Cancer Burden Is Controlled by Mural Cell-ß3-Integrin Regulated Crosstalk with Tumor Cells.

Enhanced blood vessel (BV) formation is thought to drive tumor growth through elevated nutrient delivery. However, this observation has overlooked potential roles for mural cells in directly affecting tumor growth independent of BV function. Here we provide clinical data correlating high percentages of mural-ß3-integrin-negative tumor BVs with increased tumor sizes but no effect on BV numbers. Mural-ß3-integrin loss also enhances tumor growth in implanted and autochthonous mouse tumor models with no detectable effects on BV numbers or function. At a molecular level, mural-cell ß3-integrin loss enhances signaling via FAK-p-HGFR-p-Akt-p-p65, driving CXCL1, CCL2, and TIMP-1 production. In particular, mural-cell-derived CCL2 stimulates tumor cell MEK1-ERK1/2-ROCK2-dependent signaling and enhances tumor cell survival and tumor growth. Overall, our data indicate that mural cells can control tumor growth via paracrine signals regulated by ß3-integrin, providing a previously unrecognized mechanism of cancer growth control.

Genetic and Functional Drivers of Diffuse Large B Cell Lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole-exome sequencing and transcriptome sequencing in a cohort of 1,001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.

Retinoic acid-responsive CD8 effector T cells are selectively increased in IL-23-rich tissue in gastrointestinal GVHD.

Gastrointestinal (GI) graft-versus-host disease (GVHD) is a major barrier in allogeneic hematopoietic stem cell transplantation (allo-HSCT). The metabolite retinoic acid (RA) potentiates GI-GVHD in mice via alloreactive T cells expressing the RA receptor-α (RARα), but the role of RA-responsive cells in human GI-GVHD remains undefined. Therefore, we used conventional and novel sequential immunostaining and flow cytometry to scrutinize RA-responsive T cells in tissues and blood of patients who had received allo-HSCT and to characterize the impact of RA on human T-cell alloresponses. Expression of RARα by human mononuclear cells was increased after exposure to RA. RARαhi mononuclear cells were increased in GI-GVHD tissue, contained more cellular RA-binding proteins, localized with tissue damage, and correlated with GVHD severity and mortality. By using a targeted candidate protein approach, we predicted the phenotype of RA-responsive T cells in the context of increased microenvironmental interleukin-23 (IL-23). Sequential immunostaining confirmed the presence of a population of RARαhi CD8 T cells with the predicted phenotype that coexpressed the effector T-cell transcription factor T-bet and the IL-23-specific receptor (IL-23R). These cells were increased in GI- but not skin-GVHD tissues and were also selectively expanded in the blood of patients with GI-GVHD. Finally, functional approaches demonstrated that RA predominantly increased alloreactive GI-tropic RARαhi CD8 effector T cells, including cells with the phenotype identified in vivo. IL-23-rich conditions potentiated this effect by selectively increasing ß7 integrin expression on CD8 effector T cells and reducing CD4 T cells with a regulatory cell phenotype. In summary, we have identified a population of RA-responsive effector T cells with a distinctive phenotype that is selectively expanded in human GI-GVHD and that represents a potential new therapeutic target.

KDM5 inhibition offers a novel therapeutic strategy for the treatment of KMT2D mutant lymphomas.

Loss-of-function mutations in KMT2D are a striking feature of germinal center (GC) lymphomas, resulting in decreased histone 3 lysine 4 (H3K4) methylation and altered gene expression. We hypothesized that inhibition of the KDM5 family, which demethylates H3K4me3/me2, would reestablish H3K4 methylation and restore the expression of genes repressed on loss of KMT2D. KDM5 inhibition increased H3K4me3 levels and caused an antiproliferative response in vitro, which was markedly greater in both endogenous and gene-edited KMT2D mutant diffuse large B-cell lymphoma cell lines, whereas tumor growth was inhibited in KMT2D mutant xenografts in vivo. KDM5 inhibition reactivated both KMT2D-dependent and -independent genes, resulting in diminished B-cell signaling and altered expression of B-cell lymphoma 2 (BCL2) family members, including BCL2 itself. KDM5 inhibition may offer an effective therapeutic strategy for ameliorating KMT2D loss-of-function mutations in GC lymphomas.

