IL12 integrated into the CAR exodomain converts CD8+ T cells to poly-functional NK-like cells with superior killing of antigen-loss tumors.

Chimeric antigen receptor (CAR)-redirected T cell therapy often fails to control tumors in the long term due to selecting cancer cells that downregulated or lost CAR targeted antigen. To reprogram the functional capacities specifically of engineered CAR T cells, we inserted IL12 into the extracellular moiety of a CD28-ζ CAR; both the CAR endodomain and IL12 were functionally active, as indicated by antigen-redirected effector functions and STAT4 phosphorylation, respectively. The IL12-CAR reprogrammed CD8+ T cells toward a so far not recognized natural killer (NK) cell-like signature and a CD94+CD56+CD62Lhigh phenotype closely similar, but not identical, to NK and cytokine induced killer (CIK) cells. In contrast to conventional CAR T cells, IL12-CAR T cells acquired antigen-independent, human leukocyte antigen E (HLA-E) restricted cytotoxic capacities eliminating antigen-negative cancer cells in addition to eliminating cancer cells with CAR cognate antigen. Simultaneous signaling through both the CAR endodomain and IL12 were required for inducing maximal NK-like cytotoxicity; adding IL12 to conventional CAR T cells was not sufficient. Antigen-negative tumors were attacked by IL12-CAR T cells, but not by conventional CAR T cells. Overall, we present a prototype of a new family of CARs that augments tumor recognition and elimination through expanded functional capacities by an appropriate cytokine integrated into the CAR exodomain.

Designed Ankyrin Repeat Protein (DARPin) to target chimeric antigen receptor (CAR)-redirected T cells towards CD4+ T cells to reduce the latent HIV+ cell reservoir.

Chimeric Antigen Receptor (CAR)-redirected T cells show great efficacy in the patient-specific therapy of hematologic malignancies. Here, we demonstrate that a DARPin with specificity for CD4 specifically redirects and triggers the activation of CAR engineered T cells resulting in the depletion of CD4+ target cells aiming for elimination of the human immunodeficiency virus (HIV) reservoir.

Blocking CD30 on T Cells by a Dual Specific CAR for CD30 and Colon Cancer Antigens Improves the CAR T Cell Response against CD30- Tumors.

Chimeric antigen receptor (CAR)-engineered T cells are efficacious in controlling advanced leukemia and lymphoma, however, they fail in the treatment of solid cancer, which is thought to be due to insufficient T cell activation. We revealed that the immune response of CAR T cells with specificity for carcinoembryonic antigen (CEA) was more efficacious against CEA+ cancer cells when simultaneously incubated with an anti-CD30 immunotoxin or anti-CD30 CAR T cells, although the targeted cancer cells lack CD30. The same effect was achieved when the anti-CD30 single-chain variable fragment (scFv) was integrated into the extracellular domain of the anti-CEA CAR. Improvement in T cell activation was due to interfering with the T cell CD30-CD30L interaction by the antagonistic anti-CD30 scFv HRS3; an agonistic anti-CD30 scFv or targeting the high-affinity interleukin-2 (IL-2) receptor was not effective. T cells with the anti-CD30/CEA CAR showed superior immunity against established CEA+ CD30- tumors in a mouse model. The concept is broadly applicable since anti-CD30/TAG72 CAR T cells also showed improved elimination of TAG72+ CD30- cancer cells. Taken together, targeting CD30 on CAR T cells by the HRS3 scFv within the anti-tumor CAR improves the redirected immune response against solid tumors.

CD28-ζ CAR T Cells Resist TGF-ß Repression through IL-2 Signaling, Which Can Be Mimicked by an Engineered IL-7 Autocrine Loop.

Adoptive cell therapy with chimeric antigen receptor (CAR)-redirected T cells induced spectacular regressions of leukemia and lymphoma, however, failed so far in the treatment of solid tumors. A cause is thought to be T cell repression through TGF-ß, which is massively accumulating in the tumor tissue. Here, we show that T cells with a CD28-ζ CAR, but not with a 4-1BB-ζ CAR, resist TGF-ß-mediated repression. Mechanistically, LCK activation and consequently IL-2 release and autocrine IL-2 receptor signaling mediated TGF-ß resistance; deleting the LCK-binding motif in the CD28 CAR abolished both IL-2 secretion and TGF-ß resistance, while IL-2 add-back restored TGF-ß resistance. Other γ-cytokines like IL-7 and IL-15 could replace IL-2 in this context. This is demonstrated by engineering IL-2 deficient CD28ΔLCK-ζ CAR T cells with a hybrid IL-7 receptor to provide IL-2R ß chain signaling upon IL-7 binding. Such modified T cells showed improved CAR T cell activity against TGF-ß+ tumors. Data draw the concept that an autocrine loop resulting in IL-2R signaling can make CAR T cells more potent in staying active against TGF-ß+ solid tumors.

