Anti-tumor efficacy of a combination therapy with PD-L1 targeted alpha therapy and adoptive cell transfer of PD-1 deficient melanoma-specific human T-lymphocytes.

The optimization of adoptive transfer approaches of anti-tumor T cells requires both the functional improvement of the injected T cells and the modulation of the tumor microenvironment, favoring the recruitment of these T cells and their activation. We have recently shown the therapeutic benefit of two approaches tested individually in a melanoma model wich were on one hand the adoptive transfer of specific T cells deficient for the expression of the inhibitory receptor PD-1, and on the other hand PD-L1 targeted alpha therapy (TAT). In this study, we sought to investigate the efficacy of these two therapies combined, compared to each monotherapy, in order to evaluate the synergy between these two approaches, in the same melanoma model. Here we used melanoma-specific T-cell clones, previously validated for the edition of PDCD1 gene and with previously demonstrated superior anti-tumor activity than their wild-type counterparts, after adoptive transfer in NSG mice engrafted with PD-L1 expressing human melanoma tumors. We also used a previously validated TAT approach, using a 213Bi-anti-human-PD-L1 mAb, alone or in combination with adoptive cell transfer, in the same mouse model. We confirmed previous results obtained with each monotherapy and documented the safety and the superior ability of a combination between the adoptive transfer of PD-1 deficient T cells and TAT targeting PD-L1 to control the growth of melanoma tumors in NSG mice. This study provides the first proof-of-concept of the efficacy of a combination therapy using TAT, adoptive cell transfer and genomic editing of IC-coding genes.

Production and characterization of monoclonal antibodies specific for canine CD138 (syndecan-1) for nuclear medicine preclinical trials on spontaneous tumours.

We isolated 11 antibodies specific for canine CD138 (cCD138) to validate the interest of CD138 antigen targeting in dogs with spontaneous mammary carcinoma. The affinity of the monoclonal antibodies in the nanomolar range is suitable for immunohistochemistry and nuclear medicine applications. Four distinct epitopes were recognized on cCD138 by this panel of antibodies. CD138 expression in canine healthy tissues is comparable to that reported in humans. CD138 is frequently expressed in canine mammary carcinomas corresponding to the human triple negative breast cancer subtype, with cytoplasmic and membranous expression. In canine diffuse large B-cell lymphoma, CD138 expression is associated with the 'non-germinal center' phenotype corresponding to the most aggressive subtype in humans. This homology of CD138 expression between dogs and humans confirms the relevance of tumour-bearing dogs as spontaneous models for nuclear medicine applications, especially for the evaluation of new tumour targeting strategies for diagnosis by phenotypic imaging and radio-immunotherapy.

Comparative analysis of multiple myeloma treatment by CD138 antigen targeting with bismuth-213 and Melphalan chemotherapy.

INTRODUCTION: Multiple myeloma (MM) is a B-cell malignancy of terminally differentiated plasma cells within the bone marrow. Despite intense research to develop new treatments, cure is almost never achieved. Alpha-radioimmunotherapy (RIT) has been shown to be effective in vivo in a MM model. In order to define where alpha-RIT stands in MM treatment, the aim of this study was to compare Melphalan, MM standard treatment, with alpha-RIT using a [213Bi]-anti-mCD138 antibody in a syngeneic MM mouse model. METHODS: C57BL/KaLwRij mice were grafted with 1 × 10(6) 5T33 murine MM cells. Luciferase transfected 5T33 cells were used for in vivo localization. The first step of the study was to assess the dose-response of Melphalan 21 days after engraftment. The second step consisted in therapeutic combination: Melphalan followed by RIT at day 22 or day 25 after engraftment. Toxicity (animal weight, blood cell counts) and treatment efficacy were studied in animals receiving no treatment, injected with Melphalan alone, RIT alone at day 22 or day 25 (3.7 MBq of [213Bi]-anti-CD138) and Melphalan combined with alpha-RIT. RESULTS: Fifty percent of untreated mice died by day 63 after MM engraftment. In mice treated with Melphalan alone, only the 200 µg dose improved median survival. No animal was cured after Melphalan treatment whereas 60% of the mice survived with RIT alone at day 22 after tumor engraftment with only slight and reversible hematological radiotoxicity. No therapeutic effect was observed with alpha-RIT 25 days after engraftment. Melphalan and alpha-RIT combination does not improve overall survival compared to RIT alone, and results in increased leukocyte and red blood cell toxicity. CONCLUSIONS: Alpha-RIT seems to be a good alternative to Melphalan. Association of these two treatments provides no benefit. The perspectives of this work would be to evaluate RIT impact on the regimens incorporating the novel agents bortezomide, thalidomide and lenalidomide.

