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T and B lymphocyte subsets have been not univocally associated to Graft-versus-host disease (GVHD) and relapse of hematological malignancies after stem cell transplantation (SCT). Their sequential assessment together with B and T cell neogenesis indexes has been not thoroughly analysed in relation to these changing and interrelated immunologic/clinic events yet. Lymphocyte subsets in peripheral blood (PB) and B and T cell neogenesis indexes were analysed together at different time points in a prospective study of 50 patients. Principal component analysis (PCA) was used as first step of multivariate analysis to address issues related to a high number of variables versus a relatively low number of patients. Multivariate analysis was completed by Fine-Gray proportional hazard regression model. PCA identified 3 clusters of variables (PC1-3), which correlated with acute GVHD: PC1 (pre-SCT: KRECs≥6608/ml, unswitched memory B 44%, CD8+TCM cells>4%; HR 1.9, p = 0.01), and PC3 (at aGVHD onset: CD4+TEMRA69%, switched memory CD19+ = 0 cells and KRECs

Herein we dissect mechanisms behind the dissemination of cancer cells from primary tumor site to the bone marrow, which are necessary for metastasis development, with a specific focus on multiple myeloma. The ability of tumor cells to invade vessels and reach the systemic circulation is a fundamental process for metastasis development; however, the interaction between clonal cells and the surrounding microenvironment is equally important for supporting colonization, survival, and growth in the secondary sites of dissemination. The intrinsic propensity of tumor cells to recognize a favorable milieu where to establish secondary growth is the basis of the “seed and soil” theory. This theory assumes that certain tumor cells (the “seeds”) have a specific affinity for the milieu of certain organs (the “soil”). Recent literature has highlighted the important contributions of the vascular niche to the hospitable “soil” within the bone marrow. In this review, we discuss the crucial role of stromal cells and endothelial cells in supporting primary growth, homing, and metastasis to the bone marrow, in the context of multiple myeloma, a plasma cell malignancy with the unique propensity to primarily grow and metastasize to the bone marrow.

A major problem in the field of stem cell transplantation is the difficulty to monitor the efficacy of immune reconstitution. By modifying the widely used method of measuring T-cell receptor excision circles (TRECs) and the recently proposed kappa-deleting recombination excision circles (KRECs) assay, we set up a duplex Real-Time PCR that allowed the simultaneous quantification of newly produced T and B cells in children with primary immunodeficiency undergone to transplantation. We found that lymphocyte recovery involves the mobilization of both new T and B cells from production and maturation sites, and that the increase of TRECs and KRECs can be or strictly associated or independent one from the other. Some patients showed a "lymphocyte rebound" which is followed by a progressive decrease of newly produced T and B lymphocytes starting about 2years after transplantation. In other patients, TRECs and KRECs number remained very low for the entire period of study.

The lack of adenosine deaminase (ADA) leads to the accumulation of toxic metabolites, resulting in SCID. If the disease is left untreated, it is likely to have a fatal outcome in early infancy. Because hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy with pegylated bovine ADA (PEG-ADA) are both provided in our hospital, we undertook a retrospective longitudinal comparative study of the extent of lymphocyte recovery in two groups of treated ADA-SCID children. Together with classical immunological parameters, we quantified the output of the new B and T cells from the production sites using the κ-deleting recombination excision circle and TCR excision circle assay, and we monitored T cell repertoire diversification. We found that immune reconstitution was different following the two treatments. The stable production of κ-deleting recombination excision circle+ lymphocytes sustained an increase in B cell number in HSCT-treated patients, whereas in PEG-ADA–treated patients, it was accompanied by a significant and progressive decrease in circulating CD19+ lymphocytes, which never reached the levels observed in age-matched children. The mobilization of TCR excision circle+ cells, though lower than in controls, was stable with time after HSCT treatment, leading to a constant peripheral T cell number and to the diversification of the T cell repertoire; however, it was compromised in children receiving prolonged PEG-ADA therapy, whose T cells showed progressively narrowing T cell repertoires.

