LPS consists of a relatively conserved region of lipid A and core oligosaccharide and a highly variable region of O-antigen polysaccharide. Whereas lipid A is known to bind to the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex, the role of the O-antigen remains unclear. Here we report a novel molecular interaction between dendritic cell-associated C-type lectin-2 (Dectin-2) and mannosylated O-antigen found in a human opportunistic pathogen, Hafnia alvei PCM 1223, which has a repeating unit of [-Man-α1,3-Man-α1,2-Man-α1,2-Man-α1,2-Man-α1,3-]. H. alvei LPS induced higher levels of TNFα and IL-10 from mouse bone marrow-derived dendritic cells (BM-DCs), when compared with Salmonella enterica O66 LPS, which has a repeat of [-Gal-α1,6-Gal-α1,4-[Glc-β1,3]GalNAc-α1,3-GalNAc-β1,3-]. In a cell-based reporter assay, Dectin-2 was shown to recognize H. alvei LPS. This binding was inhibited by mannosidase treatment of H. alvei LPS and by mutations in the carbohydrate-binding domain of Dectin-2, demonstrating that H. alvei LPS is a novel glycan ligand of Dectin-2. The enhanced cytokine production by H. alvei LPS was Dectin-2-dependent, because Dectin-2 knock-out BM-DCs failed to do so. This receptor cross-talk between Dectin-2 and TLR4 involved events including spleen tyrosine kinase (Syk) activation and receptor juxtaposition. Furthermore, another mannosylated LPS from Escherichia coli O9a also bound to Dectin-2 and augmented TLR4 activation of BM-DCs. Taken together, these data indicate that mannosylated O-antigens from several Gram-negative bacteria augment TLR4 responses through interaction with Dectin-2.

The nature of effector cells and the potential immunogenicity of Leishmania major excreted/secreted proteins (LmES) were evaluated using peripheral blood mononuclear cells (PBMCs) from healed zoonotic cutaneous leishmaniasis individuals (HZCL) and healthy controls (HC). First, we found that PBMCs from HZCL individuals proliferate and produce high levels of IFN-γ and granzyme B (GrB), used as a marker of activated cytotoxic T cells, in response to the parasite antigens. IFN-γ is produced by CD4(+) T cells, but unexpectedly GrB is also produced by CD4(+) T cells in response to stimulation with LmES, which were found to be as effective as soluble Leishmania antigens to induce proliferation and cytokine production by PBMCs from immune individuals. To address the question of regulatory T cell (Tregs) involvement, the frequency of circulating Tregs was assessed and found to be higher in HZCL individuals compared to that of HC. Furthermore, both CD4(+)CD25(+) and CD4(+)CD25(-) T cells, purified from HZCL individuals, produced IFN-γ and GrB when stimulated with LmES. Additional experiments showed that CD4(+)CD25(+)CD127(dim/-) Tregs were involved in GrB production. Collectively, our data indicate that LmES are immunogenic in humans and emphasize the involvement of CD4(+) T cells including activated and regulatory T cells in the immune response against parasite antigens.

We have previously demonstrated the immunogenicity of VGX-3100, a multicomponent DNA immunotherapy for the treatment of Human Papillomavirus (HPV)16/18-positive CIN2/3 in a phase 1 clinical trial. Here, we report on the ability to boost immune responses with an additional dose of VGX-3100. Patients completing our initial phase 1 trial were offered enrollment into a follow on trial consisting of a single boost dose of VGX-3100. Data show both cellular and humoral immune responses could be augmented above pre-boost levels, including the induction of interferon (IFN)γ production, tumor necrosis factor (TNF)α production, CD8+ T cell activation and the synthesis of lytic proteins. Moreover, observation of antigen-specific regulation of immune-related gene transcripts suggests the induction of a proinflammatory response following the boost. Analysis of T cell receptor (TCR) sequencing suggests the localization of putative HPV-specific T cell clones to the cervical mucosa, which underscores the putative mechanism of action of lesion regression and HPV16/18 elimination noted in our double-blind placebo-controlled phase 2B trial. Taken together, these data indicate that VGX-3100 drives the induction of robust cellular and humoral immune responses that can be augmented by a fourth "booster" dose. These data could be important in the scope of increasing the clinical efficacy rate of VGX-3100.

Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRα. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRα, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2(+) MAIT cells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAIT antigens are derived from riboflavin metabolites, this suggests that TRAV12-2(+) clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites.

Immunogenic biologics trigger an anti-drug antibody (ADA) response in patients that reduces efficacy and increases adverse reactions. Our laboratory has shown that targeting protein antigen to the liver microenvironment can reduce antigen-specific T cell responses; herein, we present a strategy to increase delivery of otherwise immunogenic biologics to the liver via conjugation to a synthetic mannose polymer, p(Man). This delivery leads to reduced antigen-specific T follicular helper cell and B cell responses resulting in diminished ADA production, which is maintained throughout subsequent administrations of the native biologic. We find that p(Man)-antigen treatment impairs the ADA response against recombinant uricase, a highly immunogenic biologic, without a dependence on hapten immunodominance or control by T regulatory cells. We identify increased T cell receptor signaling and increased apoptosis and exhaustion in T cells as effects of p(Man)-antigen treatment via transcriptomic analyses. This modular platform may enhance tolerance to biologics, enabling long-term solutions for an ever-increasing healthcare problem.

Lentiviral vectors (LVs) are highly efficient at inducing CD8+ T cell responses. However, LV-encoded antigens are processed inside the cytosol of antigen-presenting cells, which does not directly communicate with the endosomal major histocompatibility complex class II (MHC-II) presentation pathway. LVs are thus poor at inducing CD4+ T cell response. To overcome this limitation, we devised a strategy whereby LV-encoded antigens are extended at their N-terminal end with the MHC-II-associated light invariant chain (li), which contains an endosome-targeting signal sequence. When evaluated with an LV-encoded polyantigen composed of CD4+ T cell targets from Mycobacterium tuberculosis, intranasal vaccination in mice triggers pulmonary polyfunctional CD4+ and CD8+ T cell responses. Adjuvantation of these LVs extends the mucosal immunity to Th17 and Tc17 responses. A systemic prime and an intranasal boost with one of these LV induces protection against M. tuberculosis. This strategy improves the protective power of LVs against infections and cancers, where CD4+ T cell immunity plays an important role.

Psoriasis is a chronic inflammatory skin disease associated with a T helper 17 response. Yet, it has proved challenging to identify relevant peptide-based T cell antigens. Antigen-presenting Langerhans cells show a differential migration phenotype in psoriatic lesions and express constitutively high levels of CD1a, which presents lipid antigens to T cells. In addition, phospholipase A2 (PLA2) is highly expressed in psoriatic lesions and is known to generate neolipid skin antigens for recognition by CD1a-reactive T cells. In this study, we observed expression of a cytoplasmic PLA2 (PLA2G4D) in psoriatic mast cells but, unexpectedly, also found PLA2G4D activity to be extracellular. This was explained by IFN-α-induced mast cell release of exosomes, which transferred cytoplasmic PLA2 activity to neighboring CD1a-expressing cells. This led to the generation of neolipid antigens and subsequent recognition by lipid-specific CD1a-reactive T cells inducing production of IL-22 and IL-17A. Circulating and skin-derived T cells from patients with psoriasis showed elevated PLA2G4D responsiveness compared with healthy controls. Overall, these data present an alternative model of psoriasis pathogenesis in which lipid-specific CD1a-reactive T cells contribute to psoriatic inflammation. The findings suggest that PLA2 inhibition or CD1a blockade may have therapeutic potential for psoriasis.

Administration of high dose intravenous immunoglobulin (IVIg) is widely used in the clinic to treat autoimmune and severe inflammatory diseases. However, its mechanisms of action remain poorly understood. We assessed the impact of IVIg on immune cell populations using an in vivo ovalbumin (Ova)-immunization mouse model. High dose IVIg significantly reduced the Ova-specific antibody response. Intriguingly, the results obtained indicate an immediate and massive immune reaction against IVIg, as shown by the activation and expansion of B cells and CD4+ T cells in the spleen and draining lymph nodes and the production of IVIg-specific antibodies. We propose that IVIg competes at the T-cell level with the response against Ova to explain the immunomodulatory properties of IVIg. Two monoclonal antibodies did not succeeded in reproducing the effects of IVIg. This suggests that in addition to the mouse response against human constant domains, the enormous sequence diversity of IVIg may significantly contribute to this massive immune response against IVIg. While correlation of these findings to IVIg-treated patients remains to be explored, our data demonstrate for the first time that IVIg re-directs the immune response towards IVIg and away from a specific antigen response.

