Hepatitis C viraemia reversibly maintains subset of antigen‐specific T‐bet+ tissue‐like memory B cells

Background: Chronic antigen exposure and/or ageing increases the frequency of T‐box expressed in T cells (T‐bet)‐expressing B‐lymphocytes in mice. The frequency and significance of B‐cell T‐bet expression during chronic hepatitis C (HCV) infection in human subjects has never been described. Methods: Healthy controls, cirrhotic and noncirrhotic HCV‐infected patients, and non‐HCV patients with cirrhosis were recruited. Peripheral blood mononuclear cells were phenotyped for expression of T‐bet and related markers by flow cytometry. In a subset of patients who underwent antiviral therapy and were cured of HCV infection (sustained virological response), the dynamics of T‐bet expression in B cells was monitored. After cure, convalescent B cells were tested for T‐bet expression after re‐exposure to infected plasma or recombinant HCV proteins. Results: Forty‐nine patients including 11 healthy donors, 30 hepatitis C‐infected individuals (nine with liver cancer, 13 with cirrhosis, eight without cirrhosis) and eight patients with cirrhosis due to non‐HCV‐related cause were recruited. We found that B cells in patients with chronic HCV exhibited increased frequency of T‐bet+ B cells relative to noninfected individuals (median 11.5% v. 2.2%, P<.0001) but that there were no significant differences between noncirrhotic, cirrhotic and cancer‐bearing infected individuals. T‐Bet⁺ B cells expressed higher levels of CD95, CXCR3, CD11c, CD267 and FcRL5 compared to T‐bet^? B cells and predominantly exhibit a tissue‐like memory CD27^?CD21^? phenotype independent of HCV infection. T‐bet⁺ B cells in HCV‐infected patients were more frequently class‐switched IgD^?IgG⁺ (40.4% vs. 26.4%, P=.012). Resolution of HCV infection with direct‐acting antiviral (DAA) therapy leads to a marked reduction in the frequency of T‐bet⁺ B cells (median 14.1% pretreatment v. 6.7% end of treatment v. 6.1% SVR12, P≤.01). Re‐exposure of convalescent (cured) B cells to viremic plasma and recombinant HCV E2 protein led to re‐expression of T‐bet. Conclusion: Chronic antigenemia in chronic HCV infection induces and maintains an antigen‐specific T‐bet⁺ B cell. These B cells share markers with tissue‐like memory B cells. Antigen‐driven T‐bet expression may be a critical suppressor of B‐cell activation in chronic HCV infection.     

Immunophenotypic analysis of T‐acute lymphoblastic leukemia. A CD5‐based ETP‐ALL perspective of non‐ETP T‐ALL

T‐cell antigens [CD5,CD1a,CD8] define early T‐cell precursor acute lymphoblastic leukemia (ETP‐ALL). To understand immature T‐ALL of which ETP‐ALL is part, we used these antigens to subcategorize non‐ETP T‐ALL for examining expression of myeloid/stem cell antigens (M/S) and clinical features. Using CD5 (+/?) to start categorization, we studied 69 routinely immunophenotyped patients with T‐ALL. CD5^? was a homogenous (CD8,CD1a)^? M/S⁺ ETP‐ALL group (n = 9). CD5⁺ cases were (CD8,CD1a)^? pre‐T‐ALL (n = 22) or (CD8,CD1a)⁺ (n = 38) thymic/cortical T‐ALL; M/S⁺ 20/22 (90.91%) in former and 22/38 (57.89%) in latter (P = 0.007). ETP‐ and pre‐T‐ALL together (CD1a^?,CD5^?/+ immature T‐ALL group) were nearly always M/S⁺ (29/31; 93.55%). In multivariate analysis, only ETP‐ALL predicted poor overall survival (P = 0.02). We conclude (i) CD5 negativity in T‐ALL almost always means ETP‐ALL. CD1a and CD8 negativity, as much as CD5, marks immaturity in T‐ALL, and the CD5^+/?/CD1a^?/CD8^? immature T‐ALL group needs further study to understand the biology of the T‐ALL–myeloid interface. (ii) ETP‐ALL patients may be pre‐T‐ALL if CD2⁺; CD2⁺, conversely, CD5^?/CD1a^?/CD8^? pre‐T ALL patients are ETP‐ALL. (iii) Immunophenotypic workup of T‐ALL must not omit CD1a, CD5, CD8 and CD2, and positivity of antigens should preferably be defined as recommended for ETP‐ALL, so that this entity can be better evaluated in future studies of immature T‐ALL, a group to which ETP‐ALL belongs. (iv) ETP‐ALL has poor prognosis.     

