of T-cell immunity. Whether TIGIT and CD226 are expressed in patients with multiple myeloma (MM) and how TIGIT expression relates to PD-L1/PD-1 expression is unknown. Here we evaluate expression of TIGIT, CD226, PD-1 and PD-L1 in patients with MM to inform novel immunotherapy combinations. Methods:We performed multi-color flow cytometry (n = 25 patients), and multiplex qRT-PCR (n = 7) on bone marrow specimens from patients with MM to assess expression of TIGIT, CD226, PD-1, and PD-L1 on tumor and immune cells. Cells were stained with fluorescently conjugated monoclonal antibodies to label T cells (CD3, CD4, CD8), NK cells (CD56, CD3), plasma cells (CD38, CD45, CD319, CD56), inhibitory/activating receptors (PD-1, TIGIT, PD-L1, CD226), and an amine-reactive viability dye (7-AAD). Stained and fixed cells were analyzed by flow cytometry using BD FACSCanto™ and BD LSRFortessa™. Results:TIGIT, CD226 and PD-L1/PD-1 were detectable by flow cytometry in all patients with MM who were tested, with some overlapping and distinct expression patterns. TIGIT was commonly expressed by marrow-infiltrating CD8+ T cells (median, 65% of cells), CD4+ T cells (median, 12%) and NK cells. In contrast, CD226 was more commonly expressed by marrow-infiltrating CD4+ T cells (median, 74%) compared with CD8+ T cells (median, 38%). PD-1 was expressed by marrow-infiltrating CD8+ T cells (median 38%) and CD4+ T cells (median, 16%). TIGIT was co-expressed with PD-1 on CD8+ T cells (67%-97% TIGIT+ among PD-1+), although many PD-1-negative CD8+ T cells also expressed TIGIT (39%-78% of PD-1-negative). PD-L1 was also expressed by CD8+ (median, 23%) and CD4+ (median, 8%) T cells in addition to MM plasma cells (median, 95%), albeit with significantly lower intensity on T cells compared with plasma cells. The expression of TIGIT and PD-L1 mRNA was highly correlated (R2 = 0.80). Analysis of PVR expression will also be presented. Conclusions: TIGIT, CD226, PD-1, and PD-L1 were commonly expressed in MM bone marrow, but with different patterns. Among CD8+ T cells, the frequency of TIGIT+ T cells was almost twice that of PD-1+ T cells, whereas the majority of CD4+ T cells expressed CD226. TIGIT blockade may complement anti-PD-L1/PD-1 immunotherapy by activating distinct T-cell/NK-cell subsets with synergistic clinical benefit. These results provide new insight into the immune microenvironment of MM and rationale for targeting both the PD-L1/PD-1 interaction and TIGIT in MM. Disclosures Yadav: Genentech, Inc.: Employment. Green:Genentech, Inc.: Employment. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Glibicky:Makro Technologies Inc.: Employment; Genentech, Inc.: Consultancy. Nakamura:Genentech, Inc.: Employment. Sumiyoshi:Genentech, Inc.: Employment. Meng:Genentech, Inc.: Employment, Equity Ownership. Chu:Genentech Inc.: Employment. Wu:Genentech: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment. Adamkewicz:Genentech, Inc.: Employment. Grogan:Genentech, Inc.: Employment. Venstrom:Roche-Genentech: Employment.

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Resistance of Natural Killer and T Cells to Venetoclax Allows for Combination Treatment with Cancer Immunotherapy Agents

Blood ◽

10.1182/blood-2018-99-116405 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1118-1118 ◽

Cited By ~ 2

Author(s):

