Robert Kammerer

Cytotoxic Markers and Frequency Predict Functional Capacity of Natural Killer Cells Infiltrating Renal Cell Carcinoma

Purpose: Renal cell carcinoma harbors high numbers of infiltrating lymphocytes with apparent limited efficacy in tumor control. This study focused on the natural killer (NK) cells infiltrating renal cell carcinoma. Experimental Design: Tumor-infiltrating lymphocytes (TIL) were isolated from renal cell carcinoma and analyzed for NK cell frequency and phenotype (n = 34). NK cells were enriched and tested for effector function. Results: Two renal cell carcinoma subtypes were identified, one containing high (>20% of the lymphocyte population, n = 14), the other low (<20%, n = 20), NK cell numbers. NK cells of both groups were noncytolytic ex vivo but differed in CD16 and cytotoxic effector molecule expression as well as in their capacity to acquire cytotoxic activity: The majority of NK cells from tumors with high NK cell content (high NK-TIL) were CD16bright, whereas few CD16bright NK cells were found in tumors with low NK cell frequencies (low NK-TIL). The CD16 dichotomy correlated with different capacities to develop cytotoxicity after short-term activation with interleukin-2 ex vivo: Low NK-TIL remained noncytolytic against K562 and unresponsive to signals via the activating receptor NKp46 despite expression of receptor and adaptor molecules. In contrast, high NK-TIL acquired cytotoxic function. As described for peripheral CD16bright NK cells, NK cells from high-NK tumors showed high per cell expression of granzyme A, granzyme B, and perforin. NK cells from low NK-TIL resembled CD16neg/dim peripheral NK cells with few cytotoxin+ cells and lower expression of perforin. Conclusion: The extent of NK cell infiltration and the expression of markers (CD16 and cytotoxins) predict the functional capacity of NK cells infiltrating renal cell carcinoma and can be used to characterize subgroups of renal cell carcinoma.

Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Expression and Signaling in Human, Mouse, and Rat Leukocytes: Evidence for Replacement of the Short Cytoplasmic Domain Isoform by Glycosylphosphatidylinositol-Linked Proteins in Human Leukocytes

Carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1), the primordial carcinoembryonic Ag gene family member, is a transmembrane cell adhesion molecule expressed in leukocytes, epithelia, and blood vessel endothelia in humans and rodents. As a result of differential splicing, CEACAM1 occurs as several isoforms, the two major ones being CEACAM1-L and CEACAM1-S, that have long (L) or short (S) cytoplasmic domains, respectively. The L:S expression ratios vary in different cells and tissues. In addition to CEACAM1, human but not rodent cells express GPI-linked CEACAM members (CEACAM5–CEACAM8). We compared the expression patterns of CEACAM1-L, CEACAM1-S, CEACAM6, and CEACAM8 in purified populations of neutrophilic granulocytes, B lymphocytes, and T lymphocytes from rats, mice, and humans. Human granulocytes expressed CEACAM1, CEACAM6, and CEACAM8, whereas human B lymphocytes and T lymphocytes expressed only CEACAM1 and CEACAM6. Whereas granulocytes, B cells, and T cells from mice and rats expressed both CEACAM1-L and CEACAM1-S in ratios of 2.2–2.9:1, CEACAM1-S expression was totally lacking in human granulocytes, B cells, and T cells. Human leukocytes only expressed the L isoforms of CEACAM1. This suggests that the GPI-linked CEACAM members have functionally replaced CEACAM1-S in human leukocytes. Support for the replacement hypothesis was obtained from experiments in which the extracellular signal-regulated kinases (Erk)1/2 were activated by anti-CEACAM Abs. Thus, Abs against CEACAM1 activated Erk1/2 in rat granulocytes, but not in human granulocytes. Erk1/2 in human granulocytes could, however, be activated by Abs against CEACAM8. We demonstrated that CEACAM1 and CEACAM8 are physically associated in human granulocytes. The CEACAM1/CEACAM8 complex in human cells might accordingly play a similar role as CEACAM1-L/CEACAM1-S dimers known to occur in rat cells.