Differential IFN‐γ production by adult and neonatal blood CD56+ natural killer (NK) and NK‐like‐T cells in response to Trypanosoma cruzi and IL‐15

Early interferon‐gamma (IFN‐γ) release by innate cells is critical to direct type 1 immune response able to control intracellular pathogens like Trypanosoma cruzi. Although CD56^bright natural killer (NK) cells are reported to be potent early IFN‐γ producers, other CD56⁺ cells like CD56^dim NK cells and NK‐like T cells have recently been shown to also release IFN‐γ. We have here studied the contribution of each CD56⁺ lymphocyte populations in early IFN‐γ production in both adults and neonates. On this purpose, we analysed the kinetics of IFN‐γ production by RT‐PCR, ELISA and flow cytometry from 2 h onwards after T. cruzi and IL‐15 stimulation and sought for the responding CD56⁺ cells. CD56^bright and CD56^dimCD16^? NK cells were the more potent IFN‐γ early producers in response to IL‐15 and parasites in adults and neonates. In both age groups, the majority of IFN‐γ producing cells were NK cells. However, on the contrary to neonates, CD3⁺CD56⁺ NK‐like T cells and CD3⁺CD56^? ‘classical’ T cells also contributed to early IFN‐γ production in adults. Altogether, our results support that whereas NK cells responded almost similarly in neonates and adults, cord blood innate CD56⁺ and CD56^? T cells displayed major quantitative and qualitative defects that could contribute to the well‐known neonatal immune immaturity.     

Apical membrane protein 1‐specific antibody profile and temporal changes in peripheral blood B‐cell populations in Plasmodium vivax malaria

Plasmodium falciparum‐specific antibodies tend to be short‐lived, but their cognate memory B cells (MBCs) circulate in the peripheral blood of exposed subjects for several months or years after the last infection. However, the time course of antigen‐specific antibodies and B‐cell responses to the relatively neglected parasite Plasmodium vivax remains largely unexplored. Here, we showed that uncomplicated vivax malaria elicits short‐lived antibodies but long‐lived MBC responses to a major blood‐stage P vivax antigen, apical membrane protein 1 (PvAMA‐1), in subjects exposed to declining malaria transmission in the Amazon Basin of Brazil. We found that atypical (CD19⁺CD10^?CD21^?CD27^?) MBCs, which appear to share a common precursor with classical MBCs but are unable to differentiate into antibody‐secreting cells, significantly outnumbered classical MBCs by 5:1 in the peripheral blood of adult subjects currently or recently infected with P vivax and by 3:1 in healthy residents in the same endemic communities. We concluded that malaria can drive classical MBCs to differentiate into functionally impaired MBCs not only in subjects repeatedly exposed to P falciparum, but also in subjects living in areas with low levels of P vivax transmission in the Amazon, leading to an impaired B‐cell memory that may affect both naturally acquired and vaccine‐induced immunity.     

