Vandana Kalia

Functional and genomic profiling of effector CD8 T cell subsets with distinct memory fates

An important question in memory development is understanding the differences between effector CD8 T cells that die versus effector cells that survive and give rise to memory cells. In this study, we provide a comprehensive phenotypic, functional, and genomic profiling of terminal effectors and memory precursors. Using killer cell lectin-like receptor G1 as a marker to distinguish these effector subsets, we found that despite their diverse cell fates, both subsets possessed remarkably similar gene expression profiles and functioned as equally potent killer cells. However, only the memory precursors were capable of making interleukin (IL) 2, thus defining a novel effector cell that was cytotoxic, expressed granzyme B, and produced inflammatory cytokines in addition to IL-2. This effector population then differentiated into long-lived protective memory T cells capable of self-renewal and rapid recall responses. Experiments to understand the signals that regulate the generation of terminal effectors versus memory precursors showed that cells that continued to receive antigenic stimulation during the later stages of infection were more likely to become terminal effectors. Importantly, curtailing antigenic stimulation toward the tail end of the acute infection enhanced the generation of memory cells. These studies support the decreasing potential model of memory differentiation and show that the duration of antigenic stimulation is a critical regulator of memory formation.

Prolonged Interleukin-2Rα Expression on Virus-Specific CD8+ T Cells Favors Terminal-Effector Differentiation In Vivo

CD25, the high-affinity interleukin-2 (IL-2) receptor alpha chain, is rapidly upregulated by antigen-specific CD8(+) T cells after T cell receptor stimulation. Here, we demonstrate that during an acute viral infection, CD25 expression is quite dynamic-after initial upregulation, a subset of virus-specific T cells sustains CD25 expression longer than the rest. At this time when there is distinct heterogeneity in CD25 expression, examination of the in vivo fate of effector cells revealed that CD25(lo) cells, which are relatively less sensitive to IL-2, preferentially upregulate CD127 and CD62L and give rise to functional long-lived memory cells. In contrast, CD25(hi) cells perceiving prolonged IL-2 signals proliferate more rapidly, are prone to apoptosis, exhibit a more pronounced effector phenotype, and appear to be terminally differentiated. Consistent with this, sustained IL-2 receptor signaling during expansion drove terminal-effector differentiation. These data support the hypothesis that prolonged IL-2 signals during priming promote terminal-effector differentiation.

Strength of Stimulus and Clonal Competition Impact the Rate of Memory CD8 T Cell Differentiation

The developmental pathways of long-lived memory CD8 T cells and the lineage relationship between memory T cell subsets remain controversial. Although some studies indicate the two major memory T cell subsets, central memory T (TCM) and effector memory T (TEM), are related lineages, others suggest that these subsets arise and are maintained independently of one another. In this study, we have investigated this issue and examined the differentiation of memory CD8 T cell subsets by tracking the lineage relationships of both endogenous and TCR transgenic CD8 T cell responses after acute infection. Our data indicate that TCR transgenic as well as nontransgenic TEM differentiate into TCM in the absence of Ag. Moreover, the rate of memory CD8 T cell differentiation from TEM into the self-renewing and long-lived pool of TCM is influenced by signals received during priming, including Ag levels, clonal competition, and/or the duration of infection. Although some TEM appear to not progress to TCM, the vast majority of TCM are derived from TEM. Thus, long-lasting, Ag-independent CD8 T cell memory results from progressive differentiation of memory CD8 T cells, and the rate of memory T cell differentiation is governed by events occurring early during T cell priming.

Rational Site-Directed Mutations of the LLP-1 and LLP-2 Lentivirus Lytic Peptide Domains in the Intracytoplasmic Tail of Human Immunodeficiency Virus Type 1 gp41 Indicate Common Functions in Cell-Cell Fusion but Distinct Roles in Virion Envelope Incorporation

