Search

NKG2A has emerged as a new immunotherapy target and its blockade with the novel immune checkpoint inhibitor (ICI) monalizumab can boost both NK cell and CD8

Adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have led to impressive clinical outcomes in B cell malignancies. Beyond induction of remission, the persistence of CAR-T cells is required to prevent relapse and provide long-term disease control. To improve CAR-T cell function and persistence, we developed a composite co-stimulatory domain of a B cell signaling moiety, CD79A/CD40, to induce a nuclear translocating signal, NF-κB, to synergize with other T cell signals and improve CAR-T cell function. CD79A/CD40 incorporating CD19CAR-T cells (CD19.79a.40z) exhibited higher NF-κB and p38 activity upon CD19 antigen exposure compared with the CD28 or 4-1BB incorporating CD19CAR-T cells (CD19.28z and CD19.BBz). Notably, we found that CD19.79a.40z CAR-T cells continued to suppress CD19(+) target cells throughout the co-culture assay, whereas a tendency for tumor growth was observed with CD19.28z CAR-T cells. Moreover, CD19.79a.40z CAR-T cells exhibited robust T cell proliferation after culturing with CD19(+) target cells, regardless of exogenous interleukin-2. In terms of in vivo efficiency, CD19.79a.40z demonstrated superior anti-tumor activity and in vivo CAR-T cell proliferation compared with CD19.28z and CD19.BBz CD19CAR-T cells in Raji-inoculated mice. Our data demonstrate that the CD79A/CD40 co-stimulatory domain endows CAR-T cells with enhanced proliferative capacity and improved anti-tumor efficacy in a murine model.

Therapeutic antibodies that block PD-1-mediated inhibition of T cells have revolutionized cancer therapy. Murine cancer models are an essential tool for testing the efficacy of PD-1 blockers alone or in combination with other treatments. Depending on the isotype of the antibody and the host species, blocking antibodies can also exert cytotoxic activity towards cells expressing the target molecule. In the current issue of the European Journal of Immunology [Eur. J. Immunol. 2021. 51: 1473-1481], Polesso et al. demonstrate that depletion of PD-1+ T cells by "blocking" PD-1 antibodies can greatly impact the outcome of preclinical immunotherapy experiments. Whereas some PD-1 antibodies promoted activation and proliferation of PD-1-expressing murine T cells, the authors report that administration of a particular PD-1 antibody can result in a significant loss of antigen-specific CD8 T cells in different in vivo models. These findings once more highlight that a comprehensive characterization of antibodies is warranted to avoid misinterpretation of immunotherapy studies.

Upon generation of monoclonal antibodies to the T cell antigen receptor/CD3 (TCR/CD3) complex, we isolated mAb MT3, whose reactivity correlates inversely with the production of IFN‐γ by human peripheral blood T lymphocytes. Using eukaryotic expression cloning, we identified the MT3 antigen as myelin‐and‐lymphocyte (MAL) protein. Flow cytometry analysis demonstrates high surface expression of MAL on all naïve CD4+ T cells whereas MAL expression is diminished on central memory‐ and almost lost on effector memory T cells. MAL– T cells proliferate strongly in response to stimulation with CD3/CD28 antibodies, corroborating that MAL+ T cells are naïve and MAL– T cells memory subtypes. Further, resting MAL– T cells harbor a larger pool of Ser59‐ and Tyr394‐ double phosphorylated lymphocyte‐specific kinase (Lck), which is rapidly increased upon in vitro restimulation. Previously, lack of MAL was reported to prevent transport of Lck, the key protein tyrosine kinase of TCR/CD3 signaling to the cell membrane, and to result in strongly impaired human T cell activation. Here, we show that knocking out MAL did not significantly affect Lck membrane localization and immune synapse recruitment, or transcriptional T cell activation. Collectively, our results indicate that loss of MAL is associated with activation‐induced differentiation of human T cells but not with impaired membrane localization of Lck or TCR signaling capacity., Myelin‐and‐lymphocyte protein isoform‐A (MAL‐A) is highly expressed on human naive CD4+ T cells. Activation‐induced proliferation and differentiation is associated with a gradual loss of MAL expression, resulting in highly signalling‐competent effector‐memory cells with enhanced IFN‐γ‐secretion. Knockout of MAL does not affect recruitment of lymphocyte‐specific kinase Lck and TCR downstream signalling.

