Cell-mediated Immune Control Research Articles

Active Tuberculosis Is Associated with Depletion of HIV-Specific CD4 and CD8 T Cells in People with HIV.

Infection with Mycobacterium tuberculosis (Mtb) in people with HIV (PWH) is associated with depletion of Mtb-specific CD4 T cell responses, increased risk of progression to active tuberculosis (TB) disease, and increased immune activation. Although higher HIV viral loads have been reported in Mtb/HIV co-infection, the extent to which Mtb infection and TB disease impact the frequency and phenotype of HIV-specific T cell responses has not been well described. We enrolled a cohort of PWH in Kenya across a spectrum of Mtb infection states, including those with no evidence of Mtb infection, latent Mtb infection (LTBI), and active pulmonary TB disease, and evaluated the frequency, immune activation, and cytotoxicity phenotype of HIV-specific CD4 and CD8 T cell responses in peripheral blood by flow cytometry. We found evidence of depletion of HIV-specific CD4 and CD8 T cells in people with TB, but not with LTBI. Expression of the immune activation markers human leukocyte antigen-DR isotype (HLA-DR) and Ki67 and of the cytotoxic molecules granzyme B and perforin were increased in total CD4 and CD8 T cell populations in individuals with TB, although expression of these markers by HIV-specific CD4 and CD8 T cells did not differ by Mtb infection status. These data suggest that TB is associated with overall increased T cell activation and cytotoxicity and with depletion of HIV-specific CD4 and CD8 T cells, which may contribute to further impairment of T cell-mediated immune control of HIV replication in the setting of TB.

CD4 T Cell-Mediated Immune Control of Cytomegalovirus Infection in Murine Salivary Glands.

CD4 T cells are well known for their supportive role in CD8 T cell and B cell responses during viral infection. However, during murine cytomegalovirus (MCMV) infection in the salivary glands (SGs), CD4 T cells exhibit direct antiviral effector functions to control the infection. In this mucosal organ, opposed to other infected tissues, MCMV establishes a sustained lytic replication that lasts for several weeks. While the protective function of CD4 T cells is exerted through the production of the pro-inflammatory cytokines interferon gamma (IFNγ) and tumor necrosis factor alpha (TNF), the reasons for their markedly delayed control of lytic MCMV infection remain elusive. Here, we review the current knowledge on the dynamics and mechanisms of the CD4 T cell-mediated control of MCMV-infected SGs, including their localization in the SG in relation to MCMV infected cells and other immune cells, their mode of action, and their regulation.

Roles of Lytic Viral Replication and Co-Infections in the Oncogenesis and Immune Control of the Epstein-Barr Virus.

Simple SummaryThe Epstein–Barr virus (EBV) colonizes more than 95% of the adult human population. Its cancer-forming potential is usually contained by lifelong immune control. Genetic alterations and immune modulation by co-infection point towards cytotoxic lymphocytes, such as natural killer and CD8+ T cells, as the main pillars of this immune protection. In this review, we discuss how the EBV infection program that leads to infectious virion production and co-infections, such as with malaria parasites, the human immunodeficiency virus (HIV) and the Kaposi sarcoma-associated herpesvirus (KSHV), modulate this immune control.Epstein–Barr virus (EBV) is the prototypic human tumor virus whose continuous lifelong immune control is required to prevent lymphomagenesis in the more than 90% of the human adult population that are healthy carriers of the virus. Here, we review recent evidence that this immune control has not only to target latent oncogenes, but also lytic replication of EBV. Furthermore, genetic variations identify the molecular machinery of cytotoxic lymphocytes as essential for this immune control and recent studies in mice with reconstituted human immune system components (humanized mice) have begun to provide insights into the mechanistic role of these molecules during EBV infection. Finally, EBV often does not act in isolation to cause disease. Some of EBV infection-modulating co-infections, including human immunodeficiency virus (HIV) and Kaposi sarcoma-associated herpesvirus (KSHV), have been modeled in humanized mice. These preclinical in vivo models for EBV infection, lymphomagenesis, and cell-mediated immune control do not only promise a better understanding of the biology of this human tumor virus, but also the possibility to explore vaccine candidates against it.

Vaccination against the Epstein-Barr virus.