Whether Borrelia burgdorferi, the causative agent of Lyme disease, can persist for long periods in the human body has been a controversial question. The objective of this study was to see if we could find B. burgdorferi in a Lyme disease patient after a long clinical course and after long-term antibiotic treatment. Therefore, we investigated the potential presence of B. burgdorferi antigens and DNA in human autopsy tissues from a well-documented serum-, PCR-, and culture-positive Lyme disease patient, a 53-year-old female from northern Westchester County in the lower Hudson Valley Region of New York State, who had received extensive antibiotic treatments during extensive antibiotic treatments over the course of her 16-year-long illness. We also asked what form the organism might take, with special interest in the recently found antibiotic-resistant aggregate form, biofilm. We also examined the host tissues for the presence of inflammatory markers such as CD3+ T lymphocytes. Autopsy tissue sections of the brain, heart, kidney, and liver were analyzed by histological and immunohistochemical methods (IHC), confocal microscopy, fluorescent in situ hybridization (FISH), polymerase chain reaction (PCR), and whole-genome sequencing (WGS)/metagenomics. We found significant pathological changes, including borrelial spirochetal clusters, in all of the organs using IHC combined with confocal microscopy. The aggregates contained a well-established biofilm marker, alginate, on their surfaces, suggesting they are true biofilm. We found B. burgdorferi DNA by FISH, polymerase chain reaction (PCR), and an independent verification by WGS/metagenomics, which resulted in the detection of B. burgdorferi sensu stricto specific DNA sequences. IHC analyses showed significant numbers of infiltrating CD3+ T lymphocytes present next to B. burgdorferi biofilms. In summary, we provide several lines of evidence that suggest that B. burgdorferi can persist in the human body, not only in the spirochetal but also in the antibiotic-resistant biofilm form, even after long-term antibiotic treatment. The presence of infiltrating lymphocytes in the vicinity of B. burgdorferi biofilms suggests that the organism in biofilm form might trigger chronic inflammation.

The pathogenesis of cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis is dictated mainly by the immune-mediated-tissue inflammation developed. The understanding of the immunological mechanisms that generate tissue damage or resolution of lesions is the key to the development of effective vaccine protocols and proper therapeutic schemes. It is clear that the specific immune response mediated by T cells is responsible for the beneficial outcome of the disease, however, the roles of CD4+ T, CD8+ T, NK and NKT cell subpopulations in immunopathogenesis of CL need to be elucidated. Peripheral blood cells from patients before, during and after the antimonial therapy, as well as healthy individuals (HI) were cultured with (LbAgS) or without (NS) L. braziliensis antigens (LbAg). Afterwards, the frequencies of LbAg-specific-cytotoxic CD8+ T, CD4+ T, NK and CD3+CD56+ NKT cells, as well as their activation and exhaustion profiles, were defined by flow cytometry. We observed higher frequencies of CD8+ T, NK and CD3+CD56+ NKT cells and lower frequencies of CD4+ T lymphocytes in LbAgS cell cultures from patients before treatment. The specific response to LbAg resulted in an expansion of cytotoxic-activated CD4+ T, CD8+ T, and NK cells, before and during treatment, indicating specificity in the response by these cells against L. braziliensis. Furthermore, comparing the differences of frequencies of cytotoxic-activated CD4+T, CD8+T, and NK cells, among before and during treatment patients and HI groups, we conclude that these cell populations are in charge of immune response elicited by antimonial therapy. Interestingly, we also observed that NK cells were induced by LbAg to an exhaustion profile during all clinical stages of the disease. The increased antigen-specific activation and cytotoxic activity are in line with the strong inflammatory response described in this disease, a likely cause of tissue damage. These findings reinforce the involvement of these distinct cytotoxic-activated cell populations in the immunopathogenesis of CL, showing a character of specificity in this immune response.

