Cell Receptor Zeta Chain Research Articles

Impaired regulatory function of granzyme B-producing B cells against T cell inflammatory responses in lupus mice

ObjectiveRecently, a new subtype of granzyme B (GrB)-producing Breg cells has been identified, which was proven to be involved in autoimmune disease. Our recent report demonstrated that GrB-producing Breg cells...

The signaling and the metabolic differences of various CAR T cell designs

Chimeric antigen receptor (CAR) T cell therapy is introduced as an effective, rapidly evolving therapeutic to treat cancer, especially cancers derived from hematological cells, such as B cells. CAR T cell gene constructs combine a tumor-targeting device coupled to the T cell receptor (TCR) zeta chain domain with different signaling domains such as domains derived from CD28 or 4-1BB (CD137). The incorporation of each specific co-stimulatory domain targets the immunometabolic pathways of CAR T cells as well as other signaling pathways. Defining the immunometabolic and signaling pathways by which CAR T cells become and remain active, survive, and eliminate their targets may represent a huge step forward in this relatively young research field as the CAR gene can be tailored to gain optimal function also for solid tumors with elaborate immunosuppression and protective stroma. There is a close relationship between different signaling domains applied in CAR T cells, and difficult to evaluate the benefit from different tested CAR gene constructs. In this review, we attempt to collect the latest findings regarding the CAR T cell signaling pathways that affect immunometabolic pathways.

T cell fat catabolism: A novel target for kynurenine?

T cell fat catabolism: A novel target for kynurenine?

CD28 homolog is a strong activator of natural killer cells for lysis of B7H7-positive tumor cells

Abstract The CD28–B7 family of receptor–ligand pairs regulate lymphocyte responses through costimulation and coinhibition. It includes checkpoint inhibitors, such as PD-1, which limit anti-tumor and anti-virus T cell responses. CD28 homolog (CD28H) and B7H7 have been identified as a new receptor–ligand pair in this family, which has costimulatory activity in T cells. Here we show that CD28H is expressed in primary natural killer (NK) cells and that it is a strong activator of NK cells through selective synergy with receptors NKp46 and 2B4 to induce degranulation, lysis of target cells, and production of pro-inflammatory cytokines. Expression of B7H7 on target cells enhanced both natural and antibody-dependent cellular cytotoxicity (ADCC) of NK cells. Mutation of tyrosine 192 on the CD28H cytoplasmic tail abolished NK cell activation through CD28H. As B7H7 is broadly expressed in tumor tissues, we engineered a CD28H chimeric antigen receptor (CD28H-CAR) consisting of full-length CD28H fused to the cytoplasmic domain of T cell receptor zeta chain. NK cell activation is controlled by dominant “self-inhibition” mediated by inhibitory receptors for MHC class I, which has been a constraint for NK cellular cancer therapy. Remarkably, expression of CD28H-CAR in NK cells triggered lysis of tumor cells expressing HLA-E and B7H7 by overriding inhibition mediated by the HLA-E receptor NKG2A. The cytoplasmic domains of both CD28H and TCR zeta chain were required for this activity. Thus, CD28H is a powerful activation receptor of NK cells that broadens their anti-tumor activity and holds promise as a component of NK-based CARs for cancer immunotherapy.

Defined Central Memory and Stem Memory T Cell Phenotype of CD4 and CD8 CAR T Cells for the Treatment of CD19+ Acute Lymphoblastic Leukemia in an Automated Closed System