Epstein–Barr virus (EBV) was the first human tumor virus being discovered and remains to date the only human pathogen that can transform cells in vitro. 55 years of EBV research have now brought us to the brink of an EBV vaccine. For this purpose, recombinant viral vectors and their heterologous prime-boost vaccinations, EBV-derived virus-like particles and viral envelope glycoprotein formulations are explored and are discussed in this review. Even so, cell-mediated immune control by cytotoxic lymphocytes protects healthy virus carriers from EBV-associated malignancies, antibodies might be able to prevent symptomatic primary infection, the most likely EBV-associated pathology against which EBV vaccines will be initially tested. Thus, the variety of EBV vaccines reflects the sophisticated life cycle of this human tumor virus and only vaccination in humans will finally be able to reveal the efficacy of these candidates. Nevertheless, the recently renewed efforts to develop an EBV vaccine and the long history of safe adoptive T cell transfer to treat EBV-associated malignancies suggest that this oncogenic γ-herpesvirus can be targeted by immunotherapies. Such vaccination should ideally implement the very same immune control that protects healthy EBV carriers.

The Role of Dendritic Cells in Immune Control and Vaccination against γ-Herpesviruses

The two human oncogenic γ-herpesviruses, Epstein Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV), are prototypic pathogens that are controlled by T cell responses. Despite their ubiquitous distribution, persistent infections and transforming potential, most carriers’ immune systems control them for life. Therefore, they serve as paradigms of how near-perfect cell-mediated immune control can be initiated and maintained for decades. Interestingly, EBV especially quite efficiently avoids dendritic cell (DC) activation, and little evidence exists that these most potent antigen-presenting cells of the human body are involved in the priming of immune control against this tumor virus. However, DCs can be harnessed therapeutically to expand virus-specific T cells for adoptive transfer therapy of patients with virus-associated malignancies and are also currently explored for vaccinations. Unfortunately, despite 55 and 25 years of research on EBV and KSHV, respectively, the priming of their immune control that belongs to the most robust and durable immune responses in humans still remains unclear.

Eomes and IL-10 Regulate Anti-Tumor Activity of T Cells in Chronic Lymphocytic Leukemia

Introduction: Genome-wide association studies showed that a single-nucleotide polymorphism (SNP) affecting the transcription factor Eomesodermin (Eomes) is associated with a significantly increased risk to develop chronic lymphocytic leukemia (CLL). Eomes and its paralogue T-bet are known master regulators of CD8+ effector T cells and CD4+ T helper cells and critical for T cell-mediated immune responses against pathogens and cancer. While Eomes has been shown to be essential for effector function of CD8 T cells, its role in CD4 T cells is less well understood. Recent work suggested that Eomes drives the development of IL-10 and IFNγ co-producing, FoxP3-negative, regulatory T cells, named Tr1 cells, a population that was found enriched in inflamed tissues in patients with chronic inflammatory disorders. In CLL, the T cell compartment is altered with an enrichment of effector and memory T cells that were shown to control leukemia development in a mouse model, but also to acquire a dysfunctional or exhausted state. Methods: We investigated Eomes-expressing CD4 and CD8 T cells in blood and lymph node (LN) samples of patients with CLL, as well as in the Eµ-TCL1 mouse model of CLL by flow cytometry, CyTOF, mRNA sequencing, and ex vivo functional assays. The role and function of these cells was explored in bone marrow-chimeric mice harbouring an Eomes-deficient hematopoietic microenvironment that were used for adoptive transfer of TCL1 leukemia, as well as by co-transfer experiments of Eomes- or IL-10R-deficent CD4 or CD8 T cells with TCL1 leukemia cells in Rag2-/- mice lacking B and T cells. Results: We detected an accumulation of Eomes-expressing CD4 and CD8 T cells in CLL patients, which was more severe in LN compared to blood samples, and significantly stronger in CLL LN compared to reactive LN samples as non-cancer control (Fig. 1A). This was in line with an observed expansion of Eomes-positive T cells in the spleen of leukemic Eµ-TCL1 mice and upon adoptive transfer of TCL1 leukemia. Eomes expression in CD8 T cells correlated with the expression of CD69, IFNγ and PD-1, suggesting a link between Eomes and CD8 T cell activation and function in CLL. The importance of Eomes in CD8 T cell-mediated control of CLL was demonstrated in mice that were transplanted with TCL1 leukemia, where Eomes-deficient CD8 T cells failed to control leukemia development. As we detected significantly less Eomes-deficient CD8 T cells in these mice compared to respective wildtype controls, and a lower percentage of them was positive for the proliferation marker Ki-67, we conclude that Eomes drives the differentiation and expansion of CD8 T cells in mice with CLL-like disease. We further explored Eomes-expressing CD4 T cells in CLL by transcriptome analysis, flow cytometry and ex vivo functional assays, and observed increased expression of IFNγ and IL-10, as well as inhibitory receptors, like PD-1, BLIMP-1 and LAG3, features that are described for Tr1 cells. Transfer of Eomes-deficient or wildtype CD4 T cells in Rag2-/- mice that were injected with TCL1 leukemia cells, lead to a comparable expansion of CD4 T cells independent of Eomes. But even though wildtype CD4 T cells were able to control leukemia development in this setting, Eomes-deficient CD4 T cells failed to do so (Fig. 1B). As Eomes is a known driver of IL-10 expression, we tested whether IL-10R signalling in CD4 T cells is involved in the anti-tumor activity by performing respective CD4 T cell transfer experiments comparing this time wildtype with IL-10R-deficent CD4 T cells. Interestingly, lack of IL-10R in CD4 T cells lead to a reduction in anti-tumor control (Fig. 1C) and therefore suggests that IL-10 is involved in Eomes-driven regulation of CD4 T cell-mediated immune control. Conclusions: In summary, we conclude that Eomes is required for CD8 T cell-mediated control of CLL, and Eomes+ PD-1+ IL-10-producing CD4 T cells contribute to adaptive immunity in CLL. The increased risk of developing CLL in individuals harbouring a SNP in the Eomes gene might be therefore explained by a negative impact of this alteration on CD4 and/or CD8 T cell-mediated immune control of CLL. Disclosures Stilgenbauer: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Toward T Cell-Mediated Control or Elimination of HIV Reservoirs: Lessons From Cancer Immunology.

