Advantages of Papio anubis for preclinical testing of immunotoxicity of candidate therapeutic antagonist antibodies targeting CD28

Antagonist anti-CD28 antibodies prevent T-cell costimulation and are functionally different from CTLA4Ig since they cannot block CTLA-4 and PDL-1 co-inhibitory signals. They demonstrated preclinical efficacy in suppressing effector T cells while enhancing immunoregulatory mechanisms. Because a severe cytokine release syndrome was observed during the Phase 1 study with the superagonist anti-CD28 TGN1412, development of other anti-CD28 antibodies requires careful preclinical evaluation to exclude any potential immunotoxicity side-effects. The failure to identify immunological toxicity of TGN1412 using macaques led us to investigate more relevant preclinical models.

We report here that contrary to macaques, and like in man, all baboon CD4-positive T lymphocytes express CD28 in their effector memory cells compartment, a lymphocyte subtype that is the most prone to releasing cytokines after reactivation. Baboon lymphocytes are able to release pro-inflammatory cytokines in vitro in response to agonist or superagonist anti-CD28 antibodies. Furthermore, we compared the reactivity of human and baboon lymphocytes after transfer into non obese diabetic/severe combined immunodeficiency (NOD/SCID) interleukin-2rγ knockout mice and confirmed that both cell types could release inflammatory cytokines in situ after injection of agonistic anti-CD28 antibodies. In contrast, FR104, a monovalent antagonistic anti-CD28 antibody, did not elicit T cell activation in these assays, even in the presence of anti-drug antibodies. Infusion to baboons also resulted in an absence of cytokine release.

In conclusion, the baboon represents a suitable species for preclinical immunotoxicity evaluation of anti-CD28 antibodies because their effector memory T cells do express CD28 and because cytokine release can be assessed in vitro and trans vivo.     

Signaling signatures and functional properties of anti-human CD28 superagonistic antibodies

Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation of the TCR/CD3-complex. In rodents these reagents induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. Unexpectedly, the humanized CD28 superagonist TGN1412 caused severe and life threatening adverse effects during a recently conducted phase I clinical trail. The underlying molecular mechanisms are as yet unclear. We show that TGN1412 as well as the commercially available CD28 superagonist ANC28.1 induce a delayed but extremely sustained calcium response in human naïve and memory CD4⁺ T cells but not in cynomolgus T lymphocytes. The sustained Ca⁺⁺-signal was associated with the activation of multiple intracellular signaling pathways and together these events culminated in the rapid de novo synthesis of high amounts of pro-inflammatory cytokines, most notably IFN-γ and TNF-α. Importantly, sustained transmembranous calcium flux, activation of Src-kinases as well as activation of PI3K were found to be absolutely required for CD28SA-mediated production of IFN-γ and IL-2. Collectively, our data suggest a molecular basis for the severe side effects caused by TGN1412 and impinge upon the relevance of non-human primates as preclinical models for reagents that are supposed to modify the function of human T cells.     

"Cytokine storm" in the phase I trial of monoclonal antibody TGN1412: better understanding the causes to improve preclinical testing of immunotherapeutics

The CD28-specific mAb TGN1412 rapidly caused a life-threatening "cytokine storm" in all six healthy volunteers in the Phase I clinical trial of this superagonist, signaling a failure of preclinical safety testing. We report novel in vitro procedures in which TGN1412, immobilized in various ways, is presented to human white blood cells in a manner that stimulates the striking release of cytokines and profound lymphocyte proliferation that occurred in vivo in humans. The novel procedures would have predicted the toxicity of this superagonist and are now being applied to emerging immunotherapeutics and to other therapeutics that have the potential to act upon the immune system. Data from these novel procedures, along with data from in vitro and in vivo studies in nonhuman primates, suggest that the dose of TGN1412 given to human volunteers was close to the maximum immunostimulatory dose and that TGN1412 is not a superagonist in nonhuman primates.     

