Kristin G. Anderson

Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8+ T cells

Cell-mediated immunity critically depends on the localization of lymphocytes at sites of infection. While some memory T cells recirculate, a distinct lineage (resident memory T cells (TRM cells)) are embedded in nonlymphoid tissues (NLTs) and mediate potent protective immunity. However, the defining transcriptional basis for the establishment of TRM cells is unknown. We found that CD8+ TRM cells lacked expression of the transcription factor KLF2 and its target gene S1pr1 (which encodes S1P1, a receptor for sphingosine 1-phosphate). Forced expression of S1P1 prevented the establishment of TRM cells. Cytokines that induced a TRM cell phenotype (including transforming growth factor-β (TGF-β), interleukin 33 (IL-33) and tumor-necrosis factor) elicited downregulation of KLF2 expression in a pathway dependent on phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt, which suggested environmental regulation. Hence, regulation of KLF2 and S1P1 provides a switch that dictates whether CD8+ T cells commit to recirculating or tissue-resident memory populations.

Intravascular staining for discrimination of vascular and tissue leukocytes

Characterization of the cellular participants in tissue immune responses is crucial to understanding infection, cancer, autoimmunity, allergy, graft rejection and other immunological processes. Previous reports indicate that leukocytes in lung vasculature fail to be completely removed by perfusion. Several studies suggest that intravascular staining may discriminate between tissue-localized and blood-borne cells in the mouse lung. Here we outline a protocol for the validation and use of intravascular staining to define innate and adaptive immune cells in mice. We demonstrate application of this protocol to leukocyte analyses in many tissues and we describe its use in the contexts of lymphocytic choriomeningitis virus and Mycobacterium tuberculosis infections or solid tumors. Intravascular staining and organ isolation usually takes 5–30 min per mouse, with additional time required for any subsequent leukocyte isolation, staining and analysis. In summary, this simple protocol should help enable interpretable analyses of tissue immune responses.

Cutting Edge: Intravascular Staining Redefines Lung CD8 T Cell Responses

Nonlymphoid T cell populations control local infections and contribute to inflammatory diseases, thus driving efforts to understand the regulation of their migration, differentiation, and maintenance. Numerous observations indicate that T cell trafficking and differentiation within the lung are starkly different from what has been described in most nonlymphoid tissues, including intestine and skin. After systemic infection, we found that >95% of memory CD8 T cells isolated from mouse lung via standard methods were actually confined to the pulmonary vasculature, despite perfusion. A respiratory route of challenge increased virus-specific T cell localization within lung tissue, although only transiently. Removing blood-borne cells from analysis by the simple technique of intravascular staining revealed distinct phenotypic signatures and chemokine-dependent trafficking restricted to Ag-experienced T cells. These results precipitate a revised model for pulmonary T cell trafficking and differentiation and a re-evaluation of studies examining the contributions of pulmonary T cells to protection and disease.

Lymphocytic choriomeningitis virus persistence promotes effector-like memory differentiation and enhances mucosal T cell distribution.

Vaccines are desired that maintain abundant memory T cells at nonlymphoid sites of microbial exposure, where they may be anatomically positioned for immediate pathogen interception. Here, we test the impact of antigen persistence on mouse CD8 and CD4 T cell distribution and differentiation by comparing responses to infections with different strains of LCMV that cause either acute or chronic infections. We used in vivo labeling techniques that discriminate between T cells present within tissues and abundant populations that fail to be removed from vascular compartments, despite perfusion. LCMV persistence caused up to ∼30‐fold more virus‐specific CD8 T cells to distribute to the lung compared with acute infection. Persistent infection also maintained mucosal‐homing α4β7 integrin expression, higher granzyme B expression, alterations in the expression of the TRM markers CD69 and CD103, and greater accumulation of virus‐specific CD8 T cells in the large intestine, liver, kidney, and female reproductive tract. Persistent infection also increased LCMV‐specific CD4 T cell quantity in mucosal tissues and induced maintenance of CXCR4, an HIV coreceptor. This study clarifies the relationship between viral persistence and CD4 and CD8 T cell distribution and mucosal phenotype, indicating that chronic LCMV infection magnifies T cell migration to nonlymphoid tissues.