Background: The immune defects occurring in chronic lymphocytic leukemia are responsible for the frequent occurrence of infections and autoimmune phenomena, and may be involved in the initiation and maintenance of the malignant clone. Here, we evaluated the quantitative defects of newly produced B and T lymphocytes. Methods: The output of B and T lymphocytes from the production and maturation sites was analyzed in chronic lymphocytic leukemia patients and healthy controls by quantifying kappa-deleting recombination excision circles (KRECs) and T-cell receptor excision circles (TRECs) by a Real-Time PCR assay that simultaneously detects both targets. T-lymphocyte subsets were analyzed by six-color flow cytometric analysis. Data comparison was performed by two-sided Mann-Whitney test. Results: KRECs level was reduced in untreated chronic lymphocytic leukemia patients studied at the very early stage of the disease, whereas the release of TRECs + cells was preserved. Furthermore, the observed increase of CD4 + lymphocytes could be ascribed to the accumulation of CD4 + cells with effector memory phenotype. Conclusions: The decreased number of newly produced B lymphocytes in these patients is likely related to a homeostatic mechanism by which the immune system balances the abnormal B-cell expansion. This feature may precede the profound defect of humoral immunity characterizing the later stages of the disease. Background Profound defects of both humoral and cell-mediated immunity have been described in patients with chronic lymphocytic leukemia (CLL), a disease characterized by the accumulation of mature, malignant, monoclonal B lymphocytes in blood, lymph nodes, spleen, liver, and bone marrow [1]. The disease is characterized by the presence of immune defects, responsible for the frequent occurrence of infections and autoimmune phenomena, that may be involved in the initiation and maintenance of the malignant clone. The immune abnormalities include reduced immunoglobulin (Ig) levels, as well as qualitative and quantitative defects of B, T, NK cells, neutrophils, and the monocyte/

The immunomodulating activity of glatiramer acetate on T-cells of multiple sclerosis patients has only been partially clarified. The objective of this work was to investigate whether glatiramer acetate modifies thymic release of newly produced T-cells and the peripheral composition of the T-cell repertoire. T-cell receptor excision circles, thymic naive (CD4+CD45RA+CCR7 +CD31+) T helper cells, and central (CD4+CD45RA -CCR7+) and effector (CD4+CD45RA-CCR7 -) memory T-cells were evaluated in 89 untreated patients, 84 patients treated for at least 1 year, and 31 patients beginning treatment at the time of inclusion in the study and then followed-up for 12 months; controls were 81 healthy donors. The T-cell repertoire was analysed in selected samples. The percentage of thymicnaive T helper cells was diminished in untreated patients, but rose to control values in treated subjects; a decrease in central memory T-cells was also observed in treated patients. Follow-up patients could be divided into two subgroups, one showing unmodified thymicnaive T helper cells and T-cell diversity, the other in which the increased release of new T-cells was accompanied by modifications of the T-cell repertoire. Glatiramer acetate modifies the peripheral T-cell pool by activating a thymopoietic pathway of T-cell release that leads to a different setting of T-cell diversity and, likely, to a dilution of autoreactive T-cells.

Interferon-beta receptor (IFNAR) is composed of 2 subunits, IFNAR1 and IFNAR2, the latter of which is expressed as functional (IFNAR2.2), non-functional (IFNAR2.1) and soluble (IFNAR2.3) isoform. Real-Time PCR analysis of mRNA for all IFNAR components in multiple sclerosis patients naive for therapy and undergoing long-term treatment with interferon-beta shows that IFNAR1 mRNA level is lower than in healthy controls. If long-term treated patients are divided according to the production of mRNA for Myxovirus protein-A, a marker of interferon-beta bioactivity, IFNAR1 mRNA reaches the values observed in controls only in Myxovirus protein-A-induced patients. Since chronic cell stimulation by interferon-beta induces IFNAR protein down-regulation, we suggest that the increase of IFNAR1 mRNA might serve as a mechanism for counterbalancing the loss of protein receptor, enhancing, at least in this sub-group of patients, cell responsiveness to interferon-beta.