Relapsed and refractory B-precursor acute lymphoblastic leukemia (B-ALL) remains a major therapeutic problem. Chimeric antigen receptor (CAR) modified T cells targeting CD19 are promising treatment options for these patients with the potential to induce hematological remission in adult and pediatric patients with refractory B-ALL. Despite the promising data, some patients do not respond to T-cell treatment. Until now it is not possible to fully understand and predict critical factors for response or non-response, but proliferation and persistence of CAR T cells in vivo is an essential precondition for treatment efficacy. Central memory T cells (Tcm) and stem cell-like memory T cells (Tscm) are known to be the best candidates for a sustained in vivo expansion after T-cell therapy with small cell doses. Therefore, we set up a protocol for the generation of anti-CD19 CAR T cells in a closed system that is compliant to current GMP regulations. Starting samples were mononuclear cells from pediatric ALL patients at diagnosis and under chemotherapy using up to 100cc peripheral blood. After separation for CD4+/CD8+ cells, T cells were activated with anti-CD3/CD28 beads. The lentiviral vector encoded the anti-CD19 single-chain variable fragment, 4-1BB (CD137) co-stimulation and T cell receptor (TCR) zeta chain. The whole process including separation of T cells, activation, transduction and cultivation was performed in a closed and fully automated system. Despite a broad variety in cellular composition including high blast counts, low cell numbers and a rather exhausted phenotype in the starting fraction, a robust T-cell composition was achieved at day five after activation with a mean of 63% CD4+ and 37% CD8+ T cells and a transduction rate of up to 38 %. The vast majority of CAR T cells were of a Tcm (47%) and Tscm (44%) phenotype leading to a strong proliferative potential of more than 100-fold expansion. In addition, a reduced sensitivity to inhibitory signals was documented (programmed cell death protein 1 (PD-1) expression ≤10%). CAR T cells showed effective cytotoxic functionality when co-cultured with CD19+ target cells with only little background of the un-transduced control. At an effector to target ratio of 5:1 up to 80 % of the CD19+ target cells were killed. In addition, a significant release of Interferon gamma (IFN-γ), Tumor necrosis factor (TNF-α) and Interleukin-2 (IL-2) was detected upon recognition of the target cell lines, confirming a strong and target-specific Th1 response. In conclusion, generation of CAR T cells from small pediatric blood samples was feasible in a closed GMP-compatible fully automated system. Despite variety of cell numbers, cellular composition and T cell phenotype in the starting sample, a uniform T cell product of Tcm and Tscm could be produced with a balanced CD4 / CD8 ratio leading to high expansion potential and functionality of the T cell graft. DisclosuresBlaeschke:Miltenyi Biotec GmbH: Other: Miltenyi Biotec GmbH provided reagents free of charge.. Kaiser:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment.

Granzyme B Is a Key Regulator of Plasmacytoid Dendritic Cell Immunogenicity

Despite their low frequency of 0,1 - 0,5% in the peripheral blood, human plasmacytoid dendritic cells (pDC) are important modulators of adaptive immune responses and have meanwhile taken the step from the bench to promising initial tumor vaccination studies. PDC express high levels of the IL-3-receptor CD123, MHC class II and blood dendritic cell antigens (BDCA)-2 and -4. Apart from secretion of IFN-alpha and their capacity to rapidly initiate antigen-specific CD8+ T cell responses by cross-presentation, they produce large amounts of the serine protease granzyme B (GrB). GrB exhibits various non-cytotoxic functions including T cell regulation and the support of antigen processing and phagocytosis. We therefore hypothesized that pDC may use their GrB for both T cell regulation and for antigen uptake and processing. PDC were isolated using magnetically labeled antibodies against CD123, and cultured in the presence of various stimuli. Here, we demonstrate that pDC are potent producers of enzymatically active GrB, which reached maximal concentrations up to two logs higher than those produced by cytotoxic cells including CTL and NK cells. The strongest inducers of GrB in pDC were IL-3 and IL-10, whereas toll-like receptor (TLR)-7 or TLR-9 ligands such as CpG ODN or imiquimod as well as CD40 ligand suppressed GrB induction. We demonstrate that pDC-derived GrB can suppress effector T cell proliferation by GrB-mediated degradation of the T cell receptor zeta-chain. Moreover, we tested the uptake of fluorescently labeled antigenic material into pDC after lysis of tumor cells by freeze-thaw cycles and irradiation. These studies revealed a correlation between pDC GrB expression and antigen uptake, with GrBhigh pDC taking up significantly more antigenic material than GrBlow pDC. Our data support a novel concept, in which pDC acquire their maximal immunogenic potential in a two-step activation process: First, high levels of GrB are induced by IL-3 and IL-10, thereby supporting antigen processing and uptake into pDC, while preventing precocious activation of T cells. Subsequently, TLR agonists and CD40 ligand turn off GrB in pDC and initiate the transfer of MHC-antigen complexes and co-stimulatory molecules to the pDC surface, resulting in highly immunogenic pDC. Our data may be of translational relevance for the further development of novel DC vaccination strategies in tumor therapy. DisclosuresNo relevant conflicts of interest to declare.

