T cell receptor richness in peripheral blood increases after cetuximab therapy and correlates with therapeutic response.

The role of T cell receptor (TCR) signaling for adaptive immune responses is essential. The ability to respond to a broad spectrum of tumor antigens requires an adaptive selection of various TCR. So far, little is known about the role of TCR richness and clonality in the cellular immune response to head and neck cancer (HNC), though the Endothelial Growth Factor Receptor (EGFR)-specific CD8+ T cell response can be enhanced by cetuximab therapy. Therefore, we investigated differences in TCR sequences between human papillomavirus (HPV)+ and HPV- HNC patients, as well as differences in TCR sequence characteristics between T cells of peripheral blood mononuclear cells (PBMC) and tumor infiltrating lymphocytes (TIL). Additionally, we were able to investigate the TCR richness and clonality in samples pre- and post- treatment in a prospective clinical trial of neoadjuvant cetuximab. Interestingly, HPV+ and HPV- HNSCC did not significantly differ in the extent of TCR clonality and richness in PBMC or TIL. However, neoadjuvant cetuximab treatment increased the number of unique TCR sequences in PBMC (p = 0.0003), which was more prominent in the clinical responder patients compared to non-responders (p = 0.04). A trend toward TCR gene focusing was observed in TIL (p = 0.1) post-treatment. Thus, an increase in richness of TCR sequences in the periphery with a focusing at the tumor site is associated with an improved treatment response, suggesting an influence of peripheral quantity and intratumoral quality on adaptive immunity in cetuximab treated patients.

Role of dendritic cell maturation factors produced by human invariant NKT cells in immune tolerance.

In this study, we used the culture supernatant of iNKT cells to identify human myeloid DC maturation factors produced by human CD4+ iNKT cells. S100A8 had a strong maturation effect. Notably, the recombinant S100A8 protein displayed properties of DC maturation functioning, and the induction of DC differentiation by both the purified and the recombinant protein were blocked by anti-S100A8 and anti-TLR-4 mAbs. DC differentiation induced by anti-major histocompatibility complex class II/CD1d Ab, S100A8, or both was qualitatively indistinguishable from that induced by the coculture of DCs and iNKT cells or via culture supplementation with supernatants from activated CD4+ iNKT cells. S100A8 also induced CD4+/CD25+/Foxp3+ Treg cells from naïve T cells. S100A8 may contribute to DC differentiation by elevating transcription factors or activating transcription factor-2, heat shock factor-1, or both, in mature DCs. S100A8 is a novel candidate iNKT cell-dependent DC maturation factor.

Increased PD-1+ and TIM-3+ TILs during Cetuximab Therapy Inversely Correlate with Response in Head and Neck Cancer Patients.

Despite emerging appreciation for the important role of immune checkpoint receptors in regulating the effector functions of T cells, it is unknown whether their expression is involved in determining the clinical outcome in response to cetuximab therapy. We examined the expression patterns of immune checkpoint receptors (including PD-1, CTLA-4, and TIM-3) and cytolytic molecules (including granzyme B and perforin) of CD8+ tumor-infiltrating lymphocytes (TIL) and compared them with those of peripheral blood T lymphocytes (PBL) in patients with head and neck cancer (HNSCC) during cetuximab therapy. The frequency of PD-1 and TIM-3 expression was significantly increased in CD8+ TILs compared with CD8+ PBLs (P = 0.008 and P = 0.02, respectively). This increased CD8+ TIL population coexpressed granzyme B/perforin and PD-1/TIM-3, which suggests a regulatory role for these immune checkpoint receptors in cetuximab-promoting cytolytic activities of CD8+ TILs. Indeed, the increased frequency of PD-1+ and TIM-3+ CD8+ TILs was inversely correlated with clinical outcome of cetuximab therapy. These findings support the use of PD-1 and TIM-3 as biomarkers to reflect immune status of CD8+ T cells in the tumor microenvironment during cetuximab therapy. Blockade of these immune checkpoint receptors might enhance cetuximab-based cancer immunotherapy to reverse CD8+ TIL dysfunction, thus potentially improving clinical outcomes of HNSCC patients. Cancer Immunol Res; 5(5); 408-16. ©2017 AACR.

PD-1 Status in CD8+ T Cells Associates with Survival and Anti-PD-1 Therapeutic Outcomes in Head and Neck Cancer.