Functionality and Cell Senescence of CD4/ CD8-Selected CD20 CAR T Cells Manufactured Using the Automated CliniMACS Prodigy® Platform.

Clinical studies using autologous CAR T cells have achieved spectacular remissions in refractory CD19+ B cell leukaemia, however some of the patient treatments with CAR T cells failed. Beside the heterogeneity of leukaemia, the distribution and senescence of the autologous cells from heavily pretreated patients might be further reasons for this. We performed six consecutive large-scale manufacturing processes for CD20 CAR T cells from healthy donor leukapheresis using the automated CliniMACS Prodigy® platform. Starting with a CD4/CD8-positive selection, a high purity of a median of 97% T cells with a median 65-fold cell expansion was achieved. Interestingly, the transduction rate was significantly higher for CD4+ compared to CD8+ T cells and reached in a median of 23%. CD20 CAR T cells showed a good specific IFN-γ secretion after cocultivation with CD20+ target cells which correlated with good cytotoxic activity. Most importantly, 3 out of 5 CAR T cell products showed an increase in telomere length during the manufacturing process, while telomere length remained consistent in one and decreased in another process. In conclusion, this shows for the first time that beside heterogeneity among healthy donors, CAR T cell products also differ regarding cell senescence, even for cells manufactured in a standardised automated process.

Chimeric antigen receptor T cells targeting Fc µ receptor selectively eliminate CLL cells while sparing healthy B cells.

Adoptive cell therapy of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR)-modified T cells targeting CD19 induced lasting remission of this refractory disease in a number of patients. However, the treatment is associated with prolonged "on-target off-tumor" toxicities due to the targeted elimination of healthy B cells demanding more selectivity in targeting CLL cells. We identified the immunoglobulin M Fc receptor (FcµR), also known as the Fas apoptotic inhibitory molecule-3 or TOSO, as a target for a more selective treatment of CLL by CAR T cells. FcµR is highly and consistently expressed by CLL cells; only minor levels are detected on healthy B cells or other hematopoietic cells. T cells with a CAR specific for FcµR efficiently responded toward CLL cells, released a panel of proinflammatory cytokines and lytic factors, like soluble FasL and granzyme B, and eliminated the leukemic cells. In contrast to CD19 CAR T cells, anti-FcµR CAR T cells did not attack healthy B cells. T cells with anti-FcµR CAR delayed outgrowth of Mec-1-induced leukemia in a xenograft mouse model. T cells from CLL patients in various stages of the disease, modified by the anti-FcµR CAR, purged their autologous CLL cells in vitro without reducing the number of healthy B cells, which is the case with anti-CD19 CAR T cells. Compared with the currently used therapies, the data strongly imply a superior therapeutic index of anti-FcµR CAR T cells for the treatment of CLL.

Of CARs and TRUCKs: chimeric antigen receptor (CAR) T cells engineered with an inducible cytokine to modulate the tumor stroma.

Adoptive T-cell therapy recently achieved impressive efficacy in early phase trials, in particular in hematologic malignancies, strongly supporting the notion that the immune system can control cancer. A current strategy of favor is based on ex vivo-engineered patient T cells, which are redirected by a chimeric antigen receptor (CAR) and recognize a predefined target by an antibody-derived binding domain. Such CAR T cells can substantially reduce the tumor burden as long as the targeted antigen is present on the cancer cells. However, given the tremendous phenotypic diversity in solid tumor lesions, a reasonable number of cancer cells are not recognized by a given CAR, considerably reducing the therapeutic success. This article reviews a recently described strategy for overcoming this shortcoming of the CAR T-cell therapy by modulating the tumor stroma by a CAR T-cell-secreted transgenic cytokine like interleukin-12 (IL-12). The basic process is that CAR T cells, when activated by their CAR, deposit IL-12 in the targeted tumor lesion, which in turn attracts an innate immune cell response toward those cancer cells that are invisible to CAR T cells. Such TRUCKs, T cells redirected for universal cytokine-mediated killing, exhibited remarkable efficacy against solid tumors with diverse cancer cell phenotypes, suggesting their evaluation in clinical trials.