Endothelial cells co-stimulate peripheral blood mononuclear cell responses to monoclonal antibody TGN1412 in culture

The failure of preclinical testing to predict the severity of the cytokine storm experienced by the recipients of the superagonistic anti-CD28 monoclonal antibody (mAb) TGN1412 during its Phase 1 clinical trial prompted the development of new in vitro experimental approaches for mimicking in vivo cytokine release and lymphoproliferation. Peripheral blood mononuclear cells (PBMC) presented to TGN1412 immobilised on plastic has previously been shown to stimulate a pro-inflammatory cytokine response. The aim of the present study was to investigate a 'co-culture' model for the detection of TGN1412-like immunomodulatory activity in which TGN1412 was presented to PBMC in the presence of monolayers of endothelium-derived cells and other cell types, followed by measurement of cytokine levels in the culture supernatants and proliferation of PBMC. Culturing PBMC with TGN1412 over primary human umbilical vein endothelial cells (HUVEC) and HUVEC-derived cell lines retaining classic endothelial markers, but not cell lines of non-endothelial origin, mediated the specific release of IL-6, IL-8 and TNFα, and proliferation of PBMC. Low levels of IL-2 and IFNγ were also detected in supernatants with most donors of PBMC. An anti-CD28 mAb agonist, i.e., not a superagonist like TGN1412, did not stimulate cytokine release or proliferation of PBMC in co-cultures. In conclusion, co-culture experiments for TGN1412-specific cytokine release required cells of endothelial origin. However, the profile of released cytokines in co-cultures did not mirror that in the clinical trial participants or the responses from PBMC exposed to TGN1412 immobilised on plastic, suggesting that TGN1412 stimulation of PBMC can occur through more than one mechanism.     

Rapid Regulatory T-Cell Response Prevents Cytokine Storm in CD28 Superagonist Treated Mice

Superagonistic CD28-specific monoclonal antibodies (CD28SA) are highly effective activators of regulatory T-cells (Treg cells) in rats, but a first-in-man trial of the human CD28SA TGN1412 resulted in an unexpected cytokine release syndrome. Using a novel mouse anti-mouse CD28SA, we re-investigate the relationship between Treg activation and systemic cytokine release. Treg activation by CD28SA was highly efficient but depended on paracrine IL-2 from CD28SA-stimulated conventional T-cells. Systemic cytokine levels were innocuous, but depletion of Treg cells prior to CD28SA stimulation led to systemic release of proinflammatory cytokines, indicating that in rodents, Treg cells effectively suppress the inflammatory response. Since the human volunteers of the TGN1412 study were not protected by this mechanism, we also tested whether corticosteroid prophylaxis would be compatible with CD28SA induced Treg activation. We show that neither the expansion nor the functional activation of Treg cells is affected by high-dose dexamethasone sufficient to control systemic cytokine release. Our findings warn that preclinical testing of activating biologicals in rodents may miss cytokine release syndromes due to the rapid and efficacious response of the rodent Treg compartment, and suggest that polyclonal Treg activation is feasible in the presence of antiphlogistic corticosteroid prophylaxis.     

A whole blood in vitro cytokine release assay with aqueous monoclonal antibody presentation for the prediction of therapeutic protein induced cytokine release syndrome in humans

The administration of several monoclonal antibodies (mAbs) to humans has been associated with acute adverse events characterized by clinically significant release of cytokines in the blood. The limited predictive value of toxicology species in this field has triggered intensive research to establish human in vitro assays using peripheral blood mononuclear cells or blood to predict cytokine release in humans. A thorough characterization of these assays is required to understand their predictive value for hazard identification and risk assessment in an optimal manner, and to highlight potential limitations of individual assay formats.We have characterized a whole human blood cytokine release assay with only minimal dilution by the test antibodies (95% v/v blood) in aqueous presentation format, an assay which has so far received less attention in the scientific world with respect to the evaluation of its suitability to predict cytokine release in humans. This format was compared with a human PBMC assay with immobilized mAbs presentation already well-characterized by others. Cytokine secretion into plasma or cell culture supernatants after 24 h incubation with the test mAbs (anti-CD28 superagonist TGN1412-like material (TGN1412L), another anti-CD28 superagonistic mAb (ANC28.1), a T-cell depleting mAb (Orthoclone™), and a TGN1412 isotype-matched control (Tysabri™) not associated with clinically-relevant cytokine release) was detected by a multiplex assay based on electrochemiluminescent excitation.We provide proof that this whole blood assay is a suitable new method for hazard identification of safety-relevant cytokine release in the clinic based on its ability to detect the typical cytokine signatures found in humans for the tested mAbs and on a markedly lower assay background and cytokine release with the isotype-matched control mAb Tysabri™ – a clear advantage over the PBMC assay. Importantly, quantitative and qualitative differences in the relative cytokine responses to the individual mAbs, in the concentration-response relationships and the prominent cytokine signatures for individual mAbs in the two formats reflect diverging mechanisms of cytokine release and different levels of dependency on high density coating even for two anti-CD28 super-agonistic antibodies. These results clearly show that one generic approach to assessment of cytokine release using in vitro assays is not sufficient, but rather the choice of the method, i.e. applying the whole blood assay or the PBMC assay needs to be well considered depending on the target characteristics and the mechanistic features of the therapeutic mAbs being evaluated.     