Lack of reproducibility of histopathological features in MYC-rearranged large B cell lymphoma using digital whole slide images: a study from the Lunenburg lymphoma biomarker consortium.

AIMS: Subclassification of large B cell lymphoma (LBCL) is challenging due to the overlap in histopathological, immunophenotypical and genetic data. In particular, the criteria to separate diffuse large B cell lymphoma (DLBCL) and high-grade B cell lymphoma (HGBL) are difficult to apply in practice. The Lunenburg Lymphoma Biomarker Consortium previously reported a cohort of over 5000 LBCL that included fluorescence in-situ hybridisation (FISH) data. This cohort contained 209 cases with MYC rearrangement that were available for a validation study by a panel of eight expert haematopathologists of how various histopathological features are used. METHODS AND RESULTS: Digital whole slide images of haematoxylin and eosin-stained sections allowed the pathologists to visually score cases independently as well as participate in virtual joint review conferences. Standardised consensus guidelines were formulated for scoring histopathological features and included overall architecture/growth pattern, presence or absence of a starry-sky pattern, cell size, nuclear pleomorphism, nucleolar prominence and a range of cytological characteristics. Despite the use of consensus guidelines, the results show a high degree of discordance among the eight expert pathologists. Approximately 50% of the cases lacked a majority score, and this discordance spanned all six histopathological features. Moreover, none of the histological variables aided in prediction of MYC single versus double/triple-hit or immunoglobulin-partner FISH-based designations or clinical outcome measures. CONCLUSIONS: Our findings indicate that there are no specific conventional morphological parameters that help to subclassify MYC-rearranged LBCL or select cases for FISH analysis, and that incorporation of FISH data is essential for accurate classification and prognostication.

PDL1 shapes the classical Hodgkin lymphoma microenvironment without inducing T-cell exhaustion.

Classical Hodgkin lymphoma (CHL) is unusually sensitive to PD1 inhibition and PDL1 is highly expressed on CHL cells and in the tumor microenvironment. This could be interpreted as evidence of exhaustion, but paradoxically, PD1+ lymphocyte infiltration does not predict response to PD1 inhibitors and no increase in cytotoxic markers is seen after PD1 therapy as might be expected with reversal of exhaustion. In contrast to PD1, elevated PDL1 does predict response to PD1 inhibitors and recent data associate both retained CHL MHC-II expression and increased T helper (TH) T-cell receptor diversity with response, suggesting a connection to the TH compartment. We performed a phenotypic, spatial and functional assessment of T-cell exhaustion in CHL and found co-expression of an exhaustion marker and lower PD1 expression in CHL than in reactive nodes whereas the proliferative and cytokine production capacity were similar in CHL and the reactive nodes. We found no correlation between PDL1 expression and exhaustion signatures. Instead, we identified a strong association between PDL1 expression and CHL MHC-II expression, TH recruitment, and enrichment of TH1 regulatory cells. These data suggest that a dominant effect of PDL1 expression in CHL may be TH engagement and promotion of a regulatory microenvironment rather than maintenance of exhaustion.

Endothelial-cell FAK targeting sensitizes tumours to DNA-damaging therapy.