Superior Therapeutic Index in Lymphoma Therapy: CD30(+) CD34(+) Hematopoietic Stem Cells Resist a Chimeric Antigen Receptor T-cell Attack.

Recent clinical trials with chimeric antigen receptor (CAR) redirected T cells targeting CD19 revealed particular efficacy in the treatment of leukemia/lymphoma, however, were accompanied by a lasting depletion of healthy B cells. We here explored CD30 as an alternative target, which is validated in lymphoma therapy and expressed by a broad variety of Hodgkin's and non-Hodgkin's lymphomas. As a safty concern, however, CD30 is also expressed by lymphocytes and hematopoietic stem and progenitor cells (HSPCs) during activation. We revealed that HRS3scFv-derived CAR T cells are superior since they were not blocked by soluble CD30 and did not attack CD30(+) HSPCs while eliminating CD30(+) lymphoma cells. Consequently, normal hemato- and lymphopoiesis was not affected in the long-term in the humanized mouse; the number of blood B and T cells remained unchanged. We provide evidence that the CD30(+) HSPCs are protected against a CAR T-cell attack by substantially lower CD30 levels than lymphoma cells and higher levels of the granzyme B inactivating SP6/PI9 serine protease, which furthermore increased upon activation. Taken together, adoptive cell therapy with anti-CD30 CAR T cells displays a superior therapeutic index in the treatment of CD30(+) malignancies leaving healthy activated lymphocytes and HSPCs unaffected.

Stability and activity of MCSP-specific chimeric antigen receptors (CARs) depend on the scFv antigen-binding domain and the protein backbone.

Chimeric antigen receptor (CAR)-modified T cells emerged as effective tools in the immunotherapy of cancer but can produce severe on-target off-tissue toxicities. This risk can conceivably be overcome, at least partially, by transient transfection. The design of CARs, however, has so far not been optimized for use in non-permanent T cell modification. Here we compared the performance of T cells modified with three different first- and second-generation CARs, each specific for MCSP (HMW-MAA) which is commonly expressed by melanoma cells. Upon RNA transfer, the expression of all receptors was limited in time. The second-generation CARs, which combined CD28-CD3ζ signaling, were expressed at higher levels and more prolonged than first-generation CARs with CD3ζ only. The CD28 domain increased the cytokine production, but had only an indirect effect on the lytic capacity, by prolonging the CAR expression. Especially for the second-generation CARs, the scFv clearly impacted the level and duration of CAR expression and the T cell performance. Thus, we identified a CAR high in both expression and anti-tumor cell reactivity. T cells transfected with this CAR increased the mean survival time of mice after challenge with melanoma cells. To facilitate clinical application, this CAR was used to redirect T cells from late-stage melanoma patients by RNA transfection. These T cells mediated effective antigen-specific tumor cell lysis and release of pro-inflammatory cytokines, even after cryoconservation of the transfected T cells. Taken together, the analysis identified a CAR with superior anti-melanoma performance after RNA transfer which is a promising candidate for clinical exploration.

Survivin blockade sensitizes rhabdomyosarcoma cells for lysis by fetal acetylcholine receptor-redirected T cells.

