Therapeutic targeting of CK2 in acute and chronic leukemias.

CK2 is a ubiquitously expressed, constitutively active Ser/Thr protein kinase, which is considered the most pleiotropic protein kinase in the human kinome. Such a pleiotropy explains the involvement of CK2 in many cellular events. However, its predominant roles are stimulation of cell growth and prevention of apoptosis. High levels of CK2 messenger RNA and protein are associated with CK2 pathological functions in human cancers. Over the last decade, basic and translational studies have provided evidence of CK2 as a pivotal molecule driving the growth of different blood malignancies. CK2 overexpression has been demonstrated in nearly all the types of hematological cancers, including acute and chronic leukemias, where CK2 is a key regulator of signaling networks critical for cell proliferation, survival and drug resistance. The findings that emerged from these studies suggest that CK2 could be a valuable therapeutic target in leukemias and supported the initiation of clinical trials using CK2 antagonists. In this review, we summarize the recent advances on the understanding of the signaling pathways involved in CK2 inhibition-mediated effects with a particular emphasis on the combinatorial use of CK2 inhibitors as novel therapeutic strategies for treating both acute and chronic leukemia patients.

Therapeutic potential of targeting sphingosine kinases and sphingosine 1-phosphate in hematological malignancies.

Sphingolipids, such as ceramide, sphingosine and sphingosine 1-phosphate (S1P) are bioactive molecules that have important functions in a variety of cellular processes, which include proliferation, survival, differentiation and cellular responses to stress. Sphingolipids have a major impact on the determination of cell fate by contributing to either cell survival or death. Although ceramide and sphingosine are usually considered to induce cell death, S1P promotes survival of cells. Sphingosine kinases (SPHKs) are the enzymes that catalyze the conversion of sphingosine to S1P. There are two isoforms, SPHK1 and SPHK2, which are encoded by different genes. SPHK1 has recently been implicated in contributing to cell transformation, tumor angiogenesis and metastatic spread, as well as cancer cell multidrug-resistance. More recent findings suggest that SPHK2 also has a role in cancer progression. This review is an overview of our understanding of the role of SPHKs and S1P in hematopoietic malignancies and provides information on the current status of SPHK inhibitors with respect to their therapeutic potential in the treatment of hematological cancers.

Cytotoxic activity of the casein kinase 2 inhibitor CX-4945 against T-cell acute lymphoblastic leukemia: targeting the unfolded protein response signaling.

Constitutively active casein kinase 2 (CK2) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL). CK2 phosphorylates PTEN (phosphatase and tensin homolog) tumor suppressor, resulting in PTEN stabilization and functional inactivation. Downregulation of PTEN activity has an impact on PI3K/Akt/mTOR signaling, which is of fundamental importance for T-ALL cell survival. These observations lend compelling weight to the application of CK2 inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of CX-4945-a novel, highly specific, orally available, ATP-competitive inhibitor of CK2α. We show that CX-4945 treatment induced apoptosis in T-ALL cell lines and patient T lymphoblasts. CX-4945 downregulated PI3K/Akt/mTOR signaling in leukemic cells. Notably, CX-4945 affected the unfolded protein response (UPR), as demonstrated by a significant decrease in the levels of the main UPR regulator GRP78/BIP, and led to apoptosis via upregulation of the ER stress/UPR cell death mediators IRE1α and CHOP. In vivo administration of CX-4945 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth. Our findings indicate that modulation of the ER stress/UPR signaling through CK2 inhibition could be exploited for inducing apoptosis in T-ALL cells and that CX-4945 may be an efficient treatment for those T-ALLs displaying upregulation of CK2α/PI3K/Akt/mTOR signaling.

Activity of the pan-class I phosphoinositide 3-kinase inhibitor NVP-BKM120 in T-cell acute lymphoblastic leukemia.