Evaluation of antigen-specific T-cell responses to viral antigens is frequently performed on IFN-γ secreting cells. However, T-cells are capable of producing many more functions than just IFN-γ, some of which, like Perforin, are associated with immune protection in HIV-1 disease elite controllers. We evaluated the extent of missed T-cell functions when IFN-γ secretion is used as a surrogate marker for further evaluation of T-cell functions. Intracellular cytokine staining assay and flow cytometry were used to assess peripheral blood mononuclear cells (PBMCs) from 31 HIV-infected ART-naive individuals for the extent to which gated CD4+ and CD8+ IFN-γ producing and non-producing T-cells also secreted IL-2, Perforin, and TNF-α functions. Similarly, the extent of missed virus-specific responses in IFN-γ ELISpot assay negative T-cells from 5 HIV-1 uninfected individuals was evaluated. Cells from HIV-infected individuals were stimulated with pooled consensus group M (Con M) peptides; and those from healthy individuals were stimulated with pooled adenovirus (Ad) peptides. Overall, frequencies of virus-specific IFN-γ secreting CD4+ and CD8+ cells were low. Proportions of IFN-γ negative CD4+ expressing IL-2, Perforin, or TNF-α to Con M were significantly higher (5 of 7 functional profiles) than the corresponding IFN-γ positive CD4+ (0 of 7) T-cell phenotype, p = 0.02; Fisher's Exact test. Likewise, proportions of CD8+ T-cells expressing other functions were significantly higher in 4 of the 7 IFN-γ negative CD8+ T-cells. Notably, newly stimulated Perforin, identified as Perforin co-expression with IL-2 or TNF-α, was significantly higher in IFN-γ negative CD8+ T-cell than in the positive CD8+ T-cells. Using SEB, lower responses in IFN-γ positive cells were most associated with CD4+ than CD8+ T-cells. These findings suggest that studies evaluating immunogenicity in response to HIV and Adenovirus viral antigens should not only evaluate T-cell responsiveness among IFN-γ producing cells but also among those T-cells that do not express IFN-γ.

The clinical benefit of immune checkpoint blockade (ICB) therapy is often limited by the lack of pre-existing CD8+ T cells infiltrating the tumor. In principle, CD8+ T-cell infiltration could be promoted by therapeutic vaccination. However, this remains challenging given the paucity of vaccine platforms able to induce the strong cytotoxic CD8+ T-cell response required to reject tumors. A therapeutic cancer vaccine that induces a robust cytotoxic CD8+ T-cell response against shared tumor antigens and can be combined with ICB could improve the outcome of cancer immunotherapy.

In multiple tumor types, prediction of response to immune therapies relates to the presence, distribution and activation state of tumor infiltrating lymphocytes (TILs). Although such therapies are, to date, unsuccessful in gliomas, little is known on the immune contexture of TILs in these tumors. We assessed whether low and high-grade glioma (LGG and HGG, grade II and IV respectively) differ with respect to number, location and tumor reactivity of TILs; as well as expression of molecules involved in the trafficking and activation of T cells. Intra-tumoral CD8 T cells were quantified by flow cytometry (LGG: n = 12; HGG: n = 8) and immunofluorescence (LGG: n = 28; HGG: n = 28). Neoantigen load and expression of Cancer Germline Antigens (CGAs) were assessed using whole exome sequencing and RNA-seq. TIL-derived DNA was sequenced and the variable domain of the TCRβ chain was classified according to IMGT nomenclature. QPCR was used to determine expression of T cell-related genes. CD8 T cell numbers were significantly lower in LGG and, in contrast to HGG, mainly remained in close vicinity to blood vessels. This was accompanied by lower expression of chemo-attractants CXCL9, CXCL10 and adhesion molecule ICAM1. We did not observe a difference in the number of expressed neoantigens or CGAs, nor in diversity of TCR-Vβ gene usage. In summary, LGG have lower numbers of intra-tumoral CD8 T cells compared to HGG, potentially linked to decreased T cell trafficking. We have found no evidence for distinct tumor reactivity of T cells in either tumor type. The near absence of TILs in LGG suggest that, at present, checkpoint inhibitors are unlikely to have clinical efficacy in this tumor type.

