Donor T-cell Research Articles

SIRPα Mismatch Is Associated With Relapse Protection and Chronic Graft-Versus-Host Disease After Related Hematopoietic Stem Cell Transplantation for Lymphoid Malignancies.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematologic malignancies. Alloreactivity after HSCT is known to be mediated by adaptive immune cells expressing rearranging receptors. Recent studies demonstrated that the innate immune system could likewise sense the non-self signals and subsequently enhance the alloimmune response. We recently demonstrated that the donor/recipient mismatch of signal regulatory protein α (SIRPα), an immunoglobulin receptor exclusively expressed on innate cells, is associated with a higher risk of cGVHD and relapse protection in a cohort of acute myeloid leukemia patients who underwent allo-HSCT. Whether these effects also occur in other hematologic malignancies remains unclear. In the present study, we compared outcomes by SIRPα match status in a cohort of 310 patients who received allo-HSCT from an HLA matched-related donor for the treatment of lymphoid malignancies. Multivariable analysis showed that SIRPα mismatch was associated with a significantly higher rate of cGVHD (hazard ratio [HR] 1.8, P= .002), cGVHD requiring systemic immunosuppressive therapy (HR 1.9, P= .005), a lower rate of disease progression (HR 0.5, P= .003) and improved progression-free survival (HR 0.5, P= .001). Notably, the effects of SIRPα mismatch were observed only in the patients who achieved >95% of donor T-cell chimerism. The mismatch in SIRPα is associated with favorable relapse protection and concurrently increased risk of cGVHD in patients who undergo allo-HSCT for lymphoid malignancies, and the optimal donor could be selected based on the finding of the study to mitigate the risk of GVHD and relapse.

ICOS immunoPET enables visualization of activated T cells and early diagnosis of murine acute gastrointestinal GvHD

AbstractAllogeneic hematopoietic cell transplantation (HCT) is a well-established and potentially curative treatment for a broad range of hematological diseases, bone marrow failure states, and genetic disorders. Acute graft-versus-host disease (GvHD), mediated by donor T cells attacking host tissues, still represents a major cause of morbidity and mortality following allogeneic HCT. Current approaches to diagnosis of gastrointestinal acute GvHD rely on clinical and pathological criteria that manifest at late stages of disease. New strategies allowing for GvHD prediction and diagnosis, prior to symptom onset, are urgently needed. Noninvasive antibody-based positron emission tomography (PET) (immunoPET) imaging of T-cell activation post–allogeneic HCT is a promising strategy toward this goal. In this work, we identified inducible T-cell costimulator (ICOS) as a potential immunoPET target for imaging activated T cells during GvHD. We demonstrate that the use of the Zirconium-89-deferoxamine-ICOS monoclonal antibody PET tracer allows in vivo visualization of donor T-cell activation in target tissues, namely the intestinal tract, in a murine model of acute GvHD. Importantly, we demonstrate that the Zirconium-89-deferoxamine-ICOS monoclonal antibody PET tracer does not affect GvHD pathogenesis or the graft-versus-tumor (GvT) effect of the transplant procedure. Our data identify ICOS immunoPET as a promising strategy for early GvHD diagnosis prior to the appearance of clinical symptoms.

P1309: DYSREGULATED EXPRESSIONS OF INHIBITORY CHECKPOINT MOLECULES AND THEIR LIGANDS ON T-CELLS AND BLASTS IN AML RELAPSES AFTER STEM CELL TRANSPLANTATION (SCT)

