T cells armed with C-X-C chemokine receptor type 6 enhance adoptive cell therapy for pancreatic tumours.

The efficacy of adoptive cell therapy for solid tumours is hampered by the poor accumulation of the transferred T cells in tumour tissue. Here, we show that forced expression of C-X-C chemokine receptor type 6 (whose ligand is highly expressed by human and murine pancreatic cancer cells and tumour-infiltrating immune cells) in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer. In mice with subcutaneous pancreatic tumours treated with T cells with either a transgenic T-cell receptor or a murine chimeric antigen receptor targeting the tumour-associated antigen epithelial cell adhesion molecule, and in mice with orthotopic pancreatic tumours or patient-derived xenografts treated with T cells expressing a chimeric antigen receptor targeting mesothelin, the T cells exhibited enhanced intratumoral accumulation, exerted sustained anti-tumoral activity and prolonged animal survival only when co-expressing C-X-C chemokine receptor type 6. Arming tumour-specific T cells with tumour-specific chemokine receptors may represent a promising strategy for the realization of adoptive cell therapy for solid tumours.

Cytomegalovirus-specific CD8+ T-cells are associated with a reduced incidence of early relapse after allogeneic stem cell transplantation.

Leukemia relapse is the main cause for mortality after allogeneic stem cell transplantation (allo-SCT). Donor-derived allo-immune responses eliminate the residual host hematopoiesis and protect against relapse. Cytomegalovirus (CMV) reactivation (CMV-R) after allo-SCT may trigger anti-leukemic effects. The impact of CMV-specific CD8+ T-cells (CMV-CTLs) on the outcome after allo-SCT is currently unknown. Here, we studied the relationship between CMV-CTLs, overall T-cell reconstitution and relapse incidence in 103 patients with acute leukemia (n = 91) or myelodysplastic syndrome (n = 12) following CMV-seropositive recipient/donor (R+/D+) allo-SCT. Patients were subdivided based on the presence or absence of CMV-CTLs at 3 months after allo-SCT. Presence of CMV-CTLs was associated with preceding CMV-R and a fast T-cell reconstitution. Univariate analysis showed a significantly lower 1-, 2- and 5-year cumulative incidence of relapse (CIR) in patients with CMV-CTLs compared to those without CMV-CTLs. Multivariable regression analysis of the outcome performed with other relevant parameters chosen from univariate analysis revealed that presence of CMV-CTLs and chronic graft-versus-host disease (cGvHD) were the only independent factors associated with a low CIR. Onset of relapse was significantly later in patients with CMV-CTLs (median 489 days) than in in those without (median 152 days, p = 0.041) during a five-year follow-up. Presence of CMV-CTLs was associated with a lower incidence of early relapses (1 and 2-years), while cGvHD lead to a lower incidence of late relapses (2 to 5-years). In conclusion, our data show that CMV-CTLs indicate a functional immune-reconstitution protective against early relapse.

High-affinity CD16-polymorphism and Fc-engineered antibodies enable activity of CD16-chimeric antigen receptor-modified T cells for cancer therapy.

BACKGROUND: CD16-chimeric antigen receptors (CAR) T cells recognise the Fc-portion of therapeutic antibodies, which can enable the selective targeting of different antigens. Limited evidence exists as to which CD16-CAR design and antibody partner might be most effective. We have hypothesised that the use of high-affinity CD16 variants, with increased Fc-terminus antibody affinity, combined with Fc-engineered antibodies, would provide superior CD16-CAR T cell efficacy. METHODS: CD16-CAR T (wild-type or variants) cells were co-cultured with Panc-1 pancreatic cancer, Raji lymphoma or A375 melanoma cells in the presence or absence of anti-CD20, anti-MCSP, wild-type or the glycoengineered antibody variants. The endpoints were proliferation, activation, and cytotoxicity in vitro. RESULTS: The CD16 158 V variant of CD16-CAR T cells showed increased cytotoxic activity against all the tested cancer cells in the presence of the wild-type antibody directed against MCSP or CD20. Glycoengineered antibodies enhanced CD16-CAR T cell activity irrespective of CD16 polymorphisms as compared with the wild-type antibody. The combination of the glycoengineered antibodies with the CD16-CAR 158 V variant synergised as seen by the increase in all endpoints. CONCLUSION: These results indicate that CD16-CAR with the high-affinity CD16 variant 158 V, combined with Fc-engineered antibodies, have high anti-tumour efficacy.

