Activating KIR/HLA complexes in classic Kaposi's Sarcoma

Background Classic Kaposi's Sarcoma (cKS) is a rare vascular tumor associated with Human Herpesvirus 8 (KSHV) infection, nevertheless not all KSHV-infected individuals have cKS. Objective We investigated whether particular KIR/HLA receptor/ligand genotypes would be preferentially present in KSHV-infected and uninfected individuals who have or have not developed cKS. Methods KIR/HLA genotypes were analyzed by molecular genotyping in 50 KSHV-infected individuals who did or did not have cKS and in 33 age-and sex-matched KSHV seronegative individuals. Results There was no association of individual KIR, HLA or receptor ligand combinations with KSHV infection. However, activating KIR and KIR/HLA genotypes were significantly more frequent in cKS cases, specifically KIR3DS1, KIR2DS1, and KIR2DS1 with its HLA-C2 ligand. Conclusion A nonspecific inflammatory response triggered by activation of NK cells upon KIR-HLA interaction could be associated with the pathogenesis of KS.


Findings
Classic Kaposi's Sarcoma (cKS) is a rare vascular neoplasm of the skin related to Kaposi's Sarcoma-associated Herpes Virus (KSHV or Human Herpes Virus 8, HHV-8) infection. KSHV plays a prominent role in the progression of cKS from an angio-proliferative disorder to sarcoma, KSHV infection is nevertheless not sufficient to provoke such progression as not all KSHV-infected individuals develop cKS [1]. Natural killer (NK) cells are central components of the innate immune response against viral infections and tumour growth via direct and indirect mechanisms [2]. The modulation of NK activity is a complex and multi factorial phenomenon triggered by the binding of inhibitory or activating killer cell immunoglobulin-like receptors (KIR) to class I human leukocyte antigens (HLA) [3,4]. When HLA molecules bind activating KIR receptors, a potent inflammatory response finalized at NK cell-mediated destruction of target cells, including transformed tumor cells and virus-infected cells, is stimulated [5]. KIR and HLA loci are highly polymorphic and map in distinct human chromosomes (chromosomes 19 and 6, respectively); both KIR and the specific HLA ligands must be present in order to regulate NK cell activity, such that one without the other is functionally inert.
We verified whether particular KIR/HLA genotypes would be preferentially present in KSHV-infected and uninfected individuals who do or do not develop cKS.
We studied eighty-three Caucasian individuals born and living in Northern Italy; 50 of these individuals were KSHV-infected (KSHV pos ). Histologically-confirmed cKS had been diagnosed in 32 patients (cKS pos )(23 male, 9 female, mean age: 71.1 ± 7.7 years); KSHV-infection without tumour was observed in 18 other patients. (KSHV pos /cKS neg )(8 male, 10 female, mean age: 83.4 ± 11.6 years). Thirty-three KSHV-uninfected individuals (KSHV neg )(24 male, 9 female, mean age: 70.0 ± 12.6 years) were also enrolled. Subjects did not suffer from any disease that could potentially bias the analysis outcome. KSHV-infected patients were enrolled, upon signing an informed consent approved by the Institutional Review Board, by the Dermatology Unit of the Ospedale Maggiore Policlinico, in Milan. Controls subjects were elderly healthy individuals seen at the Don Gnocchi Foundation in Milan.
We analyzed antibodies against latent and lytic KSHV antigens using an in-house immunofluorescence assay (IFA) based on the body-cavity B cell lymphoma (BCBL-1) cell line [6] as previously described [7]. Monoclonal antibodies to ORF73 (LNA-1), ORF 59, and ORF K8.1 lytic proteins (Advanced Biotechnologies Inc., Maryland, USA) were utilized as fluorescent controls. Sera containing antibodies for both latent and lytic antigens were considered positives (KSHV pos ).
We performed molecular genotyping of HLA B (22 alleles), Cw (14 alleles), and KIR (18 genes) by PCR on genomic DNA using sequence specific primers (SSP) according to the manufacturer's instructions (BAG-Lich, Germany, Astra Formedic, Milan Italy). Alleles detection was done after amplification in a GeneAmp PCR 9700 thermocycler (Applied Biosystem, Foster City, CA, USA) by gel electrophoresis on 2% agarose gel.
KIR haplotype and ligands. Two broad haplotypes, termed A and B, were defined based on KIR genes. A and/or B haplotypes were classified based on the criteria adopted by Middleton http://www.allelefrequencies.net [8].
