scholarly journals LB-ARHGDIB-1R As Novel Minor Histocompatibility Antigen For Therapeutic Application

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4465-4465
Author(s):  
Margot J Pont ◽  
Willemijn Hobo ◽  
M. Willy Honders ◽  
Simone A.P. van Luxemburg-Heijs ◽  
Michel G.D. Kester ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Hematological Malignancies ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Specific Lysis ◽  
Hematopoietic Stem ◽  
Therapeutic Relevance

Patients with hematological malignancies can be successfully treated with allogeneic hematopoietic stem cell transplantation (alloSCT). Beneficial Graft-versus-Leukemia (GvL) reactivity, however, is often accompanied with undesired Graft-versus-Host Disease (GvHD). In HLA-matched alloSCT, donor T-cells can mediate GvL reactivity by recognition of minor histocompatibility antigens (MiHA) on malignant cells, but also GvHD when MiHA are recognized on non-hematopoietic tissues. To decrease the incidence and severity of GvHD, T-cells can be (partially) depleted from the graft and re-administered later as donor lymphocyte infusion (DLI). MiHA are polymorphic peptides that are presented in HLA-molecules and can be recognized as non-self by donor-derived T-cells. Only a minority of MiHA have therapeutic relevance based on hematopoietic-restricted expression and only 25% and 40% of recipients transplanted with sibling and unrelated donors, respectively, are eligible for T-cell therapies in which one of the known hematopoietic-restricted MiHA is targeted. Therefore, to increase the efficacy and limit the toxicity of T-cell therapy, more therapeutic MiHA are needed. Recently, we identified a MiHA encoded by ARHGDIB, a gene that has been described to be highly expressed on hematopoietic cells, and we here studied the therapeutic relevance of LB-ARHGDIB-1R in detail. First, we confirmed hematopoietic-restricted expression of ARHGDIB by microarray gene expression analysis and demonstrated >10-fold overexpression in the majority of malignant and healthy hematopoietic versus non-hematopoietic cells. In line with its hematopoietic-restricted gene expression profile, LB-ARHGDIB-1R in the context of HLA-B* 07:02 was specifically recognized on different hematological malignancies, but not on non-hematopoietic fibroblasts and keratinocytes cultured in the absence or presence of IFN-γ, which was added to mimick the pro-inflammatory cytokine milieu of the early post-transplantation period. Next, we investigated the cytolytic capacity of LB-ARHGDIB-1R specific T-cells, and demonstrated specific lysis of patient, but not donor, EBV-B cells and specific lysis of an ALL sample in a 10 hrs 51Cr-release assay. Specific lysis of additional ALL and AML samples could be measured after 48 hrs of co-incubation in a flowcytometry-based cytotoxicity assay. To determine the in vivo immunogenicity of LB-ARHGDIB-1R, 11 MiHA-disparate HLA-B* 07:02 positive patient-donor pairs were screened for specific CD8+ T-cells by dual color tetramer analysis. All patients received partial T-cell depleted alloSCT and sampling was done at different time points after alloSCT (and DLI). In 4 out of 11 patients, LB-ARHGDIB-1R-specific T-cells (>0.01%) could be detected directly ex vivo and in 4 additional patients after 7 days of in vitro peptide stimulation, indicating that LB-ARHGDIB-1R is highly immunogenic. High frequencies of LB-ARHGDIB-1R specific T-cells were measured ex vivo in a patient whose hematological relapse was successfully treated with DLI, without development of GvHD, further supporting the therapeutic relevance of LB-ARHGDIB-1R. In summary, we confirmed hematopoietic-restricted expression of LB-ARHGDIB-1R and demonstrated T-cell mediated lysis of primary leukemic cells in long-term co-incubation assays. Furthermore, we showed that LB-ARHGDIB-1R is highly immunogenic and that specific T-cells could be detected in a patient who responded to DLI in the absence of GvHD. Altogether, our data support the clinical relevance of LB-ARHGDIB-1R as therapeutic MiHA with the potential to shift the delicate balance between GvL and GvHD in favor of a desired anti-tumor effect. Disclosures: No relevant conflicts of interest to declare.

