Secondary Lymphoid Structures Research Articles

Abstract PO4-25-04: MHC Class I and II Expression, Functional Tertiary Lymphoid Structure (TLS), and Outcomes in Early-Stage HER2-Positive (HER2+) Breast Cancer in NCCTG N9831 (Alliance)

Abstract Background: Tertiary lymphoid structure (TLS) is an organized form of ectopic lymphoid aggregates (LA) with secondary lymphoid organ structure. Several studies showed that the presence of TLS associates with improved outcomes in multiple cancers when treated with immune checkpoint inhibitors. However, data is currently limited regarding TLS and outcomes in HER2+ breast cancer patients (pts) treated with adjuvant trastuzumab. Furthermore, distinguishing TLS and simple LA is challenging in standard hematoxylin and eosin (H&E) staining, particularly when the germinal center is absent. Emerging studies also showed the prognostic value of MHC expression in breast cancer but mainly in triple-negative breast cancer. In this study, we evaluated integrated pathological quantification and genomic data to assess functional TLS in association with MHC expression and outcomes in the N9831 trial. Methods: Pathological evaluation of LA in H&E slides from pts treated in Arm A (chemotherapy alone) and Arm C (chemotherapy with concurrent trastuzumab) in N9831 was performed. NanoString was used to quantify mRNA of MHC class I and II expression as well as TLS-related immune genes, including IFNG, ICOSLG, CXCL13, CXCR3, BCL6, IL21R, ICOS, PDCD1, CXCR5, CXCL9, TBX21, CD38, CXCL10, CLXCL11, IL21, CD200, CD19, MS4S1. Wilcoxon rank sum test, Chi-squared test, Kaplan-Meier method, and Cox regression model were used to evaluate the association between LA and baseline characteristics, MHC expression, and outcomes. Results: LA was evaluated in 526 pts in Arm A and 485 in Arm C. Greater number of LA was significantly associated with stromal tumor-infiltrating lymphocytes (sTILs), hormone receptor negative, and higher tumor grade, but not stage and age. Using multivariable Cox regression analysis, increasing numbers of LA were associated with improved recurrence-free survival in both arms combined (RFS, p 0.028). However, when evaluating each arm separately, LA was associated with improved RFS only in Arm A (HR 0.6, 95%CI 0.43-0.84, p 0.003) but not in Arm C (HR 0.72, 95%CI 0.47-1.1, p 0.134). We further evaluated TLS-related immune genes in 252 pts in Arm C with LA ≥ 1. As a continuous variable, higher expression of BCL6 (HR 0.61, 95%CI 0.41-0.92, p 0.019) and IL21R (HR 0.78, 95%CI 0.62-0.98, p 0.03) were associated with improved RFS in Arm C pts with LA ≥ 1. However, these genes were not significantly associated with outcomes in Arm C pts without LA with BCL6 (HR1.07, 95%CI 0.67-1.71, p 0.776) and IL21R (HR 0.96, 95%CI 0.71-1.29, p 0.775). Moreover, we evaluated differential gene expression between tumors with LA ≥ 1 with high BCL6 vs. LA 0. All MHC class I and II (HLA A, B, C, E, DQ, DM, DO, DP, and DQ) were significantly higher in tumors with LA ≥ 1 with high BCL6 (p < 0.001). Conclusion: A greater number of LA was associated with improved outcomes in pts with early-stage HER2+ breast cancer, particularly when treated with chemotherapy alone. Using histologic and genomic integration with the combination of pathological LA and TLS-related immune genes, we identified that pts with functional TLS with LA ≥ 1 and higher expression of BCL6 or IL21R had significantly improved outcomes when treated with trastuzumab. High MHC class I and II expressions are associated with the presence of functional TLS, underscoring the crucial role of antigen presentation in the generation of an effective adaptive immune response. Support: U10 CA180821, U10 CA180882, U24 CA196171, https://acknowledgments.alliancefound.org; Genentech; Clinicaltrials.gov Id: NCT00005970 Citation Format: Saranya Chumsri, Tracy Shachner, Zhuo Li, Nadine Norton, Alvaro Moreno-Aspitia, Gerardo Colon-Otero, Edith Perez, Aziza Nassar, E. Thompson, Keith Knutson. MHC Class I and II Expression, Functional Tertiary Lymphoid Structure (TLS), and Outcomes in Early-Stage HER2-Positive (HER2+) Breast Cancer in NCCTG N9831 (Alliance) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-04.

Adaptive immunity and atherosclerosis: aging at its crossroads.