Chemoresistance is a serious limitation of cancer treatment. Until recently, almost all the work done to study this limitation has been restricted to tumour cells. Here we identify a novel molecular mechanism by which endothelial cells regulate chemosensitivity. We establish that specific targeting of focal adhesion kinase (FAK; also known as PTK2) in endothelial cells is sufficient to induce tumour-cell sensitization to DNA-damaging therapies and thus inhibit tumour growth in mice. The clinical relevance of this work is supported by our observations that low blood vessel FAK expression is associated with complete remission in human lymphoma. Our study shows that deletion of FAK in endothelial cells has no apparent effect on blood vessel function per se, but induces increased apoptosis and decreased proliferation within perivascular tumour-cell compartments of doxorubicin- and radiotherapy-treated mice. Mechanistically, we demonstrate that endothelial-cell FAK is required for DNA-damage-induced NF-κB activation in vivo and in vitro, and the production of cytokines from endothelial cells. Moreover, loss of endothelial-cell FAK reduces DNA-damage-induced cytokine production, thus enhancing chemosensitization of tumour cells to DNA-damaging therapies in vitro and in vivo. Overall, our data identify endothelial-cell FAK as a regulator of tumour chemosensitivity. Furthermore, we anticipate that this proof-of-principle data will be a starting point for the development of new possible strategies to regulate chemosensitization by targeting endothelial-cell FAK specifically.

Tumor necrosis factor receptor signaling is a driver of chronic lymphocytic leukemia that can be therapeutically targeted by the flavonoid wogonin.

Chronic lymphocytic leukemia is a malignancy of mature B cells that strongly depend on microenvironmental factors, and their deprivation has been identified as a promising treatment approach for this incurable disease. Cytokine array screening of 247 chronic lymphocytic leukemia serum samples revealed elevated levels of tumor necrosis factor (TNF) receptor-1 which were associated with poor clinical outcome. We detected a microenvironment-induced expression of TNF receptor-1 in chronic lymphocytic leukemia cells in vitro, and an aberrantly high expression of this receptor in the proliferation centers of patients' lymph nodes. Stimulation of TNF receptor-1 with TNF-α enhanced nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) activity and viability of chronic lymphocytic leukemia cells, which was inhibited by wogonin. The therapeutic effects of wogonin were analyzed in mice after adoptive transfer of Eµ-T-cell leukemia 1 (TCL1) leukemic cells. Wogonin treatment prevented leukemia development when given early after transplantation. The treatment of full-blown leukemia resulted in the loss of the TNF receptor-1 on chronic lymphocytic leukemia cells and their mobilization to blood. Targeting TNF receptor-1 signaling is therefore proposed for the treatment of chronic lymphocytic leukemia.

PD-L1 checkpoint blockade prevents immune dysfunction and leukemia development in a mouse model of chronic lymphocytic leukemia.

Blockade of the programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint augments antitumor immunity and induces durable responses in patients with solid cancers, but data on clinical efficacy in leukemias are sparse. Chronic lymphocytic leukemia (CLL) is associated with a tumor-supportive microenvironment and a dysfunctional immune system, as shown by "exhausted" T cells, defective immunologic synapse formation, and immunosuppressive myeloid cells. These defects involve aberrant expression of PD-L1 and are closely mirrored in the Eµ-TCL1 mouse model for CLL. In this study, we treated mice after adoptive transfer of Eµ-TCL1 CLL with PD-L1-blocking antibodies, which prevented CLL development and was accompanied by a reactivation of immune effector functions. This included restoration of mature macrophages and major histocompatibility complex class II-expressing dendritic cells and prevention of aberrant and exhaustion-like T-cell phenotypes. In addition, PD-L1 blockade restored CD8 T-cell cytotoxicity and immune synapse formation and normalized T-cell cytokines and proliferation ex vivo and in vivo. Our data demonstrate that early PD-L1 blockade effectively corrects leukemia-induced immune dysfunction and thus prevents CLL development in mice. Targeting PD-L1/PD-1 interactions should therefore be further explored in clinical studies with CLL patients, ideally in combination with novel compounds to help eliminate CLL.

Arginine deprivation using pegylated arginine deiminase has activity against primary acute myeloid leukemia cells in vivo.