Constitutively active phosphoinositide 3-kinase (PI3K) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival and drug resistance. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the pan-PI3K inhibitor NVP-BKM120 (BKM120), an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in G2/M phase cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T lymphoblasts, and promoting a dose- and time-dependent dephosphorylation of Akt and S6RP. BKM120 maintained its pro-apoptotic activity against Jurkat cells even when cocultured with MS-5 stromal cells, which mimic the bone marrow microenvironment. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. Moreover, in vivo administration of BKM120 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth, thus prolonging survival time. Taken together, our findings indicate that BKM120, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment for T-ALLs that have aberrant upregulation of the PI3K signaling pathway.

Histomorphometric evaluation of implant design as a key factor in peri-implant bone response: a preliminary study in a dog model.

AIM: Primary implant stability as the establishment of a direct bone-to-implant contact (BIC) plays a major role in long-term successful implant osseointegration. Numerous factors influencing this initial stability have been studied. This preliminary in vivo study on a dog lower jaw aimed to investigate the hypothesis that primary implant stability in low density bone may be influenced by implant design. METHODS: The authors compared two different implant designs with regard to their immediate quantitative relation to host bone (BIC% and gap area, GA%). The screw-shaped implants, manufactured by Or-Vit (Castelmaggiore-Bologna, Italy), exhibited similar microroughness surface and two different thread pitches: ''narrow-pitch'' implants (NP) and ''wide-pitch'' implants (WP) with a 0.5 mm and 1.5 mm thread pitch respectively. Implants were placed in dog jaw after complete osseous healing of the extractive sockets, according to a delayed implantation procedure. Five hours after surgery the animal was sacrificed. Radiographic, histological, morphometric and ultrastructural analysis were performed. RESULTS: An inverse relation existed among the two parameters BIC and GA: GA, as a region with high osteogenetic potentiality, appeared wider in WP implants; BIC, as the expression of primary mechanical stability, was higher in NP implants. CONCLUSION: Based on this results, we could assume that NP implants might be the clinical choice in case of immediate loading.This single case study might be considered a starting point for further long term in vivo investigations aiming to establish the implant design that best favours osseointegration at different bone quality sites.

Tendon crimps and peritendinous tissues responding to tensional forces.

Tendons transmit forces generated from muscle to bone making joint movements possible. Tendon collagen has a complex supramolecular structure forming many hierarchical levels of association; its main functional unit is the collagen fibril forming fibers and fascicles. Since tendons are enclosed by loose connective sheaths in continuity with muscle sheaths, it is likely that tendon sheaths could play a role in absorbing/transmitting the forces created by muscle contraction. In this study rat Achilles tendons were passively stretched in vivo to be observed at polarized light microscope (PLM), scanning electron microscope (SEM) and transmission electron microscope (TEM). At PLM tendon collagen fibers in relaxed rat Achilles tendons ran straight and parallel, showing a periodic crimp pattern. Similarly tendon sheaths showed apparent crimps. At higher magnification SEM and TEM revealed that in each tendon crimp large and heterogeneous collagen fibrils running straight and parallel suddenly changed their direction undergoing localized and variable modifications. These fibril modifications were named fibrillar crimps. Tendon sheaths displayed small and uniform fibrils running parallel with a wavy course without any ultrastructural aspects of crimp. Since in passively stretched Achilles tendons fibrillar crimps were still observed, it is likely that during the tendon stretching, and presumably during the tendon elongation in muscle contraction, the fibrillar crimp may be the real structural component of the tendon crimp acting as shock absorber. The peritendinous sheath can be stretched as tendon, but is not actively involved in the mechanism of shock absorber as the fibrillar crimp. The different functional behaviour of tendons and sheaths may be due to the different structural and molecular arrangement of their fibrils.

Destination of titanium particles detached from titanium plasma sprayed implants.