Costimulatory signals potently promote T‐cell proliferation and effector function. Agonistic antibodies targeting costimulatory receptors of the TNFR family, such as 4‐1BB and CD27, have entered clinical trials in cancer patients. Currently there is limited information how costimulatory signals regulate antigen‐specific but also bystander activation of human CD8 T cells. Engineered antigen presenting cells (eAPC) efficiently presenting several common viral epitopes on HLA‐A2 in combination with MHC class I tetramer staining were used to investigate the impact of costimulatory signals on human CD8 T‐cell responses. CD28 costimulation potently augmented the percentage and number of antigen‐reactive CD8 T cells, whereas eAPC expressing 4‐1BB‐ligand induced bystander proliferation of CD8 T cells and massive expansion of NK cells. Moreover, the 4‐1BB agonist urelumab similarly induced bystander proliferation of CD8 T cells and NK cells in a dose‐dependent manner. However, the promotion of bystander CD8 T‐cell responses is not a general attribute of costimulatory TNF receptor superfamily (TNFRSF) members, since CD27 signals enhanced antigen‐specific CD8 T cells responses without promoting significant bystander activation. Thus, the differential effects of costimulatory signals on the activation of human bystander CD8 T cells should be taken into account when costimulatory pathways are harnessed for cancer immunotherapy., Using a novel system of engineered antigen presenting cells (eAPC), we demonstrate that 4‐1BB signals generated by 4‐1BBL or agonistic 4‐1BB antibodies promote bystander activation of human CD8 T cells

An artificial T cell adaptor molecule (ATAM) was generated to improve persistence of T cell receptor (TCR) gene-transduced T (TCR-T) cells compared to such persistence in a preceding study. ATAMs are gene-modified CD3ζ with the intracellular domain of 4-1BB inserted in the middle of CD3ζ. NY-ESO-1 TCR-T cells transduced with an ATAM with two separated virus vectors demonstrated superior proliferation upon antigen stimulation. To further develop clinically applicable ATAM-transduced TCR-T cells, we attempted to make a single virus vector to transduce the TCR and ATAM simultaneously. Because we failed to observe improved proliferation capacity upon stimulation after one virus vector (1vv) transduction, we compared TCR-T cells transduced with 1vv and two virus vector (2vv) methods to elucidate the reason. In Jurkat reporter cells, an ATAM transduced by the 2vv method demonstrated a higher intensity than by the 1vv method, and the ATAM intensity was associated with increased nuclear factor κB (NF-κB) signals upon stimulation. In ATAM-transduced primary T cells, a transduced ATAM by the 2vv method showed higher intensity and better proliferation. ATAM-transduced TCR-T cells demonstrated improved proliferation only when the ATAM was transduced at a higher intensity. To create a simpler transduction method, we need to develop a strategy to make a higher ATAM expression to prove the efficacy of ATAM transduction in TCR-T therapy., Graphical Abstract, An artificial T cell adaptor molecule (ATAM) with a 4-1BB intracellular domain in the middle of CD3ζ was developed and transduced into T cells simultaneously with the NY-ESO-1 TCR gene. We demonstrated that gene transfer of an ATAM above a certain threshold would be necessary to boost cell proliferation after stimulation.

TIM‐3 has been considered as a target in cancer immunotherapy. In T cells, inhibitory as well as activating functions have been ascribed to this molecule. Its role may therefore depend on the state of T cells and on the presence of interaction partners capable to perform functional pairing. Carcinoembryonic antigen‐related cell adhesion molecule (CEACAM1) has been proposed to bind TIM‐3 and to regulate its function. Using a T cell reporter platform we confirmed CEACAM1‐mediated inhibition, but CEACAM1 did not functionally engage TIM‐3. TIM‐3 and CEACAM1 coexpression was limited to a small subset of activated T cells. Moreover, results obtained in extensive binding studies were not in support of an interaction between TIM‐3 and CEACAM1. Cytoplasmic sequences derived from TIM‐3 induced inhibitory signaling in our human T cell reporter system. Our results indicate that TIM‐3 functions are independent of CEACAM1 and that this receptor has the capability to promote inhibitory signaling pathways in human T cells., The role of TIM‐3 and CEACAM1 was studied in a T cell reporter system. CEACAM1–CEACAM1 interaction coinhibited T cell activation, whereas CEACAM1 did not functionally engage TIM‐3 in cis and in trans. A chimeric receptor system revealed inhibitory signaling via the cytoplasmic tail of TIM‐3 .