Adaptive immunity plays a profound role in atherosclerosis pathogenesis by regulating antigen-specific responses, inflammatory signaling and antibody production. However, as we age, our immune system undergoes a gradual functional decline, a phenomenon termed "immunosenescence". This decline is characterized by a reduction in proliferative naïve B- and T cells, decreased B- and T cell receptor repertoire and a pro-inflammatory senescence associated secretory profile. Furthermore, aging affects germinal center responses and deteriorates secondary lymphoid organ function and structure, leading to impaired T-B cell dynamics and increased autoantibody production. In this review, we will dissect the impact of aging on adaptive immunity and the role played by age-associated B- and T cells in atherosclerosis pathogenesis, emphasizing the need for interventions that target age-related immune dysfunction to reduce cardiovascular disease risk.

Intra-Tumoral Secondary Follicle-like Tertiary Lymphoid Structures Are Associated with a Superior Prognosis of Overall Survival of Perihilar Cholangiocarcinoma.

Ectopic lymphoid structures termed tertiary lymphoid structures (TLSs) have an immunomodulatory function and positively affect prognosis in certain cancers. However, their clinical relevance and prognostic utility in perihilar cholangiocarcinoma (pCCA) are unknown. Therefore, determining the involvement and prognostic utility of TLSs in pCCA is the aim of this study. Ninety-three patients with surgically resected pCCA were included retrospectively. Hematoxylin and eosin and immunohistochemical staining identified and classified the TLSs, and multiplex immunofluorescence determined the TLS composition in the pCCA sample. The correlations between clinical features and TLSs were analyzed using either Fisher's exact test or the Chi-squared test. Recurrence-free survival (RFS) and overall survival (OS) correlations with TLSs were analyzed using Cox regression and Kaplan-Meier analyses. We identified TLSs in 86% of patients with pCCA, including lymphoid aggregates (6.45%), primary (13.98%) and secondary follicles (65.59%). Patients with intra-tumoral secondary follicle-like TLSs (S-TLSs) had better OS (p = 0.003) and RFS (p = 0.0313). The multivariate analysis identified the presence of S-TLSs as a good independent prognostic indicator for OS but not for RFS. Interestingly, the presence of S-TLS only indicated better 5-year OS in 54 patients without lymph node metastasis (LNM-, p = 0.0232) but not in the 39 patients with lymph node metastasis (LNM+, p = 0.1244). Intra-tumoral S-TLSs predicted longer OS in patients with surgically resected pCCA, suggesting intra-tumoral S-TLSs' contribution to effective antitumor immunity and that S-TLSs hold promise for diagnostic and therapeutic development in pCCA.

Spatial biology analysis reveals B cell follicles in secondary lymphoid structures may regulate anti-tumor responses at initial melanoma diagnosis.

IntroductionB cells are key regulators of immune responses in melanoma. We aimed to explore differences in the histologic location and activation status of B cell follicles in sentinel lymph nodes (SLN) of melanoma patients.MethodsFlow cytometry was performed on fresh tumor draining lymph nodes (LN). Paraffin slides from a separate cohort underwent NanoString Digital Spatial Profiling (DSP)®. After staining with fluorescent markers for CD20 (B cells), CD3 (T cells), CD11c (antigen presenting cells) and a nuclear marker (tumor) was performed, regions of interest (ROI) were selected based on the location of B cell regions (B cell follicles). A panel of 68 proteins was then analyzed from the ROIs.ResultsB cell percentage trended higher in patients with tumor in LN (n=3) compared to patients with nSLN (n=10) by flow cytometry. B cell regions from a separate cohort of patients with tumor in the (pSLN) (n=8) vs. no tumor (nSLN) (n=16) were examined with DSP. Within B cell regions of the SLN, patients with pSLN had significantly higher expression of multiple activation markers including Ki-67 compared to nSLN patients. Among 4 patients with pSLN, we noted variability in arrangement of B cell follicles which were either surrounding the tumor deposit or appeared to be infiltrating the tumor. The B cell follicle infiltrative pattern was associated with prolonged recurrence free survival.ConclusionThese data suggest a role for B cell follicles in coordinating effective adaptive immune responses in melanoma when low volume metastatic disease is present in tumor draining LN.

