Antigen-presenting Capacity Research Articles

Antigen-presenting epithelial cells can play a pivotal role in airway allergy

Professional antigen-presenting cells (APCs; ie, dendritic cells, macrophages, and B cells) react against exogenous antigens and initiate an adaptive immune response by presenting antigen peptides in the groove of the MHC class II molecules. During inflammation, ectopic expression of MHC class II has been reported on cells from multiple tissues, including the nasal mucosa, suggesting an antigen-presenting capacity of epithelial cells (ECs).1-4 The present investigation was designed to examine the contribution of nasal epithelial cells (NECs) to the allergic inflammatory process.

Function and expression of CD1d and invariant natural killer T-cell receptor in the cotton rat (Sigmodon hispidus).

The cotton rat (Sigmodon hispidus) belongs to the rodent family of Cricetidae and provides a powerful model to study the pathogenesis of human respiratory viruses and measles virus. Recent studies in other rodent models have suggested a role for invariant natural killer T (iNKT) cells in antiviral immunity and vaccination against respiratory virus infections. Using new experimental tools, we provide the first evidence for a functional CD1d cell molecule (crCD1d) and iNKT T-cell receptor in cotton rats. The crCD1d cDNA sequence was identified and crCD1d transductants showed that monoclonal antibody WTH-2 stains crCD1d as efficiently as mouse or rat CD1d. The expression of crCD1d was clearly weaker for thymocytes and B cells, and higher for T cells, which is different to what is found in murine species. The antigen-presenting capacity of crCD1d was demonstrated with crCD1d-immunoglobulin dimers loaded with the glycolipid PBS57, which bound iNKT T-cell receptors. Evidence for functional cotton rat iNKT cells was provided by detection of interferon-γ and interleukin-4 in cultures of splenocytes stimulated with PBS57 and α-galactosylceramide and by specific staining of about 0·2% of splenocytes with PBS57-loaded crCD1d dimers. Canonical AV14/AJ18 rearrangements were identified and found to contain multiple members of the AV14 (AV11) family. One of them was expressed and found to bind CD1d dimers. In summary, these data provide the first evidence for functional CD1d molecules and iNKT T-cell receptors in cotton rats and provide the tools to analyse them both in the cotton rat model of infectious diseases.

Phenotypic characterization and prognostic impact of circulating γδ and αβ T-cells in metastatic malignant melanoma.

Human T cells carrying γδ T-cell receptors (TCRs) represent a minor population relative to those with αβ TCRs. There has been much interest recently in the possibility of using these γδ T-cells in cancer therapy because they can kill tumor cells in vitro in an MHC-unrestricted manner, and possess potential regulatory capability and antigen-presenting capacity. The presence of γδ T-cells in late-stage melanoma patients and their relationship with survival has not been extensively explored, although relatively lower percentages of total γδ T-cells and Vδ2+ cells have been reported. Here, we present a detailed analysis of associations of γδ T-cell subsets and differentiation stages with survival in Stage IV patients, compared with CD4+ and CD8+ αβ T-cells. We found an increased Vδ1:Vδ2-ratio and a decreased CD4:CD8-ratio in patients compared to healthy controls, on the basis both of relative frequencies and absolute cell counts per μL blood. Nonetheless, Kaplan-Meier analyses showed that a higher than median frequency of Vδ1+ cells was negatively associated with survival, whereas there were no positive or negative associations with frequencies of Vδ2+ cells. Correlations of cell differentiation status with survival revealed a negative association of early-differentiated Vδ1+ T cells with survival, both on the basis of relative frequencies and absolute counts. There was also a positive correlation between the frequencies of early-differentiated CD8+ αβ T-cells and survival. Our findings suggest peripheral blood frequencies of Vδ1+ T-cells as a potential prognostic marker in melanoma. The mechanisms by which higher abundance of Vδ1+ cells are associated with poorer survival require determination.

