T-cell receptor-CD3 Research Articles

Abstract 646: Bi-specific T-cell engaging antibody activates T-cells to target the tumor associated antigen PR1

Abstract Acute (AML) and chronic (CML) myeloid leukemia together account for approximately 50% of newly diagnosed leukemias. Both AML and advanced CML have dismal survival outcomes but are susceptible to immunotherapy. We discovered PR1 (VLQELNVTV) as an effective human leukocyte antigen (HLA)-A2 restricted peptide and have targeted it in clinical trials using PR1 peptide vaccine. PR1 is derived from the serine proteases proteinase-3 and neutrophil elastase which are highly expressed in myeloid leukemia blasts. PR1 was shown to be immunogenic in myeloid malignancies, and we are currently developing several PR1 targeting therapies. We have completed the development and humanization of a T-cell receptor-like monoclonal antibody (h8F4) that targets PR1/HLA-A2 and eliminates primary human AML xenografts in NSG mice. To improve the efficacy of h8F4, we have developed a bi-specific T-cell engaging (BiTE) antibody that targets leukemia PR1/HLA-A2 and the T-cell receptor CD3. This would enhance the anti-leukemia immune response by bringing the T-cell in close proximity to the leukemia cell, and by further activating the T-cells to eliminate the target leukemia. Here we demonstrate successful construction and expression of the h8F4 BiTE in CHO cells. The antibody was purified from cell culture supernatant via Ni-NTA column chromatography, and identity was confirmed using western blot analysis. Next, flow cytometry was used to test the binding specificities of each targeting region of the BiTE antibody. The h8F4 BiTE antibody showed increased binding to T2 cells pulsed with PR1 peptide compared with T2 pulsed with irrelevant CMV peptide pp65 (MFI=627 and 72.4, respectively). In addition, the h8F4 BiTE demonstrated 6.8 fold higher binding to wild type Jurkat cells, which express CD3, in comparison with the CD3-lacking mutant Jurkat cell line J.RT3-T3.5. These results indicate that both regions of the h8F4 BiTE can efficiently recognize their intended targets. Next, we investigated whether h8F4 BiTE engagement with both PR1/HLA-A2 on T2 cells and CD3 on healthy donor PBMC could activate T-cells. We show with flow cytometry that upon 24h incubation in the presence of h8F4 BiTE and T2 cells pulsed with PR1 peptide, both CD4 and CD8 T-cells increase their surface expression of CD69 (21 fold in CD4; 10.3 fold in CD8), CD25 (35 fold in CD4; 3.5 fold in CD8) and CD71 (26.6 fold in CD4; 640 fold in CD8) compared to co-incubation in the absence of BiTE protein. In conclusion, this study shows that we have successfully developed a h8F4 BiTE which recognizes both the CD3 on T cells and PR1/HLA-A2 complexes on myeloid leukemia. Furthermore, the h8F4 BiTE can activate multiple T-cell subsets from healthy donor PBMC as early as 24h following co-incubation with PR1-pulsed T2 cells. This novel BiTE antibody could provide a safer and more potent treatment option for patients with aggressive myeloid leukemias that will improve upon currently available highly toxic standard therapies. Citation Format: Amanda Herrmann, Jin Im, Sijie Lu, Jeffrey Molldrem. Bi-specific T-cell engaging antibody activates T-cells to target the tumor associated antigen PR1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 646. doi:10.1158/1538-7445.AM2014-646

Abstract 2488: Analysis of the salivary proteome of the prolactin-inducible-protein knockout mouse

Abstract The human Prolactin-Inducible-Protein (PIP)/Gross Cystic Disease Fluid Protein 15 (GCDFP-15) gene is abnormally expressed in human invasive breast cancer and gross cystic disease of the breast. In healthy individuals PIP is highly expressed in salivary, sweat and lacrimal glands, and the secreted protein abundantly found in saliva as well as other bodily fluids which bathe various ports of entry. PIP has been shown to bind numerous bacterial strains as well as a number of immunoregulatory molecules including the CD4-T cell receptor and IgG and is therefore implicated to play an immune role in host defense. In the present study we interrogated the salivary proteome of the PIP knockout (Pip KO) mouse by mass spectrometry to determine protein changes that occur in saliva in the absence of PIP. Proteomic analysis identified 165 proteins in the mouse saliva which had a minimum of 2 unique peptides at the 99% confidence level. Nineteen proteins, including 2 proposed markers of disease, S100A9 and adenosine deaminase, were significantly differentially expressed in the saliva of the Pip KO mouse, compared to the saliva of the wild type counterparts. Functional and biological determinations revealed that the differentially expressed proteins were associated with processes that include inflammatory responses, immunity and cell death. This study provides evidence that a number of proteins associated with host defense are affected by the loss of PIP, and lends support for an immunomodulatory role for PIP. Citation Format: Anne Blanchard, Peyman Ezzati, Xiuli Ma, John Wilkins, Yvonne Myal. Analysis of the salivary proteome of the prolactin-inducible-protein knockout mouse. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2488. doi:10.1158/1538-7445.AM2014-2488