Abstract 5627: Functional tertiary lymphoid structure (TLS) and outcome in HER2-positive (HER2+) breast cancer in NCCTG N9831 (Alliance)

Abstract Background: Previous studies showed conflicting results regarding association between stromal tumor infiltrating lymphocytes (sTIL) and outcome in HER2+ breast cancer. While sTIL were not associated with outcome in patients (pts) treated with trastuzumab in N9831, immune functions genes are linked to outcome in these pts. TLS is an organized form of ectopic lymphoid aggregates (LA) with secondary lymphoid organ structure. Several studies showed that TLS associates with improved outcome in multiple cancers. However, distinguishing TLS and simple LA is challenging, particularly when germinal center is absent. In this study, we evaluated integrated pathological quantification and genomic data to assess functional TLS. Methods: Pathological evaluation of LA in H&E slides from pts treated in Arm A (chemotherapy alone) and Arm C (chemotherapy with concurrent trastuzumab) in N9831 was performed. NanoString was used to quantify mRNA. Wilcoxon rank sum test, Chi squared test, Kaplan-Meier method and Cox regression model were used to evaluate association between LA and baseline characteristics as well as outcomes. Results: LA was quantified in 1011 pts (526 Arm A, 485 Arm C). Greater number of LA was significantly associated with higher tumor grade, ER/PR negativity and increasing sTIL, but not age, tumor size or lymph node status. Increasing numbers of LA were associated with improved RFS in both arms combined (p 0.028). However, using multivariable Cox regression analysis in each treatment arm, LA (≥ 1 vs 0) was associated with improved RFS only in Arm A (HR 0.6, 95%CI 0.43-0.84, p 0.003) but not Arm C (HR 0.72, 95%CI 0.47-1.1, p 0.134). Further evaluation of expression of TLS-related immune genes including IFNG, ICOSLG, CXCL13, CXCR3, BCL6, IL21R, ICOS, PDCD1, CXCR5, CXCL9, TBX21, CD38, CXCL10, CLXCL11, IL21, CD200 was carried out among 252 pts in Arm C with LA ≥ 1. Among these 16 TLS-related immune genes, as a continuous variable, higher expression of BCL6 (HR 0.61, 95%CI 0.41-0.92, p 0.019) and IL21R (HR 0.78, 95%CI 0.62-0.98, p 0.03) were associated with improved RFS in Arm C pts with LA ≥ 1. However, these genes were not significantly associated with outcome in Arm C pts without LA with BCL6 HR1.07 (95%CI 0.67-1.71, p 0.776) and IL21R HR 0.96 (95%CI 0.71-1.29, p 0.775). Conclusion: Similar to sTIL, greater number of LA was associated with improved outcome in HER2+ pts treated with chemotherapy alone but not chemotherapy in combination with trastuzumab. Using histogenomic integration with the combination of pathological LA and TLS-related immune genes, we identified that pts with functional TLS with LA ≥ 1 and higher expression of BCL6 or IL21R had significantly improved outcome when treated with trastuzumab. Future studies are needed to further confirm these findings. Support: U10 CA180821, U24 CA196171, https://acknowledgments.alliancefound.org; Genentech; Clinicaltrials.gov Id: NCT00005970 Citation Format: Saranya Chumsri, Tracy R. Shachner, Zhuo Li, Nadine Norton, Alvaro Moreno-Aspitia, Gerardo Colon-Otero, Edith A. Perez, E. A. Thompson, Aziza Nassar, Keith L. Knutson. Functional tertiary lymphoid structure (TLS) and outcome in HER2-positive (HER2+) breast cancer in NCCTG N9831 (Alliance) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5627.

VEGFR3-driven pulmonary lymphangiogenesis exacerbates induction of bronchus-associated lymphoid tissue in allergic airway disease

Abstract Asthmatic lung samples present with both a higher density of pulmonary lymphatic vessels and a higher incidence of bronchus associated lymphoid tissue (BALT). Here, we asked whether lymphangiogenesis, stimulated by the VEGF-C/VEGFR-3 signaling axis in lymphatic endothelial cells (LECs), plays a role in promoting BALT in mouse models of allergy. First, we determined that chronic intratracheal instillation of house dust mite (HDM), a clinically relevant allergen, recapitulates both lymphangiogenesis and BALT induction. Intratracheal stimulation of VEGFR-3 in LECs exacerbated BALT, while blocking VEGFR-3 signaling reduced BALT. Furthermore, in transgenic mice with an expanded pulmonary lymphatic network (induced prior to allergen challenge), we found an exacerbated BALT response upon chronic HDM inhalation. Recent studies have determined that LEC-derived CXCL13 plays an important role in secondary lymphoid structure organogenesis, and thus we pondered whether LEC-derived CXCL13 could play a role in the development of tertiary lymphoid structures. Indeed, we observed an increase in lung infiltration by CXCR5+ cells when we used VEGF-C to modulate the chronic allergic response. Finally, we found that the VEGF-C exacerbated BALT phenomenon was indeed CXCL13 dependent. Altogether, these results suggest a causative role for pulmonary lymphatics in mediating BALT induction in chronic allergic airway inflammation.

