Restoration of immunity in lymphopenic individuals with cancer by vaccination and adoptive T-cell transfer

Immunodeficiency is a barrier to successful vaccination in individuals with cancer and chronic infection. We performed a randomized phase 1/2 study in lymphopenic individuals after high-dose chemotherapy and autologous hematopoietic stem cell transplantation for myeloma. Combination immunotherapy consisting of a single early post-transplant infusion of in vivo vaccine-primed and ex vivo costimulated autologous T cells followed by post-transplant booster immunizations improved the severe immunodeficiency associated with high-dose chemotherapy and led to the induction of clinically relevant immunity in adults within a month after transplantation. Immune assays showed accelerated restoration of CD4 T-cell numbers and function. Early T-cell infusions also resulted in significantly improved T-cell proliferation in response to antigens that were not contained in the vaccine, as assessed by responses to staphylococcal enterotoxin B and cytomegalovirus antigens (P < 0.05). In the setting of lymphopenia, combined vaccine therapy and adoptive T-cell transfer fosters the development of enhanced memory T-cell responses.     

Influence of MHC on thymus repopulation following intrathymic transfer of mouse T-cell precursors

T-cell precursors (pre-T cells) traditionally have been detected by their ability to repopulate the thymus of heavily irradiated mice following intravenous injection. Recently, an assay system involving the direct injection of pre-T cells into the thymus of sublethally irradiated animals has been described. Here we report the results of experiments designed to evaluate the ability of bone marrow cells to produce thymic repopulation following intrathymic injection in a wide range of donor-host strain combinations. Irradiated (600 R) mice were injected intrathymically with 2 X 10(6) bone marrow cells which differed from the recipient with respect to their Thy-1 allotype and the percentage of thymus cells expressing either donor- or recipient-type Thy-1 was determined 9 to 23 days after injection. The results of these experiments showed that thymocytes expressing the Thy-1 allotype derived from the donor marrow were only detected when the donor and host were matched at MHC. By contrast, thymic repopulation by MHC-mismatched donor marrow cells could readily be observed when these cells were given intravenously.     

GMP production of anti-tumor cytotoxic T-cell lines for adoptive T-cell therapy in patients with solid neoplasia

BACKGROUND: The adoptive transfer of ex vivo-induced tumor-specific T-cell lines provides a promising approach for cancer immunotherapy. We have demonstrated previously the feasibility of inducing in vitro long-term anti-tumor cytotoxic T-cell (CTL) lines directed against different types of solid tumors derived from both autologous and allogeneic PBMC. We have now investigated the possibility of producing large amounts of autologous anti-tumor CTL, in compliance with good manufacturing practices, for in vivo use. METHODS: Four patients with advanced solid tumors (two sarcoma, one renal cell cancer and one ovarian cancer), who had received several lines of anticancer therapy, were enrolled. For anti-tumor CTL induction, patient-derived CD8-enriched PBMC were stimulated with DC pulsed with apoptotic autologous tumor cells (TC) as the source of tumor Ag. CTL were then restimulated in the presence of TC and expanded in an Ag-independent way. RESULTS: Large amounts of anti-tumor CTL (range 14-20 x 10(9)), which displayed high levels of cytotoxic activity against autologous TC, were obtained in all patients by means of two-three rounds of tumor-specific stimulation and two rounds of Ag-independent expansion, even when a very low number of viable TC was available. More than 90% of effector cells were CD3(+) CD8(+) T cells, while CD4(+) T lymphocytes and/or NK cells were less than 10%. DISCUSSION: Our results demonstrate the feasibility of obtaining large quantities of anti-tumor specific CTL suitable for adoptive immunotherapy approaches.     

Anti-asialo GM1 eliminates both inflammatory process and cytotoxic T-cell function in the lymphocytic choriomeningitis adoptive transfer model

The induction of severe inflammatory process and fatal neurological disease by transfer of lymphocytic choriomeningitis virus (LCMV)-immune T cells into cyclophosphamide (Cy)-suppressed LCMV-infected mice is greatly inhibited by treatment of these recipients with antibody to the asialo GM1 ganglioside (anti-ASGM1). Examination of cytotoxic activity in lymphoid tissue of the Cy-suppressed recipients at 72 hr after cell transfer revealed that anti-ASGM1 treatment prevented the development of the cytotoxic T lymphocyte (CTL) response, even though the dose of antibody used did not significantly decrease CTL generation in unsuppressed mice. Abrogation of CTL activity was also observed following antibody treatment of NK-deficient (bg/bg) Cy-suppressed recipients, indicating that the anti-ASGM1 was unlikely to be operating via removal of NK cells that are in some way involved in the development of the CTL response. The possibility that anti-ASGM1 may act directly on T cells should be considered in all protocols involving the use of this reagent in immunosuppressed mice.     