Introduction The long-term efficacy of allogeneic haematopoietic stem cell transplantation (SCT) relies primarily on the Graft-versus-tumor (GVT) effect, which partially overlaps with Graft versus Host disease (GvHD), the most common cause of morbidity and mortality in SCT. Researches on GVHD-biomarkers are still ongoing and a set of validate markers are still lacking, especially for chronic GVHD. Furthermore, immune parameters that univocally associate with GVHD or GVT have not been identified yet. In this study, lymphocyte subsets together with TCR-repertoire analysis, and index of thymic and bone marrow output were evaluated at different time points, in order to identify possible predictors of GVHD and ineffective GVT. Methods Prospective evaluations of lymphocyte subsets, thymic and bone marrow output were performed in 40 patients before SCT, at 30, 90, 180 days and 1 year after SCT. CD4+/CD8+ naïve, central memory, effector memory, terminally differentiated effector memory (TEMRA) cells, subsets of regulatory T-lymphocytes, immature B cells, naïve, switched and unswitched memory B cells, memory double negative (IgD-CD27-) B cells were analysed by flow cytometry. Analysis of thymic and bone marrow output was performed by detection of T cell receptor excision circles (TRECs) and kappa-deleting recombination circles (KRECs). TRECs and KRECs were simultaneously quantified by a duplex quantitative Real-Time PCR. Heteroduplex assay was used to perform TCR-repertoire analysis. A 2-step multivariate analysis was performed using principal component analysis (PCA) and Cox regression analysis, to solve the problem of the high number of variables (immunological, patients- and transplant related) in comparison with the relatively limited and heterogeneous pool of patients. Results Twenty patients developed acute GVHD (median time: 28 days, range 19-120). Chronic GVHD was observed in 9 patients (median time: 6 months, range 4-10). In multivariate analysis, acute GVHD correlated positively with pre-transplant percentage of CD4+ central memory cells, and with values of regulatory effector memory T-cells and CD4+TEMRA cell at day +30 (p=0,0006). Pre-transplant percentage of unswitched memory B cells was also associated with acute GVHD, whereas pre-transplant levels of KRECs were inversely correlated (p=0,0005). Chronic GVHD was associated with matched unrelated donor and with (p -values of regulatory effector memory T-cells at +30, percentage of CD8+TEMRA cells at +90, values of immature B cells and levels of KRECs at +180 (positive correlation) -percentage of CD4+ central memory and CD8+ effector memory cells at +90 (negative correlation). The relapse rate (27%; median time: 5,5 months, range 3-12) was used as clinical index of ineffective GVT. The following cluster of immunological parameters at day +90 correlated positively with relapse: CD8+ effector memory cells, immature B cells, naïve, switched memory B cells, memory double negative (IgD-CD27-) B cells (p=0,006). Discussion Different clusters of immunological parameters at different time points were evidenced as predictors of GVHD and ineffective GVT, allowing a clear-cut distinction between these immunological reactions. Changes in pre- and post-transplant B-lymphopoietic microenvironment and specific imbalances in the subset of B-cells may be involved in acute and chronic GVHD development. The atypical association of regulatory T-cells with GVHD may be explained by the relative efficiency of different subsets of regulatory T-cells (naïve>effector memory), as shown in some experimental models. Increased values of CD8+ effector memory cells could be an early sign of ineffective GVL. Imbalance toward a lymphocyte B-response, and especially toward "senescent" memory (IgD-CD27-) B cells, could promote tolerance to tumor cells. The validation of these clusters of immunological parameters as specific early predictors of GVHD or GVT, even before SCT, could potentially allow the development of pre-emptive and targeted therapies. Disclosures No relevant conflicts of interest to declare.