Loss of vascular endothelial notch signaling promotes spontaneous formation of tertiary lymphoid structures

Tertiary lymphoid structures (TLS) are lymph node-like immune cell clusters that emerge during chronic inflammation in non-lymphoid organs like the kidney, but their origin remains not well understood. Here we show, using conditional deletion strategies of the canonical Notch signaling mediator Rbpj, that loss of endothelial Notch signaling in adult mice induces the spontaneous formation of bona fide TLS in the kidney, liver and lung, based on molecular, cellular and structural criteria. These TLS form in a stereotypical manner around parenchymal arteries, while secondary lymphoid structures remained largely unchanged. This effect is mediated by endothelium of blood vessels, but not lymphatics, since a lymphatic endothelial-specific targeting strategy did not result in TLS formation, and involves loss of arterial specification and concomitant acquisition of a high endothelial cell phenotype, as shown by transcriptional analysis of kidney endothelial cells. This indicates a so far unrecognized role for vascular endothelial cells and Notch signaling in TLS initiation.

Combined immunodeficiency with autoimmunity caused by a homozygous missense mutation in inhibitor of nuclear factor 𝛋B kinase alpha (IKKα).

Inhibitor of nuclear factor kappa B kinase alpha (IKKα) is critical for p100/NF-κB2 phosphorylation and processing into p52 and activation of the noncanonical NF-κB pathway. A patient with recurrent infections, skeletal abnormalities, absent secondary lymphoid structures, reduced B cell numbers, hypogammaglobulinemia, and lymphocytic infiltration of intestine and liver was found to have a homozygous p.Y580C mutation in the helix-loop-helix domain of IKKα. The mutation preserves IKKα kinase activity but abolishes the interaction of IKKα with its activator NF-κB–inducing kinase and impairs lymphotoxin-β–driven p100/NF-κB2 processing and VCAM1 expression. Homozygous IKKαY580C/Y580C mutant mice phenocopy the patient findings; lack marginal zone B cells, germinal centers, and antigen-specific T cell response to cutaneous immunization; have impaired Il17a expression; and are susceptible to cutaneous Staphylococcus aureus infection. In addition, these mice demonstrate a severe reduction in medullary thymic epithelial cells, impaired thymocyte negative selection, a restricted TCRVβ repertoire, a selective expansion of potentially autoreactive T cell clones, a decreased frequency of regulatory T cells, and infiltration of liver, pancreas, and lung by activated T cells coinciding with organ damage. Hence, this study identifies IKKα deficiency as a previously undescribed cause of primary immunodeficiency with associated autoimmunity.

Prognostic value of tertiary lymphoid structures in early clinical stage oral tongue squamous cell carcinoma.

Tertiary lymphoid structures have been observed in several solid tumors and suggested to be a favorable prognostic factor. However, the prognostic value of tertiary lymphoid structures for early clinical stage (cT1/2N0) oral tongue squamous cell carcinoma (OTSCC) remains unclear. In this study, we aimed to evaluate the prognostic value of tertiary lymphoid structures in cT1/2N0stage OTSCC. Ninety-seven patients with cT1/2N0 stage OTSCC were enrolled in this study. Tertiary lymphoid structures were assessed in paraffin sections with H.E and immunohistochemistry staining. Overall and disease-free survivals were evaluated by the Kaplan-Meier method. Cox proportional hazards regression models were used to assess their prognostic value. Tertiary lymphoid structures can be readily identified in cT1/2N0 stage OTSCC, including early tertiary lymphoid structures, primary follicle-like tertiary lymphoid structures, and secondary follicle-like tertiary lymphoid structures. Its frequency in OTSCC was 76.3% in the cohort. The presence of tertiary lymphoid structures was associated with favorable overall survival (p=0.0176) and disease-free survival (p=0.0183) in early clinical stage OTSCC. Cox regression models showed that tertiary lymphoid structures can serve as an independent prognostic factor for overall survival (HR=0.434, 95% CI=0.212-0.886, p=0.022) and disease-free survival (HR=0.459, 95% CI=0.235-0.897, p=0.023) in early clinical stage OTSCC. The group of low maturity of TLS combined with pN positive had the lowest overall survival (p=0.0094) and disease-free survival (p=0.0028). Tertiary lymphoid structures are frequently observed in OTSCC. It is associated with a favorable prognosis for patients with cT1/2N0 stage OTSCC and may be used to predict immunotherapy sensitivity.