Within-person variability of the ratios of urinary 2-hydroxyestrone to 16α-hydroxyestrone in Caucasian women☆ ☆

The ratio of urinary 2-hydroxyestrone (2-OHE1) to 16alpha-hydroxyestrone (16alpha-OHE1) has been suggested as a potential biomarker for breast cancer risk. We evaluated within-person variability of this biomarker in ten healthy Caucasian women aged 23-58 years. Each study participant was asked to provide an overnight fasting morning urine sample once a week for an average of 8 weeks. These urine samples were assayed for 2-OHE1 and 16alpha-OHE1 by using competitive enzyme immunoassay kits purchased from the ImmunaCare Corporation. The coefficients of variation for urinary 2-OHE1/16alpha-OHE1 over the study period ranged from 13.7 to 59.6% (mean, 33.3%) in our study participants. There was a good correlation between the level of the urinary 2-OHE1/16alpha-OHE1 ratio in any single urine sample and the average ratio over the 8-week study period from the same woman, with the mean correlation coefficient of 0.85. These results indicated that the within-person variation of the 2-OHE1 to 16alpha-OHE1 ratio for most women was moderate and the level of this ratio in a single urine sample, in general, reflects reasonably well the level of this biomarker over a 2-month period.

CpG-mediated modulation of MDSC contributes to the efficacy of Ad5-TRAIL therapy against renal cell carcinoma

Abstract Tumor progression occurs through the modulation of a number of physiological parameters, including the development of immunosuppressive mechanisms to prevent immune detection and response. Among these immune evasion mechanisms, the mobilization of myeloid-derived suppressor cells (MDSC) is a major contributor to the suppression of antitumor T-cell immunity. Patients with renal cell carcinoma (RCC) show increased MDSC, and methods are being explored clinically to reduce the prevalence of MDSC and/or inhibit their function. In the present study, we investigated the relationship between MDSC and the therapeutic potential of a TRAIL-encoding recombinant adenovirus (Ad5-TRAIL) in combination with CpG-containing oligodeoxynucleotides (Ad5-TRAIL/CpG) in an orthotopic mouse model of RCC. This immunotherapy effectively clears renal (Renca) tumors and enhances survival, despite the presence of a high frequency of MDSC in the spleens and primary tumor-bearing kidneys at the time of treatment. Subsequent analyses revealed that the CpG component of the immunotherapy was responsible for decreasing the frequency of MDSC in Renca-bearing mice; further, treatment with CpG modulated the phenotype and function of MDSC that remained after immunotherapy and correlated with an increased T-cell response. Interestingly, the CpG-dependent alterations in MDSC frequency and function did not occur in tumor-bearing mice complicated with diet-induced obesity. Collectively, these data suggest that in addition to its adjuvant properties, CpG also enhances antitumor responses by altering the number and function of MDSC.

Shortened Intervals during Heterologous Boosting Preserve Memory CD8 T Cell Function but Compromise Longevity

Developing vaccine strategies to generate high numbers of Ag-specific CD8 T cells may be necessary for protection against recalcitrant pathogens. Heterologous prime-boost-boost immunization has been shown to result in large quantities of functional memory CD8 T cells with protective capacities and long-term stability. Completing the serial immunization steps for heterologous prime-boost-boost can be lengthy, leaving the host vulnerable for an extensive period of time during the vaccination process. We show in this study that shortening the intervals between boosting events to 2 wk results in high numbers of functional and protective Ag-specific CD8 T cells. This protection is comparable to that achieved with long-term boosting intervals. Short-boosted Ag-specific CD8 T cells display a canonical memory T cell signature associated with long-lived memory and have identical proliferative potential to long-boosted T cells Both populations robustly respond to antigenic re-exposure. Despite this, short-boosted Ag-specific CD8 T cells continue to contract gradually over time, which correlates to metabolic differences between short- and long-boosted CD8 T cells at early memory time points. Our studies indicate that shortening the interval between boosts can yield abundant, functional Ag-specific CD8 T cells that are poised for immediate protection; however, this is at the expense of forming stable long-term memory.