Tracking global changes induced in the CD4 T-cell receptor repertoire by immunization with a complex antigen using short stretches of CDR3 protein sequence.

Motivation: The clonal theory of adaptive immunity proposes that immunological responses are encoded by increases in the frequency of lymphocytes carrying antigen-specific receptors. In this study, we measure the frequency of different T-cell receptors (TcR) in CD4 + T cell populations of mice immunized with a complex antigen, killed Mycobacterium tuberculosis, using high throughput parallel sequencing of the TcRβ chain. Our initial hypothesis that immunization would induce repertoire convergence proved to be incorrect, and therefore an alternative approach was developed that allows accurate stratification of TcR repertoires and provides novel insights into the nature of CD4 + T-cell receptor recognition.Results: To track the changes induced by immunization within this heterogeneous repertoire, the sequence data were classified by counting the frequency of different clusters of short (3 or 4) continuous stretches of amino acids within the antigen binding complementarity determining region 3 (CDR3) repertoire of different mice. Both unsupervised (hierarchical clustering) and supervised (support vector machine) analyses of these different distributions of sequence clusters differentiated between immunized and unimmunized mice with 100% efficiency. The CD4 + TcR repertoires of mice 5 and 14 days postimmunization were clearly different from that of unimmunized mice but were not distinguishable from each other. However, the repertoires of mice 60 days postimmunization were distinct both from naive mice and the day 5/14 animals. Our results reinforce the remarkable diversity of the TcR repertoire, resulting in many diverse private TcRs contributing to the T-cell response even in genetically identical mice responding to the same antigen. However, specific motifs defined by short stretches of amino acids within the CDR3 region may determine TcR specificity and define a new approach to TcR sequence classification.Availability and implementation: The analysis was implemented in R and Python, and source code can be found in Supplementary Data.Contact: b.chain@ucl.ac.ukSupplementary information: Supplementary data are available at Bioinformatics online.

Polyclonal CD4 TCR Affinities are not altered in chronic viral infection (VIR7P.1058)

Abstract The range of polyclonal CD4 T cell receptor (TCR) affinities that participate during the response to viral infection is currently unknown. Utilizing a highly sensitive two-dimensional micropipette adhesion frequency assay (2D), we determined the TCR affinity and frequency of CD4 T cells that expand and survive throughout multiple phases of the chronic and acute lymphocytic choriomeningitis virus (LCMV) T cell response. We discovered consistently more (4-5 fold) CD4 T cells specific for the IAb GP66-77 LCMV epitope than detected with pMHC II tetramer. The 2D identified lower affinity cells participate in the anti-viral response and are apparent at all time points of acute and chronic infection. Average TCR affinities measured at peak and later stages of the chronic response remained constant and equivalent to corresponding acute response measurements. However, programmed death 1 (PD-1) expressing CD4+ T cells late in chronic infection are functionally unresponsive despite static affinities. In contrast, memory cells in the acute response have a higher functional avidity compared to peak effectors (produce IFNγ in response to lower amount of peptide antigen) yet T cell memory is not restricted to select TCR affinities. Our findings suggest long term antigen exposure and functional enhancement/impairment exert no selective pressure in the survival or deletion of specific CD4 TCR affinities and TCR affinity alone does not always predict biological response.