The strategy of enzymatic degradation of amino acids to deprive malignant cells of important nutrients is an established component of induction therapy of acute lymphoblastic leukemia. Here we show that acute myeloid leukemia (AML) cells from most patients with AML are deficient in a critical enzyme required for arginine synthesis, argininosuccinate synthetase-1 (ASS1). Thus, these ASS1-deficient AML cells are dependent on importing extracellular arginine. We therefore investigated the effect of plasma arginine deprivation using pegylated arginine deiminase (ADI-PEG 20) against primary AMLs in a xenograft model and in vitro. ADI-PEG 20 alone induced responses in 19 of 38 AMLs in vitro and 3 of 6 AMLs in vivo, leading to caspase activation in sensitive AMLs. ADI-PEG 20-resistant AMLs showed higher relative expression of ASS1 than sensitive AMLs. This suggests that the resistant AMLs survive by producing arginine through this metabolic pathway and ASS1 expression could be used as a biomarker for response. Sensitive AMLs showed more avid uptake of arginine from the extracellular environment consistent with their auxotrophy for arginine. The combination of ADI-PEG 20 and cytarabine chemotherapy was more effective than either treatment alone resulting in responses in 6 of 6 AMLs tested in vivo. Our data show that arginine deprivation is a reasonable strategy in AML that paves the way for clinical trials.

Prognostic relevance of CD163 and CD8 combined with EZH2 and gain of chromosome 18 in follicular lymphoma: a study by the Lunenburg Lymphoma Biomarker Consortium.

In follicular lymphoma, studies addressing the prognostic value of microenvironment-related immunohistochemical markers and tumor cell-related genetic markers have yielded conflicting results, precluding implementation in practice. Therefore, the Lunenburg Lymphoma Biomarker Consortium performed a validation study evaluating published markers. To maximize sensitivity, an end of spectrum design was applied for 122 uniformly immunochemotherapy-treated follicular lymphoma patients retrieved from international trials and registries. The criteria were: early failure, progression or lymphoma-related death <2 years versus long remission, response duration of >5 years. Immunohistochemical staining for T cells and macrophages was performed on tissue microarrays from initial biopsies and scored with a validated computer-assisted protocol. Shallow whole-genome and deep targeted sequencing was performed on the same samples. The 96/122 cases with complete molecular and immunohistochemical data were included in the analysis. EZH2 wild-type (P=0.006), gain of chromosome 18 (P=0.002), low percentages of CD8+ cells (P=0.011) and CD163+ areas (P=0.038) were associated with early failure. No significant differences in other markers were observed, thereby refuting previous claims of their prognostic significance. Using an optimized study design, this Lunenburg Lymphoma Biomarker Consortium study substantiates wild-type EZH2 status, gain of chromosome 18, low percentages of CD8+ cells and CD163+ area as predictors of early failure to immunochemotherapy in follicular lymphoma treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP [-like]), while refuting the prognostic impact of various other markers.

Extracellular HMGB1 promotes differentiation of nurse-like cells in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a disease of an accumulation of mature B cells that are highly dependent on the microenvironment for maintenance and expansion. However, little is known regarding the mechanisms whereby CLL cells create their favorable microenvironment for survival. High-mobility group protein B-1 (HMGB1) is a highly conserved nuclear protein that can be actively secreted by innate immune cells and passively released by injured or dying cells. We found significantly increased HMGB1 levels in the plasma of CLL patients compared with healthy controls, and HMGB1 concentration is associated with absolute lymphocyte count. We therefore sought to determine potential roles of HMGB1 in modulating the CLL microenvironment. CLL cells passively released HMGB1, and the timing and concentrations of HMGB1 in the medium were associated with differentiation of nurse-like cells (NLCs). Higher CD68 expression in CLL lymph nodes, one of the markers for NLCs, was associated with shorter overall survival of CLL patients. HMGB1-mediated NLC differentiation involved internalization of both receptor for advanced glycation end products (RAGE) and Toll-like receptor-9 (TLR9). Differentiation of NLCs can be prevented by blocking the HMGB1-RAGE-TLR9 pathway. In conclusion, this study demonstrates for the first time that CLL cells might modulate their microenvironment by releasing HMGB1.

Trisomy 12 chronic lymphocytic leukemia cells exhibit upregulation of integrin signaling that is modulated by NOTCH1 mutations.