Editorial: Pulmonary resident memory CD8 T cells: here today, gone tomorrow

Infection with influenza A virus can result in the establishment of crossreactive protective immunity against subsequent exposure to influenza A viruses of a distinct serotype. This phenomenon, termed heterosubtypic immunity, is conferred by the adaptive arm of the immune system and is predominantly attributed to T cell detection of shared viral protein epitopes. Paradoxically, heterosubtypic immunity to influenza virus wanes over time [1], despite the fact that long-lived, influenza-specific memory T cells persist within secondary lymphoid organs (Fig. 1A). For some time, memory T cells have been characterized as TCM, which recirculate among blood, lymph, and secondary lymphoid organs, or TEM, which were all thought to recirculate among blood, lymph, and nonlymphoid tissues. Recently, a nonrecirculating subset of memory T cells that resides permanently in nonlymphoid tissues has been described in several organs, including the skin, intestine, kidney, brain, lung, and female reproductive tract [2]. This subset is most often referred to as TRM cells, which have been shown to protect against viral infections in the skin [3, 4] and lung [5] and precipitate local inflammatory cascades that recruit circulating T cells to sites of infection [6]. It has been noted that TRM cells often express the C-type lectin CD69, as well as the E 7 integrin heterodimer (which is most often identified by staining cells with antibodies specific for E, otherwise known as CD103) [2]. CD69 may be associated with retention of TRM, as expression antagonizes S1P responsiveness, and S1P promotes egress of lymphocytes into the circulation. E 7 may also contribute directly to the local maintenance of TRM by anchoring T lymphocytes to epithelial cells through interactions with E-cadherin. However, many critical questions remain: what induces CD69 and CD103 expression among T cells in different tissue compartments? What is the longevity of TRM within different locations? How are TRM established within a particular location? Under what conditions do TRM contribute to protection? In this issue of the Journal of Leukocyte Biology, Wu and colleagues [7] help address these questions by revisiting an old mystery: the enigma that heterosubtypic immunity to respiratory influenza challenge is not long-lasting. Heterosubtypic immunity was examined using two serologically distinct recombinant strains of influenza virus that express chicken ovalbumin (WSN-OVAI and X31-OVA), as well as transgenic CD8 T cells (OT-I) expressing a TCR that recognizes the OVA-derived SIINFEKL peptide when it is presented by H-2K-bearing cells. One month after challenging mice with WSN-OVAI via the respiratory route, but not the i.p. route, OT-I cells were found in the epithelial layer lining the large lung airways. This demonstrated that the site of primary challenge impacted the establishment of T cell memory in the lung. Additionally, many memory T cells in the airway epithelium expressed CD103, consistent with the phenotype of TRM. Further analysis revealed that mice challenged only a month earlier with WSN-OVAI exhibited much more rapid control upon a subsequent heterosubtypic challenge with X31-OVA. In fact, a significant reduction in X31-OVA titers preceded the recruitment of CD8 T cells from outside of the lung. This implies that TRM within the lung, rather than recirculating memory T cells, were most responsible for viral control. This interpretation was supported by the observation that CD103 CD8 T cells in the lung epithelium dissipated within 7 months, and it was their loss that correlated with the waning of heterosubtypic immunity. However, many questions remain. Why is local infection important for the establishment of TRM within the lung epithelium? Is this a result of programming of a homing phenotype during priming, or is the infectious milieu of an infected lung driving recruitment? More importantly, what is regulating the short-term maintenance of local TRM and the subsequent attrition? Interestingly, TRM do not appear to wane significantly in the skin and intestinal mucosa [3, 4, 8], suggesting that the lung is unique in this regard. Why is the lung different, teleologically and mechanistically? One possible difference between tissues is in the regulation of CD103 itself. Many studies implicate a role for TGFin driving CD103 expression. TGFis constitutively expressed in the small intestinal mucosa, where CD103 is maintained on virtually all memory CD8 T cells within the epithelium, and interfering with TGFsignaling or CD103 expression results in a gradual loss of intestinal intraepithelial memory CD8 T cells [9–11]. In contrast, CD103 is expressed by only a minority of CD8 T cells in the lung, and many of these cells are lost over time. Perhaps TGFis also the driver of CD103 expression in the respiratory tract, but available

Abstract 4980: Engineering adoptive T cell therapy for efficacy in ovarian cancer

