Clonal Response Research Articles

Choosing Tree Genotypes for Phytoremediation of Landfill Leachate Using Phyto-Recurrent Selection

Information about the response of poplar (Populus spp.) genotypes to landfill leachate irrigation is needed, along with efficient methods for choosing genotypes based on leachate composition. Poplar clones were irrigated during three cycles of phyto-recurrent selection to test whether genotypes responded differently to leachate and water, and to test whether the methodology had merit as a tool for plant selection during remediation. Fifteen below- and above-ground traits were evaluated. Twenty-five clones were tested in cycle 1, while the best 12 genotypes were evaluated in cycles 2 and 3. Eight clones were selected and subsequently tested in an in situ landfill study (cycle 4). Results from cycles 1, 2, and 3 are presented here. Overall, clones responded differently to irrigation treatments, with certain genotypes exhibiting better below- and above-ground growth with water than leachate. However, growth was greater with leachate irrigation for some clones. In addition, differences between treatments within clones decreased with days after planting (DAP). There were no treatment differences for number of leaves, height, and root length at the end of cycle 2 (45 DAP) or cycle 3 (30 DAP). These results detail the extensive variation in clonal responses to leachate irrigation, along with the need and efficacy of using phyto-recurrent selection to choose superior genotypes.

A Profile of 44 Long Term Survivors (>10yrs) with AL Amyloidosis.

AL amyloidosis has a poor outcome. Survival of only about one year was frequently reported in studies performed in the 1990s, and the diagnosis of AL amyloidosis continues to be widely regarded as incompatible with long term survival. We report here the features of patients with AL amyloidosis followed up at the UK Amyloidosis centre (NAC) for more than 10 years, who encouragingly represented more than 10% of all cases. All patients with AL amyloidosis who first attended the NAC between 1979–1997 and subsequently survived for more than 10 years were included in this study. AL type amyloidosis was confirmed in all patients histologically with genetic studies to robustly exclude hereditary amyloidosis as indicated. Organ involvement and responses were defined as per the international consensus criteria (Gertz et al 2005). I123 serum amyloid P (SAP) scintigraphy was used to identify additional organ involvement and monitor amyloid load. 361 patients with AL amyloidosis were assessed at the NAC before 1st August 1997, with a median survival of 2.4 yrs. 132 (20%) patients survived >5 yrs, and 44 (12%) patients survived >10 years with 2 patients living ≥ 16 yrs and 7 for ≥ 14 yrs. Three patients died at 10.2, 11 and 13 years due to unrelated causes with no deaths due to progressive amyloidosis. Among the 44 ten year survivors, at presentation, median age was 51 yrs (29–70), median creatinine 86μmol/L (47–400), 24 hour proteinuria 2g (<0.1g–50g), bilirubin 8μmol/L (2–130), alkaline phosphatase 82.5 units/L (41–2645), and median NT-ProBNP 19 pMol/L (1–2158). Median ECOG performance status was 1 (0–2). Organ involvement (consensus criteria) was: renal 31 (70%); liver 6 (14%); cardiac 8 (18%); autonomic neuropathy 1(2%); peripheral neuropathy - none; gastrointestinal 2(4%) and lymph nodes or other soft tissues 4 (9%). SAP scintigraphy showed renal amyloid deposits in 30 (68%), liver 11(25%), adrenals 3(7%) and bone infiltration 7 (16%). 35 (79%) had one organ involved, 8 (18%) had 2 organs and 1 (3%) patient had three organs involved by consensus criteria; SAP scintigraphy detected additional organ involvement in 12 cases (27%). The whole body amyloid load on SAP scintigraphy was small in 25(57%), moderate 2 (4%) and large 9 (21%). Median plasma cell infiltrate in the marrow was 5% (1–65). Treatment received comprised an alkylator based regimen in 11(26%), VAD chemotherapy in 14 (32%), and stem cell transplantation (SCT) in 18 (42%). 29 (67%) patients had a complete clonal response, 13 (30%) had a partial response and 1 (2%) had no response. 25(57%) had evidence of organ improvement by conventional criteria and in 26 (59%) SAP scintigraphy showed regression of amyloid. The median time to next treatment (progression free survival - PFS) has not been reached at 10 yrs. There was no significant difference in the PFS between patients treated chemotherapy or SCT, or among the complete or partial responders. 13 patients (30%) developed end stage renal disease (ESRD) a median 5.8 yrs after diagnosis and this was not significantly more frequent among partial responders compared with complete hematologic responders. Substantial improvements in diagnosis, monitoring and treatment of AL amyloidosis have occured since 1997. It is all the more encouraging that we are able to report here that 12% of patients diagnosed before this time survived for more than 10 years and patients with AL amyloidosis diagnosed more recently are likely to have even better prospect of good long term outcome in cases who achieve good and sustained clonal responses to therapy.