Molecular Drivers of Lymphocyte Organization in Vertebrate Mucosal Surfaces: Revisiting the TNF Superfamily Hypothesis.

The adaptive immune system of all jawed vertebrates relies on the presence of B and T cell lymphocytes that aggregate in specific body sites to form primary and secondary lymphoid structures. Secondary lymphoid organs include organized MALT (O-MALT) such as the tonsils and Peyer patches. O-MALT became progressively organized during vertebrate evolution, and the TNF superfamily of genes has been identified as essential for the formation and maintenance of O-MALT and other secondary and tertiary lymphoid structures in mammals. Yet, the molecular drivers of O-MALT structures found in ectotherms and birds remain essentially unknown. In this study, we provide evidence that TNFSFs, such as lymphotoxins, are likely not a universal mechanism to maintain O-MALT structures in adulthood of teleost fish, sarcopterygian fish, or birds. Although a role for TNFSF2 (TNF-α) cannot be ruled out, transcriptomics suggest that maintenance of O-MALT in nonmammalian vertebrates relies on expression of diverse genes with shared biological functions in neuronal signaling. Importantly, we identify that expression of many genes with olfactory function is a unique feature of mammalian Peyer patches but not the O-MALT of birds or ectotherms. These results provide a new view of O-MALT evolution in vertebrates and indicate that different genes with shared biological functions may have driven the formation of these lymphoid structures by a process of convergent evolution.

Tertiary lymphoid structures in cancer - considerations for patient prognosis.

Tertiary lymphoid structures (TLS) are ectopic lymphoid formations that form within nonlymphoid tissue. They share structural and functional characteristics with secondary lymphoid structures such as lymph nodes and can contain B-cell follicles and germinal centers surrounded by a T-cell region. TLS have been described in several types of cancers and are usually associated with positive patient outcomes. However, TLS differ vastly in cellular composition and location within tissue types. In this review, we discuss factors confounding the interpretation of the evidence for a prognostic role for TLS in cancer and frame these factors in the context of translation to regular clinical use.

Abstract 3271: A systemically administered, conditionally active TLR8 agonist for the treatment of HER2-expressing tumors

Abstract Clinical development of systemically administered myeloid cell agonists has been hindered by acute toxicities due to peripheral activation of the targeted cell types. Intratumoral administration, the route of delivery typically used for innate immune/myeloid cell agonists, is limited by tumor accessibility and a dependence on abscopal responses. Here, we describe an example of a new composition class, termed ImmunoTAC, that allows for systemic delivery of an immune modulator with activity localized to tumor sites and secondary lymphoid structures. Agonism of TLR8 in human myeloid cells drives anti-tumor immunity by inducing direct macrophage killing of tumor cells, repolarizing suppressive myeloid cell populations to a pro-immunogenic phenotype, and inducing dendritic cells to drive tumor-specific CTL responses. SBT6050 is an ImmunoTAC comprised of a novel, potent TLR8 agonist conjugated to a HER2-directed monoclonal antibody. SBT6050 is designed to activate human monocytes and macrophages only in the presence of HER2pos tumor cells with moderate (2+ by IHC) or high (3+ by IHC) expression levels. This activity is dependent upon the ability of the Fc domain of the antibody to engage Fcγ receptors on the surface of the myeloid cells. Additionally, SBT6050 potently activates conventional dendritic cells that, in turn, drive a Th1/CTL program in T cells. Systemic delivery of a SBT6050 surrogate in mice shows robust single agent efficacy in tumor models intrinsically resistant to checkpoint blockade, such as the previously characterized HER2+ CT26 syngeneic model. The intratumoral immune response is characterized by robust activation of tumor-associated myeloid cells, infiltration of neutrophils, persistent increases in local cytokine and chemokine production, decreases in Treg infiltrate, and the generation of a potent, neo-Ag specific anti-tumor CTL response. Mice cured with single-agent treatment are resistant to tumor rechallenge, demonstrating induction of long-lived immunological memory. In contrast to observations with small molecule TLR8 agonists, ImmunoTAC does not induce peripheral cytokine production or associated CRS-like toxicity in mice, consistent with the localized activity of the molecule. Silverback’s lead candidate, SBT6050, is currently in preclinical development for patients with moderate or high HER2-expressing tumors. More broadly, the data presented here describe a novel therapeutic modality that allows for systemic administration of immune modulators with tissue-localized activity. Citation Format: Kara Moyes, Ty Brender, Sean W. Smith, Hengyu Xu, Ben Setter, Li-Qun Fan, Rebecca Brunette, Justin Killebrew, Phil Tan, Craig Coburn, Peter Baum, Valerie Odegard. A systemically administered, conditionally active TLR8 agonist for the treatment of HER2-expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3271.