Multimer technologies for detection and adoptive transfer of antigen-specific T cells

Identification and purification of antigen-specific T cells without altering their functional status are of high scientific and clinical interest. Staining with major histocompatibility complex (MHC)-peptide multimers constitutes a very powerful method to study antigen-specific T-cell subpopulations, allowing their direct visualization and quantification. MHC-peptide multimers, such as dimers, tetramers, pentamers, streptamers, dextramers and octamers have been used to evaluate the frequency of CD8⁺ T cells, specific for tumor/leukemia-associated antigens as well as for viral antigens, e.g., CMVpp65 and EBV-EBNA. Moreover, MHC-peptide multimers have been used for rapid and efficient ex vivo isolation and expansion of T cells. A recent development in the field of MHC-peptide multimers led to the purification of CD8⁺ T cells specific for leukemia antigens. This might help to select leukemia-specific donor lymphocyte infusions (DLIs), thus allowing dissection of the noxious graft-versus-host disease (GvHD) from beneficial anti-viral and even anti-leukemic effects. This review covers different types of MHC-peptide multimers and their applications, as well as the impact that multimers might have on further development of DLIs.     

Rapid method for isolation and staining of bacterial lipopolysaccharide.

Classically bacterial lipopolysaccharide (LPS) purification and silver staining take several days. We designed a simple and fast method for LPS isolation which when combined with silver staining using Pharmacia PhastSystem both can be completed in few hours. The purity of LPS isolated by this simple method may not be comparable to that by the phenol-water method hence we recommend this rapid isolation and staining procedures for simple and fast study of LPS patterns in gels.     

Active specific immunotherapy with extracted tumor-specific transplantation antigen, cyclophosphamide, and adoptive transfer of tumor-specific cytotoxic T-cell clones

An L3T4-, Lyt2+ tumor-specific, cloned T-lymphocyte cell line (RTT-2) was isolated from a spleen cell population harvested from C3H/HeJ mice, following in vivo immunization against a syngeneic MCA-induced fibrosarcoma (MCA-F) and in vitro restimulation with 1-butanol-extracted, isoelectrophoretically purified MCA-F tumor-specific transplantation antigen (TSTA). RTT-2 required exposure to homotypic-extracted MCA-F TSTA in combination with low-dose IL-2 to maintain its specific cytotoxic activity in vitro. In vivo local adoptive transfer of RTT-2 caused specific neutralization of homotypic MCA-F, but not heterotypic MCA-D, tumor cells. Systemic in vivo transfer of RTT-2 alone augmented host resistance. In combination with a triple regimen of weekly doses of purified TSTA (1 microgram SC) and a single ip injection of CY (20 mg/kg), adoptive transfer of RTT-2 cells (1 X 10(7)) retarded the neoplastic outgrowth in and prolonged the survival of primary hosts bearing 3-, 7-, and 14-day established MCA-F tumors. In a spontaneous pulmonary metastasis model following amputation of a tumor-bearing limb, the triple regimen of TSTA/CY/RTT-2 markedly reduced the number of lung colonies. Thus RTT-2, which displays specific tumoricidal activity in vitro and in vivo, may afford a suitable tool to dissect T-cell receptors recognizing tumor markers on 1-butanol-extracted, MCA-F TSTA.     

Genetic engineering of cytolytic T lymphocytes for adoptive T-cell therapy of neuroblastoma