Chronic B-Cell Dyscrasias Are an Important Clinical Feature of LGL Leukemia.

T-cell large granular lymphocyte leukemia (T-LGL) is a chronic clonal lymphoproliferation of CTL. The context of immune-mediated diseases led to the hypothesis that T-LGL represents an exaggerated clonal immune response to a persistent antigen such as an autoantigen or viral antigen or be associated with an immune surveillance reaction to occult malignancies. Based on the structural similarity of TCR CDR3 we have demonstrated that the transformation event in T-LGL may not be random and is driven by a related antigen. Similar to the clonal evolution in LGL, B cell expansion in low grade non-Hogkin lymphoma may also not be entirely stochastic. There have been coincidental case reports of T-LGL patients with concomitant B cell dyscrasia. It is therefore possible that similar pathogenic triggers may be operative in chronic proliferation of T and B cells, which subsequently predispose to clonal outgrowth. Consequently, we systematically examined a large series of T-LGL patients for evidence of B cell dyscrasias. When our patients (N=70) were studied we found a frequent association of low grade B cell lymphoproliferative disorders (28%). In general, all clinical comparisons with reported studies suggested that our LGL cohort had a composition equivalent to that of prior series and findings with regard to B cell dyscrasias are not due to selection bias. By comparison, a total of 51 patients with concomitant B and T cell dyscrasia were previously reported in small series or case reports. In our series, MGUS was the most common of the B cell disorders identified in T-LGL (21%), B-CLL also was present in 7%. Of note, 8 patients received rituximab and notably, evolution of clonal T cell expansion after therapy with rituximab has been identified in isolated case reports. In addition to clonal B cell expansion, polyclonal hyperglobulinemia was found in 26% of T-LGL, similar to previous report. Hypoglobulinemia was identified in 12% of patients. Evidence of involvement of both the T and B cell compartments in T-LGL fits into several models of disease pathogenesis. T-LGL may represent anti-tumor surveillance reflecting an exaggerated clonal expansion in the context of polyclonal anti-tumor response. An alternative theory is that both conditions may result from an initial polyclonal immune reaction directed against an unrelated common target; one could speculate that autoimmune/viral diseases associated with T-LGL or malignancies (e.g., MDS) provide antigenic triggers. It is also conceivable that impaired humoral immune response could result in an exuberant T cell reaction against an uncleared antigen. If B cell function is insufficient to fully clear the inciting pathogen, then chronic antigenic stimulation could polarize T cell-mediated response, resulting in LGL expansion. Finally, identification of decreased immunoglobulin levels in the context of LGL leukemia may also give merit to the theory that both B and T cell compartments are governed by regulatory/compensatory feedback mechanisms and T-LGL could evolve from unchecked T cell expansion in the context of B cell dysfunction. In sum, we describe here a high frequency of B cell dyscrasias in patients with T-LGL. The association is unlikely to be coincidental and provide important insight into dysregulated expression of T cell and B cell function.

Reed clonal characteristics and response to disturbance in a semi-arid river

Clonal characteristics of Phragmites mauritianus Kunth. were studied in a semi-arid river where disturbance by flooding is a major driver of reed patch dynamics. Physical disturbance by scouring at the patch boundary restricted clonal expansion, and, contrary to our predictions, rhizomes at the boundary were not specialised for enhanced mobility. Persistence of the central part of the patch was promoted by accumulation of sediment within the patch, including fine clays. The combined effects of scouring and burial produced linear patches with a characteristic raised profile. Clonal characteristics which promoted persistence of P. mauritianus in this habitat included a large bud bank on the rhizomes, a deep, complex rhizome system, and dense, tall stems. The development of the deep, complex rhizome system was promoted by sediment accumulation within the patch. Interactions between physical processes and plant clonal characteristics deserve further attention in aquatic environments, where feedbacks among vegetation patches, flow and sediment movement are common.