Elisabeth A Lasater ◽

An D Do ◽

Luciana Burton ◽

Yijin Li ◽

Erin Williams ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Immune Cell ◽

Single Agent ◽

Employment Equity ◽

Equity Ownership

Abstract Introduction: Intrinsic apoptosis is regulated by the BCL-2 family of proteins, which consists of both anti-apoptotic (BCL-2, BCL-XL, MCL-1) and pro-apoptotic (BIM, BAX, BAK, BAD) proteins. Interaction between these proteins, as well as stringent regulation of their expression, mediates cell survival and can rapidly induce cell death. A shift in balance and overexpression of anti-apoptotic proteins is a hallmark of cancer. Venetoclax (ABT-199/GDC-0199) is a potent, selective small molecule BCL-2 inhibitor that has shown preclinical and clinical activity across hematologic malignancies and is approved for the treatment of chronic lymphocytic leukemia with 17p deletion as monotherapy and in combination with rituximab. Objective: To investigate the effects of BCL-2 inhibition by venetoclax on viability and function of immune-cell subsets to inform combinability with cancer immunotherapies, such as anti-PD-L1. Methods and Results: B cells, natural killer (NK) cells, CD4+ T cells, and CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from healthy donors (n=3) were exposed to increasing concentrations of venetoclax that are clinically achievable in patients, and percentage of live cells was assessed by flow-cytometry using Near-IR cell staining. B cells were more sensitive to venetoclax (IC50 of ~1nM) than CD8+ T cells (IC50 ~100nM), NK cells (IC50 ~200nM), and CD4+ T cells (IC50 ~500nM) (Figure A). CD8+ T-cell subset analysis showed that unstimulated naive, but not memory cells, were sensitive to venetoclax treatment (IC50 ~30nM and 240nM, respectively). Resistance to venetoclax frequently involves compensation by other BCL-2 family proteins (BCL-XL and MCL-1). As assessed by western blot in PBMCs isolated from healthy donors (n=6), BCL-XL expression was higher in NK cells (~8-fold) and CD4+ and CD8+ T cells (~2.5-fold) than in B cells (1X). MCL-1 protein expression was higher only in CD4+ T cells (1.8-fold) relative to B cells. To evaluate the effect of venetoclax on T-cell function, CD8+ T cells were stimulated ex vivo with CD3/CD28 beads, and cytokine production and proliferation were assessed. Venetoclax treatment with 400nM drug had minimal impact on cytokine production, including interferon gamma (IFNg), tumor necrosis factor alpha (TNFa), and IL-2, in CD8+ effector, effector memory, central memory, and naïve subsets (Figure B). CD8+ T-cell proliferation was similarly resistant to venetoclax, as subsets demonstrated an IC50 >1000nM for venetoclax. Taken together, these data suggest that survival of resting NK and T cells in not impaired by venetoclax, possibly due to increased levels of BCL-XL and MCL-1, and that T-cell activation is largely independent of BCL-2 inhibition. To evaluate dual BCL-2 inhibition and PD-L1 blockade, the syngeneic A20 murine lymphoma model that is responsive to anti-PD-L1 treatment was used. Immune-competent mice bearing A20 subcutaneous tumors were treated with clinically relevant doses of venetoclax, murine specific anti-PD-L1, or both agents. Single-agent anti-PD-L1 therapy resulted in robust tumor regression, while single-agent venetoclax had no effect. The combination of venetoclax and anti-PD-L1 resulted in efficacy comparable with single-agent anti-PD-L1 (Figure C), suggesting that BCL-2 inhibition does not impact immune-cell responses to checkpoint inhibition in vivo. These data support that venetoclax does not antagonize immune-cell function and can be combined with immunotherapy targets. Conclusions: Our data demonstrate that significant venetoclax-induced cell death at clinically relevant drug concentrations is limited to the B-cell subset and that BCL-2 inhibition is not detrimental to survival or activation of NK- or T-cell subsets. Importantly, preclinical mouse models confirm the combinability of BCL-2 and PD-L1 inhibitors. These data support the combined use of venetoclax and cancer immunotherapy agents in the treatment of patients with hematologic and solid tumor malignancies. Figure Figure. Disclosures Lasater: Genentech Inc: Employment. Do:Genentech Inc: Employment. Burton:Genentech Inc: Employment. Li:Genentech Inc: Employment. Oeh:Genentech Inc: Employment. Molinero:Genentech Inc: Employment, Equity Ownership, Patents & Royalties: Genentech Inc. Penuel:Genentech Inc: Employment. Sampath:Genentech Inc: Employment. Dail:Genentech: Employment, Equity Ownership. Belvin:CytomX Therapeutics: Equity Ownership. Sumiyoshi:Genentech Inc: Employment, Equity Ownership. Punnoose:Roche: Equity Ownership; Genentech Inc: Employment. Venstrom:Genentech Inc: Employment. Raval:Genentech Inc: Consultancy, Employment, Equity Ownership.