3389 Coagulation Factor Xll-mediated contact system and its role in adaptive and innate immune cell movement

OBJECTIVES/SPECIFIC AIMS: The objectives of this study are to 1) expand upon the paradigm of HK-D5 accelerated immune cell chemotaxis; 2) characterize the role of FXII in murine models of immune-mediated disease using FXII KO mice and a small molecule inhibitor of FXIIa. METHODS/STUDY POPULATION: To test whether the addition of HK-D5 peptide would accelerate C-C chemokine receptor type 2 (CCR2)-mediated chemotaxis in vitro, a real-time transwell chemotaxis assay was developed utilizing human THP-1 monocyte cell line (Fig 1). For in vivo studies, both pharmacologic FXIIa antagonism and FXll KO mice were used. Genotyping, histopathological review, FXll protein expression, and active partial thromboplastin time (aPTT) measurements were used to phenotypically characterize FXII KO mice compared to C57BL/6 wild type mice (Fig 2). Subsequently, the FXll KO mice were studied in varied models of immune-mediated disease, including LPS-induced sepsis and experimental autoimmune encephalitis (EAE), to see if and how the absence of FXll can mitigate disease severity. The EAE model involved active immunization with myelin oligodendrocyte glycoprotein (MOG) and measurement of established clinical disease severity scores. The LPS sepsis model involved an intraperitoneal injection of LPS followed by 48-hour monitoring of core body temperature using subdermal temperature transponders as a proxy for inflammatory events related to septic shock (Fig 3). RESULTS/ANTICIPATED RESULTS: HK-D5 significantly accelerates CCR2-mediated chemotaxis compared to chemokine alone (p = 0.001) similar to HK-D5’s ability to accelerate CCR7-mediated chemotaxis as previously established. The FXll KO mice were backcrossed to the C57BL/6J background and confirmed by genotyping and complete absence of FXII protein in plasma. Compared to the control, FXII KO mice have a significantly prolonged aPTT without evidence of bleeding abnormalities, which confirms the expected phenotype previously described and recapitulates what is observed in Factor XII deficiency in humans. KO mice showed no significant gross or histopathological differences in secondary lymphoid structures compared to the control. Immunohistochemistry confirmed well-organized lymphoid structures with intact B- and T-cell populations. FXll KO mice are protected in LPS-induced septic shock and EAE models. Regarding the EAE model, FXIIa inhibition significantly reduced disease severity compared to control. In the LPS model, FXll KO mice recover within 24 hours after LPS-challenge measured subjectively and objectively by core body temperature measurement. DISCUSSION/SIGNIFICANCE OF IMPACT: The current study and previous findings suggest a novel immune signaling mechanism by which a peptide fragment of high molecular-weight kininogen (HK-D5) acts as an accelerant of both innate and adaptive immune cell chemotaxis in multiple immune contexts. This has broad implications regarding a mechanism of immune-mediated inflammation in a variety of disease states, which might be amenable to the targeting this pathway for therapeutic intent.

Toxoplasma gondii infection triggers chronic cachexia and sustained commensal dysbiosis in mice.

Toxoplasma gondii is a protozoan parasite with a predation-mediated transmission cycle between rodents and felines. Intermediate hosts acquire Toxoplasma by eating parasite cysts which invade the small intestine, disseminate systemically and finally establish host life-long chronic infection in brain and muscles. Here we show that Toxoplasma infection can trigger a severe form of sustained cachexia: a disease of progressive lean weight loss that is a causal predictor of mortality in cancer, chronic disease and many infections. Toxoplasma cachexia is characterized by acute anorexia, systemic inflammation and loss of 20% body mass. Although mice recover from symptoms of peak sickness, they fail to regain muscle mass or visceral adipose depots. We asked whether the damage to the intestinal microenvironment observed at acute time points was sustained in chronic infection and could thereby play a role in sustaining cachexia. We found that parasites replicate in the same region of the distal jejunum/proximal ileum throughout acute infection, inducing the development of secondary lymphoid structures and severe, regional inflammation. Small intestine pathology was resolved by 5 weeks post-infection. However, changes in the commensal populations, notably an outgrowth of Clostridia spp., were sustained in chronic infection. Importantly, uninfected animals co-housed with infected mice display similar changes in commensal microflora but never display symptoms of cachexia, indicating that altered commensals are not sufficient to explain the cachexia phenotype alone. These studies indicate that Toxoplasma infection is a novel and robust model to study the immune-metabolic interactions that contribute to chronic cachexia development, pathology and potential reversal.