T-Cell Receptor-CD3 Signaling Complex Extracellular Interactions Characterized by Genetic Incorporation of Unnatural Amino Acid Photo-Crosslinkers

T-cells play a central role in the adaptive immune response to invading pathogens and cancer while largely avoiding self-reactivity. The interaction of T-cell receptors (TCRs) displayed on T-cells with peptide-major histocompatibility complexes (pMHCs) on the surface of antigen presenting cells leads to signaling through the TCR-CD3 signaling complexes, which result in the initiation of cellular immune responses. However, how molecular and structural events in TCR-recognition translate through TCR-CD3 complex and generate differences in intracellular signaling is still not clear. Previous studies have shown that the extracellular TCR-CD3 subunits interactions are important for T-cell responses to TCR engagement. The objective of this research is to understand structural organization and specific interactions among TCR and CD3 subunits, and to provide insights into how structural features and mechanisms initiating TCR-triggering determine the outcome of immune responses. To monitor interactions among TCR-CD3 subunits, we developed a system to site-specifically incorporate an unnatural amino acid photo-crosslinker p-Azido-L-phenylalanine (pAzpa) or p-benzoyl-L-phenylalanine (pBpa) into TCR expressed on cell surface. Subsequently, after activation of TCR with pMHC, photo-crosslinked TCR-CD3 complexes were immunoprecipitated and analyzed by Western Blotting. Our analysis showed that specific residues in the TCR DE, CC, FG loops and G strand play critical roles in TCR-CD3 subunit interactions. Moreover, cross-linked high molecular weight complexes suggested TCR-TCR dimerization during the signaling process. Together our results 1) reveal how TCR-CD3 signaling subunits are positioned together to perform their function, 2) suggest how the antigen specific recognition information gets transferred from outside to the signaling molecules inside cell to determine immune responses, 3) provide potential targets for modulating outcome of immune response.

Interview: Immunotherapeutic Manipulation of the Tumor Microenvironment

Dario AA Vignali speaks to Katie Lockwood, Assistant Commissioning Editor Dario Vignali received his PhD in 1988 from the London School of Hygiene and Tropical Medicine, University of London, where he studied immunity to Schistosoma mansoni. He then held two postdoctoral positions from 1988 to 1993; first at the Institute for Immunology and Genetics, German Cancer Research Center (Heidelberg, Germany), with Gunter Hammerling, and then at the Department of Biochemistry and Molecular Biology, Harvard University (MA, USA), with Jack Strominger. He is currently Vice Chair and Member (Full Professor equivalent) of the Department of Immunology, St Jude Children’s Research Hospital (TN, USA), where he has been for 20 years. Dr Vignali’s research focuses on molecular and cellular aspects of Treg function, immune regulation by inhibitory receptors and inhibitory cytokines (IL-35) in tumor immunity, mucosal immunity and Type 1 diabetes. He also studies proximal events in T-cell receptor–CD3 signaling.

The Topology, in Model Membranes, of the Core Peptide Derived from the T‐Cell Receptor Transmembrane Domain

The T-cell receptor-CD3 complex (TCR-CD3) serves a critical role in protecting organisms from infectious agents. The TCR is a heterodimer composed of α- and β-chains, which are responsible for antigen recognition. Within the transmembrane domain of the α-subunit, a region has been identified to be crucial for the assembly and function of the TCR. This region, termed core peptide (CP), consists of nine amino acids (GLRILLLKV), two of which are charged (lysine and arginine) and are crucial for the interaction with CD3. Earlier studies have shown that a synthetic peptide corresponding to the CP sequence can suppress the immune response in animal models of T-cell-mediated inflammation, by disrupting proper assembly of the TCR. As a step towards the understanding of the source of the CP activity, we focused on CP in egg phosphatidylcholine/cholesterol (9:1, mol/mol) model membranes and determined its secondary structure, oligomerization state, and orientation with respect to the membrane. To achieve this goal, 15-residue segments of TCRα, containing the CP, were synthesized and spin-labeled at different locations with a nitroxide derivative. Electron spin-echo envelope modulation spectroscopy was used to probe the position and orientation of the peptides within the membrane, and double electron-electron resonance measurements were used to probe its conformation and oligomerization state. We found that the peptide is predominantly helical in a membrane environment and tends to form oligomers (mostly dimers) that are parallel to the membrane plane.

AB0093 Association of cd247 polymorphisms with rheumatoid arthritis: a replication study and a meta-analysis

BackgroundThe T-cell receptor T3 zeta chain (CD3ζ), also called CD247, is essential for assembly, surface expression and signaling cascade of the T-cell receptor-CD3 complex (TCR/CD3 complex). Abnormalities in this pathway...