Selection of Epstein-Barr virus specific cytotoxic T lymphocytes can be performed with B lymphoblastoid cell lines created in serum-free media.

Epstein-Barr Virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) are currently used for numerous applications in cellular immunology. Where protocols destined for clinical application are concerned, the final choice of assay is made according to a risk/benefit ratio analysis. In this balance the use of xenogenic or allogenic serum has always been a major concern, as it carries both an infectious and an immunological risk. So far, it is unknown whether serum can be omitted from the entire BLCL selection procedure. In addition, as BLCL have been described as heterogeneous, serum deprivation may affect their antigen-presenting capacity. In the present study, BLCL were generated in the absence or presence of fetal calf serum (referred to as BLCL0 or BLCL(FCS), respectively). Next, in order to assess the antigen-presenting capacity of these cells, we compared the ability of BLCL0 and BLCL(FCS) cells to stimulate the EBV-specific repertoire of the corresponding donor's peripheral blood mononuclear cells in vitro. Our results showed that addition of serum was not essential for BLCL infection and culture, and that as far as we could determine, BLCL0 cells were as effective as BLCL(FCS) in reactivating the EBV-specific T-cell repertoire in vitro. Notably, FCS-specific T-lymphocytes can be detected among the BLCL(FCS)-specific CD4+-CTL. Not only was this latter observation unexpected for an EBV-seropositive donor, but it implied that the BLCL had captured and processed the corresponding FCS-derived solubles antigens; taken together our results emphasized the interest of the possibility to generate BLCL0, both for research and for clinical applications.

Characterization of coated vesicles that participate in endocytic recycling.

While the recycling pathway of endocytosis has been shown to participate in many cellular functions, little is known regarding the transport carriers that mediate this pathway. In this study, we overexpressed a point mutant of ADP-ribosylation factor 6 (ARF6), that perturbs its GTPase cycle, to accumulate endosome-derived coated vesicles. Characterization by their purification revealed that, upon cell homogenization, these vesicles were mostly aggregated with larger noncoated membranes, and could be released with high-salt treatment. Equilibrium centrifugation revealed that these vesicles had buoyant density similar to the COP-coated vesicles. To purify the ARF6-regulated vesicles to homogeneity, enriched fractions from equilibrium centrifugation were subjected to immunoisolation through the hemagglutinin (HA) epitope of the mutant ARF6, by using a newly developed, high-affinity, anti-HA monoclonal antibody. Surface iodination of the purified vesicles revealed multiple prominent proteins. Immunoblotting with antibodies against subunits of the currently known coat proteins suggested that these vesicles have a novel coat complex. These vesicles are carriers for endocytic recycling, because they are enriched for transferrin receptor and also the v-SNARE cellubrevin that functions in transport from the recycling endosome to the plasma membrane. Thus, we have characterized transport vesicles that participate in endocytic recycling.

Purification of Ag-specific T lymphocytes after direct peripheral blood mononuclear cell stimulation followed by CD25 selection. I. Application to CD4(+) or CD8(+) cytomegalovirus phosphoprotein pp65 epitope determination.