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Bone Marrow Derived, De Novo Generated CD4+ T Cells Are Previously Unsuspected Participants in CD8+ T Cell-Mediated Graft-Versus-Host Disease.

Blood ◽

10.1182/blood.v106.11.581.581 ◽

2005 ◽

Vol 106 (11) ◽

pp. 581-581

Author(s):

Yi Zhang ◽

Elizabeth Hexner ◽

Dale Frank ◽

Joe Gerard ◽

Frank Kung ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Acute Gvhd ◽

De Novo ◽

Memory T Cells ◽

Tissue Injury ◽

Chronic Gvhd ◽

Host Tissue

Abstract Although mature CD8+ T cells are known to be major effectors of acute GVHD, patients receiving T cell-depleted allografts remain at high risk for chronic GVHD. To what extent CD8+, CD4+ or both T cell subsets contribute to this chronic immunopathology is not known. We have recently demonstrated that alloreactive memory T cells develop in mice with acute GVHD and account for the persistence of host tissue injury (Journal of Immunology, 2005;174:3051). Based on these findings, we now ask whether de novo generated donor T cells from engrafted T-BM themselves contribute to persistent host tissue injury in GVHD. Confirming previous observations, we found that transplantation of lethally irradiated C57BL/6SJL (B6, CD45.1) mice with highly purified C3H.SW (CD45.2) CD4+ naïve T cells did not cause GVHD, but mice receiving highly purified CD8+ naïve T cells together with C3H.SW T-BM, suffered severe acute GVHD. Surprisingly, in these mice receiving only CD8+ T cells, a substantial number of donor CD4+ T cells as well as CD8+ T cells were detected in GVHD target tissues, indicating that these infiltrating CD4+ T cells had arisen de novo from the transplanted T-BM. Donor CD4+ T cells recovered from GVHD mice expressed surface markers of activated effector/effector memory T cells, including CD25, CD69, CXCR3, and CD44hiCD62Llo. In response to host DCs, purified GVHD CD4+ T cells proliferated and expanded 4-5X more, and produced 10X higher levels of IFN-γ than did CD4+ T cells derived from B6 mice receiving C3H.SW T-BM alone. Furthermore, adoptive transfer of these in vivo generated GVHD CD4+ T cells, without CD8+ T cells, into secondary irradiated B6 recipients induced clinical GVHD characterized by delayed onset, weight loss, diarrhea, and lymphopenia, but without cutaneous inflammation. Histologic examination demonstrated chronic inflammation in the liver and intestinal tract, including epithelial apoptosis. Thymic pathology was dramatic in secondary B6 recipients of GVHD CD4+ T cells, including thymic atrophy, loss of thymic cortex, and infiltration of large amount of tingible macrophages. Taken together, these results demonstrate that donor bone marrow derived, de novo generated CD4+ T cells also contribute to GVHD together with transferred mature CD8+ T cells. Moreover, they suggest that these CD4+ T cells, in concert with alloreactive memory CD8+ T cells that develop during the evolution of GVHD, cause the persistence of acute GVHD and its subsequent progression into chronic GVHD. Thus, donor BM-derived, de novo generated CD4+ T cells are the “Hidden Dragon” of CD8+ T cell-mediated GVHD. Understanding how these CD4+ T cells are generated and regulated will prove to be critical to the prevention and treatment of both acute and chronic GVHD.

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CD8-Depleted Donor Lymphocyte Infusions Convert Mixed into Complete Donor T-Cell Chimerism after T-Cell Depleted Allogeneic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v112.11.2160.2160 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2160-2160

Author(s):

Ralf G Meyer ◽

Eva M Wagner ◽

Timo Schmitt ◽

Klaus Bender ◽

Udo F Hartwig ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Allogeneic Stem Cell Transplantation ◽