Background: Upregulation of inhibitory checkpoint molecules (ICM) on T-cells and their ligands on leukemic blasts may be a mechanism of acute myeloid leukaemia (AML) relapse after allogeneic stem cell transplantation (SCT). Aims: Better understanding of relapse biology could improve treatment efficacy. Methods: We examined peripheral blood (PB) and bone marrow (BM) samples of 5 AML patients (PTs) relapsing after SCT, and PB of 5 healthy individuals (H), including 2 stem cells donors. ICM (PD-1, CTLA-4) expressions on T-cells and their ligands (CD86, PD-L1, PD-L2) on leukemic blasts were assessed through flow cytometry. PTs’ PB was cultivated with and without “KitM” (GM-CSF+PGE-1) to generate leukemia-derived dendritic cells (DCleu), followed by MLC, enriched by PTs’ or donors’ T-cells. After MLC, immune activation and functionality (degranulation, intracellular cytokine production, blast lysis) was assessed. Results: 1. Whilst all patients showed high expressions of PD-1 on their T-Cells, additional overexpression of CTLA-4 was negatively correlated with responses to relapse treatment. Expression of ICM was low on T-cells from 4/5 healthy individuals (Figure 1). 2. Influence of KitM on ICM/ligand expressions on T-cells/blasts - ICM/ligand expressions on uncultured T-cells/blasts: In contrast to H, PTs presented high co-expressions of CTLA-4 and PD-1 on PB T-cells (Figure 1). In addition, PTs showed high frequencies of PB/BM blasts co-expressing CD86. - DC/DCleu in PB: Generation of DCleu in AML as well as generation of DC in H was successful with KitM pretreated PB vs. Control. - ICM co-expression on T-cells after MLC with KitM pretreated PB: MLC of KitM treated PB enriched with unstimulated PTs T-cells resulted in reduced frequencies of ICM-positive T-cells in 3/5 PTs, and increased frequencies of activated (leukemia specific) T-cells in 3/5 PTs. Beyond, blast lysis was improved in 4/5 samples treated with KitM. Both findings were not observed when PB cells were cultivated in the absence of KitM. 3. Possible impact of ICM profiles on clinical outcome in a particular casePT1 suffered from early relapse both after 1st and 2nd SCT from her healthy father. A role of ICM in relapse mechanisms was suggested by CTLA-4/PD-1 expression on her T-cells and CD86 expression on AML blasts. In addition, >90% of the healthy father’s T-cells expressed CTLA-4/PD-1, which might have contributed to treatment failure. In contrast, T-cells from PT1’s mother presented with low ICM levels (Figure 1), suggesting that the mother might have been a better donor. Stimulation of PT1’s PB cells with KitM generated DCleu, decreased ICM-expressions and increased T-cell activity. KitM pretreated samples also revealed improved blast lysis after MLC. Image:Summary/Conclusion: T-cells and blasts of AML PTs relapsing after SCT uniformly co-expressed ICM and their ligands, which could be a reason for inferior immune responses. High aberrant ICM-expressions, in particular CTLA-4, on donor T-cells could be responsible for relapse after SCT by inactivating antileukemic immune reactions. Hence, checkpoint-inhibitory antibodies might represent a specific therapeutic option in these patients. Further, interactions between ICM-expressions on healthy (fresh) donor T-cells could have an impact in therapy responses and should be evaluated in donor selection. Finally, generation of DCleu by treatment with KitM triggers immune responses in MLC alongside with reduced ICM-expressions on T-cells, possibly reducing their inhibitory effects and therefore improving antileukemic responses.

P1312: FORODESINE AMPLIFIES HOST INNATE IMMUNE RESPONSE THROUGH TOLL-LIKE RECEPTOR 7 ACTIVATION WHILE PREVENTING EXPERIMENTAL GRAFT-VERSUS-HOST DISEASE

Background: Forodesine, a novel inhibitor of purine nucleoside phosphorylase (PNP), causes nucleotide pool imbalances by accumulating intracellular deoxyguanosine, thereby resulting in selective apoptosis of T-cells. Recent evidence indicates that specific types of nuclear acids including guanosine and its derivatives act as natural ligands for toll-like receptor 7 (TLR7). So far, it is unclear whether and how forodesine can modulate host innate immunity, including antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs), which express TLR7 abundantly. Aims: The aim of this study is to investigate the role of forodesine in host APC function as well as donor T-cell activation both in vivo graft-versus-host disease (GVHD) models and in vitro cell assays. Methods: NOG mice were subjected to 250 cGy total body irradiation followed by intravenous injection of human isolated T-cells. Mice were treated with forodesine (20 mg/kg) or vehicle daily after transplantation. Results: Despite the mice without treatment developing severe GVHD after transplantation, guanosine and deoxyguanosine were hardly detected in the plasma, suggesting that purine nucleosides released from damaged tissues were rapidly degraded by PNP. In fact, inhibition of PNP activity by forodesine markedly increased plasma guanosine levels, whereas deoxyguanosine was still undetectable. This was also the case in GVHD target organs, including liver and lungs. At the same level of guanosine in the liver, forodesine promoted TNF-a production from in vitro alveolar macrophage cultures. Preincubation with a TLR7 antagonist, ODN20958, attenuated the TNF-a production from macrophages stimulated by guanosine and forodesine. Nuclease treatment did not reduce macrophage TNF-a production, suggesting that single-strand RNA, a well-known TLR7 ligand, is not involved in this response. Similar results were observed in Flt3-ligand-induced plasmacytoid DCs. Not only TNF-a but also IL-12 production was induced by the combination of guanosine and forodesine, and was comparable to that in pDCs pulsed with R848, a potent agonist of TLR7/8. These observations suggest that a sustained high level of guanosine caused by forodesine treatment lead to continuous activation of TLR7 signaling, resulting in cytokine production from APCs. Meanwhile, as reported previously, forodesine inhibited the proliferation of human T-cells activated with CD3 and CD28 antibodies in the presence of deoxyguanosine. Of note, however, this combination did not impair but rather stimulated IFN-g production from activated T-cells cocultured with pDCs. This suggests that T-cell priming capacity mediated by the interaction with pDCs is conserved through TLR7 activation. Finally, we investigated the in vivo effects of forodesine on survival, body weight change, and disease severity of GVHD mice. Forodesine treatment significantly prolonged survival of the mice compared with control mice, with a median survival of 17.5 days vs 14 days, respectively (p = 0.029). The mean body weight loss percentage at day 9 was 11.5% vs 19.0% in the forodesine group and control, respectively (p = 0.003). Furthermore, the clinical GVHD scores were also lower in the forodesine group vs control (4.9 vs 0.6 points, p = 0.001). Image:Summary/Conclusion: This study demonstrates that forodesine amplifies host innate immune response through TLR7 signaling, while ameliorating T-cell GVH reaction. Further studies are needed to investigate whether forodesine can provide host protection against infections in a transplant setting.