Impact of oral silymarin on virus‐ and non‐virus‐specific T‐cell responses in chronic hepatitis C infection

Silymarin displays anti‐inflammatory effects on T lymphocytes in vitro. The immunomodulatory properties of oral silymarin in vivo in humans with chronic hepatitis C have not previously been characterized. We hypothesized that silymarin would suppress T‐cell proliferation and pro‐inflammatory cytokine production of virus‐ and non‐virus‐specific T cells while increasing anti‐inflammatory IL‐10 production in vivo. Patients from one site of the SyNCH‐HCV double‐masked, placebo‐controlled study of oral silymarin in prior interferon nonresponders with chronic hepatitis C provided blood samples at baseline and treatment week 20. Mononuclear cells were stimulated with recombinant HCV proteins and controls in ³H‐thymidine proliferation assays, IFNγ ELISPOT and IL‐10 ELISPOT. The frequency of CD4⁺CD25^hi and CD4⁺foxp3⁺ regulatory T cells, serum cytokine levels, serum IP‐10 and lymphocyte interferon‐stimulated gene expression were also quantified at baseline and week 20. Thirty‐two patients were recruited (10; placebo, 11; 420 mg three times a day, 11; 700 mg three times a day). Serum ALT and HCV RNA titres did not change in any group. HCV‐specific CD4⁺ T‐cell proliferation and the frequency of IFNγ‐ and IL‐10‐producing T cells were not significantly changed in silymarin‐treated subjects. However, C. albicans‐induced T‐cell IFNγ and phytohaemagglutinin‐induced T‐cell proliferation were suppressed by silymarin therapy. A trend towards augmentation of interferon‐induced ISG15 expression was present in the high‐dose silymarin group. While no effect on HCV‐specific T cells was identified, these data confirm that high‐dose oral silymarin exerts modest nonspecific immunomodulatory effects in vivo. The impact of this anti‐inflammatory effect on long‐term liver health in chronic hepatitis C merits future clinical investigation.     

Persistent hepatitis C virus (HCV) infection impairs HCV‐specific cytotoxic T cell reactivity through Mcl‐1/Bim imbalance due to CD127 down‐regulation

Summary. In persistent hepatitis C virus (HCV) infection, HCV‐specific cytotoxic T lymphocyte (CTL) reactivity is impaired and this affects HCV control. Interleukin‐7 receptor (CD127) expression on these cells could regulate CTL reactivity through Mcl‐1/Bim balance modulation. Bim is a pro‐apoptotic molecule blocked by the action of Mcl‐1. Mcl‐1/Bim expression and T cell reactivity on HCV‐specific CTLs were compared according to CD127 phenotype. Peripheral blood lymphocytes (PBL) from HLA‐A2⁺ HCV⁺ patients were obtained. HCV‐specific CTLs were visualized by staining PBL with anti‐CD8 and HLA‐A2/peptide pentameric complexes (pentamer). Mcl‐1/Bim/CD127 phenotype of HCV‐specific CTLs was tested by staining detectable CD8⁺/pentamer⁺ cells with anti‐Mcl‐1/Bim/CD127 antibodies. HCV‐specific CTL proliferation ability after specific in vitro challenge was tested in the presence and absence of pancaspase inhibitor z‐VAD‐fmk. All stained cells were analysed by flow cytometry. CD127^low‐expressing HCV‐specific CTLs associated with high HCV viraemia, while CD127^high correlated with undetectable viral loads (P < 0.001). Directly ex vivo, pentamer⁺ cell frequency was similar according to CD127 expression level. Nevertheless, CD127^low pentamer⁺ cell proliferation after specific in vitro challenge was impaired (P < 0.05), although this was corrected by z‐VAD‐fmk treatment (P < 0.05). Mcl‐1 expression was low directly ex vivo (P < 0.01), and Bim was up‐regulated after antigen encounter (P < 0.05) of CD127^low pentamer⁺ cells. The ex vivo difference between Mcl‐1 and Bim expression on pentamer⁺ cells correlated positively with CD127 expression level (P < 0.001) and with pentamer⁺ cell reactivity (P < 0.05). In summary, a low ex vivo Mcl‐1 expression and Bim up‐regulation after antigen encounter are involved in CD127^low HCV‐specific CTL hyporeactivity during chronic infection, but it can be overcome by apoptosis blockade.     