The two main constraints that currently limit a broader usage of T cell therapy against viruses are the delay required to obtain specific T cells and the safety of the selection procedure. In the present work we developed a generally applicable strategy that eliminates the need for APC for timing reasons, and the need for infectious viral strains for safety concerns. As a model, we used the selection of T lymphocytes specific for the immunodominant CMV phosphoprotein pp65. PBMC from healthy seropositive donors were first depleted of IL-2R alpha-chain CD25(+) cells and were then stimulated for 24-96 h with previously defined peptide Ags or with autologous PBMC infected with a canarypox viral vector encoding the total pp65 protein (ALVAC-pp65). Subsequent immunomagnetic purification of newly CD25-expressing cells allowed efficient recovery of T lymphocytes specific for the initial stimuli, i.e., for the already known immunodominant epitope corresponding to the peptides used as a model or for newly defined epitopes corresponding to peptides encoded by the transfected pp65 protein. Importantly, we demonstrated that direct PBMC stimulation allowed recovery not only of CD8(+) memory T lymphocytes, but also of the CD4(+) memory T cells, which are known to be crucial to ensure persistence of adoptively transferred immune memory. Finally, our analysis of pp65-specific T cells led to the identification of several new helper and cytotoxic epitopes. This work thus demonstrates the feasibility of isolating memory T lymphocytes specific for a clinically relevant protein without the need to prepare APC, to use infectious viral strains, or to identify immunodominant epitopes.

Distribution of ARF6 between membrane and cytosol is regulated by its GTPase cycle.

The ADP-ribosylation factor (ARF) subfamily of small GTPases regulates intracellular transport. Although much is known about how ARF1 regulates transport in the secretory pathways, regulation of the endocytic pathways by ARF6 remains less understood. In particular, whereas cycling of ARF1 between membrane and cytosol represents a major mechanism of regulating its function, this regulation has been questioned for ARF6. In this study, we found that ARF6 is distributed both on membranes and in the cytosol. Cytosolic ARF6 is recruited to membranes in a GTP-dependent manner that is fundamentally similar to ARF1. However, unlike ARF1, release of membrane-bound ARF6 to the cytosol requires hydrolysis of GTP that is sensitive to the level of magnesium. These findings suggest that the GTPase cycle of ARF6 also regulates its distribution between membrane and cytosol and that this form of regulation will also likely be important for the function of ARF6. Moreover, as ARF6 has little intrinsic ability to hydrolyze GTP, magnesium concentration most likely affects the release of membrane-bound ARF6 by altering the activity of its GTPase-activating protein.

Acute graft-versus-host disease after bone marrow transplantation with a single HLA-DPB1*1001 mismatch: involvement of different TCRBV subsets.

HLA-DP incompatibility is not considered as an exclusion criterion for bone marrow donors, because such incompatibility was not shown to affect significantly the risk for acute graft-versus-host disease (GVHD). In line with this clinical observation, it was proposed that in the context of bone marrow transplantation, HLA-DP determinants did not function as transplantation antigens in the same way as HLA-A, -B or -DR. In contrast to the above conclusion, we recently demonstrated the presence of HLA-DPB1*0501 specific T cell clones in a skin biopsy of a patient who developed aGVHD after receiving a bone marrow transplant (BMT) in which the only mismatched allele in the GVHD direction was HLA-DPB1*0501. At that time, this case was unique and occurred in a relatively uncommon graft setting where the patient received purified CD34+ BM cells from an unrelated donor. In the present study, we analyzed the immunological events associated with an aGVHD which occurred in the context of a 'regular' allogeneic BMT involving a single HLA-DPB1*1001 mismatch between donor and recipient in the GVHD direction. To this end, we analyzed several amplified T cell subsets present within a T cell line derived from a skin biopsy performed at the onset of GVHD. Our results demonstrated that T cell populations belonging to the TCRBV2, TCRB6.7, TCRBV14 and TCRBV17 subsets were specific for the HLA-DPB1*1001 mismatched allele. These data strengthen and generalize our first conclusion that a single HLA-DP mismatch between donor and recipient can activate a strong T cell response in vivo and consequently challenge the notion that HLA-DP incompatibility should not be taken into account in the choice of BM donors. Moreover, they also underline the idea that HLA-DP antigens may represent an interesting immune target for future therapeutic approaches.

Close phenotypic and functional similarities between human and murine alphabeta T cells expressing invariant TCR alpha-chains.