Universal allogeneic CAR T cells engineered with Sleeping Beauty transposons and CRISPR-CAS9 for cancer immunotherapy

Allogeneic CD19-specific chimeric antigen receptor (CAR) Tcells with inactivated donor Tcell receptor (TCR) expression can be used as an "off-the-shelf" therapeutic modality for lymphoid malignancies, thus offering an attractive alternative to autologous, patient-derived Tcells. Current approaches for Tcell engineering mainly rely on the use of viral vectors. Here, we optimized and validated a non-viral genetic modification platform based on Sleeping Beauty (SB) transposons delivered with minicircles to express CD19-28z.CAR and CRISPR-Cas9 ribonucleoparticles to inactivate allogeneic TCRs. Efficient TCR gene disruption was achieved with minimal cytotoxicity and with attainment of robust and stable CD19-28z.CAR expression. The CAR Tcells were responsive to CD19+ tumor cells with antitumor activities that induced complete tumor remission in NALM6 tumor-bearing mice while significantly reducing TCR alloreactivity and GvHD development. Single CAR signaling induced the similar Tcell signaling signatures in TCR-disrupted CAR Tcells and control CAR Tcells. In contrast, TCR disruption inhibited Tcell signaling/protein phosphorylation compared with the control CAR Tcells during dual CAR/TCR signaling. This non-viral SB transposon-CRISPR-Cas9 combination strategy serves as an alternative for generating next-generation CD19-specific CAR T while reducing GvHD risk and easing potential manufacturing constraints intrinsic to viral vectors.

74-OR: Adoptively Transferred T Lymphocytes with Reduced iPLA2b Expression Delays Type 1 Diabetes Development in NOD Mice

Type 1 diabetes (T1D) is a T-cell mediated autoimmune disease characterized by the destruction of insulin-producing pancreatic islet β-cells. An understudied area is the role lipid signaling plays in this process. We find that the Ca2+-independent phospholipase A2β (iPLA2β) is induced under a diabetic milieu and mitigation of iPLA2β activity attenuates β-cell death. iPLA2β, a member of the PLA2 family, hydrolyzes the sn-2 substituent from glycerophospholipid substrates to yield a free fatty acid. When the fatty acid is arachidonic acid, it can be metabolized to eicosanoids, many of which are pro-inflammatory. To address our overarching hypothesis that iPLA2β-derived lipids (iDLs) contribute to T1D development, we investigated the role of immune cell-iDLs by utilizing a T-cell adoptive transfer. We isolated CD4+/8+ T-cells from splenocytes prepared from 11-week-old donor female NOD and NOD.iPLA2 β +/- (HET) . These were transferred i.p. to 4-week-old NOD.scid recipient mice. We find that NOD4/8-recipient mice develop T1D between 14-16 weeks of age. In contrast, T1D onset in HET4/8-recipient mice is delayed by 12 weeks and this was associated with better glucose tolerance, compared to NOD4/8 T-cell recipients. This finding is also supported by histology analysis of the pancreatic islet, which revealed less islet infiltration, more islet abundance, and the frequency of CD4 T cells were increased in the pancreas of HET4/8-recipient mice compared to NOD4/8 recipients. Further, lipidomics analyses revealed that HET donor T-cell production of proinflammatory lipids (i.e., PGE2 and DHETs) is decreased, relative to NOD T-cells. These findings suggest that pro-inflammatory iDLs generated by CD4 T-cells are critical contributors T1D development and can be targeted to counter T1D onset. Disclosure T.White: None. C.Chalfant: None. T.P.Dilorenzo: None. S.Ramanadham: None. D.Stephenson: None. Funding Training Program in Cell, Molecular, and Developmental Biology (5T32GM008111-34) NIH/NIDDK (R01DK110292) NIH/NIAID R21 (AI 146743) Cell, Developmental, & Integrative Biology (CDIB) , the University of Alabama at Birmingham

Combining CD34+ stem cell selection with prophylactic pathogen and leukemia directed T-cell immunotherapy to simultaneously reduce graft versus host disease, infection, and leukemia recurrence after allogeneic stem cell transplant.

We designed a trial to simultaneously address the problems of graft versus host disease (GVHD), infection, and recurrence of malignancy after allogeneic stem cell transplantation. CD34+ stem cell isolation was used to minimize the development of acute and chronic GVHD. Two prophylactic infusions, one combining donor-derived cytomegalovirus, Epstein-Barr virus, and Aspergillus fumigatus specific T-cells and the other comprising donor-derived CD19 directed chimeric antigen receptor (CAR) bearing T-cells, were given 21-28 days after transplant. Two patients were transplanted for acute lymphoblastic leukemia from HLA identical siblings using standard doses of cyclophosphamide and total body irradiation without antilymphocyte globulin. Patients received no post-transplant immune suppression and were given no pre-CAR T-cell lymphodepletion. Neutrophil and platelet engraftment was prompt. Following adoptive T-cell infusions, there was rapid appearance of antigen-experienced CD8+ and to a lesser extent CD4+ T-cells. Tetramer-positive T-cells targeting CMV and EBV appeared rapidly after T-cell infusion and persisted for at least 1 year. CAR T-cell expansion occurred and persisted for up to 3months. T-cell receptor tracking confirmed the presence of product-derived T-cell clones in blood targeting all three pathogens. Both patients are alive over 3 years post-transplant without evidence of GVHD or disease recurrence. Combining robust donor T-cell depletion with directed T-cell adoptive immunotherapy targeting infectious and malignant antigens permits independent modulation of GVHD, infection, and disease recurrence. The combination may separate GVHD from the graft versus tumor effect, accelerate immune reconstitution, and improve transplant tolerability.