Introduction Phosphoinositide-3-kinases (PI3Ks) are pivotal in various cellular functions including proliferation and survival, cell differentiation, intracellular trafficking and immunity. The PI3Kδ isoform is highly expressed in cells of hematopoietic origin, and is often dysregulated in various hematologic malignancies. New drugs targeting PI3Ks have emerged as promising treatment options for patients (pts) with CLL and indolent NHL, but a clear understanding of their effects on pts' immunity is lacking. Herein we performed detailed immunological analysis in two follicular lymphoma (FL) pts treated with PI3K inhibitors and developing severe immune-mediated side effects. Patients and Methods Two of 9 pts with relapsed FL treated with PI3Kδ (pt1) and PI3Kα/δ (pt2) inhibitors developed severe adverse events (SAE). Both pts were heavily pretreated including autologous transplantation and discontinued therapy due to SAE. Pt1 showed grade 3 transaminase elevation, esophagitis and diarrhea requiring hospitalization after 3 cycles of therapy. Diarrhea worsened with fever and weight loss, in spite of transient discontinuation and dose reduction of PI3k inhibitor. Colonoscopic biopsy showed GVHD-like colitis. He died 3 months later. Pt2 has developed, after the first cycle and over a period of one year, febrile neutropenia, erythroderma, ulcerative keratitis requiring corneal transplant, pure red cell aplasia, endocrine abnormalities, grade 3 diarrhea with weight loss, and inflammatory bowel disease-like colitis. Detailed T-cell subpopulations immunophenotyping and T-cell receptor (TCR) spectratyping were performed in samples obtained at 6 months and 1 year after the start of treatment. Results Compared to healthy controls (HC) both pts showed marked abnormalities in T-cell subsets including a very low percentage of CD4+CD45RA+CCR7+CD31+ recent T-emigrants (RTE) and of CD4+CD45RA+CCR7+ naïve T cells in both CD4+ and CD8+ populations; CD4+CD45RA-CCR7+ central memory were significantly decreased, while CD4+CD45RA-CCR7- effector memory T cells were increased. Natural CD4+CD25+CD127low/- regulatory T cell (Treg) were low in both pts and the defect equally involved naïve, central and effector memory Treg. On the contrary, inducible Treg (Tr1) did not differ between FL pts and HC. The predominance of effector T cells strongly biased the TCR repertoire. Cells expressing all TCR beta variable (BV) chains were clonally or oligoclonally expanded at the spectratyping analysis because their profiles deviated from the Gaussian patterns found in polyclonal repertoires of HC. Discussion The clinical picture of the two FL pts was reminiscent of congenital immunodeficiencies with defects of Treg activity (e.g. IPEX caused by FoxP3 gene mutation). Similar symptoms and T-cell abnormalities are also present in pts with inadequate thymic function and immunologic reconstitution due to severe chronic GVHD. The associated T-cell immunological derangement was characterized by: 1) a marked reduction of the thymic output; 2) a clear defect in natural Treg; 3) an increase of peripheral oligoclonal effector memory T cells. Preclinical data on the role of PI3kδ support its possible pathogenetic involvement: PI3Kδ is known to play critical roles during thymopoiesis and its deletion/inactivation results in a block at the CD4 CD8 double negative stage of T-cell development. In mice with a mutation of PI3Kδ subunit, Treg were increased in the thymus but decreased in all peripheral immune organs, suggesting an impaired emigration of these cells from the thymus. Moreover, mice expressing an inactive form of PI3Kδ develop inflammatory bowel disease. These preliminary data suggest that small molecule-mediated inhibition of PI3Kδ may cause severe alterations within the T-cell compartment in selected pts, which may derive from a defect in the thymic output, including that of Treg. The generation of mature, oligoclonal T cells can be either the result of an homeostatic T-cell proliferation or of a strong (auto)antigenic stimulation. To better elucidate such mechanisms, further studies are in progress on a larger number of pts showing different levels of PI3k inhibitor toxicity. Nevertheless, we suggest that a close follow-up of lymphocyte subpopulation changes should be worthwhile since it may anticipate the onset of these severe clinical manifestations. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Background. One of the major concern for high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT) for HIV-associated lymphoma is that posttransplant immunosuppression might worsen immune defects of HIV+ individuals. Since the introduction of highly active antiretroviral therapy has made HSCT possible also in these patients, we analyzed whether the immune system already compromised by HIV infection might support an efficient T-cell recovery after HSCT. Methods. The kinetics and the extent of T-cell reconstitution were investigated before and after HSCT in four patients with HIV-related lymphoma (one with Hodgkin's Disease and three with non-Hodgkin's lymphoma) by measuring the thymic output, the level of IL-7 and the heterogeneity of T-cell repertoire. T-cell competence was gauged at two functional levels: by determining the number of T-cell divisions and by measuring IFN-γ production. Results. The thymus of transplanted patients can be capable of generating new T cells, but there is no relationship between increasing number of newly produced lymphocytes and modification of IL-7 level. Various T-cell subsets, expressing different T-cell receptor variable beta genes, were preferentially expanded in CD8 population and most of them showed a restricted diversity. Furthermore, CD3+ lymphocytes showed heterogeneous behaviors in terms of proliferative capability and IFN-γ production. Conclusions. High-dose therapy and HSCT in HIV+ patients under highly active antiretroviral therapy does not worsen the immune defects. On the contrary, in the presence of some conditions (including the type of hematologic malignancy, the therapy compliance, and the immune status before transplantation), high-dose therapy and HSCT might support the improvement of immune conditions.