Teaching an old dog new tricks: next-generation CAR T cells.

Adoptive T cell therapy (ACT) refers to the therapeutic use of T cells. T cells genetically engineered to express chimeric antigen receptors (CAR) constitute the most clinically advanced form of ACT approved to date for the treatment of CD19-positive leukaemias and lymphomas. CARs are synthetic receptors that are able to confer antigen-binding and activating functions on T cells with the aim of therapeutically targeting cancer cells. Several factors are essential for CAR T cell therapy to be effective, such as recruitment, activation, expansion and persistence of bioengineered T cells at the tumour site. Despite the advances made in CAR T cell therapy, however, most tumour entities still escape immune detection and elimination. A number of strategies counteracting these problems will need to be addressed in order to render T cell therapy effective in more situations than currently possible. Non-haematological tumours are also the subject of active investigation, but ACT has so far shown only marginal success rates in these cases. New approaches are needed to enhance the ability of ACT to target solid tumours without increasing toxicity, by improving recognition, infiltration, and persistence within tumours, as well as an enhanced resistance to the suppressive tumour microenvironment.

Characterization of High-Avidity Cytomegalovirus-Specific T Cells with Differential Tetramer Binding Coappearing after Allogeneic Stem Cell Transplantation.

CMV reactivation is a major complication after allogeneic stem cell transplantation (SCT). Immune reconstitution of CMV-specific CTLs (CMV-CTLs) is essential for virus control. During CMV-CTL monitoring using mutated HLA/CMV tetramers selectively detecting high-avidity T cells, we observed coappearance of CMV-CTLs with low (CMV tetlow CTLs) and high tetramer binding (CMV tethigh CTLs) in 53/115 CMV IgG+ patients stem cell transplanted from CMV IgG+ donors. However, the relevance of these coappearing differentially tetramer binding ("dual") CMV-CTLs was unclear. In this study, we investigated the kinetics, properties, and clinical impact of coappearing CMV tetlow and tethigh CTLs after allogeneic SCT. Patients with dual CMV-CTLs had more CMV tethigh than tetlow CTLs. Chimerism analysis of isolated CMV tetlow and tethigh CTLs revealed their exclusive donor origin. CMV tetlow and tethigh CTLs had an identical effector memory CD45RA-CCR7- and CD45RA+CCR7- T cell distribution, equal differentiation, senescence, and exhaustion marker expression and were negative for regulatory CD8+ T cell markers. Isolated CMV tetlow and tethigh CTLs were equally sensitive to CMV peptides in IFN-γ release and cytotoxicity assays. However, CMV tethigh CTLs proliferated more in response to low CMV peptide concentrations than tetlow CTLs. TCR repertoire analysis revealed that CMV tetlow and tethigh CTLs use different TCRs. Finally, dual CMV-CTLs were not associated with CMV antigenemia. In conclusion, these data show for the first time, to our knowledge, that both CMV tetlow and tethigh CTLs are functional effector T cells differing by proliferation, numbers in peripheral blood, and probably by their precursors without increasing the CMV reactivation risk after allogeneic SCT.

Human CD8+ CD57- TEMRA cells: Too young to be called "old".