Rapid single-cell identification of Epstein–Barr virus-specific T-cell receptors for cellular therapy

Background and aimsEpstein–Barr virus (EBV) is associated with solid and hematopoietic malignancies. After allogeneic stem cell transplantation, EBV infection or reactivation represents a potentially life-threatening condition with no specific treatment available in clinical routine. In vitro expansion of naturally occurring EBV-specific T cells for adoptive transfer is time-consuming and influenced by the donor's T-cell receptor (TCR) repertoire and requires a specific memory compartment that is non-existent in seronegative individuals.The authors present highly efficient identification of EBV-specific TCRs that can be expressed on human T cells and recognize EBV-infected cells. Methods and ResultsMononuclear cells from six stem cell grafts were expanded in vitro with three HLA-B*35:01- or four HLA-A*02:01-presented peptides derived from six EBV proteins expressed during latent and lytic infection. Epitope-specific T cells expanded on average 42-fold and were single-cell-sorted and TCRαβ-sequenced. To confirm specificity, 11 HLA-B*35:01- and six HLA-A*02:01-restricted dominant TCRs were expressed on reporter cell lines, and 16 of 17 TCRs recognized their presumed target peptides. To confirm recognition of virus-infected cells and assess their value for adoptive therapy, three selected HLA-B*35:01- and four HLA-A*02:01-restricted TCRs were expressed on human peripheral blood lymphocytes. All TCR-transduced cells recognized EBV-infected lymphoblastoid cell lines. ConclusionsThe authors’ approach provides sets of EBV epitope-specific TCRs in two different HLA contexts. Resulting cellular products do not require EBV-seropositive donors, can be adjusted to cell subsets of choice with exactly defined proportions of target-specific T cells, can be tracked in vivo and will help to overcome unmet clinical needs in the treatment and prophylaxis of EBV reactivation and associated malignancies.

Mixed T cell CHIMERISM in bone marrow at day+30 after allogeneic stem cell transplantation: Correlation with acute GVHD grades III-IV and no impact on relapse and overall survival.

Early chimerism analysis is important to assess engraftment in allogeneic hematopoietic stem cell transplantations. We retrospectively investigated the impact of T-cell chimerism at day 30 in bone marrow on acute graft-versus-host disease (aGVHD), relapse, and overall survival in 142 adult allo-transplanted patients. The majority of patients (89%) received myeloablative conditioning and 90% have undergone T-cell replete donor graft. At day 30, 103 patients showed T-complete chimerism with prevalence in haploidentical transplants, whereas 39 cases had CD3+ mixed chimerism, including 30 patients transplanted with HLA identical donors, and 21 with T-cell donors<90%. T-cell chimerism at day 30 was weakly inversely related to aGVHD grades II-IV (p=.078) with no cases of grades III-IV aGVHD in patients with CD3+ <95%. Mixed T-cell chimerism did not impact on relapse (p = .448) and five of the seven patients who relapsed had T-cell chimerism ≤90%. Older age and active disease at transplant had a statistically significant negative effect on overall survival (p = .01 and p = .0001, respectively), whereas mixed CD3+ chimerism did not. T lymphocyte chimerism analysis at day +30 in bone marrow could identify allo-transplanted patients at major risk of aGVHD grades III-IV (CD3+ donors >95%) mainly post-myeloablative conditioning regimen.

Is pathogen reduction an acceptable alternative to irradiation for risk mitigation of transfusion-associated graft versus host disease?

Transfusion-associated graft versus host disease (TA-GVHD) is a highly morbid and often fatal adverse event associated with transfusion of cellular blood products [platelets, red blood cells (RBCs) and whole blood] and more rarely with never-frozen plasma products. It is caused by residual viable donor T-lymphocytes that proliferate and actively target recipient tissues. Selective or universal irradiation of blood components using gamma-irradiation and more recently, X-ray irradiation, are the most commonly applied interventions and have been validated by the demonstration of in vitro T-lymphocyte inactivation, in murine models of TA-GVHD and by years of clinical experience. Irradiation, however, has multiple limitations including a sharp dose-response curve that renders quality control of dosage critically important, the use of radioactive radiation sources that are a terrorism risk, and selective implementation in many countries that leads to inadvertent omission and patient risk exposure. Certain pathogen reduction technologies (PRT) for platelets have been approved by regulatory authorities and endorsed by professional societies as an alternative to irradiation for reducing the risk of TA-GVHD, and PRT for RBCs and whole blood are in development. While the mechanism of action of T-lymphocyte inactivation differs from gamma/X-ray irradiation, the impact on T-lymphocyte inactivation for PRT is equivalent or superior to that of irradiation as demonstrated by sensitive in vitro lymphocyte proliferation assays and in vivo mouse models that approximate human TA-GVHD. Clinical trials and cumulative routine-use experience attest to the efficacy of PRT when used as an alternative to irradiation. While T-lymphocyte inactivation efficacy varies between PRT platforms, the implementation of PRT for platelets increases blood safety for patients beyond the mitigation of TA-GVHD, by decreasing the risk of transfusion transmitted infections with known viruses, bacteria and parasites as well as emerging pathogens.