Abstract 2353 Poster Board II-330 Background Chronic lymphocytic leukemia (CLL) results in accumulation of mature, malignant, monoclonal B cells in blood, lymph nodes, spleen, liver, and bone marrow. Patients with CLL have fundamental defects in both humoral and cell-mediated immunity that significantly impact on its clinical course. It is still not known if these immune abnormalities include a decrease of new B- and T-cell mobilization from their production sites. During early lymphocyte development and differentiation, occurring in bone marrow and in thymus, B- and T-cells undergo rearrangements of B-cell and T-cell receptor genes whereby specific chromosomal sequences are excised to produce episomal DNA products identified respectively as T-cell receptor excisional circles (TRECs) and Kappa-deleting recombination excision circles (KRECs). Being stable circular fragments of DNA, TRECs and KRECs do not replicate during the mitotic process required for cell proliferation and, therefore, they are diluted out by cell divisions and are lost when the cells die. Since T-cells leaving the thymus are 70% TRECs+ and KRECs are present randomly in about 50% of neo-produced normal B-cells, the quantification of TRECs and KRECs allows a good estimation of the thymic and bone marrow output. Aim To investigate the extent of neo-synthesis of normal B and T cells in untreated patients with CLL. Patients and Methods Twelve previously untreated CLL patients were enrolled in this study. M:F ratio was 5:1; median age was 66; 7 patients had mutated IgVH genes, while 5 patients had unmutated IgVH genes; all patients were stage A Binet; FISH analysis including del(13q), del(11q), del(17p) and trisomy 12 detected 13q deletion in 6 patients while the other 6 patients presented no abnormalities. TRECs and KRECs were measured in mononuclear cells isolated from peripheral blood by duplex quantitative Real-Time PCR. This new assay, based on the use of a standard curve prepared with a plasmid containing fragments of TRECs, KRECs and of a reference gene, allows to quantify neo-produced B and T lymphocytes. T-cell subpopulations were determined by flow cytometry as follows: recent thymic emigrants (RTE) as cells with CD4+CD45RA+CCR7+CD31+ phenotype, regulatory T cells (Treg), as CD4+CD25int/highCD127low, RTE-Treg as Treg expressing CCR7+CD31+CD25int/high markers, effector memory (TEM) and central memory (TCM) T cells as lymphocytes displaying CD4+CD45RA-CCR7- and CD4+CD45RA-CCR7+ phenotype. Results The overall number of CD4+ lymphocytes was increased in chemotherapy-naïve patients with CLL (1585/μl vs 953/μl; p=0.02). TRECs were significantly higher in CLL patients compared to age-matched healthy controls (2.9/ml vs 1.4/ml; p=0.04), while the proportion of RTE was lower (24.9% vs 32.4%; p=0.02). No significant differences were observed in the percentage of Treg and RTE-Treg as well as of TCM. On the contrary, the percentage of TEM was higher (22.4% vs 10.4%; p=0.01) in CLL patients. The number of KRECs was lower in CLL patients than in controls (3.8/ml vs 7.4/ml; p=0.004). No correlation between IgVH gene mutational status, Ig levels and KRECs was found. Conclusions The neo-synthesis of normal T, with the exception of Treg, and B cells is reduced in patients with CLL compared to controls, even in a good prognosis, chemotherapy-naïve subset. The increased number of TRECs+ cells and TEM together with low RTE could be ascribed to the increased number of circulating CD4+ lymphocytes which do not appear to be recently mobilized from the thymus (CD31- cells) or to undergo peripheral expansion, but to accumulate in the peripheral blood. The low number of KRECs compared to normal controls could be interpreted as a consequence of the abnormal leukemic B-cell expansion which may impair normal B cell neo-synthesis. The level of KRECs did not seem to correlate to the mutational status of IgVH genes or the Ig level but a larger number of patients is needed to confirm these preliminary data and to establish whether the analysis of TRECs and KRECs may help clarify the complex immune abnormalities in CLL. Disclosures: No relevant conflicts of interest to declare.