Eric Braakman

A randomized multicenter comparison of CD34+-selected progenitor cells from blood vs from bone marrow in recipients of HLA-identical allogeneic transplants for hematological malignancies

OBJECTIVE Peripheral blood progenitor cells (PBPC) have been established as an alternative source of hematopoietic stem cells for allogeneic transplantation, but an increased incidence of both acute and chronic graft-vs-host disease (GVHD) has become apparent. We performed a prospective randomized trial comparing bone marrow transplantation (BMT) vs PBPC transplantation (PBPCT) using CD34(+) selection for T-cell depletion (TCD) in both study arms. PATIENTS AND METHODS Between January 1996 and October 2000, 120 patients with a diagnosis of acute leukemia, myelodysplasia, multiple myeloma, or lymphoma were randomized to receive either filgrastim-mobilized PBPC or BM from HLA-identical sibling donors after standard high-dose chemoradiotherapy. Patient characteristics did not differ between study arms. RESULTS Recipients of PBPC received more CD3(+) T cells (median: 3.0 vs 2.0 x 10(5)/kg, p<0.0001) and more CD34(+) cells (median: 3.6 vs 0.9 x 10(6)/kg, p<0.0001). Neutrophil and platelet recoveries occurred significantly faster after PBPCT. The cumulative incidence of acute GVHD grades II-IV was 37% after BMT vs 52% after PBPCT and was most significantly (p=0.007) affected by the number of CD3(+) T cells in the graft. Acute GVHD appeared strongly associated with increased treatment-related mortality (TRM) in a time-dependent analysis. Higher numbers of CD34(+) cells were associated with less TRM. With a median follow-up of 37 months (range: 12-75), overall survival at 4 years from transplantation was 60% after BMT and 34% for recipients of PBPCT (p=0.04), which difference was largely due to increased GVHD and TRM in PBPC recipients receiving T-cell dosages greater than 2 x 10(5)/kg. CONCLUSION Outcome following T cell-depleted PBPCT critically depends on the number of CD3(+) T cells, whereby high T-cell numbers may blunt a favorable effect of higher CD34(+) cell numbers.

CD16 on human γδ T lymphocytes: Expression, function, and specificity for mouse IgG isotypes☆

Abstract We examined the expression, the signal transduction capacity and mouse IgG-isotype specificity of CD16 on human γδ T cells. CD16 is expressed by the majority of γδ T cells in peripheral blood and by part of the γδ T cell clones. The amount of CD16 expressed on γδ T cell clones varied considerably with passaging of the cells, but was always significantly less than on freshly isolated γδ T cells. Like CD16 on CD3−CD16+ natural killer (NK) cells, CD16 on γδ T cells can act as an activation site triggering cytotoxic activity. CD16+ γδ T cell clones exerted antibody-dependent cellular cytotoxicity (ADCC) which could be blocked by anti-CD16 mAb. ADCC activity of γδ T cell clones was also inhibited by anti-CD3 mAb, suggesting a functional linkage between the CD16 and CD3 activation pathways. MAb directed against CD16 induced lysis of FcγR+ target cells by CD16+ γδ T cell clones. The mouse IgG-isotype specificity of CD16 on γδ T cells was analyzed using isotype switch variants of a murine anti-glycophorin A mAb in EA rosette assays, and was found to be identical to that of CD16 on CD3−CD16+ NK cells, i.e., highest affinity for mIgG2a, intermediate affinity for mIgG2b, and undetectable binding of mIgG1-sensitized erythrocytes. CD16 was partly modulated from the cell surface of both γδ T cells and NK cells after rosette formation with mIgG2a-sensitized erythrocytes, indicating that the rosette formation was indeed mediated via the CD16 molecule.

Flt3 Ligand Expands Lymphoid Progenitors Prior to Recovery of Thymopoiesis and Accelerates T Cell Reconstitution after Bone Marrow Transplantation

Deficient thymopoiesis and retarded recovery of newly developed CD4+ T cells is one of the most important determinants of impaired immunocompetence after hemopoietic stem cell transplantation. Here we evaluated whether Fms-like tyrosine kinase 3 (Flt3) ligand (FL) alone or combined with IL-7 affects T cell recovery, thymopoiesis, and lymphoid progenitor expansion following bone marrow transplantation in immunodeficient mice. FL strongly accelerated and enhanced the recovery of peripheral T cells after transplantation of a low number of bone marrow cells. An additive effect on T cell recovery was not observed after coadministration of IL-7. Lineage−sca-1+c-kit+flt3+ lymphoid progenitor cell numbers were significantly increased in bone marrow of FL-treated mice before recovery of thymopoiesis. Thymocyte differentiation was advanced to more mature stages after FL treatment. Improved T cell recovery resulted in better immunocompetence against a post-bone marrow transplantation murine CMV infection. Collectively, our data suggest that FL promotes T cell recovery by enhanced thymopoiesis and by expansion of lymphoid progenitors.