EP397: Inadvertent allogeneic hematopoietic stem cell transplant from a lung allograft

Graft-versus-host disease (GVHD) following solid-organ transplant occurs when donor T-cell lymphocytes residing in the transplanted organ evade elimination by the suppressed immune system of the recipient. These cells can then proliferate, engraft, and induce an immune response attacking host tissues. GVHD following lung transplantation is uncommon but is well described. In contrast, reports of bone marrow engraftment by donor-derived hematopoietic stems cells after solid organ transplant are exceedingly rare.

Low-Dose 5-Aza and DZnep Alleviate Acute Graft-Versus-Host Disease With Less Side Effects Through Altering T-Cell Differentiation.

ObjectivePrevious studies showed that hypomethylating agents (HMAs) could alleviate acute graft-versus-host disease (aGvHD), but affect engraftment after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The combination of two different HMAs in lower doses might overcome this problem. This study aimed to evaluate the treatment effect of the combination of two HMAs—azacitidine (5-Aza) and histone H3K27 methyltransferase inhibitor 3-deazaneplanocin (DZNep)—for the prophylaxis of aGvHD after allo-HSCT and to explore the possible mechanisms.MethodsWe first optimized the concentrations of individual and combinational 5-Aza and DZNep treatments to ensure no obvious toxicities on activated T cells by evaluating T-cell proliferation, viability, and differentiation. A mouse model of aGvHD was then established to assess the prophylactic efficacy of 5-Aza, DZNep, and their combination on aGvHD. The immunomodulatory effect on T cells and the hematopoietic reconstruction were assessed. Additionally, RNA sequencing (RNA-seq) was performed to identify the underlying molecular mechanisms.ResultsCompared with single treatments, the in vitro application of 5-Aza with DZNep could more powerfully reduce the production of T helper type 1 (Th1)/T cytotoxic type 1 (Tc1) cells and increase the production of regulatory T cells (Tregs). In an allo-HSCT mouse model, in vivo administration of 5-Aza with DZNep could enhance the prophylactic effect for aGvHD compared with single agents. The mechanism study demonstrated that the combination of 5-Aza and DZNep in vivo had an enhanced effect to inhibit the production of Th1/Tc1, increase the proportions of Th2/Tc2, and induce the differentiation of Tregs as in vitro. RNA-seq analysis revealed the cytokine and chemokine pathways as one mechanism for the alleviation of aGvHD with the combination of 5-Aza and DZNep.ConclusionThe combination of 5-Aza and DZNep could enhance the prophylactic effect for aGvHD by influencing donor T-cell differentiation through affecting cytokine and chemokine pathways. This study shed light on the effectively prophylactic measure for aGvHD using different epigenetic agent combinations.

Promiscuity of Peptides Presented in HLA-DP Molecules from Different Immunogenicity Groups Is Associated With T-Cell Cross-Reactivity.

In the context of HLA-DP-mismatched allogeneic stem cell transplantation, mismatched HLA-DP alleles can provoke profound allo-HLA-DP-specific immune responses from the donor T-cell repertoire leading to graft-versus-leukemia effect and/or graft-versus-host disease in the patient. The magnitude of allo-HLA-DP-specific immune responses has been shown to depend on the specific HLA-DP disparity between donor and patient and the immunogenicity of the mismatched HLA-DP allele(s). HLA-DP peptidome clustering (DPC) was developed to classify the HLA-DP molecules based on similarities and differences in their peptide-binding motifs. To investigate a possible categorization of HLA-DP molecules based on overlap of presented peptides, we identified and compared the peptidomes of the thirteen most frequently expressed HLA-DP molecules. Our categorization based on shared peptides was in line with the DPC classification. We found that the HLA-DP molecules within the previously defined groups DPC-1 or DPC-3 shared the largest numbers of presented peptides. However, the HLA-DP molecules in DPC-2 segregated into two subgroups based on the overlap in presented peptides. Besides overlap in presented peptides within the DPC groups, a substantial number of peptides was also found to be shared between HLA-DP molecules from different DPC groups, especially for groups DPC-1 and -2. The functional relevance of these findings was illustrated by demonstration of cross-reactivity of allo-HLA-DP-reactive T-cell clones not only against HLA-DP molecules within one DPC group, but also across different DPC groups. The promiscuity of peptides presented in various HLA-DP molecules and the cross-reactivity against different HLA-DP molecules demonstrate that these molecules cannot be strictly categorized in immunogenicity groups.

Sitagliptin: A promising DPP-4 inhibitor for prevention of acute graft versus host disease.

