Person:

Preffer, Frederic

Acute undiffentiated leukemia (AUL) is an acute leukemia with no more than one membrane marker of any given lineage. Blasts often express HLA-DR, CD34, and/or CD38 and may be positive for terminal deoxynucleotidyl transferase (TdT). The expression of CD34, HLA-DR, and CD38 has been shown in pro-T-ALL, although in this case, blasts should also express CD7 and cyCD3. However, some cases of T-ALL without CD3 in the cytoplasm and all TCR chain genes in germ line configuration are reported, features that fit well with a very early hematopoietic cell. We report a case of acute leukemia CD34+/−HLADR+CD7+CD38+cyCD3− in which a diagnosis of AUL was considered. However the blasts were also positive for CD99 and TCR delta gene rearrangement which was found on molecular studies. Therefore a differential diagnosis between AUL and an early cyCD3 negative T-ALL was debated.

Five patients with end-stage renal disease received combined bone marrow and kidney transplants from HLA single-haplotype mismatched living related donors, with the use of a nonmyeloablative preparative regimen. Transient chimerism and reversible capillary leak syndrome developed in all recipients. Irreversible humoral rejection occurred in one patient. In the other four recipients, it was possible to discontinue all immunosuppressive therapy 9 to 14 months after the transplantation, and renal function has remained stable for 2.0 to 5.3 years since transplantation. The T cells from these four recipients, tested in vitro, showed donor-specific unresponsiveness and in specimens from allograft biopsies, obtained after withdrawal of immunosuppressive therapy, there were high levels of P3 (FOXP3) messenger RNA (mRNA) but not granzyme B mRNA.

The zinc finger transcriptional repressor Gfi-1b is essential for erythroid and megakaryocytic development in the embryo. Its roles in the maintenance of bone marrow erythropoiesis and thrombopoiesis have not been defined. We investigated Gfi-1b’s adult functions using a loxP-flanked Gfi-1b allele in combination with a novel doxycycline-inducible Cre transgene that efficiently mediates recombination in the bone marrow. We reveal strict, lineage-intrinsic requirements for continuous adult Gfi-1b expression at two distinct critical stages of erythropoiesis and megakaryopoiesis. Induced disruption of Gfi-1b was lethal within 3 wk with severely reduced hemoglobin levels and platelet counts. The erythroid lineage was arrested early in bipotential progenitors, which did not give rise to mature erythroid cells in vitro or in vivo. Yet Gfi-1b−/− progenitors had initiated the erythroid program as they expressed many lineage-restricted genes, including Klf1/Eklf and Erythropoietin receptor. In contrast, the megakaryocytic lineage developed beyond the progenitor stage in Gfi-1b’s absence and was arrested at the promegakaryocyte stage, after nuclear polyploidization, but before cytoplasmic maturation. Genome-wide analyses revealed that Gfi-1b directly regulates a wide spectrum of megakaryocytic and erythroid genes, predominantly repressing their expression. Together our study establishes Gfi-1b as a master transcriptional repressor of adult erythropoiesis and thrombopoiesis.

Immune responses are modified by a diverse and abundant repertoire of carbohydrate structures on the cell surface, which is known as the glycome. In this study, we propose that a unique glycome that can be identified through the binding of galectin-4 is created on local, but not systemic, memory CD4+ T cells under diverse intestinal inflammatory conditions, but not in the healthy state. The colitis-associated glycome (CAG) represents an immature core 1–expressing O-glycan. Development of CAG may be mediated by down-regulation of the expression of core-2 β1,6-N-acetylglucosaminyltransferase (C2GnT) 1, a key enzyme responsible for the production of core-2 O-glycan branch through addition of N-acetylglucosamine (GlcNAc) to a core-1 O-glycan structure. Mechanistically, the CAG seems to contribute to super raft formation associated with the immunological synapse on colonic memory CD4+ T cells and to the consequent stabilization of protein kinase C θ activation, resulting in the stimulation of memory CD4+ T cell expansion in the inflamed intestine. Functionally, CAG-mediated CD4+ T cell expansion contributes to the exacerbation of T cell–mediated experimental intestinal inflammations. Therefore, the CAG may be an attractive therapeutic target to specifically suppress the expansion of effector memory CD4+ T cells in intestinal inflammation such as that seen in inflammatory bowel disease.