Small titanium particles may detach from titanium plasma sprayed (TPS) implants during implant insertion, when no preliminary tapping is used, probably for the frictional force between titanium coating and host bone. Aim of this study was to investigate the destination of these titanium particles observed in the peri-implant environment. Twenty-four TPS screws were implanted in tibiae of two sheep. Fourteen and 90 days after implantation the implants with the surrounding bone were removed and processed to be analyzed by light microscope and scanning electron microscope (secondary electron and back-scattered electron probes). Small titanium particles detached from the unloaded TPS implants were observed both in the newly-formed bone matrix and in marrow tissue. Histomorphometric analysis showed that both at 14 and 90 days after implantation the titanium particles appeared more concentrated in marrow tissue than in calcified bone matrix, decreasing by 66.4% over time. In particular, smaller particles (<250 microm(2)) decreased by 81.5%, whereas the larger ones (250-2000 microm(2)) did not show any significant variations over time, suggesting that most of the smaller particles may undergo to ionic dissolution, probably migrating into the peri-implant marrow lacunae. A slight migration of titanium particles from the implant surface towards the more distant peri-implant tissues was also demonstrated over time.

Biological fixation of endosseous implants.

Primary implant stability is ensured by a mechanical fixation of implants. However, during implant healing a biological anchorage is necessary to achieve final osseointegration. Aim of this study was to investigate the histological aspects of biological fixation around titanium screws. Forty-eight titanium screws with different surfaces (smooth, plasma sprayed, sand blasted) were inserted in tibiae and femura of sheep and analyzed by light microscope and SEM 1 hour, 14 and 90 days after implantation. One hour after implantation the implant-bone gap was filled with a blood clot and host bone chips arising from burr surgical preparation or friction during implant insertion. Fourteen days after implantation new trabecular bone and enveloped bone chips were observed in the gap: no osteogenesis developed where implant threads were in contact with host bone. Ninety days after surgery all trabecular bone and most of the bone chips were substituted by a mature lamellar bone with few marrow spaces. Our results suggest that the trabecular bone and bone chips represent a three-dimensional network ensuring a biological implant fixation in all different implant surfaces 2 weeks after surgery. Host bone chips could favour the peri-implant osteogenesis. Inter-trabecular and implant-trabecular marrow spaces of both trabecular and lamellar bone may favour the peri-implant bone turnover.

Early detachment of titanium particles from various different surfaces of endosseous dental implants.

Titanium (Ti) endosseous dental screws with different surfaces (smooth titanium--STi, titanium plasma-sprayed-TPS, alumina oxide sandblasted and acid-etched--Al-SLA, zirconium oxide sandblasted and acid etched--Zr-SLA) were implanted in femura and tibiae of sheep to investigate the biological evolution of the peri-implant tissues and detachment of Ti debris from the implant surfaces in early healing. Implants were not loaded. Sections of the screws and the peri-implant tissues obtained by sawing and grinding were analysed by light microscopy immediately after implantation (time 0) and after 14 days. All samples showed new bone trabeculae and vascularised medullary spaces in those areas where gaps between the implants and host bone were visible. In contrast, no osteogenesis was induced in the areas where the implants were initially positioned in close contact with the host bone. Chips of the pre-existing bone inducing new peri-implant neo-osteogenesis were surrounded by new bone trabeculae. The threads of some screws appeared to be deformed where the host bone showed fractures. Ti granules of 3-60 microm were detectable only in the peri-implant tissues of TPS implants both immediately after surgery and after 14 days, thus suggesting that this phenomenon may be related to the friction of the TPS coating during surgical insertion.

Time-domain analysis of exercise-induced ST-segment elevation in Q-wave myocardial infarction: a useful tool for the screening of myocardial viability.