End-stage differentiation of antigen-specific T-cells may precede loss of immune responses against e.g. viral infections after allogeneic stem cell transplantation (SCT). Antigen-specific CD8+ T-cells detected by HLA/peptide multimers largely comprise CD45RA-/CCR7- effector memory (TEM) and CD45RA+/CCR7- TEMRA subsets. A majority of terminally differentiated T-cells is considered to be part of the heterogeneous TEMRA subset. The senescence marker CD57 has been functionally described in memory T-cells mainly composed of central memory (TCM) and TEM cells. However, its role specifically in TEMRA cells remained undefined. Here, we investigated the relevance of CD57 to separate human CD8+ TEMRA cells into functionally distinct subsets. CD57- CD8+ TEMRA cells isolated from healthy donors had considerably longer telomeres and showed significantly more BrdU uptake and IFN-γ release upon stimulation compared to the CD57+ counterpart. Cytomegalovirus (CMV) specific T-cells isolated from patients after allogeneic SCT were purified into CD57+ and CD57- TEMRA subsets. CMV specific CD57- TEMRA cells had longer telomeres and a considerably higher CMV peptide sensitivity in BrdU uptake and IFN-γ release assays compared to CD57+ TEMRA cells. In contrast, CD57+ and CD57- TEMRA cells showed comparable peptide specific cytotoxicity. Finally, CD57- CD8+ TEMRA cells partially changed phenotypically into TEM cells and gained CD57 expression, while CD57+ CD8+ TEMRA cells hardly changed phenotypically and showed considerable cell death after in vitro stimulation. To the best of our knowledge, these data show for the first time that CD57 separates CD8+ TEMRA cells into a terminally differentiated CD57+ population and a so far functionally undescribed "young" CD57- TEMRA subset with high proliferative capacity and differentiation plasticity.

Possible Impact of Cytomegalovirus-Specific CD8+ T Cells on Immune Reconstitution and Conversion to Complete Donor Chimerism after Allogeneic Stem Cell Transplantation.

Complete donor chimerism is strongly associated with complete remission after allogeneic stem cell transplantation (allo-SCT) in patients with hematologic malignancies. Donor-derived allo-immune responses eliminate the residual host hematopoiesis and thereby mediate the conversion to complete donor chimerism. Recently, cytomegalovirus (CMV) reactivation was described to enhance overall T cell reconstitution, to increase graft-versus-host disease incidence, and to reduce the leukemia relapse risk. However, the link between CMV and allo-immune responses is still unclear. Here, we studied the relationship between CMV-specific immunity, overall T cell reconstitution, and residual host chimerism in 106 CMV-seropositive patients transplanted after reduced-intensity conditioning including antithymocyte globulin. In accordance with previous reports, the recovery of CMV-specific cytotoxic T cells (CMV-CTLs) was more frequent in CMV-seropositive recipients (R) transplanted from CMV-seropositive than from seronegative donors (D). However, once CMV-CTLs were detectable, the reconstitution of CMV-specific CTLs was comparable in CMV R+/D- and R+/D+ patients. CD3+ and CD8+ T cell reconstitution was significantly faster in patients with CMV-CTLs than in patients without CMV-CTLs both in the CMV R+/D- and R+/D+ setting. Moreover, CMV-CTL numbers correlated with CD3+ and CD8+ T cell numbers in both settings. Finally, presence of CMV-CTLs was associated with low host chimerism levels 3 months after allo-SCT. In conclusion, our data provide a first indication that CMV-CTLs in CMV-seropositive patients might trigger the reconstitution of T cells and allo-immune responses reflected by the conversion to complete donor chimerism.

Immune Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation.

The timely reconstitution and regain of function of a donor-derived immune system is of utmost importance for the recovery and long-term survival of patients after allogeneic hematopoietic stem cell transplantation (HSCT). Of note, new developments such as umbilical cord blood or haploidentical grafts were associated with prolonged immunodeficiency due to delayed immune reconstitution, raising the need for better understanding and enhancing the process of immune reconstitution and finding strategies to further optimize these transplant procedures. Immune reconstitution post-HSCT occurs in several phases, innate immunity being the first to regain function. The slow T cell reconstitution is regarded as primarily responsible for deleterious infections with latent viruses or fungi, occurrence of graft-versus-host disease, and relapse. Here we aim to summarize the major steps of the adaptive immune reconstitution and will discuss the importance of immune balance in patients after HSCT.

Recent Developments in Cellular Immunotherapy for HSCT-Associated Complications.