An idiopathic capillary leak syndrome (“engraftment syndrome”) often occurs in recipients of hematopoietic cells, manifested clinically by transient azotemia and sometimes fever and fluid retention. Here we report the renal pathology in 10 recipients of combined bone marrow and kidney allografts. Nine developed graft dysfunction on day 10–16 and renal biopsies showed marked acute tubular injury, with interstitial edema, hemorrhage and capillary congestion, with little or no interstitial infiltrate (≤10%) and marked glomerular and peritubular capillary (PTC) endothelial injury and loss by electron microscopy. Two had transient arterial endothelial inflammation; and 2 had C4d deposition. The cells in capillaries were primarily CD68+MPO+ mononuclear cells and CD3+CD8+ T cells, the latter with a high proliferative index (Ki67+). B cells (CD20+) and CD4+ T cells were not detectable, and NK cells were rare. XY FISH showed that CD45+ cells in PTCs were of recipient origin. Optimal treatment remains to be defined; two recovered without additional therapy, six were treated with anti-rejection regimens. Except for one patient, who later developed thrombotic microangiopathy and one with acute humoral rejection, all fully recovered within 2–4 weeks. Graft endothelium is the primary target of this process, attributable to as yet obscure mechanisms, arising during leukocyte recovery.

Background

We have previously reported operational tolerance in patients receiving HLA-mismatched combined kidney and bone marrow transplantation (CKBMT). We now report on transient multilineage hematopoietic chimerism and lymphocyte recovery in five patients receiving a modified CKBMT protocol, and evidence for early donor-specific unresponsiveness in one of these patients.

Methods

Five patients with end-stage renal disease received CKBMT from HLA-mismatched, haploidentical living related donors following modified non-myeloablative conditioning. Polychromatic flow cytometry (FCM) was used to assess multilineage chimerism where evaluable and lymphocyte recovery post-transplant. Limiting dilution analysis was used to assess helper-T-lymphocyte reactivity to donor antigens.

Results

Transient multilineage mixed chimerism was observed in all patients but chimerism became undetectable by 2 weeks post-CKBMT. A marked decrease in T and B lymphocyte counts immediately following transplant was followed by gradual recovery. Initially recovering T cells were depleted of CD45RA+/CD45RO− “naïve-like” cells, which have shown strong recovery in two patients and CD4/CD8 ratios increased immediately following transplant but then declined markedly. NK cells were enriched in the peripheral blood of all patients following transplant.

For Subject 2, a pre-transplant limiting dilution assay revealed T helper cells recognizing both donor and third-party PBMCs. However, the anti-donor response was completely undetectable by Day 24, while third-party reactivity persisted.

Conclusion

These results characterize the transient multilineage mixed hematopoietic chimerism and recovery of lymphocyte subsets in patients receiving a modified CKBMT protocol. The observations are relevant to the mechanisms of donor-specific tolerance in this patient group.

Women with late-stage ovarian cancer usually develop chemotherapeutic-resistant recurrence. It has been theorized that a rare cancer stem cell, which is responsible for the growth and maintenance of the tumor, is also resistant to conventional chemotherapeutics. We have isolated from multiple ovarian cancer cell lines an ovarian cancer stem cell-enriched population marked by CD44, CD24, and Epcam (3+) and by negative selection for Ecadherin (Ecad−) that comprises less than 1% of cancer cells and has increased colony formation and shorter tumor-free intervals in vivo after limiting dilution. Surprisingly, these cells are not only resistant to chemotherapeutics such as doxorubicin, but also are stimulated by it, as evidenced by the significantly increased number of colonies in treated 3+Ecad− cells. Similarly, proliferation of the 3+Ecad− cells in monolayer increased with treatment, by either doxorubicin or cisplatin, compared with the unseparated or cancer stem cell-depleted 3−Ecad+ cells. However, these cells are sensitive to Mullerian inhibiting substance (MIS), which decreased colony formation. MIS inhibits ovarian cancer cells by inducing G1 arrest of the 3+Ecad− subpopulation through the induction of cyclin-dependent kinase inhibitors. 3+Ecad− cells selectively expressed LIN28, which colocalized by immunofluorescence with the 3+ cancer stem cell markers in the human ovarian carcinoma cell line, OVCAR-5, and is also highly expressed in transgenic murine models of ovarian cancer and in other human ovarian cancer cell lines. These results suggest that chemotherapeutics may be stimulative to cancer stem cells and that selective inhibition of these cells by treating with MIS or targeting LIN28 should be considered in the development of therapeutics.

