Alloreactive T-cell Clones Research Articles

A strategy to reconstitute immunity without GVHD via adoptive allogeneic Tscm therapy.

Adoption of allogeneic T cells directly supplements the number of T cells and rapidly induces T-cell immunity, which has good efficacy for treating some tumors and immunodeficiency diseases. However, poor adoptive T-cell engraftment and graft-versus-host disease (GVHD) limit the application of these methods. Alloreactive T-cell clones were eliminated from the donor T-cell repertoire, and the remaining T-cell clones were prepared as Tscm for T-cell adoptive treatment to reconstruct recipient T-cell immunity without GVHD. The subjects in this study included three different strains of mice. Lymphocytes from mice (C57BL/6) were used as the donor T-cell repertoire, from which the Tscm allo-reactive T cell clone was depleted (ATD-Tscm). This was confirmed by showing that the Tscm was not responsive to the alloantigen of the recipient (BALB/c). To prepare ATD-Tscm cells, we used recipient lymphocytes as a simulator, and coculture of mouse and recipient lymphocytes was carried out for 7 days. Sorting of non-proliferative cells ensured that the prepared Tscm cells were nonresponsive. The sorted lymphocytes underwent further expansion by treatment with TWS119 and cytokines for an additional 10 days, after which the number of ATD-Tscm cells increased. The prepared Tscm cells were transferred into recipient mice to observe immune reconstitution and GVHD incidence. Our protocol began with the use of 1×107 donor lymphocytes and resulted in 1 ×107 ATD-Tscm cells after 17 days of preparation. The prepared ATD-Tscm cells exhibited a nonresponse upon restimulation of the recipient lymphocytes. Importantly, the prepared ATD-Tscm cells were able to bind long and reconstitute other T-cell subsets in vivo, effectively recognizing and answering the "foreign" antigen without causing GVHD after they were transferred into the recipients. Our strategy was succeeded to prepare ATD-Tscm cells from the donor T-cell repertoire. The prepared ATD-Tscm cells were able to reconstitute the immune system and prevent GVHD after transferred to the recipients. This study provides a good reference for generating ATD-Tscm for T-cell adoptive immunotherapy.

Characteristics of Alloreactive T-Cell Clones That Predict Early Development of Acute GvHD Following Allogeneic HCT

Acute Graft-versus-Host Disease (GvHD) is mediated by T-cells recognizing alloantigens. Despite advances in prophylactic regimens that broadly deplete T-cells, incidence of acute GvHD remains high, suggesting that there is a need to further characterize alloreactive T-cells specifically. Using mixed-lymphocytes reactions (MLR) and high-throughput T cell receptor (TCR) sequencing of third complementarity-determining region (CDR3) of the TCRβ chain, we identified and characterized alloreactive T-cell clones in donor-recipient pairs of allogeneic hematopoietic cell transplantation. We sequenced and identified a total of 34,903 unique donor-derived CDR3β functional sequences (FS) from 20 donor-recipient in vitro MLRs using peripheral blood mononuclear cells (PBMC) collected from donors and patients pre-transplant. We also sequenced the respective unstimulated donor PBMC samples (Donor Type: 13 HLA-Matched/7 Mismatched, GvHD prophylaxis: 11 Tacrolimus (Tac)-Methotrexate/8 Tac-MMF/1 Sirolimus, PTCy: 12 Yes/8 No). On average, our identified alloreactive FS make up a small fraction of the sampled donor repertoire in comparison to nonalloreactive FS. (Mean fraction: 0.004 vs. 0.996, p < 0.0001) Surprisingly, we found that alloreactive FS have significantly shorter CDR3β length compared to nonalloreactive FS (mean nt 43.50 vs. 44.40, p < 10E-15). We also found that alloreactive FS which can be located within the unstimulated donor sample (n= 4522) are shorter in comparison to all derived alloreactive FS (mean nt 42.43 vs. 43.50, p < 10E-15) and to nonalloreactive FS (mean nt 42.43 vs. 44.40, p < 10E-15) (Figure 1A). Publicly shared FS have, on average, shorter CDR3β length and are overrepresented among FS associated with autoreactivity and cross-reactivity (Khosravi-Maharlooei et al., J Clin Invest, 2019). Thus, we hypothesized that alloreactive FS may be also publicly shared. We derived a list of shared FS from our donor samples, where if a FS is found in more than one donor, it is considered shared. Alloreactive clones were significantly more likely to be shared compared to nonalloreactive (OR 1.94; CI 95%: 1.88 to 2.01). In addition, using a publicly available set of cross-reactive FS (https://vdjdb.cdr3.net/search), we found that alloreactive sequences are also highly cross-reactive compared to nonalloreactive sequences (OR 6.6; CI 95%: 5.18 to 8.42). FS with greater levels of sharedness appear more frequently within a repertoire (Madi et al., Genome Res, 2014). As alloreactive sequences are highly shared, we hypothesized that donor repertoires with greater proportion of shared and alloreactive FS will increase the odds of developing clinically significant GvHD (Grade 2-4) in the recipient. Indeed, within donor repertoires, we found a higher fraction of alloreactive sequences amongst the highly expressed FS (The top 50% of FS in repertoire frequency) which correlated with development of GvHD in recipients post-transplant (mean fraction: GvHD+ group 0.013 vs GvHD- group 0.004, p < 10E-7). Due to the higher portion of shared and alloreactive FS in the top half of the donor T-cell repertoire, we used R50, an index that estimates the diversity of the top 50% of a donor repertoire, to predict the development of GvHD. Utilizing an initial validation cohort of 12 donors, we found that donors whose corresponding recipients developed GvHD within the first 3 months post-transplant had significantly lower R50 compared to those that did not develop GvHD (R50 of 0.096 vs 0.345 p < 0.0001). We also conducted a time-to-event analysis regarding GvHD development in the first 90 days post-transplant. Using the average R50 of the validation cohort as the cutoff for low and high R50 (Average R50: 0.20), we found, in an expanded bi-institutional cohort (n=57), that those with lower R50 values had a significantly greater risk of GvHD compared to those with higher R50 (Figure 1B). In conclusion, our data suggests that functional alloreactive T lymphocytes are characterized by shorter TCRβ CDR3 length. These FS are highly shared and cross-reactive, suggesting a common mechanism underlying the selection of alloreactive and autoreactive clones. From there, we were able to develop and validate a diversity metric that predicts GvHD development pre-transplant based on the donor T-cell repertoire. These findings have implications for donor selection and need to be examined prospectively.