The leukocyte adhesion cascade is important in chronic lymphocytic leukemia (CLL), as it controls migration of malignant cells into the pro-survival lymph node microenvironment. Circulating trisomy 12 CLL cells have increased expression of the integrins CD11a and CD49d, as well as CD38, but the tissue expression of these and other molecules, and the functional and clinical sequelae of these changes have not been described. Here, we demonstrate that circulating trisomy 12 CLL cells also have increased expression of the integrins CD11b, CD18, CD29, and ITGB7, and the adhesion molecule CD323. Notably, there was reduced expression of CD11a, CD11b, and CD18 in trisomy 12 cases with NOTCH1 mutations compared with wild type. Trisomy 12 cells also exhibit upregulation of intracellular integrin signaling molecules CALDAG-GEFI, RAP1B, and Ras-related protein ligand, resulting in enhanced very late antigen-4 [VLA-4] directed adhesion and motility. CD38 expression in CLL has prognostic significance, but the increased CD38 expression in trisomy 12 CLL cells must be taken into account in this subgroup, and the threshold of CD38 positivity should be raised to 40% for this marker to retain its prognostic value. In conclusion, trisomy 12 CLL cells exhibit functional upregulation of integrin signaling, with ß2-integrin expression being modulated by NOTCH1 mutation status.

P110α-mediated constitutive PI3K signaling limits the efficacy of p110δ-selective inhibition in mantle cell lymphoma, particularly with multiple relapse.

Phosphoinositide-3 kinase (PI3K) pathway activation contributes to mantle cell lymphoma (MCL) pathogenesis, but early-phase studies of the PI3K p110δ inhibitor GS-1101 have reported inferior responses in MCL compared with other non-Hodgkin lymphomas. Because the relative importance of the class IA PI3K isoforms p110α, p110ß, and p110δ in MCL is not clear, we studied expression of these isoforms and assessed their contribution to PI3K signaling in this disease. We found that although p110δ was highly expressed in MCL, p110α showed wide variation and expression increased significantly with relapse. Loss of phosphatase and tensin homolog expression was found in 16% (22/138) of cases, whereas PIK3CA and PIK3R1 mutations were absent. Although p110δ inhibition was sufficient to block B-cell receptor-mediated PI3K activation, combined p110α and p110δ inhibition was necessary to abolish constitutive PI3K activation. In addition, GDC-0941, a predominantly p110α/δ inhibitor, was significantly more active compared with GS-1101 against MCL cell lines and primary samples. We found that a high PIK3CA/PIK3CD ratio identified a subset of primary MCLs resistant to GS-1101 and this ratio increased significantly with relapse. These findings support the use of dual p110α/p110δ inhibitors in MCL and suggest a role for p110α in disease progression.

The microenvironment of AIDS-related diffuse large B-cell lymphoma provides insight into the pathophysiology and indicates possible therapeutic strategies.

Despite the use of highly active antiretroviral therapy (HAART), AIDS-related lymphoma remains common. We investigated the tumor, microenvironment, and viral components in 41 AIDS-related diffuse large B-cell lymphomas (AR-DLBCLs) in the pre- and post-HAART era. The outcome has improved and the frequency of the prognostically unfavorable immunoblastic histology has decreased after HAART. Compared with sporadic cases, AR-DLBCL demonstrated increased hyperproliferation (P < .001) and c-Myc rearrangements, reduced CD4(+) (P < .001) and FOXP3(+) T cells (P < .001), increased activated cytotoxic cells (P < .001), but no difference in tumor-associated macrophages. Our analysis showed that AR-DLBCL is highly angiogenic with higher blood-vessel density than sporadic cases (P < .001) and highlighted the role of Epstein-Barr virus in angiogenesis. We recognized viral profiles and as a second step examined the reactive cytotoxic cell infiltrates. Our observation of markedly higher numbers of cytotoxic cells in AR-DLBCL with LMP1 and/or p24 compared with cases lacking viral antigens (P < .001) has important clinical implications, implicitly linked to the immunosurveillance theory. Whereas early initiation of HAART should improve immunosurveillance and reduce the incidence of LMP1-positive AR-DLBCL, cases without viral antigens appear able to avoid immunologic reaction and likely require additional strategies to improve surveillance.