We assessed differences between groups by the chisquare test for categorical variables. P value with Yates correction (p y ) or Fisher exact test (p f ) and Bonferroni correction for multiple test (p c ) were applied. The association of each polymorphism with disease (cKS) was measured by the Odd Ratio (OR) and a 95% Confidence Interval (95%CI). For each independent variable, crude and adjusted odds ratios and 95% confidence intervals were calculated.
Multivariate analysis were performed with binary logistic regression model to assess KIR-HLA genotypes associated with cKS, considering cKS pos versus cKS neg as dependent variable, gender and age as covariates. Analyses were carried out using a SPSS 16.0 for Windows.
KIR gene distribution was evaluated in the three groups studied (Additional file 1: Table S1); no significant difference was detected between KSHV pos / cKS neg infected and KSHV neg individuals.
Because distribution of KIR and HLA genotypes were similar between the two control groups, we thought it was appropriate to combine them in subsequent analyses.
Evaluations focusing on these two groups showed the presence of higher frequencies of activating KIR genes ( Figure 1, Panel a) and of activating KIR/HLA receptor/ ligand genotypes (Figure 1, Panel b) in KSHV pos /cKS pos patients compared to subjects without cKS.
The frequency of KIR3DS1 gene distribution was quite similar to that of KIR2DS1, probably due to their linkage disequilibrium (LD).
Additionally, the activating KIR3DS1+ gene was statistically more frequent in cKS pos compared to cKS neg individuals (p y = 0.006; OR:4.00).
Finally KIR2DS2 gene and KIR2DS2/C1+ genotypes were quite equally distributed in all the studied groups.
Finally, no differences were observed in the genotype distribution of the other examined inhibitory and activating KIR genes (Additional file 1: Table S1) or in the frequency of HLA-B and HLA-Cw alleles (not shown).
In this work we investigated whether specific KIR genes and their HLA class I ligands would be associated with classic Kaposi sarcoma in KSHV pos infected subjects.
Results herein, albeit stemming from analyses performed in a limited number of individuals, indicate that a statistically higher frequency of activating KIR genes together  with their HLA ligands is present in KSHV-infected patients who have cKS compared to individuals including those with KSHV infection without cKS Importantly, whereas activating KIR genes were more frequent in cKS patients, the distribution of KIR genes was similar between KSHV pos and KSHV neg healthy controls; this allowed us to combine them in a single control group. The KIR2DS1+ and KIR3DS1+ genes were prevalent in cKS patients; This similarity of distribution may be secondary to the fact that these genes are in linkage disequilibrium. However, whereas the activating KIR2DS1+/C2+ genotype was positively associated with cKS development, HLA ligand distribution did not reveal any statistical association with cKS. This observation, together with the fact that the OR of KIR2DS1 association increased when the KIR2DS1+/C2+ genotype was evaluated, may suggest that the association of KIR2DS1 +/C2+ with cKS is mostly due to KIR-HLA interaction and not to either KIR or to HLA genes separately.
Data herein indicate that a KIR/HLA "activating milieu" is present in cKS; such milieu may be a risk factor for the development of this tumor. Our results add cKS to the list of virus-associated cancers including cervical neoplasia [12,13] and nasopharyngeal carcinoma [14] where an increased presence of activating KIR/HLA receptor/ligand combinations is observed and is suggested to play a role in oncogenesis.
Genotypes that increase the activation of NK cells are possibly beneficial in viral infections such as HIV and HCV [15]; inflammatory components associated with NK cell activation might nevertheless have a detrimental effect, possibly facilitating the oncogenic process for cancers that, as is the case with cKS, have an inflammatory component [11,16]. Thus, the elicitation of inflammation and NK cells activity via KIR/HLA interactions might be directly involved in malignant transformation and tumor progression as a consequence of the production of cytokines such as TNF-α, IL-6, and TGF-β that contribute to the proliferation and survival of tumor cells [17]. Non-specific inflammatory responses, such as oxidative DNA damage, may be triggered by activated NK upon KIR-HLA interaction as well, possibly facilitating the growth of oncogenic viruses [18].
The small sample size is a significant limitation of this study. We tried to bypass this problem by sampling together KSHV-infected and -uninfected healthy individuals, once we verified that the distribution of KIR and HLA molecules was comparable.
It is important to underline that these data stem from analyses performed in a relatively small group of individuals and will need to be validated using bigger cohorts of KS patients.