Epstein–Barr virus‐associated pneumonia in patients with post‐transplant lymphoproliferative disease after hematopoietic stem cell transplantation

Q.‐F. Liu, Z.‐P. Fan, X.‐D. Luo, J. Sun, Y. Zhang, Y.‐Q. Ding. Epstein–Barr virus‐associated pneumonia in patients with post‐transplant lymphoproliferative disease after hematopoietic stem cell transplantation.
Transpl Infect Dis 2010: 12: 284–291. All rights reserved. Abstract: Epstein–Barr virus (EBV) reactivation or infection after hematopoietic stem cell transplantation (HSCT) most often induces post‐transplant lymphoproliferative disease (PTLD), but it also may be associated with clinical symptoms such as pneumonia. Our aim was to investigate and describe the clinical manifestations of PTLD and PTLD accompanied by EBV‐associated pneumonia in 323 patients after HSCT. PTLD within extravisceral lymphoid tissue was identified in 7 cases (5 with CD20⁺ diffuse large B‐cell lymphoma, 1 with CD20⁺ polymorphic B‐cell hyperplasia, and 1 with CD3⁺CD45RO⁺ peripheral T‐cell lymphoma unspecified). Six of the patients with PTLD were EBV positive. Three patients had EBV‐associated pneumonia, and chest computed tomography revealed multifocal patchy and diffuse ground‐glass attenuation in both lungs. EBV‐DNA was positive in bronchoalveolar lavage (BAL) fluid, which contained mainly CD3⁺ T cells but no CD19⁺ or CD20⁺ B cells. Lung biopsy showed interstitial intra‐alveolar infiltrates of mainly CD3⁺ T cells and some CD68⁺ macrophages without CD19⁺ and CD20⁺ B cells. The patients with PTLD accompanied by EBV‐associated pneumonia developed hyperpyrexia and dyspnea, which progressed rapidly, and eventually all died within 2 weeks of the onset of PTLD. EBV‐associated PTLD accompanied by EBV‐associated pneumonia after HSCT is rare. Cytology of BAL fluid and lung biopsy may help establish the diagnosis.     

Activating killer immunoglobulin‐like receptor incompatibilities enhance graft‐versus‐host disease and affect survival after allogeneic hematopoietic stem cell transplantation

Objectives: Killer immunoglobulin‐like receptors (KIRs) regulate function of natural killer (NK) cells and a subset of T cells. In this study, we prospectively evaluated the impact of donor and recipient activating KIR genes on outcome of allogeneic hematopoietic stem cell transplantation (alloHSCT) for patients with hematological malignancies. Methods: One‐hundred consecutive recipients of myeloablative transplantation and their donors were tested for KIR genotype as well as for immune reconstitution, including activating KIR expression on NK cells and T cells. Results: In a multivariate analysis, mismatches of particular activating KIRs such that the patient was negative and the donor was positive (P^?D⁺) resulted in increased risk of acute (KIR2DS1) and chronic (KIR2DS3) graft‐versus‐host disease (GVHD) as well as relapse (KIR2DS5). KIR2DS1 incompatibility in the same direction in the presence of HLA‐C‐group 2 ligand in recipient was associated with reduced overall (risk ratio, RR = 3.01; P = 0.01) and disease‐free survival (RR = 2.92, P = 0.03). Activating mismatches in P^?D⁺ direction resulted in decreased CD4⁺ : CD8⁺ T‐cell ratio up to 1 yr after alloHSCT, as a consequence of decreased CD3⁺CD4⁺ number within the first 100 d and increased CD3⁺CD8⁺ number in later time‐points. Among six evaluated patients, expression of activating KIRs on NK cells and T cells was particularly prominent for those developing intestinal GVHD. Conclusion: Our findings indicate that the presence of particular activating KIRs in donor with their absence in recipient enhances GVHD, which is not accompanied by graft‐versus‐leukemia effect. Evaluation of activating KIR genotype may allow optimization of both donor selection and transplantation procedure in order to avoid GVHD.     