Manipulation of human early T lymphopoiesis by coculture on human bone marrow stromal cells: Potential utility for adoptive immunotherapy

T cell precursors are an attractive target for adoptive immunotherapy. We examined the regulation of human early T lymphopoiesis by human bone marrow stromal cells to explore invitro manipulation of human T cell precursors in a human-only coculture system. The generation of CD7(+)CD56(-)cyCD3(-) proT cells from human hematopoietic progenitors on telomerized human bone marrow stromal cells was enhanced by stem cell factor, flt3 ligand, and thrombopoietin, but these stimulatory effects were suppressed by interleukin 3. Expression of Notch ligands Delta-1 and -4 on stromal cells additively promoted T cell differentiation into the CD7(+)cyCD3(+) pre-T cell stage, while cell growth was strongly inhibited. By combining these coculture systems, we found that initial coculture with telomerized stromal cells in the presence of stem cell factor, flt3 ligand, and thrombopoietin, followed by coculture on Delta-1- and -4-coexpressing stromal cells led to a higher percentage and number of pre-T cells. Adoptive immunotherapy using peripheral blood T cells transduced with a tumor antigen-specific T cell receptor (TCR) is a promising strategy but has several limitations, such as the risk of forming a chimeric TCR with the endogenous TCR. We demonstrated that incubation of TCR-transduced hematopoietic progenitors with the combination of coculture systems gave rise to CD7(+)TCR(+)CD3(+)CD1a(-) T cell precursors that rapidly proliferated and differentiated under the culture condition to induce mature T cell differentiation. These data show the regulatory mechanism of early T lymphopoiesis on human stromal cells and the potential utility of engineered human stromal cells to manipulate early T cell development for clinical application.

CD8 Suppressor Cells Can Be Induced by Pre-B Acute Lymphoblastic Leukemia As a Potential Mechanism of Immune Escape

AbstractAbstract 4797Suppressor CD8 T-cells have been associated with poor antiviral immunity and, more recently, poor prognosis in hematologic malignancy. We have previously observed that nonprofessional antigen presenting cells, including B-lymphocytes, can induce IL10-secreting CD8 suppressors in the setting of lymphopenia. In this study, we hypothesized that pre-B cell acute lymphoblastic leukemia could also induce CD8 suppressors as a potential mechanism of immune escape.To test this hypothesis, naïve murine (B6) CD8 T-cells (CD45.2) specific for the immunodominant HY peptide (HY-CD8), were cultured with a B6 HY-expressing murine pre-B cell acute lymphoblastic leukemia (CD45.2) carrying the human E2A-PBX transgene (E2APBX-ALL). Male B6 CD45.1+ dendritic cells and B-cells served as controls, as did female (HY-) E2APBX-ALL. Production of IFNγ and IL10 was determined by enzyme-linked immunoadsorbent assay (ELISA) at 48–72 hours, and specific identification of IFNγ, IL10 and FoxP3 expression by HY-CD8 was measured by intracellular flow cytometry by gating on live, CD8a+CD45.2+ cells. Surface expression of B7 family costimulatory molecules such as PD1 was also measured.As expected, coculture of activated male dendritic cells (DC) with HY-CD8 generated high levels of IFNγ (1036 pg/ml +/− 40), with 67.2 +/− 9.4% CD8a+CD45.2+IFNγ+ cells at 72 hours. HY-CD8 did not produce detectable IL10 or express FoxP3 by either immunoassay or intracellular stain by 72 hours of culture in the presence of male DC. In contrast, HY-expressing E2APBX ALL induced IL10 production in HY-CD8, significantly less interferon gamma production by immunoassay (576 pg/ml +/− 150, p<0.0034) and intracellular flow cytometry (26.8% +/− 9.4), with 72.8% + 4.7 HY-CD8 expressing FoxP3 and nearly 100% expressing PD1. Interestingly, although female E2APBX-ALL did not stimulate IFNγ or IL10 production, suggesting antigen-specific effects, a similar induction of PD-1 on HY-specific T cells was produced by exposure to either male or female E2APBX-ALL.In conclusion, our data indicates that pre-B acute lymphoblastic leukemia is capable of inducing an IL10-producing, FoxP3+ suppressive phenotype in CD8 T-cells. In this in vitro system, the suppressive phenotype is only induced when ALL bears an antigen recognized by the CD8 T-cell receptor, suggesting antigen-specific regulatory blockade as a potential target. Further studies that address the in vivo function and molecular signature of ALL-induced CD8 suppressors in a preclinical model are ongoing. Disclosures:No relevant conflicts of interest to declare.