In situ Delivery of Antigen to DC-SIGN+CD14+ Dermal Dendritic Cells Results in Enhanced CD8+ T-Cell Responses

CD14(+) dendritic cells (DCs) present in the dermis of human skin represent a large subset of dermal DCs (dDCs) that are considered macrophage-like cells with poor antigen (cross)-presenting capacity and limited migratory potential to the lymph nodes. CD14(+) dDC highly express DC-specific ICAM-3-grabbing non-integrin (DC-SIGN), a receptor containing potent endocytic capacity, facilitating intracellular routing of antigens to major histocompatibility complex I and II (MHC-I andII) loading compartments for the presentation to antigen-specific CD8(+) and CD4(+) T cells. Here we show using a human skin explant model that the in situ targeting of antigens to DC-SIGN using glycan-modified liposomes enhances the antigen-presenting capacity of CD14(+) dDCs. Intradermal vaccination of liposomes modified with the DC-SIGN-targeting glycan Lewis(X), containing melanoma antigens (MART-1 or Gp100), accumulated in CD14(+) dDCs and resulted in enhanced Gp100- or MART-1-specific CD8(+) T-cell responses. Simultaneous intradermal injection of the cytokines GM-CSF and IL-4 as adjuvant enhanced the migration of the skin DCs and increased the expression of DC-SIGN on the CD14(+) and CD1a(+) dDCs. These data demonstrate that human CD14(+) dDCs exhibit potent cross-presenting capacity when targeted in situ through DC-SIGN.

Immunoscoring by correlating MHC class II and TCR expression: high level immune functions represented by the KIRP dataset of TCGA.

The Cancer Genome Atlas (TCGA) is primarily oriented towards revealing the status of cancer cells but TCGA RNASeq data have the potential of representing gene expression for a variety of cells that are included during RNA preparation, i.e., cells that cannot be removed from the microenvironment during cancer sample isolation. Thus, we seek to determine whether RNASeq data can be used to pioneer greater precision for immunoscoring. We obtained the RNASeq results for HLA class II genes, the class II transactivator (CIITA) and T-cell receptor (TCR) alpha segments. The data indicated strong degrees of correlation of HLA class II expression with TCR expression. Furthermore, biomarkers of professional antigen-presenting cells also correlated with TCR expression, with the kidney renal papillary cell carcinoma (KIRP) dataset indicative of the highest level, immune function microenvironment. These analyses indicate that an immune function signature, with probable internal HLA class II-TCR verifications, can be obtained from individual TCGA samples; this in turn indicates that such signatures might provide a basis for correlations with prognosis or for convenient indications of therapy options, such as tumor-infiltrating lymphocyte availability for ex-vivo amplification. Although tumor immunoscoring has been proposed, the above analysis represents the first immunoscoring approach that correlates antigen presentation capacity with TCR mRNA expression.

Abstract POSTER-THER-1418: PD-1 mediated paralysis of ovarian cancer infiltrating dendritic cells

Abstract Purpose: It is well known that the immune system impacts the clinical course of ovarian cancer suggesting that immune-based approaches may effective in treating the disease. This has led to several clinical trials of novel treatments such as antibody, vaccine, and adoptive T cell therapy. PD-1:PD-L1 axis is a major immune regulatory pathway that blunts immune effectors in the tumor microenvironment. It is well known that high surface expression of PD-1 on tumor infiltrating T cells is a sign of their exhaustion. However, the complete mechanism behind the PD-1 regulation of T cell exhaustion is yet to be explored. Recently we identified that PD-1 is also expressed on ovarian tumor-infiltrating myeloid dendritic cells (DCs) that exhibit an immunosuppressive phenotype. Our goal in this study was to understand the mechanism by which PD-1 mediates the paralysis of ovarian cancer infiltrating DCs. Experimental procedures: PD-1+ DCs obtained from ID8 mouse model of ovarian cancer were used in this study. Data from this preclinical model was confirmed by using myeloid DCs obtained from ovarian cancer patient’s samples. Using standard flow cytometry, immunoassays (ELISA, multiplexed cytokine assay), PCR array, western blot and confocal microscopy, PD-1 mediated paralysis of ovarian cancer DCs was determined. Results: Our data shows that blockade of surface PD-1 on ovarian cancer infiltrating DCs results in their activation by, enhancing their surface co-stimulatory molecules expression, increasing their motility, enhancing their antigen presentation capacity, and increasing their production of immunostimulatory cytokines. Also, our results suggest that PD-1 expressed on tumor DCs mediates the suppression of NFkB which occurs through SHP-2 and IKK dependent mechanisms. Conclusion: Our study reveals that blockade of PD-1 on ovarian cancer infiltrating DCs results in the reversal of their paralysis to such an extent that it overrides their B7-H1 mediated suppression of T cells and this occurs through the reversal of PD-1 mediated tonic suppression of NFkB. Citation Format: Lavakumar Karyampudi James Krempski, Purushottam Lamichhane, Kimberly R. Kalli, Marshall D. Behrens, Doris M. Vargas, Lynn C Hartmann, Karen E Hedin, Ellen L. Goode, Keith L. Knutson. PD-1 mediated paralysis of ovarian cancer infiltrating dendritic cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1418.