Acquisition of t(11;14) in a patient with chronic lymphocytic leukemia carrying both t(14;19)(q32;q13.1) and +12

A rare recurrent chromosomal translocation, t(14;19)(q32;q13), has been identified in a variety of B‐cell malignancies, including chronic lymphocytic leukemia (CLL). We report a unique case of CLL in a patient carrying both trisomy 12 and t(14;19) (q32;q13.1), in whom t(11;14)(q13;q32) developed at relapse. The patient was a 77‐yr‐old woman, and her lymphoma cells at presentation showed CD5⁺, CD10^?, CD19⁺, CD20⁺(dim), CD23⁺, CD38⁺, and CD11c⁺. At relapse, the patient's lymphoma cells showed positive staining for cyclin D1 in addition to CD5, CD20, and CD23. Lymphoma cells in specimens at both presentation and relapse were positive for lymphoid enhancer factor 1 (LEF1) and negative for sex‐determining region Y‐box 11 (SOX11). IGH‐BCL1 FISH became positive at relapse. Split FISH assay using BCL1, BCL3, IGH, and CCND1 probes on lymph node specimens obtained at presentation and at autopsy confirmed that the translocation of BCL3 was solely detected in the lymph node at presentation and detected BCL3 and CCND1 translocations in the specimen at autopsy. These observations indicated that IGH‐BCL3 and IGH‐CCND1 had occurred in the same clone after treatment of the disease. In line with immunohistochemical and cytogenetic studies, additional PCR analysis of the FR3‐JH region showed the same sequence derived from IGHV4‐34 in specimens obtained at disease onset and relapse.     

Very early onset of an acute myeloid leukemia in an adult patient with B‐cell lymphoblastic leukemia

We report on a case of a 30‐year‐old male with acute B‐lymphoblastic leukemia (B‐ALL) with immunophenotype CD19⁺, CD22⁺, CD20⁺, CD10⁺, with aberrant expression of CD13 and CD117, and IgH gene rearrangements. Three months after treatment with GMALL‐2003 and Ida/FLAG protocols bone marrow showed predominance of blasts with myeloid morphology and phenotype MPO⁺, CD13⁺, CD33⁺, CD64⁺, CD15⁺, CD56⁺, EVI‐1 gene overexpression and lack of IgH rearrangements. The case is the first report of a very early emergence of myeloid leukemia during the induction treatment for B‐ALL in an adult patient. Different pathogenetic mechanisms are discussed – clonal evolution or selection, lineage switch or development of a de novo or therapy‐induced leukemia.     

IL‐6 promotes CD4+ T‐cell and B‐cell activation during Plasmodium infection

Humoral immunity develops in the spleen during blood‐stage Plasmodium infection. This elicits parasite‐specific IgM and IgG, which control parasites and protect against malaria. Studies in mice have elucidated cells and molecules driving humoral immunity to Plasmodium, including CD4⁺ T cells, B cells, interleukin (IL)‐21 and ICOS. IL‐6, a cytokine readily detected in Plasmodium‐infected mice and humans, is recognized in other systems as a driver of humoral immunity. Here, we examined the effect of infection‐induced IL‐6 on humoral immunity to Plasmodium. Using P. chabaudi chabaudi AS (PcAS) infection of wild‐type and IL‐6^?/? mice, we found that IL‐6 helped to control parasites during primary infection. IL‐6 promoted early production of parasite‐specific IgM but not IgG. Notably, splenic CD138⁺ plasmablast development was more dependent on IL‐6 than germinal centre (GC) B‐cell differentiation. IL‐6 also promoted ICOS expression by CD4⁺ T cells, as well as their localization close to splenic B cells, but was not required for early Tfh‐cell development. Finally, IL‐6 promoted parasite control, IgM and IgG production, GC B‐cell development and ICOS expression by Tfh cells in a second model, Py17XNL infection. IL‐6 promotes CD4⁺ T‐cell activation and B‐cell responses during blood‐stage Plasmodium infection, which encourages parasite‐specific antibody production.     