Gene Expression Profiling and Signaling Pathway Analysis of Tolerant T cells Circulating in Adult Patients after PTCy Haplotransplantation

Abstract The mechanisms for the signaling pathways of T cell-dependent tolerance after haploidentical hematopoietic stem cell transplantation (haplo-HSCT), are incompletely understood. We tested 8 patients (Pt) with leukemia or myelodysplastic syndromes (median age:51-yrs old) following IRB approved consenting. All received Flu+TBI (+/− Cy) pre-conditioning and haplo-HSCT with post-transplant Cytoxan (PTCy) and FK506 as GvHD prophylaxis. The circulating T cells were hyporeactive in mixed lymphocyte reaction (MLR) assay to recipient DC in Pts at 1yr (or 2yrs in 1 Pt) post-haplo-HSCT (~6m post-ISD withdrawal), while these T cells responded vigorously to 3rd party APC. Neither depletion of Tregs, nor blockade of IL10R or PD1 could reverse T-cell hyporeactivity. Low-dose IL2 triggered mild anti-host responses. Cytokine profiling further confirmed the MLR responses. ImmunoSEQ™ revealed the disappearance of alloreactive T-cell clones from peripheral pool with time, even though a few alloreactive T clones remained. Low dose IL2 had minimal impact to reverse the diminishment. Unlike graft T-cell responses against host DC, tolerant T cells in circulation against hDC showed relative downregulation of allograft rejection, T-helper response, co-stimulation, and cell proliferation pathway, that was paired with upregulation of co-inhibition, PD1-PDL1, Ferroptosis and CREB pathways. In summary, hyporeactivity of donor-derived circulating T cells to recipient DC were evident. We found Treg, Tr1 activity to be insignificant in maintaining long-term tolerance. T-cell clonal deletion appears to be the dominant mechanism, along with systemically altered signaling pathways in tolerant T-cells.

Graft-versus-host disease is locally maintained in target tissues by resident progenitor-like T cells

In allogeneic hematopoietic stem cell transplantation, donor αβ Tcells attack recipient tissues, causing graft-versus-host disease (GVHD), a major cause of morbidity and mortality. A central question has been how GVHD is sustained despite Tcell exhaustion from chronic antigen stimulation. The current model for GVHD holds that disease is maintained through the continued recruitment of alloreactive effectors from blood into affected tissues. Here, we show, using multiple approaches including parabiosis of mice with GVHD, that GVHD is instead primarily maintained locally within diseased tissues. By tracking 1,203 alloreactive Tcell clones, we fitted a mathematical model predicting that within each tissue a small number of progenitor Tcells maintain a larger effector pool. Consistent with this, we identified a tissue-resident TCF-1+ subpopulation that preferentially engrafted, expanded, and differentiated into effectors upon adoptive transfer. These results suggest that therapies targeting affected tissues and progenitor Tcells within them would be effective.

Gene Expression Profiling and Signaling Pathway Analysis of Tolerant T Cells Circulating in Adult Patients after Ptcy Haplotransplantation