Phenotypic and functional analysis of CD1a+ dendritic cells from cats chronically infected with feline immunodeficiency virus.

Numerous studies suggest dendritic cell (DC) dysfunction is central to the dysregulated immune response during HIV infection; however, in vivo studies are lacking. In the present study we used feline immunodeficiency virus (FIV) infection of cats as a model for HIV-1 infection to assess the maturation and function of dendritic cells, in vivo and in vitro. We compared CD1a+ DC migration, surface phenotype, endocytosis, mixed leukocyte reaction (MLR) and regulatory T cell (Treg) phenotype induction by CD1a+ cells isolated from lymph nodes of FIV-infected and control cats. Results showed that resident CD1a+ DC in lymph nodes of chronically FIV-infected cats are phenotypically mature, can stimulate normal primary T cell proliferation, override Treg suppression and do not skew toward Treg induction. In contrast, FIV infection had deleterious effects on antigen presentation and migratory capacity of CD1a+ cells in tissues.

Abstract 2490: CD40 ligand expressing recombinant vaccinia virus (rVV40L) modulation of central memory CD8-mediated immune response

Abstract In cancer immunotherapy, induction of tumor-reactive CD8+ T cells displaying phenotypic and functional profile of central memory T cells (TCM) is associated with favorable prognosis. A key element in the generation of TCM CD8+ cells is represented by the help provided by CD4+ T cells during the priming of naïve CD8+ T cells. In particular CD40 ligand (CD40L) expressed and/or secreted by activated CD4+ T cells triggers CD40 receptor expressed on antigen presenting cells (APCs) thereby enhancing their antigen presentation capacity. In order to bypass the requirement of CD4+ T cells we generated a non-replicating recombinant vaccinia virus encoding human CD40L (rVV40L) and compared its ability to shape CD8-mediated immune response to soluble CD40L recombinant protein (sCD40L). In this regard, our data clearly underline the different biological properties of membrane-bound CD40L, as provided by rVV40L infection, as compared to its soluble form, in CD14+ APCs activation. Notably, considering expression of IL-12p40, IFN-a and -b genes, cytokines of proven relevance for memory T cell induction, rVV40L-infection was much more potent than s40L-treatment alone or combine with WT infection. In parallel, s40L-stimulation induced a much more significant expression of IL-10 and indoleamine-2, 3-dioxygenase (IDO) genes encoding immunosuppressive factors. Considering a panel of molecules involved in the generation of the immunological synapse with T cells on CD14+ cells, rVV40L appeared to promote a less intense up regulation of CD80 co-stimulatory molecules but, most importantly, only a minor increase of programmed death ligand-1 (PD-L1). Therefore, gene expression and phenotypic profiles suggested that rVV40L-infected CD14+ cells might be highly effective APC in the induction of TCM CD8+ cells. Indeed, a single in vitro stimulation of naïve CD8+ T cells by rVV40L-infected CD14+ cells, in the absence of CD4+ T cells, was able to promote the rapid generation of central memory TAA (MAGE and MART-1) specific CD8+ T cells. These TCM were characterized by the typical central memory phenotype, as indicated by co-expression of CD45RO, CD62L and IL-7Ra, and by the high proliferative potential upon antigen recognition. Collectively our data indicate that rVV40L efficiently modulates the quality of different APC signals delivered during the formation of the immunological synapse with CD8+ T cells. These in-vitro observations validate the strong clinical potential of our recombinant vaccinia virus constructs co-expressing CD40L for cancer immunotherapies. Citation Format: Emanuele Trella, Evangelos Panopoulos, Nermin Raafat, Chantal Mengus, Emmanuel Traunecker, Swantje Heidtmann, Michael Heberer, Daniel Oertli, Giulio Cesare Spagnoli, Paul Zajac. CD40 ligand expressing recombinant vaccinia virus (rVV40L) modulation of central memory CD8-mediated immune response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2490. doi:10.1158/1538-7445.AM2015-2490