TGN1412 Induces Lymphopenia and Human Cytokine Release in a Humanized Mouse Model

Therapeutic monoclonal antibodies (mAbs) such as the superagonistic, CD28-specific antibody TGN1412, or OKT3, an anti-CD3 mAb, can cause severe adverse events including cytokine release syndrome. A predictive model for mAb-mediated adverse effects, for which no previous knowledge on severe adverse events to be expected or on molecular mechanisms underlying is prerequisite, is not available yet. We used a humanized mouse model of human peripheral blood mononuclear cell-reconstituted NOD-RAG1^-/-Aβ^-/-HLADQ^{(tg+ or tg-)}IL-2Rγc^-/- mice to evaluate its predictive value for preclinical testing of mAbs. 2–6 hours after TGN1412 treatment, mice showed a loss of human CD45⁺ cells from the peripheral blood and loss of only human T cells after OKT3 injection, reminiscent of effects observed in mAb-treated humans. Moreover, upon OKT3 injection we detected selective CD3 downmodulation on T cells, a typical effect of OKT3. Importantly, we detected release of human cytokines in humanized mice upon both OKT3 and TGN1412 application. Finally, humanized mice showed severe signs of illness, a rapid drop of body temperature, and succumbed to antibody application 2–6 hours after administration. Hence, the humanized mouse model used here reproduces several effects and adverse events induced in humans upon application of the therapeutic mAbs OKT3 and TGN1412.     

Failure to upregulate cell surface PD-1 is associated with dysregulated stimulation of T cells by TGN1412-like CD28 superagonist

The CD28 superagonist (CD28SA) TGN1412 was administered to humans as an agent that can selectively activate and expand regulatory T cells but resulted in uncontrolled T cell activation accompanied by cytokine storm. The molecular mechanisms that underlie this uncontrolled T cell activation are unclear. Physiological activation of T cells leads to upregulation of not only activation molecules but also inhibitory receptors such as PD-1. We hypothesized that the uncontrolled activation of CD28SA-stimulated T cells is due to both the enhanced expression of activation molecules and the lack of or reduced inhibitory signals. In this study, we show that anti-CD3 antibody-stimulated human T cells undergo time-limited controlled DNA synthesis, proliferation and interleukin-2 secretion, accompanied by PD-1 expression. In contrast, CD28SA-activated T cells demonstrate uncontrolled activation parameters including enhanced expression of LFA-1 and CCR5 but fail to express PD-1 on the cell surface. We demonstrate the functional relevance of the lack of PD-1 mediated regulatory mechanism in CD28SA-stimulated T cells. Our findings provide a molecular explanation for the dysregulated activation of CD28SA-stimulated T cells and also highlight the potential for the use of differential expression of PD-1 as a biomarker of safety for T cell immunostimulatory biologics.     

Failure to upregulate cell surface PD-1 is associated with dysregulated stimulation of T cells by TGN1412-like CD28 superagonist