Naive and Ex Vivo Activated Human T Cells Generate Consistent Engraftment and Lethal Graft-Versus-Host Disease (GvHD) in NOD SCID β 2M Null Mice: A New Xenogeneic Model for GvHD.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3106-3106
Author(s):  
Bruno Nervi ◽  
Michael P. Rettig ◽  
Julie K. Ritchey ◽  
Gerhard Bauer ◽  
Jon Walker ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Conditioning Regimen ◽  
Activation Markers ◽  
In Vivo Models ◽  
Hematopoietic Stem ◽  
Human T Cells ◽  
The Impact

Abstract GvHD remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion. The human GvHD pathophysiology includes recipient tissue destruction and proinflammatory cytokine production associated with the conditioning regimen; donor T cells become allo-activated, proliferate, and mediate tissue injury in various organs, including the liver, skin, and gut. Modern therapeutic strategies to control GvHD while maintaining the beneficial graft-versus-leukemia effects require ex vivo T cell stimulation and expansion. Multiple studies have demonstrated that these ex vivo expanded T cells exhibit decreased survival and function in vivo, including reduced alloreactivity and GvHD potential. Unfortunately no in vivo models exist to consistently examine the impact of ex vivo manipulation of human T cells (HuT) on T cell function. Naive HuT were compared to HuT activated using CD3/28 beads (XcyteTMDynabeads) with 50 U/ml IL-2 for 4 days (Act). We initially evaluated the HuT engraftment and GvHD potential of naive and Act in RAG2γ null mice (n=22) conditioned with clodronate liposomes on day −1 and 350cGy on day 0, as previously described by others. We injected 107 and 1.5x107 naive or Act HuT intravenously (iv). All mice exhibited low HuT engraftment and no lethal GvHD. NOD SCIDβ 2M null mice (β 2M) were next conditioned with 250cGy on day −1 (n=34), or 300cGy on day 0 (n=21). 107 naive vs Act HuT were injected retroorbitaly (ro). Lower HuT doses or iv injection resulted in no expansion or GvHD. Engraftment of HuT in peripheral blood of recipient mice was evaluated weekly by FACS and euthanasia was performed if mice lost > 20% body weight. 60% of the mice conditioned with 250cGy that received naive HuT developed lethal GvHD, in comparison to 75% of mice that received 300cGy and nave HuT, and 100% of mice that received 300cGy and Act HuT. Table 1 250cGy 300cGy Naive (n=34) Naive (n=8) Activated (n=13) *p<0.02 PB engraftment (%HuT) 20%±15 33%±21 59%±19 Lethal GvHD 60% 75% 100% All mice receiving 300cGy had well preserved CD4/CD8 ratios (1–1.5). Tissue infiltration was greatest in mice that had received 300cGy and Act HuT (spleen, liver, lung, kidney: 50–70%). Of interest, serum levels of hu IFNγ dramatically increased over time in all mice who went on to develop lethal GvHD (day 3=270 ug/ml and day 15=36,000 ug/ml) compared to mice that did not develop lethal GvHD (day 10=40 ug/ml and day 17=1,020 ug/ml)(p<0.05). Interestingly, the up-regulation of the activation markers CD25 and CD30 in HuT, and IFNγ production predicted lethal GvHD in β 2M null mice. In summary, we developed a xenogeneic model of lethal GvHD where naive or ex vivo Act HuT injected ro in sublethaly irradiated β 2M not only engraft, expand in vivo, but also infiltrate and damage different mouse target organs. HuT are allo-activated against mouse antigens and damage the target tissues, sharing the major characteristics of human GvHD and causing the death of mice. This model will allow us to study the effects of specific ex vivo T cell manipulation including transduction, selection, expansion, and the depletion or addition of various T cells and other cellular subsets on the outcome of GvHD, to determine improved therapeutic interventions.

Repertoire Analysis of Ex Vivo Polyclonally Expanded Regulatory T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3222-3222
Author(s):  
Jenny Zilberberg ◽  
Kira Goldgirsh ◽  
Robert Korngold ◽  
Thea M. Friedman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Clinical Situation ◽  
Post Transplantation ◽  
Cell Repertoire ◽  
Hematopoietic Stem ◽  
Pcr Products