Madam, allogeneic hematopoietic stem cell transplant (HSCT) remains a life altering cure to exceptionally challenging diseases ranging from hematologic malignancies to primary immunodeficiencies and blood cancers. One of its adversities includes graft versus host disease (GVHD), a lethal immunologically mediated condition wherein the donor's T-cells attack the host's tissues. The presentation can be classified as acute or chronic. Acute GVHD is divided into four grades. Despite current prophylaxis, including a combination of calcineurin inhibitor with either methotrexate or sirolimus, the incidence of grade-II or higher GVHD is between 30% to 80%, with grade-IV GVHD approaching mortality rates up to 90%.1 A recent phase II trial by Farag et al. addresses the issue by introducing a novel combination of sitagliptin – a specific DPP-4 inhibitor used to manage type 2 diabetes in adults – along with tacrolimus and sirolimus for prevention of GVHD.2 Using the new blend, an overall marked reduction in the risks of grade-II to IV (5%) and grade-III to IV (3%) acute GVHD was shown, compared to previous studies using only sirolimus and tacrolimus.3 Moreover, no adverse effects were associated with the drug during the period of study. Advancements in the field of HSCTs are particularly crucial in developing countries such as Pakistan. The high prevalence of consanguineous marriages and insufficient genetic counselling make the country especially vulnerable to diseases like thalassemia, bone marrow failure syndromes and immunodeficiency states. Up to 9000 children require transplantation each year due to the high prevalence of beta- thalassemia major.4 The situation is further complicated by limited resources and few stem cell transplant centres, which are limited to major cities. Although HSCT efficiently treats numerous life-threatening conditions, only 719 patients underwent HSCT till mid-2017 cumulatively in Pakistan.4 In accordance with the aforementioned factors, the number of transplants performed heavily outnumbers their demand, making it essential to tackle a complication as deadly as acute GVHD. In a 2005 study conducted in Rawalpindi by Hashmi et al., the overall incidence of acute GVHD grade-II to IV after standard immunosuppressive therapy was 44.2%.5 While the results were comparable to other studies, the high f morbidity and mortality rates necessitates alternate treatment courses. Sitagliptin, a locally manufactured, cost-effective drug, will be an appropriate choice of medication in Pakistan where average income amounts to a meagre US$1000 per year.4 However, randomized trials are needed to further investigate its role in the prevention of acute GVHD. Continue....

T cell receptor diversity as a biomarker for immune reconstitution after bone marrow transplantation for Severe Combined Immunodeficiency

Severe combined immunodeficiency (SCID) describes several profound defects of lymphocyte development that require hematopoietic cell transplantation (HCT) for survival. Depending on genotype and conditioning regimen, 10-40% of patients fail to achieve immune recovery post-HCT and remain at risk for life-threatening infections. It is difficult to predict which patients will fail immune reconstitution, but we hypothesize that these patients have lower T-cell diversity as early as 3 months post-HCT, when donor T-cells can first be detected.

MicroRNA-31 regulates T-cell metabolism via HIF1α and promotes chronic GVHD pathogenesis in mice

AbstractChronic graft-versus-host disease (cGVHD) remains a major obstacle impeding successful allogeneic hematopoietic cell transplantation (HCT). MicroRNAs (miRs) play key roles in immune regulation during acute GVHD development. Preclinical studies to identify miRs that affect cGVHD pathogenesis are required to develop these as potential lifesaving interventions. Using oligonucleotide array, we identified miR-31, which was significantly elevated in allogeneic T cells after HCT in mice. Using genetic and pharmacologic approaches, we demonstrated a key role for miR-31 in mediating donor T-cell pathogenicity in cGVHD. Recipients of miR-31–deficient T cells displayed improved cutaneous and pulmonary cGVHD. Deficiency of miR-31 reduced T-cell expansion and T helper 17 (Th17) cell differentiation but increased generation and function of regulatory T cells (Tregs). MiR-31 facilitated neuropilin-1 downregulation, Foxp3 loss, and interferon-γ production in alloantigen-induced Tregs. Mechanistically, miR-31 was required for hypoxia-inducible factor 1α (HIF1α) upregulation in allogeneic T cells. Therefore, miR-31–deficient CD4 T cells displayed impaired activation, survival, Th17 cell differentiation, and glycolytic metabolism under hypoxia. Upregulation of factor-inhibiting HIF1, a direct target of miR-31, in miR-31–deficient T cells was essential for attenuating T-cell pathogenicity. However, miR-31–deficient CD8 T cells maintained intact glucose metabolism, cytolytic activity, and graft-versus-leukemia response. Importantly, systemic administration of a specific inhibitor of miR-31 effectively reduced donor T-cell expansion, improved Treg generation, and attenuated cGVHD. Taken together, miR-31 is a key driver for T-cell pathogenicity in cGVHD but not for antileukemia activity. MiR-31 is essential in driving cGVHD pathogenesis and represents a novel potential therapeutic target for controlling cGVHD.