Enhancing the Efficacy of T Cell Receptor (TCR) Gene Therapy by Co-Transfer of TCR and Additional CD3 Molecules Into CD4+ T Cells

AbstractAbstract 2044 Introduction:The specificity of T cells can be redirected using retroviral T cell receptor (TCR) gene transfer. This has the potential to generate tumour specific T cells that can be adoptively transferred to target defined tumour antigens. The majority of TCR gene therapy studies have focused on the transfer of TCR genes into CD8 T cells. However the transfer of antigen specific CD8 T cells in the absence of antigen specific CD4 T cells leads to impaired anti-tumour responses and impaired memory development in vivo. Class I restricted TCR can be used to transduce CD4 T cells for use in adoptive transfer. The majority of class I restricted TCRs are CD8 dependent and thus require co-transduction of CD8 to be fully functional in CD4 T cells. CD4 T cells transduced with the class I restricted F5-TCR (specific for influenza peptide NP presented by H2-Dbclass I molecules) produce IL-2 and proliferate in vitro in response to class II negative tumour cells expressing NP peptide but these cells were not able to generate an IFN-γ response. In vivo, F5-TCR CD4 T cells could provide help for F5-TCR CD8 T cell mediated tumour eradication. These F5-TCR CD4 T cells persisted in vivo for up to 90 days post tumour regression and were able to re-expand following tumour challenge. In order to improve the function of class I restricted TCR expressing CD4 T cells, we co-transduced a vector containing all 4 chains of the CD3 complex. High surface expression of TCR has been shown to correlate with increased responsiveness to specific antigen. When additional TCR is introduced into a T cell, the introduced T cell must compete with the endogenous TCR for binding to CD3. The amount of CD3 within the cell will thus be rate limiting for the level of surface expression of the introduced TCR. Method:The retroviral vectors pMP71-F5α-2A-F5β (F5-TCR) and pMP71-CD3-ζ-2A-ε-2A-δ-2A-γ-IRES-GFP (CD3) were used for retroviral transduction. CD4 splenocytes obtained from C57BL/6 mice were activated with CD3/CD28 magnetic beads for 24 hours prior to transduction with either F5-TCR alone or F5-TCR and CD3. 5 days post transduction, transduced T cells were stimulated with C57BL/6 splenocytes loaded with NP (relevant) peptide or WT1 (irrelevant) peptide and cytokine production was measured by ELISA and intracellular cytokine staining and proliferation by [3H] thymidine incorporation. For in vivo tumour challenge, C57BL/6 recipient mice were irradiated with 5.5Gy and injected subcutaneously with 1 × 106 EL4-NP-luciferase cells (a lymphoma cell line stably transfected with NP peptide and luciferase) on day 0. On day 1, mice received 1 × 106 F5-TCR CD3 CD4 T cells or 1 × 106 F5-TCR CD4 T cells or 1 × 106 Mock Transduced T cells. Tumour area was measured by calipers and by bioluminescence imaging. For T cell trafficking experiments, the experimental set up was as above but transgenic CD4 luciferase T cells were used for adoptive transfer and EL4-NP luciferase negative cells were used for tumour challenge. Results:CD4 T cells transduced with F5-TCR and CD3 had a 5-fold higher expression of F5-TCR compared to cells transduced with F5-TCR alone. In vitro, F5-TCR CD3 CD4 T cells showed increased proliferation and increased production of IL-2 and IFN-γ in response to specific antigen compared to F5-TCR CD4 T cells. F5-TCR CD3 CD4 T cells responded to at a 2-fold lower concentration of specific peptide than F5-TCR CD4 T cells. Following adoptive transfer in murine models, F5-TCR CD3 CD4 T cells eradicated NP expressing EL4 tumours but transfer of equivalent doses of F5-TCR CD4 T cells did not lead to tumour regression. Using bioluminescence imaging, F5-TCR CD3 CD4 T cells trafficked to tumour site faster and accumulated in greater numbers than F5-TCR CD4 T cells. Following tumour challenge, there were higher numbers of F5-TCR CD3 CD4 T cells persisting in bone marrow, lymph node and peripheral blood than in mice that received F5-TCR CD4 T cells. Conclusion:Increased surface expression of class I restricted TCR in CD4 T cells leads to increased sensitivity to peptide in vitro and higher levels of proliferation and cytokine production in response to specific peptide. This translates in vivo to enhanced persistence of F5-TCR CD3 CD4 T cells and more efficient trafficking to tumour site and superior tumour protection. Therefore, the co-transduction of additional CD3 can improve the function of class I restricted TCR in CD4 T cells. Disclosures:Stauss:Cell Medica: Scientific Advisor Other.