Funding Information: The authors thank Laura Turunen, Jani Saarela, Katja Suomi, and Maria Nurmi of the High-Throughput Biomedicine Unit and the personnel of the Sequencing Laboratory at the Institute of Molecular Medicine Finland. The authors also thank Caroline Heckman, Sirpa Lepp?, and Olli Lohi for collaboration by providing the cell lines used in the study. The Biomedicum Helsinki Flow Cytometry Core Unit and the Biomedicum Virus Core are acknowledged for their services. The primary B-ALL samples were provided by the Finnish Hematology Registry and Clinical Biobank (FHRB). We thank all the patients for their generous participation. The FHRB biobank is supported by the Finnish Association of Hematology, Finnish Red Cross Blood Service, Institute for Molecular Medicine Finland, and participating hospitals in Finland. This study was supported by the Cancer Foundation Finland, the Sigrid Juselius Foundation, the Relander Foundation, state funding for university-level health research in Finland, and HiLife fellow funds from the University of Helsinki. O.D. was supported by grants from the Biomedicum Helsinki Foundation and the Finnish Medical Society. J. Koski was supported by grants from the Finnish Pediatric Cancer Foundation, the V?re Pediatric Research Foundation, the Cancer Foundation Finland, the Orion Research Foundation, and state funding for university-level health research in Finland. Funding Information: This study was supported by the Cancer Foundation Finland, the Sigrid Juselius Foundation, the Relander Foundation, state funding for university-level health research in Finland, and HiLife fellow funds from the University of Helsinki. O.D. was supported by grants from the Biomedicum Helsinki Foundation and the Finnish Medical Society. J. Koski was supported by grants from the Finnish Pediatric Cancer Foundation, the Väre Pediatric Research Foundation, the Cancer Foundation Finland, the Orion Research Foundation, and state funding for university-level health research in Finland. Funding Information: The authors thank Laura Turunen, Jani Saarela, Katja Suomi, and Maria Nurmi of the High-Throughput Biomedicine Unit and the personnel of the Sequencing Laboratory at the Institute of Molecular Medicine Finland. The authors also thank Caroline Heckman, Sirpa Leppä, and Olli Lohi for collaboration by providing the cell lines used in the study. The Biomedicum Helsinki Flow Cytometry Core Unit and the Biomedicum Virus Core are acknowledged for their services. The primary B-ALL samples were provided by the Finnish Hematology Registry and Clinical Biobank (FHRB). We thank all the patients for their generous participation. The FHRB biobank is supported by the Finnish Association of Hematology, Finnish Red Cross Blood Service, Institute for Molecular Medicine Finland, and participating hospitals in Finland. Publisher Copyright: © 2020 American Society of Hematology Chimeric antigen receptor (CAR) T-cell therapy has proven effective in relapsed and refractory B-cell malignancies, but resistance and relapses still occur. Better understanding of mechanisms influencing CAR T-cell cytotoxicity and the potential for modulation using small-molecule drugs could improve current immunotherapies. Here, we systematically investigated druggable mechanisms of CAR T-cell cytotoxicity using >500 small-molecule drugs and genome-scale CRISPR-Cas9 loss-of-function screens. We identified several tyrosine kinase inhibitors that inhibit CAR T-cell cytotoxicity by impairing T-cell signaling transcriptional activity. In contrast, the apoptotic modulator drugs SMAC mimetics sensitized B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma cells to anti-CD19 CAR T cells. CRISPR screens identified death receptor signaling through FADD and TNFRSF10B (TRAIL-R2) as a key mediator of CAR T-cell cytotoxicity and elucidated the RIPK1-dependent mechanism of sensitization by SMAC mimetics.Death receptor expression varied across genetic subtypes of B-cell malignancies, suggesting a link between mechanisms of CAR T-cell cytotoxicity and cancer genetics. These results implicate death receptor signaling as an important mediator of cancer cell sensitivity to CAR T-cell cytotoxicity, with potential for pharmacological targeting to enhance cancer immunotherapy. The screening data provide a resource of immunomodulatory properties of cancer drugs and genetic mechanisms influencing CAR T-cell cytotoxicity. Key Points: • Survey of immunomodulatory effects of >500 drugs identifies SMAC mimetics as sensitizers to CAR T-cell cytotoxicity. • Genome-scale CRISPR screen reveals essentiality of death receptor signaling for CAR T-cell cytotoxicity.

