Maria A. Lagarkova

Distinct Role of Surface Lymphotoxin Expressed by B Cells in the Organization of Secondary Lymphoid Tissues

In order to definitively ascertain the functional contribution of lymphotoxin (LT) expressed by B cells, we produced mice with the LTbeta gene deleted from B cells (B-LTbeta KO mice). In contrast to systemic LTbeta deletion, in B-LTbeta KO mice only splenic microarchitecture was affected, while lymph nodes and Peyers patches (PP) were normal, except for PPs reduced size. Even though B-LTbeta KO spleens retained a small number of follicular dendritic cells (FDC) which appeared to be dependent on LTbeta produced by T cells, IgG responses to sheep red blood cells were markedly reduced. Thus, the organogenic function of B-LTbeta is almost entirely restricted to spleen, where it supports the correct lymphoid architecture that is critical for an effective humoral immune response.

Cloning and characterization of TNKL, a member of tankyrase gene family

By serological screening of a breast tumor cDNA library we have identified a novel human gene, tnkl, encoding an ankyrin-related protein with a high degree of similarity to tankyrase, the poly(ADP-ribose)polymerase associated with human telomeres (Smith et al, Science 282: 1484). The tnkl gene maps to chromosome 10, while the tnks gene encoding tankyrase is located on chromosome 8. The predicted 1166-aa protein product of the tnkl gene is 78% identical to human tankyrase and 62% to a putative D. melanogaster protein. Since the proteins have essentially identical domain structures, the corresponding genes form a distinct gene family. The possible link between TNKL and cancer justifies its further functional analysis.

Novel Lymphotoxin Alpha (LTα) Knockout Mice with Unperturbed Tumor Necrosis Factor Expression: Reassessing LTα Biological Functions

ABSTRACT Lymphotoxin alpha (LTα) can exist in soluble form and exert tumor necrosis factor (TNF)-like activity through TNF receptors. Based on the phenotypes of knockout (KO) mice, the physiological functions of LTα and TNF are considered partly redundant, in particular, in supporting the microarchitecture of the spleen and in host defense. We exploited Cre-LoxP technology to generate a novel neomycin resistance gene (neo) cassette-free LTα-deficient mouse strain (neo-free LTα KO [LTαΔ/Δ]). Unlike the “conventional” LTα−/− mice, new LTαΔ/Δ animals were capable of producing normal levels of systemic TNF upon lipopolysaccharide (LPS) challenge and were susceptible to LPS/d-galactosamine (D-GalN) toxicity. Activated neutrophils, monocytes, and macrophages from LTαΔ/Δ mice expressed TNF normally at both the mRNA and protein levels as opposed to conventional LTα KO mice, which showed substantial decreases in TNF. Additionally, the spleens of the neo-free LTα KO mice displayed several features resembling those of LTβ KO mice rather than conventional LTα KO animals. The phenotype of the new LTαΔ/Δ mice indicates that LTα plays a smaller role in lymphoid organ maintenance than previously thought and has no direct role in the regulation of TNF expression.

Evaluation of humoral response to tumor antigens using recombinant expression-based serological mini-arrays (SMARTA)

Screening of expression cDNA libraries derived from human neoplasms with autologous sera (SEREX) is an established method for defining antigens immunogenic in individual cancer patients. Although the majority of SEREX-derived cDNA clones encode autoantigens, some of them represent shared cancer antigens with cancer-related serological profiles. Routine evaluation of multiple SEREX-derived clones in serological assays using panels of allogeneic sera from cancer patients is an important step towards defining disease parameters of diagnostic and prognostic significance. Here we show how the seroreactivity of multiple SEREX-derived antigens can be simultaneously evaluated using a rapid semi-quantitative protocol of allogeneic screening, which we call SMARTA (serological mini-arrays of recombinant tumor antigens).

Antibody response to a non-conserved C-terminal part of human histone deacetylase 3 in colon cancer patients†

Antibodies to cancer antigens can often be detected in the sera of patients, although the mechanism of the underlying humoral immune response is poorly understood. Using immunoscreening of tumor‐derived cDNA expression libraries (SEREX), we identified human histone deacetylase 3 (HDAC3) as serologically defined antigen in colon cancer. Closely related HDAC1 and HDAC2 do not elicit humoral response in colon cancer patients. We show that the C‐terminal region of HDAC3 protein lacking the homology to other Class I HDAC contains at least 3 distinct B‐cell epitopes that are recognized by the serum antibodies. HDAC3 in combination with other SEREX antigens may become a useful molecular biomarker with diagnostic or prognostic value for a subset of colon cancer patients. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience. wiley.com/jpages/0020‐7136/suppmat/index.html.

Human cortactin as putative cancer antigen.

Humoral immune response against overexpressed oncogenic or tumor supressor proteins has been demonstrated for many types of cancer. In this study we report on the detection of the autologous antibody response to putative oncogene, human cortactin using serological analysis of breast carcinoma expression library. Cortactin maps to chromosome 11q13, the region amplified in about 15% of primary breast carcinomas and 30% of head and neck squamous cell carcinomas. Cortactin overexpression due to such amplification might affect adhesive properties of human cancer cells and is associated with poor disease prognosis. Accordingly, we detected overexpression of cortactin transcript in autologous tumor and amplification/overexpression of cortactin in tumors of breast cancer patients serologically positive for this marker. We demonstrate that 15% of breast cancer patients elicit humoral immune response against human cortactin.

Serological Analysis of the Repertoire of Human Cancer Antigens and Autoantigens

The spectrum of human antigens allows a monitoring of various pathological processes such as autoimmune disorders and tumorigenesis. Serological analysis of cDNA expression libraries (SEREX) is now used to search for new cancer-associated antigens, which are potential diagnostic markers or targets for immunotherapy of cancer. The results obtained for several solid tumors are reviewed. Groups of antigens detectable by SEREX, causes of immunogenicity of autoantigens, and prospective implication of antigens in diagnostics and monitoring of cancer are discussed.