Reduced Graft-Versus-Host Disease-Inducing Capacity of T Cells After Activation, Culturing, and Magnetic Cell Sorting Selection in an Allogeneic Bone Marrow Transplantation Model in Rats

Graft-versus-host disease (GvHD), a major complication of allogeneic bone marrow transplantation, has been ascribed to mature T cells in the graft. Because T cells play an important role in engraftment of the bone marrow and decrease the probability of relapse of leukemia, a treatment strategy was developed to preserve the benefits of T cells in the graft and to control the severe complications of GvHD. This can be accomplished by the genetic modification of donor T cells with a suicide gene that allows their selective in vivo elimination and subsequently the abrogation of GvHD. For clinical benefit the alloreactivity of herpes simplex virus thymidine kinase (HSV-TK) gene-transduced T cells should be retained. Therefore, we investigated the influence of gene transduction and the selection procedure on T cells. We demonstrated that activation and culturing of T cells reduce their capacity to induce lethal GvHD in an allogeneic rat bone marrow transplantation model. Furthermore, positive immunomagnetic selection of gene-transduced T cells resulted in loss of the GvHD-inducing capacity of HSV-TK(+) T cells directly after MACS (magnetic cell sorting) selection; this loss could be recovered by a 1-day expansion of the selected T cells. No effect on alloreactivity was observed to be caused by the gene transduction procedure. Our study resulted in the development of an optimized culture and gene transduction protocol with preservation of T cell alloreactivity. Treatment of transplanted rats with ganciclovir resulted in a rapid reduction in the number of HSV-TK(+) T cells in the peripheral blood and in increased survival of the animals.

Quantification of newly developed T cells in mice by real-time quantitative PCR of T-cell receptor rearrangement excision circles

OBJECTIVE Thymic output of newly developed ab T cells in humans can be measured via signal joint T-cell receptor rearrangement excision circles (sjTRECs). Deletion of the TCRD locus via dRec to psiJa recombination during TCRA rearrangement results in the production of such sjTRECs. The deleting elements dRec and psiJa are highly conserved between humans and mice and used in a comparable manner. We developed and evaluated a real-time quantitative PCR (RQ-PCR) to detect and quantify dRec-psiJa sjTRECs in murine peripheral blood leukocytes for estimation of thymic output of newly developed ab T cells in mice. METHODS The threshold cycle (Ct) of the sjTREC RQ-PCR was related to the Ct value of an endogenous reference gene. The difference in Ct value (DCt) was correlated to the absolute numbers of CD45+ and CD3+ cells per mL of blood, as obtained by a single platform flow cytometric assay, resulting in the frequency of sjTRECs in CD45+ and CD3+ cells. RESULTS The RQ-PCR proved to be sensitive with a detection level of approximately one sjTREC copy in 100 ng of DNA. SjTRECs could not be detected in peripheral blood leukocytes of RAG-1(-/-) mice, demonstrating the specificity of the assay. As in humans and primates, sjTREC levels declined in aging and thymectomized mice. Remarkably, significant mouse strain-dependent differences in sjTREC levels were observed. 129Sv and C57BL/6 mice had significantly lower sjTREC levels in blood than Balb/c and DBA2 mice. CONCLUSION Quantification of murine sjTRECs by RQ-PCR may allow for accurate assessment of thymic output in mice.

T cell targeting in cancer therapy

SummaryTargeting of immune cells by bispecific antibodies has proven a powerful tool for the investigation of cellular cytotoxicity, lymphocyte activation and induction of cytokine production, as well as to represent an innovative form of immunotherapy for the treatment of cancer. The hallmark of this approach is the use of the specificity of monoclonal antibodies to join target and immune cells by virtue of the dual specificity of bispecific antibodies for the two entities. More precisely the bispecific antibody has two different binding sites, which are capable of recognizing tumor associated antigens on the one hand and lymphocyte activation sites on the other. This process of crosslinking results in the activation of the lymphocyte and triggering of its lytic machinery, as well as lymphokine production. A major advantage of this therapeutic modality is, that use is made of the normal cellular immune defence system and therefore is only associated with minor toxicity. The distinct lymphocyte populations, which can be used for adoptive immunotherapy and the various bispecific antibody preparations, as well as the chimeric immunoglobulin/T cell receptor construction are the major topics of this review.

Insufficient recovery of thymopoiesis predicts for opportunistic infections in allogeneic hematopoietic stem cell transplant recipients

Background Recovery of thymopoiesis after allogeneic hematopoietic stem cell transplantation is considered pivotal for full immune competence. However, it is still unclear to what extent insufficient recovery of thymopoiesis predicts for subsequent opportunistic infections and non-relapse mortality. Design and Methods A detailed survey of all post-engraftment infectious complications, non-relapse mortality and overall survival during long-term follow-up was performed in 83 recipients of allogeneic stem cell grafts after myeloablative conditioning. Recovery of thymopoiesis was assessed using analysis of signal joint T-cell receptor rearrangement excision circles. The impact of recovery of thymopoiesis at 2, 6, 9 and 12 months post-transplantation on clinical outcome beyond those time points was evaluated by univariate and multivariate Cox regression analyses. Results The cumulative incidence of severe infections at 12 months after transplantation was 66% with a median number of 1.64 severe infectious episodes per patient. Patients in whom thymopoiesis did not recover were at significantly higher risk of severe infections according to multivariable analysis. Hazard ratios indicated 3- and 9-fold increases in severe infections at 6 and 12 months, respectively. Impaired recovery of thymopoiesis also translated into a higher risk of non-relapse mortality and outweighed pre-transplant risk factors including age, donor type, and disease risk-status. Conclusions These results indicate that patients who fail to recover thymopoiesis after allogeneic hematopoietic stem cell transplantation are at very high risk of severe infections and adverse clinical outcome.