Expression of Indoleamine 2,3-Dioxygenase in Tumor Endothelial Cells Correlates with Long-term Survival of Patients with Renal Cell Carcinoma

Purpose: The inflammatory enzyme indoleamine 2,3-dioxygenase (IDO) participates in immune tolerance and tumor immune escape processes by degradation of the essential amino acid tryptophan and formation of toxic catabolites. Here, we analyzed the role of IDO in tumor growth and disease progression in patients with clear cell renal cell carcinoma (RCC). Experimental Design: Expression of IDO mRNA was analyzed by quantitative reverse transcription-PCR in 55 primary and 52 metastatic RCC, along with 32 normal kidneys. Western blot and immunohistochemistry analyses were used to semiquantitatively determine IDO proteins in a subset of tumor samples, in RCC cell lines, and microvessel endothelial cells. IDO expression was correlated with expression of the proliferation marker Ki67 in tumor cells and survival of patients with tumor. Results: More than 75% of the clear cell RCC in comparison to normal kidney contained elevated levels of IDO mRNA, which correlated with their IDO protein content. Low IDO mRNA levels in primary tumors represented an unfavorable independent prognostic factor (hazard ratio, 3.8; P = 0.016). Unexpectedly, immunohistochemical analyses revealed that IDO is nearly exclusively expressed in endothelial cells of newly formed blood vessels and is virtually absent from tumor cells, although RCC cells could principally synthesize IDO as shown by in vitro stimulation with IFN-γ. A highly significant inverse correlation between the density of IDO-positive microvessels and the content of proliferating Ki67-positive tumor cells in primary and metastatic clear cell RCC was found (P = 0.004). Conclusions: IDO in endothelial cells might limit the influx of tryptophan from the blood to the tumor or generate tumor-toxic metabolites, thus restricting tumor growth and contributing to survival.

CEACAM1 (CD66a) mediates delay of spontaneous and fas ligand-induced apoptosis in granulocytes

Granulocytes form the first and fastest line of defense against pathogenic infections. Their survival is limited by apoptosis, a process that is critical for the resolution of inflammation. Pro‐apoptotic and pro‐inflammatory cytokines, as well as several receptors, can alter the lifespan of granulocytes. Here we report that the carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1, CD66a) is involved in the regulation of granulocyte survival. Until now CEACAM1 is described to control cell proliferation, cell migration, tumor growth, angiogenesis and diverse leukocyte functions. However, very little is known about its role in granulocytes. We found that CEACAM1 expression in resting rat granulocytes is significantly higher than in other leukocyte subtypes. Stimulation led to a strongly increased CEACAM1 cell surface expression and to release of soluble CEACAM1. DNA fragmentation assays and annexin V staining revealed that binding of CEACAM1‐specific antibodies, Fab fragments and soluble CEACAM1‐Fc constructs to cell surface‐expressed CEACAM1 causes a delay of spontaneous and Fas ligand (CD95L)‐induced apoptosis. Tyrosine phosphorylation of CEACAM1‐L, its association with SHP‐1, the activation of Erk1/2 and caspase‐3 appeared to be crucial for the CEACAM1‐mediated anti‐apoptotic effect. These findings provide evidence that CEACAM1 influences the resolution of inflammation by prolonging the survival of rat granulocytes.

Coevolution of activating and inhibitory receptors within mammalian carcinoembryonic antigen families

