Oligoclonal T-cell Expansions Research Articles

Somatic and Germline HLA Determinants of Immune Surveillance and Escape in Myelodysplastic Syndromes

While the contribution of canonical myeloid genetic drivers to myelodysplastic syndromes/neoplasms (MDS) pathobiology is well established, other genetic events operative under special circumstances, and by itself not constituting intrinsic drivers, may affect the disease course. These include genetic defects conveying resistance to specific therapies, clonally restricted DNA repair alterations favoring the accumulation of additional mutations, and/or lesions facilitating the resistance to inflammation or immune escape. Here, we focus on this latter aspect as it may shed light on the pressure exerted by immune tumor surveillance or acquired resistance to cancer immune therapies, a field that has lagged in myeloid neoplasia as compared to the dramatic changes obtained in solid tumors. We hypothesize that during MDS ontogeny and leukemia evolution a spectrum of immunological mechanisms may shape the oncogenic landscape and facilitate the emergence of clones with diverse immunogenic potential as targets, leading to selective immunotherapeutic liabilities. We accrued a clinically and molecularly well annotated cohort of 101 MDS patients at various stages of the disease spectrum [N=51 low risk (LR)-MDS, N=32 high risk (HR)-MDS, N=18 secondary AML-sAML)]. This cohort included specific clinical sub-entities wherein the contribution of immune forces appears to be a prominent feature i.e., hypocellular MDS (hMDS, N=9) or MDS with co-occurring T large granular lymphocytosis (LGL) (N=5). By combining human leukocyte antigen (HLA) and T cell receptor (TCR) immunosequencing at disease onset, we leveraged our bioinformatics expertise to impute HLA mutations and losses, HLA evolutionary divergence (HED), and characteristics of T cell repertoires. The median age of the whole cohort was 71.2 years (IQR: 63.4-77.7), and median bone marrow (BM) blast count was 3% (1-11%). Cytogenetics revealed normal karyotype in 41 patients (40%), while complex karyotype was observed in 21 (20%) and partial or complete loss of chromosome 7 in 10 cases (10%). Comparisons with a cohort of 784 ethnicity-matched healthy individuals (HC), showed no differences in terms of locus-specific HED configurations. However, when HED scores were analyzed across the disease spectrum, we observed a heterogeneous distribution in MDS patients for the HLA-C locus, with higher values in hypoplastic and LGL-associated MDS as compared to HR diseases (p= 0.0068) and sAML (p= 0.0409). The screening for immune evasion highlighted the presence of both somatic mutations and losses in the HLA region in 14 patients, with both type of alterations found in 1 case. Of those, 6 patients (20% LGL-MDS, 5% LR-MDS and 17% sAML) harbored HLA mutations (in HLA-A, B, C and DPB1 loci), whereas HLA losses were observed in 9 patients (22% hMDS, 5% LR-MDS, 10% HR MDS, 11% sAML) almost exclusively in class II alleles (chiefly DPB1, DRB1 and DPA1). When exploring the degree of immunocompetence by TCR immunosequencing as a proxy, we found a higher degree of diversity (imputed via inverse Simpson index) as compared to healthy controls (p=1.5e-07), possibly in relationship to diverse T cell responses across the disease spectrum. However, no difference between MDS sub-entities was found. Of interest is that the maximal productive frequency (an indicator of the degree of T cell expansion) was higher in patients with hMDS and LGL-associated MDS as compared to HR-MDS (p=0.0079). Finally, inter-sample comparisons of shared clonotypes showed a high degree of overlap with regards to TCR repertoires among MDS as compared to healthy subjects. Our data emphasize the adaptive immune-molecular network underpinning MDS development. HLA constellations and their interactions with TCR repertoires may create selective immune pressure. Somatic HLA aberrations may serve as a marker of ongoing immune pressure and correspond to oligoclonal T-cell expansions and variations in TCR diversity. While virtually no HLA mutations have been previously found in AML/MDS, we show a relatively higher frequency of HLA aberrations in hMDS and MDS-LGL, immunologically stressed scenarios reminiscent of aplastic anemia and post-allogenic transplant AML/MDS cases, whereby respectively 36% 1 and 38% 2 of patients harbored HLA lesions as a mean of escape from cytotoxic lymphocytes or allogeneic Graft-vs-Leukemia reactions.

Hemophagocytic Lymphohistiocytosis Susceptibility (HLH) Factors IL-18 and Cytotoxic Impairment Converge on Pathologic CD8 T-cell Hyperactivation