The mechanisms of T cell-dependent tolerance, especially its signaling pathways after haploidentical hematopoietic stem cell transplantation (haplo-HSCT), are incompletely understood. Both central and peripheral regulation mechanisms are suspected. Subjects and Methods: We tested 8 patients with leukemia (5) or myelodysplastic syndromes (MDS, 3) (age 35-73-years old, median: 51-years old) following IRB approved consenting. All received Flu+TBI (+/- Cy) pre-conditioning and haplo-HSCT with post-transplant Cytoxan (PTCy) and FK506 as GvHD prophylaxis. Chimerism at tolerance studies was donor dominant (97-100%, mean: 99% for both CD33+ myeloid cells and CD3+T cells). Immunosuppression drug (ISD) was withdrawn ~6 months before the time of the in vitro tolerance study described below. We described previously Treg depletion from purified T cells with IL2-immunotoxin (Denileukin Diftitox, Seragen Inc™) to test the involvement of these cells in long-term tolerance. The contribution of Tr1 or anergy was examined by either blocking IL10R or adding low dose IL2, respectively. T-cell exhaustion was tested by blocking the PD1 pathway with anti-PD1 MoAb (OPDIVO, Bristol Myers Squibb™). Purified T cell responses to recipient dendritic cells (DC) were measured in mixed lymphocyte reaction (MLR) assay, and further tested by quantifying cytokine secretion in MLR culture supernatants using Bio-Plex immunoassays (Bio-Rad Lab™). To test for clonal deletion, alloreactive T-cell clones with frequency >0.1% identified in vitro from the graft pre-infusion were tracked by monitoring their T-cell receptor CDR3 sequence serially, in an ImmunoSEQ™ assay (Adaptive Biotech™). Gene expression profiling and signaling pathway analysis on alloreactive or tolerant T-cells were performed by collecting T cells from in vitro MLR reactions, purifying total RNAs from these cells, running RNA-sequencing and analyzing with software of CLC Genomic Workbench®v21, GSEA®v4.1.0 and IPA®. Results: We have previously shown that circulating T cells were hyporeactive to recipient DC in patients at 1yr (or 2yrs in 1 patient) post-haplo-HSCT [6 months, (or 2-5 months in 3 patients) post-ISD withdrawal], while these T cells responded vigorously to 3rd party APC, As positive control, host DC triggered robust 3rd party T-cell response. Neither depletion of Tregs, nor blockade of IL10R or PD1 could reverse T-cell hypo-reactivity. Low-dose IL2 triggered mild anti-host proliferation responses. Similar scenarios were detected in graft T cells against self-DC, (negative control in autologous set up), while vigorous responses were shown in the graft T cells against host-DC pre-haplo-HSCT. Cytokine profiling further confirmed the MLR responses. ImmunoSEQ™ revealed disappearance of alloreactive T-cell clones from peripheral blood with time. Low dose IL2 had minimal impact to reverse hyporeactivity. While most alloreactive T clones were undetectable, a few remained in the peripheral pool in tolerant patients tested (Nee, T et al, Transplantation and Cellular Therapy, 2022;28(3):S44-S45). Unlike graft T-cell responses against host DC, tolerant T-cells in circulation (including remaining alloreactive T cells) against hDC showed distinct patterns of gene expressions, with relative downregulation of allograft rejection (GSEA-Hallmark) (Fig 1A, B, C) and T-helper response, co-stimulation, cell proliferation pathway, that was paired with upregulation of co-inhibition, PD1-PDL1, Ferroptosis, PTEN and CREB pathways (Fig1D). The profiles suggested systemic alterations in the signaling pathways on tolerant T cells post-haplo-HSCT. Conclusion: Hypo-reactivity of donor-derived circulating T cells to recipient DC and graft T cells to graft DC (negative control) were evident. We found Treg, Tr1 activity or T-cell exhaustion to be insignificant as their blockade or removal did not lead to 'flare’ in circulating tolerant T-cell reactivity against patient DC. T-cell clonal deletion appears to be the dominant mechanism explaining the in vitro hyporeactive proliferation, cytokine secretion, and alloreactive TCR clonotype attrition, along with systemically altered signaling pathways in tolerant T-cells including the alloreactive T cells remained. Anergy may play a minor supportive role. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

425.3: Single-Cell Immune Profiling of Human Intestinal Allografts Reveals Differential Phenotypes of Alloreactive T-Cell Clones in Quiescent vs Chronically Rejecting Allografts

Introduction: The success of intestinal transplantation (ITx) is limited by high rejection rates. We have demonstrated that host-vs-graft (HvG) alloreactive T-cell clones are enriched in intestinal allografts during early rejection and persist despite rejection resolution. Recipient T cells in the mucosa eventually take on resident memory T cell (TRM) features, possibly posing a constant threat of late rejection. Methods: We integrated clonotype, alloreactivity and gene expression profiles of FACS-sorted allograft recipient T cells to assess the functional phenotype of donor-reactive mucosal T cells in association with graft outcomes using the 10x single cell RNA sequencing platform and our method for identifying donor-reactive TCRs from pre- and post-Tx mixed lymphocyte reactions (MLRs) (Fig.1A). Results: Recipient mucosal T cells from 6 quiescent and 5 chronically rejecting allograft specimens and a non-transplant control intestine shared at least five transcriptionally-defined clusters (Fig.1B-C): multifunctional (IL17A+, IL22+, TNF+) TRMs (CD69+, ITGAE+, CXCR6+); cytotoxic γδ and CD8 αβ T cells with mixed effector T cell (Teff) and TRM features (CD69dim, RUNX3+, TBX21+, GZMB+); follicular helper T cells (Tfh: CXCR5+, PDCD1+, Bcl6+); nonTRMs (CCR7+, KLF2+, S1PR1+); and regulatory T cells (FOXP3+, CTLA4+). Pre-Tx MLR-defined HvG clones were mainly detected in TRM, Teff/TRM and Tfh clusters in both quiescent and chronically rejecting allografts. Hyporesponsiveness of circulating recipient T cells to donor vs third-party antigens in post-Tx MLR indicated partial tolerance of circulating recipient T cells to donor antigens post-Tx (Fig.1D). There was a significantly decreased likelihood of detecting HvG clones defined by post- compared to pre-Tx MLR in late quiescent (but not chronically rejecting) allografts (Fig.1E). Quiescent allografts contained a significantly greater percentage of pre-Tx HvG clones that became tolerant in post-Tx MLR (tolerant HvG) compared to chronically rejecting allografts (Fig.2A-C). Tolerant HvG among quiescent allografts were dominated by TRM profiles. The fraction of tolerant HvG cells in TRM clusters was significantly higher in quiescent compared to rejecting allografts (Fig. 2D). Patients with quiescent allografts tended to show a higher level and longer persistence of tolerant HvG clones in ileum compared to PBMCs (Fig.2E). Cells classified as missing HvG (HvG in pre-Tx MLR and not detected in post-Tx MLR or circulation) and de novo H’vG (unmappable in pre-Tx MLR and unstimulated cells but HvG in post-Tx MLR) in chronically rejecting allografts showed significantly higher expression of genes related to cytotoxic Teff functions compared to those in quiescent allografts (Fig.2F). Conclusion: Single cell immune profiling reveals distinct contributions of pre-existing HvG-reactive T cells that potentially became tolerant in allografts with TRM transcriptional profiles in quiescent vs chronically rejecting allografts.

Analysis of T-Cell Receptor Repertoire in Transplantation: Fingerprint of T Cell-mediated Alloresponse.