In this work, we report on two novel monoclonal antibodies, specific for porcine CD9. CD9 is a tetraspanin that is expressed on a wide variety of cells. We phenotyped porcine immune cell subsets and found that CD9 was expressed on all monocytes as well as a subset of B cells. CD9 was variably expressed on T cells, with CD4 T cells containing the highest frequency of CD9+ cells. CD9 expression positively correlated with the frequency of central memory CD4 T cells in ex vivo PBMC. Therefore, we proceeded to explore CD9 as a marker of T cell function. Here we observed that CD9 was expressed on the vast majority of long-lived influenza A virus-specific effector cells that retained the capacity for cytokine production in response to in vitro recall antigen. Therefore, the new antibodies enable the detection of a cell surface molecule with functional relevance to T cells. Considering the importance of CD9 in membrane remodelling across many cell types, they will also benefit the wider field of swine biomedical research.

Antibody-based therapies blocking the programmed cell death-1/ligand-1 (PD-1/PD-L1) axis have provided unprecedent clinical success in cancer treatment. Acquired resistance, however, frequently occurs, commonly associated with the upregulation of additional inhibitory molecules. Diacylglycerol kinase (DGK) α limits the extent of Ras activation in response to antigen recognition, and its upregulation facilitates hypofunctional, exhausted T cell states. Pharmacological DGKα targeting restores cytotoxic function of chimeric antigen receptor and CD8+ T cells isolated from solid tumors, suggesting a mechanism to reverse T cell exhausted phenotypes. Nevertheless, the contribution of DGKα downstream of the PD-1/PD-L1 inhibitory axis in human T cells and the consequences of combining DGKα and anti-PD-1/PD-L1 inhibitors are still unresolved relevant issues. We used a human triple parameter reporter cell line to investigate DGKα contribution to the PD-1/PD-L1 inhibitory pathway. We also addressed the impact of deleting DGKα expression in the growth dynamics and systemic tumor-derived effects of a PD-1-related tumor model, the MC38 colon adenocarcinoma. We identify DGKα as a contributor to the PD-1/PD-L1 axis that strongly limits the Ras/ERK/AP-1 pathway. DGKα function reinforces exhausted T cell phenotypes ultimately promoting tumor growth and generalized immunosuppression. Pharmacological DGKα inhibition selectively enhances AP-1 transcription and, importantly, cooperates with antibodies blocking the PD-1/PD-L1 interrelation. Our results indicate that DGKα inhibition could provide an important mechanism to revert exhausted T lymphocyte phenotypes and thus favor proper anti-tumor T cell responses. The cooperative effect observed after PD-1/PD-L1 and DGKα blockade offers a promising strategy to improve the efficacy of immunotherapy in the treatment of cancer.

Background Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is a novel treatment option for patients with B-cell lineage derived cancers. Other cancer entities are less successfully targeted due to the lack of antigens that are expressed on cancer cells but not healthy tissue cells. One way to address this issue is the concept of inhibitory chimeric antigen receptors (iCARs) that deliver an inhibitory signal upon antigen encounter on off-target cells. The activating effect of CARs depends on a multitude of factors including expression levels, affinity, different signaling domains and steric effects. It has to be expected that co-expression of an iCAR would result in even more complexity. Consequently, there is a demand for a robust high-throughput cellular system to evaluate iCAR-formats. Materials and Methods Our approach is based on a previously published Jurkat based triple parameter reporter cell (TPR) system. This setup allows for molecular monitoring of the T cell activation state by measuring fluorescent reporter gene expression via flow cytometry. Inhibitory effects of receptors are determined as the ratio of cellular geometric mean fluorescent intensity (gMFI) in the presence of the inhibitory ligand versus without inhibitory ligand. Results To test if inhibitory receptors would measurably reduce reporter activation, PD-1 as a well-characterized inhibitory receptor was expressed on Jurkat TPRs. When PD-L1 was present during stimulation reporter activation was reduced by 26–34% proving feasibility of our approach. Intracellular domains of other inhibitory receptors including BTLA, ILT-2 and KIR2DL1 were evaluated. All three domains outperformed PD-1 in a series of experiments with a mean reduction of gMFI by 48–57%, 50–53% and 38–41% respectively. To assess if the reporter platform could be used to study downstream signaling pathways of inhibitory constructs we created a SHP-2 knock-out reporter cell line using the CRISPR/Cas9 gene editing technique. To simulate different degrees of activating signal strength, peptide-MHC complex recognizing activating receptors were created. The reporter activation correlated with the concentrations of peptide in the stimulation cultures. Co-expression of iCAR and CAR could be achieved using selection for two separate antibiotic resistance genes introduced into the respective vector. Preliminary experiments showed greatly reduced inhibitory efficacy of iCAR molecules due to an adhesion effect resulting from the high affinity extracellular domain of the iCARs that lead to tighter cell-cell contact and stronger stimulation through the CAR. Conclusions We present a highly flexible and controllable Jurkat-based reporter cell platform for the thorough study of inhibitory signaling mechanisms. This project was supported by the Austrian Science Fund, FWF; Project P32411 Disclosure Information M.A. Funk: None. A. De Sousa Linhares: None. C. Battin: None. K. Radakovics: None. J. Leitner: None. P. Steinberger: None.

