IL-12 and IL-18 induce interferon-γ production and de novo CD2 expression in porcine γδ T cells

γδ T cells are highly abundant in the blood and spleen of pigs but little is known about their functional differentiation. In this study the potential of the type-1 polarizing cytokines IL-12 and IL-18 in combination with IL-2 and Concanavalin A (ConA) to stimulate porcine γδ T cells was investigated. Stimulation of purified γδ T cells with ConA and IL-2 induced a strong proliferation of CD2⁻ γδ T cells, whereas additional stimulation with IL-12 and IL-18 caused a stronger proliferation of CD2⁺ γδ T cells. IFN-γ could only be detected in supernatants of γδ T-cell cultures supplemented with IL-12 and IL-18. Experiments with sorted CD2/SWC5-defined γδ T-cell subsets revealed that CD2⁺SWC5⁻ γδ T cells are the main producers of IFN-γ following stimulation with IL-2/IL-12/IL-18. Additional stimulation with ConA led to an upregulation of CD2 within the CD2⁻ γδ T cell subsets, indicating a previously unnoticed plasticity of CD2-defined γδ T cell subsets.     

Murine CD5+, CD8+ Normal Fetal Liver Cells Enhance an Immune Response: Benzo(α) Pyrene-Exposed CD5+ Fetal Liver Cells are Inhibitors

The effects of 150 ug benzo(α)pyrene/gm body weight given intraperitoneally to the pregnant mouse at mid-gestation leads to long-lasting (> 18 months post-birth) immune deficiency in the progeny. Although the progeny are immunodeficient their T cell subsets induced marked enhancement and/or inhibition of cell proliferation in an immune response. Earlier we saw a striking increase (up to 7-fold) in CD8⁺ cells in the 18 day gestation liver of benzo(α)pyrene-exposed fetuses. We hypothesized that immune deficiency in carcinogen-exposed progeny could be attributed to an increase in classical CD8⁺ suppressor T-cells. Surprisingly, however, we found that CD8⁺ and CD5⁺ (Lyt 1⁺) cells of normal fetal liver enhance cell proliferation in an immune response. However, liver CD5⁺ cells from benzo(α)pyrene-exposed fetuses led to a dramatic reduction of the enhancing effect. Thus, as a novel finding, it appears that the profile of CD5⁺ cells, under the ontogenic influence of benzo(α)pyrene, transforms from cells that normally augment cell proliferation in an immune response to cells that are inhibitors. On the other hand the functional status of CD8⁺ cells is not affected. This change in physiological status of CD5⁺ cells may have important implications on T and B cell interactions, and the role of CD5⁺cells in T cell receptor signaling.     

Cytomegalovirus‐seropositivity has a profound influence on the magnitude of major lymphoid subsets within healthy individuals

Cytomegalovirus (CMV) infects most individuals and elicits a strong CMV‐specific immune response. We have studied the influence of CMV‐seropositivity on the size of lymphoid subsets in healthy donors and demonstrate that the virus substantially modulates the peripheral lymphoid pool. CD8⁺ T cell numbers are increased in all CMV‐seropositive individuals because of a striking 60% increment in the CD8⁺ T cell memory pool. The CD45RA⁺ resting memory pool is doubled after CMV infection and increases further with age. The magnitude of the naïve CD8⁺ T cell pool is dramatically reduced in CMV‐seropositive individuals at all ages, and this accelerates the physiological decline by approximately 40 years. The number of CD4⁺ effector memory T cells is increased in CMV‐seropositive individuals and is differentially accommodated by a reduction in the number of naïve and central memory CD4⁺ T cells in young and elderly donors respectively. CMV‐seropositivity also increases the total number of B cells in older donors and suppresses the number of CD5⁺ B cells. These data reveal that CMV has a profound influence on the immune system of all healthy individuals and add to growing concern regarding the clinical and immunomodulatory significance of CMV infection in healthy donors.     