Multiple malignancies may occur in the same patient, and a few reports describe cases with multiple hematologic and non-hematologic neoplasms. We report the case of a patient who showed the sequential occurrence of four different lymphoid neoplasms together with a squamous cell carcinoma of the lung. A 62-year-old man with adenopathy was admitted to the hospital, and lymph node biopsy was positive for low-grade follicular lymphoma. He achieved a partial remission with chemotherapy. Two years later, a PET-CT scan showed a left hilar mass in the lung; biopsy showed a squamous cell carcinoma. Simultaneously, he was diagnosed with diffuse large B cell lymphoma in a neck lymph node; after chemo- and radiotherapy, he achieved a complete response. A restaging PET-CT scan 2 years later revealed a retroperitoneal nodule, and biopsy again showed a low-grade follicular lymphoma, while a biopsy of a cutaneous scalp lesion showed a CD30-positive peripheral T cell lymphoma. After some months, a liver biopsy and a right cervical lymph node biopsy showed a CD30-positive peripheral T cell lymphoma consistent with anaplastic lymphoma kinase-negative anaplastic large cell lymphoma. Flow cytometry and cytogenetic and molecular genetic analysis performed at diagnosis and during the patient’s follow-up confirmed the presence of two clonally distinct B cell lymphomas, while the two T cell neoplasms were confirmed to be clonally related. We discuss the relationship between multiple neoplasms occurring in the same patient and the various possible risk factors involved in their development.

Flow cytometry and cell sorting are critical tools in stem cell research. Recent advances in flow cytometric hardware, reagents, and software have synergized to permit the stem cell biologist to more full identify and isolate rare cells based on their immunofluorescent and light scatter characteristics. Some of these improvements include physically smaller air-cooled lasers, new designs in optics, new fluorescent conjugate-excitation pairs, and improved software to visualize data, all which combine to open up new horizons in the study of stem cells, by enhancing the resolution and specificity of inquiry. In this review, these recent improvements in technology will be outlined and important cell surface and functional antigenic markers useful for the study of stem cells described.

Cancer stem cells are proposed to be tumor-initiating cells capable of tumorigenesis, recurrence, metastasis, and drug resistance, and, like somatic stem cells, are thought to be capable of unlimited self-renewal and, when stimulated, proliferation and differentiation. Here we select cells by expression of a panel of markers to enrich for a population with stem cell-like characteristics. A panel of eight was initially selected from 95 human cell surface antigens as each was shared among human ovarian primary cancers, ovarian cancer cell lines, and normal fimbria. A total of 150 combinations of markers were reduced to a panel of three—CD44, CD24, and Epcam—which selected, in three ovarian cancer cell lines, those cells which best formed colonies. Cells expressing CD44, CD24, and Epcam exhibited stem cell characteristics of shorter tumor-free intervals in vivo after limiting dilution, and enhanced migration in invasion assays in vitro. Also, doxorubicin, cisplatin, and paclitaxel increased this enriched population which, conversely, was significantly inhibited by Müllerian inhibiting substance (MIS) or the MIS mimetic SP600125. These findings demonstrate that flow cytometry can be used to detect a population which shows differential drug sensitivity, and imply that treatment of patients can be individualized to target both stem/progenitor cell enriched and nonenriched subpopulations. The findings also suggest that this population, amenable to isolation by flow cytometry, can be used to screen for novel treatment paradigms, including biologic agents such as MIS, which will improve outcomes for patients with ovarian cancer.