BACKGROUND: Exercise-induced ST-segment elevation in Q-wave leads has been traditionally associated with passive stretching of the infarct wall, perinecrotic ischemia and, according to recent scintigraphic studies, with myocardial viability. At present, however, no definitive conclusions are available. We evaluated the potential role of a time-domain analysis of exercise-induced ST-segment elevation for the identification of viable myocardium and residual ischemia in patients with previous Q-wave myocardial infarction. METHODS: Sixty patients with a previous Q-wave myocardial infarction underwent a bicycle exercise stress test, dobutamine stress echocardiography, coronary arteriography and left ventriculography. RESULTS: Patients with exercise-induced ST-segment elevation in Q-wave leads (n = 36) showed more severe impairment of resting left ventricular function, when evaluated in terms of wall motion score index at echocardiography (1.62 +/- 0.33 vs 1.41 +/- 0.22, p < 0.01) and in terms of wall motion score at ventriculography (5.9 +/- 1.6 vs 4.1 +/- 1.5, p < 0.03), compared to patients without ST-segment shift (n = 24). No differences between the two groups were seen in the severity and extension of coronary artery disease. The two groups of patients did not differ in the overall incidence of viability (50% in patients with vs 62% in those without ST-segment elevation, p = NS) and homozonal ischemia (39 vs 26%, p = NS), when evaluated with dobutamine echocardiography. However, a time-domain analysis of the ST-segment changes during exercise showed that the duration of exercise up to 0.1 mV ST-segment elevation was significantly lower in patients with viability (6.2 +/- 3.3 min) than in those without (10.2 +/- 2.2 min) (p < 0.001). Accordingly, ST-segment elevation occurred within 3 and 6 min of exercise in 7/18 and in 12/18 patients with viability respectively, but in only 0/18 (p < 0.01) and in 1/18 (p < 0.01) patients without viability. Thus, ST-segment elevation occurring within the first two stages of the exercise test was, respectively, 39 and 67% sensitive and 100 and 94% specific for viability. Early onset ST-segment elevation (within 3 and 6 min) was also more frequent in patients with high-dose dobutamine-induced homozonal ischemia than in those without (sensitivity for ischemia 50 and 67%; specificity 95 and 74%, respectively). CONCLUSIONS: After myocardial infarction, ST-segment elevation in Q-wave leads at the peak of exercise is associated with severe resting left ventricular dysfunction but fails to identify patients with a viable myocardium or residual ischemia. Instead, ST-segment elevation occurring in the early phases of exercise is a highly specific, although not very sensitive marker of dobutamine-assessed viability in the infarct area and may be indicative of residual ischemia.

Developmental approaches in immunological control of acute myelogenous leukaemia.

After many years of hope and disillusionment, the possibility of utilizing immune-mediated approaches to control neoplastic clones has become a reality in various haematological malignancies. This is largely a consequence of the continuous advances in knowledge and the progressive development of more refined technologies that have led to a better understanding of the biology of the malignant cells and of the host immune system, to a more precise definition of disease entities and to the design of innovative therapeutic programmes. In this chapter, we will review different immunological strategies that have reached clinical practice in patients with acute myelogenous leukaemia (AML), the focus of this volume, and discuss pre-clinical developments that may in the near future translate into the design of new immunotherapeutic protocols for the management of AML. Treatment of AML with antibody directed therapy will also be discussed.

A CD4+ T cell clone selected from a CML patient after donor lymphocyte infusion recognizes BCR-ABL breakpoint peptides but not tumor cells.

BACKGROUND: In patients with chronic myelocytic leukemia (CML), the breakpoint cluster region and fusion between the BCR and the c-ABL genes (BCR-ABL) oncogen product is a potential tumor-specific antigen. Previous studies have shown that T cells specific for the junctional region peptides of the BCR-ABL oncoprotein can be detected in healthy individuals as well as in patients with CML in chronic phase. We assessed whether BCR-ABL- specific T cells could be found in a patient achieving a complete cytogenetic remission after CD4+ donor lymphocyte infusion. METHODS: Using dendritic cells pulsed with BCR-ABL breakpoint peptides as antigen-presenting cells, we stimulated patient peripheral blood lymphocytes to isolate peptide-specific T cell clones present at the time of the cytogenetic response. T cell clones were isolated and the cellular specificity of these cells was examined. RESULTS: A CD3+ CD4+ T cell clone (1F7) that recognizes overlapping p210 junctional peptides presented by HLA-DR molecules was identified and expanded in vitro. Clone 1F7 failed to recognize autologous tumor cells as well as dendritic cells derived from patient CML cells. Clone 1F7 did not inhibit the growth and differentiation of CML precursor cells in a standard colony formation assay. Finally, using a clone-specific probe, 1F7 cells could not be detected in patient peripheral blood at the time of the donor lymphocyte infusion response. CONCLUSIONS: These results suggest that clone 1F7 was selected in vitro using highly potent peptide pulsed dendritic cells but was not representative of the anti-leukemia immune response in vivo. Based on these findings, CD4+ T cells with BCR-ABL specificity do not appear to be mediators of the anti-leukemia response in vivo after donor lymphocyte infusion.