Characterization of species-specific genes regulated by E2-2 in human plasmacytoid dendritic cells.

Dendritic cells (DCs) are sentinels of the immune system and comprise two distinct subsets: conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Human pDCs are distinguished from mouse pDCs phenotypically and functionally. Basic helix-loop-helix protein E2-2 is defined as an essential transcription factor for mouse pDC development, cell fate maintenance and gene programe. It is unknown whether E2-2 regulation contributes to this species-specific difference. Here we investigated the function of E2-2 in human pDCs and screened human-specific genes regulated by E2-2. Reduced E2-2 expression in human pDC cell line GEN2.2 resulted in diminished IFN-α production in response to CpG but elevated antigen presentation capacity. Gene expression profiling showed that E2-2 silence down-regulated pDC signature genes but up-regulated cDC signature genes. Thirty human-specific genes regulated by E2-2 knockdown were identified. Among these genes, we confirmed that expression of Siglec-6 was inhibited by E2-2. Further more, Siglec-6 was expressed at a higher level on a human pDC subset with drastically lower expression of E2-2. Collectively, these results highlight that E2-2 modulates pDC function in a species-specific manner, which may provide insights for pDC development and functions.

TLR9-induced miR-155 and Ets-1 decrease expression of CD1d on B cells in SLE.

B cells present lipid antigens to CD1d-restricted invariant natural killer T (iNKT) cells to maintain autoimmune tolerance, and this process is disrupted in systemic lupus erythematosus (SLE). Inflammation may inhibit CD1d expression to exacerbate the pathology of lupus. However, how inflammation regulates CD1d expression on B cells is unclear in SLE. In the present study, we showed that the surface expression of CD1d on B cells from SLE mice was decreased and that stimulation of inflammatory responses through TLR9 decreased the membrane and total CD1d levels of CD1d on B cells. Moreover, inflammation-related microRNA-155 (miR-155) negatively correlated with the expression of CD1d in B cells. miR-155 directly targeted the 3'-untranslated region (3'-UTR) of CD1d upon TLR9 activation in both humans and mice. The inhibitory effects of miR-155 on CD1d expression in B cells impaired their antigen-presenting capacity to iNKT cells. In addition, Ets-1, a susceptibility gene of SLE, also directly regulated the expression of the CD1d gene at the transcriptional level. These findings provide new insight into the mechanism underlying decreased CD1d expression on B cells in SLE, suggesting that inhibition of inflammation may increase CD1d expression in B cells to ameliorate SLE via modulating iNKT cells.

Reinforcing the 'gauntlet' of thymic negative selection via exosomal transfer of self-antigens.

Reinforcing the ‘gauntlet’ of thymic negative selection via exosomal transfer of self-antigens

Function and regulation of self-reactive marginal zone B cells in autoimmune arthritis.

Polyreactive innate-type B cells account for many B cells expressing self-reactivity in the periphery. Improper regulation of these B cells may be an important factor that underlies autoimmune disease. Here we have explored the influence of self-reactive innate B cells in the development of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis. We show that splenic marginal zone (MZ), but not B-1 B cells exhibit spontaneous IgM reactivity to autologous collagen II in naı¨ve mice. Upon immunization with heterologous collagen II in complete Freund's adjuvant the collagen-reactive MZ B cells expanded rapidly, while the B-1 B cells showed a modest anti-collagen response. The MZ B cells were easily activated by toll-like receptor (TLR) 4 and 9-ligands in vitro, inducing proliferation and cytokine secretion, implying that dual engagement of the B-cell receptor and TLRs may promote the immune response to self-antigen. Furthermore, collagen-primed MZ B cells showed significant antigen-presenting capacity as reflected by cognate T-cell proliferation in vitro and induction of IgG anti-collagen antibodies in vivo. MZ B cells that were deficient in complement receptors 1 and 2 demonstrated increased proliferation and cytokine production, while Fcγ receptor IIb deficiency of the cells lead to increased cytokine production and antigen presentation. In conclusion, our data highlight self-reactive MZ B cells as initiators of the autoimmune response in CIA, where complement and Fc receptors are relevant in controlling the self-reactivity in the cells.