Deep Sequencing of Antiviral T-Cell Responses to HCMV and EBV in Humans Reveals a Stable Repertoire That Is Maintained for Many Years

CD8+ T-cell responses against latent viruses can cover considerable portions of the CD8+ T-cell compartment for many decades, yet their initiation and maintenance remains poorly characterized in humans. A key question is whether the clonal repertoire that is raised during the initial antiviral response can be maintained over these long periods. To investigate this we combined next-generation sequencing of the T-cell receptor repertoire with tetramer-sorting to identify, quantify and longitudinally follow virus-specific clones within the CD8+ T-cell compartment. Using this approach we studied primary infections of human cytomegalovirus (hCMV) and Epstein Barr virus (EBV) in renal transplant recipients. For both viruses we found that nearly all virus-specific CD8+ T-cell clones that appeared during the early phase of infection were maintained at high frequencies during the 5-year follow-up and hardly any new anti-viral clones appeared. Both in transplant recipients and in healthy carriers the clones specific for these latent viruses were highly dominant within the CD8+ T-cell receptor Vβ repertoire. These findings suggest that the initial antiviral response in humans is maintained in a stable fashion without signs of contraction or changes of the clonal repertoire.

CD3-T Cell Receptor Co-stimulation through SLAMF3 and SLAMF6 Receptors Enhances RORγt Recruitment to the IL17A Promoter in Human T Lymphocytes

Th17 lymphocytes play a key role during immune responses against bacteria and fungi and are involved in the pathophysiology of multiple autoimmune disorders. The co-stimulatory molecules SLAMF3 and SLAMF6 have been implicated in the formation of Th17 phenotypes and IL-17A expression. Increased surface expression of SLAMF3 and SLAMF6 has been linked with disease activity in systemic lupus erythematosus. Here we demonstrate that in human total T lymphocytes the canonical CD28 and the non-canonical SLAMF3/SLAMF6 co-stimulatory pathways cooperate in the recruitment of the transcription factor NFAT1 to the IL17A promoter. Furthermore, the dominance of the SLAMF3/SLAMF6 pathway in inducing IL-17A production can be attributed to an increased nuclear abundance and recruitment of RORγt to the IL17A promoter. Thus, we have identified an additional mechanism that may be central for the specific control of IL17A gene regulation in systemic lupus erythematosus T lymphocytes.

Assessing a Theoretical Risk of Dolutegravir-Induced Developmental Immunotoxicity in Juvenile Rats

HIV-1 integrase inhibitors (INIs) are a promising class of antiretrovirals for the treatment of HIV in adults; there is interest in expanding their use into pediatric populations. A theoretical concern for developmental immunotoxicity was raised after a publication suggested that two HIV INI tool compounds inhibited in vitro cleavage activity of recombination activating genes 1 and 2 (RAG1/2) through the inhibition of their binding to recombination signal sequences. RAG1/2 are required for the development of mature B and T lymphocyte populations. The potential effects of the investigational INI dolutegravir on RAG1/2 were addressed by developing assays in juvenile rats to measure T cell receptor (TCR) Vβ usage by flow cytometry as an indicator of TCR repertoire diversity and a T cell dependent antibody response (TDAR) as an indicator of immunosuppression. These endpoints were incorporated into a juvenile rat toxicity study, along with immunophenotyping, hematology, and histopathology of immunologic organs. Dose levels of 0, 0.5, 2, or 75mg/kg/day dolutegravir were given via oral gavage from postnatal day 4 through 66. At the highest dose, there was decreased body weight gain and two preweanling deaths; however, there were no treatment-related effects on developmental parameters. There were no effects on immunologic competence, as measured by TDAR, and no effects on lymphocyte subsets or CD4 and CD8 TCR Vβ usage in peripheral blood. Histopathology of immunologic organs (spleen, thymus, lymph nodes) and hematology evaluation revealed no effects. The no observed adverse effect level for immunotoxicity endpoints was 75mg/kg/day.