Upregulated TCR Zeta and Improved the IFN-γ Secretion and Cytotoxicity in T Cell from Patients with AML after IL-2, IL-7 and IL-12 Stimulation

T cells from patients with acute myeloid leukemia (AML) display antigen receptor–mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain, a subunit of the TCR/CD3 complex. Up-regulate TCR signaling activation by TCR zeta recombinant vector transfection was showed to reverse IL-2 production and increased the activation in T cells from patients with chronic myeloid leukemia. This study was undertaken to explore the possibility that increased TCR zeta gene expression stimulated with immune cytokines may upregulate T cell receptor signaling activation in T cells from patients with AML. CD3+ T cells sorted from peripheral blood of 8 cases with AML were induced with different immune cytokines, including IL-2, IL-7, IL-12, anti-CD3 and anti-CD28 antibodies in vitro. The result showed that TCR zeta chain protein was significantly upregulated after stimulation with IL-2 + IL-7 at 72 hours (p<0.05) by western-blot analysis, the expression of Zap-70 which is TCR zeta-associated protein was increased apparently after stimulation by combined IL-7 and IL-12 at 72 hours (p<0.05). A higher level of interferon (IFN)-γ secretion was showed obviously with single stimulating factor or combination with different cytokines (p<0.05), especially in IL-12 + IL-7, CD3 + CD28 + IL-2 or CD3 + CD28 + IL-7 combination. In addition, the cytotoxicity of T cell from patients with AML was achieved after stimulation with different cytokines combinations at 7 days, especially in CD3 + CD28 + IL-2 group and CD3 + CD28 + IL-2 + IL-12 group. These result showed that downregulation of the TCR zeta chain in T cell from patients with AML is a reversible event. Combined different cytokines could restore TCR zeta chain deficiency and enhance IFN-γ production and cytotoxicity in T cells from AML patients. It is possible that upregulated TCR zeta chain expression in T cell from AML patients will effectively increase or recover their activation of antileukemia cytotoxicity. DisclosuresChen:National Natural Science Foundation of China (no. 81100353, 81270604), the Fundamental Research Funds for the Central Universities (No. 21611447, 21612116), And Medical Science Foundation of Guangdong Province(A2011325): Research Funding.

The fate of B cells in HIV infection - shifting the balance from plasma cell towards regulatory B cell (Breg) differentiation (LYM6P.776)

Abstract Certain regulatory lymphocyte populations suppress T cell expansion in a granzyme B (GrB)-dependent manner. Recently, we found that human B cells also contribute to immune regulation by interleukin (IL-)21-dependent expression of GrB. GrB-expressing Bregs are induced by incompletely activated CD4+ T cells, which express high levels of IL-21, but only low CD40 ligand. In contrast, fully activated CD4+ T cells strongly induce plasma cell differentiation. Here we demonstrate that up to 60% of B cells from HIV-infected patients with high viremia exhibit a regulatory (Breg) phenotype with expression of GrB and potent suppression of T cell proliferation by GrB-dependent degradation of the T cell receptor zeta-chain (TCR-zeta). These results are supported by additional findings showing that T cells in HIV-infected individuals produce high levels of IL-21, while expressing only low levels of CD40L and TCR-zeta. Importantly, soluble CD40L multimers can restore the differentiation of Bregs derived from HIV-infected individuals into plasma cells. Our results suggest that incompletely activated CD4+ T cells in HIV patients lead to a misrouting of B cell differentiation into Bregs at the cost of fully functional plasma cells. Apart from a disturbed humoral immune response these B cells may further aggravate the immune status of HIV patients. The use of CD40L multimers may disrupt the defective B cell-T cell interactions and may prove beneficial for future HIV vaccination approaches.

Infusion Of Donor-Derived CD19-Redirected-Virus-Specific T Cells For B-Cell Malignancies Relapsed After Allogeneic Stem Cell Transplant: A Phase I Study