Myeloid molecular characteristics of human γδ T cells support their acquisition of tumor antigen-presenting capacity.

Human T cells expressing γδ T cell receptor have a potential to show antigen-presenting cell-like phenotype and function upon their activation. However, the mechanisms that underlie the alterations in human γδ T cells remain largely unclear. In this study, we have investigated the molecular characteristics of human γδ T cells related to their acquisition of antigen-presenting capacity in comparison with activated αβ T cells. We found that activated γδ but not αβ T cells upregulated cell surface expression of a scavenger receptor, CD36, which seemed to be mediated by signaling through mitogen-activated protein kinase and/or NF-κB pathways. Confocal microscopical analysis revealed that activated γδ T cells can phagocytose protein antigens. Activated γδ T cells could induce tumor antigen-specific CD8(+) T cells using both apoptotic and live tumor cells as antigen resources. Furthermore, we detected that C/EBPα, a critical transcription factor for the development of myeloid-lineage cells, is expressed much higher in γδ T cells than in αβ T cells. These results unveiled the molecular mechanisms for the elicitation of antigen-presenting functions in γδ T cells and would also help designing new approaches for γδ T cell-mediated human cancer immunotherapy.

HGF/Met-Signaling Contributes to Immune Regulation by Modulating Tolerogenic and Motogenic Properties of Dendritic Cells.

Hepatocyte growth factor (HGF)-signaling via Met can induce mitogenic, morphogenic, and motogenic activity in various cell types. Met expression in the immune system is limited to cells with antigen-presenting capacities, including dendritic cells (DCs). Thus, it appears highly conceivable that Met-signaling impacts on adaptive immune responses. However, the mechanisms by which HGF imparts its effects on immunological responses are not yet fully understood. DCs possess unique functionalities that are critically involved in controlling both tolerance and immunity. HGF conveys immunoregulatory functions, which strongly correlate with that of DCs orchestrating the apt immune response in inflammation. Therefore, this review focuses on the current knowledge of Met-signaling in DCs with specific emphasis on the morphogenic and motogenic activities. HGF has been identified to play a role in peripheral immune tolerance by directing DC differentiation towards a tolerogenic phenotype. In skin immunity, Met-signaling was shown to drive mobilization of DCs by regulating matrix metalloproteinase activities. This is strikingly reminiscent of the role of Met for regulating a cell fate program during embryonic development, wound healing, and in tumor invasion known as epithelial-mesenchymal transition (EMT). Thus, the concept emerges that an EMT program is executed by Met-signaling in DCs, which will be also discussed.

Monocyte-derived dendritic cells from cirrhotic patients retain similar capacity for maturation/activation and antigen presentation as those from healthy subjects

Few studies have investigated the impact of liver cirrhosis on dendritic cell function. The purpose of this study was to compare the activation and antigen-presentation capacity of monocyte-derived dendritic cells (MoDC) from cirrhotic patients (CIR) relative to healthy donors (HD). MoDC from CIR and HD were matured, phenotyped, irradiated and pulsed with 15mer peptides for two hepatocellular carcinoma-related antigens, alphafetoprotein and glypican-3, then co-cultured with autologous T-cells. Expanded T-cells were evaluated by interferon-gamma ELISPOT and intracellular staining. 15 CIR and 7 HD were studied. While CD14+ monocytes from CIR displayed enhanced M2 polarization, under MoDC-polarizing conditions, we identified no significant difference between HD and CIR in maturation-induced upregulation of co-stimulation markers. Furthermore, no significant differences were observed between CIR and HD in subsequent expansion of tumor antigen-specific IFNγ+ T-cells. Conclusion: MoDCs isolated from cirrhotic individuals retain similar capacity for in vitro activation, maturation and antigen-presentation as those from healthy donors.