Skewed T-Cell Receptor Variable Beta Repertoire and Massive T-Cell Activation in Idiopathic Orofacial Granulomatosis

Orofacial granulomatosis (OFG) is a clinicopathologic entity describing oral lesions with noncaseating granulomas including a spectrum of diseases such as the Melkersson-Rosenthal syndrome. The involvement of abnormal T-cell responses has been suggested in the pathogenesis of OFG although few and contrasting data are currently available on this issue. In a patient with OFG, we observed virtually complete CD4 and CD8 T-cell receptor (TCR) β-chain variable region (BV) repertoires at the lesion level and in circulation. However, oligoclonal profiles were found in CD4 and, to a greater extent, in CD8 subsets. These findings were seen in association with a massive peripheral T-cell activation, decreased naive T cells, reduced thymic output, altered cytokine production, and increased apoptosis. Our data, pointing to a random influx of T cells at the site of inflammation, argue against the hypothesis of a main allergen acting at the level of oral mucosa. The profound dysregulation of the peripheral T-cell compartment suggests that OFG should be regarded as a systemic disorder with localized manifestations.

Primary cutaneous CD56 positive lymphoma: a diagnostic conundrum in an unusual case of lymphoma

We present an unusual case of a CD56-positive T-cell lymphoma exhibiting immunophenotypic characteristics of both γδ T-cell lymphoma and extranodal NK/T-cell lymphoma, nasal-type. The patient presented with a 2-month history of rapidly progressive, pruritic and cutaneous nodules on his arms. A biopsy showed a dense pan-dermal infiltrate of markedly atypical CD3-positive lymphocytes, compatible with tumor stage cutaneous T-cell lymphoma. Retrospective review of a preceding biopsy and flow cytometric analysis, performed at an outside institution, showed strong expression of surface CD3, CD7, CD43 and γδ T-cell receptor (TCR), findings consistent with a diagnosis of cutaneous γδ T-cell lymphoma. In light of these data, we performed additional studies that showed diffuse positive staining of the atypical lymphocytes for CD56, CD4 and CD43 as well as Epstein-Barr virus-encoded small nonpolyadenylated RNA (EBER). Interestingly, this case displays characteristic features of γδ T-cell lymphoma, with strong surface expression of CD3 and γδ-TCR, as well as characteristics of natural killer (NK)/T-cell lymphoma, including expression of CD4 and EBER positivity, that represent two separate categories in the current classification of cutaneous lymphomas. Taken together, these findings underscore the difficulty of rendering an unambiguous classification of the presented neoplasm given the close ontogenetic relationship between NK and cytotoxic T-cells and highlight the need for continued reevaluation of the current classification system.

Lipid Membrane Association of the T Cell Antigen Receptor ζ Subunit: Affinities and Structure

The T cell antigen receptor (TCR) is a cell surface protein complex that binds peptide antigen fragments presented by the Major Histocompatibility Complex (MHC). The interaction of TCR with MHC leads to T cell activation and initiation of an immune response,1 but the molecular details of the transmembrane signaling process remain unknown. TCRζ is the 115 amino acid long cytosolic signaling domain of the CD3-T cell receptor complex that plays a central role in this process. TCRζ is predominantly unstructured2 and carries several copies of the immunoreceptor tyrosine-based activation motif (ITAM). ITAMs become phosphorylated upon receptor engagement, constituting an early and obligatory event in the signaling cascade that blocks lipid association and lipid-associated conformational rearrangements3 of TCRζ. We present a characterization of the affinity of TCRζ to tethered lipid bilayers by SPR and a structural study of its association with such membranes using neutron reflectometry (NR). We studied the binding of unphosphorylated TCRζ to highly charged stBLMs rich in anionic phosphatidylserine (PS) or phosphatidylglycerol (PG) and observed affinity constants of Kd ≈ 10 μM. Minute amounts of phosphoinositides (PI(4,5)P2) increase the membrane affinity of the protein by more than 10-fold. An NR characterization of TCRζ in the membrane-associated state shows the major portion of the protein interfacially associated with the bilayer surface and a minor protein penetrating the bilayer deeply. This is consistent with a model in which an α-helical structure is aligned on the membrane surface that may be connected via a hinge with another α-helical segment inserted into the bilayer.[1] Call, M.E., and Wucherpfennig, K.W., Annu. Rev. Immunol. 23:101 (2005).[2] Sigalov, A.B., et al., Biochemistry 45:15731 (2006).[3] Aivazian, D.A., et al., Nat. Struct. Biol. 7:1023 (2000).