As the AIDS epidemic unfolded, the appearance of opportunistic infections in at-risk persons provided clues to the underlying problem: a dramatic defect in cell-mediated immunity associated with infection and depletion of CD4+ T lymphocytes. Moreover, the emergence of HIV-associated malignancies in these same individuals was a clear indication of the significant role effective cellular immunity plays in combating cancers. As research in the HIV field progressed, advances included the first demonstration of the role of PD-1 in human T cell exhaustion, and the development of gene-modified T cell therapies, including chimeric antigen receptor (CAR) T cells. In the intervening years, the oncology field has capitalized on these advances, effectively mobilizing the cellular immune response to achieve immune-mediated remission or cure of previously intractable cancers. Although similar therapeutic advances have not yet been achieved in the HIV field, spontaneous CD8+ T cell mediated remission or functional cure of HIV infection does occur in very small subset of individuals in the absence of anti-retroviral therapy (ART). This has many similarities to the CD8+ T cell mediated functional control or elimination of cancers, and indicates that immunotherapy for HIV is a rational goal. In HIV infection, one major barrier to successful immunotherapy is the small, persistent population of infected CD4+ T cells, the viral reservoir, which evades pharmacological and immune-mediated clearance, and is largely maintained in secondary lymphoid tissues at sites where CD8+ T cells have limited access and/or function. The reservoir-enriched lymphoid microenvironment bears a striking resemblance to the tumor microenvironment of many solid tumors–namely high levels of anti-inflammatory cytokines, expression of co-inhibitory receptors, and physical exclusion of immune effector cells. Here, we review the parallels between CD8+ T cell-mediated immune control of HIV and cancer, and how advances in cancer immunotherapy may provide insights to direct the development of effective HIV cure strategies. Specifically, understanding the impact of the tissue microenvironment on T cell function and development of CAR T cells and therapeutic vaccines deserve robust attention on the path toward a CD8+ T cell mediated cure of HIV infection.

Are we there yet? The never-ending quest for an Epstein-Barr virus vaccine.

The Epstein-Barr virus (EBV) is estimated to infect a large part of the population and is associated with a variety of human tumors; therefore, EBV is an important target for vaccine development. In this issue of the JCI, Rühl et al. developed a promising heterologous prime-boost vaccination strategy for EBV-associated malignancies and symptomatic primary infection. The authors show that two prime-boost regimens, using either dendritic cells or an adenovirus approach targeting nuclear antigen EBNA1 followed by a modified vaccinia virus Ankara (MVA) booster, induced significant T cell-mediated, EBV-specific immune control and Ab production. These findings suggest that administration of heterologous prime-boost vaccinations targeting EBNA1 may result in potent CD4+ and CD8+ T cell-mediated EBV immune control and may be a promising clinical approach.