Excess IL-18 and graded defects in cytotoxicity are fundamental susceptibility factors for HLH and MAS. In mice, neither excess IL-18 (18tg mice) nor perforin-deficiency individually cause immunopathology without inflammatory challenge. However, 18tg mice lacking perforin (18tg; Prf1-/-) develop spontaneous lethal hyperinflammation, while subclinical MAS was found in 18tg;Prf1+/- mice. HLH and MAS patients demonstrate CD8 T-cell activation in tissue & peripheral blood. We sought to understand how IL-18 and cytotoxic impairment may interact in driving spontaneous hyperinflammation.We examined mice bearing single- and dual-susceptibility factors for “clinical” features of hyperinflammation, cytokine levels, and flow cytometric, transcriptional, functional, and TCR-seq phenotypes.Flow cytometric analysis of dual susceptibility mice showed spontaneous expansion of CD8 T-cells (and not CD4 T- or NK-cells), which expressed the IL-18 receptor and multiple inhibitory receptors (PD-1, Lag-3, CD39), yet overproduced IFNg. Such “hyperinflammatory CD8 T-cells” were present in reticuloendothelial organs (spleen, bone marrow, and liver) but not lymph nodes, peripheral blood, or thymus. Transcriptional analysis demonstrated hyperactivated CD8 T cells showed features of both terminal effector function and exhaustion when compared to well-described datasets. This analysis also revealed a strong signature for recent antigen activation. BulkCD8 TCR sequencing showed dramatic oligoclonal hyper-expansion of TCR sequences from dual susceptibility mice with no significant clonal overlap between biologic replicate samples. In vitro, TCR stimulation was necessary for IL-18 to promote IFNg production and restimulation-induced cell death (RICD), whereas perforin-deficiency prevented RICD. Broad-spectrum antibiotics did not rescue spontaneous hyperinflammation in dual-susceptibility mice, suggesting T-cell responses were not directed at commensal bacteria. Attempts to circumvent CD8 T-cell activation, using inducible CD8 T-cell-specific deletion of Il18r1 or by promoting allelic exclusion by fixing the T-cell receptor, showed breakthrough expansion of hyperactivated CD8 T-cells.Thus, IL-18 and cytotoxicity drive progressive, post-thymic, oligoclonal CD8 T-cell expansion that may underly life-threatening immunopathology in HLH and MAS. Expansion of hyperactivated CD8T cells in reticuloendothelial organs aligns with sites of hemophagocytosis. These data together implicate HLH/MAS susceptibility factors interact with TCR stimulation, whether by infectious triggers or as-yet undetermined self antigens, to drive potentially-targetable mechanisms of T-cell activation. [Display omitted] Supporting figures.

Use of Immunosuppressive Medications in Patients With Congenital Athymia Following Treatment With Allogeneic Processed Thymus Tissue-agdc

Congenital athymia is an ultra-rare condition characterized by profound T-cell immunodeficiency. Patients who have elevated T-cell proliferative response to mitogens or oligoclonal T-cell expansion may require immunosuppressive medications to treat and/or prevent graft-vs-host disease (GVHD/autologous GVHD). We evaluated immunosuppressive therapy use in clinical trials of allogeneic processed thymus tissue-agdc.

Oligoclonal T-cell expansion in a patient with bone marrow failure after CD19 CAR-T therapy for Richter-transformed DLBCL

The advent of chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment landscape of refractory B-cell malignancies. Real-world evidence has highlighted the high incidence of hematological toxicity, including prolonged and profound cytopenia. Here, we present a case of bone marrow (BM) failure in a 57-year old man with DLBCL-type Richter transformation receiving tisagenlecleucel in a standard-of-care setting. His clinical course was notable for underlying BM infiltration, severe cytokine release syndrome, and multiple infectious complications in the setting of prolonged, profound neutropenia. Cytokine profiling revealed a signature consistent with acquired aplastic anemia - including downregulation of CD40-L and EGF. Flow cytometric analyses demonstrated migration of CAR T-cells to the BM, as well as peripheral blood expansion of both CAR and non-CAR bearing CD8+CD57+ T-cells, an immunophenotype linked to oligoclonality in aplastic anemia. From single-cell level transcriptomics, this cytotoxic T-cell population was characterized by T-cell receptor Vβ oligoclonal expansion and post-CAR clonal drift. Gene expression profiling revealed upregulation of exhaustion markers on (CAR) T-cells and decreased expression of genes involved in STAT signalling and inflammatory response. In conclusion, this case highlights the complex nature of CAR-T-related hematological toxicity and introduces oligoclonal (CAR) T-cell expansion as a potential contributing pathophysiologic mechanism.

Pegilodecakin Combined with Anti-PD-1 Monoclonal Antibodies for Patients with Advanced Renal Cell Carcinoma (IVY): A Multicentre, Open-Label, Phase 1b Trial

Pegilodecakin Combined with Anti-PD-1 Monoclonal Antibodies for Patients with Advanced Renal Cell Carcinoma (IVY): A Multicentre, Open-Label, Phase 1b Trial

Overall responses and survival in RCC on pegilodecakin with anti-PD-1

Background: Interleukin 10 (IL-10) has anti-inflammatory activities and stimulates CD8+ T cell cytotoxicity and antigen specific CD8+ T cell proliferation. Pegilodecakin (Pegylated IL-10) induces oligoclonal T-cell expansion and has single agent activity in advanced renal cell cancers. In a phase 1 expansion cohort we assessed the safety and efficacy of the combination of pegilodecakin and anti PD-1 immune checkpoint inhibitors in advanced renal cell cancer patients.