Growth and biomass of Populus irrigated with landfill leachate

Resource managers are challenged with waste disposal and leachate produced from its degradation. Poplar ( Populus spp.) trees offer an opportunity for ecological leachate disposal as an irrigation source for managed tree systems. Our objective was to irrigate Populus trees with municipal solid waste landfill leachate or fertilized well water (control) (N, P, K) during the 2005 and 2006 growing seasons and test for differences in tree height, diameter, volume, and biomass of leaves, stems, branches, and roots. The trees were grown at the Oneida County Landfill located 6 km west of Rhinelander, Wisconsin, USA (45.6°N, 89.4°W). Eight clones belonging to four genomic groups were tested: NC13460, NC14018 [( Populus trichocarpa Torr. & Gray × Populus deltoides Bartr. ex Marsh) × P. deltoides ‘BC 1’]; NC14104, NC14106, DM115 ( P. deltoides × Populus maximowiczii A. Henry ‘DM’); DN5 ( P. deltoides × Populus nigra L. ‘DN’); NM2, NM6 ( P. nigra × P. maximowiczii ‘NM’). The survival rate for each of the irrigation treatments was 78%. The total aboveground biomass ranged from 0.51 to 2.50 Mg ha −1, with a mean of 1.57 Mg ha −1. The treatment × clone interaction was not significant for tree diameter, total volume, dry mass of the stump or basal roots, or root mass fraction ( P > 0.05). However, the treatment × clone interaction was significant for height, total tree dry mass, aboveground dry mass, belowground dry mass, and dry mass of the leaves, stems + branches (woody), and lateral roots ( P < 0.05). There was broad clonal variation within the BC 1 and DM genomic groups, with genotypes performing differently for treatments. In contrast, the performance of the NM and DN genomic groups was relatively stable across treatments, with clonal response to irrigation being similar regardless of treatment. Nevertheless, selection at the clone level also was important. For example, NC14104 consistently performed better when irrigated with leachate compared with water, while NC14018 responded better to water than leachate. Overall, these data will serve as a basis for researchers and resource managers making decisions about future leachate remediation projects.

Safety and efficacy of risk-adapted cyclophosphamide, thalidomide, and dexamethasone in systemic AL amyloidosis

AbstractHigh-dose melphalan with stem-cell transplantation is believed to be the most effective treatment for systemic light-chain (AL) amyloidosis, but many patients are ineligible because of the extent of their disease, and treatment-related mortality (TRM) remains substantial. We report the use of a risk-adapted oral regimen of cyclophosphamide, thalidomide, and dexamethasone (CTD) or attenuated CTD (CTDa) in 75 patients with advanced AL amyloidosis, including 44 patients with clonal relapse after prior therapy. Fifty-one (68%) patients received CTD and 24 (32%) received CTDa. A hematologic response occurred in 48 (74%) of 65 evaluable patients, including complete responses in 14 (21%) and partial responses in 34 (53%) cases. Median estimated overall survival (OS) from commencement of treatment was 41 months, and from diagnosis median was not reached with a median follow-up of 22 months. Three-year estimated OS was 100% and 82% among complete and partial hematologic responders, respectively. Toxicity necessitating cessation of therapy occurred in 8% and was at least grade 2 in 52% of patients. TRM was 4%. The clonal response rates to CTD reported here are higher than any previously reported nontransplantation regimen in AL amyloidosis, and risk adaptation allows its use in poorer risk patients. CTD merits prospective randomized study.

Clonal regeneration of an arrow bamboo, Fargesia qinlingensis, following giant panda herbivory