Objective Transgenic re‐expression enables unbiased investigation of T‐cell receptor (TCR)‐intrinsic characteristics detached from its original cellular context. Recent advancements in TCR repertoire sequencing and development of protocols for direct cloning of full TCRαβ constructs now facilitate large‐scale transgenic TCR re‐expression. Together, this offers unprecedented opportunities for the screening of TCRs for basic research as well as clinical use. However, the functional characterisation of re‐expressed TCRs is still a complicated and laborious matter. Here, we propose a Jurkat‐based triple parameter TCR signalling reporter endogenous TCR knockout cellular platform (TPRKO) that offers an unbiased, easy read‐out of TCR functionality and facilitates high‐throughput screening approaches. Methods As a proof‐of‐concept, we transgenically re‐expressed 59 human cytomegalovirus‐specific TCRs and systematically investigated and compared TCR function in TPRKO cells versus primary human T cells. Results We demonstrate that the TPRKO cell line facilitates antigen‐HLA specificity screening via sensitive peptide‐MHC‐multimer staining, which was highly comparable to primary T cells. Also, TCR functional avidity in TPRKO cells was strongly correlating to primary T cells, especially in the absence of CD8αβ co‐receptor. Conclusion Overall, our data show that the TPRKO cell lines can serve as a surrogate of primary human T cells for standardised and high‐throughput investigation of TCR biology., A highly sensitive triple parameter T‐cell receptor (TCR) reporter cell line was adapted for high‐throughput testing of transgenically expressed TCRs by CRISPR/Cas9‐mediated deletion of the endogenous TCR and introduction of the CD8αβ co‐receptor. Using 59 CMV‐specific TCRs, we demonstrated a high similarity with primary T cells in terms of multimer staining, functional avidity and CD8‐dependence. In conclusion, we present a T‐cell reporter platform excellently suited for high‐throughput functional preclinical testing of TCRs.

Chimeric antigen receptor (CAR)-T cell immunotherapy is under intense preclinical and clinical investigation, and it involves a rapidly increasing portfolio of novel target antigens and CAR designs. We established a platform that enables rapid and high-throughput CAR-screening campaigns with reporter cells derived from the T cell lymphoma line Jurkat. Reporter cells were equipped with nuclear factor kappa B (NF kappa B) and nuclear factor of activated T cells (NFAT) reporter genes that generate a duplex output of enhanced CFP (ECFP) and EGFP, respectively. As a proof of concept, we modified reporter cells with CD19-specific and ROR1-specific CARs, and we detected high-level reporter signals that allowed distinguishing functional from non-functional CAR constructs. The reporter data were highly reproducible, and the time required for completing each testing campaign was substantially shorter with reporter cells (6 days) compared to primary CAR-T cells (21 days). We challenged the reporter platform to a large-scale screening campaign on a ROR1-CAR library, and we showed that reporter cells retrieved a functional CAR variant that was present with a frequency of only 6 in 1.05 x 10(6). The data illustrate the potential to implement this reporter platform into the preclinical development path of novel CAR-T cell products and to inform and accelerate the selection of lead CAR candidates for clinical translation.

Complement regulation leads to the generation of complement split products (CSPs) such as complement component (C)4d, a marker for disease activity in autoimmune syndromes or antibody-mediated allograft rejection. However, the physiologic role of C4d has been unknown. By screening murine thymoma BW5147 cells expressing a cDNA library generated from human monocyte-derived dendritic cells with recombinant human C4d, we identified Ig-like transcript (ILT)4 and ILT5v2 as cellular receptors for C4d. Both receptors, expressed on monocytes, macrophages, and dendritic cells, also interacted with the CSPs C3d, C4b, C3b, and iC3b. However, C4d did not bind to classic complement receptors (CRs). Interaction between cell surface-resident ILT4 and soluble monomeric C4d resulted in endocytosis of C4d. Surprisingly, binding of soluble ILT4 to C4d covalently immobilized to a cellular surface following classic complement activation could not be detected. Remarkably, C4d immobilized to a solid phaseviaits intrinsic thioester conferred a dose-dependent inhibition of TNF-α and IL-6 secretion in monocytes activatedviaFc-cross-linking of up to 50% as compared to baseline. Similarly, C4d conferred an attenuation of intracellular Ca(2+)flux in monocytes activatedviaFc-cross-linking. In conclusion, ILT4 represents a scavenger-type endocytotic CR for soluble monomeric C4d, whereas attenuation of monocyte activation by physiologically oriented C4d on a surface appears to be dependent on a yet to be identified C4d receptor.-Hofer, J., Forster, F., Isenman, D. E., Wahrmann, M., Leitner, J., Hölzl, M. A., Kovarik, J. K., Stockinger, H., Böhmig, G. A., Steinberger, P., Zlabinger, G. J. Ig-like transcript 4 as a cellular receptor for soluble complement fragment C4d.