BACKGROUND: Disease relapse is the leading cause of mortality for children diagnosed with disseminated neuroblastoma. The adoptive transfer of tumor-specific T cells is an attractive approach to target minimal residual disease following conventional therapies. We describe here the genetic engineering of human cytotoxic T lymphocytes (CTL) to express a chimeric immunoreceptor for re-directed HLA-independent recognition of neuroblastoma. METHODS: The CE7R chimeric immunoreceptor was constructed by PCR splice overlap extension and is composed of a single-chain antibody extracellular domain (scFv) derived from the L1-CAM-specific murine CE7 hybridoma fused to human IgG1 hinge-Fc, the transmembrane portion of human CD4, and the cytoplasmic tail of huCD3-zeta chain (scFvFc:zeta). Primary human T cells were genetically modified by naked DNA electrotransfer of plasmid expression vector CE7R-pMG then analyzed by Western blotting, flow cytometry for CE7R expression and cell surface trafficking, 4-h chromium release assay for re-directed neuroblastoma lysis, and ELISA for tumor-specific activation of cytokine production. RESULTS: CE7R is expressed as an intact chimeric protein that trafficks to the cell surface as a type I transmembrane protein. Primary human CE7R-expressing CD8(+) CTL clones specifically recognize human neuroblastoma tumor cells and are activated for tumor cell lysis and T(c)1 cytokine production. CONCLUSIONS: These data demonstrate the utility of CE7R for re-directing the effector function of CTL to neuroblastoma and have provided the rationale to initiate a FDA-authorized (BB-IND#9149) pilot clinical trial to establish the feasibility and safety of adoptive transfer of autologous CE7R(+)CD8(+) CTL clones to children with recurrent/refractory neuroblastoma.     

Optimized adoptive T-cell therapy for the treatment of residual mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive B-cell neoplasm with few patients achieving long-term survival with current treatment regimens. High-dose therapy is effective in reducing the tumor burden; however, patients eventually relapse due to minimal residual disease. Having demonstrated efficacy in other malignancies, the effectiveness of dendritic cell-based immunotherapy for minimal residual MCL was examined. We demonstrated that dendritic cells (DC) primed with MCL antigens stimulated the activation of MCL-specific T cells that recognized and destroyed both MCL cell lines and primary MCL in vitro. In addition, in vivo studies demonstrated that adoptively transferred MCL-specific T cells were able to significantly inhibit tumor growth in mice with minimal residual MCL. Subsequently, when combined with CHOP chemotherapy, adoptive T-cell therapy was able to significantly extend the survival of the mice by further reducing the tumor burden. These results clearly show that MCL-specific cellular immunotherapy is effective in treating minimal residual MCL, paving the way for future clinical studies.     

NNAlign_MA; MHC Peptidome Deconvolution for Accurate MHC Binding Motif Characterization and Improved T-cell Epitope Predictions

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Highlights

• •NNAlign_MA enables full deconvolution of single MHC specificities from MS assays.
• •NNAlign_MA expands MHC allelic coverage, improving identification of T-cell epitopes.
• •NNAlign_MA was benchmarked on MHC classes I and II, outperforming current methods.
• •NNAlign_MA offers a universal solution to analyze and exploit MHC peptidomics data.

     

Methods for diversity and overlap analysis in T-cell receptor populations

The paper presents some novel approaches to the empirical analysis of diversity and similarity (overlap) in biological or ecological systems. The analysis is motivated by the molecular studies of highly diverse mammalian T-cell receptor (TCR) populations, and is related to the classical statistical problem of analyzing two-way contingency tables with missing cells and low cell counts. The new measures of diversity and overlap are proposed, based on the information-theoretic as well as geometric considerations, with the capacity to naturally up-weight or down-weight the rare and abundant population species. The consistent estimates are derived by applying the Good–Turing sample-coverage correction. In particular, novel consistent estimates of the Shannon entropy function and the Morisita–Horn index are provided. Data from TCR populations in mice are used to illustrate the empirical performance of the proposed methods vis a vis the existing alternatives.     

T-cell receptor gene transfer for treatment of leukemia

The therapeutic efficacy of donor lymphocyte infusions has been proven for patients with relapsed hematologic malignancies after allogeneic stem cell transplantation. The beneficial effect of donor lymphocytes, however, is often accompanied by graft-versus-host-disease (GvHD). Adoptive transfer of antigen (Ag)-specific T-cell lines may eradicate the relapsed hematological malignancy, and may separate the anti-leukemic effect from GvHD. The main drawback of adoptive therapy of defined T-cell populations is the difficulty in producing sufficient quantities of these Ag-specific T cells. In addition, the specificity of the infused T cells is difficult to control. As the T-cell receptor (TCR) solely determines the specificity of T cells, transfer of relevant TCR genes into appropriate T-cell populations may provide a potent therapeutic reagent. With this strategy, donor-derived T-cell populations would be equipped with a TCR of defined specificity in short-term in vitro procedures, and infusion of the redirected cells would result in T-cell reactivity against the defined Ag. In this review we discuss the current status of TCR gene transfer for the treatment of hematological malignancies.     