▪Donor lymphocyte infusions (DLI) following hematopoietic stem cell transplantation may reduce or control opportunistic infections and leukemia/lymphoma relapse, but the associated graft versus host disease (GvHD) limits the clinical success of this procedure. Since T cell immunotherapy may be a safer alternative to DLI we have now used a single T cell platform that mediates both antileukemic and antiviral activity. Autologous T cells modified to express CD19-specific chimeric antigen receptors (CD19.CAR) have had clinical activity against CD19-expressing malignancies, but it is unknown if similarly modified allogeneic T cells will be equally effective. Allogeneic virus specific T cells (VSTs) directed to cytomegalovirus (CMV), adenovirus (Adv), and Epstein Barr virus (EBV) have been shown to be safe and effective in preventing and treating life-threatening viral infections post HSCT. Therefore, we sought to determine whether allogeneic VSTs could be engineered to express CD19.CAR and would retain the safety and effectiveness of unmodified VSTs whilst gaining anti-tumor activity.VSTs were expanded ex vivo using antigen presenting cells engineered to express adenovirus and cytomegalovirus (using an Ad5f35 adenoviral vector expressing the CMV pp65 gene), and Epstein Barr virus (using EBV-infected lymphoblastoid cell lines) antigens. After 3 stimulations, the VST's were modified to express CD19.CAR.28ζ using a retroviral vector encoding the CAR-CD19 receptor coupled to the CD28 co-stimulatory molecule and the T cell receptor zeta (ζ) chain. Nine CD19.CAR-modified virus specific T cell (CD19.CAR-VSTs) products were generated for infusion. All VST lines recognized at least one viral antigen as determined by Elispot or chromium release assays and 20% to 48% of cells expressed the CD19.CAR. All lines killed CD19-expressing cells in vitro.We treated nine patients with these CD19.CAR-VSTs, 3 months to 13 years after HSCT. Six patients received CD19.CAR-VSTs for relapsed disease and 3 patients received the T cells as adjuvant therapy to prevent viral infection and relapse after HSCT. Safety. There were no infusion-related toxicities. One patient presented with gastrointestinal symptoms following infusion subsequently determined to be unrelated to the T cells. Persistence. VSTs persisted a median of 8 weeks in the peripheral blood and up to 9 weeks at disease sites. In three patients (#1, #3 and #5), CD19.CAR signals were detectable in the bone marrow or the lymph nodes (44.8, 25.85, and 32 copies/1000 ng DNA) even when no signal was measurable in peripheral blood, indicating preferential accumulation of the infused T cells at the disease site. Anti-Tumor Activity. During the period of CD19.CAR-VST persistence, objective anti-tumor activity was evident in 2/6 patients with relapsed disease (patient # 1 had detectable blasts in the peripheral blood which disappeared within 1-2 weeks following infusion, patient # 2 had 16% circulating CLL cells which decreased within 2 weeks of T cell infusion) but disease recurred after 3 and 2 months, respectively. The two patients who received cells while in remission remain disease-free >3 and >9 months later. Anti-Viral Activity. In two patients with EBV reactivation, donor CD19.CAR-VSTs expanded concomitant with an increase in virus-specific T cell responses, and decreased viral load. A third patient had a rise in adenovirus specific VSTs during an episode of adenovirus associated diarrhea. Although the infection was controlled, there was no concomitant rise in CD19-CAR expressing T cells in this patient. No other patient had viral disease.In conclusion, allogeneic CD19.CAR-VSTs administered after allogeneic HSCT are safe and can exert both anti-tumor and anti-viral activity in the absence of GvHD. Earlier administration of CD19.CAR-VSTs after HSCT, when the host is lymphodepleted and the incidence of viral infection is higher, may allow these cells to better capture the potential advantages of native TCR stimulation (and associated co-stimulation) for expansion and persistence, and thereby produce a higher frequency of sustained tumor responses. Alternatively, intentional stimulation of the native TCRs by viral vaccines may produce equal benefit, with greater predictability. Disclosures:Savoldo:Celgene: Patents & Royalties, Research Funding. Heslop:Celgene: Patents & Royalties, Research Funding; Cell Medica: Patents & Royalties. Rooney:Cell Medica: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celgene: Patents & Royalties, Research Funding. Brenner:Celgene: Patents & Royalties, Research Funding. Dotti:Celgene: Patents & Royalties, Research Funding.