Large cell neuroendocrine lung carcinoma induces peripheral T-cell repertoire alterations with predictive and prognostic significance

ObjectivesThis study was performed to evaluate for a potentially important role of T cells in the pathophysiology and treatment sensitivity of large cell neuroendocrine lung carcinoma (LCNEC), an orphan disease with poor prognosis and scarce data to guide novel therapeutic strategies. Materials and methodsWe performed T-cell receptor (TCR) β-chain spectratyping on blood samples of patients treated within the CRAD001KDE37 trial (n = 35) using age-matched current or former (n = 11) and never smokers (n = 10) as controls. The data were analyzed in conjunction with the complete blood counts of the probands as well as the data about response to treatment and overall survival in the clinical trial. Results and conclusionUntreated stage IV LCNEC patients had significant T-cell repertoire alterations (p < 0.001) compared to age-matched smokers. These changes correlated positively with blood lymphocyte counts (r = 0.49, p < 0.01), suggesting antigen-induced T-cell proliferation as the causative mechanism. At the same time, LCNEC patients showed mild lymphopenia (1.54 vs. 2.51/nl in median, p < 0.01), which reveals a second, antigen-independent mechanism of systemic immune dysregulation. More pronounced T-cell repertoire alterations and higher blood lymphocyte counts at diagnosis were associated with a better treatment response by RECIST and with a longer overall survival (441 vs. 157 days in median, p = 0.019). A higher degree of T-cell repertoire normalization after 3 months of therapy also distinguished a patient group with more favourable prognosis (median overall survival 617 vs. 316 days, p = 0.036) independent of radiological response. Thus, LCNEC induces clinically relevant changes of the T-cell repertoire, which are measurable in the blood and could be exploited for prognostic, predictive and therapeutic purposes. Their pathogenesis appears to involve antigen-induced oligoclonal T-cell expansions superimposed on TCR-independent lymphopenia.

Association of changes in vaginal microbiome with oligoclonal T-cell expansion and early response to chemoradiation for cervical cancer.

8 Background: The composition of the vaginal microbiome has been shown to affect clearance of HPV virus and transformation to invasive cancer. Clinical studies correlating the vaginal microbiome with immune activation and response to cancer treatment are lacking. We profiled intratumoral T-cell clonality during radiation therapy and correlated it with the diversity of the vaginal flora. Methods: Thirty patients with newly diagnosed locally advanced cervical cancer were enrolled on a prospective study to characterize changes in the cervical microbiome during chemoradiation. Cervical samples were obtained before radiation therapy and during the 1st, 3rd, and 5th week of radiation therapy. The vaginal microbiome was characterized using 16 sRNA gene sequencing to produce operational taxonomic units (OTU’s) representing individual bacterial species. Disease response was categorized as early response (ER), late response (LR), or nonresponse (NR) on the basis of clinical examination at brachytherapy and 3-month PET/CT. Twenty patients had T-cell receptor β sequencing of DNA performed using the ImmunoSEQ platform. The maximum productive frequency of the top three clones (MP3) was used to assess T-cell clonality. Results: Early response was associated with clonal T-cell expansion with an increase of MP3 of 11.1% during treatment as compared to a decline of 6.1% in patients with LR/NR (p = 0.05). Early response was also associated with lower quantity of observed OTU’s of vaginal microbiota (25.0 [SD 12.68]) vs patients with LR/NR (41.15 [SD = 23.3]) (p = 0·03). Increased MP3 was associated with increased abundance of Corynebacteriales (R = 0.90; p &lt; .0001) , Actinomycetales (R = 0.83; p &lt; .0001) and Bifidobacteriales (R = 0.82; p &lt; .0001) . Decreased MP3 was associated with increased abundance of lactobacillus (R = -0.61; p &lt; .0001). Conclusions: Increased diversity of the vaginal microbiome is negatively associated with outcome, supporting previous clinical studies in non-cancer settings. Specific vaginal bacterial species are associated with increased or decreased T-cell clonality at completion of radiation.

Abstract SY31-02: The interplay of radiotherapy with the tumour microenvironment: Novel opportunities to overcome adaptive resistance

Abstract SY31-02: The interplay of radiotherapy with the tumour microenvironment: Novel opportunities to overcome adaptive resistance

Viral-induced CD28 loss evokes costimulation independent alloimmunity

BackgroundBelatacept, a B7-specific fusion protein, blocks CD28-B7 costimulation and prevents kidney allograft rejection. However, it is ineffective in a sizable minority of patients. Although T-cell receptor and CD28 engagement are known to initiate T-cell activation, many human antigen-experienced T-cells lose CD28, and can be activated independent of CD28 signals. We posit that these cells are central drivers of costimulation blockade resistant rejection (CoBRR) and propose that CoBRR might relate to an accumulation of CD28− T-cells resulting from viral antigen exposure. Materials and methodsWe infected C57BL/6 mice with polyomavirus (a BK virus analog), murine cytomegalovirus (a human cytomegalovirus analog), and gammaherpesvirus (HV68; an Epstein–Barr virus analog) and assessed for CD28 expression relative to mock infection controls. We then used mixed lymphocyte reaction (MLR) assays to assess the alloreactive response of these mice against major histocompatibility complex-mismatched cells. ResultsWe demonstrated that infection with polyomavirus, murine CMV, and HV68 can induce CD28 downregulation in mice. We showed that these analogs of clinically relevant human viruses enable lymphocytes from infected mice to launch an anamnestic, costimulation blockade resistant, alloreactive response against major histocompatibility complex-mismatched cells without prior alloantigen exposure. Further analysis revealed that gammherpesvirus–induced oligoclonal T-cell expansion is required for the increased alloreactivity. ConclusionsVirus exposure results in reduced T-cell expression of CD28, the target of costimulation blockade therapy. These viruses also contribute to increased alloreactivity. Thus, CD28 downregulation after viral infection may play a seminal role in driving CoBRR.