Over 20,000 women are diagnosed with ovarian cancer annually - more than half will die within 5 years and this rate has changed very little in the last 20 years, highlighting the need for innovative therapies. One promising new treatment strategy has the potential to control tumor growth without toxicity to healthy tissues, by employing immune T cells engineered to target proteins uniquely overexpressed in tumors. Recent technological advances have helped identify and validate Wilms’ Tumor Antigen 1 (WT1) and mesothelin (MSLN) as valid antigen targets for ovarian cancer, as these proteins contribute to malignant and invasive phenotypes and have limited expression in healthy cells. In preclinical studies using either patient-derived cell lines or the mouse ID8 ovarian tumor model, we found that T cells engineered to express either a WT1- or MSLN- specific high-affinity T cell receptor (TCR) can kill human and murine ovarian tumor cells in vitro. Moreover, in a disseminated in vivo murine model, adoptively transferred TCR-engineered T cells preferentially accumulated within established ID8 tumors, delayed ovarian tumor growth and prolonged mouse survival. However, our data also revealed that the tumor microenvironment (TME) can limit engineered T cell persistence and killing capacity. Cellular and molecular analyses showed human therapy will face similar TME-mediated obstacles. The ovarian cancer TME is a nutrient- and oxygen-deprived milieu, and adaptive metabolic responses by infiltrating T cells have protean effects on T cell function. Thus, strategies that modulate T cell metabolic pathways, and thereby influence activity in the TME, might enhance T cell function and improve anti-tumor efficacy by overcoming a critical component of immune evasion by solid tumors. Ongoing studies will be discussed that are exploring strategies to overcome elements common to the human and murine TME, including direct modulation of the environment and T cell engineering to promote T cell survival and function. Citation Format: Kristin G. Anderson, Breanna M. Bates, Edison Y. Chiu, Philip D. Greenberg. Engineering adoptive T cell therapy for efficacy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4980. doi:10.1158/1538-7445.AM2017-4980

Abstract SY31-03: Employing TCRs in engineered T cells to develop therapeutic reagents for effectively targeting malignancies