T cells play a key role in determining allograft function by mediating allogeneic immune responses to cause rejection, and recent work pointed their role in mediating tolerance in transplantation. The unique T-cell receptor (TCR) expressed on the surface of each T cell determines the antigen specificity of the cell and can be the specific fingerprint for identifying and monitoring. Next-generation sequencing (NGS) techniques provide powerful tools for deep and high-throughput TCR profiling, and facilitate to depict the entire T cell repertoire profile and trace antigen-specific T cells in circulation and local tissues. Tailing T cell transcriptomes and TCR sequences at the single cell level provides a full landscape of alloreactive T-cell clones development and biofunction in alloresponse. Here, we review the recent advances in TCR sequencing techniques and computational tools, as well as the recent discovery in overall TCR profile and antigen-specific T cells tracking in transplantation. We further discuss the challenges and potential of using TCR sequencing-based assays to profile alloreactive TCR repertoire as the fingerprint for immune monitoring and prediction of rejection and tolerance.

Integrated Analysis Toolset for Defining and Tracking Alloreactive T-cell Clones After Human Solid Organ and Hematopoietic Stem Cell Transplantation.

We have developed a suite of tools for integrated analysis of T-Cell-Receptor Sequencing data to define and track alloreactive T-cells in human transplant studies. This has enabled discovery of sequences and patterns of T-cell enrichment and deletion associated with clinical outcomes such as transplant rejection and tolerance. The codebase includes user-friendly default analyses with customizable parameters which greatly accelerate computational workflows and provide robust statistics comparing post-transplant specimens to pre-transplant baseline. It also includes helper functions for robust characterization of T-cell-repertoire diversity, sample-to-sample divergence, resolution of sample-of-origin ambiguity in pooled assays, and functions to output all sequences defined as alloreactive.

Impact of Post-Transplant High-Dose Cyclophosphamide on Reconstitution in Bone Marrow Recipients

Introduction: Hematopoetic stem cell transplantation (HSCT) is the only curative therapy for many patients with hematologic malignancies. Occurrence of complications and mortality after allo HSCT is still high and it's strongly associated with immune reconstitution. Despite the wide-spread of Post-Transplant High-Dose Cyclophosphamide (PTCy) immune reconstitution and immunological safety of this method is still poorly understood.Aim: to evaluate immune reconstitution profile in patients who was underwent HSCT with a bone marrow as a stem cell source and with or without PTCy.Patients and methods: In National Research Center for Hematology 41patients after allogeneic HSCT were analyzed in 2 groups; patients with PTCy (n=17) and without PTCy (n=24). The total cohort had 12 males and 29 females, and had a median age of 33 years (range 19-61). All patients were diagnosed with a hematological malignancy. 14 patients were underwent myeloablative conditioning and 27 patients - non-myeloalative prior HSCT.The GVHD prophylaxis consisted of ATG-PTCy-CsA-MMF (n=10), ATG-PTCy (n=5), Mono-PTCy (n=1), ATG-CsA-MMF-MTX (n=20), CsA-MTX (n=2), ATG-CsA-MTX (n=2), CsA-MMF-MTX(n=1). The patients who only had received ATG were included on the analysis.Immune reconstitution profile was tested via serial flow cytometry analysis of peripheral blood on day +14, +30, +60 and +90 after allo-HSCT. Anti-CD3 FITC, anti-CD16PE, anti-CD56 PE, anti-CD45 Per-CP-CYTM5.5, anti-CD4 PE-CyTM7, anti-CD19 APC, anti-CD8 APC-Cy7; anti-CD62L FITC (BD Biosciences, USA); anti-CD14PE, anti-CD16PE, anti-HLA-DR APC (eBiosciences, USA) were used to define white blood cell subsets.Results: Reported detailed data is presented in Table 1. In bone marrow recipients the number of CD4+- cells was significantly lower on day +14 and +30, NK-cells on day +30 and CD8+-cells on day +60 in case of using PTCy versus immunosuppression without PTCy.The number of classical monocytes on day +30 and patrol monocytes on day +30 and +90 was significantly lower in case of using PTCy versus a standard protocol.Conclusion:Lymphocyte and monocyte reconstitution was impaired for the PTCy groups in the immediate post-HSCT period but quickly recovered. The mechanism of tolerance induction with PTCy on day+ 3, + 4 is not limited only to deletion of alloreactive T-cell clones, but also affects other leukocyte subpopulations (B cells, monocytes, granulocytes). The use of PTCy on day+ 3, + 4 is an immunologically safe method for prevention of GVHD.Table 1. Short-term reconstitution in BM recipients with and without PTCy [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Haploidentical CD34-Selected Peripheral Blood Stem Cell Transplantation (PBSCT) from Related Donors in Pediatric Patients with Malignant and Non-Malignant Disorders

