Caroline Arber

Ex Vivo Expanded Dendritic Cells Home to T-Cell Zones of Lymphoid Organs and Survive in Vivo after Allogeneic Bone Marrow Transplantation

Little is known about adoptive transfer of allogeneic ex vivo expanded dendritic cells (eDCs). We investigated the trafficking pattern of eDCs in mice after allogeneic bone marrow transplantation by using bioluminescence imaging. eDCs were expanded from bone marrow precursors in the presence of GM-CSF, interleukin-4, and Flt3L and retrovirally transduced to express luciferase (luc) and green fluorescence protein (gfp). Flow cytometry showed polyclonal DC populations after expansion that consisted of CD11c+CD11b+ and CD11c-CD11b+ cells that co-expressed CD40, CD80, CD86, and MHCII. eDCs were functional in mixed lymphocyte reactions and produced tumor necrosis factor-alpha on phytohemagglutinin stimulation. The eDCs were then injected intravenously into BALB/c recipient mice that had received allogeneic bone marrow transplantation 6 weeks previously. On day 1 after transfer, eDCs were detected by bioluminescence imaging throughout the lungs and spleen. In the later course, signals were observed throughout thymus, lower abdomen, and spleen throughout a period of more than 42 days. Immunofluorescence microscopy confirmed CD11c positivity on the gfp+ donor cells, which localized in T-cell zones of mesenteric lymph nodes, Peyers patches, spleen, and thymus. These findings are important for adoptive immunotherapies because they indicate that eDCs migrate efficiently in vivo and are capable of surviving long term.

Patient and product factors affecting platelet transfusion results

BACKGROUND: Providing patients with platelet (PLT) transfusions requires important logistic resources and represents a considerable cost factor. Optimizing PLT transfusions is in the interest of not only patient safety but also economic importance. Only few studies have evaluated factors associated with transfusion results.

Survivin-specific T cell receptor targets tumor but not T cells

Survivin is a tumor-associated antigen (TAA) that inhibits apoptosis and is widely overexpressed in cancer cells; therefore, survivin has potential as a target for cancer immunotherapy. Application of HLA-A2-restricted survivin-specific T cell receptors (TCRs) isolated from allogeneic HLA-mismatched TCR repertoires has, however, been impeded by the inability of these TCRs to distinguish healthy cells expressing low levels of survivin from cancer cells with high survivin expression levels. Here, we identified an HLA-A2-restricted survivin-specific TCR isolated from autologous TCR repertoires that targets tumor cells in vitro and in vivo but does not cause fratricidal toxicity. Molecular modeling of the TCR-peptide-HLA ternary complexes and alanine scanning revealed that the autologously derived TCRs had tighter interactions with the survivin peptide than did fratricidal TCRs. Similar recognition patterns were observed among 7 additional TAA-specific TCRs isolated from allogeneic versus autologous repertoires. Together, the results from this study indicate that maximal peptide recognition is key for TCR selectivity and likely critical for reducing unwanted off-target toxicities. Moreover, isolating TCRs from autologous repertoires to maximize TCR selectivity has potential as a useful strategy to identify and select other shared tumor- and self-antigen-specific TCRs and ensure selective antitumor activity.

The immunogenicity of virus-derived 2A sequences in immunocompetent individuals.

Genetic engineering of T cells for adoptive immunotherapy in cancer patients has shown significant promise. To ensure optimal antitumor activity and safety, the simultaneous expression of multiple genes is frequently required, and short viral-derived 2A sequences are increasingly preferred for this purpose. Concerns exist, however, that these virus-derived sequences may induce unwanted immune responses, and thus diminish persistence of the gene-modified cells after adoptive transfer. Whereas such responses were absent in immunocompromised recipients, potential immunogenicity in immunocompetent individuals remains a concern. We now address whether ex vivo T cell responses can be elicited against the most widely used 2A sequences (2A-Thosea asigna virus (TAV) or 2A-equine rhinitis virus (ERAV), specifically) in immunocompetent individuals. We used a potent ex vivo culture system previously validated to induce T cell responses even against weakly immunogenic antigens. Of the sixteen donors tested, only five released very low levels of interferon-γ in response to 2A-TAV peptide mixtures (single peptide specificity in three donors, adjacent self-antigen peptide specificity in one donor and nonspecific reactivity in one donor). None of them produced cytotoxic activity or responded to 2A-ERAV. These results suggest that exposure to viral-derived 2A sequences is unlikely to produce unwanted T cell responses in immunocompetent individuals and further supports their continued use for studies of human gene therapy.