Cd4 T Cells ◽

Acute Gvhd ◽

T Cell Reconstitution ◽

The Impact ◽

Donor Lymphocyte Infusions

Abstract Donor lymphocyte infusions (DLI) are increasingly used to treat minimal residual disease or mixed hematopoietic donor-recipient chimerism in T-cell depleted allogeneic stem cell transplantation (SCT). In addition, several clinical trials currently investigate the prophylactic application of DLI to promote donor T-cell reconstitution after transplantation. However, DLI carry a substantial risk of inducing graft-versus-host disease (GVHD). We investigate DLI heavily depleted of CD8 T cells using a clinical grade immunomagnetic in vitro procedure in an ongoing clinical study [Meyer et al., Blood2007, 109:374]. These DLI are administered in a prophylactic setting to patients with hematological malignancies who are off immunosuppressive treatment and free of GVHD early after allogeneic SCT. The reduced-intensity conditioning regimen consists of fludarabine and melphalan and in vivo T-cell depletion (TCD) by the anti-CD52 antibody Alemtuzumab. Up to now, 24 patients have been treated with 1 to 4 increasing doses of CD8-depleted DLI starting with 1x10^6 CD4+ T cells/kg bodyweight. The median time between SCT and first DLI was 119 days (range, 60–194). Seven of 24 patients (29%) developed acute GVHD of grade 2 to 4 or extensive chronic GVHD following CD8-depleted DLI. We did a longitudinal analysis of lineage-specific T-cell chimerism in 20 patients who received CD8-depleted DLI in comparison to 17 patients who did not qualify for DLI due to spontaneously occurring acute GVHD (n=14) or unavailable donors (n=3). The patients’ characteristics in both groups were comparable with a median age of 55 (range, 35–64) years in the DLI group and of 57 (range, 29–67) years in the non-DLI group. The donor types were matched sibling (DLI: n=6; non-DLI: n=2), matched unrelated (DLI: n=8; non-DLI: n=8), and unrelated with 1 HLA-mismatch (DLI: n=6; non-DLI: n=7), respectively. Twelve of the 20 patients in the DLI group and 6 of 17 patients in the non-DLI group showed a secondary decrease of donor T-cell chimerism to a median of 52% (range, 10–90%) between 7 and 35 weeks after transplantation (median, 12 weeks). Only one of the latter spontaneously reconverted to a full donor T-cell chimera. Of the remaining 5 non-DLI patients, 4 patients subsequently relapsed with their underlying disease and one patient still had a mixed T-cell chimerism of 50% two years after transplantation. In contrast, in patients receiving CD8-depleted DLI the proportion of donor T cells significantly increased and 11 of 12 patients converted to durable full donor chimerism. Three patients of the DLI group subsequently developed disease relapse. By monitoring CD52-expression on reconstituting T cells by flow cytometry, we were able to demonstrate the impact of CD8-depleted DLI on post-transplant T-cell reconstitution: In the non-DLI group, the majority of CD4 T cells remained CD52-negative 9 months after transplantation. Simultaneously, the proportion of CD52-expressing CD4 T cells was significantly higher in the DLI group (mean: 42% versus 86%; t-test, p<0.01). A similar difference was not observed in the CD8 T-cell lineage. Our results show that the impact of adoptively transferred CD4 T cells after anti-CD52 antibody-mediated TCD can be readily demonstrated by CD52 staining of reconstituting T cells. In addition, CD8-depleted DLI can be efficiently applied to switch mixed into complete donor T-cell chimerism. On the basis of our data we propose a prospective randomized trial in patients with a secondary decrease of donor T-cell chimerism that investigates if CD8-depleted versus non-depleted DLI lead to an improved outcome with regard to disease control and GVHD.

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Hepatocyte growth factor preserves graft-versus-leukemia effect and T-cell reconstitution after marrow transplantation

Blood ◽

10.1182/blood-2003-12-4309 ◽

2004 ◽

Vol 104 (5) ◽

pp. 1542-1549 ◽

Cited By ~ 29

Author(s):

Takehito Imado ◽

Tsuyoshi Iwasaki ◽

Yasuro Kataoka ◽

Takanori Kuroiwa ◽

Hiroshi Hara ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Growth Factor ◽

Acute Gvhd ◽

Gene Transfection ◽

T Cell Reconstitution ◽

Graft Versus Leukemia ◽

Hepatocyte Growth ◽

Graft Versus Leukemia Effect

Abstract Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation (BMT). When GVHD is controlled by T-cell–depleted grafts or immunosuppressants, BM transplant recipients often suffer from an increased rate of leukemic relapse and impaired reconstitution of immunity. Using a mouse BMT model, we investigated the effects of hepatocyte growth factor (HGF) gene transfection on the severity of GVHD, the graft-versus-leukemia effect, and the reconstitution of T cells after BMT. After HGF gene transfer, acute GVHD was reduced, while mature donor T-cell responses to host antigens were preserved, resulting in a significant improvement of leukemia-free survival. HGF gene transfer promoted regeneration of bone marrow–derived T cells and the responsiveness of these cells to alloantigens. Furthermore, HGF preserved the thymocyte phenotype and thymic stromal architecture in mice with GVHD. This suggested that HGF exerts a potent protective effect on the thymus, which in turn promotes reconstitution of bone marrow–derived T cells after allogeneic BMT. These results indicate that HGF gene transfection can reduce acute GVHD preserving the graftversus-leukemia effect, while promoting thymic-dependent T-cell reconstitution after allogeneic BMT.