Immune checkpoint inhibitors targeting coinhibitory pathways in T cells possess efficacy in combating cancer. In addition to PD-1/PD-L1 and CTLA-4 antibodies which are already established in tumor immunotherapy, immune checkpoints such as LAG-3 or BTLA are emerging, which may have the potential to enhance T-cell responses alone or in combination with PD-1 blockers. CD4+ T cells play a central role in the immune system and contribute to productive immune responses in multiple ways. The effects of immune checkpoint inhibitors on this cell subset may thus critically influence therapeutic outcomes. Here, we have used in vitro responses to tetanus toxoid (TT) as a model system to study the effects of immune checkpoint inhibitors on CD4+ T-cell responses. CFSE-labeled PBMCs of 65 donors were stimulated with TT in the presence of blocking antibodies to PD-L1, CTLA-4, LAG-3, or BTLA for 7 days. We found that the PD-L1 antibody greatly enhanced cytokine production and antigen-specific CD4+ T-cell proliferation, whereas blocking antibodies to BTLA or LAG-3 did not augment responses to TT. Surprisingly, the presence of the therapeutic CTLA-4 antibody ipilimumab resulted in a significant reduction of CD4+ T-cell proliferation and cytokine production. Stimulation experiments with an IgG4 variant of ipilimumab indicated that the inhibitory effect of ipilimumab was dependent on its IgG1 isotype. Our results indicate that the therapeutic CTLA-4 antibody ipilimumab can impair CD4+ effector T-cell responses and that this activity is mediated by its Fc part and CD16-expressing cells. Electronic supplementary material The online version of this article (10.1007/s00262-019-02369-x) contains supplementary material, which is available to authorized users.

The B7-like protein family members play critical immunomodulatory roles and constitute attractive targets for the development of novel therapies for human diseases. We identified Ig-like domain–containing receptor (ILDR)2 as a novel B7-like protein with robust T cell inhibitory activity, expressed in immune cells and in immune-privileged and inflamed tissues. A fusion protein, consisting of ILDR2 extracellular domain with an Fc fragment, that binds to a putative counterpart on activated T cells showed a beneficial effect in the collagen-induced arthritis model and abrogated the production of proinflammatory cytokines and chemokines in autologous synovial-like cocultures of macrophages and cytokine-stimulated T cells. Collectively, these findings point to ILDR2 as a novel negative regulator for T cells, with potential roles in the development of immune-related diseases, including autoimmunity and cancer.

A substantial proportion of CD8+ T cells in adults lack the expression of the CD28 molecule, and the aging of the immune system is associated with a steady expansion of this T cell subset. CD28−CD8+ T cells are characterized by potent effector functions but impaired responses to antigenic challenge. CD28 acts as the primary T cell costimulatory receptor, but there are numerous additional receptors that can costimulate the activation of T cells. In this study, we have examined such alternative costimulatory pathways regarding their functional role in CD28−CD8+ T cells. Our study showed that most costimulatory molecules have a low capacity to activate CD28-deficient T cells, whereas the engagement of the CD2 molecule by its ligand CD58 clearly costimulated proliferation, cytokine production, and effector function in this T cell subset. CD58 is broadly expressed on APCs including dendritic cells. Blocking CD58 mAb greatly reduced the response of human CD28−CD8+ T cells to allogeneic dendritic cells, as well as to viral Ags. Our results clearly identify the CD58/CD2 axis as the primary costimulatory pathway for CD8 T cells that lack CD28. Moreover, we show that engagement of CD2 amplifies TCR signals in CD28−CD8+ T cells, demonstrating that the CD2–CD58 interaction has a genuine costimulatory effect on this T cell subset. CD2 signals might promote the control of viral infection by CD28−CD8+ T cells, but they might also contribute to the continuous expansion of CD28−CD8+ T cells during chronic stimulation by persistent Ag.