The CD28 superagonist (CD28SA) TGN1412 was administered to humans as an agent that can selectively activate and expand regulatory T cells but resulted in uncontrolled T cell activation accompanied by cytokine storm. The molecular mechanisms that underlie this uncontrolled T cell activation are unclear. Physiological activation of T cells leads to upregulation of not only activation molecules but also inhibitory receptors such as PD-1. We hypothesized that the uncontrolled activation of CD28SA-stimulated T cells is due to both the enhanced expression of activation molecules and the lack of or reduced inhibitory signals. In this study, we show that anti-CD3 antibody-stimulated human T cells undergo time-limited controlled DNA synthesis, proliferation and interleukin-2 secretion, accompanied by PD-1 expression. In contrast, CD28SA-activated T cells demonstrate uncontrolled activation parameters including enhanced expression of LFA-1 and CCR5 but fail to express PD-1 on the cell surface. We demonstrate the functional relevance of the lack of PD-1 mediated regulatory mechanism in CD28SA-stimulated T cells. Our findings provide a molecular explanation for the dysregulated activation of CD28SA-stimulated T cells and also highlight the potential for the use of differential expression of PD-1 as a biomarker of safety for T cell immunostimulatory biologics.     

Immunobiology of the IL-15/IL-15Rα complex as an antitumor and antiviral agent

Interleukin (IL)-15 is essential for natural killer (NK), NKT and memory (m) CD8⁺ T cell development and function, and is currently under investigation as an immunotherapeutic agent for the treatment of cancer. Recently, the creation of IL-15 superagonist by complexing IL-15 and its high affinity receptor alpha (IL-15 Rα) in solution, inspired by the natural trans-presentation of IL-15, advances the potential of IL-15-based tumor immunotherapy. IL-15 superagonist shows promising advantages over monomeric IL-15 such as sustaining high circulating concentrations due to prolonged half-life and more potently stimulating NK and CD8⁺ T effector lymphocytes. So far, there are three different forms of recombinant IL-15 superagonist fusion protein based on configurational modifications. Gene therapy using engineered cells co-expressing IL-15/IL-15 Rα complex for cancer treatment is also emerging. All forms have demonstrated efficacy in causing tumor regression in animal studies, which provides strong rationale for advancing IL-15 superagonist through clinical trials. To date, there are fourteen phase I/II IL-15 superagonist trials in cancer patients and one phase I trial in HIV patients. Information generated by ongoing trials regarding the toxicity and efficacy of IL-15 superagonist is awaited. Finally, we elaborate on immunotoxicity caused by IL-15 superagonist in preclinical studies and discuss important safety considerations.     

CC chemokine receptor 9 expression defines a subset of peripheral blood lymphocytes with mucosal T cell phenotype and Th1 or T-regulatory 1 cytokine profile

The chemokine receptor CCR9 is expressed on most small intestinal lamina propria and intraepithelial lymphocytes and on a small subset of peripheral blood lymphocytes. CCR9-expressing lymphocytes may play an important role in small bowel immunity and inflammation. We studied the phenotype and functional characteristics of CCR9(+) lymphocytes in blood from normal donors. A subset of CCR9(+) T cells have a phenotype of activated cells and constitutively express the costimulatory molecules CD40L and OX-40. In contrast to CCR9(-), CCR9(+)CD4(+) peripheral blood T cells proliferate to anti-CD3 or anti-CD2 stimulation and produce high levels of IFN-gamma and IL-10. IL-10-producing cells were exclusively detected within the CCR9(+) subset of CD4(+) T cells by intracellular staining and were distinct from IL-2- and IFN-gamma-producing cells. Moreover, memory CCR9(+)CD4(+) lymphocytes respond to CD2 stimulation with proliferation and IFN-gamma/IL-10 production, whereas memory CCR9(-)CD4(+) cells were unresponsive. In addition, memory CCR9(+)CD4(+) T cells support Ig production by cocultured CD19(+) B cells in the absence of prior T cell activation or addition of exogenous cytokines. Our data show that the memory subset of circulating CCR9(+)CD4(+) T cells has characteristics of mucosal T lymphocytes and contains cells with either Th1 or T-regulatory 1 cytokine profiles. Studies on the cytokine profile and Ag specificity of this cell subset could provide important insight into small intestinal immune-mediated diseases and oral tolerance in humans.     