Several studies have demonstrated the existence of a murine NK1.1+ alphabeta T cell subset expressing V alpha14+ TCR alpha-chains with highly conserved invariant junctional sequences and able to secrete Th2 cytokines when exposed to CD1+ stimulator cells. In humans, alphabeta T cells carrying invariant V alpha24+ TCR alpha-chains highly homologous to those expressed by murine NK1.1 cells have been recently described. Here we show that these cells (referred to as V alpha24inv T cells) and murine NK1.1+ alphabeta T cells resemble each other in several ways. First, like their murine counterparts, T cells expressing high levels of V alpha24inv TCRs can be either CD4- CD8- double negative (DN) or CD4+, but they never express heterodimeric CD8 molecules. Second, most V alpha24inv T cells are brightly stained by NKRP1-specific mAb but not by mAb directed against other type II transmembrane proteins of the NK complex. Third, DN and particularly CD4+ V alpha24inv T cells are greatly enriched for IL-4 producers. The concomitant expression of highly conserved TCRs of a particular set of NK markers and of Th2 cytokines in human and murine alphabeta T cells suggests a coordinate acquisition of these phenotypic and functional properties. Furthermore, the relatively high frequency of human V alpha24inv T cells, which are presently shown to represent on average 1/500 PBL, and the high interindividual variations of the size of this cell subset under physiologic conditions go for a major role played by alphabeta T cells carrying invariant TCR in a large array of immune responses.

Selection of T cells reactive against autologous B lymphoblastoid cells during chronic rheumatoid arthritis.

The repertoire and Ag specificity of T cells infiltrating inflamed joints from a chronic rheumatoid arthritis (RA) patient were studied in detail. Repertoire analysis demonstrated a reduced clonality of joint-infiltrating lymphocytes (JIL) as compared with patient's PBL, which was presumably due to an intra-articular expansion of T cell clones with recurrent TCR features. Strikingly, a large fraction of these JIL T cell clones, which were predominantly CD8+, proliferated in vitro when exposed to autologous B lymphoblastoid cells (BLC), unlike randomly chosen PBL clones derived from the same patient. This proliferative response was HLA-restricted, which confirmed a classical TCR-mediated recognition of BLC and was not observed against autologous PHA blasts, suggesting recognition of either EBV or B cell-specific Ags. Finally, a preliminary analysis of synovial lymphocytes derived from another chronic RA patient demonstrated a similar enrichment for T cells reactive against autologous BLC within JILs as compared with patient's PBLs. Taken together, these results, which suggest frequent expansions of autologous BLC-reactive T cells within inflamed joints of chronic RA patients, provide a basis for future studies evaluating the fine specificity and pathogenicity of these lymphocytes.

Human HLA-specific T-cell clones with stable expression of a suicide gene: a possible tool to drive and control a graft-versus-host- graft-versus-leukemia reaction?

Allogeneic bone marrow transplantation is still limited by the morbidity and mortality caused by graft-versus-host disease (GVHD), resulting from host recognition by donor T lymphocytes. It is possible to drastically reduce the T-cell content of the graft. However, transplanted T cells can also have a beneficial effect by graft enhancement and the graft-versus-leukemia effect. How can we keep the beneficial GVL effect while protecting the patient from possible GVHD? A recent report proposed the ex vivo transfer of the herpes simplex thymidine kinase (HSv-tk) gene into donor T cells before their infusion with hematopoietic stem cells. This procedure is expected to allow selective donor T-cell depletion with ganciclovir should GVHD occur, but it has two major drawbacks: reinjection of a fraction of untransfected T cells cannot be avoided and heterogeneity of the transfected population results in increased risks such as HSv-tk gene instability or dysfunction of some of the transfected T cell. Alternative approaches must be considered. We demonstrate here the feasibility of generating HSv-tk transfected HLA-specific CD4+ cytotoxic T-cell clonal populations, in which 100% of the cells have the HSv-tk gene inserted at a single site within their genome. These clones retained their specificity, their function, and their sensitivity to ganciclovir treatment. Our approach is not limited to bone marrow transplantation. Indeed, this procedure represents a useful alternative to retroviral gene transduction and is applicable to every circumstance where clinical use of gene modified T-cell clones is to be considered.

Acute graft versus host disease due to T lymphocytes recognizing a single HLA-DPB1*0501 mismatch.