Characteristics of giant panda herbivory sites and clonal regeneration of an arrow bamboo Fargesia qinlingensis following giant panda grazing were studied in the Qinling Mountains of China. Three types of plots were located in a pandas’ summer habitat in 2002: herbivory (naturally grazed by giant pandas), clipped (simulated panda herbivory), and control. Average area of herbivory sites was 2.92 m2 and average distance from herbivory sites to the closest tree (dbh > 10 cm) was 1.0 m. Pandas avoided thin bamboo culms with basal diameters <5 mm. Average height of stumps of culms grazed by panda was 0.67 m and average density of grazed culms was 9.0 culms m−2. Annual culm mortality rate was significantly greater in herbivory and clipped plots than in control plots while annual recruitment rate was not significantly different among the three plot types in 2003. Neither recruitment rate nor mortality rate were significantly different among the three plot types in 2004. Annual recruitment rate was significantly greater than annual mortality rate only in control plots in both 2003 and 2004, suggesting static ramet dynamics in disturbed plots (herbivory and clipped). Density of new shoots was not significantly different, but basal diameter of new shoots was significantly less in herbivory plots compared to control plots in 2002. Differences of annual mortality rate and growth of new shoots found between control plots and herbivory plots suggest that clonal regeneration of F. qinlingensis culms was negatively affected by giant panda grazing. Therefore, no evidence of a clonal integration compensatory response to panda herbivory was found in F. qinlingensis.

Molecular strategies for detection and quantitation of clonal cytotoxic T-cell responses in aplastic anemia and myelodysplastic syndrome

AbstractImmune mechanisms are involved in the pathophysiology of aplastic anemia (AA) and myelodysplastic syndrome (MDS). Immune inhibition can result from cytotoxic T cell (CTL) attack against normal hematopoiesis or reflect immune surveillance. We used clonally unique T-cell receptor (TCR) variable β-chain (VB) CDR3 regions as markers of pathogenic CTL responses and show that while marrow failure syndromes are characterized by polyclonal expansions, overexpanded clones exist in these diseases and can serve as investigative tools. To test the applicability of clonotypic assays, we developed rational molecular methods for the detection of immunodominant clonotypes in blood and in historic marrow biopsies of 35 AA, 37 MDS, and 21 paroxysmal nocturnal hemoglobinuria (PNH) patients, in whom specific CDR3 sequences and clonal sizes were determined. CTL expansions were detected in 81% and 97% of AA and MDS patients, respectively. In total, 81 immunodominant signature clonotypes were identified. Based on the sequence of immunodominant CDR3 clonotypes, we designed quantitative assays for monitoring corresponding clones, including clonotypic Taqman polymerase chain reaction (PCR) and clonotype-specific sequencing. No correlation was found between clonality and disease severity but in patients treated with immunosuppression, truly pathogenic clones were identified based on the decline that paralleled hematologic response. We conclude that immunodominant clonotypes associated with marrow failure may be used to monitor immunosuppressive therapy.

Population and clonal level responses of a perennial grass following fire in the northern Chihuahuan Desert

Relationships involving fire and perennial grasses are controversial in Chihuahuan Desert grasslands of southern New Mexico, USA. Research suggests that fire delays the resprouting of perennial grasses well after two growing seasons. However, such results are confounded by livestock grazing, soil erosion, and drought. Additionally, post-fire grass responses may depend on initial clone size. We evaluated the effects of fire, grazing, and clone size on Bouteloua eriopoda (black grama) in southern New Mexico grasslands. Four 2-ha plots were established in each of four sites. Fire and grazing were applied or not applied in 1999 such that four treatment combinations were assigned randomly to plots within each site. Within each plot, small (0-10 cm(2) basal area), medium (10-30 cm(2)), and large ( > 30 cm(2)) clones were initially mapped in five 0.91-m(2) quadrats where grass attributes and litter cover were evaluated before and at the end of two growing seasons following fire. Maximum fire temperature was also measured. At a population level, canopy and litter cover were each approximately 50% less in burned than unburned areas. However, compared to initial levels, canopy height had increased by 10% at the end of the study, regardless of fire. At a clonal level, basal cover reductions were attributed mostly to large clones that survived fire. Smaller clone densities had decreased by as much as 19% in burned compared to unburned areas, and fire reduced the basal cover of medium clones. Basal and canopy cover, recruitment, and clone basal area decreased with increased fire temperatures. Almost all responses were independent of grazing, and interactive effects of grazing and fire were not detected. Fire did not kill all perennial grass clones, regardless of size. However, rapid responses were likely influenced by above-average precipitation after fire. Future studies in desert grasslands should examine how perennial grass dynamics are affected by fire, precipitation patterns, and interactions with grazing.