CD4+ memory T cells: functional differentiation and homeostasis

Summary: CD4⁺ T cells are central regulators of both humoral and cellular immune responses. There are many subsets of CD4⁺ T cells, the most prominent being T‐helper 1 (Th1), Th2, Th‐17, and regulatory T cells, specialized in regulating different aspects of immunity. Without participation by these CD4⁺ T‐cell subsets, B cells cannot undergo isotype switching to generate high‐affinity antibodies, the microbicidal activity of macrophages is reduced, the efficiency of CD8⁺ T‐cell responses and CD8⁺ T‐cell memory are compromised, and downregulation of effector responses is impaired. It therefore stands to reason that memory CD4⁺ T cells are likely to fulfill an important facilitator role in the maintenance and control of protective immune responses. This review discusses some issues of importance for the generation of memory CD4⁺ T cells and focuses in particular on their heterogeneity and plasticity, with respect to both phenotypic characteristics and function. Finally, we discuss a number of factors that affect long‐term maintenance of memory CD4⁺ T cells.     

Direct recognition of LPS by human but not murine CD8+ T cells via TLR4 complex

LPS comprises a major PAMP and is a key target of the immune system during bacterial infection. While LPS can be recognised by innate immune cells via the TLR4 complex, it is unknown whether T lymphocytes, especially CD8⁺ T cells are also capable of doing so. We report here that naïve human CD8⁺ T cells, after activation by TCR stimulation, express surface TLR4 and CD14. These activated CD8⁺ T cells can then secrete high concentrations of IFN‐γ, granzyme and perforin in response to LPS. These effects can be specifically inhibited using siRNA for TLR4. Furthermore, LPS can synergise with IL‐12 to polarise the CD8⁺ T cells into cytotoxic T‐cell 1 (Tc1) that produce IFN‐γ but not IL‐4, with or without TCR activation. Moreover, CD8⁺CD45RO⁺ memory T cells constitutively expressed TLR4 and markedly enhanced IFN‐γ production when stimulated with LPS. In contrast, activated murine CD8⁺ T cells lack TLR4 and CD14 expression and fail to respond to LPS for proliferation and cytokine production. Thus, human but not murine CD8⁺ T cells are able to directly recognise bacterial LPS via LPS receptor complex and TLR4 provides a novel signal for the activation of effector and memory human CD8⁺ T cells.     

ICAM‐1 and B7‐1 provide similar but distinct costimulation for CD8+ T cells,while CD4+ T cells are poorly costimulated by ICAM‐1

The function of purified ICAM‐1 in costimulating CD4⁺ and CD8⁺ T cell responses has been directly compared to that of B7‐1 in a model system that minimizes contributions of other receptor‐ligand interactions. While B7‐1 costimulates both subsets of T cells, ICAM‐1 is much more effective in the costimulation of CD8⁺ cells. ICAM‐1 also synergizes with B7‐1 for the induction of IL‐2 production in CD8⁺ but not CD4⁺ T cells. These differences are not explained by differences in LFA‐1 receptor expression on the two subsets of T cells. The CD8⁺ T cell response to ICAM‐1 costimulation is associated with increased proliferation and IL‐2 production at levels similar to those seen with B7‐1 costimulation, but clonal expansion in response to ICAM‐1 is not as great due to decreased cell survival. ICAM‐1‐mediated costimulation is effective for both naive and memory CT8⁺ T cells, is independent of CD28 engagement, and does not appear to be due solely to effects on adhesion. These results suggest that ICAM‐1‐dependent, B7‐independent costimulation may be important in initiating a CTL response to class I antigen presented by cells that are not professional APC.     

JNK and p38 MAP kinases in CD4+ and CD8+ T cells

Summary: The c‐Jun aminoterminal kinase (JNK) and p38 mitogen‐activated protein (MAP) kinase signaling pathways have been associated with cell death, differentiation and proliferation. CD4⁺ and CD8⁺ T cells have different effector functions after antigen stimulation and control specific aspects of the immune response. The studies carried out in our group indicate that the role of JNK and p38 MAP kinases in CD4⁺ T cells is different from their role in CD8⁺ T cells. Moreover, these two pathways are not redundant in either T cell population. We have also shown that p38 MAP kinase regulates early stages of T cell development in the thymus. It is therefore important to consider the specific function of these kinases in each T cell population when pharmacological inhibitors of JNK and p38 MAP kinases are used for therapeutic purposes to control the immune response.     