Defining characteristics of classical Hodgkin lymphoma microenvironment T-helper cells.

CD4(+) T-helper cells (THs) dominate the classical Hodgkin lymphoma (CHL) microenvironment, but their role is poorly understood. Advances in flow cytometry and immunohistochemistry permit more detailed investigation of this aspect of CHL pathophysiology. To address the hypothesis that the TH-infiltrate, rather than being TH2-enriched, senescent and hypofunctional, is TH1 and activation marker-rich, cytokine-secretory and proliferative, we applied comprehensive flow cytometric immunophenotyping and functional assays of cytokine secretion/proliferation to TH cells from 18 CHL-derived single-cell suspensions (SCSs) compared to reactive lymph nodes (RLNs). CHL-derived TH cells express TH1-associated CXCR3/CCR5 and TNFα/IFNγ/interleukin-2 (IL-2) and less TH2-associated CCR3/CCR4, with no IL-4/IL-13. They lack exhaustion-/suppression-associated PD1, CD57 and terminally differentiated effector memory cells, with more central memory cells, activation-associated partners of Hodgkin Reed Sternberg (HRS) cell-expressed CD30/OX40-L/ICOS-L, and other activation markers. TH cell lines established from CHL and RLN-derived SCSs remain cytokine-secretory. We confirmed and extended these studies using tissue microarray immunohistochemistry (TMA-IHC) from a large CHL tissue bank (n = 122) and demonstrate TH1-associated TBET is abundant in CHL, and TH2-associated CMAF/GATA3 and exhaustion-associated PD1 expressed at significantly lower levels. These molecular insights into the CHL-associated TH offer potential diagnostic, prognostic and pharmacologically modifiable therapeutic targets and do not support the established view of a TH2-enriched, senescent/exhausted, hypofunctional, hypoproliferative infiltrate.

EZH2 mutations are frequent and represent an early event in follicular lymphoma.

Gain of function mutations in the H3K27 methyltransferase EZH2 represent a promising therapeutic target in germinal center lymphomas. In this study, we assessed the frequency and distribution of EZH2 mutations in a large cohort of patients with follicular lymphoma (FL) (n = 366) and performed a longitudinal analysis of mutation during the disease progression from FL to transformed FL (tFL) (n = 33). Mutations were detected at 3 recurrent mutation hot spots (Y646, A682, and A692) in 27% of FL cases with variant allele frequencies (VAF) ranging from 2% to 61%. By comparing VAF of EZH2 with other mutation targets (CREBBP, MLL2, TNFRSF14, and MEF2B), we were able to distinguish patients harboring clonal EZH2 mutation from rarer cases with subclonal mutations. Overall, the high incidence of EZH2 mutations in FL and their stability during disease progression makes FL an appropriate disease to evaluate EZH2 targeted therapy.

Revisiting the immune microenvironment of diffuse large B-cell lymphoma using a tissue microarray and immunohistochemistry: robust semi-automated analysis reveals CD3 and FoxP3 as potential predictors of response to R-CHOP.

Gene expression studies have identified the microenvironment as a prognostic player in diffuse large B-cell lymphoma. However, there is a lack of simple immune biomarkers that can be applied in the clinical setting and could be helpful in stratifying patients. Immunohistochemistry has been used for this purpose but the results are inconsistent. We decided to reinvestigate the immune microenvironment and its impact using immunohistochemistry, with two systems of image analysis, in a large set of patients with diffuse large B-cell lymphoma. Diagnostic tissue from 309 patients was arrayed onto tissue microarrays. Results from 161 chemoimmunotherapy-treated patients were used for outcome prediction. Positive cells, percentage stained area and numbers of pixels/area were quantified and results were compared with the purpose of inferring consistency between the two semi-automated systems. Measurement cutpoints were assessed using a recursive partitioning algorithm classifying results according to survival. Kaplan-Meier estimators and Fisher exact tests were evaluated to check for significant differences between measurement classes, and for dependence between pairs of measurements, respectively. Results were validated by multivariate analysis incorporating the International Prognostic Index. The concordance between the two systems of image analysis was surprisingly high, supporting their applicability for immunohistochemistry studies. Patients with a high density of CD3 and FoxP3 by both methods had a better outcome. Automated analysis should be the preferred method for immunohistochemistry studies. Following the use of two methods of semi-automated analysis we suggest that CD3 and FoxP3 play a role in predicting response to chemoimmunotherapy in diffuse large B-cell lymphoma.