Choroid plexus carcinomas and rhabdoid tumors: phenotypic and genotypic overlap.

Five of six poorly differentiated choroid plexus carcinomas identified at our institution contained cells displaying a rhabdoid phenotype. Immunoperoxidase stains showed focal positivity for cytokeratin, epithelial membrane antigen, glial fibrillary acidic protein, S100, and vimentin. The MIB-1 proliferative index ranged from 7.0% to 27.1%. All six tumors were p53 positive. Only the one child with Li-Fraumeni syndrome had a p53 germline mutation. Electron microscopy verified choroid plexus differentiation and the co-existence of rhabdoid cells. Of the five studied, four had deletions of chromosome 22 [three with monosomy 22 and one with del(22)(q12)]. Thus, there was a phenotypic and genotypic overlap between choroid plexus carcinomas and rhabdoid tumors.

Defective signaling to Fyn by a T cell antigen receptor lacking the alpha -chain connecting peptide motif.

A key role in the communication between the alphabetaTCR and the CD3/zeta complex is played by a specific motif within the connecting peptide domain of the TCR alpha chain (alpha-CPM). T cell hybridomas expressing an alpha-CPM-mutated TCR show a dramatic impairment in antigen-driven interleukin-2 production. This defect can be complemented by a calcium ionophore, indicating that activation of the calcium pathway is impaired. Several lines of evidence implicate Fyn in the regulation of calcium mobilization, at least in part through the activation of phospholipase Cgamma. Here we have investigated the potential involvement of Fyn in the TCR alpha-CPM signaling defect. Using T cell hybridomas expressing either a wild-type TCR or an alpha-CPM mutant, we show that Fyn fails to be activated by the mutant receptor following SEB binding and fails to generate tyrosine-phosphorylated Pyk2, a member of the focal adhesion kinase family. This defect correlated with an impairment in phospholipase Cgamma phosphorylation. Production of interlukin-2 and activation of the transcription factor NF-AT in response to triggering of the TCR alpha-CPM mutant with SEB were fully restored in the presence of constitutively active Fyn. Hence the signaling defect generated by the TCR alpha-CPM mutation results at least in part from an impaired coupling of the TCR.CD3 complex to Fyn activation.

Conversion to full donor chimerism following donor lymphocyte infusion is associated with disease response in patients with multiple myeloma.