T cell receptor Zeta chain (TCRζ) expression in patients with idiopathic thrombocytopenic purpura

T cell receptor Zeta chain (TCRζ) expression in patients with idiopathic thrombocytopenic purpura

HIV-Infected T Cells Induce Granzyme B-Secreting Regulatory B Cells in An Interleukin 21-Dependent Fashion,

Abstract 3222We and others have recently provided evidence that a series of lymphocyte subsets including, plasmacytoid dendritic cells, B cells and regulatory T cells are able to secrete the cytotoxic serine protease granzyme B (GrB) into the extracellular compartment, where it contributes to the suppression of T cell proliferation by a so far undefined GrB-dependent mechanism. For B cells, we found that viral antigens in the context of the acute phase cytokine interleukin (IL-) 21 can potently induce GrB. Here, we demonstrate that infection of CD4+ T cells with HIV-1 (NL4-3), but not mock infection, induces strong expression of IL-21 on both mRNA and protein levels in CD4+ T cells. Moreover, we found that HIV-infected CD4+ T cells are able to induce high levels of GrB in co-cultured B cells and that inhibition of IL-21 with specific antibodies abrogates T cell-mediated GrB induction in B cells. In support of these data, serum levels of both IL-21 and GrB are significantly higher in patients acutely infected with HIV as compared to healthy control subjects. Surprisingly, co-culture of CD4+ T cells with B cells during HIV-1 infection strongly suppressed both, proliferation of T cells as well as virus replication as indicated by significantly reduced p24 levels in culture supernatants. Notably, this effect was enhanced by external stimulation of B cells with IL-21, and was reduced by inhibition of GrB using specific GrB inhibitors. To further explore the underlying mechanisms of our findings, we performed confocal microscopy of T cell-B cell co-cultures and demonstrated that GrB-secreting B cells directly interact with CD4+ T cells, thereby transferring active GrB to them. Moreover, we found that GrB+ B cells decreased CD4+ T cell expression of the T cell receptor-zeta chain, a known GrB target, which is required for T cell proliferation, and known to be suppressed in HIV patients. In summary, we provide evidence that HIV induces the acute phase cytokine IL-21 in infected CD4+ T cells, thereby indirectly triggering the expression of GrB by B cells. GrB-secreting B cells may play a so far unappreciated role in decelerating the expansion of HIV, particularly in the early phase of acutely infected patients. Our study reveals a novel pathogenetic mechanism in HIV infection with potential relevance for the development of novel immunotherapeutic approaches. Disclosures:No relevant conflicts of interest to declare.

Recognition of a dendritic cell ligand by an MCMV-encoded MHC-I-like protein (154.17)

Abstract Mouse cytomegalovirus (MCMV), a valuable model for studying virus-host interactions, encodes several glycoproteins with predicted MHC-I structure, which may function as immunoevasins. We recently determined the crystallographic structure of a member of this family, m153, and showed that this is a cell surface homodimeric protein with structural similarity to MHC-I, which is expressed independently of beta-2m or peptide. To determine the role of m153 in the viral infection of the host, we wished to identify host proteins with which m153 interacts. We constructed both a birA-tagged version of m153 for staining experiments as well as a reporter cell containing the m153 extracellular domain fused to the human T cell receptor zeta chain. Using both approaches we show that CD11c+ splenic dendritic cells (DCs) and bone marrow-derived DCs express a cell surface ligand for m153. To our knowledge this is the first example of a putative viral immunoevasin that targets a DC expressed molecule. We describe our efforts to identify this molecule using a variety of approaches, including antibody blocking, cDNA library screening, and pull-down experiments on DC lysates. Identification of the m153 ligand on DCs offers to elucidate mechanisms the host employs to defend against viral infection, and also to clarify the evolution of viral immunoevasion of the immune response. This research was supported by the Intramural Research Program of the NIH, NIAID.

K33-Linked Polyubiquitination of T Cell Receptor-ζ Regulates Proteolysis-Independent T Cell Signaling

Tagging the cell surface receptor with ubiquitin is believed to provide a signal for the endocytic pathway. E3 ubiquitin ligases such as Cbl-b and Itch have been implicated in T cell activation and tolerance induction. However, the underlying mechanisms remain unclear. We describe that in mice deficient in the E3 ubiquitin ligases Cbl-b and Itch, T cell activation was augmented, accompanied by spontaneous autoimmunity. The double-mutant T cells exhibited increased phosphorylation of the T cell receptor-zeta (TCR-zeta) chain, whereas the endocytosis and stability of the TCR complex were not affected. TCR-zeta was polyubiquitinated via a K33-linkage, which affected its phosphorylation and association with the zeta chain-associated protein kinase Zap-70. The juxtamembrane K54 residue in TCR-zeta was identified to be a primary ubiquitin conjugation site, whose mutation increased its phosphorylation and association of TCR-zeta and Zap-70. Thus, the present study reveals unconventional K33-linked polyubiquitination in nonproteolytic regulation of cell-surface-receptor-mediated signal transduction.