ABSTRACT Two highly conserved cationic amphipathic α-helical motifs, designated lentivirus lytic peptides 1 and 2 (LLP-1 and LLP-2), have been characterized in the carboxyl terminus of the transmembrane (TM) envelope glycoprotein (Env) of lentiviruses . Although various properties have been attributed to these domains, their structural and functional significance is not clearly understood. To determine the specific contributions of the Env LLP domains to Env expression, processing, and incorporation and to viral replication and syncytium induction, site-directed LLP mutants of a primary dualtropic infectious human immunodeficiency virus type 1 (HIV-1) isolate (ME46) were examined. Substitutions were made for highly conserved arginine residues in either the LLP-1 or LLP-2 domain (MX1 or MX2, respectively) or in both domains (MX4). The HIV-1 mutants with altered LLP domains demonstrated distinct phenotypes. The LLP-1 mutants (MX1 and MX4) were replication defective and showed an average of 85% decrease in infectivity, which was associated with an evident decrease in gp41 incorporation into virions without a significant decrease in Env expression or processing in transfected 293T cells. In contrast, MX2 virus was replication competent and incorporated a full complement of Env into its virions, indicating a differential role for the LLP-1 domain in Env incorporation. Interestingly, the replication-competent MX2 virus was impaired in its ability to induce syncytia in T-cell lines. This defect in cell-cell fusion did not correlate with apparent defects in the levels of cell surface Env expression, oligomerization, or conformation. The lack of syncytium formation, however, correlated with a decrease of about 90% in MX2 Env fusogenicity compared to that of wild-type Env in quantitative luciferase-based cell-cell fusion assays. The LLP-1 mutant MX1 and MX4 Envs also exhibited an average of 80% decrease in fusogenicity. Altogether, these results demonstrate for the first time that the highly conserved LLP domains perform critical but distinct functions in Env incorporation and fusogenicity.

Interleukin-21 Is a Critical Cytokine for the Generation of Virus-Specific Long-Lived Plasma Cells

ABSTRACT Long-lived plasma cells that reside in the bone marrow constitutively produce antibody in the absence of antigen and are the cellular basis of durable humoral immunity. The generation of these long-lived plasma cells depends upon a series of highly orchestrated interactions between antigen-specific CD4 T cells and B cells and the formation of germinal centers (GCs). In this study, we have examined the role of the cytokine interleukin-21 (IL-21) in regulating humoral immunity during acute viral infections. Using IL-21 receptor-deficient (IL-21R−/−) mice, we found that virus-specific CD4 T cells were generated after infection with lymphocytic choriomeningitis virus (LCMV) and that these CD4 T cells differentiated into T follicular helper (TFH)-like cells in the absence of IL-21 signaling. There was also no defect in the formation of GCs, although after day 15 these GCs disappeared faster in IL-21R−/− mice than in wild-type mice. Isotype switching and the initial LCMV-specific IgG response were normal in IL-21R−/− mice. However, these mice exhibited a profound defect in generating long-lived plasma cells and in sustaining antibody levels over time. Similar results were seen after infection of IL-21R−/− mice with vesicular stomatitis virus and influenza virus. Using chimeric mice containing wild-type or IL-21R−/− CD4 T cells and B cells, we showed that both B and CD4 T cells need IL-21 signaling for generating long-term humoral immunity. Taken together, our results highlight the importance of IL-21 in humoral immunity to viruses.

Antibody Neutralization Escape Mediated by Point Mutations in the Intracytoplasmic Tail of Human Immunodeficiency Virus Type 1 gp41

ABSTRACT The persistence of human immunodeficiency virus type 1 (HIV-1) infection in the presence of robust host immunity has been associated in part with variation in viral envelope proteins leading to antigenic variation and escape from neutralizing antibodies. Previous studies of natural neutralization escape mutants have predominantly focused on gp120 and gp41 ectodomain sequence variations that alter antibody binding via changes in conformation or glycosylation pattern of the Env, likely due to the immune pressure exerted on the exposed ectodomain component of the glycoprotein. Here, we show for the first time a novel mechanism by which point mutations in the intracytoplasmic tail of the transmembrane component (gp41) of envelope can render the virus resistant to neutralization by monoclonal antibodies and broadly neutralizing polyclonal serum antibodies. Point mutations in a highly conserved structural motif within the intracytoplasmic tail resulted in decreased binding of neutralizing antibodies to the Env ectodomain, evidently due to allosteric changes both in the gp41 ectodomain and in gp120. While receptor binding and infectivity of the mutant virus remained unaltered, the changes in Env antigenicity were associated with an increase in neutralization resistance of the mutant virus. These studies demonstrate the structurally integrated nature of gp120 and gp41 and underscore a previously unrecognized potentially critical role for even minor sequence variation of the intracytoplasmic tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.

MicroRNA-17~92 regulates effector and memory CD8 T-cell fates by modulating proliferation in response to infections.

The precise microRNAs and their target cellular processes involved in generation of durable T-cell immunity remain undefined. Here we show a dynamic regulation of microRNAs as CD8 T cells differentiate from naïve to effector and memory states, with short-lived effectors transiently expressing higher levels of oncogenic miR-17-92 compared with the relatively less proliferating memory-fated effectors. Conditional CD8 T-cell-intrinsic gain or loss of expression of miR-17-92 in mature cells after activation resulted in striking reciprocal effects compared with wild-type counterparts in the same infection milieu-miR-17-92 deletion resulted in lesser proliferation of antigen-specific cells during primary expansion while favoring enhanced IL-7Rα and Bcl-2 expression and multicytokine polyfunctionality; in contrast, constitutive expression of miR-17-92 promoted terminal effector differentiation, with decreased formation of polyfunctional lymphoid memory cells. Increased proliferation upon miR-17-92 overexpression correlated with decreased expression of tumor suppressor PTEN and increased PI3K-AKT-mTOR signaling. Thus, these studies identify miR17-92 as a critical regulator of CD8 T-cell expansion and effector and memory lineages in the physiological context of acute infection, and present miR-17-92 as a potential target for modulating immunologic outcome after vaccination or immunotherapeutic treatments of cancer, chronic infections, or autoimmune disorders.