MicroRNAs affect dendritic cell function and phenotype.

MicroRNA (miRNA) are small, non-coding RNA molecules that have been linked with immunity through regulating/modulating gene expression. A role for these molecules in T-cell and B-cell development and function has been well established. An increasing body of literature now highlights the importance of specific miRNA in dendritic cell (DC) development as well as their maturation process, antigen presentation capacity and cytokine release. Given the unique role of DC within the immune system, linking the innate and adaptive immune responses, understanding how specific miRNA affect DC function is of importance for understanding disease. In this review we summarize recent developments in miRNA and DC research, highlighting the requirement of miRNA in DC lineage commitment from bone marrow progenitors and for the development of subsets such as plasmacytoid DC and conventional DC. In addition, we discuss how infections and tumours modulate miRNA expression and consequently DC function.

Silence STAT3 in the procancer niche… and activate CD8+ T cells to kill premetastatic myeloid intruders.

Several recent studies have implicated myeloid cells in providing a microenvironment that promotes tumor cell survival and metastasis, therefore preparing a "premetastatic niche" for cancer progression. In this issue of the European Journal of Immunology, Zhang et al. [Eur. J. Immunol. 2015. 45: 71-81] address the regulation of immune cells in premetastatic lymph nodes in experimental mouse models. The authors show that signal transducer and activator of transcription 3 (STAT3) ablation in murine myeloid cells, which renders the premetastatic niche less receptive to metastasis by B16 melanoma cells, also leads to local activation in the niche of CD8(+) T cells with increased expression of IFN-γ and granzyme B. Data further suggest that STAT3 activation in the myeloid population leads to poor tumor antigen presenting capacity as well as resistance to CD8(+) T-cell killing. Based on these studies in mice and observations in human cancer patients, the authors propose treatments designed to regulate STAT3 activation, which are correlated with increased cytolytic activity of CD8(+) T cells in mouse models.

The CD21low B Cell Population Is Composed of an Anergic and an Immunostimulatory Germinal Center-like B Cell Subset

In recent years we have gained an increased understanding of the complexity of B cell biology and function. It has become increasingly recognized that apart from antibody production B cells exert many more function. B cells serve as antigen-presenting cells (APC), they contribute to immunoregulation and represent an important source of cytokines and chemokines. A deeper understanding of the role of B cells in the pathophysiology of human diseases has been hampered by the lack of well-defined functional B cell subsets. We therefore aimed to identify novel human B cell subsets which could serve as biomarkers or targets of therapeutic intervention. Using a transcriptomic approach combined with flowcytometric immune assessment of healthy human subjects and patients we were able to identify several functional B cell subpopulations with relevance to human disease. We were able to define a CD21low CD86pos human B cell subset with strong antigen-presenting capacity which gradually develops from conventional resting B cells under the continuous stimulation via CD40. These cells were phenotypically and functionally distinct from CD21low CD86neg B lymphocytes, which represent anergic B cells. Using calcium flux assays and phospho-specific flow cytometry we were able to show that the CD21low B cell subsets displayed distinct signaling states. Both CD21low B cell subpopulations had an impaired response to B cell receptor stimulation. However, CD21low CD86pos B cells had higher basal calcium levels and basal phosphorylation of BCR-associated signaling molecules such as Syk and Erk. Contrary to CD21low CD86neg B cells, which demonstrated poor antigen-presenting capacity, CD21low CD86pos B cells were potent immunostimulatory antigen-presenting cells. CD21low CD86pos B cells were increased in acute inflammation and autoimmune diseases such as rheumatoid arthritis. CD21low CD86neg B cells, on the other hand, were increased in chronic inflammatory conditions such as chronic HIV infection. The balance between the CD21low B cell subsets varied with the functional state of the B cell compartment in inflammatory conditions and could be used to classify the functional state of the B cell compartment. In summary, we have identified several novel human B cell subsets with distinct functions. Given the large number of B cell-directed drugs which are in clinical development or already approved it seems likely that an increased knowledge of the human B cell subsets will not only provide important insights into the pathology of immune-mediated diseases but will also result in novel therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