Paradoxical effects of interleukin-10 on the maturation of murine myeloid dendritic cells

The immunoregulatory cytokine, interleukin‐10 (IL‐10), has been shown to inhibit the maturation of human myeloid dendritic cells (DC). In the present study, we demonstrate that IL‐10 has paradoxical effects on the maturation of murine myeloid bone marrow‐derived DC. On the one hand, IL‐10 inhibits the maturation of murine myeloid DC. The addition of IL‐10 to granulocyte–macrophage colony‐stimulating factor (GM‐CSF)‐supported murine BM‐derived DC cultures reduced the frequency of major histocompatibility complex (MHC) class IIbright cells. These IL‐10‐pretreated DC have a reduced capacity to stimulate T cells in an allogeneic mixed leucocyte reaction. On the other hand, however, and in contrast to the effects of IL‐10 on human DC, we found that the addition of IL‐10 from the initiation of the culture onwards induced an up‐regulation of the expression of the costimulatory molecules CD40, CD80 and CD86 on murine myeloid DC, as compared to DC generated with GM‐CSF only. Moreover, a subpopulation of IL‐10‐pretreated MHC class IIdim DC lacked the capacity to take up dextran‐fluorescein isothiocyanate (FITC), a feature of DC maturation. Taken together, our data demonstrate that the generation of murine myeloid DC in the presence of IL‐10 results in a population of incompletely matured MHC class IIdim CD80+ CD86+ DC. These DC lack T‐cell stimulatory capacity, suggesting a role for IL‐10 in conferring tolerogenic properties on murine myeloid DC.

Keratinocyte Growth Factor Induces Expansion of Murine Peripheral CD4+Foxp3+ Regulatory T Cells and Increases Their Thymic Output

Keratinocyte growth factor (KGF) has been shown to reduce the incidence and severity of graft-versus-host disease by prevention of epithelial damage and by modulating alloreactivity. Since regulatory T cells (Treg) play a crucial role in immune modulation, we evaluated the effects of exogenous KGF on peripheral CD4+Foxp3+ Treg and the generation of Treg in the thymus of normal mice. A 3-day course of KGF induced a rapid selective increase in the number of highly suppressive CD4+Foxp3+ Treg. Blood Treg numbers remained elevated for >2 mo, but the frequency normalized after 2 wk due to a concomitant increase in CD4+Foxp3− T cells. Analysis of single joint TCR excision circles frequency and Ki-67 expression in peripheral blood Treg showed that the early selective increase of Treg was predominantly accounted for by peripheral expansion. Thymectomy before KGF administration did not affect the early selective increase of Treg but abrogated the late increase in CD4+ T cell numbers, thereby showing its dependence on thymic output. Collectively, these results show that KGF induces an increase in blood CD4+Foxp3+ Treg numbers via two independent mechanisms. First by selective peripheral expansion of Treg and thereafter by enhanced thymic output of newly developed Treg.

Second gene expression in bicistronic constructs using short synthetic intercistrons and viral IRES sequences

In this study, we describe the efficiency of second gene translation in bicistronic constructs containing either a short (36bp) synthetic intercistron or known internal ribosomal entry sites (IRES). Experiments were performed using two different gene combinations: Herpes simplex virus-thymidine kinase (HSV-TK) and neomycine (NEO) or human glucocerebrosidase (hGC) and a methotrexate (MTX) resistant mutant dihydrofolate reductase (DHFR). We demonstrate that upon transfection, second gene translation is efficient using either an IRES or a 36-bp intercistron. Infection with retrovirus carrying the TK and NEO genes linked via a 36-bp intercistron resulted in both G418R (NEO expression) and gancyclovir (GCV) sensitivity (TK expression), indicating that both genes were expressed and thus that the genomic DNA and RNA of this bicistronic construct were intact. Likewise, retrovirus carrying the hGC and mutant DHFR gene separated by a short intercistron was harvested from MTXR murine PsiCRE cells. However, infection of PA317 cells with this virus supernatant did not result in the presence of hGC enzyme activity in these murine cells. Proviral DNA and RNA analyses indicated that the hGC coding region was lost from the original construct in the infected PA317 cells. In contrast, retrovirus carrying the hGC and DHFR cDNAs was linked via an IRES functioned as expected. Based on these results, we conclude that the efficiency of second gene translation using short synthetic intercistrons might prove useful in bicistronic constructs, depending on the gene combination used.