Sustained B7/CD28 interactions and resultant phosphatidylinositol 3‐kinase activity maintain G1→S phase transitions at an optimal rate

Twenty-four hours of TCR engagement and CD28 costimulation was found sufficient to elicit an optimal rate of cell division over a 72-h period only when a high concentration of IL-2 was produced in the culture and remained readily available to the CD4(+) T cells. The cell division response could be aborted following 24 h of stimulation by the simultaneous abrogation of IL-2R signaling and the blockade of CD28 or TCR ligands. Biochemical and pharmacologic studies indicated that a phosphatidylinositol 3-kinase-Akt signaling cascade costimulated by the TCR and CD28 maintained the blasting cell division rate at a maximal level beyond 24 h even when IL-2 was withdrawn, neutralized, or exhausted. These data show that CD4(+) T cells remain sensitive to antigens (Ag) and costimulatory signals throughout the clonal expansion response. Furthermore, only those T cells that perceive the presence of a continued threat in the form of Ag/MHC complexes and B7 costimulatory ligands or a high concentration of a growth factor are directed to remain in cell cycle.

Comparative immunology

As a discipline, comparative immunology enhances zoology and has gained wide acceptance in the biological sciences. It is an offshoot of the parent field, immunology, and is an amalgam of immunology and zoology. All animals from protozoans to humans have solved the threat of extinction by having evolved an immune-defense strategy that ensures the capacity to react against foreign, non-self microorganisms and cancers that disturb the homeostatic self. Invertebrate-type innate immune responses evolved first and they characterize the metazoans. These rapid natural responses act immediately and are often essential for the occurrence of slower, more specific, adaptive vertebrate-type immune responses. As components of the innate immune system, there is an emphasis on several major steps in the evolutionary process: (i) recognition; (ii) the phagocytic cell; and (iii) the natural killer cell. When vertebrates evolved, beginning with fish, thymus-controlled T cells first appeared, as did bone marrow-derived B cells (first found in amphibians with long bones). These were the precursors of the plasma cells that synthesize and secrete antibodies. Confirming the concept of self/non-self, invertebrates possess natural, non-adaptive, innate, non-clonal, non-anticipatory immune responses, whereas vertebrates possess adaptive, acquired, clonal, and anticipatory responses. This symposium concerns: (i) aspects of the immune spectrum in representative groups; (ii) specific findings (in particular models; e.g. earthworms); (iii) clues as to the possible biomedical application of relevant molecules derived from animals, notably invertebrates; and (iv) some views on the more practical applications of understanding immune systems of invertebrates and ectotherms, and their possible role in survival.

The PPD-specific T-cell clonal response in UK and Malawian subjects following BCG vaccination: A new repertoire evolves over 12 months

Mycobacterium bovis bacille Calmette Guerin vaccination protects against pulmonary tuberculosis in the United Kingdom but not in Malawi. We investigated whether a difference in the clonal T-cell response to BCG vaccination might account for this. The results of clonal analysis were compared to those obtained by skin testing and in a whole blood interferon gamma assay. Pre-vaccination antigen specific T-cell clones were detected, but the majority of clones present 12 months after vaccination were not present earlier. The magnitude of the clonal response did not correlate well with results of the other assays. These data indicate that single assays may not be reliable and that a stable memory T-cell repertoire is slow to develop.

Correlation of Suppression of FDG PET Uptake with Serum Free Light Chain Levels - Both FDG PET-CT and Serum Clonal Free Light Chain Response Precede and Predict the Likelihood of Subsequent Complete Remission in Newly Diagnosed Multiple Myeloma.