Analysis of a large number of unrelated bone marrow transplantations (BMT) has shown that HLA-DP incompatibility did not detectably influence the risk for acute graft-versus-host disease (aGVHD). Accordingly, it was proposed that HLA-DP determinants did not function as transplantation antigens in the same way as HLA-A, -B, or -DR. We have previously shown that HLA-DP (as well as HLA-A, -B, -DQ, or -DR)-specific T cells could be isolated from skin biopsies of patients who developed an aGVHD after semiallogeneic BMT. Nevertheless, whether a single HLA-DP mismatched allele could induce a detectable allo-specific reaction in vivo after BMT remained to be established. To directly address this issue we studied one patient who presented aGVHD after receiving purified CD34+ bone marrow (BM) cells from an unrelated donor with a single HLA-DP mismatch in the GVHD direction. To characterize the immunological events associated with GVHD, we analyzed the peripheral T cell repertoire, the T cell receptor Vbeta diversity, and the specificity of T cells invading a skin biopsy at the onset of GVHD. Our results demonstrated that a large fraction of skin-infiltrating lymphocytes, which expressed diverse T cell receptors, were reactive against this single HLA-DPB1 *0501 mismatch and consequently that a single HLA-DP mismatch between BM donor and recipient can activate a strong T cell response in vivo.

HLA-target antigens and T-cell receptor diversity of activated T cells invading the skin during acute graft-versus-host disease.

To study the repertoire and specificity of T lymphocytes infiltrating skin lesions during graft-versus-host disease (GVHD), we performed an exhaustive molecular and functional analysis of 146 T-cell clones derived from the skin of three patients undergoing an acute GVHD after allogeneic bone marrow transplantation (BMT) from HLA-mismatched related donors. Analysis of T-cell receptor (TCR) rearrangement and TCR chain junctional sequences demonstrated the presence of 11 distinct clones among the 64 derived from patient UPN1, six among the 58 derived from patient UPN2, and seven among the 24 derived from patient UPN3. Three of the 11 T-cell clones from patient UPN1, and all clones from patients UPN2 and UPN3 reacted with mismatched HLA alleles between the bone-marrow donor and recipient. Moreover, both HLA class I (HLA-A2 and -B27) and class II (HLA DP101, DP401, DP1301, DQ8, and DR402) molecules were recognized during this early antihost response. Finally, both TCR alpha and beta chains turned out to be extremely diverse, even within populations of clones derived from the same patient and directed against the same HLA allele. Taken together, these results indicate that any HLA mismatch is potentially targeted during early GVHD, and that the T-cell response at the onset of GVHD is both oligoclonal and highly diversified.

Alterations of T cell repertoire after bone marrow transplantation: characterization of over-represented subsets.

We recently demonstrated that frequencies of T cell receptor-V (TcR-V)-specific subsets are frequently altered after both allogeneic and autologous BMT. The data reported here describe several characteristics of altered T cell subsets: (i) their capacity to endure peripherally, (ii) their correspondence to clonal donor T cell subsets, (iii) the origin of the clone (in one case amenable to analysis) from a mature T cell and not from new lymphopoiesis, and (iv) the presence of such a clone throughout a year of follow-up in a patient with chronic graft-versus-host disease (GVHD) in whom it represented up to 1/10th of CD3+ peripheral blood lymphocytes (PBL) and was found to be host-reactive. Taken together, these findings provide direct evidence for the oligoclonality of a large proportion of the peripheral T cell repertoire in patients subsequent to bone marrow transplantation, possibly accounting for their frequent depressed immune status. Moreover, the anti-host reactivity demonstrated in a clone from the patient with chronic GVHD strongly suggests that an oligoclonal response can be linked to a pathological process.

Surface expression of functional T cell receptor chains formed by interlocus recombination on human T lymphocytes.