Effective cellular therapy for human malignancies requires first identifying and validating an appropriate antigenic target, and then establishing in each patient a tumor-reactive T cell response of high avidity and high magnitude that is safe and can infiltrate and retain function in the tumor microenvironment. We have been exploring in preclinical models and clinical trials methods to reproducibly provide such responses by transfer of genetically engineered T cells that acquire target specificity by virtue of an introduced high affinity TCR. To identify candidate antigens in leukema, we examined purified human leukemic stem cells for over-expression of genes based on comparisons to purified human hematopoietic stem cells as well as normal somatic tissues. Our analysis revealed that WT1, a gene known to be associated with promoting leukemic transformation, is expressed in comparative abundance in human leukemic stem cells. Preclinical studies were then performed in a mouse model, and revealed that CD8 T cells specific for this oncogene with even higher avidity than can be detected in normal repertoires could be safely administered, with no evidence of toxicity to the normal tissues known to express low but detectable levels of WT1. For our initial clinical trial, poor prognosis leukemia patients who relapsed after hematopoietic cell transplant (HCT) were treated with transfer of WT1-specific CD8 T cells clones isolated and expanded in vitro from the HCT donor. This study demonstrated that such T cells were safe, mediated in vivo anti-leukemic activity, and were associated with maintenance of long-term remissions in some patients, but generating sufficient numbers of WT1-specific CD8 T cells with high avidity for the target in each patient represented a substantive problem. Therefore, to create a more predictably effective standardized reagent for treatment of patients with a tumor that expresses the target antigen and shares the associated MHC restricting allele, we pursued methods to genetically engineer patient T cells to acquire high avidity for the tumor target. This requires identifying a high affinity TCR and producing a vector that can achieve high-level expression of the genes encoding the Vα and Vβ genes of a TCR demonstrated to have high affinity for the target epitope. Therefore, we screened a large number of normal repertoires for the presence of high avidity WT1-specific CD8 T cells, and selected the T cell clone expressing the highest affinity TCR. We then incorporated changes in the TCR genes such as codon optimization to enhance expression, and introduced a point mutation in each chain to create a disulfide bond that minimizes the potential problem of mispairing of the introduced TCR chains with the endogenous TCR chains. We have now have now initiated a trial in which this high affinity, WT1-specific, HLA-A2-restricted TCR is being introduced into patient CD8 T cells with a lentiviral vector and the transduced cells are being infused into the patient. The early results from this trial appear promising in terms of both evidence of antileukemic activity and the capacity for the transferred cells to persist in patients, and we plan to begin very shortly another trial in patients with non-small cell lung cancer (NSCLC) utilizing this same TCR, as WT1 is also commonly overexpressed in NSCLC as well as many other malignancies. For many candidate target antigens that are also normal self-antigens, isolating high affinity TCRs may not be readily achieved from normal repertoires. Therefore, we have developed strategies to enhance the affinity of isolated TCRs with retention of specificity, including saturation mutagenesis of CDR3 regions and an in vitro thymic selection system that allows for capture of a more diverse set of high affinity specific TCR genes during TCR gene rearrangement. These approaches induce modifications to the TCR region that predominantly makes contacts with the peptide epitope rather than MHC, which is necessary to minimize the risk of off-target toxicity from promiscuous peptide/MHC recognition. However, it remains essential that such modified TCRs do not induce unanticipated tissue damage, and we are using bioinformatics as well as modeling in the mouse to uncover any potential for off-target toxicity. Unfortunately, providing a high avidity T cell response does not necessarily result in tumor eradication, as there are other substantive obstacles that can preclude even a T cell expressing a high affinity TCR from being effective. These impediments include the development of T cell dysfunction, particularly within the microenvironment of solid tumors, and we are using genetically engineered mouse models to elucidate the cellular and molecular pathways that need to be modulated to achieve meaningful therapeutic benefit in a variety of solid tumor settings, including pancreatic and ovarian cancer. Our preclinical therapy studies, particularly in a pancreatic ductal adenocarcinoma (PDA) model, already appear very promising, as we have demonstrated that T cells expressing a high affinity TCR targeting a tumor antigen expressed by PDA cells can infiltrate the tumor, mediate tumor lysis, modify the tumor stroma, and provide therapeutic benefit. We have already identified high affinity human TCRs specific for this tumor antigen, and plan to use the insights derived from these studies to initiate within the next 1-2 years clinical trials in human pancreatic and ovarian cancers. The genetically-engineered mouse models of spontaneously developing tumors we are using, which recapitulate many aspects of the analogous human cancer, are also making it possible to assess strategies to improve the efficacy of T cell therapy. These models have helped elucidate the importance of not only cell extrinsic mechanisms of regulation and dysfunction that render T cells unresponsive, particularly via inhibitory cells commonly present in the tumor microenvironment that interfere with an effector response such as the accumulation of regulatory CD4 T cells (Treg), myeloid derived suppressor cells (MDSC), and tumor-associated macrophages (TAM), but also the cell intrinsic mechanisms that derive in large part from persistent stimulation by the tumor antigen and ultimately can render T cells progressively dysfunctional, leading to epigenetic modifications that eventually result in non-responsive cells that cannot be readily rescued. These cumulative mechanisms highlight the difficulties eliciting and/or sustaining responses to tumor antigens. Strategies to disrupt inhibitory pathways by systemic administration of mAbs or cytokines are currently being pursued clinically, but such reagents globally disrupt inhibitory pathways which can have significant toxicity to the host. Therefore, we are evaluating strategies to sustain function and anti-tumor activity by genetically modifying T cells to enhance function and to be resistant to obstacles that prevent tumor eradication. As different tumor types exhibit unique characteristics and are capable of engaging distinct inhibitory pathways, improved understanding of the immunobiology of the tumor type to be treated will likely prove essential for designing effective therapies. However, the relatively straightforward means to use synthetic biology to genetically engineer T cells to acquire novel capacities to overcome inhibitory signals and function in the tumor microenvironment suggests that cancer therapy with engineered T cells will likely find an increasing role in the treatment of human cancers. Citation Format: Philip D. Greenberg, Tom M. Schmitt, Andrea Schietinger, Ingunn M. Stromnes, Sunil R. Hingorani, Shannon K. Oda, Rachel Perret, Kristin G. Anderson, Merav Bar, Aude G. Chapuis. Employing TCRs in engineered T cells to develop therapeutic reagents for effectively targeting malignancies. [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 SY31-03. doi:10.1158/1538-7445.AM2015-SY31-03