Abstract CD4+CD25+ regulatory T cells (Treg) are essential for the maintenance of self-tolerance and have also been implicated in the control of alloreactive immune responses. Several studies using murine models of graft-vs.-host disease (GVHD) have shown that addition of equivalent numbers of Treg to the donor T cell inoculum at time of hematopoietic stem cell transplantation can significantly reduce the incidence of GVHD. In addition, in an MHC-matched, minor histocompatibility disparate model, the infusion of Treg ten days post-transplantation was shown to ameliorate the progression of GVHD while permitting a graft-versus-leukemia effect. However, because Treg constitute <5% of peripheral CD4+ T cells in humans, the use of freshly isolated Treg to treat and/or prevent GVHD, as well as other diseases in the clinical situation, is limited. Therefore, much effort is now under way to expand Treg in order to have sufficient numbers for therapeutic use. There is little available information regarding the repertoire complexity of ex vivo, polyclonally expanded regulatory T cells. We hypothesize that like their CD4+CD25− T cell counterparts, the diversity of the Treg T cell receptor (TCR) repertoire will also be complex. To this end, CD4+CD25− and CD4+CD25+ T cells from B10.BR mice were purified using fluorescence activated cell sorting; both populations were polyclonally expanded using CD3/CD28 paramagnetic microbeads in combination with high levels (100 IU/ml) of hrIL-2. After achieving a greater than 50 fold expansion, RNA from 1–1.5×107 cells was isolated for RT-PCR. The complexity of the T cell repertoire of expanded CD4+CD25− and CD4+CD25+ was determined using TCR Vb CDR3-size spectratype analysis. The PCR products were run on a sequencing gel and analyzed by the GeneMapper Software from Applied Biosystems. This comparison revealed that the number of resolvable Vb families is more heterogeneous in the CD25− populations. Whether this reflected a lack of complexity in the regulatory repertoire warrants further investigation. However, for the resolvable Vb families there were no significant differences in the complexity indexes between these two groups.

Rapid T Cell Recovery and Possible Auto-GVHD Following Adoptive Transfer of Ex-Vivo Costimulated Autologous T Cells at Day +2 Post-Transplant.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 769-769 ◽  
Author(s):  
Aaron P. Rapoport ◽  
Stephan A. Grupp ◽  
Edward A. Stadtmauer ◽  
Robert H. Vonderheide ◽  
Bruce L. Levine ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Ex Vivo ◽  
Cell Recovery ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Cell Counts ◽  
Engraftment Syndrome ◽  
The Impact

Abstract Retrospective studies suggest that rapid lymphocyte recovery following autologous stem cell transplants (SCT) may be associated with better outcomes. Previously we showed that adoptive transfer of in-vivo vaccine-primed and ex-vivo (anti-CD3/anti-CD28) costimulated autologous T cells (ex-T) at about day 14 post-transplant increased CD4 and CD8 T cell counts at day 42 post-transplant and induced pneumococcal conjugate vaccine-directed T and B-cell responses [Rapoport et al, Nature Medicine, 2005]. In 2 current studies, we are further investigating the impact of ex-vivo costimulated autologous T cells on vaccine responses after SCT. In the first study, we are investigating whether a similar strategy of pre- and post-transplant immunizations along with an early infusion of vaccine-primed ex-T can induce responses to a putative tumor vaccine composed of 4 HLA-A2-restricted peptides derived from survivin and hTERT in pts undergoing SCT for myeloma. In the second (randomized) trial, the impact of early ex-T on immune recovery and vaccine reponses is being tested in pediatric neuroblastoma pts. Compared to the previous study, two methodologic changes were made: The target number of T cells infused was raised 5-fold to 5 x 1010 (109/kg) T cells were infused on day + 2 to take greater advantage of homeostatic expansion mechanisms. Patients were monitored for delayed hematopoietic recovery because of this switch to early ex-T and the fact that survivin and hTERT are also expressed in hematopoietic stem cells. At the time of submission, 16 adult and 30 pediatric patients have been enrolled on these trials of whom 11 and 21, respectively, are evaluable for post-transplant hematopoietic and T-cell recovery. On the myeloma trial, the mean # of T cells infused was 3.95 x 1010 with 96% viability and a CD4/CD8 ratio of 1.8:1. At day 14 post-transplant, the median CD4 count was 1951/mcl (range 651–7668) and the median CD8 count was 4117/mcl (range 1499–39,354). The median # days to achieve an absolute neutrophil count (ANC) > 500 was 12 (range 11–14) and the median # days to achieve a PLT count >20,000/mcl was 13 days (range 0–28). Similarly, in the pediatric cohort, median CD4 and CD8 counts at day 30 were 1500 and 2100/mcl, respectively, compared to 22 and 14 in a group of pts who did not receive d+2 ex-T, with no impact on engraftment. 1 adult and 3 pediatric pts also developed an “engraftment syndrome” characterized by GHVD-like features with or without fever. The adult pt with day 14 CD4 and CD8 counts of 2,724 and 11,571 cells/mcl had clinical and histologic features of (autologous) gut GVHD. 3 pediatric pts developed pruritic rashes clinically and pathologically indistiguishable from GVHD within 14 d of ex-T infusion, with fever seen in 1. In the adult and 1 pediatric pt, steroid treatment led to complete resolution of symptoms. These combined data sets demonstrate that robust CD4 and CD8 T cells counts can be achieved as early as day 14 post-SCT when adults or children receive ex-T at day +2 post-SCT without exogenous IL-2 or other cytokine support. It appears that a subset of patients develop a T cell “engraftment syndrome” similar to autologous GVHD. The mechanisms responsible for this rapid immune cell recovery are currently under investigation.