Donor lymphocyte infusions (DLIs) have been demonstrated to induce clinical responses in patients with relapsed multiple myeloma after allogeneic bone marrow transplantation, but the immunologic mechanisms involved have not been well characterized. In patients with chronic myelocytic leukemia (CML), remissions following DLI are invariably associated with conversion to complete donor hematopoiesis, suggesting that the target antigens of this response are expressed on both normal and CML-derived hematopoietic stem cells. In the present study, we examined hematopoietic chimerism and the complexity of the T-cell receptor (TCR) repertoire in 4 patients with relapsed multiple myeloma who received infusions of donor CD4+ lymphocytes. Three of 4 patients had a clinical response that began 1 to 2 months after DLI. All 3 responding patients developed lymphocytosis at the initiation of response that was due to a 2- to 4.5-fold increase in the number of CD3+ T cells. In 1 patient, this was due primarily to increases in CD3+ and CD8+ cells; in 2 patients, to increased numbers of CD3+ and CD8+ and CD3+ and CD4+ T cells. In all responding patients, conversion to complete donor hematopoiesis occurred in the first 2 months after DLI. The single nonresponding patient remained it 100% recipient hematopoiesis. The TCR repertoire complexity was examined by polymerase chain reaction amplification of complementary-determining region 3 (CDR3) derived from 24 Vbeta gene subfamilies. In 2 patients, the initiation of myeloma response and conversion to complete donor hematopoiesis was associated with normalization of TCR complexity. Complete donor chimerism and normal TCR complexity remained stable in all patients and did not change with subsequent relapse or development of graft-versus-host disease (GVHD). Thus, conversion to full donor chimerism was temporally associated with the antimyeloma effect of DLI but not with the development of GVHD. Nevertheless, the maintenance of stable donor hematopoiesis did not prevent disease relapse and was not associated with prolonged remission. The selective relapse of myeloma cells without concomitant return of mixed hematopoietic chimerism suggests that myeloma tumor cells in some patients develop resistance to immune destruction.

Beneficial effects of intracoronary adenosine as an adjunct to primary angioplasty in acute myocardial infarction.

BACKGROUND: The benefits of vessel recanalization in acute myocardial infarction (AMI) are limited by reperfusion damage. In animal models, adenosine limits reperfusion injury, reducing infarct size and improving ventricular function. The aim of this study was to evaluate the safety and feasibility of adenosine adjunct to primary PTCA in AMI. METHODS AND RESULTS: Fifty-four AMI patients undergoing primary PTCA were randomized to intracoronary adenosine or saline. The 2 groups were similar for age, sex, and infarct location. Adenosine administration was feasible and well tolerated. PTCA was successful in all patients and resulted in TIMI 3 flow in all patients given adenosine and in 19 given saline (P<0.05). The no-reflow phenomenon occurred in 1 adenosine patient and in 7 saline patients (P=0.02). Creatine kinase was lower in the adenosine group, and a Q-wave MI developed in 16 adenosine patients and in 23 saline patients (P=0.04). Sixty-four percent of dyssynergic segments improved in the adenosine group and 36% in the saline group (P=0. 001). Function worsened in 2% of dysynergic segments in the adenosine group and in 20% in the saline group (P=0.0001). Adverse cardiac events occurred in 5 patients in the adenosine group and in 13 patients in the saline group (P=0.03). CONCLUSIONS: Intracoronary adenosine administration is feasible and well tolerated in AMI. Adenosine adjunct to primary PTCA ameliorates flow, prevents the no-reflow phenomenon, improves ventricular function, and is associated with a more favorable clinical course.

Adenosine and angiotensin system interact in the regulation of renal microcirculation in humans.

We sought to evaluate the possible interaction between the adenosine and angiotensin systems in the regulation of renal microcirculation in humans. Twenty normotensive patients entered the study. Ten patients (group 1) were pretreated with 50 mg of captopril, an inhibitor of angiotensin-converting enzyme, whereas 10 patients (group 2) were pretreated with placebo. Incremental doses of adenosine (from 10(-5) to 1 mg) were injected into a renal artery to all patients at 5-min intervals. Adenosine injection reduced mean renal blood flow velocity in both groups (from 17.3+/-2.8 and 16.7+/-2 cm/s to 5.1+/-1.1 and 3.8+/-0.8 cm/s, in groups 1 and 2, respectively). The decrease in flow velocity was immediate after adenosine, and its duration was proportional to dosage (y = 3.05 x -2.7; R2 = 0.46; p < 0.01). However, group 1 had a slope of regression lower than group 2 (2.37 vs. 3.82 s; p < 0.03). The index of renal resistance (mean arterial pressure/mean blood flow velocity) increased linearly in both groups with adenosine, but group I showed a lower slope of increment (2.77 vs. 5.57 mm Hg/cm/s; p < 0.01). Adenosine administration induced a marked and transient increase in human renal resistance. This vasoconstrictive effect of adenosine was blunted by captopril pretreatment.

