Recognition of renal cell carcinoma by CD8⁺ and CD4⁺ TCR-engineered T lymphocytes.

Immunotherapy using T cells is a new approach that is being explored for the treatment of metastatic melanoma. For renal cell carcinoma (RCC), adoptive T cell therapy (ATT) is currently hampered by the lack of T cells expressing suitable T cell receptors (TCR). A tumor-infiltrating T cell population (TIL) was identified in our group (TIL-53) that showed a pattern of tumor recognition consistent with the requirements of a TCR applicable for immunotherapy. With the advent of genetic TCR engineering it was possible to further define the TIL specificity which was previously precluded by the failure to cultivate TIL-53. To achieve high expression levels and functionality, the TCR53 required the exchange of the human TCR constant region by the TCR murine constant region (TCR53m). The B3Z T cell hybridoma which stably expressed TCR53m after retroviral transduction was used to analyze a large panel of tumor lines and non-malignant cell cultures for expression of the TCR53 ligand. The analysis included 34 RCC cell lines, 55 tumor cell lines of different histologies and 30 non-tumor cell lines. 65 % of the HLA-A2+ RCC cells and 25 % of other HLA-A2+ tumor lines were recognized by the B3Z-TCR53m cells. Among the non-RCC tumors, the TCR53 ligand was frequently found in malignant B cell lines and EBV-transformed B-lymphoblastoid cell lines (5/13, 38 %). Of 25 HLA-A2+ non-tumor cells only 2 were marginally recognized. The TCR53 ligand expression could be increased with IFN-a but not IFN-g treatment on cell lines that already had some TCR53 ligand expression. De novo induction in cell lines that had no prior expression of the TCR53 ligand was not observed. The B3Z-TCR53m cell line could detect the TCR53 ligand on fresh tumor material and, if used for therapy, B3Z-TCR53m could be used to identify those patients whose tumors are positive for the TCR53 ligand and thus could benefit from the therapy. To achieve high expression levels and functionality of the TCR53m on human PBLs, the TCR53 a and b chain sequences had to be optimized for codon usage. PBLs expressing these recombinant TCR sequences (TCR53mc) showed very low formation of hybrid TCRs between the TCR53mc b chain and endogenous TCR a chains. TCR53mc-expressing T cells of RCC patients and healthy donors showed specific killing of tumor cell lines and had a polyfunctional profile, defined by the detection of T cells that simultaneously secreted cytokines (IFN-g, TNF-a or IL-2) and performed granule exocytosis when recognizing targets. The functional response of TCR53mc-expressing T cells depended on the expression of HLA-A2 on the target cells. Analysis of RCC tumors using multicolor fluorescence immunohistology allowed the detection and localization of CD8+ T cells in relation to blood vessels. The majority of CD8+ T cells were found extra-luminal, indicating strong extravasation of T cells into RCC tumors. The majority of the T cells in the lumen of the blood vessel had perforin (~ 90 %), while T cells that were outside the blood vessels were to a large percentage perforin negative (~ 60 %). Thus, CD8+ T cells apparently arrive at the tumor being perforin-positive and lose perforin when outside the blood vessels. The three-dimensional growth of cells in spheroids was used to mimic the tumor milieu in vitro and to evaluate the functional capacity of T cells with transgenic RCC specific TCR expression. T cells infiltrated the spheroids and preferentially accumulated in the rim of the spheroid (~ 100 μm). The killing capacity of TCR53mcexpressing T cells in the 3-D environment in a 4 h assay was similar to that observed in a standard 4 h chromium release assay with RCC cells in suspension. However, after being cultured for 24 h in the spheroids, the T cells were no longer able to secrete cytokines upon stimulation with target cells and were negative for perforin, granzyme B and CD28. The presence of CD4+ T cells in the spheroids significantly increased the number of CD8+ T cells infiltrating the 3-D tumors. Moreover, the CD8+ T cell response was enhanced with more degranulating T cells and T cells secreting cytokines, which was not seen in the absence of CD4+ T cells. The functional improvement of the CD8+ T cell response required the CD4+ T cells to be activated, as it was not observed when CD4+ T cells were used that lacked TCRs specific for the spheroid tumor cells

T-cell receptor gene-modified T cells with shared renal cell carcinoma specificity for adoptive T-cell therapy.