Analysis of TCR Vgamma and Vdelta Repertoire and Clonality of T Cells in MDS Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4833-4833
Author(s):  
SuXia Geng ◽  
Xin Du ◽  
Yangqiu Li ◽  
Jianyu Weng ◽  
Liye Zhong ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Clonal Expansion ◽  
Refractory Anemia ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Discriminative Value ◽  
Skew Distribution

Abstract Abstract 4833 Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and frequent progression to acute myeloid leukemia. The pathophysiology of these syndromes remains poorly explained. The immune system also seems to contribute to the progressive cytopenias observed in MDS in some cases. We investigated the frequency and the discriminative value of TCR gamma and delta CDR3 clonality using RT-PCR and genescan technique in 40 MDS patients (24 male, 16 female),which included refractory anemia(n=1), refractory anemia with with ringed sideroblasts(n=6), refractory anemia with excess of blasts I (n=16)and II (n=17) respectively. The median age was 60 years (range 15-84).The results showed that 35(87.5%) MDS patients expressed all three TCR Vgamma subfamilies. Four(10%) and one(2.5%) MDS patients expressed two and one TCR Vgamma subfamilies respectively. Clonal expansion of T cells in some Vgamma subfamilies could be identified in 31 patients. The clonal expansion of Vgamma2 subfamily were identified most frequently. All the Vdelta subfamilies were absent in 2 MDS patients. The remaining 38 (95%) MDS cases expressed 0-6 Vdelta subfamilies. Vdelta1 and Vdelta2 were expressed most frequently followed by Vdelta8. While only 3(7.5%) patients expressed Vdelta5 subfamilies. Clonal expansion of Vdelta T cells were found in all MDS patients. The most frequent clonal expansion T cell was Vdelta3. In addition, 12 patients expressed monoclonal T cell Vdelta subfamily. Monoclonal expanded T cells were found in Vdelta3, Vdelta4, Vdelta6, Vdelta7 and Vdelta8 subfamilies. In summary,we found that MDS patients showed TCR Vgamma and delta skew distribution and clonal expansion. The absent of TCR Vdelta subfamily was more evident than TCR Vgamma subfamily. These findings provided further evidence that T cell mediated immune processes were a feature of MDS. Disclosures No relevant conflicts of interest to declare.

Anti-Thymocyte Globulin (ATG) Differentially Depletes naïve and Memory T Cells and Permits Memory-Type Regulatory T Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4670-4670
Author(s):  
Chang-Qing Xia ◽  
Anna Chernatynskaya ◽  
Clive Wasserfall ◽  
Benjamin Looney ◽  
Suigui Wan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Memory T Cells ◽  
Conflicts Of Interest ◽  
T Cell Subsets ◽  
Hematopoietic Stem