Background Hematopoietic stem cell transplantation (HSCT) from HLA-haploidentical related donors is increasingly considered for transplant candidates who lack a histocompatible related or unrelated donor. Since October 2015, we have been performing haploidentical PBSCT, utilizing a two-step approach first described by Grosso et al. (Biol Blood Marrow Transplant 21:646, 2015), in which the lymphoid and CD34+ cell populations are administered separately. Selective depletion of alloreactive T-cell clones is accomplished by administering high-dose cyclophosphamide (CY) after the donor lymphocyte infusion but prior to infusion of the CD34-selected peripheral blood stem cell product. Methods As of June 2019, 15 pts have undergone haploidentical PBSCT from related donors for treatment of AML (n=1), ALL (n=8), MDS (n=3), CML (n=1) JMML (n=1) and HLH (n=1). Median age at transplant was 11 years (range, 0.7-23.5 years). Twelve pts were conditioned with TBI 1200 cGy and CY 60 mg/kg IV x 2, while 3 pts received fludarabine 35 mg/m2 IV x 4, busulfan 0.8-1 mg/kg IV q6h x 16 doses, and CY 60 mg/kg IV x 2. The mother served as the donor for 8 pts, the father for 6 pts and an adult sister for 1 pt. Donor lymphocytes containing 2 × 108 CD3+ cells/kg were infused after completion of TBI or busulfan, depending on the conditioning regimen utilized, followed 66 hours later by administration of CY 60 mg/kg daily x 2. CD34+ selected peripheral blood stem cells were infused 48 hours after the second dose of CY. GvHD prophylaxis consisted of tacrolimus and mycophenolate, both begun on Day -1. In the absence of GvHD, mycophenolate was discontinued at Day +30 and tacrolimus was tapered over 1 month, beginning on Day +60. Results Median CD34+ cell dose was 7.9 × 106/kg (range, 4.1-12.3). Neutrophil and platelet (>20,000/microliter) engraftment occurred at a median of 9 days (range, 8-10 days) and 9.5 days (range, 8-20 days), respectively. All pts achieved full donor chimerism, with no secondary graft failure. Pts were discharged a median of 22 days post-transplant (range, 15-77 days). Grade II-III acute GvHD (mostly transient skin involvement) was observed in 5 pts and 1 pt developed Stage IV acute liver GvHD. Chronic skin GvHD (1 mild, 1 extensive) was observed in 2 pts. Two pts have died, one from progressive disease and one from multiorgan failure in the setting of severe acute liver GvHD. Six pts (1 JMML, 1 AML, 1 MDS, 3 ALL) have suffered disease relapse, 68-446 days post-transplant. All 3 ALL pts subsequently received CAR T-cell therapy. Eight pts remain relapse-free survivors a median of 2.4 years (range, 0.3-3.8 years) post-transplant. Conclusion Haploidentical HSCT using this two-step methodology is a safe and effective approach for pts lacking a suitably compatible donor. Relapse of disease remains the most frequent obstacle to successful treatment.

CD4 Donor Lymphocyte Infusion Can Cause Conversion of Chimerism Without GVHD by Inducing Immune Responses Targeting Minor Histocompatibility Antigens in HLA Class II.

Under non-inflammatory conditions HLA class II is predominantly expressed on hematopoietic cells. Therefore, donor CD4 T-cells after allogeneic stem cell transplantation (alloSCT) may mediate graft-vs.-leukemia reactivity without graft-vs.-host disease (GVHD). We analyzed immune responses in four patients converting from mixed to full donor chimerism without developing GVHD upon purified CD4 donor lymphocyte infusion (DLI) from their HLA-identical sibling donor after T-cell depleted alloSCT. In vivo activated T-cells were clonally isolated after CD4 DLI. Of the alloreactive T-cell clones, 96% were CD4 positive, illustrating the dominant role of CD4 T-cells in the immune responses. We identified 9 minor histocompatibility antigens (MiHA) as targets for alloreactivity, of which 8 were novel HLA class II restricted MiHA. In all patients, MiHA specific CD4 T-cells were found that were capable to lyse hematopoietic cells and to recognize normal and malignant cells. No GVHD was induced in these patients. Skin fibroblasts forced to express HLA class II, were recognized by only two MiHA specific CD4 T-cell clones. Of the 7 clones that failed to recognize fibroblasts, two targeted MiHA were encoded by genes not expressed in fibroblasts, presentation of one MiHA was dependent on HLA-DO, which is absent in fibroblasts, and T-cells recognizing the remaining 4 MiHA had an avidity that was apparently too low to recognize fibroblasts, despite clear recognition of hematopoietic cells. In conclusion, purified CD4 DLI from HLA-identical sibling donors can induce conversion from mixed to full donor chimerism with graft-vs.-malignancy reactivity, but without GVHD, by targeting HLA class II restricted MiHA.

HLA monomers as a tool to monitor indirect allorecognition.

BackgroundRecognition of donor antigens can occur through two separate pathways: the direct pathway (non-self HLA on donor cells) and the indirect pathway (self-restricted presentation of donor derived peptides on recipient cells). Indirect allorecognition is important in the development of humoral rejection; therefore, there is an increasing interest in the monitoring of indirect alloreactive T-cells. We have used an in vitro model to determine the optimal requirements for indirect presentation and assessed the risk for semidirect presentation in this system.MethodsHLA-typed monocyte-derived dendritic cells (moDCs) were incubated with cellular fragments or necrotic cells and incubated with either indirect or direct alloreactive T-cell clones. T-cell reactivity was measured through proliferation or cytokine secretion. HLA-typed moDC, monocytes, or PBMCs were incubated with HLA class I monomers, in combination with either direct/indirect T-cell clones.ResultsAlthough both were efficiently taken up, alloreactivity was limited to the semi-direct pathway, as measured by allospecific CD4 (indirect) and CD8 T-cell clones (direct) when cells were used. In contrast, HLA-A2 monomers were not only efficiently taken up but also processed and presented by HLA-typed moDC, monocytes, and PBMCs. Activation was shown by a dose-dependent induction of IFN-γ production and proliferation by the CD4 T-cell clone. Antigen presentation was most efficient when the monomers were cultured for longer periods (24–48 hr) in the presence of the T-cells. Using this method, no reactivity was observed by the CD8 T-cell clone, confirming no semidirect alloreactivity.ConclusionWe have developed a system that could be used to monitor indirect alloreactive T-cells.