18F-labeled Fluorodeoxyglucose Positron Emission Tomography - computed tomography (PET-CT) imaging results from newly diagnosed patients with multiple myeloma (MM) were correlated with serum free light chain (FLC) results from Total Therapy 3 (TT3). Patients had baseline PET-CT scans and serum kappa and lamda FLC analysis at diagnosis and at 2, 14 and 30 days post first cycle of VDTPACE and at pre-transplant 1 of two planned autologous stem cell transplants (TX1), correlating PET-CT response, clonal and nonclonal FCL response, and likelihood of subsequent complete clinical response (CR). Patients were selected with serum clonal FLC levels above the normal range that had been enrolled for sufficient time for a complete response (CR) to be recorded. Correlations between the serum clonal and nonclonal FLC levels and PET-CT response defined by both reduction in number of PET-defined focal lesions (PET-FL) (5 mm or larger areas of circumscribed uptake visually definable above background activity) and reduction in the maximum standardized uptake value normalized to the patients' lean body mass (max SUV) were performed.Results: Reduction in FLC and PET-FL and max SUV are correlated, as was normalization of clonal FLC levels with likelihood of subsequent CR (Fig 1). The Spearman's correlation coefficient between percent reduction in clonal FLC and percent reduction in max SUV was 0.04 (n=49, p=0.79) at 2 days post-VDTPACE, 0.32 (n=48, p=0.028) at 30 days post-VDTPACE, and 0.52 (n=33, p=0.002) prior to TX1. The greatest reduction in max SUV occurred by 2 days post-VDTPACE while the maximum reduction in clonal FLC occurred at 30 days post-VDTPACE, indicating that the PET response lead and predicted the FLC response in near “real time.” While normalization of the clonal FLC levels at 30 days was strongly predictive of subsequent CR (p<0.001, n=125), normalization of the non-clonal FLC levels was not (p=0.41, n=124).Conclusion: We conclude that normalization of serum clonal FLC correlates both with improvement in FDG PET-CT scanning in both max SUV and number of PET-defined focal lesions and with likelihood of subsequent CR, though it “lags” behind FDG PET response by nearly a month, with FDG PET following the clinical course in near real time. [Display omitted]

The Role of Renal Transplantation in Systemic AL Amyloidosis.

Kidneys are the commonest site of amyloid deposition in AL amyloidosis (AL) and many patients present in advanced renal failure. Successful treatment of the underlying plasma cell dyscrasia may not prevent development of end stage renal failure and dialysis dependence. Even in the presence of good clonal response the prognosis of these patients on dialysis is poor with mortality being 3 times that for patients with diabetes (UK renal registry data 2003). Renal transplantation is controversial due to the systemic and progressive nature of AL amyloidosis. We report the experience with renal transplantation in 16 patients with AL seen at the National Amyloidosis Centre UK between 1986 and 2000. All patients had histologically proven amyloidosis with an underlying plasma cell dycrasia. None of the patients had extra-renal organ dysfunction. Median age at diagnosis of AL was 64 years (range 49–74) with 3 males and 13 females. 15 (93%) had monoclonal gammopathy as the underlying clonal disorder while 1 (7%) patient had Waldenstroms macroglobulinaemia. The median time from diagnosis of renal failure to renal transplantation was 5.5 yrs (range 0.3–17) including a median of 1.7 yrs on dialysis. Median age at renal transplantation was 56yrs (42–67). There were 2 (12%) live related renal transplants and 14 (88%) from cadaveric donors; there were no procedure-related deaths and none had primary graft non-function. Graft function remains well-preserved with the median creatinine clearance at last follow-up was 41.6 ml/min (range 25.1–68); the only cause of graft failure was death. 13 patients received cytotoxic treatment as follows: autologous stem cell transplantation (ASCT) 3 (18%), VAD chemotherapy 5 (31%), melphalan and prednisone 4 (25%), chlorambucil 1 (6%). 2 (15%) had a partial response to treatment while 11 (84%) had no response (this included the three patients with ASCT). The median age at death was 71.4 yrs. Six patients have died, all of progressive multi-organ amyloidosis, at a median 7.5yrs from renal transplantation (which is 12.2 yrs and 12.9 yrs respectively from the diagnosis of amyloidosis and ESRF). Four of these patients had evidence of amyloid accumulation in their renal graft. There was no significant difference in the median survival of patients with recurrent amyloid in the graft versus those without (p=0.78) and the only cause of graft loss was death with a functioning graft. The patients without amyloid in the graft had a trend for better OS (median not reached vs 12.9 yrs) and survival from renal transplantation (7.59 yrs vs. 5.5 yrs). In summary, this study shows that the overall survival of patients with predominant renal AL who underwent renal transplantation was remarkably good. Renal allograft survival was 7.4 yrs (median) despite a very poor haematological response to treatment in many patients. This study suggests that renal transplantation may have been underutilized in AL amyloidosis and should be considered in selected patients.