Costimulation of CD3/TcR complex with either integrin or nonintegrin ligands protects CD4+ allergen-specific T-cell clones from programmed cell death

An optimal stimulation of CD4⁺ cells in an immune response requires not only signals transduced via the TcR/CD3 complex, but also costimulatory signals delivered as a consequence of interactions between T-cell surface-associated costimulatory receptors and their counterparts on antigen-presenting cells ‘APC). The intercellular adhesion molecule-1 ‘ICAM-1, CD54) efficiently costimulates proliferation of resting, but not antigen-specific, T cells. In contrast, CD28 and CD2 support interleukin ‘IL)-2 synthesis and proliferation of antigen-specific T cells more efficiently than those of resting T cells. The molecular basis for this differential costimulation of T cells is poorly understood. Cypress-specific T-cell clones ‘TCC) were generated from four allergic subjects during in vivo seasonal exposure to the allergen. Purified cypress extract was produced directly from fresh collected pollen and incubated with the patients' mononuclear cells. Repeated allergen stimulation was performed in T-cell cultures supplemented with purified extract and autologous APC. The limiting-dilution technique was then adopted to generate allergen-specific TCC, which were also characterized by their cytokine secretion pattern as ThO ‘IL-4 plus interferon-gamma) or Th2 ‘IL-4). Costimulation-induced proliferation or apoptosis was measured by propidium iodide cytofluorometric assay. By cross-linking cypress-specific CD4⁺ and CD8⁺ T-cell clones with either anti-CD3 or anti-CD2, anti-CD28, and anti-CD54 monoclonal antibodies, we demonstrated that CD4+ clones ‘with ThO- or Th2-type cytokine production pattern) undergo programmed cell death only after anti-CD3 stimulation, whereas costimulation with either anti-CD54 or anti-CD28 protects target cells from apoptosis. The costimulation-induced protection from apoptotic death was associated with a significant rise in IL-4 secretion in both Th0 and Th2-type clones. In contrast, cypress-specific Th0 CD8⁺ clones were more susceptible to stimulation-induced apoptosis via either anti-CD3 or anti-CD2, alone or in combination with anti-CD54 or anti-CD28, thus displaying only slight but nonsignificant modifications in the pattern of IL-4 secretion. The death-promoting costimulatory effects were not observed with highly purified normal resting CD4⁺ or CD8⁺ lymphocytes. Taken together, these results suggest that TcR engagement by an allergen in the context of functionally active APC induces activation-dependent cell death of some, perhaps less specific, cells, and this may be an important homeostatic mechanism through which functional expansion of allergen-specific T cells is regulated during an ongoing immune response.     

Maintenance of CD8+ memory T lymphocytes in the spleen but not in the bone marrow is dependent on proliferation

It is current belief that numbers of CD8⁺ memory T lymphocytes in the memory phase of an immune response are maintained by homeostatic proliferation. Here, we compare the proliferation of CD8⁺ memory T lymphocytes, generated by natural infections and by intentional immunization, in spleen and bone marrow (BM). Fifty percent of CD8⁺ memory T lymphocytes in the spleen are eliminated by cyclophosphamide within 14 days, indicating that numbers of at least 50% of splenic CD8⁺ memory T lymphocytes are maintained by proliferation. The numbers of CD8⁺ memory T lymphocytes in the BM, however, were not affected by cyclophosphamide. This stability was independent of circulating CD8⁺ memory T cells, blocked by FTY720, showing that BM is a privileged site for the maintenance of memory T lymphocytes, as resident cells, resting in terms of proliferation.     