Optimization of isolation and further characterization of umbilical cord blood‐derived very small embryonic/ epiblast‐like stem cells (VSELs)

Because of their small size and density, umbilical cord blood (UCB)‐derived very small embryonic/epiblast‐like stem cells (VSELs) are usually lost at various steps of UCB preparation. Accordingly, we noticed that a significant number of these cells, which are smaller than erythrocytes, are lost during gradient centrifugation over Ficoll‐Paque as well as during routine volume depletion of UCB units before freezing. To preserve these cells in final UCB preparations, we propose a relatively short and economical three‐step isolation protocol that allows recovery of approximately 60% of the initial number of Lin^?/CD45^?/CD133⁺ UCB‐VSELs present in freshly harvested UCB units. In this novel approach (i) UCB is lysed in a hypotonic ammonium chloride solution to deplete erythrocytes; (ii) CD133⁺ including VSELs cells are enriched by employing immunomagnetic beads; and subsequently (iii) Lin^?/CD45^?/CD133⁺ cells are sorted by fluorescence‐activated cell sorting. The whole isolation procedure takes approximately 2–3 h per UCB unit and isolated cells are highly enriched for an Oct‐4⁺ and SSEA‐4⁺ population of small Lin^?/CD45^?/CD133⁺ cells.     

Myeloma cells act as tolerogenic antigen‐presenting cells and induce regulatory T cells in vitro

Regulatory T cells (Tregs) are essential for maintenance of self‐tolerance; however, tumor cells can exploit the tolerance to escape the immune system. We investigated the Tregs frequency in patients with multiple myeloma (MM) and in those with monoclonal gammopathy of undetermined significance (MGUS), and found that CD4⁺FoxP3⁺ and CD8⁺FoxP3⁺ Tregs were significantly increased in patients with MM and correlated with the active phase. Both Tregs subsets were expanded in cocultures of CD3⁺ lymphocytes and fresh CD138⁺ MM plasma cells or RPMI8226 and U266 cell lines and functioned as natural (n) and inducible (i) Tregs insofar as they inhibited the proliferation of stimulated CD3 lymphocytes via contact‐dependent and contact‐independent pathways. Induction of Tregs by MM plasma cells required a contact‐dependent pathway, implying antigen recognition by T cells. MM plasma cells acted as immature and tolerogenic antigen‐presenting cells (APCs), in that they displayed low CD80/CD86 expression associated with a phagocytic activity. By acting as immature APCs, MM plasma cells plausibly expand (n)Tregs and (i)Tregs both through conversion of CD3⁺FoxP3^? into CD3⁺FoxP3⁺ T cells and proliferation of CD3⁺FoxP3⁺ T cells, which may suppress the anti‐MM immune response.     

T-cell-rich B-cell lymphoma of the spleen presenting with severe hypersplenism

We report a 19-year-old woman who was presented with B-symptoms, massive splenomegaly, hepatomegaly and hypersplenism. She underwent diagnostic/therapeutic splenectomy. Microscopically, the spleen showed a vaguely micronodular and diffuse proliferation of lymphoid cells in the white pulp that also involved the red pulp. On immunohistochemical staining, this proliferation consisted predominantly of CD3(+), CD7(+) small T cells with the presence of a minor population of CD15(-),CD30(-), CD20(+) large atypical B cells. A liver biopsy also showed a similar morphology to that seen in the spleen. After splenectomy, only the pancytopenia improved. A combined immunochemotherapy regimen (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone) was utilized, which resulted in a complete remission.     