Abstract Abstract 4670 Anti-thymocyte globulin (ATG) has been used in clinic for the treatment of allograft rejection and autoimmune diseases. However, its mechanism of action is not fully understood. To our knowledge, how ATG therapy affects naïve and memory T cells has not been well investigated. In this study, we have employed nonobese diabetic mouse model to investigate how administration of anti-thymocyte globulin (ATG) affects memory and naïve T cells as well as CD4+CD25+Foxp3+ regulatory T cells in peripheral blood and lymphoid organs; We also investigate how ATG therapy affects antigen-experienced T cells. Kinetic studies of peripheral blood CD4+ and CD8+ T cells post-ATG therapy shows that both populations decline to their lowest levels at day 3, while CD4+ T cells return to normal levels more rapidly than CD8+ T cells. We find that ATG therapy fails to eliminate antigen-primed T cells, which is consistent with the results that ATG therapy preferentially depletes naïve T cells relative to memory T cells. CD4+ T cell responses post-ATG therapy skew to T helper type 2 (Th2) and IL-10-producing T regulatory type 1 (Tr1) cells. Intriguingly, Foxp3+ regulatory T cells (Tregs) are less sensitive to ATG depletion and remain at higher levels following in vivo recovery compared to controls. Of note, the frequency of Foxp3+ Tregs with memory-like immunophenotype is significantly increased in ATG-treated animals, which might play an important role in controlling effector T cells post ATG therapy. In summary, ATG therapy may modulate antigen-specific immune responses through modulation of naïve and memory T cell pools and more importantly through driving T cell subsets with regulatory activities. This study provides important data for guiding ATG therapy in allogenieic hematopoietic stem cell transplantation and other immune-mediated disorders. Disclosures: No relevant conflicts of interest to declare.

The T-Cell/CLL/Macrophage Triad Shapes a Supportive Tumor Microenvironment in CLL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1715-1715
Author(s):  
Martijn H.A. van Attekum ◽  
Sanne Terpstra ◽  
Emilie Reinen ◽  
Marieke Von Lindern ◽  
Erik Slinger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Expression ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Cellular Interactions ◽  
Activated T Cells ◽  
Cd40 Activation

Abstract Survival of CLL cells critically depends on heterotypic communication with benign bystanders cells in micro-environmental niches such as lymph node (LN) tissue. Here, mesenchymal stromal cells and macrophages, in concert with CD40L expressing T cells, are thought to participate in the dialog with the neoplastic B cells, but the mechanisms of this intricate interplay remain largely unknown. Moreover, whether CLL cells actively participate in shaping their prosurvival niche is poorly understood. We aimed to study 1) whether CD40 stimulation initiates active recruitment of monocytes by CLL cells, 2) whether CLL cells are able to differentiate these monocytes towards a supporting phenotype and 3) by which mechanism macrophages induce CLL survival. We first studied the chemokinome of CLL cells after T cell stimulation using both microarray and Luminex techniques. Co-culture of autologous activated T cells with CLL cells resulted in induction of mRNA expression of CCL2,3,4,5,22 and IL10, which are known chemo-attractants for monocytes. These effects could be mimicked by CD40 activation of CLL cells. Protein screens of supernatants of CD40 activated CLL cells by Luminex assays confirmed increased protein expression of these chemo-attractants. Indeed, transwell assays showed enhanced migration of primary monocytes towards supernatants of CD40L stimulated CLL cells. Inhibitor experiments furthermore showed that the migratory effects of these chemokines was largely governed via the CCR2 and CCR3 receptors. We next examined and compared polarization patterns of monocytes after differentiation with serum derived from CLL patients (N=25) or pooled healthy donor serum and found that CLL serum was able to differentiate macrophages towards a tumor supporting M2 phenotype. This finding was confirmed ex vivo by IHC, as M2 marker CD206 co-localizes with CD68 cells in CLL LNs, while the majority of macrophages in non-CLL derived LNs are CD80+ (M1 type). Lastly, we examined how these macrophages exert their pro-survival effect on CLL. From a variety of Bcl-2 family proteins investigated, only Mcl-1 protein expression levels increased after interaction with macrophages. The relevance of Mcl-1 upregulation was verified by MCL-1 siRNA interference studies. The mechanism of induction of Mcl-1 was independent on NF-κB signaling, Mcl-1 mRNA transcription levels or protein stability, but rather unexpectedly appeared as a result of recruitment of polysomes to Mcl-1 mRNA, resulting in an increase in translation. This increase was accompanied by an increased phosphorylation of the rate-limiting translation initiation factor 4E-BP1 and ribosomal protein S6. The increase in Mcl-1 translation could be attributed to macrophage-induced Akt signaling. In conclusions, these studies shed light on reciprocal cellular interactions in the CLL LN that shape pro-tumor differentiation of supporting cells, that in turn cause survival by changing the apoptotic balance. These interactions can be targeted at different levels, creating new treatment venues for this still incurable disease. Disclosures No relevant conflicts of interest to declare.

