Adriana Baz

Progressive Differentiation and Commitment of CD8+ T Cells to a Poorly Cytolytic CD8low Phenotype in the Presence of IL-4

Exposure to IL-4 during activation of naive murine CD8+ T cells leads to generation of IL-4-producing effector cells with reduced surface CD8, low perforin, granzyme B and granzyme C mRNA, and poor cytolytic function. We show in this study that maximal development of these cells depended on exposure to IL-4 for the first 5 days of activation. Although IL-4 was not required at later times, CD8 T cell clones continued to lose surface CD8 expression with prolonged culture, suggesting commitment to the CD8low phenotype. This state was reversible in early differentiation. When single CD8low cells from 4-day cultures were cultured without IL-4, 65% gave rise to clones that partly or wholly comprised CD8high cells; the proportion of reverted clones was reduced or increased when the cells were cloned in the presence of IL-4 or anti-IL-4 Ab, respectively. CD8 expression positively correlated with perforin and granzyme A, B, and C mRNA, and negatively correlated with IL-4 mRNA levels among these clones. By contrast, most CD8low cells isolated at later time points maintained their phenotype, produced IL-4, and exhibited poor cytolytic function after many weeks in the absence of exogenous IL-4. We conclude that IL-4-dependent down-regulation of CD8 is associated with progressive differentiation and commitment to yield IL-4-producing cells with little cytolytic activity. These data suggest that the CD4−CD8− cells identified in some disease states may be the product of a previously unrecognized pathway of effector differentiation from conventional CD8+ T cells.

Interferon-γ and interleukin-4 reciprocally regulate CD8 expression in CD8+ T cells

The CD8 co-receptor can modulate CD8+ T cell function through its contributions to T cell receptor (TCR) binding and signaling. Here we show that IFN-γ and IL-4 exert opposing effects on the expression of CD8α mRNA and surface CD8 protein during CD8+ T cell activation. IL-4 caused down-regulation of surface CD8 on ovalbumin (OVA)257–264-specific TCR-transgenic OT-I CD8+ T cells activated with OVA257–264-coated antigen presenting cells or polyclonal stimuli, and on wild type CD8+ T cells activated with polyclonal stimuli. This effect was enhanced in each case when the cells lacked a functional IFN-γ or IFN-γR gene. When WT or IFN-γ-deficient OT-I CD8+ T cells were analyzed 9 days after co-injection with control or IL-4-expressing OVA+ tumor cells into RAG-2−/−γc−/− mice, CD8 levels were highest on WT donor cells from mice that received the control tumor and lowest on IFN-γ-deficient donor cells from mice that received the IL-4-expressing tumor. The latter CD8low cells displayed markedly impaired binding of OVA257–264/MHC tetramers and peptide/MHC-dependent degranulation. The data reveal an unexpected role for IFN-γ in tuning the CD8 co-receptor during primary CD8+ T cell activation both in vitro and in vivo.

Profiling the CD8low phenotype, an alternative career choice for CD8 T cells during primary differentiation

A CD8+ T cell of naive phenotype has multiple career choices during its primary differentiation into an effector cell population. One of these career options is becoming a CD8low T cell. We have previously shown by in vitro studies that CD8low T cells have lost expression of CD8 surface protein and mRNA and are poorly cytolytic. In line with poor cytolytic function, CD8low T cells express low levels of perforin and granzyme B and C, mediators of the granule‐exocytosis machinery. However, CD8low T cells express IFN‐γ and substantial amounts of IL‐4, the signature cytokines of type 1 and type 2 T‐cell polarization, respectively. Here, we argue that the CD8low phenotype is an alternative career choice for any naive CD8+ T cell during primary activation but that the probability of choosing this option is greatly enhanced by both IL‐4 and strong activation conditions. CD8low T cells have downregulated CD8α/β heterodimers and no preferential CD8α/α homodimer expression. As shown by anti‐CD8 Ab blocking experiments, surface CD8 substantially contributes to the CD8 T cells effector function (i.e. cytokine expression and cytolytic activity). The distinct effector profile of CD8low T cells gives an example of the complexity of different CD8 T cell careers during primary effector differentiation.

IFN-γ Inhibits IL-4–Induced Type 2 Cytokine Expression by CD8 T Cells In Vivo and Modulates the Anti-Tumor Response

Activation of naive CD8 T cells in vitro in the presence of IL-4 induces type 2 cytokine expression, loss of CD8 expression, and reduced cytolytic potential. This represents a major shift from the canonical phenotype of effector CD8 T cells. It has not been established, however, whether IL-4 can induce comprehensive type 2 cytokine expression by CD8 T cells in vivo, nor whether the effects of IL-4 on type 2 cytokine production by CD8 T cells can be inhibited by IFN-γ. Furthermore, disparate results have been reported regarding the anti-tumor ability of type 2 polarized effector CD8 T cells, and the effects of IFN-γ in this respect remain unknown. To address these questions, wild-type or IFN-γ–deficient OVA-specific CD8+ T cells were activated in RAG-2−/− γc−/− recipients with control or IL-4–expressing OVA+ tumor cells, and then transferred to secondary recipients for tumor challenge. Tumor-derived IL-4 induced the expression of type 2 cytokines and the transcription factor GATA-3 by responding CD8 T cells while reducing their CD8 coreceptor expression and ability to eliminate a secondary tumor challenge. Each of these effects of IL-4 was exaggerated in IFN-γ–deficient, compared with wild-type, CD8 T cells. The results demonstrate that endogenous IFN-γ counteracts the induction of type 2 cytokines and the downregulation of both CD8 coreceptor levels and the anti-tumor response in CD8 T cells exposed to IL-4 during activation in vivo. These findings may explain the anomalies in the reported functional phenotype of type 2 polarized CD8 T cells.