High-resolution repertoire analysis reveals a major bystander activation of Tfh and Tfr cells

T follicular helper (Tfh) and regulatory (Tfr) cells are terminally differentiated cells found in germinal centers (GCs), specialized secondary lymphoid organ structures dedicated to antibody production. As such, follicular T (Tfol) cells are supposed to be specific for immunizing antigens, which has been reported for Tfh cells but is debated for Tfr cells. Here, we used high-throughput T cell receptor (TCR) sequencing to analyze the repertoires of Tfh and Tfr cells, at homeostasis and after immunization with self- or foreign antigens. We observed that, whatever the conditions, Tfh and Tfr cell repertoires are less diverse than those of effector T cells and Treg cells of the same tissues; surprisingly, these repertoires still represent thousands of different sequences, even after immunization with a single antigen that induces a 10-fold increase in Tfol cell numbers. Thorough analysis of the sharing and network of TCR sequences revealed that a specific response to the immunizing antigen can only, but hardly, be detected in Tfh cells immunized with a foreign antigen and Tfr cells immunized with a self-antigen. These antigen-specific responses are obscured by a global stimulation of Tfh and Tfr cells that appears to be antigen-independent. Altogether, our results suggest a major bystander Tfol cell activation during the immune response in the GCs.

Peptide Antigen Concentration Modulates Digital NFAT1 Activation in Primary Mouse Naive CD8+ T Cells as Measured by Flow Cytometry of Isolated Cell Nuclei.

Circulating naive T cells exist in a quiescent state. After TCR contact with the cognate peptide presented by APCs in secondary lymphoid structures, T cells undergo a period of rapid transcriptional changes that set the stage for fate-determining effector or memory programming. We describe a novel method to analyze TCR signaling pathway activation in nuclei isolated from primary mouse naive T cells after stimulation with natural peptide Ags. We prelabeled cells with cell tracking dye to easily distinguish CD8+ T cell nuclei from APC nuclei by conventional flow cytometry. Using this approach, we observed clear digital activation of NFAT1 transcription factor in OT-I T cells stimulated with OVA peptide presented by bulk splenocytes. OVA concentration had discrete control over the fraction of the cells that translocated NFAT1, indicating that a distinct threshold amount of TCR signaling is required to switch on NFAT1 in naive T cells. This behavior was cell contact dependent and qualitatively more exact than the NFAT1 response in ionomycin-stimulated naive T cells. These data contribute to our understanding of the digital behavior of TCR signaling components documented in other studies and indicate how T cells might discriminate log-fold changes in Ag availability during an actual infection. Overall, these results highlight the potential of this coculture nuclei isolation protocol to address stimulation-dependent translocation of proteins in primary lymphocytes.

Predicting the response to CTLA-4 blockade by longitudinal noninvasive monitoring of CD8 T cells.

Immunotherapy using checkpoint-blocking antibodies against targets such as CTLA-4 and PD-1 can cure melanoma and non-small cell lung cancer in a subset of patients. The presence of CD8 T cells in the tumor correlates with improved survival. We show that immuno-positron emission tomography (immuno-PET) can visualize tumors by detecting infiltrating lymphocytes and, through longitudinal observation of individual animals, distinguish responding tumors from those that do not respond to therapy. We used 89Zr-labeled PEGylated single-domain antibody fragments (VHHs) specific for CD8 to track the presence of intratumoral CD8+ T cells in the immunotherapy-susceptible B16 melanoma model in response to checkpoint blockade. A 89Zr-labeled PEGylated anti-CD8 VHH detected thymus and secondary lymphoid structures as well as intratumoral CD8 T cells. Animals that responded to CTLA-4 therapy showed a homogeneous distribution of the anti-CD8 PET signal throughout the tumor, whereas more heterogeneous infiltration of CD8 T cells correlated with faster tumor growth and worse responses. To support the validity of these observations, we used two different transplantable breast cancer models, yielding results that conformed with predictions based on the antimelanoma response. It may thus be possible to use immuno-PET and monitor antitumor immune responses as a prognostic tool to predict patient responses to checkpoint therapies.

Genome-Wide Exome Analysis of Cmv5-Disparate Mouse Strains that Differ in Host Resistance to Murine Cytomegalovirus Infection.