Infection and immune control of human oncogenic γ-herpesviruses in humanized mice.

Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) comprise the oncogenic human γ-herpesvirus family and are responsible for 2-3% of all tumours in man. With their prominent growth-transforming abilities and high prevalence in the human population, these pathogens have probably shaped the human immune system throughout evolution for near perfect immune control of the respective chronic infections in the vast majority of healthy pathogen carriers. The exclusive tropism of EBV and KSHV for humans has, however, made it difficult in the past to study their infection, tumourigenesis and immune control in vivo. Mice with reconstituted human immune system components (humanized mice) support replication of both viruses with both persisting latent and productive lytic infection. Moreover, B-cell lymphomas can be induced by EBV alone and KSHV co-infection with gene expression hallmarks of human malignancies that are associated with both viruses. Furthermore, cell-mediated immune control by primarily cytotoxic lymphocytes is induced upon infection and can be probed for its functional characteristics as well as putative requirements for its priming. Insights that have been gained from this model and remaining questions will be discussed in this review. This article is part of the theme issue 'Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses'.

Human γ-Herpesvirus Infection, Tumorigenesis, and Immune Control in Mice with Reconstituted Human Immune System Components.

The human γ-herpesviruses Epstein–Barr virus (EBV or HHV4) and Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) are each associated with around 2% of all tumors in humans worldwide. However, investigations into their infection, oncogenesis, and immune responses that protect from the associated tumors have been hampered by the exclusive tropism of these pathogens for humans. Mice with reconstituted human immune system components (HIS mice) provide the unique opportunity to study persistent infection, virus associated lymphoma formation, and cell-mediated immune control of EBV and KSHV. Moreover, since these pathogens are unique stimuli for cytotoxic human lymphocyte responses, they also allow us to characterize long-lasting cell-mediated immune control and the requirements for its initiation, which would also be desirable to achieve during antitumor vaccination in general. Thus, human γ-herpesvirus infection of HIS mice provides unique insights into the biology of these important human pathogens and human cell-mediated immune responses that are considered to be the main protective entity against tumors.

Cognate HLA absence in trans diminishes human NK cell education.

NK cells are innate lymphocytes with protective functions against viral infections and tumor formation. Human NK cells carry inhibitory killer cell Ig-like receptors (KIRs), which recognize distinct HLAs. NK cells with KIRs for self-HLA molecules acquire superior cytotoxicity against HLA- tumor cells during education for improved missing-self recognition. Here, we reconstituted mice with human hematopoietic cells from donors with homozygous KIR ligands or with a mix of hematopoietic cells from these homozygous donors, allowing assessment of the resulting KIR repertoire and NK cell education. We found that co-reconstitution with 2 KIR ligand-mismatched compartments did not alter the frequency of KIR-expressing NK cells. However, NK cell education was diminished in mice reconstituted with parallel HLA compartments due to a lack of cognate HLA molecules on leukocytes for the corresponding KIRs. This change in NK cell education in mixed human donor-reconstituted mice improved NK cell-mediated immune control of EBV infection, indicating that mixed hematopoietic cell populations could be exploited to improve NK cell reactivity against leukotropic pathogens. Taken together, these findings indicate that leukocytes lacking cognate HLA ligands can disarm KIR+ NK cells in a manner that may decrease HLA- tumor cell recognition but allows for improved NK cell-mediated immune control of a human γ-herpesvirus.

KLRG1 and PD-1 expression are increased on T-cells following tuberculosis-treatment and identify cells with different proliferative capacities in BCG-vaccinated adults

In cancer and chronic infectious diseases, immune checkpoint-blockade of inhibitory receptors can enhance T-cell immunity. In tuberculosis (TB), a chronic infectious disease, prolonged antigen exposure can potentially drive terminal T-cell differentiation towards functional 'exhaustion': in human TB T-cells express PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T-lymphocyte-associated protein-4). However, in murine TB not PD-1 but rather killer cell lectin-like receptor subfamily-G1 (KLRG1) was a superior indicator of terminal T-cell differentiation. We therefore compared expression of KLRG1, PD-1 and CTLA-4 on T-cells in different stages of human TB, and also analysed their induction following BCG-vaccination. KLRG1, PD-1 and CTLA-4-expression were highest on in vitro BCG-stimulated CD4(+) T-cells following recent TB-treatment; KLRG1 and PD-1-expression on CD4(+) T-cells in active--but not latent--TB were only slightly increased compared to healthy donors. BCG-vaccination induced KLRG1-expression on BCG-stimulated CD8(+) but not CD4(+) T-cells, while neither PD-1 nor CTLA-4-expression increased. KLRG1-expressing CD8(+) T-cells exhibited markedly decreased proliferation, whereas PD-1(+) T-cells proliferated after in vitro BCG-stimulation. Thus, we demonstrate the presence of increased KLRG1-expressing T-cells in TB-treated individuals, and present KLRG1 as a marker of decreased human T-cell proliferation following BCG-vaccination. These results expand our understanding of cell-mediated immune control of mycobacterial infections.