Polyfunctional response by ImmTAC (IMCgp100) redirected CD8+ and CD4+ T cells

The success of immune system‐based cancer therapies depends on a broad immune response engaging a range of effector cells and mechanisms. Immune mobilizing monoclonal T cell receptors (TCRs) against cancer (ImmTAC™ molecules: fusion proteins consisting of a soluble, affinity enhanced TCR and an anti‐CD3 scFv antibody) were previously shown to redirect CD8⁺ and CD4⁺ T cells against tumours. Here we present evidence that IMCgp100 (ImmTAC recognizing a peptide derived from the melanoma‐specific protein, gp100, presented by HLA‐A*0201) efficiently redirects and activates effector and memory cells from both CD8⁺ and CD4⁺ repertoires. Using isolated subpopulations of T cells, we find that both terminally differentiated and effector memory CD8⁺ T cells redirected by IMCgp100 are potent killers of melanoma cells. Furthermore, CD4⁺ effector memory T cells elicit potent cytotoxic activity leading to melanoma cell killing upon redirection by IMCgp100. The majority of T cell subsets belonging to both the CD8⁺ and CD4⁺ repertoires secrete key pro‐inflammatory cytokines (tumour necrosis factor‐α, interferon‐γ, interleukin‐6) and chemokines (macrophage inflammatory protein‐1α‐β, interferon‐γ‐inducible protein‐10, monocyte chemoattractant protein‐1). At an individual cell level, IMCgp100‐redirected T cells display a polyfunctional phenotype, which is a hallmark of a potent anti‐cancer response. This study demonstrates that IMCgp100 induces broad immune responses that extend beyond the induction of CD8⁺ T cell‐mediated cytotoxicity. These findings are of particular importance because IMCgp100 is currently undergoing clinical trials as a single agent or in combination with check point inhibitors for patients with malignant melanoma.     

CD38+ CD45RBlow CD4+ T cells: a population of T cells with immune regulatory activities in vitro

An antibody reactive with CD38 revealed both phenotypic and functional heterogeneity amongst CD45RB^low cells. Functional analysis of the CD38⁺ and CD38⁻ fractions showed that the latter contained T cells which responded to recall antigens and produced high levels of cytokine in response to polyclonal stimulation. In contrast, the CD38⁺ population failed to proliferate or to produce detectable levels of cytokines. Despite appearing unresponsive, the CD38⁺ population significantly inhibited anti-CD3-induced proliferation and cytokine secretion by the reciprocal CD38⁻ population. Immune suppression required stimulation through the TCR and was dependent on a physical interaction between regulatory and responding CD4⁺ populations. It did not involve killing of the responding T cells or secretion of IL-10 or TGF-β. Despite some similarities there is no direct correlation between the in vitro suppression characteristic of the CD38⁺ CD45RB^low subset and in vivo suppression which has been shown to be mediated by unseparated CD45RB^low CD4⁺ T cells. However, these results demonstrate that two functionally distinct subsets of T cells reside within the antigen-exposed or CD45RB^low CD4⁺ T cell population and are thus generated in vivo: (1) conventional memory T cells which proliferate and secrete cytokines in response to activation and (2) a population of regulatory T cells which inhibit T cell activation in vitro. Antibodies reactive with CD38 may provide a useful tool with which to study the role of these T cell subsets in the induction and regulation of the immune response.     

Molecular mechanisms of apoptosis in the cells of the immune system in human aging

Summary: Aging is associated with progressive decline in immune functions and increased frequency of infections, autoimmunity, and cancer. Among immune functions, a decline in T‐cell functions during aging predominates. In this review, I discuss the molecular signaling of three distinct pathways of apoptosis, namely the death receptor pathway, the mitochondrial pathway, and the most recently described endoplasmic reticulum stress pathway, and the relative sensitivity of naïve, central memory, and effector memory CD8⁺ T‐cell subsets to apoptosis. In addition, I review apoptosis, especially via death receptor pathway, in naïve and various memory subsets of CD4⁺ and CD8⁺ T cells (with primary emphasis on CD8⁺ naïve and memory subsets) in human aging and discuss the role of apoptosis in immune senescence.     