Improved understanding of expression of immune checkpoint receptors (ICR) on tumor-infiltrating lymphocytes (TIL) may facilitate more effective immunotherapy in head and neck cancer (HNC) patients. A higher frequency of PD-1+ TIL has been reported in human papillomavirus (HPV)+ HNC patients, despite the role of PD-1 in T-cell exhaustion. This discordance led us to hypothesize that the extent of PD-1 expression more accurately defines T-cell function and prognostic impact, because PD-1high T cells may be more exhausted than PD-1low T cells and may influence clinical outcome and response to anti-PD-1 immunotherapy. In this study, PD-1 expression was indeed upregulated on HNC patient TIL, and the frequency of these PD-1+ TIL was higher in HPV+ patients (P = 0.006), who nonetheless experienced significantly better clinical outcome. However, PD-1high CD8+ TILs were more frequent in HPV- patients and represented a more dysfunctional subset with compromised IFN-γ secretion. Moreover, HNC patients with higher frequencies of PD-1high CD8+ TIL showed significantly worse disease-free survival and higher hazard ratio for recurrence (P < 0.001), while higher fractions of PD-1low T cells associated with HPV positivity and better outcome. In a murine HPV+ HNC model, anti-PD-1 mAb therapy differentially modulated PD-1high/low populations, and tumor rejection associated with loss of dysfunctional PD-1high CD8+ T cells and a significant increase in PD-1low TIL. Thus, the extent of PD-1 expression on CD8+ TIL provides a potential biomarker for anti-PD-1-based immunotherapy. Cancer Res; 77(22); 6353-64. ©2017 AACR.

Tumor-infiltrating Tim-3+ T cells proliferate avidly except when PD-1 is co-expressed: Evidence for intracellular cross talk.

Programmed Death 1 (PD-1) and T cell Ig and mucin domain-3 protein (Tim-3) are immune checkpoint receptors highly expressed on tumor infiltrating T lymphocytes (TIL). PD-1 inhibits T cell activation and type-1 T cell responses, while Tim-3 is proposed to mark more extensively exhausted cells, although the mechanisms underlying Tim-3 function are not clear. Trials of anti-PD-1 therapy have identified a large subset of non-responder patients, likely due to expression of alternative checkpoint molecules like Tim-3. We investigated the phenotypic and functional characteristics of T cells with differential expression of PD-1 (high/low) and Tim-3 (positive/negative), using TIL directly isolated from head and neck squamous cell carcinomas (HNSCC). Unexpectedly, we found that expression of Tim-3 alone does not necessarily mark TIL as dysfunctional/exhausted. In Tim-3-TIL, PD-1 levels correlate with T cell dysfunction, with a PD-1low/intermed phenotype identifying recently activated and still functional cells, whereas PD-1hiTim-3- T cells are actually exhausted. Nonetheless, PD-1intermed cells are still potently suppressed by PD-L1. PD-1 expression was associated with reduced phosphorylation of ribosomal protein S6 (pS6), whereas Tim-3 expression was associated with increased pS6. Using a novel mouse model for inducible Tim-3 expression, we confirmed that expression of Tim-3 does not necessarily render T cells refractory to further activation. These results suggest the existence of PD-1 and Tim-3 crosstalk in regulating antitumor T cell responses, with important implications for anti-PD-1 immunotherapy.

CTLA-4⁺ Regulatory T Cells Increased in Cetuximab-Treated Head and Neck Cancer Patients Suppress NK Cell Cytotoxicity and Correlate with Poor Prognosis.

The EGFR-targeted antibody cetuximab is effective against head and neck cancer (HNSCC), but in only 15% to 20% of patients, and the variability and extent of cetuximab-mediated cellular immunity is not fully understood. We hypothesized that regulatory T cells (Treg) may exert a functional and clinical impact on antitumor immunity in cetuximab-treated individuals. The frequency, immunosuppressive phenotype, and activation status of Treg and natural killer (NK) cells were analyzed in the circulation and tumor microenvironment of cetuximab-treated patients with HNSCC enrolled in a novel neoadjuvant, single-agent cetuximab clinical trial. Notably, cetuximab treatment increased the frequency of CD4(+)FOXP3(+) intratumoral Treg expressing CTLA-4, CD39, and TGFß. These Treg suppressed cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and their presence correlated with poor clinical outcome in two prospective clinical trial cohorts. Cetuximab expanded CTLA-4(+)FOXP3(+) Treg in vitro, in part, by inducing dendritic cell maturation, in combination with TGFß and T-cell receptor triggering. Importantly, cetuximab-activated NK cells selectively eliminated intratumoral Treg but preserved effector T cells. In ex vivo assays, ipilimumab targeted CTLA-4(+) Treg and restored cytolytic functions of NK cells mediating ADCC. Taken together, our results argue that differences in Treg-mediated suppression contribute to the clinical response to cetuximab treatment, suggesting its improvement by adding ipilimumab or other strategies of Treg ablation to promote antitumor immunity.