Genotype, phenotype, and outcomes of nine patients with T‐B+NK+ SCID

There are few reports of clinical presentation, genotype, and HCT outcomes for patients with T-B+NK+ SCID. Between 1981 and 2007, eight of 84 patients with SCID who received and/or were followed after HCT at UCSF had the T-B+NK+ phenotype. One additional patient with T-B+NK+ SCID was identified as the sibling of a patient treated at UCSF. Chart reviews were performed. Molecular analyses of IL7R, IL2RG, JAK3, and the genes encoding the CD3 T-cell receptor components δ (CD3D), ε (CD3E), and ζ (CD3Z) were carried out. IL7R mutations were documented in four patients and CD3D mutations in two others. Three patients had no defects found. Only two of nine patients had an HLA-matched related HCT donor. Both survived, and neither developed GVHD. Five of seven recipients of haploidentical grafts survived. Although the majority of reported cases of T-B+NK+ SCID are caused by defects in IL7R, CD3 complex defects were also found in this series and should be considered when evaluating patients with T-B+NK+ SCID. Additional genes, mutations in which account for T-B+NK+ SCID, remain to be found. Better approaches to early diagnosis and HCT treatment are needed for patients lacking an HLA-matched related donor.

Change in expression pattern of TCR–CD3 complex in patients with multiple myeloma

In haematological malignancy, cell-mediated immunity has been shown to be suppressed in advanced disease. This immune dysfunction may be due, in part, to altered expression of the T cell receptor (TCR)–CD3 complex. The distribution and clonality of the TCR Vbeta repertoire and the expression levels of CD3gamma, CD3delta, CD3epsilon, and CD3zeta genes in T cells from patients with multiple myeloma (MM) were investigated. Specific Vbeta subfamily primers, reverse transcription polymerase chain reaction, and the GeneScan® technique were used to analyse the expression of the TCR Vbeta subfamily and the clonality of Vbeta T cells in 11 patients with MM. Real-time reverse transcription polymerase chain reaction was used to detect the expression levels of CD3gamma, CD3delta, CD3epsilon, and CD3zeta genes in peripheral blood mononuclear cells from 19 patients with MM. The beta2-microglobulin gene was used as an endogenous reference. A total of 5–22 Vbeta subfamily T cells were detected in different patients (mean value of expressed Vbeta subfamilies was 12·55±6·11), whereas all 24 Vbeta genes were identified in all control samples. The most frequently expressed Vbeta subfamilies were Vbeta1 (100%), Vbeta2, Vbeta3, Vbeta9, Vbeta13, and Vbeta16 (81·8%), while the expression of Vbeta20 was undetectable in all MM samples. Oligoclonal expansion of one or more Vbeta subfamily of T cells was detected in all patients. Such expansions involved different MM stages, and the numbers of expanded clonal Vbeta subfamilies seemed higher in stage I/II groups than in stage III; however, there was no significant difference. Among MM samples, of the Vbeta subfamily members, Vbeta13, Vbeta1, and Vbeta21, were expanded most frequently. A significant decrease in the expression level of the CD3gamma gene was observed in MM samples; in contrast, a higher expression of CD3epsilon was found in the MM group than in the healthy group. The expression pattern of the four CD3 chains was epsilon>zeta>delta>gamma in peripheral blood mononuclear cells from MM, while a gamma>epsilon>zeta>delta expression pattern was found in healthy controls. In conclusions, the present study presents precise data on changes in the variability of Vbeta patterns and expression of TCR signal transduction molecules in MM patients compared to controls, which may be associated with immune dysfunction. This study contributes to a better understanding of the cellular immune features in MM patients.

Animal models of toxic epidermal necrolysis

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe acute exfoliative skin diseases developing extensive epidermal detachment and mucosal damage. Although SJS and TEN are mostly caused by drugs, an animal model of TEN using drugs has not been established yet. We have established an autoimmune skin disease model mouse reproducing the devastating skin damage of TEN by a combination of transgenic mice expressing an epidermal model antigen and its specific CD8(+) T-cell receptor. In this model mouse, we found that the thymus-derived CD4(+) CD25(+) regulatory T cell (Treg) is a critical regulator of cytotoxic T lymphocytes (CTL) causing TEN. Indeed, loss of Treg function was recently demonstrated by human studies of TEN patients. Although how drug-reactive CTL is activated in vivo is still unknown, this model elucidated the immunological pathomechanism of TEN after CTL obtained cytotoxicity against epidermal keratinocyte. In this review, roles of CTL, Treg, cytotoxic granules and antigen-presenting cells were discussed on pathogenesis of TEN.