The impact of biological sex on T-cell immunity to Cytomegalovirus (CMV) has not been investigated in detail with only one published study comparing CMV-specific T-cell responses in men and women. Many studies, however, have shown an association between CMV infection and immunosenescence, with broad effects on peripheral blood lymphocyte subsets as well as the T and B-cell repertoires. Here, we provide a detailed analysis of CMV-specific T-cell responses in (n=94) CMV+ older people, including 47 women and 47 men aged between 60 and 93 years. We explore sex differences with respect to 16 different CMV proteins arranged in 14 peptide pools (overlapping peptides). Following ex vivo stimulation, CD4 and CD8 T-cells producing IFN-γ, TNF, and IL-2 were enumerated by flow-cytometry (intracellular cytokine staining). T-cell responses were evaluated in terms of each cytokine separately or in terms of cytokines produced simultaneously (polyfunctionality). Surface memory phenotype and CD3 downmodulation were assessed in parallel. The polyfunctionality index and a memory subset differentiation score were used to identify associations between response size, cytokine production, polyfunctionality, and memory subset distribution. While no significant sex differences were found with respect to overall CMV target protein selection, the T-cell response in men appeared more focused and accompanied by a more prominent accumulation of CMV-specific memory CD4 and CD8 T-cells. T-cell polyfunctionality and differentiation were similar in the sexes, however, CMV-specific T-cells in men produced more pro-inflammatory cytokines. Particularly, TNF production by CD4 T-cells was stronger in men than in women. Also, compared with women, men had larger responses to CMV proteins with immediate-early/early kinetics than women, which might have been driven by CMV reactivation. In conclusion, the CMV-specific T-cell response in men was larger and more pro-inflammatory than in women. Our findings may help explain sex differences in CMV-associated pathologies.

In allergic asthma, allergen inhalation leads to local Th2 cell activation and peribronchial inflammation. However, the mechanisms for local antigen capture and presentation remain unclear. By two-photon microscopy of the mouse lung, we established that soluble antigens in the bronchial airway lumen were efficiently captured and presented by a population of CD11c+ interstitial macrophages with high CX3CR1-GFP and MHC class II expression. We refer to these cells as Bronchus-Associated Macrophages (BAMs) based on their localization underneath the bronchial epithelium. BAMs were enriched in collagen-rich regions near some airway branchpoints, where inhaled antigens are likely to deposit. BAMs engaged in extended interactions with effector Th2 cells and promoted Th2 cytokine production. BAMs were also often in contact with dendritic cells (DCs). After exposure to inflammatory stimuli, DCs migrated to draining lymph nodes, whereas BAMs remained lung resident. We propose that BAMs act as local antigen presenting cells in the lung and also transfer antigen to DCs.

CD8+ T cells specific to caspase-cleaved antigens derived from apoptotic T cells (apoptotic epitopes) represent a principal player in chronic immune activation, which is known to amplify immunopathology in various inflammatory diseases. The purpose of the present study was to investigate the relationship involving these autoreactive T cells, the rheumatoid arthritis immunopathology, and the response to tumor necrosis factor-α inhibitor therapy. The frequency of autoreactive CD8+ T cells specific to various apoptotic epitopes, as detected by both enzyme-linked immunospot assay and dextramers of major histocompatibility complex class I molecules complexed with relevant apoptotic epitopes, was longitudinally analyzed in the peripheral blood of rheumatoid arthritis patients who were submitted to etanercept treatment (or other tumor necrosis factor inhibitors as a control). The percentage of apoptotic epitope-specific CD8+ T cells was significantly higher in rheumatoid arthritis patients than in healthy donors, and correlated with the disease activity. More important, it was significantly more elevated in responders to tumor necrosis factor-α inhibitor therapy than in non-responders before the start of therapy; it significantly dropped only in the former following therapy. These data indicate that apoptotic epitope-specific CD8+ T cells may be involved in rheumatoid arthritis immunopathology through the production of inflammatory cytokines and that they may potentially represent a predictive biomarker of response to tumor necrosis factor-α inhibitor therapy to validate in a larger cohort of patients.