CD8+ T‐cell memory in tumor immunology and immunotherapy

Summary: The cellular and molecular mechanisms underlying the formation of distinct central, effector, and exhausted CD8⁺ T‐cell memory subsets were first described in the setting of acute and chronic viral diseases. The role of these T‐cell memory subsets are now being illuminated as relevant to the tumor‐bearing state. The generation and persistence of productive CD8⁺ T‐cell memory subsets is determined, in part, by antigen clearance, costimulation, responsiveness to homeostatic cytokines, and CD4⁺ T‐helper cells. By contrast, chronic exposure to antigen, negative costimulation, and immunomodulation by CD4⁺ T regulatory cells corrupt productive CD8⁺ T memory formation. It has become clear from human and mouse studies that the mere generation of CD8⁺ T‐cell memory is not a ‘surrogate marker’ for cancer vaccine efficacy. Some current cancer vaccine strategies may fail because they amplify, rather than correct or reset, the corrupted CD8⁺ memory population. Thus, much of the present effort in the development of vaccines for cancer and chronic infectious diseases is aimed at creating effective memory responses. Therapeutic vaccines for cancer and chronic infectious diseases may achieve consistent efficacy by ablation of the dysfunctional immune state and the provision of newly generated, non‐corrupted memory cells by adoptive cell transfer.     

Study of the NKT cell subsets with superantigen SEB-activated tolerance

AIM: To study the NKT cell subsets and their differentiation. METHODS: Splenic lymphocytes from C57BL/J mice that had received SEB treatment were collected as effector cells on the 10(th) day. The cells were cultured in medium containing ConA, LPS and IL-2 for 3 days and measured their response to mitogens and cytokine. The inhibitory action of the effector cells was examined. The effector cells were cultured with normal lymphocytes and above mitogens or cytokine for 3 day. The cells proliferation was assessed with MTT method.The NKT cell subsets among these effector cells with the tolerance function were analyzed and their differentiation sources and correlation of functions were detected by flow cytometry. RESULTS: The response of SEB-activated effector cells to ConA, LPS and IL-2 was significantly decreased compared with that of normal lymphocytes. The A values of cell proliferation were decreased from 0.80+/-0.04, 0.60+/-0.03 and 0.55+/-0.07 in control groups to 0.60+/-0.05, 0.30+/-0.05 and 0.27+/-0.04 in effector groups, respectively (P<0.01, n=3).The inhibitory ability of effectors cells against the response of normal lymphocytes to ConA, LPS and IL-2 were clearly observed. They inhibited the response of normal lymphocytes to several mitogens and cytokine. And the A values of cell proliferation were decreased to 0.26+/-0.02, 0.48+/-0.04 and 0.34+/-0.02, respectively (P<0.01, n=3). The CD4(+)NK1.1(+), CD8(+)NK1.1(+), TcRV8(+)NK1.1(+) NKT cell subsets among SEB-activated effector cells with tolerance function were significantly increased and shown that they come from T cell population. And the CD4(-)CD8(-)/NK1.1(+)CD3(+)NKT cells by ConA or SEB-activated were shown coming from NK cell population. CONCLUSION: The effector cells with tolerance function activated by superantigen SEB relate to CD4(+)NK1.1(+), CD8(+)NK1.1(+), TcRVbeta8(+)NK1.1(+) NKT cell subsets. The NKT cell subsets come from T cells. The CD4(-)CD8(-)/NK1.1(+)CD3(+)NKT cells differentiating from NK cells are not involved in the regulation of tolerance.     

Enhancing the pro-inflammatory anti-cancer T cell response via biomanufactured,secretome-based,immunotherapeutics

T lymphocytes play a critical role in the pro-inflammatory anti-cancer response; hence, significant pharmacologic efforts have been made to enhance the endogenous T cell response. Unfortunately, significant toxicity arises consequent to pan T cell activation. In contrast, the less robust T cell alloresponse has also demonstrated an anti-cancer effect, but poses an inherent risk of GvHD. To overcome the GvHD risk, an acellular pro-inflammatory agent (IA1) has been biomanufactured from the secretome of the allorecognition response. To assess IA1’s immunomodulatory activity, T cell proliferation and differentiation were determined in vitro. The pro-inflammatory properties of the IA1 therapeutic were mediated by the miRNA-enriched fractions. Moreover, cross-species efficacy was observed consequent to the evolutionary conservation of miRNA. IA1 exerted no toxicity to resting PBMC but induced significant proliferation of resting CD3⁺ (CD4⁺ and CD8⁺) T cells and skewed the response towards a pro-inflammatory state (i.e., increased Teff:Treg ratio). Crucially, IA1-activated PBMC demonstrated a potent inhibition of cancer cell (HeLa and SH-4 melanoma) proliferation relative to the resting PBMC. The anti-proliferation effect of IA1-activated PBMC was noted within ?12?h versus 4–5 days for resting cells. A second biomanufactured therapeutic (IA2; produced using HeLa cells) surprisingly demonstrated direct toxicity to cancer cells but was less effective than IA1 in inducing a cell-mediated response. This study demonstrates that miRNA-enriched therapeutics can be biomanufactured from the secretome and can induce a potent pro-inflammatory, anti-cancer, effect on resting lymphocytes.     