Activated pancreatic stellate cells sequester CD8+ T cells to reduce their infiltration of the juxtatumoral compartment of pancreatic ductal adenocarcinoma.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic microenvironment that contains many different immune cells. Activated pancreatic stellate cells (PSCs) contribute to the desmoplasia. We investigated whether distinct stromal compartments are differentially infiltrated by different types of immune cells. METHODS: We used tissue microarray analysis to compare immune cell infiltration of different pancreaticobiliary diseased tissues (PDAC, ampullary carcinoma, cholangiocarcinoma, mucinous cystic neoplasm, chronic inflammation, and chronic pancreatitis) and juxtatumoral stromal (<100 µm from tumor) and panstromal compartments. We investigated the association between immune infiltrate and patient survival times. We also analyzed T-cell migration and tumor infiltration in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice and the effects of all-trans retinoic acid (ATRA) on these processes. RESULTS: Juxtatumoral compartments in PDAC samples from 2 independent groups of patients contained increased numbers of myeloperoxidase(+) and CD68(+) cells compared with panstromal compartments. However, juxtatumoral compartments of PDACs contained fewer CD8(+), FoxP3(+), CD56(+), or CD20(+) cells than panstromal compartments, a distinction absent in ampullary carcinomas and cholangiocarcinomas. Patients with PDACs that had high densities of CD8(+) T cells in the juxtatumoral compartment had longer survival times than patients with lower densities. In KPC mice, administration of ATRA, which renders PSCs quiescent, increased numbers of CD8(+) T cells in juxtatumoral compartments. We found that activated PSCs express cytokines, chemokines, and adhesion molecules that regulate T-cell migration. In vitro migration assays showed that CD8(+) T cells, from patients with PDAC, had increased chemotaxis toward activated PSCs, which secrete CXCL12, compared with quiescent PSCs or tumor cells. These effects could be reversed by knockdown of CXCL12 or treatment of PSCs with ATRA. CONCLUSIONS: Based on studies of human PDAC samples and KPC mice, activated PSCs appear to reduce migration of CD8(+) T cells to juxtatumoral stromal compartments, preventing their access to cancer cells. Deregulated signaling by activated PSCs could prevent an effective antitumor immune response.

Multiple inhibitory ligands induce impaired T-cell immunologic synapse function in chronic lymphocytic leukemia that can be blocked with lenalidomide: establishing a reversible immune evasion mechanism in human cancer.

Cancer immune evasion is an emerging hallmark of disease progression. We have demonstrated previously that impaired actin polymerization at the T-cell immunologic synapse is a global immune dysfunction in chronic lymphocytic leukemia (CLL). Direct contact with tumor cells induces defective actin polarization at the synapse in previously healthy T cells, but the molecules mediating this dysfunction were not known. In the present study, we show via functional screening assays that CD200, CD270, CD274, and CD276 are coopted by CLL cells to induce impaired actin synapse formation in both allogeneic and autologous T cells. We also show that inhibitory ligand-induced impairment of T-cell actin dynamics is a common immunosuppressive strategy used by both hematologic (including lymphoma) and solid carcinoma cells. This immunosuppressive signaling targets T-cell Rho-GTPase activation. Of clinical relevance, the immunomodulatory drug lenalidomide prevented the induction of these defects by down-regulating tumor cell-inhibitory molecule expression. These results using human CLL as a model cancer establish a novel evasion mechanism whereby malignant cells exploit multiple inhibitory ligand signaling to down-regulate small GTPases and lytic synapse function in global T-cell populations. These findings should contribute to the design of immunotherapeutic strategies to reverse T-cell tolerance in cancer.