Uncoupling of T Cell Receptor Zeta Chain Function during the Induction of Anergy by the Superantigen, Staphylococcal Enterotoxin A

Staphylococcus aureus enterotoxins have immunomodulatory properties. In this study, we show that Staphylococcal enterotoxin A (SEA) induces a strong proliferative response in a murine T cell clone independent of MHC class II bearing cells. SEA stimulation also induces a state of hypo-responsiveness (anergy). We characterized the components of the T cell receptor (TCR) during induction of anergy by SEA. Most interestingly, TCR zeta chain phosphorylation was absent under SEA anergizing conditions, which suggests an uncoupling of zeta chain function. We characterize here a model system for studying anergy in the absence of confounding costimulatory signals.

Monocyte Dependent Regulation of Autoimmune Inflammation

In chronic inflammation, across a number of quite different pathological conditions, monocytes accumulate. In autoimmune disease, these cells are widely recognised to play an inflammatory and tissue destructive role. But these cells also inhibit T cell proliferation by a range of different mechanisms that are accompanied by the depletion of specific amino acids in the local microenvironment and the downregulation of the T cell receptor zeta chain. This occurs within the pro-inflammatory environment and in the presence of Th1 (IFNgamma) and Th17 (IL-17) cytokines. In tumours, related cells are part of a population called myeloid-derived suppressor cells (MDSC) and they are associated with immunosuppression. Their depletion can lead to clinical improvement. In organ specific autoimmune disease, where such cells can be found in the spleen and in target organs, recent evidence indicates that they may play a role in limiting the T cell response to autoantigens in the target tissue. This occurs by a targeted disruption of T cell division. In this review we discuss evidence for the presence on MDSC in murine and human autoimmune disease and the mechanisms by which such cells inhibit T cell proliferation.

Mechanisms involved in the down‐regulation of TCR ζ chain in tumor versus peripheral blood of oral cancer patients

Immune dysfunction is the hallmark of patients with oral cancer. Down-regulation of T cell receptor (TCR) zeta chain expression was observed in T cells from patients with oral squamous cell carcinoma. In peripheral blood, the decrease in TCR zeta chain showed an inverse correlation with the tumor stage as demonstrated by western blotting, confocal microscopy and flow cytometry. The mechanism of TCR zeta chain degradation in the peripheral blood involves ubiquitination and subsequent targeting of TCR zeta for degradation in the lysosome. Decreased expression of PKC theta and the subsequent decrease of TCR zeta chain transcription factor Elf-1 and its binding to DNA may contribute to the decreased/or absent TCR zeta chain transcripts in the tumor infiltrating lymphocytes. Oral cancer patients exhibiting TCR zeta chain defect also showed impaired lymphocyte proliferation, cytokine profile and intracellular calcium release upon stimulation with anti CD3 mAb. Our data shows that posttranslational degradation is primarily responsible for decreased TCR zeta chain expression in the peripheral blood, while a transcriptional defect is observed in the tumor compartment. The down-regulation of TCR zeta chain culminates into impaired lymphocyte responses in these patients.

Post-Transcriptional Regulation of T Cell Receptor zeta Chain in Systemic Lupus Erythematosus

Post-Transcriptional Regulation of T Cell Receptor zeta Chain in Systemic Lupus Erythematosus

Adoptive Transfer of Chimeric FcεRI Receptor Gene-Modified Human T Cells for Cancer Immunotherapy