Hematopoietic Bone Marrow Transplantation (BMT) for Patients with High-Risk Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), or Myelodysplastic Syndrome (MDS) Using HLA-Haploidentical Related Donors: A Trial Using Radiolabeled Anti-CD45 Antibody Combined with Immunosuppression Before and After BMT

Abstract 4164Patients with advanced leukemia or high-risk MDS undergoing conventional hematopoietic cell transplantation (HCT) have a significant risk of both relapse and non-relapse mortality. We previously conducted a series of studies in patients with high-risk AML, ALL or MDS using radiolabeled anti-CD45 antibody as part of an allogeneic HCT preparative regimen employing HLA-matched donors. This approach has made it possible to deliver high doses of radiation relatively specifically to sites of disease while sparing normal organs. This advance may also allow us to maximize the anti-tumor effect and minimize toxicity of a preparative regimen. We have now extended this therapeutic strategy to patients without HLA-matched donors, often patients of mixed ethnicity or belonging to ethnic minority groups. We and others have utilized a combination of immunosuppressive agents, including cyclophosphamide (CY) administered before and after HCT with non-T-cell depleted bone marrow (BM) from HLA-haploidentical donors, to facilitate engraftment and delete highly alloreactive T-cell clones presumably involved in graft-versus-host disease (GvHD). We report a Phase I/II study using 131I-BC8 antibody at escalating doses in combination with a less toxic reduced-intensity conditioning regimen in the setting of haploidentical BMT.This dose escalation study was designed to determine the maximum tolerated dose of 131I-anti-CD45 (BC8) antibody that can be combined with fludarabine (FLU) and 2Gy total body irradiation (TBI) in addition to pre-and post-BMT CY. Ten patients over 18 years of age with advanced AML, ALL or high-risk MDS (>5% blasts) have been treated with a median of 508 mCi (range: 300–818) of 131I delivering a median of 7.9 Gy (range: 1.8–42) to the BM, a median of 74.6 Gy (range: 45.6–178.8) to the spleen, and a median of 15.8 Gy (range: 12.2–22.1) to the liver (does-limiting organ). Patients then received FLU (30 mg/m2 daily for 5 days), 2 Gy TBI, CY (14.5 mg/kg daily for 2 days), and HLA-haploidentical BM grafts, with GvHD prophylaxis consisting of post-transplant CY, tacrolimus and mycophenolate mofetil. The median age of patients was 47.2 years (range: 31–64). Six patients had AML: one patient was in second complete remission, 2 patients had MDS that progressed to AML, 2 had primary refractory disease, and 1 patient had relapsed after previous HCT. One patient had high-risk MDS, one patient had ALL in second remission with minimal residual disease (MRD) and two patients had CML presenting in blast crisis (one in myeloid blast phase and one in lymphoid blast phase). All AML patients had intermediate or high-risk disease by cytogenetic and molecular criteria. Treatment with the 131I-BC8 Ab/FLU/TBI regimen produced a complete remission in all 10 patients, and 8 had 100% donor CD3+ and CD33+ cell engraftment by day 28 post-transplant. The remaining 2 patients were not tested on day 28, however they achieved 100% donor CD3+ and CD33+ by day 56 chimerism evaluation. The absolute neutrophil count surpassed 500/μL at a median of 14 days (range: 12–16) after BMT, and platelets became self-sustained at ≥ 20,000/μL at a median of 22 days (range: 12–35) after BMT. Four patients are surviving relapse-free for a median of 2.8 years (range: 1.6–4.2) after BMT. Five patients experienced disease relapse at 1.8 to 10.7 months after transplant. The overall day-100 non-relapse mortality was 10%, with one death attributed to sepsis in the setting of steroid-refractory GvHD. This study demonstrates that, in addition to a reduced intensity transplant regimen, 131I labeled anti-CD45 antibody can deliver an average of 14.2 Gy of targeted radiotherapy to BM, and an average of 89.2 Gy to the spleen, without a marked increase in 100-day mortality. The maximum tolerated dose of absorbed radiation has not been estimated, and we continue to escalate the radiation dose, with the most recently enrolled patient receiving 24Gy to the liver. This study shows that anti-CD45 radioimmunotherapy yields encouraging results with haploidentical BMT for patients who lack an HLA-matched donor. Disclosures:No relevant conflicts of interest to declare.

Allo-HLA–reactive T cells inducing graft-versus-host disease are single peptide specific

AbstractT-cell alloreactivity directed against non–self-HLA molecules has been assumed to be less peptide specific than conventional T-cell reactivity. A large variation in degree of peptide specificity has previously been reported, including single peptide specificity, polyspecificity, and peptide degeneracy. Peptide polyspecificity was illustrated using synthetic peptide-loaded target cells, but in the absence of confirmation against endogenously processed peptides this may represent low-avidity T-cell reactivity. Peptide degeneracy was concluded based on recognition of Ag-processing defective cells. In addition, because most investigated alloreactive T cells were in vitro activated and expanded, the previously determined specificities may have not been representative for alloreactivity in vivo. To study the biologically relevant peptide specificity and avidity of alloreactivity, we investigated the degree of peptide specificity of 50 different allo-HLA–reactive T-cell clones which were activated and expanded in vivo during GVHD. All but one of the alloreactive T-cell clones, including those reactive against Ag-processing defective T2 cells, recognized a single peptide allo-HLA complex, unique for each clone. Down-regulation of the expression of the recognized Ags using silencing shRNAs confirmed single peptide specificity. Based on these results, we conclude that biologically relevant alloreactivity selected during in vivo immune response is peptide specific.