BackgroundMost rapidly evolving gene families are involved in immune responses and reproduction, two biological functions which have been assigned to the carcinoembryonic antigen (CEA) gene family. To gain insights into evolutionary forces shaping the CEA gene family we have analysed this gene family in 27 mammalian species including monotreme and marsupial lineages.ResultsPhylogenetic analysis provided convincing evidence that the primordial CEA gene family in mammals consisted of five genes, including the immune inhibitory receptor-encoding CEACAM1 (CEA-related cell adhesion molecule) ancestor. Our analysis of the substitution rates within the nucleotide sequence which codes for the ligand binding domain of CEACAM1 indicates that the selection for diversification is, perhaps, a consequence of the exploitation of CEACAM1 by a variety of viral and bacterial pathogens as their cellular receptor. Depending on the extent of the amplification of an ancestral CEACAM1, the number of CEACAM1-related genes varies considerably between mammalian species from less than five in lagomorphs to more than 100 in bats. In most analysed species, ITAM (immunoreceptor tyrosine-based activation motifs) or ITAM-like motif-containing proteins exist which contain Ig-V-like, ligand binding domains closely related to that of CEACAM1. Human CEACAM3 is one such protein which can function as a CEACAM1 decoy receptor in granulocytes by mediating the uptake and destruction of specific bacterial pathogens via its ITAM-like motif. The close relationship between CEACAM1 and its ITAM-encoding relatives appears to be maintained by gene conversion and reciprocal recombination. Surprisingly, secreted CEACAMs resembling immunomodulatory CEACAM1-related trophoblast-specific pregnancy-specific glycoproteins (PSGs) found in humans and rodents evolved only in a limited set of mammals. The appearance of PSG-like genes correlates with invasive trophoblast growth in these species.ConclusionsThese phylogenetic studies provide evidence that pathogen/host coevolution and a possible participation in fetal-maternal conflict processes led to a highly species-specific diversity of mammalian CEA gene families.

Transcript signature predicts tissue NK cell content and defines renal cell carcinoma subgroups independent of TNM staging.

Clear cell renal cell carcinoma (ccRCC) is an aggressive and difficult to manage cancer. Immunotherapy has the potential to induce long-lasting regression in a small group of patients. However, severe side effects limit broad application which highlights the need for a marker to distinguish responder from nonresponder. TNMG staging, referring to tumor size, lymph node involvement, presence of metastasis, and grade of tumor differentiation, represents an important prognostic system but is not useful for predicting responders to immunotherapy. NK cells are potent antitumor effector cells, and a role as prognostic marker in some solid tumors has been suggested. As NK cells are responsive to various immune modifiers, they may be important mediators of patient response to immunotherapies, in particular those including IL-2. We report that the NK cell percentage within RCC-infiltrating lymphocytes, as determined by flow cytometry, allows ccRCC subgrouping in NKhigh/NKlow tissues independent of TNMG classification. Quantitative reverse transcriptase polymerase chain reaction using whole-tissue RNA identified four markers (NKp46, perforin, CX3CL1, and CX3CR1) whose transcript levels reproduced the NKhigh/NKlow tissue distinction identified by flow cytometry with high selectivity and specificity. Combined in a multiplex profile and analyzed using neural network, the accuracy of predicting the NKhigh/NKlow groups was 87.8%, surpassing that of each single marker. The tissue transcript signature, based on a robust high-throughput methodology, is easily amenable to archive material and clinical translation. This now allows the analysis of large patient cohorts to substantiate a role of NK cells in cancer progression or response to immunotherapy.

Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

CEACAM1, CEA/CEACAM5, and CEACAM6 are cell adhesion molecules (CAMs) of the carcinoembryonic antigen (CEA) family that have been shown to be deregulated in lung cancer and in up to 50% of all human cancers. However, little is known about the functional impact of these molecules on undifferentiated cell growth and tumor progression. Here we demonstrate that cell surface expression of CEACAM1 on confluent A549 human lung adenocarcinoma cells plays a critical role in differentiated, contact-inhibited cell growth. Interestingly, CEACAM1-L, but not CEACAM1-S, negatively regulates proliferation via its ITIM domain, while in proliferating cells no CEACAM expression is detectable. Furthermore, we show for the first time that CEACAM6 acts as an inducer of cellular proliferation in A549 cells, likely by interfering with the contact-inhibiting signal triggered by CEACAM1-4L, leading to undifferentiated anchorage-independent cell growth. We also found that A549 cells expressed significant amounts of non-membrane anchored variants of CEACAM5 and CEACAM6, representing a putative source for the increased CEACAM5/6 serum levels frequently found in lung cancer patients. Taken together, our data suggest that post-confluent contact inhibition is established and maintained by CEACAM1-4L, but disturbances of CEACAM1 signalling by CEACAM1-4S and other CEACAMs lead to undifferentiated cell growth and malignant transformation.

The tumour suppressor gene CEACAM1 is completely but reversibly downregulated in renal cell carcinoma.