A nonhuman primate scrub typhus model: protective immune responses induced by pKarp47 DNA vaccination in cynomolgus macaques.

We developed an intradermal (ID) challenge cynomolgus macaque (Macaca fascicularis) model of scrub typhus, the leading cause of treatable undifferentiated febrile illness in tropical Asia, caused by the obligate intracellular bacterium, Orientia tsutsugamushi. A well-characterized animal model is required for the development of clinically relevant diagnostic assays and evaluation of therapeutic agents and candidate vaccines. We investigated scrub typhus disease pathophysiology and evaluated two O. tsutsugamushi 47-kDa, Ag-based candidate vaccines, a DNA plasmid vaccine (pKarp47), and a virus-vectored vaccine (Kp47/47-Venezuelan equine encephalitis virus replicon particle) for safety, immunogenicity, and efficacy against homologous ID challenge with O. tsutsugamushi Karp. Control cynomolgus macaques developed fever, classic eschars, lymphadenopathy, bacteremia, altered liver function, increased WBC counts, pathogen-specific Ab (IgM and IgG), and cell-mediated immune responses. Vaccinated macaques receiving the DNA plasmid pKarp47 vaccine had significantly increased O. tsutsugamushi–specific, IFN-γ–producing PBMCs (p = 0.04), reduced eschar frequency and bacteremia duration (p ≤ 0.01), delayed bacteremia onset (p < 0.05), reduced circulating bacterial biomass (p = 0.01), and greater reduction of liver transaminase levels (p < 0.03) than controls. This study demonstrates a vaccine-induced immune response capable of conferring sterile immunity against high-dose homologous ID challenge of O. tsutsugamushi in a nonhuman primate model, and it provides insight into cell-mediated immune control of O. tsutsugamushi and dissemination dynamics, highlights the importance of bacteremia indices for evaluation of both natural and vaccine-induced immune responses, and importantly, to our knowledge, has determined the first phenotypic correlates of immune protection in scrub typhus. We conclude that this model is suitable for detailed investigations into vaccine-induced immune responses and correlates of immunity for scrub typhus.

Determination of EBV serostatus prior to kidney transplantation: Comparison of VIDAS®, LIAISON® and immunofluorescence assays

Immunosuppression following solid organ transplantation reduces T cell-mediated immune control of Epstein–Barr Virus (EBV), which may then drive development of post-transplant lymphoproliferative disease. Serology plays a key role in determination of risk of outgrowth of such lesions following transplantation.The study compared the VIDAS® (bioMérieux) and LIAISON® (DiaSorin) enzyme immunoassays (EIAs) and immunofluorescence assays (IFA; MBL-Bion) in the kidney transplantation setting. Sera from 100 live kidney donors [51 males; age range 20–82 years (mean 51.2 years)] and 100 cadaveric kidney recipients [70 males; age range 17–77 years (mean 51.0 years)] were tested. Overall proportional agreement ranged from 96% to 100% for VIDAS® and LIAISON®. Sensitivity ranged from 91% to 100% and 92% to 100% for VIDAS®/IFA and LIAISON®/IFA, respectively.The VIDAS® and LIAISON® approaches gave similar results. Such automated random access EIAs are well suited to busy clinical virology laboratories and rapid determination of donor and recipient EBV serostatus prior to transplantation.