Cellular immunotherapy may provide a strategy to overcome the poor prognosis of metastatic and recurrent rhabdomyosarcoma (RMS) under the current regimen of polychemotherapy. Because little is known about resistance mechanisms of RMS to cytotoxic T cells, we investigated RMS cell lines and biopsy specimens for expression and function of immune costimulatory receptors and anti-apoptotic molecules by RT-PCR, Western blot analysis, IHC, and cytotoxicity assays using siRNA or transfection-modified RMS cell lines, together with engineered RMS-directed cytotoxic T cells specific for the fetal acetylcholine receptor. We found that costimulatory CD80 and CD86 were consistently absent from all RMSs tested, whereas inducible T-cell co-stimulator ligand (ICOS-L; alias B7H2) was expressed by a subset of RMSs and was inducible by tumor necrosis factor α in two of five RMS cell lines. Anti-apoptotic survivin, along with other inhibitor of apoptosis (IAP) family members (cIAP1, cIAP2, and X-linked inhibitor of apoptosis protein), was overexpressed by RMS cell lines and biopsy specimens. Down-regulation of survivin by siRNA or pharmacologically in RMS cells increased their susceptibility toward a T-cell attack, whereas induction of ICOS-L did not. Treatment of RMS-bearing Rag(-/-) mice with fetal acetylcholine receptor-specific chimeric T cells delayed xenograft growth; however, this happened without definitive tumor eradication. Combined blockade of survivin and application of chimeric T cells in vivo suppressed tumor proliferation during survivin inhibition. In conclusion, survivin blockade provides a strategy to sensitize RMS cells for T-cell-based therapy.

Arming cytokine-induced killer cells with chimeric antigen receptors: CD28 outperforms combined CD28-OX40 "super-stimulation".

Cytokine-induced killer (CIK) cells raised interest for use in cellular antitumor therapy due to their capability to recognize and destroy autologous tumor cells in a HLA-independent fashion. The antitumor attack of CIK cells, predominantly consisting of terminally differentiated CD8(+)CD56(+) cells, can be improved by redirecting by a chimeric antigen receptor (CAR) that recognizes the tumor cell and triggers CIK cell activation. The requirements for CIK cell activation were, however, so far less explored and are likely to be different from those of "younger" T cells. We revealed that CD28 and OX40 CARs produced higher interferon- secretion as compared with the first-generation ζ-CAR; CD28-ζ and the third-generation CD28-ζ-OX40 CAR, however, performed similar in modulating most CIK cell effector functions. Compared with the CD28-ζ CAR, however, the CD28-ζ-OX40 CAR accelerated terminal maturation of CD56(+) CIK cells producing high frequencies in activation-induced cell death (AICD) and reduced antitumor efficiency in vivo. Consequently, CD28-ζ CAR CIK cells of CD56(-) phenotype were superior in redirected tumor cell elimination. CAR-mediated CIK cell activation also increased antigen-independent target cell lysis; the CD28-ζ CAR was more efficient than the CD28-ζ-OX40 CAR. Translated into therapeutic strategies, CAR-redirected CIK cells benefit from CD28 costimulation; "super-costimulation" by the CD28-ζ-OX40 CAR, however, performed less in antitumor efficacy due to increased AICD.

Eradication of melanomas by targeted elimination of a minor subset of tumor cells.

Proceeding on the assumption that all cancer cells have equal malignant capacities, current regimens in cancer therapy attempt to eradicate all malignant cells of a tumor lesion. Using in vivo targeting of tumor cell subsets, we demonstrate that selective elimination of a definite, minor tumor cell subpopulation is particularly effective in eradicating established melanoma lesions irrespective of the bulk of cancer cells. Tumor cell subsets were specifically eliminated in a tumor lesion by adoptive transfer of engineered cytotoxic T cells redirected in an antigen-restricted manner via a chimeric antigen receptor. Targeted elimination of less than 2% of the tumor cells that coexpress high molecular weight melanoma-associated antigen (HMW-MAA) (melanoma-associated chondroitin sulfate proteoglycan, MCSP) and CD20 lastingly eradicated melanoma lesions, whereas targeting of any random 10% tumor cell subset was not effective. Our data challenge the biological therapy and current drug development paradigms in the treatment of cancer.

T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice.

BACKGROUND & AIMS: New treatment approaches are needed for patients with pancreatic adenocarcinoma. Carcinoembryonic antigen (CEA) is highly expressed on the surface of pancreatic adenocarcinoma cells; we investigated the effects of cytolytic T cells that recognize CEA in a mouse model of pancreatic carcinoma. METHODS: Immune-competent mice that expressed the CEA transgene (CEAtg) in the intestinal and pulmonary tracts were given intrapancreatic injections of Panc02 CEA(+) cells (express CEA and click beetle luciferase) and tumors were grown for 10 days. Mice were then given single intravenous injections of T cells engineered to express a chimeric antigen receptor (CAR) with high specificity, but moderate affinity, for CEA and a luminescence marker. RESULTS: Injection of the anti-CEA CAR T cells reduced the size of pancreatic tumors to below the limit of detection in all mice and produced long-term tumor eradication in 67% of mice. T cells also eradicated CEA(+) fibrosarcoma cells injected 45 days later. Bioluminescence imaging revealed the accumulation and persistence of the T cells at the tumor site. The efficacy of the T cells did not require lymphodepletion and was not reduced by soluble CEA. Mice developed some noninflammatory infiltrations of CAR(+) T cells in intestine and lung, but there was no evidence of destruction of CEA(+) healthy tissues. CONCLUSIONS: Injection of T cells that target CEA can eradicate tumors grown from CEA(+) pancreatic carcinoma cells in the pancreas of CEAtg mice without autoimmune effects.