High-Throughput Profiling of the T-Cell Receptor Gene Repertoire Supports Antigen Drive in the Pathogenesis of Chronic Idiopathic Neutropenia

Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by prolonged neutropenia, mainly affecting middle-age females of the HLA-DRB1*1302 type. The defective hematopoiesis in CIN can be mainly attributed to accelerated Fas-mediated death of the CD34+/CD33+ granulocytic progenitors, secondary to an inflammatory bone marrow (BM) microenvironment. Crucial to CIN pathogenesis are the increased numbers of activated T cells identified in both peripheral blood (PB) and BM of CIN patients, supporting an immune pathogenesis. Using Sanger sequencing, we previously reported that the T-cell receptor (TR) gene repertoire in CIN is skewed, indicating antigen selection in CIN ontogeny. However, the analytical depth afforded by Sanger sequencing is limited, hindering definitive conclusions. In order to obtain a truly comprehensive view into the role of antigen drive in CIN, using next generation sequencing (NGS) we interrogated the TR repertoire of 13 patients (8 females, 5 males) included in our previous study as well as a healthy female. TRBV-TRBD-TRBJ gene rearrangements were amplified according to the BIOMED2 protocol on either genomic DNA or cDNA isolated from CD8+ cells of PB (n=4) or BM (n=10) samples. PCR products were used as a substrate for paired-end sample preparation (Illumina) and subjected to NGS on the MiSeq Illumina Platform. The raw NGS data were preprocessed with a dedicated pipeline for this purpose, including: (i) quality filtering of each read; (ii) merging of paired-end reads via local alignment; (iii) preparation of fasta files for submission to the IMGT/High V-QUEST tool; and, (iv) IMGT/High V-QUEST metadata analysis, interpretation and visualization. Overall, 6,196,980 TRBV-TRBD-TRBJ gene rearrangements were analyzed (130,020-1,037,680 /case) of which 5,317,609 were productive since they used functional TRBV genes and also carried in-frame CDR3. Rearrangements with identical TRBV gene usage and CDR3 sequence were defined as clonotypes. For repertoire analyses, clonotypes rather than single rearrangement sequences were considered. Overall, 553,145 unique clonotypes were identified (median 39,510; range 7,732-172,253/case), of which 408,744 represented singletons. All clonotypes from the Sanger analysis were detected by NGS as well. Among the 46 functional TRBV genes identified, the most frequent were: TRBV29-1 (13.9%), TRBV19 (6.7%), TRBV12-3 (5.6%), TRBV6-5 (5.4%), TRBV27 (4.9%) and TRBV6-1 (4.0%), collectively accounting for 40,5% of the TRBV repertoire; the TRBV29-1 gene predominated in 9/13 CIN cases. All CIN cases were found to carry distinct expanded clonotypes (median 10,314; range 2,279-40,245/case). The predominant clonotype ranged in frequency from 5.25 to 20.2% of the total clonotypes observed in each case. This contrasts significantly (p<0.001) with a 0.47% frequency of the dominant clonotype in the healthy control. Cluster analysis of the sequences of all CIN cases identified 9034 different clonotypes shared by different patients and, thus, deemed as public. Notably, public clonotypes of a given CDR3 length could show high sequence similarity, further underscoring the restricted nature of the repertoire. As an example, 1632/2665 (61.2%) public clonotypes with 12 aminoacid-long CDR3 were grouped into 168 distinct communities, populated with 2-280 highly similar sequences, each linked with 1 aminoacid distance with at least another member of the community. Overall, the present study offers conclusive evidence that the TR repertoire in CIN is remarkably skewed. The finding of oligoclonal T-cell expansions and public clonotypes strongly indicates that antigen-driven immune responses are very likely implicated in the pathogenesis of CIN. DisclosuresNo relevant conflicts of interest to declare.

Expansion of Memory-Type CD8+ T Cells Correlates With the Failure of Early Immunosuppression Withdrawal After Cadaver Liver Transplantation Using High-Dose ATG Induction and Rapamycin

We report on a pilot study investigating the feasibility of early immunosuppression withdrawal after liver transplantation (LT) using antithymocyte globulin (ATG) induction and rapamycin. LT recipients received 3.75 mg/kg per day ATG from days 0 to 5 followed by rapamycin-based immunosuppression. In the absence of acute rejection (AR), rapamycin was withdrawn after month 4. Immunomonitoring included analysis of peripheral T-cell phenotypes and clonality, cytokine production in mixed lymphocyte reaction, and characterization of intragraft infiltrating cells. Ten patients were enrolled between October 2009 and July 2010. In the first three patients, complete withdrawal of immunosuppression after month 4 led to AR. No further withdrawals of immunosuppressive were attempted. Two AR occurred in the remaining seven patients. ATG induced profound T-cell depletion followed by CD8(+) T-cell reexpansion exhibiting memory/effector-like phenotype associated with progressive oligoclonal T-cell expansion (Vβ/HPRT ratio) and gradually enhanced anti-cytomegalovirus and anti-Epstein-Barr virus T-cell frequencies. Patients developing AR were characterized by decreased TCAIM expression. AR were associated with increased donor-specific production of interferon (IFN)-γ and interleukin (IL)-17, increased intragraft expression of IFN-γ mRNA, and significant CD8(+) T-cell infiltrates colocalizing with IL-17(+) cells. High-dose ATG followed by short-term rapamycin treatment failed to promote early operational tolerance to LT. AR correlates with expansion of memory-type CD8(+) T cells and increased levels of IFN-γ and IL-17 in mixed lymphocyte reaction and in the graft. This suggests that resistance and preferential expansion of effector memory T-cell in lymphopenic environment could represent the major barrier for establishment of tolerance to LT in approaches using T-cell-depleting induction.