Tissue Specificity of Cross-Reactive Allogeneic Responses by EBV EBNA3A-Specific Memory T Cells

The crossreactivity of Epstein-Barr virus (EBV Epstein-Barr virus nuclear antigen 3A [EBNA3A])-specific CD8 T cells against allogeneic human leukocyte antigen (HLA)-B*44:02 has been shown to be dependent on presentation of self-peptide EEYLQAFTY by the target antigen. In this study, we report that allogeneic HLA-B*44:02 proximal tubular epithelial cells (PTECs) and human umbilical vein endothelial cells (HUVECs) are poor targets for EBV EBNA3A-specific T cells. The EEY peptide was exogenously loaded onto HLA-B*44:02 and HLA-B*44:03-expressing PTECs and HUVECs. EEY-peptide-loaded, and unloaded, PTECs and HUVECs were then incubated with serial dilutions of our EBNA3A T-cell clone, in a cytotoxicity assay. Although HLA-B*44:02-expressing PTECs were specifically lysed in proportion to the effector/target ratio by the EBNA3A T-cell clone, without peptide loading, lysis was greatly increased by exogenous EEY peptide loading (15% vs. 75%; P<0.0001). HLA-B*44:02-expressing HUVECs were only lysed when loaded with exogenous EEY peptide (0% vs. 64%; P<0.0001). Lack of HLA expression and lack of ABCD3 gene expression were excluded as a cause for these results. PTECs and HUVECs were specifically targeted by another alloreactive T-cell clone without exogenous peptide loading, suggesting that the lack of recognition of HLA-B*44:02 epithelial and endothelial cells by the EBV EBNA3A T-cell clone was due to lack of EEYLQAFTY peptide presentation. Tissue-specific (peptide dependent) alloreactivity may have important implications for transplantation monitoring and rejection.

Leukemic Blasts Acting as Host Antigen Presenting Cells Trigger a Combined CD4 and CD8 Allo-Immune Response Directed against Mismatched HLA Class I

HLA disparity between patient and donor increases the risk and the severity of graft versus host disease (GVHD). In order to safely administer HLA mismatched stem cell transplantation (SCT) and donor lymphocyte infusion (DLI), it is important to understand the mechanisms underlying GVHD in HLA mismatched settings. The degree of patient chimerism, especially of the antigen presenting cells, at the time of DLI has been shown to influence the development of GVHD. This is suggested by the correlation between the time after SCT of DLI administration and the occurrence of GVHD. In mouse models the underlying role of the presence of host APCs in the occurrence of GVHD has been demonstrated in both MHC matched and mismatched models. In this study, the alloimmune response in a patient suffering from GVHD after an HLA class I mismatched DLI was characterized. The patient received a T-cell depleted SCT followed by two DLIs from the same donor. The SCT and first DLI, administered for mixed chimerism, did not result in any signs of GVHD. In contrast, the second DLI led to severe GVHD. Patient chimerism in different cell subsets present prior to the two DLIs showed no significant difference in patient T-cell chimerism and there were no patient B-cell, monocytes or dendritic cells present prior to each DLI. However, a significant difference in the presence of patient leukemic blasts was found at the time of the two DLIs. Prior to the first DLI no leukemic cells could be demonstrated, whereas 9% leukemic blasts were present in bone marrow prior to the second DLI. We showed that the leukemic blasts expressed HLA class II. Correlating with the absence of GVHD, the first DLI did not lead to activation of donor T-cells while the second DLI led to severe GVHD accompanied by a vigorous CD8 and CD4 T-cell response. Activated donor derived CD8 and CD4 T-cells from this response were isolated by single cell sort based on HLA-DR expression. Expanded clones were characterized for alloreactivity, HLA restriction, specificity and clonality. We showed that 50 of the 53 expanded CD8 T-cells were alloreactive and that all alloreactive CD8 T-cells were restricted to the mismatched HLA-A2. Additionally, by sequencing the T-cell receptor (TCR) Vβ CDR3 region, we showed that all alloreactive CD8 clones expressed a different CDR3 region, illustrating polyclonality. Of the 88 expanded CD4 clones, 21 were alloreactive and the majority of the alloreactive CD4 T-cell clones were directed against an HLA-A2 derived peptide presented in HLA-DR1. By Vβ analysis we showed that the CD4 T-cell response was also polyclonal. Finally, to investigate the role of the leukemic blasts in the initiation of the GVHD, the capacity of the different cell subsets present prior to DLI 1 or 2 to stimulate donor CD8 and CD4 T-cells was analyzed. This showed that only the leukemic blasts were able to highly stimulate both the CD8 and the CD4 T-cells. Our data indicate that the leukemic blasts by acting as host APCs triggered a combined CD4 and CD8 allo-immune response directed against the mismatched HLA, and thus initiated not only anti-leukemic reactivity but also lethal GVHD.

New Insights in CD28‐Independent Allograft Rejection

CD28 costimulatory blockade induces tolerance in most murine transplant models but fails to do so in stringent transplant models, such as skin transplantation. The precise immunological mechanisms of CD28-independent rejection remain to be fully defined. Using two novel mouse strains in which both CD28 and either CD4 or CD8 are knocked out (CD4(-/-)CD28(-/-) or CD8(-/-)CD28(-/-) mice), we examined mechanisms of CD28-independent CD4(+) or CD8(+) T-cell-mediated allograft rejection. CD4(-/-)CD28(-/-) and CD8(-/-)CD28(-/) deficient mice rejected fully allogeneic skin allografts at a tempo comparable with that in wild-type mice. Rejection proceeded despite significant reduction in alloreactive T-cell clone sizes suggesting the presence of a subset of T cells harnessing alternate CD28-independent costimulatory pathways. Blockade of CD40-CD154 and CD134-CD134L, but not ICOS-B7h pathways in combination significantly prolonged allograft survival in CD8(-/-)CD28(-/-) recipients and to a lesser extent in CD4(-/-)CD28(-/-) recipients. Prolongation in allograft survival was associated with reduced effector-memory T-cell generation, decreased allospecific Th1 cytokine generation and diminished alloreactive T-cell proliferation in vivo. In aggregate, the data identify these two pathways as critical mediators of CD28-independent rejection by CD4(+) and to a lesser extent CD8(+) T cells, and provide novel mechanistic insights into functions of novel T-cell co-stimulatory pathways in vivo.