Pathologic clonal cytotoxic T-cell responses: nonrandom nature of the T-cell–receptor restriction in large granular lymphocyte leukemia

AbstractT-cell large granular lymphocyte (T-LGL) leukemia is a clonal lymphoproliferation of cytotoxic T cells (CTLs) associated with cytopenias. T-LGL proliferation seems to be triggered/sustained by antigenic drive; it is likely that hematopoietic progenitors are the targets in this process. The antigen-specific portion of the T-cell receptor (TCR), the variable beta (VB)–chain complementarity-determining region 3 (CDR3), can serve as a molecular signature (clonotype) of a T-cell clone. We hypothesized that clonal CTL proliferation develops not randomly but in the context of an autoimmune response. We identified the clonotypic sequence of T-LGL clones in 60 patients, including 56 with known T-LGL and 4 with unspecified neutropenia. Our method also allowed for the measurement of clonal frequencies; a decrease in or loss of the pathogenic clonotype and restoration of the TCR repertoire was found after hematologic remission. We identified 2 patients with identical immunodominant CDR3 sequence. Moreover, we found similarity between multiple immunodominant clonotypes and codominant as well as a nonexpanded, “supporting” clonotypes. The data suggest a nonrandom clonal selection in T-LGL, possibly driven by a common antigen. In contrast, the physiologic clonal CTL repertoire is highly diverse and we were not able to detect any significant clonal sharing in 26 healthy controls.

Mammalian Toll-like receptors: to immunity and beyond

Toll-like receptors (TLRs) constitute an archetypal pattern recognition system. Their sophisticated biology underpins the ability of innate immunity to discriminate between highly diverse microbial pathogens and self. However, the remarkable progress made in describing this biology has also revealed new immunological systems and processes previously hidden to investigators. In particular, TLRs appear to have a fundamental role in the generation of clonal adaptive immune responses, non-infectious disease pathogenesis and even in the maintenance of normal mammalian homeostasis. Although an understanding of TLRs has answered some fundamental questions at the host-pathogen interface, further issues, particularly regarding therapeutic modulation of these receptors, have yet to be resolved.

Direct processing and presentation of antigen from malaria sporozoites by professional antigen‐presenting cells in the induction of CD8+ T‐cell responses

Irradiated malaria sporozoites induce better protection than viable untreated sporozoites. We observed early differences between irradiated and viable untreated sporozoites in priming responses in vivo to a protective CD8 T-cell epitope, pb9, of the circumsporozoite protein of Plasmodium berghei. Sporozoites were processed for MHC class I presentation by dendritic cells (DC) to prime pb9-specific IFN-gamma-producing CD8 T cells. DC pulsed with untreated and irradiated sporozoites were similarly capable of priming central memory T-cell responses, detectable by the IFN-gamma cultured ELISPOT assay. However, irradiation significantly enhanced sporozoites' ability to prime effector T-cell responses detectable by the IFN-gammaex vivo ELISPOT assay. Irradiation also enhanced the ability of splenic APC to process and present sporozoites in order to re-stimulate pb9-specific polyclonal and clonal T-cell responses. Sporozoites did not stimulate T cells in the absence of APC. Over-irradiation decreased the sporozoites' T-cell stimulating capacity in vitro at high parasite doses, which may indicate that an optimal irradiation dose is necessary to induce protective immunity by sporozoite inoculation. The induction of sporozoite-specific CD8 T-cell responses without the need for liver stage infection identifies a potentially important mechanism in the development of pre-erythrocytic immunity.

Analysis of T-Cell Receptor Vβ Gene Repertoires after Immune Stimulation and in Malignancy by Use of Padlock Probes and Microarrays

Detection of expanded T-cell clones, identified by their receptor (TCR) repertoires, can assist diagnosis and guide therapy in infectious, inflammatory, and autoimmune conditions as well as in tumor immunotherapy. Analysis of tumor-infiltrating lymphocytes often reveals preferential use of one or a few TCR V beta genes, compared with peripheral blood, indicative of a clonal response against tumor antigens. To simultaneously measure the relative expression of all V beta gene families, we combined highly specific and sensitive oligonucleotide reagents, called padlock probes, with a microarray read-out format. T-Cell cDNA was combined with a pool of V beta subfamily-specific padlock probes. Reacted probes were selectively amplified and the products hybridized to a microarray, from which the V beta subfamily distribution in each sample could be determined relative to a control sample. In lymphocytes stimulated with the superantigen staphylococcal enterotoxin B, we detected expansions at the mRNA level of TCR subfamilies previously shown to respond to staphylococcal enterotoxin B. Expansions of the same V beta families could also be detected by flow cytometry. In samples from two bladder cancer patients, we detected predominant representations of specific V beta subfamilies in both tumor-infiltrating lymphocytes and in the draining lymph nodes, but not in non-tumor-draining lymph nodes or peripheral blood. Several expression profiles from draining lymph nodes in patients with malignant melanoma were divergent from profiles seen in non-tumor-draining lymph nodes. Padlock probe-based parallel analysis of TCR V beta gene distributions provides an efficient method for screening multiple samples for T-cell clonal expansions with reduced labor and time of analysis compared with traditional methods.