Immunotherapeutic approaches involving genetic modification of T cells show promise in generating highly specific tumor-reactive effector cells for cancer treatment. Given the high affinity of FcRI (the subtype I Fc receptor for IgE) for IgE monoclonal antibody (mAb), modification of T cells with chimeric FcRI in combination with tumor-specific IgE mAbs is potentially a powerful and effective strategy to specifically target T cells to tumor cells. In this study, we retrovirally transduce human primary T cells with a cDNA encoding the extracellular domain of FcRI linked to the hinge and transmembrane domains of FcRI and the cytoplasmic domains of CD28 and T cell receptor zeta chain (FcRI-CD28-zeta). We demonstrate that human T cells expressing FcRI-CD28-zeta, in the presence of tumor-specific IgE mAb recognizing mouse CD8 antigen (Ly- 2.1+), can specifically secrete cytokine, proliferate, and mediate cytotoxic function after antigen ligation. Furthermore, adoptive transfer of FcRI-CD28-zeta cells incubated with anti-Ly-2.1 IgE mAb significantly enhances the survival of irradiated nonobese diabetic-severe combined immunodeficiency mice bearing Ly-2.1+ tumor compared with control mice. Thus, this set of experiments demonstrates that Fc gene-engineered human T cells mediate effector function in vitro and in vivo in an IgE-dependent manner and thus a novel and valid approach for cancer therapy can now be further developed.

Effect of melanoma on immune function in regional lymph nodes (LN)

8000 Background: Metastatic melanoma is associated with various measures of systemic and local immune suppression. We hypothesized that immune suppression in the regional LN basin exists and may facilitate melanoma progression. Methods: We collected portions of regional LNs from tumor uninvolved sentinel LNs (USLN) (remote from primary melanoma removal) and from macroscopically involved nodes (TILN) from therapeutic node dissections in successive melanoma patients. Lymphocytes were extracted, stained using surface and internal antibodies to T cell receptor zeta chain (TCRζ), T regulatory cells (CD4+CD25+ Fox 3p+) (Tregs) and tumor associated myeloid cells (CD11b+ CD14- CD15+) (TAMC), and analyzed via flow cytometer. Results from USLN specimens were compared with TILN. Results: 30 LNs (14 USLN/16 TILN) from 30 patients were evaluated. Median age of patients was 48 years (range 30–83); 20 were male. USLN patients were stage IB (12) or IIA (2); TILN patients were stage IIIB (5), IIIC (5), and IV (6). 7 patients with TILN and none with USLN have progressed. TILN had significantly less TCRζ chain expression than SLN (62% vs. 85%; p = 0.0001). This amounts to a localized decrease of 23% of TCRζ expression (95% CI 13%; 34%). No significant relationship between lymph node involvement and Treg or TAMC was documented. Conclusion: Regional LN involvement with melanoma is associated with a significant reduction in lymphocyte TCRζ expression indicating immune suppression. The mechanism for this is uncertain, but does not appear to be related to changes in Tregs or TAMC (arginase producing cells). We plan to further investigate TCRζ expression in microscopically involved SLNs and SLNs removed at time of primary excision to see if immune changes precede macroscopic tumor involvement. Supported by Harvard Skin SPORE Project 5 and Immune Monitoring Core P50CA93683–01 No significant financial relationships to disclose.

Stability and Translation of T Cell Receptor zeta Chain are regulated by the Splice Deleted 562‐bp 3′UTR in Systemic Lupus Erythematosus Patients

The molecular mechanisms involved in the diminished expression of the TCRζ mRNA of patients with systemic lupus erythematosus (SLE) remains unknown. Previously we demonstrated that while normal T cells express high levels of TCRζ with wild-type (WT) 3′ UTR, SLE T cells display significantly high levels of ζ chain with an alternatively spliced (AS) 3′ UTR form which is derived by splice deletion of 672 to 1233 of TCRζ chain transcript. The 3′UTRs of eukaryotic mRNAs are known to play a crucial role in post-transcriptional regulation of gene expression, stability and rates of degradation. Selective expression of TCRζ with very short AS 3′ UTR in SLE suggests that it plays an important role in the down regulation of TCRζ expression in SLE T cells. Recently we have reported that stability of TCRζ mRNA with an AS is low compared to WT 3′ UTR in SLE. Therefore, we conducted studies to determine the stability of TCRζ mRNA with a splice deleted (SD) 3′ UTR in SLE T cells, and the transport and subsequent translation of TCRζ with WT or AS 3′ UTR or SD in normal and SLE T cells. In this communication we demonstrate that SD 3′ UTR confers stability to TCRζ and contributes to increased levels of TCRζ mRNA. In addition, we show that the TCRζ mRNA with SD 3′ UTR displayed high level of translation efficiency leading to the production of high amounts of protein. Taken together our findings confirm that splice deleted 562-bp of TCRζ plays a critical role in its stability and translational regulation in human T cells.