Structural diversity of lymphocyte antigen receptors (the immunoglobulin [Ig] of B cells and the alpha/beta or gamma/delta T cell receptor [TCR] of T cells) is generated through somatic rearrangements of V, D, and J gene segments. Classically, these recombination events involve gene segments from the same Ig or TCR locus. However, occurrence of "trans" rearrangements between distinct loci has also been described, although in no instances was the surface expression of the corresponding protein under normal physiological conditions demonstrated. Here we show that hybrid TCR genes generated by trans rearrangement between V gamma and (D) J beta elements are translated into functional antigen receptor chains, paired with TCR alpha chains. Like classical alpha/beta T cells, cells expressing these hybrid TCR chains express either CD4 or CD8 coreceptors and are frequently alloreactive. These results have several implications in terms of T cell repertoire selection and relationships between TCR structure and specificity. First, they suggest that TCR alloreactivity is determined by the repertoire selection processes operating during lymphocyte development rather than by structural features specific to V alpha V beta regions. Second, they suggest the existence of close structural relationships between gamma/delta and alpha/beta TCR and more particularly, between V gamma and V beta regions. Finally, since a significant fraction of PBL (at least 1/10(4)) expressed hybrid TCR chains on their surface, these observations indicate that trans rearrangements significantly contribute to the combinatorial diversification of the peripheral immune repertoire.

Alteration of the T cell repertoire after bone marrow transplantation.

We investigated T cell receptor (TCR) alpha/beta and gamma/delta repertoire reconstitution after autologous and allogeneic bone marrow transplantation (BMT) in humans using 13 monoclonal antibodies directed at constant and variable regions of the TCR. The TCR gamma/delta repertoire was studied kinetically during the first month and then 1 year post-BMT whereas alpha/beta peripheral blood T lymphocytes (PBTL) were studied within the first 3 months and 1 year post-BMT. Through these two studies, we found 7 of 26 patients with over-represented TCR gamma/delta subsets during the early period post-BMT. Moreover, during this period the V gamma 9V delta 2 frequency among gamma/delta T cells was found to be higher than among normal donors. Study on TCR alpha/beta T cells also revealed abnormally expanded V-specific subset (5 of 10 patients within 3 months following BMT) demonstrating that repertoire alteration early after BMT is a general phenomenon concerning potentially all T cell subsets. More surprisingly, the alpha/beta T cell repertoire was also found to be altered late after BMT (7 of 15 patients after 1 year post-BMT presented one or more overepresented alpha/beta TCR subset). These alterations of TCR combinatorial diversity should be taken into account in understanding the immunological status of patients after BMT.

Close correlation between Daudi and mycobacterial antigen recognition by human gamma delta T cells and expression of V9JPC1 gamma/V2DJC delta-encoded T cell receptors.

Recent studies have demonstrated that a large fraction of human gamma delta PBL recognize Ag of prokaryotic and eukaryotic origins, respectively found in hydrosoluble mycobacterial extracts and on the Daudi Burkitt's lymphoma cells. The structural basis of the recognition of these Ag have been presently studied in detail, through analysis of a large panel of thymus- and peripheral blood-derived gamma delta T-cell clones. Our results suggest that Daudi and mycobacteria-reactive gamma delta subsets are strictly overlapping and hence that gamma delta T-cell responses against these two Ag are closely related. Daudi cells and mycobacteria were recognized by V gamma 9+V delta 2+, but not by V gamma 9+V delta 2-, V gamma 9-V delta 2+, or V gamma 9-V delta 2- PBL clones. However, not all V gamma 9+V delta 2+ clones were reactive and, in particular: 1) the proportion of Ag-reactive lymphocytes was much lower among thymus- than PBL-derived clones (respectively 24/36 vs 72/73); 2) none of the V gamma 9+V delta 2+ clones expressing a V9J2C2 gamma chain (n = 4) were reactive to Daudi or mycobacteria, indicating that expression of a disulfide-linked TCR is probably a prerequisite for recognition of these Ag; and 3) among V gamma 9+V delta 2+ clones bearing disulfide-linked TCR, almost all (50/53) clones expressing a V9JPC1 gamma chain were reactive, whereas a large fraction (6/10) of those expressing a V9J1C1 gamma chain were weakly or nonreactive. Together, these observations suggest that germline residues specific to V gamma 9, V delta 2, and J gamma P elements directly contribute to recognition of Daudi and mycobacterial Ag. Furthermore, these findings may provide an explanation for coordinate use of these gene elements by a large fraction of gamma delta PBL, through peripheral selection events mediated by ligands identical or structurally related to the above Ag.