CLL Is Associated with Development of Subclinical Cytomegalovirus Viraemia and Accumulation of Large Populations of “exhausted” CMV-Specific CD4+ T Cells

Chronic Lymphocytic Leukaemia (CLL) is associated with T cell dysfunction and increased expression of markers of T cell exhaustion. Cytomegalovirus is a common herpesvirus infection and is associated with development of accelerated immune senescence in older adults. Within patients with CLL, CMV leads to marked expansion of virus-specific CD4 and CD8 T cells and development of oligoclonal T cell populations. However these features are not known to be associated with clinical symptoms and CMV viral load remains essentially undetectable by conventional qPCR. In this study we used digital PCR to determine CMV viral load in patients with CLL and correlated this with the magnitude and phenotype of the CMV-specific T cell immune response. In particular, we utilised HLA class II tetramers for the first time, in order to assess the contribution of CMV-specific T cell populations to the profile of T cell exhaustion seen in this disease.68 CMV-seropositive CLL patients and 19 age-matched healthy donors (HD) were recruited for study. All patients were either untreated or had not received chemotherapy for at least 6 months. CMV viral load was determined within purified monocyte populations using a digital droplet PCR method. Viral load per monocyte was increased in CLL patients compared to HD (p=0.04), with the highest viral loads detected in stage C patients.In order to investigate the CMV-specific immune response, nine HLA class I tetramers were generated, containing viral epitopes from pp65, pp50 and IE-1, and two class II tetramers were also available, with epitopes from glycoprotein B and pp65.CMV-specific CD4 T cell responses were increased in CLL patients (n=14) compared to HD (n=11) (4.1 % vs. 0.9 %; p=0.049). Remarkably, in one patient more than half (50.9%) of all CD4 T cells were directed against a single CMV epitope. CD4 CMV-specific T cells were nearly always effector memory in phenotype (78%), somewhat in contrast to CD8 populations where the memory phenotype is split between effector memory (CCR7-, CD45RA-) and terminally differentiated memory cells (CCR7-, CD45RA+).PD1 is an important marker of T cell exhaustion and expression was increased on both CD8 T cells (16.9% Vs 9.6%; p=0.003); and CD4 T cells (16.2 % Vs 8.7 %; p=0.0007) in CLL patients compared to HD. Interestingly, PD1 expression was much higher on CMV-specific CD4 T cells compared to the total CD4 T cell repertoire (50.6% Vs 21%; p=0.01), whereas the opposite profile was observed in relation to CD8 populations.CMV-specific CD4 T cells demonstrated a Th1 cytotoxic phenotype with production of TNF-alpha, IFN-y and Granzyme B. Production of IFN-y and TNF-alpha was reduced in PD1+ populations, consistent with an exhausted phenotype. No CD25+FoxP3 + regulatory T cells were observed within the CMV-specific CD4 T cell population which is of interest given the well documented expansion of this population in patients with CLL.In order to investigate the replicative history of CMV-specific T cells we investigated the telomere length of antigen-specific cells using single cell telomere length analysis. CMV-specific cells had markedly shortened telomere lengths compared to background CD4 and CD8 T cells, with the mean difference of 0.911 kb (maximum 1.836 kb) indicating that they have undergone extensive proliferation in vivo.This work is the first to use digital PCR to measure the subclinical CMV load within peripheral blood and also to examine CMV-specific CD4 T cells using HLA class II tetramers. Our results indicate that immune control of CMV viral load is impaired during the clinical progression of CLL and that a proportion of virus-specific CD4 T cells show signs of exhaustion. These data may reflect ‘cross presentation’ of viral protein by B-CLL tumour cells, which are known to have poor capacity for antigen presentation. The potential clinical importance of these observations is now being addressed. DisclosuresNo relevant conflicts of interest to declare.