Exacerbation of murine cutaneous leishmaniasis by adoptive transfer of parasite-specific helper T cell populations capable of mediating Leishmania major-specific delayed-type hypersensitivity

The effect of adoptive transfer of in vitro-propagated Leishmania major-specific T cell populations on the course of experimentally induced cutaneous leishmaniasis was studied in mice. The L. major-specific T cells expressed the T helper/inducer phenotype and were able in vitro to a) mount a specific proliferative response, b) provide specific helper activity for antibody responses, c) activate parasitized macrophages resulting in L. major destruction, and d) secrete macrophage-activating factors as tested in a tumoricidal assay. These T cells were also found capable of transferring parasite-specific delayed-type hypersensitivity responses to normal syngeneic mice. Results indicated that the i.v. transfer of these L. major-specific T cell populations into normal syngeneic mice exacerbated cutaneous lesions induced by infection with L. major. This effect on the disease process appeared to be dependent upon recognition of parasite antigens by the injected T cells because no exacerbation of the disease process was seen after the transfer of similar T cell populations specific for an antigen unrelated to the parasite, namely ovalbumin. However, the inclusion of ovalbumin in the L. major infecting inoculum resulted in an exacerbating effect of ovalbumin-specific T cells on cutaneous leishmaniasis. These unexpected results were supported by observations showing that immunization of mice with L. major antigens in complete Freund's adjuvant 7 days before infection with L. major led to exacerbated lesions. A similar aggravation of L. major-induced cutaneous lesions was also observed in mice previously immunized with an unrelated antigen provided that this antigen was included in the L. major infecting inoculum.     

Thermodynamics of T-cell receptor-peptide/MHC interactions: progress and opportunities

alphabeta T-cell receptors (TCRs) recognize peptide antigens presented by class I or class II major histocompatibility complex molecules (pMHC). Here we review the use of thermodynamic measurements in the study of TCR-pMHC interactions, with attention to the diversity in binding thermodynamics and how this is related to the variation in TCR-pMHC interfaces. We show that there is no enthalpic or entropic signature for TCR binding; rather, enthalpy and entropy changes vary in a compensatory manner that reflects a narrow free energy window for the interactions that have been characterized. Binding enthalpy and entropy changes do not correlate with structural features such as buried surface area or the number of hydrogen bonds within TCR-pMHC interfaces, possibly reflecting the myriad of contributors to binding thermodynamics, but likely also reflecting a reliance on van't Hoff over calorimetric measurements and the unaccounted influence of equilibria linked to binding. TCR-pMHC binding heat capacity changes likewise vary considerably. In some cases, the heat capacity changes are consistent with conformational differences between bound and free receptors, but there is little data indicating these conformational differences represent the need to organize disordered CDR loops. In this regard, we discuss how thermodynamics may provide additional insight into conformational changes occurring upon TCR binding. Finally, we highlight opportunities for the further use of thermodynamic measurements in the study of TCR-pMHC interactions, not only for understanding TCR binding in general, but also for understanding specifics of individual interactions and the engineering of TCRs with desired molecular recognition properties.     

MHCcluster, a method for functional clustering of MHC molecules

The identification of peptides binding to major histocompatibility complexes (MHC) is a critical step in the understanding of T cell immune responses. The human MHC genomic region (HLA) is extremely polymorphic comprising several thousand alleles, many encoding a distinct molecule. The potentially unique specificities remain experimentally uncharacterized for the vast majority of HLA molecules. Likewise, for nonhuman species, only a minor fraction of the known MHC molecules have been characterized. Here, we describe a tool, MHCcluster, to functionally cluster MHC molecules based on their predicted binding specificity. The method has a flexible web interface that allows the user to include any MHC of interest in the analysis. The output consists of a static heat map and graphical tree-based visualizations of the functional relationship between MHC variants and a dynamic TreeViewer interface where both the functional relationship and the individual binding specificities of MHC molecules are visualized. We demonstrate that conventional sequence-based clustering will fail to identify the functional relationship between molecules, when applied to MHC system, and only through the use of the predicted binding specificity can a correct clustering be found. Clustering of prevalent HLA-A and HLA-B alleles using MHCcluster confirms the presence of 12 major specificity groups (supertypes) some however with highly divergent specificities. Importantly, some HLA molecules are shown not to fit any supertype classification. Also, we use MHCcluster to show that chimpanzee MHC class I molecules have a reduced functional diversity compared to that of HLA class I molecules. MHCcluster is available at www.cbs.dtu.dk/services/MHCcluster-2.0.     