Clinical and immunophenotypic features of atypical complete DiGeorge syndrome

DiGeorge syndrome is a congenital malformation characterized by variable defects of the thymus, heart and parathyroid glands. Athymic patients are classified as exhibiting complete DiGeorge syndrome. Some of these patients may also exhibit oligoclonal T-cell expansion, generalized rash and lymphadenopathy at some point after birth. This rare condition is known as atypical complete DiGeorge syndrome, resembles Omenn syndrome, and has not been fully characterized. The clinical and immunophenotypic features of atypical complete DiGeorge syndrome were assessed in two affected Japanese infants. T-cell receptor (TCR) Vβ repertoire was analyzed on flow cytometry and complementarity-determining region 3 spectratyping. Both patients had no detectable thymus tissue and profound T-cell lymphopenia soon after birth. Progressive increase of activated T cells, however, as well as eosinophilia, high serum IgE level, generalized rash, and lymphadenopathy were observed during early infancy. A highly restricted TCR Vβ repertoire was demonstrated both in CD4(+) and CD8(+) T cells. The Omenn syndrome-like manifestations might be associated with the oligoclonal proliferation of activated T cells. Analysis of the immunophenotype and TCR Vβ repertoire is helpful to establish the early diagnosis of atypical complete DiGeorge syndrome.

Immune reconstitution after haploidentical hematopoietic cell transplantation: impact of reduced intensity conditioning and CD3/CD19 depleted grafts

Haploidentical hematopoietic cell transplantation (HHCT) using CD34 selected grafts is complicated by slow engraftment and immune reconstitution. Engraftment and immune reconstitution might be improved using CD3/CD19-depleted grafts and reduced intensity conditioning (RIC). We report on 28 patients after HHCT with CD3/CD19-depleted grafts using RIC, which were prospectively evaluated for engraftment and immune reconstitution. Engraftment was rapid with full chimerism reached on day +15 after HHCT. T-cell reconstitution was delayed with a median of 205 CD3+ cells/μl, 70 CD3+CD4+ cells/μl and 66 CD3+ CD8+ cells/μl on day +100, respectively. A skewed T-cell receptor-Vβ repertoire with oligoclonal T-cell expansions to day +100 and normalization after day +200 was observed. B-cell reconstitution was slow with a median of 100 CD19+ CD20+ cells/μl on day +150. Natural killer (NK) cell engraftment was fast reaching normal values on day +20. An increased natural cytotoxicity receptor and NKG2A, but decreased NKG2D and KIR expressions were observed on NK cells until day +100. We observed a positive impact of donor lymphocyte infusions on immune reconstitution. In conclusion, after HHCT, using CD3/CD19-depleted grafts and RIC, T- and B-cell reconstitution is delayed, whereas NK-cell reconstitution occurs early and fast.

Results of a Phase II Study of Haploidentical Hematopoietic Cell Transplantation (HHCT) in Adults Using Reduced Intensity Conditioning and CD3/CD19-Depleted Grafts: Clinical Outcome and Immune Reconstitution.