CD4+T Cells: Guardians of the Phagosome

CD4(+) T cells are key cells of the adaptive immune system that use T cell antigen receptors to recognize peptides that are generated in endosomes or phagosomes and displayed on the host cell surface bound to major histocompatibility complex molecules. These T cells participate in immune responses that protect hosts from microbes such as Mycobacterium tuberculosis, Cryptococcus neoformans, Leishmania major, and Salmonella enterica, which have evolved to live in the phagosomes of macrophages and dendritic cells. Here, we review studies indicating that CD4(+) T cells control phagosomal infections asymptomatically in most individuals by secreting cytokines that activate the microbicidal activities of infected phagocytes but in a way that inhibits the pathogen but does not eliminate it. Indeed, we make the case that localized, controlled, persistent infection is necessary to maintain large numbers of CD4(+) effector T cells in a state of activation needed to eradicate systemic and more pathogenic forms of the infection. Finally, we posit that current vaccines for phagosomal infections fail because they do not produce this "periodic reminder" form of CD4(+) T cell-mediated immune control.

A Novel Model of Immune-Mediated Disease Equilibrium in Acute Lymphoblastic Leukemia

AbstractAbstract 3540Acute lymphoblastic leukemia (ALL) is a challenging target for immunotherapy due to its poor antigen presentation, lack of costimulatory molecule expression, and resistance to cytotoxic killing by immune effector cells. In order to develop new strategies to generate autologous immune-mediated protection against ALL progression, we sought to understand more about the immune requirements that must be met in order to achieve disease control. To do this, we have transfected B cell precursor ALL cell lines derived from leukemic Eμ-ret transgenic BALB/c mice with a lentiviral construct encoding green fluorescent protein (GFP) and firefly luciferase (luc) in tandem. The luciferase protein serves a dual purpose as a potent neoantigen capable of stimulating leukemia-specific immune responses and as a bioluminescent reporter for highly sensitive detection of minimal residual disease (MRD).Intravenous injection of 106 non-transfected Eμ-ret-derived leukemia cells is consistently lethal in wild type BALB/c mice within 3–5 weeks. However, adoptive transfer of 106 GFP/luc-expressing leukemia cells into wild type BALB/c mice does not lead to morbidity or mortality (p=0.025, logrank for survival). These mice show detectable disease in bone marrow by bioluminescent imaging below the limit of detection by peripheral blood flow cytometry that increases until day 7–10, followed by a spontaneous decrease in leukemia burden to undetectable levels by imaging. BALB/c mice lacking B and T cells (RAG1−/−) or B, T and NK cells (gamma common−/−) injected with GFP/luc ALL cells develop leukemia that is lethal by day 25 (p=0.037, logrank). Early T cell-depletion in wild type BALB/c mice on days 0, 3, 6, and 9 with CD4- and/or CD8-depleting antibodies also results in complete breakthrough of disease, while NK cell-depletion with asialo-GM1 on the same days does not significantly alter the timeframe of leukemia clearance (p<0.0001, 2-way ANOVA; Figure 1). These results demonstrate that the presence of a neoantigen is sufficiently immunogenic to generate effective T cell-mediated control of the disease.We have followed immunocompetent mice that received GFP/luc ALL for up to 12 months. While most mice have long-term remissions both by imaging and peripheral blood sampling, a subset have evidence of persistent MRD by bioluminescent imaging that waxes and wanes in luminosity but may not break through to a fulminant relapse. Relapses in these mice have occurred up to 11 months from injection. Late T cell-depletion with CD4 and CD8 antibodies (weekly x12, beginning at day 28) leads to an increased rate of leukemia breakthroughs and occurs more frequently in mice who had cleared their leukemia in the setting of early NK cell depletion (p<0.001, 2-way ANOVA), suggesting early crosstalk between NK and T cells to develop a sustained immune response. In addition, many mice relapse with GFP/luc-expressing leukemia, while a proportion of mice have late relapses with GFP/luc-negative leukemia, suggesting antigen loss is one of several potential mechanisms of immune evasion in ALL.Finally, BALB/c mice transgenic for GFP and luciferase do not generate T cell protection against outgrowth of injected GFP/luc ALL cells and succumb to disease by day 20 (p = 0.027 versus wild type mice, logrank). This result suggests that tolerance mechanisms, rather than antigen presentation deficiencies, may undermine host immune responses against ALL.We present a novel model of immune equilibrium in ALL, made evaluable by exquisitely sensitive bioluminescent imaging. Importantly, despite its well-documented lack of immunogenicity, we have shown ALL can be a sensitive target for long-term leukemia-specific T cell-mediated immune control. We are currently investigating additional cellular mechanisms of immune protection, the role of antigen spreading in control of ALL, and the mechanisms allowing leukemia to escape from immune control. Our approach will enable the development of strategies to break immune tolerance towards leukemia antigens and generate control of MRD in ALL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Loss of Mismatched HLA At Leukemia Relapse After Hematopoietic Stem Cell Transplantation Is Significantly Associated with Clinical and Immunogenetic Hallmarks of Donor-Versus-Host Alloreactivity