B cell superantigens: a microbe?s answer to innate-like B cells and natural antibodies

Marginal zone B cells and B-1 cells have been termed innate-like B cells as they express limited repertoires that play special roles in immune defenses against common infections. These B cells are the sources of natural antibodies and are capable of highly accelerated clonal responses that help counter blood-borne infections. We have characterized a class of microbial product with highly adapted binding interactions with host immunoglobulins/B cell receptors (BCRs), which enable the targeting of large supra-clonal sets of B cells for activation-associated apoptotic death. In recent studies, we have shown that all B cells with V region-targeted BCRs are susceptible. However, compared to follicular B cells, in vivo exposure preferentially causes innate-like B cells to undergo induced death with subsequent long-lasting supra-clonal depletion and immune tolerance. Based on these properties, it is likely that B cell superantigens influence the pathogenesis of some common infections, but also may provide novel therapeutic opportunities to treat B cell neoplastic and autoimmune diseases.

Intermediate Dose Intravenous Melphalan and Dexamethasone Treatment in 144 Patients with Systemic AL Amyloidosis.

The optimal intensity of chemotherapy in AL amyloidosis remains contentious. Traditional low dose oral melphalan and prednisolone therapy is ineffective in most patients, whereas high dose therapy with autologous stem cell rescue carries substantial procedure related mortality. We report here the outcome of intermediate dose intravenous melphalan (IDM) (25mg/m2 d1 + dexamethasone 20mg po d1-4, repeated every 21-28d) among 144 patients with amyloidosis (83M:61F, median age 62yrs, range 30–79). The median number of organs involved by amyloid was 2, including kidneys in 74% of patients, heart in 56%, neuropathy in 25% and liver in 20%. Median follow-up was 13mo (0.5–53). Patients received a median of 3 cycles of treatment (range 1–8). Dexamethasone was omitted in 51 cases, principally due to concern about fluid retention associated with nephrotic syndrome or cardiac amyloidosis. There were 52 deaths of which 3 (2%) were considered treatment-related (1 neutropenic sepsis, 2 dexamethasone-induced pulmonary oedema). Serum free light chain (sFLC) measurements were available before and after at least one cycle of IDM in 140 patients; 23% achieved complete response (CR, sustained normalization of sFLC ratio), 31% a partial response (PR, sustained >50% reduction in pre-treatment clonal isotype) and 46% did not respond (NR). Rates of sFLC CR+PR were higher among patients who also received dexamethasone at 64% vs 37% (p<0.0004). Paraproteins were detectable by electrophoresis and immunofixation of serum and/or urine in 115pts (80%); 85 of these pts had post-treatment data, showing CR (complete resolution of serum and urine paraprotein) in 17 (20%), PR (50% reduction) in 24 (28%) and NR in 44 (52%). Cycle by cycle assays of sFLC allowed rapid assessment of early clonal response, which was evident within 2 cycles in almost all responders. Maximal response was usually achieved by the fourth cycle. Median survival was 44 months among patients with CR or PR, and 18 months among non-responders (p<0.003). Amyloidotic organ dysfunction improved in 14%, remained stable in 51%, and deteriorated in 19% of evaluable cases. The findings support the use of IDM coupled with cycle by cycle sFLC measurements as a valid treatment strategy in patients with AL amyloidosis. Although clonal response rates in this series may be lower than selected patients treated with high dose therapy and autologous stem cell rescue, IDM can be delivered to a wider range of patients, in whom the rapidity of its action, relative simplicity of the regimen, and much lower treatment related mortality favor its continued use and further investigation.