Abstract 1203Poster Board I-225HHCT using reduced intensity conditioning (RIC) and immunomagnetic CD3/CD19 graft depletion is of low toxicity and enables fast engraftment even with grafts of low CD34 content. Immune reconstitution may be improved compared to graft CD34 selection due to the cotransplantation of facilitating cells, CD34- progenitors, dendritic cells and natural killer (NK)-cells. A prospective multicenter phase II study of HHCT using CD3/CD19-depleted grafts after RIC with fludarabine (150-200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2), OKT-3 (5 mg/day, day -5 to +14) was initiated. No post grafting immunosuppression was applied if the graft contained <5×104 CD3+ cells/kg. 60 adults with median age of 45 years (range, 19-61) have been enrolled from 2002 to 2009. Diagnoses were AML (n=37), ALL (n=8), NHL (n=6), myeloma (n=4), CML (n=3), CLL (n=1) and MDS (n=1). Patients were “high risk” because of refractory disease (n=6), cytogenetics (n=5), chemosensitive relapse (n=26) or relapse after prior HCT (allo=16, auto=6, both=1). At HHCT, 30 patients were in CR and 30 in PR. Grafts contained median of 6.8×106 (range, 3.5-22) CD34+ cells/kg, 4.0×104 (range, 0.9-44) CD3+ T cells/kg and 2.8×107 (range, 0.00-37.3) CD56+ cells/kg. Engraftment was rapid with median of 12 days to >500 granulocytes/μL (range, 9-50) and 11 days to >20.000 platelets/μL (range, 7-38). Incidence of grade II-IV acute GVHD and chronic GvHD (cGvHD) was 47% and 15%, respectively. Non-relapse-mortality on day 100 was 25% and 44% for the whole study period. Current overall survival (OS) is 18 of 60 patients (30%) with median follow-up of 525 days (range, 21-1542) of patients alive resulting in a Kaplan-Meier estimate 1-year OS of 41% and 2-year OS of 24%. Kaplan-Meier estimate 1-year OS was 40 % in AML, 38 % in ALL and 67% in NHL patients. No positive impact of KIR-mismatch on survival even in the subgroup of patients with AML was observed. Patients with cGVHD had a trend toward better survival with 56% vs. 39% (p=0.09). 14 patients died because of infections. Detailed immune reconstitution was analyzed in 24 patients. NK-cell engraftment was fast with median of 248 CD16+56+CD3- cells/μl (range, 1-886) on day 20. Increased natural cytotoxicity receptors (NKP30, NKP44, NKP46) and NKG2A, but decreased NKG2D and KIR-expression was observed on NK-cells in the first 3 months. T-cells regenerated delayed with median of 191 CD3+ cells/μl (range, 38-799) on day 100. A slow reconstitution of CD8+ and CD4+ T-cells with median of 66 CD8+ (range, 8-170) vs. 70 CD4+ cells/μl (range, 12-301) on day 100 and 157 (range, 32-379) vs. 181 cells/μl (range, 19-980) on day 400 was observed. The subset of memory T-cells regenerated faster compared to naïve T-cells with median of 25 CD4+45RA+ (range, 4-109) vs. 80 CD4+45RO+ cells/μl (range, 0 to 255) and 46 (range 7-191) vs. 164 cells/μl (range, 66-323) on days 80 and 250, respectively. T-cell repertoire was skewed with oligoclonal T-cell expansion to day 100 and normalization after day 200. B-cell reconstitution was slow with median of 8 (range, 0-407) CD19+20+ cells/μl on day 100. 6 of these 24 patients received donor-lymphocyte-infusions for relapse or mixed chimerism resulting in acceleration of immune recovery in T- and NK-cells. In conclusion, HHCT using RIC and CD3/CD19 depleted grafts is of low toxicity and allows fast engraftment even with low doses of CD34 cells. Overall survival seems promising in a high risk patient cohort. Recovery of NK cells occurs early and fast. KIR receptor expression remains low in the first 3 months. T- and B-cell reconstitution is delayed but seems faster compared to published data after CD34 selection. To evaluate the treatment protocol earlier during the course of disease a new study has just been initiated. Disclosures:Off Label Use: The use of Fludarabine, Thiotepa, Melphalan and OKT-3 in the conditioning is off-label-use.

T-Cell Receptor Complementarity Determining Region Analysis of Peripheral Blood and Bone Marrow T-Lymphocyte Subsets and Quantitative Evaluation of T-Regulatory Cells in Patients with Chronic Idiopathic Neutropenia

Clonal/oligoclonal expansions of cytotoxic T-lymphocytes (CTLs) and quantitative/qualitative abnormalities of T-regulatory cells (Tregs) have been implicated in the pathophysiology of acquired bone marrow (BM) failure syndromes such as aplastic anemia, myelodysplastic syndromes and large granular lymphocyte proliferative disease. Chronic Idiopathic Neutropenia (CIN) is an acquired disorder of granulopoiesis characterised by increased apoptosis of BM CD34+/CD33+ granulocytic progenitor cells. We have previously provided evidence for oligoclonal T-cell expansions with possible pathogenetic significance in CIN. The aim of the current study is (a) to define and compare the magnitude of CD4+ and CD8+ T-cell responses and (b) to evaluate the number of Tregs, in the peripheral blood (PB) and BM of CIN patients. Eighty five CIN patients and 85 healthy controls, age- and sex-matched to the patients, were studied after informed consent. All patients had PB neutrophil counts below 1800/μL (mean 1410 ± 330 neutrophils/μL, range 100–1799 neutrophils/μL), displayed negative anti-neutrophil antibody activity and were satisfying the previously reported diagnostic criteria for the disease.The T-cell receptor (TCR)-Vβ repertoire was analysed by flow cytometry (Vβ spectratyping) in the PB and BM CD3+, CD4+ and CD8+ cells. The size distribution of the TCR-Vβ complementarity determining region 3 (CDR3) was analysed in immunomagnetically sorted PB and BM CD4+ and CD8+ cells by PCR according to the -2 protocol using a fluorescence-based DNA sequencer (CDR3 spectratyping). Vβ family expansions were defined as above of 2 standard deviations from the mean values of the 85 healthy controls. CDR3 oligoclonal/monoclonal patterns were defined upon comparison with the normal Gaussian-type size distribution. The Treg cell frequency was defined as the proportion of FOXP3+ cells within the CD4+/CD25high PB and BM cell fraction by flow cytometry. We found that 69.41% and 82.61% of CIN patients displayed one or more predominant TCR-Vβ family expansions within the CD3+ cell fraction of PB and BM, respectively. None of the controls displayed clonal/oligoclonal expansions in either PB or BM (P<0.001 and P<0.001, respectively). Further analysis showed that 58.62% and 74.19% of CIN patients displayed a skewed TCR-Vβ repertoire in PB CD4+ and CD8+ cells, respectively. A higher proportion of patients i.e. 93.75% and 75.0% displayed a skewed TCR-Vβ repertoire in BM CD4+ and CD8+ cells, respectively. The total sum of predominant TCR-Vβ clones was statistically significant increased in patients' BM compared to PB in all subpopulations studied, namely the CD3+, CD4+ and CD8+ cells (P<0.05, P<0.05, P<0.05, respectively). Interestingly, CDR3 spectratyping identified a skewed (oligoclonal) pattern in PB and/or BM CD8+ cells in 100% of patients. PCR analysis of PB and BM CD4+ cells revealed a normal Gaussian-like CDR3 size distribution in all but three patients. None of the controls displayed a skewed CDR3 profile in either CD4+ or CD8+ cells. The percentage of FOXP3+ cells within the PB CD4+CD25high cell fraction was significantly decreased in CIN patients (57.77%±15.77%) compared to controls (72.95±12.23%; P=0.0004). A parallel analysis of Treg cell proportion in patients' PB and BM showed statistically significant increased percentage of FOX3+ cells within the CD4+/CD25high T-cells of BM (68.51%±11.88%) compared to PB (52.20%±12.77%) (P=0.0005). No statistically significant difference was found in the percentage of FOX3+ cells within the CD4+/CD25high T-cells of BM compared to PB in healthy controls. These data suggest that oligoclonal CTL expansions are uniformly identified in CIN patients. The polarized cell subsets are more frequent in BM than the PB suggesting the possible existence of “target” within the BM. Whether, however, the CD34+/CD33+ BM cells represent the target or the innocent bystanders of this immune process, remains elusive. The low frequency of Tregs in the PB and the accumulation of these cells in patients' BM represents probably a compensatory control mechanism aiming to suppress the local immune reactions. All these data substantiate further the concept that CIN belongs to the spectrum of T-cell mediated BM failure syndromes.