Limited long-term naive CD4+ T cell reconstitution in patients experiencing viral load rebounds during HAART

Long-term (3.5 years) immune reconstitution in relation to viral load response was determined. Plasma HIV-1 RNA was suppressed in 40 patients (full responders) up to 42 months, and 17 patients achieved partial response. The measurements of CD4⁺ and CD8⁺ T lymphocyte subsets (CD45RA, CD45RACD62L, CD45RO, CD28, CD38) were carried out by flow cytometry. Full responders had a significant increase of CD4⁺ and all CD4⁺ T subsets both up to 6 and from 6 to 42 months, while the increase for partial responders was only up to 6 months. By 6 months, higher slopes were observed in full versus partial responders in the % of CD28 on CD4⁺ and the % of CD4⁺ memory subset and in both naïve and memory CD4⁺ subsets from 6 to 42 months. The percentage of CD8⁺ and its subsets was decreased significantly in full responders both up to 6 and from 6 to 42 months (except for an increase in the CD8⁺CD45RA⁺ CD62L⁺ cells), while in partial responders this decrease was only up to 6 months. Lower slopes were observed in full versus partial responders from 6 to 42 months in the percentages of CD8⁺, CD8⁺CD45RO⁺, CD8⁺CD28⁻, and CD8⁺CD38⁺ T cells. In conclusion, full responders have a stronger long-term naive CD4⁺ T cell subset reconstitution than partial responders. J. Med. Virol. 73:235–243, 2004. © 2004 Wiley-Liss, Inc.     

Mycobacterium bovis BCG Vaccination Induces Divergent Proinflammatory or Regulatory T Cell Responses in Adults

Mycobacterium bovis bacillus Calmette-Guérin (BCG), the only currently available vaccine against tuberculosis, induces variable protection in adults. Immune correlates of protection are lacking, and analyses on cytokine-producing T cell subsets in protected versus unprotected cohorts have yielded inconsistent results. We studied the primary T cell response, both proinflammatory and regulatory T cell responses, induced by BCG vaccination in adults. Twelve healthy adult volunteers who were tuberculin skin test (TST) negative, QuantiFERON test (QFT) negative, and BCG naive were vaccinated with BCG and followed up prospectively. BCG vaccination induced an unexpectedly dichotomous immune response in this small, BCG-naive, young-adult cohort: BCG vaccination induced either gamma interferon-positive (IFN-γ⁺) interleukin 2-positive (IL-2⁺) tumor necrosis factor α-positive (TNF-α⁺) polyfunctional CD4⁺ T cells concurrent with CD4⁺ IL-17A⁺ and CD8⁺ IFN-γ⁺ T cells or, in contrast, virtually absent cytokine responses with induction of CD8⁺ regulatory T cells. Significant induction of polyfunctional CD4⁺ IFN-γ⁺ IL-2⁺ TNF-α⁺ T cells and IFN-γ production by peripheral blood mononuclear cells (PBMCs) was confined to individuals with strong immunization-induced local skin inflammation and increased serum C-reactive protein (CRP). Conversely, in individuals with mild inflammation, regulatory-like CD8⁺ T cells were uniquely induced. Thus, BCG vaccination either induced a broad proinflammatory T cell response with local inflammatory reactogenicity or, in contrast, a predominant CD8⁺ regulatory T cell response with mild local inflammation, poor cytokine induction, and absent polyfunctional CD4⁺ T cells. Further detailed fine mapping of the heterogeneous host response to BCG vaccination using classical and nonclassical immune markers will enhance our understanding of the mechanisms and determinants that underlie the induction of apparently opposite immune responses and how these impact the ability of BCG to induce protective immunity to TB.