Adoptive T cell therapy using TCR transgenic autologous T cells has shown great potential for the treatment of tumor patients. Thorough characterization of genetically reprogrammed T cells is necessary to optimize treatment success. Here, we describe the generation of triple parameter reporter T cells based on the Jurkat 76 T cell line for the evaluation of TCR and chimeric antigen receptor functions as well as adoptive T cell strategies. This Jurkat subline is devoid of endogenous TCR alpha and TCR beta chains, thereby circumventing the problem of TCR miss-pairing and unexpected specificities. The resultant reporter cells allow simultaneous determination of the activity of the transcription factors NF-κB, NFAT and AP-1 that play key roles in T cell activation. Human TCRs directed against tumor and virus antigens were introduced and reporter responses were determined using tumor cell lines endogenously expressing the antigens of interest or via addition of antigenic peptides. Finally, we demonstrate that coexpression of adhesion molecules like CD2 and CD226 as well as CD28 chimeric receptors represents an effective strategy to augment the response of TCR-transgenic reporters to cells presenting cognate antigens.

Antithymocyte globulin (ATG) preparations are used for treatment and prevention of graft rejection episodes, graft versus host disease and aplastic anemia. The immunomodulatory and immuosuppressive properties of ATGs are mediated by their interaction with a large variety of antigens expressed on immune and nonimmune cell populations. We have conducted a comprehensive analysis on antibody specificities contained in rabbit ATGs in clinical use, ATG-Fresenius (ATG-F) and Thymoglobulin (THG). We have used retroviral expression cloning to identify novel ATG antigens and demonstrate that together with ATG antigens described earlier, these molecules account for the majority of ATG antibodies directed to human cells. Moreover, we have employed cell lines engineered to express antigens at high levels to quantify the antibodies directed to each ATG antigen. We have used cell lines expressing the T cell receptor complex, CD2 and CD28 to remove antibodies to these antigens from ATG preparations and demonstrate that this treatment abrogated the ability of ATGs to induce activation and forkhead box P3 expression in T cells. Comprehensive information and differences on the antigens targeted by ATG-F and THG as well as novel approaches to assess their functional properties are the basis for a better understanding of their immunomodulatory capacities and might eventually translate into improved ATG-based regimen.

Langerhans cell histiocytosis (LCH) is an enigmatic disease defined by the accumulation of Langerhans cell-like dendritic cells (DCs). In the present study, we demonstrate that LCH cells exhibit a unique transcription profile that separates them not only from plasmacytoid and myeloid DCs, but also from epidermal Langerhans cells, indicating a distinct DC entity. Molecular analysis revealed that isolated and tissue-bound LCH cells selectively express the Notch ligand Jagged 2 (JAG2) and are the only DCs that express both Notch ligand and its receptor. We further show that JAG2 signaling induces key LCH-cell markers in monocyte-derived DCs, suggesting a functional role of Notch signaling in LCH ontogenesis. JAG2 also induced matrix-metalloproteinases 1 and 12, which are highly expressed in LCH and may account for tissue destruction in LCH lesions. This induction was selective for DCs and was not recapitulated in monocytes. The results of the present study suggest that JAG2-mediated Notch activation confers phenotypic and functional aspects of LCH to DCs; therefore, interference with Notch signaling may be an attractive strategy to combat this disease.

The cytoplasmic tail of CD45 (ct‐CD45) is proteolytically cleaved and released upon activation of human phagocytes. It acts on T cells as an inhibitory, cytokine‐like factor in vitro. Here, we show that ct‐CD45 is abundant in human peripheral blood plasma from healthy adults compared with plasma derived from umbilical cord blood and plasma from patients with rheumatoid arthritis or systemic lupus erythematosus. Plasma depleted of ct‐CD45 enhanced T‐cell proliferation, while addition of exogenous ct‐CD45 protein inhibited proliferation and reduced cytokine production of human T lymphocytes in response to TCR signaling. Inhibition of T‐cell proliferation by ct‐CD45 was overcome by costimulation via CD28. T‐cell activation in the presence of ct‐CD45 was associated with an upregulation of the quiescence factors Schlafen family member 12 (SLFN12) and Krueppel‐like factor 2 (KLF2) as well as of the cyclin‐dependent kinase (CDK) inhibitor p27kip1. In contrast, positive regulators of the cell cycle such as cyclin D2 and D3 as well as CDK2 and CDK4 were found to be downregulated in response to ct‐CD45. In summary, we demonstrate that ct‐CD45 is present in human plasma and sets the threshold of T‐cell activation.