TNFRSF14 aberrations in Follicular Lymphoma B Cells Result in Increased Alloresponses in Vitro and in Vivo

IntroductionGenetic aberrations of Tumor Necrosis Factor Receptor Superfamily 14 (TNFRSF14, also known as HVEM) have been shown to occur at high frequencies in patients (pts) with follicular lymphoma (FL). HVEM is a ligand for B and T lymphocyte attenuator (BTLA) which negatively regulates T cell responses and BTLA stimulation reduces acute graft-versus-host disease (aGvHD) in murine allogeneic hematopoietic cell transplantation (AHCT) models. As activated FL B cells are potent alloantigen presenting cells, we hypothesized that TNFRSF14 aberrations in FL B cells would reduce expression of HVEM and potentiate capacity of FL B cells to stimulate allogeneic T cell responses. We therefore sought to determine the functional effect of TNFRSF14 aberrations on FL B cell-stimulated donor T cell alloresponses in vitro. We also examined the impact of TNFRSF14 aberrations on the outcome of FL pts after HLA-matched reduced intensity conditioning (RIC) AHCT.ResultsFL B cells from lymph nodes were FACS-sorted (>90% purity and > 95% light chain restriction), activated and used as stimulators in mixed lymphocyte reactions with purified allogeneic responder CD3+ T cells. HVEM expression on FL B cells from pts with biallelic TNFRSF14 aberrations (Mut/Del cases) was undetectable whereas 40% of FL B cells from TNFRSF14 WT cases expressed HVEM (Fig 1 A). In contrast, FL B cells from Mut/Del and WT cases expressed similar levels of MHC class I/II, CD80, CD86 and CD58 before and after activation. Allostimulation with Mut/Del FL B cells resulted in significantly greater expression of activation markers on responder CD4+ T cells, increased secretion of pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-2) measured by ELISA and increased frequencies of cytokine-secreting CD4+ and CD8+ T cells enumerated by intracellular cytokine staining. Responder T cell proliferation by thymidine incorporation was significantly greater after stimulation with Mut/Del FL B cells compared to WT FL B cells. CFSE labeling studies demonstrated that this effect resulted from increased proliferation of CD4+ and CD8+ responder T cells after both primary (Fig 1B) and secondary allostimulation. To determine if the increased alloresponses we observed using FL B cells from TNFRSF14 Mut/Del cases was due to reduced HVEM-BTLA signaling, we performed allogeneic co-cultures in the presence of antagonist or agonist BTLA antibodies (ab). Antagonist anti-BTLA ab increased proliferation of responder T cells after stimulation with WT FL B cells confirming that BTLA limits alloresponses in our in vitro model. Importantly, agonist BTLA ab reduced alloresponses stimulated by Mut/Del FL B cells.We next sought to determine if the increased alloresponses we detected in vitro in FL pts with TNFRSF14 aberrations resulted in an increase in clinical alloreactivity after AHCT. DNA from lymph nodes from FL pts undergoing T-cell replete RIC AHSCT was screened for TNFRSF14 mutations and deletions by Sanger sequencing and multiplex ligation-probe amplification respectively. Cumulative incidences (CI) of aGvHD and GvHD-related death were calculated with FL progression as a competing risk. TNFRSF14 aberrations were identified in 10/21 pts prior to RIC AHCT (4 Mut/Del, 1 Del/Del, 1 Mut/WT, 4 Del/WT). Most (18/21) pts had evidence of ongoing FL pre-transplant. Disease and donor characteristics were similar in pts with and without aberrations. There was no significant difference in CI of aGvHD in pts with or without TNFRSF14 aberrations. However there was a significantly higher CI of fatal aGvHD in patients with TNFRSF14 aberrations (45%) compared to those without aberrations (0%, p<0.01). Interestingly, relapse was less frequent in patients with TNFRSF14 aberrations consistent with increased graft-versus-tumor effects, although this did not reach statistical significance.ConclusionThis study is the first to describe the impact of TNFRSF14 aberrations on the allostimulatory capacity of FL B cells. TNFRSF14 aberrations were associated with enhanced T-cell alloresponses in vitro and increased death from aGvHD. Importantly, our results suggest FL patients with TNFRSF14 aberrations may benefit from more aggressive immunosuppression to prevent fatal aGvHD after AHCT. The increased antigen-presenting capacity of FL B cells with TNFRSF14 aberrations could also influence autologous anti-tumor responses and impact outcome after other treatment modalities. [Display omitted] DisclosuresGribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.