Branched and linear lipopeptide vaccines have different effects on primary CD4+ and CD8+ T-cell activation but induce similar tumor-protective memory CD8+ T-cell responses.

We compared murine T-cell responses to synthetic lipopeptide vaccines in which the TLR2 ligand Pam(2)Cys was attached to co-linear CD4+ and CD8+ T-cell epitopes of ovalbumin (OVA) in a linear or branched configuration. Mice received OVA-specific transgenic CD8+ and CD4+ T-cells followed by one injection of vaccine. Although the branched lipopeptide was more potent in activating OVA-specific CD4+ and CD8+ T-cells in the primary response, both vaccines induced cytolytic T lymphocytes (CTL) that expressed perforin, granzyme A-C, and IFN-gamma mRNAs and conferred long-term protection of most mice against challenge with OVA-expressing tumor cells. OVA epitope display was reduced in tumors that developed in some mice, suggesting CD8+ T-cell dependent selection.

Towards new immunotherapies: targeting recombinant cytokines to the immune system using live attenuated Salmonella.

We have used Salmonella as a delivery system for eukaryotic expression plasmids encoding cytokines, and assessed its capacity to modulate immune responses in different experimental models. Plasmids encoding mouse IL-4 and IL-18 under cytomegalovirus promoter were constructed and transformed into live attenuated Salmonella enterica serovar Typhi strain CVD 908-htrA, and Salmonella enterica serovar Typhimurium strain SL3261. We have shown that systemic as well as mucosal immunization with such constructs can influence the antibody and cytokine responses to the Salmonella carrier and to co-administered bystander antigens, as well as the specific immune response elicited during a parasitic infection. Further, we have shown that oral cytokine-therapy using Salmonella as gene vector induce antitumoral effect as demonstrated by extended survival time in melanoma-bearing mice. This approach may be particularly suited for the development of new immunotherapies with applications in parasitic infections and cancer, were alterations of the hosts immune responses are usually found, and therapy-induced modulation of the immune response is likely to be required.

Memory cytolytic T-lymphocytes: induction, regulation and implications for vaccine design

The design of vaccines that protect against intracellular infections or cancer remains a challenge. In many cases, immunity depends on the development of antigen-specific memory CD8+ T-cells that can express cytokines and kill antigen-bearing cells when they encounter the pathogen or tumor. Here, the authors review current understanding of the signals and cells that lead to memory CD8+ T-cell differentiation, the relationship between the primary CD8+ T-cell response and the memory response and the regulation of memory CD8+ T-cell survival and function. The implications of this new knowledge for vaccine design are discussed, and recent progress in the development of lipidated peptide vaccines as a promising approach for vaccination against intracellular infections and cancer is reviewed.

Interleukin-4-induced loss of CD8 expression and cytolytic function in effector CD8 T cells persists long term in vivo

Activation of naive CD8+ T cells in the presence of interleukin‐4 modulates their CD8 co‐receptor expression and functional differentiation, resulting in the generation of CD8low cells that produce type 2 cytokines and display poor cytolytic and anti‐tumour activity. Although this CD8low phenotype becomes stable after about a week and can persist with further stimulation in vitro, it is not known whether it can be maintained long term in vivo. Here we report that CD8low cells derived from oval‐bumin257–264‐specific T‐cell receptor‐transgenic CD8+ T cells activated in the presence of interleukin‐4 could be detected in the spleen for at least 4 months after adoptive transfer into normal mice. A significant proportion of the long‐term surviving cells retained their CD8low phenotype in vivo and after clonal re‐activation in vitro. Although long‐term surviving CD8low cells lacked detectable cytolytic activity or perforin expression, they showed some anti‐tumour function in vivo. The persistence of functional cells with a CD8low phenotype in vivo raises the possibility that such cells can contribute to effector or regulatory responses to tumours or pathogens.

Quantitative assessment of the functional plasticity of memory CD8+ T cells

While the functional plasticity of memory CD4+ T cells has been studied extensively, less is known about this property in memory CD8+ T cells. Here, we report the direct measurement of plasticity by paired daughter analysis of effector and memory OT‐I CD8+ T cells primed in vivo with ovalbumin. Naïve, effector, and memory OT‐I cells were isolated and activated in single‐cell culture; then, after the first division, their daughter cells were transferred to new cultures with and without IL‐4; expression of IFN‐γ and IL‐4 mRNAs was measured 5 days later in the resultant subclones. Approximately 40% of clonogenic memory CD8+ T cells were bipotential in this assay, giving rise to an IL‐4− subclone in the absence of IL‐4 and an IL‐4+ subclone in the presence of IL‐4. The frequency of bipotential cells was lower among memory cells than naïve cells but markedly higher than among 8‐day effectors. Separation based on high or low expression of CD62L, CD122, CD127, or Ly6C did not identify a phenotypic marker of the bipotential cells. Functional plasticity in memory CD8+ T‐cell populations can therefore reflect modulation at the level of a single memory cell and its progeny.