Immune Reconstitution after Haploidentical Hematopoietic Cell Transplantation: Impact of Reduced Intensity Conditioning and CD3/CD19-Depleted Grafts

Haploidentical hematopoietic cell transplantation (HHCT) using CD3/CD19 depleted grafts may lead to faster engraftment and immune reconstitution since grafts contain also graft-facilitating-cells, CD34− progenitors, NK cells, and dendritic cells. Reduced intensity conditioning may also have a positive impact on immune reconstitution following HHCT. 26 adults received CD3/CD19 depleted HHCT after RIC (150–200 mg/m2 fludarabine, 10mg/kg thiothepa, 120 mg/m2 melphalan and 5mg/day OKT-3 (day −5 to +14)) at our institution between 2005–2008. We prospectively evaluated engraftment and immune reconstitution. B-, NK-, T- and T-cell subsets (CD3/8, CD4/8, CD4/45RA/RO), TCR-Vβ repertoire and NK-cell receptors (NKP30, NKP44, NKP46, NKG2D, CD158a/b/e, CD85j, NKG2A, CD161) were analyzed by FACS. Grafts contained 8.8×106 CD34+ (range, 4.3–18.0 ×106), 2.9×104 CD3+ (range, 1.2–9.2×104) and 3.6×107 CD56+ (range, 0.02–23.0 ×107) cells/kg. Engraftment was rapid with a median time to >500 granulocytes/μl of 11 days (range, 9–15) and a median time to >20 000 platelets/μl of 11 days (range, 8–23). Full chimerism was reached on day 14 (median; range, 6–26). NK-cell engraftment was rapid, reaching normal values on day 20 (median of 247 CD16+CD56+CD3− cells/μl (range, 1–886)) with NK cells comprising up to 70% of lymphocytes. B-cell reconstitution was delayed with 81 (range, 0–280) and 335 (range, 11–452) CD19+20+ cells/μl on days 150 and 400, respectively. T-cell reconstitution was impaired with 49 (range, 0–586) and 364 (range, 35–536) CD3+ cells/μl on day 60 and day 150, respectively. We observed an increase of CD3+CD8+ cells in contrast to CD3+CD4+ cells early after HHCT with a median of 24 (range, 0–399) vs 16 (range, 0–257) and 159 (range, 1–402) vs 96 (range, 18–289) cells/μl on day 50 and day 200, respectively. CD4+CD45RA+ T cells increased slowly while CD4+CD45RO+ T cells reconstituted faster with a median of 61 CD4+CD45RO+ cells/μl (range, 0–310) vs 24 CD4+CD45RA+ (range, 0 to 152) on day 100. Within the CD4+CD25+ regulatory T cells there was a slow regeneration with median of 14 CD4+CD25+ cells/μl (range, 0–96) on day 100 and 28 CD4+CD25+ cells/μl (range, 19–160) on day 200. CD14+CD45+ monocytes did not reach normal values within the time of observation with 7 CD14+CD45+ cells/μl (range, 0–21) on day 120 and 7 CD14+CD45+ cells (range, 2–381) on day 400. TCR-Vβ repertoire and NK-cell receptor reconstitution was analyzed so far in 7 and 8 patients, respectively. We found a skewed T-cell repertoire with oligoclonal T-cell expansions to day 100 and normalization after day 200. An increased natural cytotoxicity receptor (NKP30, NKP44, NKP46) and NKG2A, but decreased NKG2D and KIR-expression was observed on NK-cells until day 100. In conclusion, T- and B-cell reconstitution is delayed after HHCT using CD3/CD19 depleted grafts and RIC. However, T-cell reconstitution is faster compared to data published with CD34 selected grafts and myeloablative conditioning. A fast NK-cell reconstitution early after HHCT was observed. Thus a combination of reduced intensity conditioning with CD3/CD19 depleted grafts appears to accelerate the immune recovery after haploidentical stem cell transplantation.