A CD19-Anti-ErbB2 scFv Engager Protein Enables CD19-Specific CAR T Cells to Eradicate ErbB2+ Solid Cancer.

The efficacy of CD19-specific CAR T cells in the treatment of leukemia/lymphoma relies, at least in part, on the unique properties of the particular CAR and the presence of healthy B cells that enhance the target cell lysis and cytokine secretion through repetitive stimulation. Here, we report to apply the same CAR to target solid tumors, such as ErbB2+ carcinoma. CD19 CAR T cells are redirected towards the ErbB2+ cells by a fusion protein that is composed of the herceptin-derived anti-ErbB2 scFv 4D5 linked to the CD19 exodomain. The CD19-4D5scFv engager enabled CD19 CAR T cells to recognize the ErbB2+ cancer cells and to suppress the ErbB2+ tumor growth. The primary killing capacity by the ErbB2-redirected CD19 CAR T cells was as efficient as by the ErbB2 CAR T cells, however, adding CD19+ B cells furthermore reinforced the activation of the CD19 CAR T cells, thereby improving the anti-tumor activities. The ErbB2-redirected CD19 CAR T cells, moreover, showed a 100-fold superior selectivity in targeting cancer cells versus healthy fibroblasts, which was not the case for the ErbB2 CAR T cells. The data demonstrate that the CD19 CAR T cells can be high-jacked by a CD19-scFv engager protein to attack specifically solid cancer, thereby expanding their application beyond the B cell malignancies.

CAR and TCR form individual signaling synapses and do not cross-activate, however, can co-operate in T cell activation.

In engineered T cells the CAR is co-expressed along with the physiological TCR/CD3 complex, both utilizing the same downstream signaling machinery for T cell activation. It is unresolved whether CAR-mediated T cell activation depends on the presence of the TCR and whether CAR and TCR mutually cross-activate upon engaging their respective antigen. Here we demonstrate that the CD3ζ CAR level was independent of the TCR associated CD3ζ and could not replace CD3ζ to rescue the TCR complex in CD3ζ KO T cells. Upon activation, the CAR did not induce phosphorylation of TCR associated CD3ζ and, vice versa, TCR activation did not induce CAR CD3ζ phosphorylation. Consequently, CAR and TCR did not cross-signal to trigger T cell effector functions. On the membrane level, TCR and CAR formed separate synapses upon antigen engagement as revealed by total internal reflection fluorescence (TIRF) and fast AiryScan microscopy. Upon engaging their respective antigen, however, CAR and TCR could co-operate in triggering effector functions through combinatorial signaling allowing logic "AND" gating in target recognition. Data also imply that tonic TCR signaling can support CAR-mediated T cell activation emphasizing the potential relevance of the endogenous TCR for maintaining T cell capacities in the long-term.

Improved activation toward primary colorectal cancer cells by antigen-specific targeting autologous cytokine-induced killer cells.

Adoptive therapy of malignant diseases with cytokine-induced killer (CIK) cells showed promise in a number of trials; the activation of CIK cells from cancer patients towards their autologous cancer cells still needs to be improved. Here, we generated CIK cells ex vivo from blood lymphocytes of colorectal cancer patients and engineered those cells with a chimeric antigen receptor (CAR) with an antibody-defined specificity for carcinoembryonic antigen (CEA). CIK cells thereby gained a new specificity as defined by the CAR and showed increase in activation towards CEA⁺ colon carcinoma cells, but less in presence of CEA⁻ cells, indicated by increased secretion of proinflammatory cytokines. Redirected CIK activation was superior by CAR-mediated CD28-CD3ζ than CD3ζ signaling only. CAR-engineered CIK cells from colon carcinoma patients showed improved activation against their autologous, primary carcinoma cells from biopsies resulting in more efficient tumour cell lysis. We assume that adoptive therapy with CAR-modified CIK cells shows improved selectivity in targeting autologous tumour lesions.