Septic polyarthritis with Ureaplasma urealyticum in a patient with prolonged agammaglobulinemia and B-cell aplasia after allogeneic HSCT and rituximab pretreatment

A recent study in this journal1 raised the issue of prolonged hypogammaglobulinemia in patients receiving autologous hematopoietic stem cell transplantation (HSCT) for high-risk B-cell lymphoma and maintenance therapy with rituximab. Despite significant hypogammaglobulinemia, these patients did not experience significant infectious complications. With the following case report, we aim to increase the awareness of the potential for severe infectious complications associated with the hypogammaglobulinemia observed in lymphoma patients given lymphocytotoxic chemotherapy in combination with rituximab before allogeneic HSCT.2

Protection against Lethal Aspergillus fumigatus Infection in Mice by Allogeneic Myeloid Progenitors Is Not Major Histocompatibility Complex Restricted

Invasive fungal infections are a leading cause of morbidity and mortality after myelotoxic chemotherapy or radiation exposure. The resulting depletion of myeloid precursors under these conditions appears to be the factor that limits approaches to accelerate immune reconstitution. In a murine model of myeloablation after radiation exposure, we demonstrated that highly purified common myeloid and granulocyte-monocyte progenitors (CMPs/GMPs) accelerated myeloid recovery and, thus, enhanced innate immunity as measured by survival after a lethal challenge with Aspergillus fumigatus. Of greatest significance was the demonstration that the protection afforded by CMPs/GMPs was not major histocompatibility complex restricted. Furthermore, the effect of CMP/GMP cellular therapy was additive with that of liposomal amphotericin B treatment. These observations greatly expand the potential donor pool and, thus, the clinical utility of CMP/GMP cellular therapy in patients with myeloid depletion.

C-reactive protein and fever in neutropenic patients.

The predictive value of daily C-reactive protein (CRP) monitoring to distinguish causes of fever in neutropenic patients was studied retrospectively. A total of 143 fever episodes during 113 consecutive hospitalizations were studied in 71 patients who had been referred for chemotherapy or haemopoietic stem cell transplantation (HSCT). There were, on average, 1.3 fever episodes per hospital stay, attributed to: infection (55, 27 invasive bacterial, 5 fungal, 3 viral and 20 probable infections); acute graft vs host disease (GvHD) (20); drugs (22); transfusions (7); and not attributable (39). 130 (91%) fever episodes were accompanied by a rise in CRP, 6 (4%) episodes were fatal. Maximal CRP levels (CRPmax) and maximal temperature (Tmax) were higher in invasive bacterial infections than in aGvHD and higher in aGvHD than in drug- or transfusion-related fever (p < 0.0001). Temperature and CRP rose in parallel. A total of 16 patients developed grade II-IV aGvHD by day 11 (9-21) (median, range) after allogeneic HSCT. Acute GvHD was preceded by fever for 3 d (1-7), and by CRP increase for 5 d (0-15) (p < 0.0001). CRP monitoring may be useful to distinguish between causes of fever. Very high CRP levels tend to be associated with invasive bacterial infections. CRP is not an early warning sign. An increase in CRP and fever may precede other clinical manifestations of aGvHD.The predictive value of daily C-reactive protein (CRP) monitoring to distinguish causes of fever in neutropenic patients was studied retrospectively. A total of 143 fever episodes during 113 consecutive hospitalizations were studied in 71 patients who had been referred for chemotherapy or haemopoetic stem cell transplantation (HSCT). There were, on average, 1.3 fever episodes per hospital stay, attributed to: infection (55, 27 invasive bacterial, 5 fungal, 3 viral and 20 probable infections); acute graft vs host disease (GvHD) (20); drugs (22); transfusions (7); and not attributable (39). 130 (91%) fever episodes were accompanied by a rise in CRP, 6 (4%) episodes were fatal. Maximal CRP levels (CRPmax) and maximal temperature (Tmax) were higher in invasive bacterial infections than in aGvHD and higher in aGvHD than in drug- or transfusion-related fever ( p B 0.0001). Temperature and CRP rose in parallel. A total of 16 patients developed grade II-IV aGvHD by day 11 (9-21) (median, range) after allogeneic HSCT. Acute GvHD was preceded by fever for 3 d (1-7), and by CRP increase for 5 d (0-15) ( p B 0.0001). CRP monitoring may be useful to distinguish between causes of fever. Very high CRP levels tend to be associated with invasive bacterial infections. CRP is not an early warning sign. An increase in CRP and fever may precede other clinical manifestations of aGvHD.