T-cells from advanced atherosclerotic lesions recognize hHSP60 and have a restricted T-cell receptor repertoire

Atherosclerosis is a multifactorial, chronic-inflammatory disease for which the underlying cause remains unknown. It is also well documented that T-cells are among the first cells to migrate into the arterial intimal vessel layer, but their function there is still unexplained. Clinical and experimental data have provided evidence that atherosclerosis starts as an autoimmune reaction based on humoral and cellular immunity against a phylogenetically highly conserved stress protein, heat shock protein 60 (HSP60). In the present study, we phenotypically characterized T-cells from endarterectomized specimens of the carotid artery, and tested their reactivity to human HSP60. In addition, the T-cell receptor repertoire of the T-cell lines was defined by immunoscope analysis. We found a mixed population of CD4 + and CD8 + intralesional T-cells, with a slight predominance of CD8 + cells. IFN-γ production prevailed over IL-4 production. The T-cell reaction against human HSP60 was significantly increased in intralesional cells compared to peripheral T-cells. The lesion-derived T-cells showed an oligoclonally-restricted repertoire, in contrast to the polyclonal pattern of PBMC. These results clearly show that HSP60 is a major antigenic candidate, and that an oligoclonal T-cell expansion takes place in advanced human atherosclerotic lesions.

TAP1–/– mice present oligoclonal BV‐BJ expansions following the rejection of grafts bearing self antigens

Our previous work showed that transporter associated with antigen processing 1 (TAP1)-/- (H-2b) mice rejected grafts from H-2b mice which display a normal density of class I major histocompatibility complex (MHC) molecules at the cell surface. Our results indicated that H-2b molecules themselves may be a target in this kind of rejection and that CD4+ T cells play a major role in this autoreactive process. Our data also suggested that TAP1-/- mice, in addition to the well-recognized phenotype of class I and CD8+ T-cell deficiency, present a functional alteration in their autoreactive CD4+ T-cell repertoires. In this model of inflammatory autoreactivity to modified self, we have analysed T-cell receptor (TCR) V-beta-J-beta (BV-BJ) usage by complementarity determining region 3 (CDR3) length spectratyping in splenocytes from naïve TAP1-/- mice and transplanted TAP1-/- mice that rejected B6 heart grafts or responded to synthetic self H-2Kb peptides. Importantly, oligoclonal T-cell expansions shared by different animals were detected in the peripheral T-cell repertoire of transplanted TAP1-/- mice. Such public expansions were also induced in vitro by H-2Kb peptides, suggesting that dominant class I peptides can induce preferential expansions of restricted T-cell populations during rejection. Some of these public T-cell expansions were also detected in transplanted mice even before in vitro stimulation with peptides, indicating that post-transplantation expansion of these populations had occurred in vivo. The functional activity of these T-cell populations awaits elucidation, as do the underlying mechanisms involved in the inflammatory autoreactive process, in TAP1-/- mice.

Shared blood and muscle CD8+ T-cell expansions in inclusion body myositis

Inclusion body myositis (IBM) is the most frequent inflammatory myopathy over the age of fifty. Pathological findings suggest that two processes may contribute to IBM pathogenesis: a primary degenerative process affecting muscle fibre and/or an autoimmune process mediated by major histocompatibility complex (MHC) class-I-restricted cytotoxic CD8+ T cells. Previous studies have demonstrated that muscle-infiltrating CD8+ T cells in IBM display restricted expression of T-cell receptor (TCR)-BV families or evidenced oligoclonal T-cell expansions. This study was performed to investigate whether blood T cells similarly exhibit clonal expansions due to the recirculation of muscle-infiltrating T cells in the periphery. For this, we studied the T-cell repertoire of 17 IBM patients by complementarity-determining-region (CDR) 3 length distribution (immunoscope) analysis of TCR-B transcripts. Mean age was 68 years (range 53-88) and mean duration of the disease was 6.5 years (2-20). Oligoclonal T-cell expansions were observed in the blood of IBM patients. The quantitative average perturbation D index was significantly increased in IBM patients [D = 13.7% +/- 1.2%, mean +/- standard error of measurement (SEM)] as compared with 17 age-matched controls suffering from connective tissue diseases not associated with T-cell repertoire perturbation, that is, dermatomyositis (DM) and systemic sclerosis (9.3 +/- 0.6%, P < 0.005). Nevertheless, there was no correlation between the level of blood perturbation and muscle inflammation. Sorting experiments showed that these perturbations were due to oligoclonal expansions of CD8+ T cells. In the three IBM patients analysed, we could relate the blood expansions to T-cell clones also found in muscle. The clonally expanded blood T cells dramatically responded to interleukin-2 (IL-2) in vitro, suggesting that they had been primed in vivo, presumably in response to yet unknown muscle auto-antigens. Together, our results indicate that clonally expanded muscle-infiltrating CD8+ T cells re-circulate in the blood and support the concept of a CD8+ T-cell-mediated autoimmune component in IBM, similarly to what is observed in polymyositis (PM).