Tick bites have been shown to transmit a novel form of severe food allergy, the galactose-α-1,3-galactose (α-Gal) syndrome (AGS). Cellular responses to α-Gal in patients with AGS have, to date, not been thoroughly scrutinized. Therefore, we investigated T and B cell proliferation, activation, and cytokine profiles in response to tick protein extract (TE) and α-Gal-free TE in patients with AGS and in healthy controls. T and B cells from both patients and controls proliferated in response to TE, but significantly more in patients with AGS. B cell proliferation, but not T cell proliferation, in patients with AGS was reduced by removing α-Gal from the TE. In addition, TE induced a clear Th2 cytokine profile in patients with AGS. Expression of CD23 by B cells correlated only to T cell proliferation. However, both B cell proliferation and CD23 expression were reduced when CD40L and IL-4 were blocked. A large portion of the IgG1 and IgE antibodies binding TE in patients with AGS were directed against the α-Gal epitope. We have, for what we believe to be the first time, investigated T and B cell responses to α-Gal carrying tick proteins in patients with AGS, which will be essential for the understanding of the immune response against an allergenic carbohydrate transmitted by ticks.

T cell responses play an important role in protection against beta-coronavirus infections, including SARS-CoV-2, where they associate with decreased COVID-19 disease severity and duration. To enhance T cell immunity across epitopes infrequently altered in SARS-CoV-2 variants, we designed BNT162b4, an mRNA vaccine component that is intended to be combined with BNT162b2, the spike-protein-encoding vaccine. BNT162b4 encodes variant-conserved, immunogenic segments of the SARS-CoV-2 nucleocapsid, membrane, and ORF1ab proteins, targeting diverse HLA alleles. BNT162b4 elicits polyfunctional CD4+ and CD8+ T cell responses to diverse epitopes in animal models, alone or when co-administered with BNT162b2 while preserving spike-specific immunity. Importantly, we demonstrate that BNT162b4 protects hamsters from severe disease and reduces viral titers following challenge with viral variants. These data suggest that a combination of BNT162b2 and BNT162b4 could reduce COVID-19 disease severity and duration caused by circulating or future variants. BNT162b4 is currently being clinically evaluated in combination with the BA.4/BA.5 Omicron-updated bivalent BNT162b2 (NCT05541861).

Advances within cancer immunotherapy have fueled a paradigm shift in cancer treatment, resulting in increasing numbers of cancer types benefitting from novel treatment options. Despite originally being considered an immunologically silent malignancy, recent studies encourage the research of breast cancer immunogenicity to evaluate immunotherapy as a treatment strategy. However, the epitope landscape in breast cancer is minimally described, limiting the options for antigen-specific, targeted strategies. Aromatase, never in mitosis A-related kinase 3 (NEK3), protein inhibitor of activated STAT3 (PIAS3), and prolactin are known as upregulated proteins in breast cancer. In the present study, these four proteins are identified as novel T cell targets in breast cancer. From the four proteins, 147 peptides were determined to bind HLA-A*0201 and -B*0702 using a combined in silico/in vitro affinity screening. T cell recognition of all 147 peptide-HLA-A*0201/-B*0702 combinations was assessed through the use of a novel high-throughput method utilizing DNA barcode labeled multimers. T cell recognition of sequences within all four proteins was demonstrated in peripheral blood of patients, and significantly more T cell responses were detected in patients compared to healthy donors for both HLA-A*0201 and -B*0702. Notably, several of the identified responses were directed toward peptides, with a predicted low or intermediate binding affinity. This demonstrates the importance of including low-affinity binders in the search for epitopes within shared tumor associated antigens (TAAs), as these might be less subject to immune tolerance mechanisms. The study presents four novel TAAs containing multiple possible targets for immunotherapy of breast cancer.

Tumour-derived exosomes can be released to serum and provide information on the features of the malignancy, however, in order to perform systematic studies in biological samples, faster diagnostic techniques are needed, especially for detection of low abundance proteins. Most human cancer cells are positive for at least one ligand for the activating immune receptor NKG2D and the presence in plasma of NKG2D-ligands can be associated with prognosis.