Changes in T cell receptor repertoire associated with graft-versus-tumor effect and graft-versus-host disease in patients with relapsed multiple myeloma after donor lymphocyte infusion.

Recent reports of clinical responses following donor lymphocyte infusions (DLI) in patients with relapsed multiple myeloma (MM) after allogeneic BMT have demonstrated the ability of allogeneic cells to mediate a graft-versus-myeloma (GVM) effect, but the mechanisms involved have not been determined. To identify changes in the T cell compartment associated with DLI, we performed a molecular analysis of the T cell receptor (TCR) repertoire in four patients with relapsed MM who received infusions of CD4+ lymphocytes from HLA-identical sibling donors. Three of the four patients demonstrated a clinical anti-myeloma response following DLI but also developed graft-versus-host disease (GVHD). The TCR repertoire was examined after PCR amplification of 24 Vbeta gene subfamilies. This method determines the relative utilization of each Vbeta gene subfamily and also allows the identification of clonal and oligoclonal T cell populations through analysis of CDR3 regions for each TCR Vbeta gene subfamily. Serial blood samples were obtained over at least a 1 year period before and after DLI and results compared to 10 normal donors. Serial analysis of CDR3 size profiles demonstrated the appearance of clonal T cell populations after DLI in each of the three responding patients. The appearance of some clones was noted within the first 3 months after DLI and coincided with decreasing levels of monoclonal paraprotein indicating an ongoing GVM response. Other T cell clones appeared at later time points and coincided with the development of GVHD. These findings demonstrate that T cell clones with different patterns of onset can be identified in the peripheral blood of MM patients following DLI. Further functional characterization of these distinct clonal expansions will be required to determine whether these T cell clones are mediators of either anti-myeloma or anti-host activity.

Reconstitution of T-cell receptor repertoire diversity following T-cell depleted allogeneic bone marrow transplantation is related to hematopoietic chimerism.

CDR3 spectratyping was used to analyze the complexity of the T-cell repertoire and to define the mechanisms and kinetics of the reconstitution of T-cell immunity after allogeneic bone marrow transplantation (BMT). This method, which is based on polymerase chain reaction amplification of all CDR3 regions using the T-cell receptor (TCR) Vbeta genes, was used to examine serial samples of peripheral blood lymphocytes from 11 adult patients with chronic myelogenous leukemia (CML) who underwent T-cell-depleted allogeneic BMT. In contrast to 10 normal donors who display highly diverse and polyclonal spectratypes, patient samples before and early after BMT revealed markedly skewed repertoires, consisting of absent, monoclonal, or oligoclonal profiles for the majority of Vbeta subfamilies. To quantify changes in TCR repertoire over time, we established an 8-point scoring system for each Vbeta subfamily. The mean complexity score for patient samples before transplant (130.8) was significantly lower than that for normal donors (183; P = 0. 0007). TCR repertoire complexity was abnormal in all patients at 3 months after BMT (mean score = 87). Normalization of repertoire began in 4 patients at 6 months after BMT, but the majority of patients continued to display abnormal repertoires for up to 3 years after BMT. To determine whether the reconstituted T-cell repertoire was derived from the donor or recipient, unique microsatellite loci were examined to establish chimeric status. At 3 months after BMT, 7 patients demonstrated mixed chimerism; 4 had complete donor hematopoiesis (CDH). CDH strongly correlated with likelihood of restoration of T-cell repertoire complexity (P = 0.003). In contrast, patients who demonstrated persistence of recipient hematopoiesis failed to reconstitute a diverse TCR repertoire. These findings suggest that the reconstitution of a normal T-cell repertoire from T-cell progenitors in adults is influenced by interactions between recipient and donor hematopoietic cells. (Blood. 2000;95: 352-359)