Abstract 1957 Background:In spite of the considerable progress achieved during the last decade in the field of allogeneic Hematopoietic Stem Cell Transplantation (HSCT), disease relapse remains a crucial unsolved issue, due to our limited insights into the biology of the interplay between leukemia and donor immunity. We and others have shown that genomic loss of patient-specific mismatched HLA is a frequent mechanism by which residual leukemic cells can evade donor T cell-mediated immune control and determine a clinical relapse. HLA loss relapses have been reported both after family haploidentical and after volunteer unrelated donor HSCT, but the actual incidence and risk factors for these peculiar relapses are to date unknown. Methods:We retrospectively evaluated 230 consecutive transplants performed over the last ten years in a single institution. All donors were partially HLA mismatched (family mismatched: 170; volunteer unrelated: 60). All patients were affected by high-risk myeloid malignancies (Acute Myeloid Leukemia (AML): 179; Myelodysplastic Syndrome (MDS): 27; Myeloproliferative Neoplasms: 17; Others: 7), received a fully myeloablative conditioning regimen, and infusion of donor T cells, either as part of the graft or as post-transplantation add-back. In addition to standard bone marrow morphological examination, post-transplantation follow-up comprised genomic HLA typing of bone marrow aspirate samples, aimed to identify HLA loss relapses. Results:A total of 83 relapses occurred, 72 and 11 after related and unrelated donor HSCT, respectively. We documented 21 relapses with genomic loss of the patient-specific mismatched HLA (25% of relapses). HLA loss occurred predominantly in patients with AML (n=19), but was also observed in patients with MDS (n=1) and primary myelofibrosis (n=1). None of the 11 cases of relapse after volunteer unrelated donor HSCT displayed HLA loss, whereas it was evident in 21 out of 72 (29%) relapses after family mismatched donor HSCT. Accordingly, the presence of multiple (more than 4) HLA mismatches between donor and recipient represented a significant risk factor for HLA loss (HR: 4.17, 95% CI: 0.88–19.82, p=0.07). None of the disease-related factors tested (morphological subtype, presence of dysplasia, hyperleukocytosis, cytogenetic and molecular profile) correlated significantly with HLA loss. In line with the hypothesis that selection of these leukemic variants may be elicited more frequently in the presence of a strong alloreactive immune pressure, HLA loss occurred more frequently in patients with clinical Graft-versu-Host Disease (GvHD), either acute (p=0.02) or chronic (p=0.007). Interestingly, HLA loss relapses occurred later than their “classical” counterparts (median time to relapse 307 vs 116 days, range 56–784 vs 10–579, p<0.0001), suggesting the existence of a long phase of equilibrium between leukemic cells and donor T cells before the occurrence of a de novo mutation and the outgrowth of immune escape variants. Of notice, HLA loss relapses shared several features with post-transplantation extramedullary relapses, suggesting a common biological drive leading to the two immune escape mechanisms. Conclusions:Loss of the mismatched HLA is a frequent mechanism of relapse for patients with high-risk myeloid malignancies, and is associated with donor-versus-host alloreactivity, as demonstrated by its significant correlation with the number of donor-recipient HLA mismatches and with the occurrence of GvHD. Ultimately, given the documented ineffectiveness of lymphocyte infusions from the original stem cell donor in patients with HLA loss relapses, efforts should be aimed towards the optimization of early detection tools and targeted therapeutic strategies specific for these peculiar and frequent relapse variants. Disclosures:Bordignon:MolMed SpA: Employment. Bonini:MolMed S.p.A.: Consultancy.