In situ RHAMM protein expression in acute myeloid leukemia blasts suggests poor overall survival

Treatment options for patients with high-risk acute myeloid leukemia (AML) include high-dose chemotherapy regimens in combination with allogeneic hematopoietic stem cell transplantation, which takes advantage of the donor T-cell-mediated graft-versus-leukemia effect. Together with beneficial responses observed in assays targeted at leukemia-associated antigens (LAA), this encouraged research on cancer vaccines and adoptive cellular therapies in AML. The receptor for hyaluronic acid-mediated motility (RHAMM, CD168) was identified as one of the most promising LAA in AML. Thus far, little is known about in situ expression in leukemic bone marrow blasts or the prognostic role of RHAMM and its interaction partners in AML. We immunohistochemically analyzed the expression and prognostic significance of RHAMM on trephine bone marrow biopsies from 71 AML cases that had been evaluated for cytogenetics and presence of FLT3-internal tandem duplications and NPM1 mutations. Fifty-five patients (77%) were treated with curative intent, while 16 (23%) received the most appropriate supportive care. Twenty of 71 (28%) AML cases were considered RHAMM+. Receiver operating characteristic curves showed significant discriminatory power considering overall survival (OS) in AML patients treated curatively for RHAMM (p = 0.015). Multivariable analysis revealed that expression of RHAMM in >5% of leukemic blasts identifies a subgroup of curatively treated cases with adverse OS independent of failures to achieve complete remission. RHAMM not only represents a promising LAA with specific T-cell responses in AML but, if assessed in situ on blasts, also a probable prognostic factor.

Impaired B-cell reconstitution in lymphoma patients undergoing allogeneic HSCT: an effect of pretreatment with rituximab?

Allogeneic hematopoietic SCT (HSCT) is increasingly considered an option in refractory or relapsing lymphoma. Today, most patients with B-cell lymphoma are treated with the monoclonal anti-CD20 antibody rituximab before HSCT. We hypothesized that prior therapy with rituximab might alter immune reconstitution after allogeneic transplantation due to in vivo depletion of B cells at the time of graft infusion. We studied B-cell immune reconstitution in 12 patients with lymphoma receiving rituximab 1–12 months before HSCT. Compared to an age- and sex-matched population of patients transplanted for myeloid malignancies, lymphoma patients with rituximab pretreatment showed significantly reduced B-cell counts at time of HSCT at +3, +6 and +12 months; B-cell counts reached values comparable to controls only 24 months after HSCT. In parallel, levels of immunoglobulins were markedly reduced for up to 2 years post transplant in patients with prior rituximab treatment. Two patients suffered from severe late bacterial infections to which the impaired humoral immunity may have contributed. In contrast, T- and NK-cell reconstitution was not different compared to control patients.In conclusion, B-cell reconstitution can be significantly delayed in allogeneic HSCT recipients with prior rituximab treatment. Rituximab appears to have clinical consequences beyond the immediate early post-transplant period.

High-dose chemotherapy using BEAM without autologous rescue followed by reduced-intensity conditioning allogeneic stem-cell transplantation for refractory or relapsing lymphomas: a comparison of delayed versus immediate transplantation.

Patients with refractory/relapsing lymphoma are rarely cured by chemotherapy. High-dose chemotherapy (HDC) for tumor debulking followed by reduced-intensity conditioning (RIC) hematopoietic stem-cell transplantation (HSCT) has been advocated as a concept. We previously treated 10 patients (group A) with BEAM chemotherapy followed by delayed RIC HSCT at day 28. We now report on the subsequent 11 patients receiving BEAM followed immediately by fludarabine/total body irradiation and allogeneic HSCT (group B), and compare the outcome to group A patients. Non-hematological toxicity before engraftment was comparable, only gut toxicity was higher in group B. Days in aplasia, days on antibiotics and length of hospital stay were significantly longer in group A. Cumulative incidence of acute (GvHD) ⩾grade II and incidence of chronic GvHD were lower in group B. At last follow-up, seven patients in group A were alive, with six of them in complete remission. In group B, nine patients were alive, seven of them in complete remission. No significant difference in estimated 3-year overall survival was seen. These data challenge the initial concept of debulking first and delaying allogeneic RIC HSCT. Allogeneic HSCT with standard BEAM conditioning is a valid alternative for patients with resistant/relapsed lymphoma, which might be considered earlier in the disease course.