120 POSTER Transduction of tumor cell lines with adenoviral vectors expressing TLR ligands and scFv anti-IL-10 activates dendritic cell maturation and IL-12 secretion in the presence of IL-10

To investigate whether anti-idiotypic (anti-Id) antibodies activate T cells either directly or indirectly, we examined the ability of syngeneic anti-Id monoclonal antibodies (mAbs) to regulate idiotype (Id) expression, antigen-binding antibody production, and T-cell reactivity to antigen. Our idiotypic system consists of an anti-I-A mAb that carries an infrequently expressed Id. Using three syngeneic anti-Id mAbs (Ab2), we previously defined the idiotype of the 11-5.2.1.9 (11-5) anti-I-Ak mAb. Two of these mAbs, IIIDI and IA2, recognize the same or closely related epitopes on 11-5 and cross react with two additional anti-I-Ak mAbs, 8B and 39J; the third anti-Id mAb, VC6, recognizes a distinct epitope shared by 11-5 and 8B. In the present study, BALB/c (H-2d) mice were primed with varying doses of these anti-Ids and were then boosted with C3H (H-2k) spleen cells. Among 130 such primed mice, the syngeneic anti-Ids when tested at priming doses between 10 ng and 10 μg were unable to induce Id production. The priming anti-Id mAbs persisted in the serum of the mice and were detectable as late as 40 days after priming. Ab1 expression was not modulated in BALB/c mice immunized with KLH-coupled Ab2, however, this immunization elicited the production of Ab3 which shared idiotypes with 11-5, 8B, and 39J. BALB/c anti-C3H alloreactive T-cell clones were also not induced by anti-Id priming, nor could they be shown to bind directly to the three Ab2 used. Nevertheless, the proliferative response of one anti-I-Ak specific T-cell clone that recognizes the same epitope as 11-5, 8B, and 39J, was inhibited by the IIIDI and IA2 Ab2. Thus, a T cell can express an idiotype shared by a B cell, but the linked recognition of an Id-associated carrier determinant(s) by an alloreactive T cell is required to elicit an anti-Id antibody response. These results favor the possibility that the activation of T cells is not dependent upon their ability to bind to anti-Id, but rather on their capacity to respond to epitopes of Id-anti-Id antigen-antibody complexes formed on B cells.

Reconstitution of adaptive and innate immunity following allogeneic hematopoietic stem cell transplantation in humans

Allogeneic hematopoietic stem cell transplantation is a potentially curative treatment modality for a number of hematologic malignancies, as well as inherited immunodeficiencies and hemoglobinopathies, and may also have a role in selected acquired autoimmune disorders. The complete or near-complete ablation of host immunity and subsequent establishment of donor-derived immunity that is required for successful engraftment and long-term outcomes provide a major obstacle to such transplantation approaches. A delicate balance exists between the need for the reconstituted donor-derived immunity to provide both protection against pathogenic challenges and graft-versus-malignancy activity, and the potentially harmful expansion of alloreactive T-cell clones mediating GvHD. The search for interventions that would allow more rapid and selective reconstitution of beneficial immune specificities continues to be informed by the development of new tools enabling a more precise dissection of the kinetics of reconstituting populations. This review summarizes more recent data on immune reconstitution following allogeneic transplantation in humans.

934 POSTER Phase II study of irinotecan and oral etoposide in patients with platinum/taxane-resistant ovarian carcinoma

To investigate whether anti-idiotypic (anti-Id) antibodies activate T cells either directly or indirectly, we examined the ability of syngeneic anti-Id monoclonal antibodies (mAbs) to regulate idiotype (Id) expression, antigen-binding antibody production, and T-cell reactivity to antigen. Our idiotypic system consists of an anti-I-A mAb that carries an infrequently expressed Id. Using three syngeneic anti-Id mAbs (Ab2), we previously defined the idiotype of the 11-5.2.1.9 (11-5) anti-I-Ak mAb. Two of these mAbs, IIIDI and IA2, recognize the same or closely related epitopes on 11-5 and cross react with two additional anti-I-Ak mAbs, 8B and 39J; the third anti-Id mAb, VC6, recognizes a distinct epitope shared by 11-5 and 8B. In the present study, BALB/c (H-2d) mice were primed with varying doses of these anti-Ids and were then boosted with C3H (H-2k) spleen cells. Among 130 such primed mice, the syngeneic anti-Ids when tested at priming doses between 10 ng and 10 μg were unable to induce Id production. The priming anti-Id mAbs persisted in the serum of the mice and were detectable as late as 40 days after priming. Ab1 expression was not modulated in BALB/c mice immunized with KLH-coupled Ab2, however, this immunization elicited the production of Ab3 which shared idiotypes with 11-5, 8B, and 39J. BALB/c anti-C3H alloreactive T-cell clones were also not induced by anti-Id priming, nor could they be shown to bind directly to the three Ab2 used. Nevertheless, the proliferative response of one anti-I-Ak specific T-cell clone that recognizes the same epitope as 11-5, 8B, and 39J, was inhibited by the IIIDI and IA2 Ab2. Thus, a T cell can express an idiotype shared by a B cell, but the linked recognition of an Id-associated carrier determinant(s) by an alloreactive T cell is required to elicit an anti-Id antibody response. These results favor the possibility that the activation of T cells is not dependent upon their ability to bind to anti-Id, but rather on their capacity to respond to epitopes of Id-anti-Id antigen-antibody complexes formed on B cells.