Allogeneic hematopoietic stem cell transplantation is associated with serious complications, and improvement of the overall clinical outcome of patients with hematological malignancies is necessary. During the last decades, posttransplant donor-derived adoptive cellular immunotherapeutic strategies have been progressively developed for the treatment of graft-versus-host disease (GvHD), infectious complications, and tumor relapses. To date, the common challenge of all these cell-based approaches is their implementation for clinical application. Establishing an appropriate manufacturing process, to guarantee safe and effective therapeutics with simultaneous consideration of economic requirements is one of the most critical hurdles. In this review, we will discuss the recent scientific findings, clinical experiences, and technological advances for cell processing toward the application of mesenchymal stromal cells as a therapy for treatment of severe GvHD, virus-specific T cells for targeting life-threating infections, and of chimeric antigen receptors-engineered T cells to treat relapsed leukemia.

Milestones of Hematopoietic Stem Cell Transplantation - From First Human Studies to Current Developments.

Since the early beginnings, in the 1950s, hematopoietic stem cell transplantation (HSCT) has become an established curative treatment for an increasing number of patients with life-threatening hematological, oncological, hereditary, and immunological diseases. This has become possible due to worldwide efforts of preclinical and clinical research focusing on issues of transplant immunology, reduction of transplant-associated morbidity, and mortality and efficient malignant disease eradication. The latter has been accomplished by potent graft-versus-leukemia (GvL) effector cells contained in the stem cell graft. Exciting insights into the genetics of the human leukocyte antigen (HLA) system allowed improved donor selection, including HLA-identical related and unrelated donors. Besides bone marrow, other stem cell sources like granulocyte-colony stimulating-mobilized peripheral blood stem cells and cord blood stem cells have been established in clinical routine. Use of reduced-intensity or non-myeloablative conditioning regimens has been associated with a marked reduction of non-hematological toxicities and eventually, non-relapse mortality allowing older patients and individuals with comorbidities to undergo allogeneic HSCT and to benefit from GvL or antitumor effects. Whereas in the early years, malignant disease eradication by high-dose chemotherapy or radiotherapy was the ultimate goal; nowadays, allogeneic HSCT has been recognized as cellular immunotherapy relying prominently on immune mechanisms and to a lesser extent on non-specific direct cellular toxicity. This chapter will summarize the key milestones of HSCT and introduce current developments.

Cytomegalovirus-Specific T Cells Isolated by IFN-γ Secretion Assay Do Not Induce Significant Graft-Versus-Host Reactions In Vitro.

BACKGROUND: Graft-versus-host (GvH) disease (GvHD) remains a serious concern for patients undergoing antiviral cellular therapy. Despite the major improvements in cellular immunotherapy, the immunogenicity of virus-specific T cells has not yet been fully defined. This present study aims to examine how cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTLs) respond to allogeneic antigen stimulation and whether they give rise to GvHD target tissue damage. METHODS: Cytomegalovirus-specific CTLs were isolated by the IFN-γ secretion assay (gamma-catch) from healthy seropositive volunteers and expanded in vitro. The levels of intracellular IFN-γ, cytotoxic activity, IFN-γ and granzyme B secretion, and CD25 expression were measured using flow cytometry (fluorescence-activated cell sorting). The ability of CMV-CTLs to induce GvHD target tissue damage was evaluated using the human in vitro skin explant assay (skin explant assay). RESULTS: Cytomegalovirus-specific CTLs responded specifically to CMV-phosphoprotein 65 stimulation by secreting IFN-γ and killing virus peptide loaded autologous phytohemagglutinin (PHA) blasts. Compared with unselected peripheral blood mononuclear cells, CMV-CTLs induced significantly less severe cutaneous GvH tissue damage. This observation coincided with low levels of CD25 expression, as well as IFN-γ and granzyme B secretion after allogeneic antigen stimulation in both the mixed lymphocyte reaction and in the skin explant assay. CONCLUSIONS: Cytomegalovirus-specific CTLs isolated by the IFN-γ secretion assay from HLA-unmatched healthy donors exhibited a high level of anti-CMV potency without inducing significant cutaneous GvH tissue damage in vitro. This finding provides novel evidence supporting the safe use of in vitro expanded CMV-CTLs as an antiviral therapy in transplant patients with refractory CMV infections.