Experimental Study of the In Vitro and In Vivo Functional Activity of NKG2D Chimeric Antigen Receptor

Aim. To study antitumor cytotoxic effect of CAR-T NKG2D and CAR-T anti-CD19 in vitro and in vivo in order to compare antitumor activity of chimeric antigen receptors (CAR) with different structural and functional properties. Materials &amp; Methods. CAR constructions were produced by molecular cloning. CAR-T cell populations were obtained by transduction of healthy donor T-lymphocytes with recombinant lentiviral particles coding CAR NKG2D or CD19 target antigen CAR sequences. CAR-T cell proportion was assessed by FusionRed fluorescence and EGFR membrane receptor imaging. Specific in vitro cytotoxic activity of CAR-T effector cells was analyzed by Real-Time Cytotoxicity Assay (RTCA) during co-cultivation with HeLa_CD19 target cell line using xCELLigence. Interferon-Y (IFN-y) synthesis in vitro and in vivo along with the degree of cytotoxic effect were analyzed by immunoassay of culture medium of co-cultivated effector cells and target cells as well as isolated auto-plasma from the peripheral blood of mice. To assess the in vivo functional activity, CAR-T cell populations were infused into immunodeficient NSG-SGM3 mice (10 000 000 cells/mouse) 12 days after HeLa_CD19 cell injection and confirmation of engraftment and tumor growth. Upon euthanasia, tumors were removed and fixed in paraffin to prepare histological sections. CAR-T cell tumor infiltration was assessed by CD3 antigen immunohistochemical staining. Results. The highest ligand (molecules MICA, ULBP1/2/3/4/5/6) expression levels were detected in HeLa cell line. The obtained NKG2D CAR-T cells showed a considerable cytotoxic activity against HeLa_CD19 target line (cell index [CI] = 1.27), which was, however, twice as low as that of CAR-T anti-CD19 (CI = 0.60) (p = 0.0038). IFN-y level during co-cultivation of CAR-T anti-CD19 with HeLa_CD19 at the ratio of Е/Т = 1:1 was 64,852 pcg/mL, which was 3.5 times higher than IFN-y level during co-cultivation of CAR-T NKG2D with HeLa_CD19 (18,635 pcg/mL) (p = 0.0360). The degree of tumor infiltration by CAR-T anti-CD19 cells was higher than that by CAR-T NKG2D. The absence of NKG2D proliferating CAR-T cells in mice peripheral blood confirms their low persistence. IFN-y concentration in mice auto-plasma was 11.89 pcg/mL after CAR-T anti-CD19 infusion and 0.57 pcg/mL after CAR-T NKG2D infusion (p = 0.0079). The mean weight of tumor xenografts in experimental groups 10 days after CAR-T anti-CD19 injection was 0.72 g (p = 0.0142), after Т-lymphocyte and NKG2D CAR-T cell infusions it was 2.12 g and 1.2 g, respectively. Conclusion. CAR-T anti-CD19 cells are characterized by more pronounced cytotoxic effect under both in vitro and in vivo experimental conditions compared with CAR-T NKG2D cells. The degree of CAR-T anti-CD19 proliferation and their infiltration in mice xenograft models is considerably higher than the levels reached with NKG2D CAR-T cell injections. A single CAR-T NKG2D injection results only in short-term tumor reduction.

T-Cell-Replete Versus ex vivo T-Cell-Depleted Haploidentical Haematopoietic Stem Cell Transplantation in Children With Acute Lymphoblastic Leukaemia and Other Haematological Malignancies.

Allogeneic haematopoietic stem cell transplantation (HSCT) represents a potentially curative option for children with high-risk or refractory/relapsed leukaemias. Traditional donor hierarchy favours a human leukocyte antigen (HLA)-matched sibling donor (MSD) over an HLA-matched unrelated donor (MUD), followed by alternative donors such as haploidentical donors or unrelated cord blood. However, haploidentical HSCT (hHSCT) may be entailed with significant advantages: besides a potentially increased graft-vs.-leukaemia effect, the immediate availability of a relative as well as the possibility of a second donation for additional cellular therapies may impact on outcome. The key question in hHSCT is how, and how deeply, to deplete donor T-cells. More T cells in the graft confer faster immune reconstitution with consecutively lower infection rates, however, greater numbers of T-cells might be associated with higher rates of graft-vs.-host disease (GvHD). Two different methods for reduction of alloreactivity have been established: in vivo T-cell suppression and ex vivo T-cell depletion (TCD). Ex vivo TCD of the graft uses either positive selection or negative depletion of graft cells before infusion. In contrast, T-cell-repleted grafts consisting of non-manipulated bone marrow or peripheral blood grafts require intense in vivo GvHD prophylaxis. There are two major T-cell replete protocols: one is based on post-transplantation cyclophosphamide (PTCy), while the other is based on anti-thymocyte globulin (ATG; Beijing protocol). Published data do not show an unequivocal benefit for one of these three platforms in terms of overall survival, non-relapse mortality or disease recurrence. In this review, we discuss the pros and cons of these three different approaches to hHSCT with an emphasis on the significance of the existing data for children with acute lymphoblastic leukaemia.