PD-1/SHP-2 inhibits Tc1/Th1 phenotypic responses and the activation of T cells in the tumor microenvironment.

Immune rejection of tumors is mediated by IFNγ production and T-cell cytolytic activity. These processes are impeded by PD-1, a coinhibitory molecule expressed on T cells that is elevated in tumor-infiltrating lymphocytes (TIL). PD-1 elevation may reflect T-cell exhaustion marked by decreased proliferation, production of type I cytokines, and poor cytolytic activity. Although anti-PD-1 antibodies enhance IFNγ secretion after stimulation of the T-cell receptor (TCR), the mechanistic link between PD-1 and its effects on T-cell help (Tc1/Th1 skewing) remains unclear. In prospectively collected cancer tissues, we found that TIL exhibited dampened Tc1/Th1 skewing and activation compared with peripheral blood lymphocytes (PBL). When PD-1 bound its ligand PD-L1, we observed a marked suppression of critical TCR target genes and Th1 cytokines. Conversely, PD-1 blockade reversed these suppressive effects of PD-1:PD-L1 ligation. We also found that the TCR-regulated phosphatase SHP-2 was expressed higher in TIL than in PBL, tightly correlating with PD-1 expression and negative regulation of TCR target genes. Overall, these results defined a PD-1/SHP-2/STAT1/T-bet signaling axis mediating the suppressive effects of PD-1 on Th1 immunity at tumor sites. Our findings argue that PD-1 or SHP-2 blockade will be sufficient to restore robust Th1 immunity and T-cell activation and thereby reverse immunosuppression in the tumor microenvironment.

The role of NK cells and NK cell receptors in autoimmune disease.

Natural killer (NK) cells are the first line of defense against infection and transformation. Additionally, NK cells can play seemingly opposite roles in autoimmune disease. Here, we summarize the functions of NK cells as both regulators and inducers of autoimmune disease. The role NK cells play depends on which cells become targets for NK cell attack. The activity of NK cells is controlled by inhibitory receptors specific for MHC Class I molecules, and by activating receptors with diverse specificities. The ligands for both activating and inhibitory receptors are present on potential target cells. It is the balance in expression of these different ligands that determines NK cell activation and therefore whether the cell becomes a target for NK cell-mediated killing. We further discuss the roles of NK cell receptors and their ligands in autoimmune disease.

Tumor antigen-specific monoclonal antibodies and induction of T-cell immunity.

For decades the primary available cancer therapies were relatively nonspecific cytotoxic agents which, while effective in some patients, were limited by narrow therapeutic indices, extensive toxicity and development of resistance, likely due to tumor heterogeneity. Although these chemotherapies remain common tools of conventional treatment, the approval of a growing number of tumor antigen (TA)-specific monoclonal antibodies (mAbs) by the US Food and Drug Administration has driven a shift in the paradigm of cancer therapy. For a subset of patients with lymphoma, colorectal, head and neck, and breast cancers, the inclusion of rituximab (anti-CD20), cetuximab (anti-human epidermal growth factor 1), and trastuzumab (anti-human epidermal growth factor 2) has resulted in overall improved clinical response rates and survival advantages. The mechanisms that contribute to these effects are limited not only to inhibition of signaling pathways but also include cell-mediated cytotoxicity by innate immune cells and priming of effector cells of adoptive immunity triggered by the TA-specific mAb. However, as the use of these therapeutic mAbs has become more widespread, it has been observed that there is significant variability of response in patients treated with these agents. Thus, the factors that mediate this variability in clinical responses must be elucidated to optimize the use of TA-specific mAbs.

Cetuximab-activated natural killer and dendritic cells collaborate to trigger tumor antigen-specific T-cell immunity in head and neck cancer patients.