Caspase-mediated apoptosis and cell death of rhesus macaque CD4+ T-cells due to cryopreservation of peripheral blood mononuclear cells can be rescued by cytokine treatment after thawing.

Cryopreservation of peripheral blood mononuclear cells (PBMC) from animal model studies and clinical trials is utilized as a primary method for long-term storage of PBMC for future in vitro and in vivo applications. The objective of this study was to define the mechanistic pathways involved in cryopreservation-induced apoptosis of CD4+ T-cells in PBMC, and to evaluate a cytokine treatment of the cryopreserved samples to rescue apoptosis for the potential future use of the cryopreserved PBMC. Using cryopreserved PBMC samples isolated from naïve and Simian immunodeficiency virus (SIV)-infected rhesus macaques as a model, frozen PBMC showed significantly increased levels of apoptosis-induced CD4+ T-cell death compared to fresh PBMC over a 5-day culture period as detected by Annexin V/PI and trypan blue staining. Mechanistic studies using a broad-spectrum caspase inhibitor z-VAD demonstrated a crucial involvement of caspases in cryopreservation-induced apoptosis of CD4+ T-cells. Furthermore, the ability of z-VAD to inhibit both mitochondrial membrane perturbation and apoptotic cell death implicated the involvement of caspase-mediated mitochondrial membrane damage in cryopreservation-induced apoptosis of CD4+ T-cells. Due to their known properties to promote T-cell survival and inhibit apoptosis, we evaluated the ability of IL-2, IL-4, and IL-7 combination cytokine treatment of the cryopreserved cells to rescue apoptosis of the CD4+ T-cells. The cytokine treatment resulted in a significant inhibition (p<0.01) of apoptosis-induced cell death and rescued CD4+ T-cell survival (p<0.01) in the cryopreserved cells. Efficient rescue of cryopreserved CD4+ T-cells has clinical significance in immune function analysis of longitudinal samples and in various long-term protocols requiring cryopreservation, including bone marrow and stem cell transplantation.

CD8 T-Cell Memory Differentiation during Acute and Chronic Viral Infections

CD8 T cell responses play an important role in protection against intracellular parhogens. Memory CD8 T cells mediate rapid clearance of pathogens upon secondary infection owing to their elevated frequency, ready localization to peripheral sites of infection and their ability to rapidly expand and mount effector functions. Such potent long-lasting protective memory CD8 T cells develop in acute infections where antigen is effectively cleared. In contrast, chronic infections with persistently high viral loads are characterized by CD8 T-cell dysfunction. In this chapter we present our current understanding of signals and mechanisms that regulate the development of functional long-lived memory CD8 T cells during acute infections. This is discussed in the context of proposed models of memory differentiation and compared with CD8 T-cell exhaustion and altered T-cell homeostasis, as occurs during persistent viral infections.

Quiescence of Memory CD8+ T Cells Is Mediated by Regulatory T Cells through Inhibitory Receptor CTLA-4

Immune memory cells are poised to rapidly expand and elaborate effector functions upon reinfection yet exist in a functionally quiescent state. The paradigm is that memory Txa0cells remain inactive due to lack of Txa0cell receptor (TCR) stimuli. Here, we report that regulatory T (Treg) cells orchestrate memory Txa0cell quiescence by suppressing effector and proliferation programs through inhibitory receptor, cytotoxic-T-lymphocyte-associated protein-4 (CTLA-4). Loss of Treg cells resulted in activation of genome-wide transcriptional programs characteristic of effector Txa0cells and drove transitioning as well as established memory CD8(+) Txa0cells toward terminally differentiated KLRG-1(hi)IL-7Rα(lo)GzmB(hi) phenotype, with compromised metabolic fitness, longevity, polyfunctionality, and protective efficacy. CTLA-4 functionally replaced Treg cells in trans to rescue memory Txa0cell defects and restore homeostasis. These studies present the CTLA-4-CD28-CD80/CD86 axis as a potential target to accelerate vaccine-induced immunityxa0and improve Txa0cell memory quality in current cancer immunotherapies proposing transient Treg cell ablation.