A prospective study of T‐ and B‐lymphocyte subpopulations,CD81 expression levels on B cells and regulatory CD4+CD25+CD127low/−FoxP3+ T cells in patients with chronic HCV infection during pegylated interferon‐alpha2a plus ribavirin treatment

Summary. Resolution of hepatitis C virus (HCV) infection requires a complex interplay between innate and adaptative immune responses. The role of lymphocyte subpopulations during combined antiviral treatment remains to be defined. This study was conducted to assess the effect of pegylated interferon‐alpha2a (pegIFN‐α2a) and ribavirin treatment on peripheral blood lymphocytes, mainly on CD81 expression on B cells and CD4⁺CD25⁺CD127^low/?FoxP3⁺ regulatory T cells (Tregs) in patients with chronic HCV infection. Thirty‐five patients with chronic HCV infection who started pegIFN‐α2a and ribavirin treatment were enrolled. Peripheral blood mononuclear cells (PBMC) were obtained at baseline before treatment (BT), mid‐treatment (MT), the end of treatment (ET) and 24 weeks post‐treatment (PT). During combined antiviral treatment, a significant decrease in the percentage of CD3⁺, CD8⁺, CD3⁺gamma/delta (γδ)⁺, CD19⁺ lymphocyte subpopulations and Tregs was observed. There was also a significant increase in the percentage of the CD4⁺ lymphocyte subpopulation and in CD81 expression levels on CD19⁺ B cells when BT was compared with ET (all P < 0.05). Seventeen patients were nonresponders (NR) and 18 had a sustained virological response (SVR). At baseline, NR patients had higher CD81 expression levels on CD19⁺ B cells (P = 0.017) and a higher Tregs percentage (P = 0.025) than SVR patients. Our results suggest that immunomodulation fluctuates during antiviral treatment and that percentage CD81 expression levels on B cells and Tregs might be useful as an immunological prognostic factor for pegIFN‐α2a and ribavirin treatment response in chronic HCV infection.     

Compartmentalization and its implication for peripheral immunologically‐competent cells to the liver in patients with HBV‐related acute‐on‐chronic liver failure

Aims: This study attempts to characterize the feature of immunologically competent cells (ICCs) and evaluate its clinical implication in patients with acute‐on‐chronic liver failure (ACLF) in relation to chronic hepatitis B virus (HBV) infection. Methods: Circulating ICCs were examined in ACLF patients (n = 75), as well as in patients with hepatitis B (CHB, n = 31), CHB‐related liver cirrhosis (LC, n = 36), and normal controls (NC, n = 30). Intrahepatic ICCs in some patients were further analyzed via immunohistochemical and flow cytometric assays. Results: Total lymphocytes, CD4⁺ T cells, CD8⁺ T cells, and NK cells in circulation were numerically lower in the ACLF and LC groups compared to the CHB and NC groups. Importantly, the number of these cells was significantly lower in non‐surviving ACLF patients compared with surviving ACLF patients. In comparison to NC, ACLF patients displayed a significantly higher ratio of liver‐infiltrating CD4⁺ T‐cell frequency than its circulating counterpart, suggesting that the possiblility of the ICCs compartmentalization from the peripheral blood into the liver in ACLF. Immunohistochemical analysis showed that intrahepatic CD4⁺ cells, CD8⁺ cells, and CD56⁺ cells were significantly higher in the ACLF group compared with the other three groups, suggesting a stronger cellular immune response‐mediated inflammation in ACLF group than other patient groups. Conclusions: The abnormal prevalence of circulating and intrahepatic ICCs possibly acts as an important factor that may drive the progression of HBV‐related ACLF.     