B-Cell-Based and Soluble Biomarkers in Body Liquids for Predicting Acute/Chronic Graft-versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the main curative therapy for hematological malignancy such as leukemias, lymphomas, or multiple myelomas and some other hematological disorders. In this therapy, cure of hematological diseases relies on graft-versus-malignancy effects by allogenic immune cells. However, severe posttransplant treatment-associated complications such as acute graft-versus-host disease (aGvHD) and chronic graft-versus-host disease (cGvHD) limit this approach. Most research into GvHD has concentrated on the aGvHD, while the more complex and multifaceted chronic form has been largely poorly investigated. cGvHD is a multi-organ autoimmune disorder and is the major cause of non-relapse morbidity and mortality following allo-HSCT, occurring in about 50% of patients, or 13,000-15,000 patients per year worldwide. Therefore, there is a high medical need for an early prediction of these therapy-associated toxicities. Biomarkers have gained importance over the last decade in diagnosis, in prognosis, and in prediction of pending diseases or side effects. Biomarkers can be cells, factors isolated from target tissues, or soluble factors that can be detected in body fluids. In this review, we aim to summarize some of the recent developments of biomarkers in the field of allo-HSCT. We will focus on cell-based biomarkers (B-cell subsets) for cGvHD and soluble factors including microRNA (miRNA), which are excreted into serum/plasma and urine. We also discuss the potential role of cytosolic and extracellular 70 kDa heat shock proteins (HSP70) as potential biomarkers for aGvHD and their role in preclinical models. Proteomic biomarkers in the blood have been used as predictors of treatment responses in patients with aGvHD for many years. More recently, miRNAs have been found to serve as a biomarker to diagnose aGvHD in the plasma. Another development relates to urine-based biomarkers that are usually detected by capillary electrophoresis and mass spectrometry. These biomarkers have the potential to predict the development of severe aGvHD (grades III-IV), overall mortality, and the pending development of cGvHD in patients posttransplant.

Multimer monitoring of CMV-specific T cells in research and in clinical applications.

Multimer monitoring has become a standard technique for detection of antigen-specific T cells. The term "multimer" refers to a group of reagents based on the multimerisation of molecules in order to raise avidity and thus stabilize binding to their ligand. Multimers for detection of antigen-specific T-cell responses are based on major histocompatibility complex class I peptide complexes. Multimer staining enables fast and direct visualization of antigen-specific T cells; thus, it is widely applied to assess antiviral immunity, e.g., monitor patients in vaccination trials or confirm purity of cell products for adoptive transfer. Assessment of T-cell immunity against persistent pathogens like cytomegalovirus (CMV) is of major importance in immunosuppressed patients. Recent advancements of multimers facilitate reversible labeling and allow isolation of epitope-specific T cells for adoptive transfer. Here, we give an overview on the different multimers and their applications, with an emphasis on CMV-specific T-cell responses.

ADAM17 silencing in mouse colon carcinoma cells: the effect on tumoricidal cytokines and angiogenesis.

ADAM17 (a disintegrin and metalloprotease 17) is a major sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules and is often overexpressed in malignant cells. It is generally accepted that ADAM17 promotes tumor development via activating growth factors from the EGF family, thus facilitating autocrine stimulation of tumor cell proliferation and migration. Here we show, using MC38CEA murine colon carcinoma model, that ADAM17 also regulates tumor angiogenesis and cytokine profile. When ADAM17 was silenced in MC38CEA cells, in vivo tumor growth and in vitro cell motility were significantly diminished, but no effect was seen on in vitro cell proliferation. ADAM17-silencing was accompanied by decreased in vitro expression of vascular endothelial growth factor-A and matrix metalloprotease-9, which was consistent with the limited angiogenesis and slower growth seen in ADAM17-silenced tumors. Among the growth factors susceptible to shedding by ADAM17, neuregulin-1 was the only candidate to mediate the effects of ADAM17 on MC38CEA motility and tumor angiogenesis. Concentrations of TNF and IFNγ, cytokines that synergistically induced proapoptotic effects on MC38CEA cells, were significantly elevated in the lysates of ADAM17-silenced tumors compared to mock transfected controls, suggesting a possible role for ADAM17 in host immune suppression. These results introduce new, complex roles of ADAM17 in tumor progression, including its impact on the anti-tumor immune response.