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Early T Cell Recovery of Thymus-Derived Naive T Cells and NK Cells in Pediatrics Patients after T-Cell Depleted HLA-Haploidentical Stem Cell Transplantation for Thalassemia.

Blood ◽

10.1182/blood.v110.11.3816.3816 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3816-3816 ◽

Cited By ~ 1

Author(s):

Antonella Isgro’ ◽

Pietro Sodani ◽

Marco Marziali ◽

Buket Erer ◽

Cecilia Alfieri ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Stromal Cells ◽

Cd4 T Cells ◽

Th Cells ◽

Post Transplant

Abstract Delayed immune recovery post transplant remains a significant obstacle and results in increased risk of infections. T cells are regenerated via 2 pathway, thymus-derived and peripheral expansion, processes for which IL-7 is critical. To analyse the mechanisms involved in immunological reconstitution, we studied six thalassemia patients after 20 and 60 days post T-cell-depleted HLA-haploidentical stem cell transplantation. The mean age ranged from 14 to 5 years. As controls, 6 healthy donors matched by sex and age with the patients were included. We analysed T cell subsets by flow cytometry. Stromal cells, obtained from long term culture of bone marrow mononuclear cells were analysed by immunohystochemistry and the stromal IL-7 production was analysed by ELISA. Day + 20 post transplant, the patients had significantly lower CD4+ T cells in comparison to the controls (1.9 ± 1.4% vs. 47.5 ± 6% respectively), and this reduced number was mainly observed in CD45RA+CD62L+ (naive phenotype) subset (1.3 ± 2% in patients vs. 52 ± 12% in controls). A significant decrease of peripheral CD45RA+CD31+ Th cells (thymic naive Th cells) (on average 0.5 ± 0.3% in patients vs. 37 ± 10% in controls) was observed, whereas CD8+ T cells numbers did not statistically differ between patients and controls (24.2 ± 33.7% vs. 20 ± 7%). NK cells were among the first lymphocytes to repopulate the peripheral blood, and up to 70% of these cells were CD56 bright whereas CD56dim CD16+ NK cells were reduced. Day + 60 post transplant an increase in the percentages of CD4+ T cells, naïve CD4+ cells and in thymic naïve Th cells were observed (3 ± 1.2%, 2.9 ± 2.1%, 2.7 ± 1%, respectively). CD8+ T cells were also increased (in mean 35 ± 27.5%). Compared with normal subjects, thalassemia patients showed a significant increase of CD4+ cell activation markers (CD95, HLA-DR and CCR5) and this was observed after 60 days post transplant, in parallel with the increase of the CD56dim CD16+ NK cells especially in the patients with full engraftment. Stromal cells secreted lower IL-7 levels (0.3 + 0.1 pg/mL vs. 0.8 + 0.1 pg/mL, in controls) and displayed by immunohistochemistry an altered phenotype (“macrophage-like” morphology). A significant decrease in total lymphocyte counts and depletion of CD4+ T cells expressing predominantly the CD45RA+CD62L+ phenotype were observed after 60 days post transplant. Also the CD4+CD45RA+CD31+ T cell subset was initially reduced but an increase has been observed at day + 60 post transplant, suggesting a thymus involvement in these patients. An IL7/IL7R pathway dysregulation has been also observed, possibly involving bone marrow stromal cells. NK cells were among the earliest lymphocytes to repopulate the peripheral blood, but. CD56dim CD16+ NK cells were increased after 60 days post transplant, especially in the patients with full engraftment, suggesting a role of donor NK cells on bone marrow engraftment. We hypothesize that the recovery of T cell compartment may be due to a deregulated production of new T cells starting from haematopoietic stem cells under the influence of stromal cytokines production.

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