PURPOSE: Adoptive therapy with genetically engineered T cells carrying redirected antigen specificity is a new option for the treatment of cancer. This approach is not yet available for metastatic renal cell carcinoma (RCC), due to the scarcity of therapeutically useful reagents. We analyzed tumor-infiltrating lymphocytes (TIL) from RCC to identify T-cell specificities with shared tumor-specific recognition to develop T-cell receptor (TCR)-engineered T lymphocytes for adoptive therapy of RCC. EXPERIMENTAL DESIGN: We established a T-cell clone from TIL that recognized a human leukocyte antigen (HLA)-A2-restricted tumor antigen. The TCR alpha- and beta-chain genes were isolated, modified by codon optimization and murinization, and retrovirally transduced into peripheral blood lymphocytes (PBL). A TCR-expressing indicator line (B3Z-TCR53) was established to screen for antigen prevalence in RCC, other malignancies, and normal cell counterparts. RESULTS: TCR53-engineered PBL recapitulated the specificity of the TIL and showed tumor-specific HLA-A2-restricted effector activities (IFN-gamma, tumor necrosis factor-alpha, interleukin-2, macrophage inflammatory protein-1beta, cytotoxicity). PBL-TCR53 of healthy donors and RCC patients exhibited similar transduction efficiency, expansion, and polyfunctional profile. Using B3Z-TCR53 cells, 130 tumor and normal cells were screened and shared TCR53 peptide: MHC expression was found in >60% of RCC and 25% of tumor lines of other histology, whereas normal tissue cells were not recognized. CONCLUSIONS: To date, TCR53 is the only TCR with shared HLA-A2-restricted recognition of RCC. It fulfills the criteria for utilization in TCR gene therapy and advances T cell-based immunotherapy to patients with RCC and other malignancies expressing the TCR ligand

Human endogenous retrovirus transcription profiles of the kidney and kidney-derived cell lines.

The human genome comprises approximately 8-9 % of human endogenous retroviruses (HERVs) that are transcribed with tissue specificity. However, relatively few organs have been examined in detail for individual differences in HERV transcription pattern, nor have tissue-to-cell culture comparisons been frequently performed. Using an HERV-specific DNA microarray, a core HERV transcription profile was established for the human kidney comparing 10 tissue samples. This core represents HERV groups expressed uniformly or nearly so in non-tumour kidney tissue. The profiles obtained from non-tumour tissues were compared to 10 renal tumour tissues (renal cell carcinoma, RCC) derived from the same individuals and additionally, to 22 RCC cell lines. No RCC cell line or tumour-specific differences were observed, suggesting that HERV transcription is not altered in RCC. However, when comparing tissue transcription to cell line transcription, there were consistent differences. The differences were irrespective of cancer state and included cell lines derived from non-tumour kidney tissue, suggesting that a specific alteration of HERV transcription occurs when establishing cell lines. In contrast to previous publications, all known HERV-derived tumour antigens, including those identified in RCC, were expressed both in multiple RCC cell lines and several non-tumour tissue-derived cell lines, a result that contrasts with findings from patient samples. The results establish the core kidney transcription pattern of HERVs and reveal differences between cell culture lines and tissue samples

Data from: The extracellular domains of IgG1 and T cell-derived IL-4/IL-13 are critical for the polyclonal memory IgE response in vivo

IgE-mediated activation of mast cells and basophils contributes to protective immunity against helminths but also causes allergic responses. The development and persistence of IgE responses are poorly understood, which is in part due to the low number of IgE-producing cells. Here, we used next generation sequencing to uncover a striking overlap between the IgE and IgG1 repertoires in helminth-infected or OVA/alum-immunized wild-type BALB/c mice. The memory IgE response after secondary infection induced a strong increase of IgE+ plasma cells in spleen and lymph nodes. In contrast, germinal center B cells did not increase during secondary infection. Unexpectedly, the memory IgE response was lost in mice where the extracellular part of IgG1 had been replaced with IgE sequences. Adoptive transfer studies revealed that IgG1+ B cells were required and sufficient to constitute the memory IgE response in recipient mice. T cell-derived IL-4/IL-13 was required for the memory IgE response but not for expansion of B cells from memory mice. Together, our results reveal a close relationship between the IgE and IgG1 repertoires in vivo and demonstrate that the memory IgE response is mainly conserved at the level of memory IgG1+ B cells. Therefore, targeting the generation and survival of allergen-specific IgG1+ B cells could lead to development of new therapeutic strategies to treat chronic allergic disorders