Enhancing Cytotoxicity of Autologous T Cells in AML By Blockade of CTLA-4 and PD-1

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4057-4057 ◽  
Author(s):  
Kirsten Marie Boughan ◽  
Xiaohua Chen ◽  
Paul Szabolcs
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Response ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
T Cell Response ◽  
Inhibitory Receptors

Abstract Background: AML remains a disease diagnosed in the aging population with chemotherapy followed by bone marrow transplant in some cases being the standard of care. Although response rates remain around 50-60%, treatment related mortality and disease relapse remain high. Adoptive immunotherapy, especially those targeting T cell co-inhibitory receptors, has proven successful in solid malignancies however, AML remains less explored. Our laboratory has previously demonstrated the feasibility to generate autologous AML reactive T cells in vitro (Mehta/Szabolcs; Immunotherapy 2016). It was noted that "resistant" AML blasts over expressed a number of genes associated with immunosuppressive characteristics. Over expression of these genes may induce T cell functional exhaustion. Therefore, we hypothesized that blocking PD-1 and/or CTLA-4 during co-culture with IFNg activated AML blasts, may enhance T cell activation and cytotoxicity. To test this hypothesis, we tested CTL responses against AML blasts and IFNg ELISpot formation after blocking with PD-1, CTLA-4 or both receptors, and compared the response in untreated T cells. Gene expression profiles of co-stimulatory/co-inhibitory receptors were also monitored to test for correlation. Methods: We evaluated 12 patients with newly diagnosed AML under an IRB approved protocol with written informed consent of patients. Mononuclear cell preparation was generated from fresh marrow samples or drawn from a biorepository of previously cryopreserved leukophereses. T cells were then purified using immunomagnetic CD3/CD28 beads (Life technologies) and cultivated in media with IL-2 and IL-7 for 2 weeks. AML blasts were cultured over a supporting layer of mesenchymal stromal cells (MSCs) derived from healthy BM donors for 1 week and then cryopreserved. T cells were then co-cultured with restored and irradiated autologous AML cells at an effector: target (E: T) ratio of 5:1 to 40:1. AML and T cells were co-cultured in the presence of Ipilimumab (anti-CTLA-4), or Nivolumab (anti-PD-1), or a combination of both drugs. T cells and AML were re stimulated in X-vivo 15 with IL-12, IL-15 and IL-2 weekly x 3weeks. T cell response to AML was quantitated by IFNg ELISpot assay and Europium TDA (EuTDA) CTL assays independently. Co-stimulatory/co-inhibitory expression on T cells was examined with RT-q PCR assay. Paired-sample student t test was used for statistical analysis with p<0.05. Results and Discussion: Out of 12 samples, 10 (83%) yielded viable AML cells available for cytotoxicity assay. One third (33%) of co-cultures exhibited a positive T cell response in CTL assays ("killers"). There was no difference in CTL activity by blockade of either PD-1 or CTL-4 (Fig 1). IFN-ɣ spot formation in ELISpot was observed in 4/10 samples (40%) with statistical significance noted in cells blocked with PD-1 as compared to all other blockade types (Fig 2). The results indicated that in vitro priming with autologous AML blasts or together with blocking PD-1 can enhance T cell response in 33-40%. By gene expression analysis, the ratio of co-stimulatory to co-inhibitory genes was calculated. In PD-1 blocked cells, the ratio of activation/inhibition was not impacted in T cells from "killers" (0.9; p=0.1), however, T cells from "non-killer cells" had a diminished ratio due to higher expression of co-inhibitory molecules (0.4; p=0.04) (Fig 3). This trend was also present in CTLA-4 blocked cells (0.85; p=0.4 in killers vs 0.54; p=0.03 in non-killers) (data not shown). Interestingly, dual blockage failed to influence gene expression ratio, data not shown. Conclusion: The above studies demonstrate that cytotoxicity can be achieved in T cells when primed against autologous AML. PD-1 blockade can enhance IFNg production and cytotoxic responses, but CTLA-4 and dual blockade failed to enhance T cell function. The upregulation of an inhibitory pattern of genes in T cells that did not express cytotoxicity (non-killers) could allude to an "inhibitory phenotype" that may be resistant to immunotherapy drug blockade and requires further study. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sting Negatively Regulates Allogeneic T-Cell Responses By Constraining Antigen-Presenting Cell Function