CD28 costimulation Impairs the efficacy of a redirected t-cell antitumor attack in the presence of regulatory t cells which can be overcome by preventing Lck activation.

Adoptive T-cell transfer showed promising efficacy in recent trials raising interest in T cells with redirected specificity against tumors. T cells were engineered with a chimeric antigen receptor (CAR) with predefined binding and CD3ζ signaling to initiate T-cell activation. CD28 costimulation provided by a CD28-CD3ζ signaling CAR moreover improved T cell activation and persistence; however, it failed to meet the expectations with respect to mounting attacks against solid tumors infiltrated with regulatory T (Treg) cells. We revealed that a CD28 CAR-redirected T-cell attack is accompanied by higher numbers of Treg cells infiltrating the tumor and is less efficient against cancer cells in presence of Treg cells than a CD3ζ CAR T-cell attack. Deletion of the lck binding moiety in the CD28 CAR endodomain, however, improved redirected anti-tumor activity in presence of Treg cells without impairing interferon-γ (IFN-γ) secretion, proliferation, and cytolysis. CD28 modification abrogated interleukin-2 (IL-2) induction upon CAR engagement which in turn is no longer available to sustain Treg cell persistence. CARs with the modified CD28 endodomain thereby expedite the implementation of adoptive T-cell therapy in patients with a variety of cancer types that are heavily infiltrated by Treg cells.

Rational development of high-affinity T-cell receptor-like antibodies.

T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1(157-165) peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW "peg" dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2-4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1(157-165) target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes.

Costimulation by chimeric antigen receptors revisited the T cell antitumor response benefits from combined CD28-OX40 signalling.

The therapeutic success of adoptive therapy with chimeric antigen receptor (CAR) engineered T cells depends on the appropriate costimulation of CD3ζ to induce full T cell activation. Costimulatory endodomains of the CD28 family are therefore fused with CD3ζ in a dual signalling CAR. Serious adverse events in two most recent trials; however, highlight the need to analyse in more detail the impact of each costimulatory endodomain on individual effector functions of redirected T cells. We therefore performed a thoroughly controlled side-by-side comparison of the most frequently used endodomains with respect to their impact on CD4(+) and CD8(+) T cell effector functions. CD28 reinforced T cell proliferation and is mandatory to induce IL-2. In the absence of added IL-2, CD28 and OX40 (CD137) but not 4-1BB (CD134) enhanced specific cytolysis. While CD28, 4-1BB and OX40 similarly improved pro-inflammatory cytokine secretion, OX40 most efficiently prevented activation induced cell death of CD62L(-) effector memory T cells. CD28 was superior to initiate the T cell response, OX40 and 4-1BB sustained the response in long term with OX40 being most effective. We consequently combined the beneficial functions in a 3rd generation CD28-OX40 CAR which substantially improved the antitumor response without loosing specificity.

Regulatory T cells with reduced repressor capacities are extensively amplified in pulmonary sarcoid lesions and sustain granuloma formation.

Sarcoidosis can evolve into a chronic disease with persistent granulomas accompanied by progressive fibrosis. While an unlimited inflammatory response suggests an impaired immune control in sarcoid lesions, it stands in contrast to the massive infiltration with CD4(+)CD25(high)FoxP3(+) regulatory T cells. We here revealed that those Treg cells in affected lung lesions were mainly derived from activated natural Treg cells with GARP (LRRC32)-positive phenotype but exhibited reduced repressor capacities despite high IL-10 and TGF-beta 1 levels. The repressive capacity of blood Treg cells, in contrast, was not impaired compared to age-matched healthy donors. Treg derived cells in granuloma lesions have undergone extensive rounds of amplifications indicated by shortened telomeres compared to blood Treg cells of the same patient. Lesional Treg derived cells moreover secreted pro-inflammatory cytokines including IL-4 which sustains granuloma formation through fibroblast amplification and the activation of mast cells, the latter indicated by the expression of membrane-bound oncostatin M.