Up to now for Swine Workshop Cluster 2 (SWC2) the orthologous human CD molecule was unknown. By use of the SWC2-specific mAb b30c7 and a retroviral cDNA expression library derived from stimulated porcine peripheral blood mononuclear cells we could identify SWC2 as porcine CD27. Phenotypic analyses of lymphocytes isolated from blood and lymphatic organs revealed that mature T cells in thymus and T cells in the periphery with a naive phenotype were CD27+. However, within CD8α+ T helper and CD8α+ γδ T cells also CD27− cells were present, indicating a down-regulation after antigen contact in vivo. B cells lacked CD27 expression, whereas NK cells expressed intermediate levels. Furthermore, plate-bound mAb b30c7 showed a costimulatory capacity on CD3-activated T cells for proliferation, IFN-γ and TNF-α production. Hence, our data indicate an important role of porcine CD27 for T-cell differentiation and activation as described for humans and mice.

Background Antithymocyte globulins (ATGs) are used to prevent and treat allograft rejection and graft versus host disease. They are purified IgG fractions derived from rabbits immunized with the Jurkat T-cell line (ATG-Fresenius) or thymus cells (Thymoglobulin). Differences not only in the amounts of leukocyte reactive antibodies but also in the antigens targeted by ATGs could potentially affect the clinical efficacy of different batches of these polyclonal antibody preparations. Methods Four batches of ATG-Fresenius and Thymoglobulin were compared regarding their capacity to interact with human leukocytes from healthy donors and kidney transplant recipients. Using flow cytometric assays, we analyzed the reactivity of these ATG preparations with Jurkat cells and with primary leukocytes. In addition, ATGs derived from different batches were probed with a panel of cell lines expressing high levels of ATG antigens. Their ability to mediate complement-mediated lysis of human monocytes and lymphocytes was also compared. Results Binding studies to leukocyte antigens and functional analysis pointed to a high conformity among different batches in both ATG preparations. Conclusions From our in vitro data, it can be expected that ATGs derived from different batches will not differ in their clinical efficacy. Furthermore, the methods described in this study allow for a reliable analysis of ATG batches.

PD-1 expression on T cells correlates with T-cell exhaustion and disease progression in HIV-infected patients. Previous studies have shown that combinational antiretroviral therapy induced viral suppression results in immune restoration and reduced PD-1 expression. However, a significant number of patients fail to restore CD4 T cells despite suppression of HIV replication below limit of quantification. In this study, we have analyzed PD-1 expression on CD4 and CD8 T cells in patients with poor immune reconstitution despite successful highly active antiretroviral therapy. We found that T cells of such patients express significantly higher levels of PD-1 than patients who had normal recovery of CD4 cells after treatment. In contrast, failing immune reconstitution was not associated with the expression of activation markers, indicating that PD-1 is a unique marker for failing immune reconstitution despite viral suppression. Furthermore, we show that T cells from patients with poor immune recovery differ from T cells of elderly in respect of their marker profile. PD-1 expression negatively correlated with individual CD4 cell counts, and PD-1 expressing T cells were more prone to programmed death ligand-mediated inhibition of T-cell proliferation, indicating that PD-1-mediated T-cell suppression may have a role in impaired immune reconstitution in HIV patients.

The polyclonal rabbit antithymocyte globulins (ATGs), Thymoglobulin and ATG-Fresenius S, are widely used for prevention and therapy of allograft rejection and graft versus host disease. Dendritic cells (DC) govern immune responses and thus the interaction of ATGs with these cells could potentially contribute to the clinical effects of ATG therapy. Currently there is little information on the DC-antigens targeted by ATGs. In this study we have used a new methodology to identify DC surface antigens recognized by ATGs. By screening an eukaryotic expression library generated from DC with ATGs we could identify several novel ATG antigens including CD81, CD82, CD98, CD99 and CD147. Furthermore, we engineered cells to express previously described ATG antigens and probed them with Thymoglobulin and ATG-Fresenius S. Our results demonstrated strong binding to some but not all of these molecules. We show that previously described antigens and antigens identified in this study account for around 80% of the DC reactivity of ATGs. Analysis of molecules induced by ATG-DC interaction are more in support for an activation of these cells by ATGs than for a specific induction of a tolerogenic DC phenotype.