CD43 expression is associated with inferior survival in the non‐germinal centre B‐cell subgroup of diffuse large B‐cell lymphoma

Cytomegalovirus (CMV) infections post‐haematopoietic stem cell transplantation (HSCT) can be effectively controlled through the adoptive transfer of donor‐derived CMV‐specific T cells (CMV‐T). Current strategies involve a second leukapheresis collection from the original donor to manufacture CMV‐T, which is often not possible in the unrelated donor setting. To overcome these limitations we have investigated the use of a small aliquot of the original granulocyte‐colony stimulating factor (G‐CSF) mobilized HSCT graft to manufacture CMV‐T. We explored the T cell response to CMVpp65 peptide stimulation in G‐CSF mobilized peripheral blood mononuclear cells (PBMC) and subsequently examined isolation of CMV‐T based on the activation markers CD154 and CD25. CD25⁺ enriched CMV‐T from G‐CSF mobilized PBMC contained a higher proportion of FoxP3 expression than non‐mobilized PBMC and showed superior suppression of T cell proliferation. Expanded CMV‐T enriched through CD154 were CD4⁺ and CD8⁺, demonstrated a high specificity for CMV, secreted cytotoxic effector molecules and lysed CMVpp65 peptide‐loaded phytohaemagglutinin‐stimulated blasts. These data provide the first known evidence that CMV‐T can be effectively manufactured from G‐CSF mobilized PBMC and that they share the same characteristics as CMV‐T isolated in an identical manner from conventional non‐mobilized PBMC. This provides a novel strategy for adoptive immunotherapy that abrogates the need for successive donation.     

Abnormal expansion of naïve B lymphocytes after unrelated cord blood transplantation – a case report

A 33‐year‐old woman underwent unrelated cord blood transplantation (U‐CBT) for myelodysplastic syndrome (MDS)‐related secondary AML. She showed impressive increases in the number of CD19⁺ B cells in bone marrow and CD19⁺27^?IgD⁺ B cells in peripheral blood from about 1 month to 3 months after U‐CBT. The serum level of IL‐6 temporarily increased after transplantation, and this increase seemed to be correlated with the expansion of CD19⁺ B cells. Although, compared with BMT, little is known about the kinetics of hematological and immunological reconstitution in U‐CBT, there was initial B‐cell recovery after CBT as some described. This B cell recovery may be associated with a high number of B‐cell precursors present in cord blood (CB). The phenomenon of naïve B lymphocyte expansion that we found might be associated with a high number of B‐cell precursors present in CB.     

Prevention of acute graft‐versus‐host disease by humanized anti‐CD26 monoclonal antibody

CD26 (DPP4) is a T cell costimulatory molecule as well as T cell activation marker, and CD26⁺ T cells are accumulated in inflamed tissues, such as rheumatoid synovitis and autoimmune thyroiditis. In the present study, we found accumulation of CD26⁺ T cells in graft‐versus‐host disease (GVHD) target organs. To expand our in vitro findings to an in vivo system, we examined CD26‐dependent organ injury in a xenogeneic GVHD (x‐GVHD) murine model. Following intraperitoneal injection of human peripheral blood mononuclear cells into non‐obese diabetic severe combined immunodeficiency/γ_c^?/? mice (hu‐PBL‐NOG mice), the mice exhibited the onset of GVHD symptoms associated with the presence of CD26^high human lymphocytes in the peripheral blood and GVHD target tissues. Administration of humanized anti‐human CD26 monoclonal antibody (mAb) decreased x‐GVHD severity and prolonged survival in hu‐PBL‐NOG mice without loss of engraftment of human T cells, while increasing doses of CTLA4‐ immunoglobulin fusion protein diminished engraftment of human lymphocytes. Importantly, anti‐CD26 mAb treatment preserved the graft‐versus‐leukaemia effects in studies using cotransplantation of P815 murine leukaemic cells. In addition, CD26⁺ lymphocytes infiltrated the GVHD patients' target tissues. Altogether, our data indicate a role for CD26 in the regulation of GVHD and point to CD26 as a novel target for therapeutic intervention in this disease.