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 37-38
Author(s):  
Yongxia Wu ◽  
Chih-Hang Anthony Tang ◽  
Corey Mealer ◽  
David Bastian ◽  
Mohammed Hanief Sofi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
T Cell Responses ◽  
Receptor Expression ◽  
Constitutive Activation ◽  
Cell Responses ◽  
And Function

The endoplasmic-reticulum-resident protein STING (Stimulator of IFN genes) is a downstream signaling effector of cytosolic DNA sensor cGAS (cyclic GMP-AMP synthase). STING-mediated innate immune activation plays a key role in tumor- and self-DNA elicited anti-tumor immunity and autoimmunity, respectively, yet the mechanism remains largely unclear. We utilized murine models of allogeneic hematopoietic cell transplantation (allo-HCT) to study the biology of STING in antigen-presetting cells (APCs) and T cells. STING expression in donor T cells was dispensable for their ability to induce graft-versus-host disease (GVHD), a major complication of allo-HCT in the clinic. However, when STING-deficient mice were used as recipients, more severe disease was induced after allo-HCT. Using bone marrow (BM) chimeras where STING was absent in different compartments, we found that STING-deficiency on host hematopoietic cells (Fig. A), but not on non-hematopoietic cells, was primarily responsible for exacerbating the disease. Furthermore, STING expression on host CD11c+ cells played a dominant role in the regulation of allogeneic T-cell responses (Fig. B). Mechanistically, STING deficiency resulted in increased survival, activation and function of irradiated APCs, including macrophages and dendritic cells (DCs, fig. C-D). To further determine the role of STING in APCs, we generated a STING V154M knock-in mouse model, in which V154M mutation in TMEM173 causes constitutive activation of STING. Consistently, constitutive activation of STING attenuated the survival, activation and function of APCs isolated from STING V154M knock-in mice. In addition, STING-deficient APCs augmented donor T-cell expansion, chemokine receptor expression and migration into intestinal tissues (Fig. E), resulting in accelerated/exacerbated disease. Using pharmacologic approaches, we demonstrate that systemic administration of a STING agonist (c-di-GMP) to recipient mice before transplantation significantly reduced GVHD mortality (Fig. F). In conclusion, we report an inhibitory role of STING in regulating survival and T-cell priming function of hematopoietic APCs, especially CD11c+ cells, after allo-HCT. We validate that pharmacological activation of STING may serve as a potential therapeutic strategy to constrain APCs and induce immune tolerance. Figure Disclosures No relevant conflicts of interest to declare.

Molecular Follow-Up of Patients Treated with HSV-TK/ΔLNGFR Engineered Donor Lymphocytes after Allogeneic Stem Cell Transplantation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1741-1741
Author(s):  
Chiara Bonini ◽  
Zulma Magnani ◽  
Alessandra Recchia ◽  
Fabrizia Urbinati ◽  
Sara Muraro ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Pcr Analysis ◽  
Hematopoietic Stem ◽  
T Cell Clones ◽  
Cell Clones

Abstract Donor lymphocyte infusions (DLI) play a crucial role in promoting immune reconstitution and anti-tumor activity in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). The efficacy of DLI is, however, limited by the occurrence of graft-versus-host disease (GvHD). In three different clinical trials, we showed that the infusion of lymphocytes transduced by a retroviral vector expressing a suicide gene (HSV-TK) and a surface marker (ΔLNGFR) allows to control GvHD in 100% of cases, while preserving anti-tumor and antiviral activities. In >40 patients treated with TK-cells in the context of HLA identical and haplo-identical HSCT, we observed consistent expansion (up to 40% of circulating cells) and long-term persistence (>10 years) of transduced cells. No acute or chronic adverse or toxic effect due to the gene transfer procedure was observed in these patients, who were treated with a total of >1011 cells generated by >60 independent transductions. Analysis of vector integration sites was preformed by LM-PCR on lymphocytes obtained up to 10 years after treatment from 4 patients, and selected for ΔLNGFR expression. 60% of the sequences met our validity criteria and were unambiguously mapped onto the human genome by Ensembl BLAST analysis. Transduced T-cells were highly polyclonal, with vector integrations occurring preferentially within genes (52%), and particularly within the first intron (25%). Quantitative PCR analysis of selected integrations was performed to follow the dynamics of individual T-cell clones during time. Microchip analysis of the gene expression profile showed that <200 out of >22,000 genes (0.9%) were differentially expressed in ΔLNGFR+ vs. ΔLNGFR- T-cells from two different patients, suggesting that no significant perturbation was induced by retroviral transduction or HSV-TK/ΔLNGFR expression in human lymphocyte populations. Interestingly, these data also show the substantial biological identity of T-cell population generated by HSCT and those administered as DLI. Finally, the influence of proviral integration on the expression of targeted genes was studied in individual T-cell clones transduced in vitro or obtained ex vivo from treated patients by quantitative gene expression analysis.