Host resistance to murine cytomegalovirus (MCMV) varies in different strains of laboratory mice due to differences in expression of determinants that control and clear viral infection. The major histocompatibility complex class I Dk molecule is one such determinant that controls MCMV through the action of natural killer (NK) cells. However, the extent of NK cell–mediated Dk-dependent resistance to infection varies in different mouse strains. The molecular genetic basis of this variation remains unclear. Previous work to examine the Dk effect on MCMV resistance in MA/My × C57L offspring discovered multiple quantitative trait loci (QTL) that may serve to modify NK cells or their capacity to respond during MCMV infection. One QTL in particular, Cmv5, was found to regulate the frequency of NK cells and secondary lymphoid organ structure in spleen during MCMV infection. Cmv5 alleles, however, have not been identified. We therefore sequenced and analyzed genome-wide exome (GWE) variants, including those aligned to the critical genetic interval, in Cmv5-disparate mouse strains. Their GWE variant profiles were compared to assess strain-specific sequence data integrity and to analyze mouse strain relatedness across the genome. GWE content was further compared against data from the Mouse Genomes Project. This approach was developed as a platform for using GWE variants to define genomic regions of divergence and similarity in different mouse strains while also validating the overall quality of GWE sequence data. Moreover, the analysis provides a framework for the selection of novel QTL candidate sequences, including at the Cmv5 critical region.

Abstract B18: Noninvasive imaging of antitumor immune responses

Abstract By using the body's own immune response, immunotherapy may prove to be a turning point in the treatment of cancer. Predictors of a patient's response to immunotherapy remain to be defined, as actual responses are heterogeneous and difficult to study in real-time. Is it possible to predict the outcome of therapy for an individual patient? What is the behavior and what are the dynamics of immune cells in the tumor microenvironment? We need new methods to noninvasively explore the microenvironment of tumors and enumerate relevant cell types. Imaging the extent of infiltration of immune cells into tumors and identifying the relevant lymphocyte subsets is therefore essential. We developed single domain antibodies (VHHs, ~15 kDa) against a variety of lymphocyte surface antigens to image antitumor immune responses. VHHs were engineered to contain a sortase recognition tag, X-LPETGG, at their C-terminus to enable site-specific modification with tags of choice. We synthesized a substrate that contains both a click handle and a metal chelator, where the click handle can be used to attach a polymer, and the chelator is used to chelate a radiometal onto the VHH of interest. We thus produced VHHs equipped with a polyethyleneglycol (PEG) molecule to increase circulatory half-life and a chelator. C57/BL6 mice were injected with 89Zr-PEGylated VHHs, and images were acquired 24 h post injection. Images of the PEGylated-VHHs showed specific staining with low background in kidney and intestine. When we used 89Zr-labeled PEGylated-VHHs specific for CD8 or Class II MHC, secondary lymphoid structures, including the mesenteric lymph node, were clearly visible. Knock-out mice were used to establish specificity of PET imaging. Mice bearing panc02 tumors were imaged at 24 h p.i. and showed CD8 T cells infiltrating the tumor. We monitored the CD8-T cell response to immunotherapy in the B16 melanoma model by using the antiCD8 VHH. For animals that showed a response that led to regression of the tumor, the distribution of the signal was homogeneous throughout the tumor. It is thus possible to monitor antitumor immune responses. Noninvasive monitoring could therefore change how therapy is applied. Citation Format: Mohammad Rashidian, Michael Dougan, Jessica Ingram, Hidde Ploegh. Noninvasive imaging of antitumor immune responses. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B18.

Genomic modifiers of natural killer cells, immune responsiveness and lymphoid tissue remodeling together increase host resistance to viral infection

Abstract The MHC class I Dk molecule supplies vital host resistance during murine cytomegalovirus (MCMV) infection. Natural killer (NK) cells expressing the Ly49G2 inhibitory receptor, which specifically binds Dk, are required to control viral spread. The extent of Dk-dependent host resistance, however, differs significantly amongst related strains of mice, C57L and MA/My. As a result, we predicted that relatively small-effect modifier genetic loci might together shape immune cell features, NK cell reactivity, and the host immune response to MCMV. A robust Dk-dependent genetic effect, however, has so far hindered attempts to identify additional host resistance factors. Thus, we applied genomic mapping strategies and multicolor flow cytometric analysis of immune cells in naive and virus-infected hosts to identify genetic modifiers of the host immune response to MCMV. We discovered and validated many quantitative trait loci (QTL); these were mapped to at least 19 positions on 16 chromosomes. Intriguingly, one newly discovered non-MHC locus (Cmv5) controlled splenic NK cell accrual, secondary lymphoid organ structure, and lymphoid follicle development during MCMV infection. We infer that Cmv5 aids host resistance to MCMV infection by expanding NK cells needed to preserve and protect essential tissue structural elements, to enhance lymphoid remodeling and to increase viral clearance in spleen.