Cytolytic T lymphocytes from HLA-B8+ donors frequently recognize the Hodgkin's lymphoma associated latent membrane protein 2 of Epstein Barr virus

Epstein Barr virus (EBV) is a lymphotrophic γ-herpesvirus that infects more than 90% of the adult human population. It transforms B cells in vitro and is associated with lymphomas in vivo. In most EBV carriers the emergence of these malignancies, however, is prevented by T cell mediated immune control. Part of this control is mediated by CD8+ T cells, which mainly target a subset of viral nuclear antigens, EBNA3A, B and C, in healthy EBV carriers. In HLA-B8 positive individuals, the dominant CTL response is biased towards recognition of EBNA3A. However, spontaneously arising EBV-associated malignancies, such as Hodgkin's lymphoma and nasopharyngeal carcinoma do not express EBNA3s and instead express latent membrane protein 2 (LMP2) as well as LMP1 and EBNA1. Here we describe the new HLA-B8 restricted, LMP2 derived CD8+ T cell epitope, LMP2345-352. Although the frequency of LMP2345-352 specific CD8+ T cells is usually lower than immunodominant EBNA3A specific CD8+ T cells in fresh blood, the former can be expanded in the majority of HLA-B8+ EBV carriers after 1 week co-culture with peptide pulsed dendritic cells. We demonstrate that LMP2345-352 specific CD8+ T cells secrete IFN-γ and kill both peptide pulsed targets as well as HLA-B8 matched LCL and LMP2 expressing Hodgkin's lymphoma cells. We suggest that cytotoxic CD8+ T cell responses against LMP2 coexist with the immunodominant EBNA3 specific responses in healthy EBV carriers and help to resist EBV associated malignancies.

PD-1 expression on peripheral CD8+ TEM/TEMRA subsets closely correlated with HCV viral load in chronic hepatitis C patients

BackgroundTight correlation between host circulating CD8+ T cell-mediated immune response and control of viral replication is classical characteristic of long-term HCV infection. CD8+ T cell maturation/activation markers are expected to be associated with viral replication and disease progression in chronic HCV infection. The aim of the present study was to explore novel markers on CD8+ T cells with ability to evaluate HCV viral replication and disease progression.MethodsPBMCs were isolated from 37 chronic HCV-infected patients and 17 healthy controls. Distributed pattern of CD8+ T cells subsets and expression of PD-1, CD38, HLA-DR and CD127 were analyzed by flow cytometry. The correlation between expression of surface markers and HCV viral load or ALT was studied.ResultsDeclined naïve and increased TEMRA CD8+ T subsets were found in HCV-infected individuals compared with healthy controls. Percentage and MFI of PD-1, CD38 and HLA-DR on all CD8+ T cell subsets were higher in HCV-infected patients than healthy controls. In contrast, CD127 expression on CD8+ TCM showed an opposite trend as PD-1, CD38 and HLA-DR did. In chronic HCV infection, MFI of PD-1 on CD8+ TEM (p < 0.0001) and TEMRA (p = 0.0015) was positively correlated with HCV viral load while HLA-DR expression on non-naive CD8+ T cell subsets (p < 0.05) was negatively correlated with HCV viral load.ConclusionPD-1 level on peripheral CD8+ TEM/TEMRA was highly correlated with HCV viral load in chronic HCV-infected patients, which made PD-1 a novel indicator to evaluate HCV replication and disease progression in chronic hepatitis C patients.

Vaccine-Induced Memory CD8+ T Cells Cannot Prevent Central Nervous System Virus Reactivation

Noncytopathic viruses use multiple strategies to evade immune detection, challenging a role for vaccine induced CTL in preventing microbial persistence. Recrudescence of neurotropic coronavirus due to loss of T cell-mediated immune control provided an experimental model to test T cell vaccination efficacy in the absence of Ab. Challenge virus was rapidly controlled in vaccinated Ab-deficient mice coincident with accelerated recruitment of memory CD8+ T cells and enhanced effector function compared with primary CD8+ T cell responses. In contrast to primary effectors, reactivated memory cells persisted in the CNS at higher frequencies and retained ex vivo cytolytic activity. Nevertheless, despite earlier and prolonged T cell-mediated control in the CNS of vaccinated mice, virus ultimately reactivated. Apparent loss of memory CD8+ effector function in vivo was supported by a prominent decline in MHC expression on CNS resident target cells, presumably reflecting diminished IFN-gamma. Severely reduced MHC expression on glial cells at the time of recrudescence suggested that memory T cells, although fully armed to exert antiviral activity upon Ag recognition in vitro, are not responsive in an environment presenting few if any target MHC molecules. Paradoxically, effective clearance of viral Ag thus affords persisting virus a window of opportunity to escape from immune surveillance. These studies demonstrate that vaccine-induced T cell memory alone is unable to control persisting virus in a tissue with strict IFN-dependent MHC regulation, as evident in immune privileged sites.