Adenovirus Specific T-Cell Lines Devoid of Alloreactivity Against Haploidentical Recipients Can Be Obtained Using a Set of Adenovirus Hexon Peptides.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3273-3273
Author(s):  
Patrizia Comoli ◽  
Marco W. Schilham ◽  
Sabrina Basso ◽  
Tamara van Vreeswijk ◽  
Rita Maccario ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Peptide Pool ◽  
Specific Lysis ◽  
Hematopoietic Stem ◽  
Hexon Protein ◽  
T Cell Lines

Abstract Human Adenovirus (HAdV) infection/reactivation may cause life-threatening complications in recipients of hematopoietic stem cell transplantation (HSCT), the highest risk being observed in pediatric recipients of a T-cell depleted allograft from haploidentical family donor. The effectiveness of pharmacological therapy for HAdV infection is still suboptimal. It has been recently demonstrated that cell therapy may offer a unique opportunity to restore antiviral immune surveillance, leading to clearance of infection and prevention/treatment of disease. However, infusion of HAdV-specific T-cells in the haplo-HSCT cohort poses the concern that GVHD may ensue as a consequence of T-cell transfer. We have conducted scale-up experiments to validate a method of in vitro culture to expand T-cells specific for HAdV, based on stimulation of donor peripheral blood mononuclear cells (PBMC) with a pool of 5 30-mer peptides derived from HAdV5 hexon protein, for use in recipients of haplo-HSCT (Veltrop-Duits et al, Eur J Immunol36, p2410; 2006). A total of 20 T-cell lines were generated, starting from a median of 20 × 106 donor PBMC, that yielded a median of 80 × 106 cells. Most of the cell lines obtained included a majority of CD4+ T-lymphocytes, with a lower % CD8+ cells (median and range: 78, 19–94 and 18, 5–58, respectively) but 5/20 lines contained a high number of CD8+ T cells (ranging between 43% and 58%), which were CD56+ and/or TCRγδ+, and in 1 case also 44% NK cells. Eighteen of the 20 T-cell lines were HAdV-specific, since they showed a median proliferation to the HAdV hexon peptide pool and inactivated HAdV of 14615 (95%CI 8924–31532) and 11103 (95%CI 8805–30174) cpm/105 cells after subtraction of background (responders+irradiated autologous PBMC), respectively. HAdV-specific lysis >10% at a 2:1 effector to target (E:T) ratio was observed in 50% of the T-cell lines. The 2 non-specific, as well as the 3 T-cell lines with lower specific activity, included >40% CD8+ T-cells. Production of IFNγ in an ELIspot assay to HAdV hexon peptide pool above 40 SFU/105 cells was observed in 9 out of 13 tested T-cell lines. Evaluation of specific response to hexon peptides in showed a majority of responses to II42 (80%), with 50–60% responses to II50, II57, II61, and II64. Only 2 out of the 20 T-cell lines tested were prevalently alloreactive against the recipient. Of the 18 HAdV-specific lines, 1 showed higher proliferation to patient PBMC than to HAdV (13518 vs 11717 mean cpm), and would have thus been discarded as unsuitable for in vivo use, while the other 17 showed no alloreactivity (14) or alloreactivity between 10 and 23% of specific proliferation (3). None of these 18 T-cell lines showed lysis >5% against recipient PHA blasts in the cytotoxicity assay. Our data show that PBMC stimulation with HAdV hexon protein-derived 30-mer peptides is able to reproducibly induce the generation of HAdV-specific CD4+ T-cell lines with efficient in vitro antiviral response in most HLA-mismatched HSCT donors. The majority of these T-cell lines show low/undetectable alloreactivity against recipient targets, and could therefore be safely employed for adoptive treatment of HAdV complications developing after HSCT from a HLA-haploidentical donor.

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