PURPOSE: Tumor antigen-specific monoclonal antibodies (mAb) block oncogenic signaling and induce Fcγ receptor (FcγR)-mediated cytotoxicity. However, the role of CD8(+) CTL and FcγR in initiating innate and adaptive immune responses in mAb-treated human patients with cancer is still emerging. EXPERIMENTAL DESIGN: FcγRIIIa codon 158 polymorphism was correlated with survival in 107 cetuximab-treated patients with head and neck cancer (HNC). Flow cytometry was carried out to quantify EGF receptor (EGFR)-specific T cells in cetuximab-treated patients with HNC. The effect of cetuximab on natural killer (NK) cell, dendritic cell (DC), and T-cell activation was measured using IFN-γ release assays and flow cytometry. RESULTS: FcγRIIIa polymorphism did not predict clinical outcome in cetuximab-treated patients with HNC; however, elevated circulating EGFR(853-861)-specific CD8(+) T cells were found in cetuximab-treated patients with HNC (P < 0.005). Cetuximab promoted EGFR-specific cellular immunity through the interaction of EGFR(+) tumor cells and FcγRIIIa on NK cells but not on the polymorphism per se. Cetuximab-activated NK cells induced IFN-γ-dependent expression of DC maturation markers, antigen processing machinery components such as TAP-1/2 and T-helper cell (T(H)1) chemokines through NKG2D/MICA binding. Cetuximab initiated adaptive immune responses via NK cell-induced DC maturation, which enhanced cross-presentation to CTL specific for EGFR as well as another tumor antigen, MAGE-3. CONCLUSION: Cetuximab-activated NK cells promote DC maturation and CD8(+) T-cell priming, leading to tumor antigen spreading and TH1 cytokine release through "NK-DC cross-talk." FcγRIIIa polymorphism did not predict clinical response to cetuximab but was necessary for NK-DC interaction and mAb-induced cross-presentation. EGFR-specific T cells in cetuximab-treated patients with HNC may contribute to clinical response.

Porcine Fc gammaRIII isoforms are generated by alternative splicing.

The Fc gammaRIII plays an essential role in antibody-mediating effector functions of immune cells. Here, we report that transcripts encoding porcine Fc gammaRIII isoforms are generated by alternative splicing. Fc gammaRIII a.1 is expressed on the cell surface while Fc gammaRIII a.2 is secreted from the transfected cells due to a partial deletion of the transmembrane domain. Interestingly, a putative soluble Fc gammaRIII (sCD16) is detected in circulating plasma. Both Fc gammaRIII a.2 and sCD16 exhibit a similar molecular mass (approximately 35 kDa) and contain the extracellular D2 domains that are structurally intact. Despite the D2 domain deletion, Fc gammaRIII a.3 associates with Fc gammaRIII a.1. Hence, these results suggest one possibility that three Fc gammaRIII isoforms differentially modulate Fc gammaRIII-mediated immune responses in the porcine system. Furthermore, we demonstrate that a cytolytic triggering G7 monoclonal antibody recognizes the D2 domain that is responsible for interacting with the immune complexes and subsequent activations of porcine innate immune cells. This result suggests that the D2 domain is a target region to develop therapeutic antibodies that regulate the FcR-mediated immune responses.

Activation of plasmacytoid dendritic cells with TLR9 agonists initiates invariant NKT cell-mediated cross-talk with myeloid dendritic cells.

CD1d-restricted invariant NK T (iNKT) cells and dendritic cells (DCs) have been shown to play crucial roles in various types of immune responses, including TLR9-dependent antiviral responses initiated by plasmacytoid DCs (pDCs). However, the mechanism by which this occurs is enigmatic because TLRs are absent in iNKT cells and human pDCs do not express CD1d. To explore this process, pDCs were activated with CpG oligodeoxyribonucleotides, which stimulated the secretion of several cytokines such as type I and TNF-alpha. These cytokines and other soluble factors potently induced the expression of activation markers on iNKT cells, selectively enhanced double-negative iNKT cell survival, but did not induce their expansion or production of cytokines. Notably, pDC-derived factors licensed iNKT cells to respond to myeloid DCs: an important downstream cellular target of iNKT cell effector function and a critical contributor to the initiation of adaptive immune responses. This interaction supports the notion that iNKT cells can mediate cross-talk between DC subsets known to express mutually exclusive TLR and cytokine profiles.

Molecular cloning, expression pattern and chromosomal mapping of pig CD69.

CD69 is a type II membrane protein belonging to C-type lectin family receptor, and expressed on activated leukocytes. Pig CD69 was cloned by RT-PCR using degenerate primers. Pig CD69 cDNA contains a 600 bp open reading frame with its predicted polypeptide sequence of 200 amino acids. Pig CD69 has 75%, 67%, and 57% sequence identity with cow, human, and mouse CD69, respectively. A splicing isoform, which lacks exon 2 encoding the transmembrane domain, was detected. Pig CD69 gene is located on Chromosome (Chr) 5q25 where the NKG2D gene was mapped. In RT-PCR analysis, pig CD69 mRNA was detected in activated PBL, NK cells, macrophages, monocytes, and granulocytes, but not in resting cells. The inducers for CD69 gene expression were PMA, PHA, LPS, G7 mAb, PNK-E mAb, PM16-6 mAb and the K562 cell line. Moreover, CD69 mRNA is expressed in bone marrow, spleen, thymus and lymph nodes but not in muscle, mammary gland, or the pig kidney cell line (LLC-PK(1)). These results indicate that pig Chr 5q25 contains the NK gene complex and CD69 can be used as an activation marker in pig cells of innate as well as acquired immune systems.