CEACAM1 acts as a tumour suppressor in various epithelial tumours. On the other hand, de novo expression of CEACAM1 is strongly associated with reduced disease‐free survival of melanoma and non‐small cell lung carcinoma patients. Since effector functions of natural killer and T cells are inhibited by homophilic CEACAM1 interaction, immune escape could be responsible for the poor prognosis of these patients. Here, we describe CEACAM1 expression in normal kidney, renal adenomas and renal cell carcinomas (RCC) using a novel antibody generated by genetic immunization. In normal kidney, CEACAM1 was found in epithelial cells of proximal tubules and in endothelial cells. In contrast, tumour cells of 30 clear cell, three chromophobic, and two chromophilic RCCs were completely devoid of CEACAM1. Renal adenomas also lacked CEACAM1 expression. Similarly, RCC cell lines CaKi1, CaKi2, A498, and RCC26 exhibited no or low‐level CEACAM1 expression. However, CEACAM1 expression was transiently induced in A498 cells upon contact with allogeneic CD8+ T cells, mediated at least in part by interferon‐γ. Furthermore, the majority of tumour‐infiltrating T and NK cells expressed CEACAM1 upon stimulation. Thus, transient expression of the tumour suppressor CEACAM1 by tumour cells and subsequent homophilic interaction with CEACAM1 on tumour‐infiltrating lymphocytes could represent a novel immune escape mechanism in RCC. Copyright

The Critical Role of Residues 43R and 44Q of Carcinoembryonic Antigen Cell Adhesion Molecules-1 in the Protection from Killing by Human NK Cells

The multifunctional carcinoembryonic Ag cell adhesion molecule (CEACAM)1 protein has recently become the focus of intense immunological research. We have previously shown that the CEACAM1 homophilic interactions inhibit the killing activity of NK cells. This novel inhibitory mechanism plays a key role in melanoma immune evasion, inhibition of decidual immune response, and controlling NK autoreactivity in TAP2-deficient patients. These roles are mediated mainly by homophilic interactions, which are mediated through the N-domain of the CEACAM1. The N-domain of the various members of the CEACAM family shares a high degree of similarity. However, it is still unclear which of the CEACAM family members is able to interact with CEACAM1 and what are the amino acid residues that control this interaction. In this study we demonstrate that CEACAM1 interacts with CEACAM5, but not with CEACAM6. Importantly, we provide the molecular basis for CEACAM1 recognition of various CEACAM family members. Sequence alignment reveals a dichotomy among the CEACAM family members: both CEACAM1 and CEACAM5 contain the R and Q residues in positions 43 and 44, respectively, whereas CEACAM3 and CEACAM6 contain the S and L residues, respectively. Mutational analysis revealed that both 43R and 44Q residues are necessary for CEACAM1 interactions. Implications for differential expression of CEACAM family members in tumors are discussed.

CEACAM1: a key regulator of vascular permeability

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) is an immunoglobulin-like cell surface co-receptor expressed on epithelial, hematopoietic and endothelial cells. CEACAM1 functions as an adhesion molecule, mainly binding to itself or other members of the CEA family. We and others have previously shown that CEACAM1 is crucial for in vivo vascular integrity during ischemic neo-vascularization. Here, we have deciphered the roles of CEACAM1 in normal and pathological vascularization. We have found that Ceacam1−/− mice exhibit a significant increase in basal vascular permeability related to increased basal Akt and endothelial nitric oxide synthase (eNOS) activation in primary murine lung endothelial cells (MLECs). Moreover, CEACAM1 deletion in MLECs inhibits VEGF-mediated nitric oxide (NO) production, consistent with defective VEGF-dependent in vivo permeability in Ceacam1−/− mice. In addition, Ceacam1-null mice exhibit increased permeability of tumor vasculature. Finally, we demonstrate that CEACAM1 is tyrosine-phosphorylated upon VEGF treatment in a SHP-1- and Src-dependent manner, and that the key residues of the long cytoplasmic domain of CEACAM1 are crucial for CEACAM1 phosphorylation and NO production. This data represents the first report, to our knowledge, of a functional link between CEACAM1 and the VEGFR2/Akt/eNOS-mediated vascular permeability pathway.