A technique for the effective enrichment and isolation of Bacillus thuringiensis

Abstract The Neurospora crassa exo -1 mutant produced maximum extracellular glucoamylase activity in media supplemented with starch as the sole carbon source. The apparent molecular mass of the enzyme was 82 kDa (SDS-PAGE and gel filtration). The enzyme was a glycoprotein with 5.1 % carbohydrate content and exhibited a temperature optimum of 60 °C. The pH optima were 5.4 and 5.0 for glucoamylase and maltase activities, respectively. Cu²⁺ inhibited maltase activity while Mn²⁺ stimulated glucoamylase activity. The purified enzyme hydrolyzed branched substrates more efficiently than linear substrates. Starch was the best substrate utilized and amylose was hydrolyzed faster than maltose. Kinetic experiments suggested that maltose and starch were hydrolyzed at the same catalytic site.     

Anti-tumor activity of class II MHC antigen-restricted cloned autoreactive T cells. II. Novel immunotherapy of B16 melanomas by local and systemic adoptive transfer

Lyt-1+, L3T4a+ autoreactive cloned T cells, producing lymphotoxin (LT) and interferon-gamma (IFN-gamma) in response to self-class II major histocompatibility complex antigen in vitro were examined for their anti-tumor effect in vivo against B16 melanomas. Without the aid of exogenous interleukin 2, the autoreactive T cells, when injected immediately and at an equal cell number into the site of s.c. inoculated B16 melanoma cells inhibited tumor growth in sublethally irradiated and nonirradiated syngeneic mice. The autoreactive T cells also induced regression of tumors established 3 days earlier. Normal spleen cells or class II-restricted cloned T cells specific for chicken gamma-globulin (CGG) had no inhibitory effect on tumor growth. A single injection of autoreactive T cells delayed tumor growth and prolonged the survival of mice that had received a lethal dose of B16 melanoma cells. The autoreactive T cells caused extensive necrosis at the injection site. A treatment regime consisting of two successive injections of anti-I-Ab monoclonal antibody 3JP prevented the inhibition of tumor growth, supporting the hypothesis that the autoreactive T cells inhibited the growth of melanomas by releasing LT and IFN-gamma upon recognition of I-A antigen-bearing cells at the injection site. The CGG-specific control T cells did not cause necrosis and survived within the nests of uninhibited tumor cells. Autoreactive T cells administered i.v. immediately after i.v. injection of B16 melanoma cells markedly reduced pulmonary metastases, whereas CGG-specific T cells did not. These results indicate that autoreactive T cells can function in vivo as inhibitors of tumor growth.     

Protoplats isolation and regeneration and nuclear staining of mycoparasitic Gliocladium species

Protoplast isolation and regeneration from 24 h germinating conidia of Gliocladium catenulatum, G. roseum and G. virens have been optimized. The number of nuclei per cell of four cell types of the Gliocladium spp. was determined. The optimal enzyme combination for protoplast production contained chitinase, lyticase and cellulase onuzaku in 0.5 M mannitol osmoticum. The quantity of protoplasts produced was dependent on the duration of enzymatic digestion, the concentration of germinating condidia and the species of Gliocladium being assayed. A maximum of 11 to 55% of the protoplasts of wild-type Gliocladium spp. and of two double amino acid autoxotrophic mutants of G. roseum (both Met⁻ Leu⁻) regenerated to mature, conidiating colonies depending on the regeneration medium utilized and the strain assayed. Protoplasts of G. roseum and G. catenulatum were approximately 4% multinucleate, 56% uninucleate and 40% anucleate. Protoplasts of G. virens were approximately 81% multinucleate, 6% uninucleate and 13% anucleate. Regenerating protoplasts and germinating conidia of G. catenulatum and G. roseum were predominantly uninucleate; those of G. virens were predominantly multinucleate. Conidia of all three Gliocladium spp. were predominantly uninucleate.     

The simultaneous isolation of multiple high and low frequent T-cell populations from donor peripheral blood mononuclear cells using the major histocompatibility complex I-Streptamer isolation technology

Background

Adoptive transfer of donor-derived T cells can be applied to improve immune reconstitution in immune-compromised patients after allogeneic stem cell transplantation. The separation of beneficial T cells from potentially harmful T cells can be achieved by using the major histocompatibility complex (MHC) I-Streptamer isolation technology, which has proven its feasibility for the fast and pure isolation of T-cell populations with a single specificity. We have analyzed the feasibility of the simultaneous isolation of multiple antigen-specific T-cell populations in one procedure by combining different MHC I-Streptamers.