CCL8 deficiency in the host abrogates early mortality of acute graft-versus-host disease in mice with dysregulated IL-6 expression

Although allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for diverse malignant and nonmalignant diseases, acute graft-versus-host disease (aGVHD) is strongly linked to mortality caused by HSCT. We previously reported that CC chemokine ligand 8 (CCL8) is closely correlated to aGVHD mortality in both humans and mice. To study the role of CCL8 in aGVHD, CCL8 knockout (CCL8-/-) mice were transplanted with fully allogeneic marrow grafts. These mice exhibited a significant reduction in mortality (90.0% vs. 23.4% survival for CCL8-/- vs. wild-type recipients at day 28, p < 0.0001). As a result, apparent prolonged median survival from 9 days in wild-type mice to 45 days in CCL8-/- mice was observed. Acute GVHD pathology and liver dysfunction in CCL8-/- mice were significantly attenuated compared with those in wild-type mice. In association with the reduced mortality, a surge of plasma interleukin (IL)-6 was observed in CCL8-/- recipients with allogeneic marrow, which was significantly increased compared with wild-type mice that received allografts. Donor T-cell expansion and plasma levels of interferon-γ and TNF-α during aGVHD were similar in both types of mice. Collectively, these findings indicate that CCL8 plays a major role in aGVHD pathogenesis with possible involvement of an IL-6 signaling cascade.

Donor Plasmacytoid Dendritic Cells Regulate GvHD in a VIP Dependent Manner in Allogeneic BMT Recipients

Introduction: Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide known to induce differentiation of regulatory dendritic cells and regulatory T cells. Using allogeneic hematopoietic stem cell transplantation (allo-HSCT) models, we have shown that donor bone marrow (BM) plasmacytoid dendritic cells (pDCs) facilitate HSC engraftment and attenuate pathogenesis of graft vs. host disease (GvHD) through regulation of recipient T cells. However, the mechanism by which pDCs mitigate the GvHD activity of recipient T cells is not clearly understood. Here, we report that donor pDCs limit pathogenic T cell inflammation by VIP production.Methods: To study VIP production by pDCs, FACS-sorted pDCs from B6 mouse BM were cultured with or without PMA/ionomycin in-vitro. After activation and cytospin slide preparation, pDCs were labeled with anti-PDCA1 (pDC marker) and anti-VIP antibodies for confocal fluorescence microscopy. To investigate the effects of VIP production on T cell proliferation, an in-vitro co-culture assay was performed using R848 and CpG-activated WT or VIP-KO pDCs with anti-CD3-activated, CFSE-labeled syngeneic T cells. For GvHD experiments, irradiated B10.BR (H-2K k) mice received 5x10 3 HSCs, 5x10 4 pDCs and 1x10 6 T cells from WT B6 (H-2K b) or VIP-KO B6 (H-2K b) mice. H&E histology of intestine and colon was performed for GvHD scoring 7 days post-transplant. Graft vs. leukemia (GvL) effects were tested by inoculating recipient mice with 5x10 5 LBRM 33-5A4 cells in the same model. Recipient mice were monitored twice weekly using a 10-point GvHD scoring system. Gene expression analysis of FACS-sorted donor T-cells from recipient spleens was performed using the Nanostring Myeloid Innate Immunity Panel at days 8 and 15 post-transplant.Results: Confocal microscopic images of PMA/ionomycin stimulated or unstimulated sorted pDCs show that VIP is synthesized by pDCs (anti-VIP, green; anti-PCDA-1, red; DAPI counterstain, blue) (Fig 1). After in-vitro culture, VIP expression and frequencies of VIP + pDCs were similar in PMA/ionomycin treated or untreated cells (not shown). VIP-KO mice have significantly higher percentages of pDCs in BM compared to WT (Fig 2a). T cells co-cultured with VIP-KO pDCs showed higher proliferation than T cells co-cultured with WT pDCs, demonstrating that VIP secreted by pDCs reduces T cell proliferation (Fig 2b). Moreover, VIP-KO pDCs induce significantly greater proliferation of IFN-gamma + CD8 T cells compared to WT, indicating that pDCs lacking VIP promote Th1 polarization in-vitro (Fig 2c). The data are consistent with results from GvHD experiments showing increased frequencies of Th1 polarized T cells and fewer regulatory T cells in recipients of VIP-KO pDCs compared with recipients of WT pDCs. Intestinal GvHD scores and crypt apoptosis in the colon were higher in recipient groups transplanted without pDCs or with VIP-KO pDCs compared with recipients of WT pDCs (Fig 3a, b, c). These results indicate that VIP secreted from pDCs limits GvHD in the gut. In the GvL model, administration of pDCs lacking VIP did not alter the anti-tumor effect of donor T cells. Nanostring analysis of T cells recovered from VIP-KO pDC recipients had increased expression of the pro-inflammatory transcription factor Bhlhe40 during the first two weeks post-transplant, and higher transcription levels of the inflammatory mediator Cyclophilin A at day 15 post-transplant than T cells from recipients of WT pDCs.Conclusion: Data from in vitro and in vivo experiments suggest that VIP secreted by pDCs limits pathogenic T cell proliferation. In murine allo-BMT, increased gut GvHD scores and crypt apoptosis in recipients transplanted without pDCs or with VIP-KO pDCs indicates that VIP secreted by pDCs consolidates gut integrity without altering GvL. Gene expression analysis also supports a mechanism by which VIP-secreting donor pDCs reduce T cell inflammation through negative regulation of Bhlhe40. Our findings suggest paracrine VIP signaling is a novel immune checkpoint pathway by which donor pDCs limit T cell activation, Th1 polarization, and inflammation, and improve outcomes of allo-BMT by reducing GvHD activity. [Display omitted] DisclosuresWaller: Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Verastem Oncology: Consultancy, Research Funding.