Extracellular purine metabolism and signaling of CD73-derived adenosine in murine Treg and Teff cells

CD73-derived adenosine acts as potent inhibitor of inflammation, and regulatory T cells (Treg) have been shown to express CD73 as a novel marker. This study explored the role of endogenously formed adenosine in modulating NF-κB activity and cytokine/chemokine release from murine Treg and effector T cells (Teff) including key enzymes/purinergic receptors of extracellular ATP catabolism. Stimulating murine splenocytes and CD4(+) T cells with anti-CD3/anti-CD28 significantly upregulated activated NF-κB in CD73(-/-) T cells (wild type: 4.36 ± 0.21; CD73(-/-): 6.58 ± 0.75; n = 4; P = 0.029). This was associated with an augmented release of proinflammatory cytokines IL-2, TNF-α, and IFN-γ. Similar changes were observed with the CD73 inhibitor APCP (50 μM) on NF-κB and IFN-γ in wild-type CD4(+) T-cells. Treatment of stimulated CD4(+) T-cells with adenosine (25 μM) potently reduced IFN-γ release which is mediated by adenosine A2a receptors (A2aR). AMP (50 μM) also reduced cytokine release which was not inhibited by APCP. In Teff, A2aR activation (CGS21680) potently inhibited the release of IL-1, IL-2, IL-3, IL-4, IL-12, IL-13, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), CCL3, and CCL4. However, in Treg, CGS21680 did not alter cytokine/chemokine release. In summary, CD73-derived adenosine tonically inhibits active NF-κB in CD4(+) T-cells, thereby modulating the release of a broad spectrum of proinflammatory cytokines and chemokines. Downregulation of P2X7 and upregulation of CD73 in Treg after antigenic stimulation may be an important mechanism to maintain the ability of Treg to generate immunosuppressive adenosine.     

Generation of CD4(+)CD45RA(+) effector T cells by stimulation in the presence of cyclic adenosine 5'-monophosphate-elevating agents

After TCR cross-linking, naive CD4(+)CD45RA(+) T cells switch to the expression of the CD45RO isoform and acquire effector functions. In this study we have shown that cAMP-elevating agents added to anti-CD3- and anti-CD28-stimulated cultures of T lymphocytes prevent acquisition of the CD45RO(+) phenotype and lead to the generation of a new subpopulation of primed CD4(+)CD45RA(+) effector cells (cAMP-primed CD45RA). These cells displayed a low apoptotic index, as the presence of dibutyryl cAMP (dbcAMP)-rescued cells from CD3/CD28 induced apoptosis. Inhibition of CD45 splicing by dbcAMP was not reverted by addition of exogenous IL-2. cAMP-primed CD45RA cells had a phenotype characteristic of memory/effector T lymphocytes, as they showed an up-regulated expression of CD2, CD44, and CD11a molecules, while the levels of CD62L Ag were down-regulated. These cells also expressed the activation markers CD30, CD71, and HLA class II Ags at an even higher level than CD3/CD28-stimulated cells in the absence of dbcAMP. In agreement with this finding, cAMP-primed CD45RA cells were very efficient in triggering allogenic responses in a MLR. In addition, cAMP-primed CD45RA cells produce considerable amounts of the Th2 cytokines, IL-4, IL-10, and IL-13, whereas the production of IFN-gamma and TNF-alpha was nearly undetectable. The elevated production of IL-13 by neonatal and adult cAMP-primed CD45RA cells was specially noticeable. The cAMP-dependent inhibition of CD45 splicing was not caused by the production of immunosuppressor cytokines. These results suggest that within the pool of CD4(+)CD45RA(+) cells there is a subpopulation of effector lymphocytes generated by activation in the presence of cAMP-elevating agents.     

Direct stimulation of human T cells via TLR5 and TLR7/8: flagellin and R-848 up-regulate proliferation and IFN-gamma production by memory CD4+ T cells

TLRs are involved in innate cell activation by conserved structures expressed by microorganisms. Human T cells express the mRNA encoding most of TLRs. Therefore, we tested whether some TLR ligands may modulate the function of highly purified human CD4+ T lymphocytes. We report that, in the absence of APCs, flagellin (a TLR5 ligand) and R-848 (a TLR7/8 ligand) synergized with suboptimal concentrations of TCR-dependent (anti-CD3 mAb) or -independent stimuli (anti-CD2 mAbs or IL-2) to up-regulate proliferation and IFN-gamma, IL-8, and IL-10 but not IL-4 production by human CD4+ T cells. No effect of poly(I:C) and LPS, ligands for TLR3 and TLR4, respectively, was detected. We also observed that CD4+CD45RO+ memory T cell responses to TLR ligands were more potent than those observed with CD4+CD45RA+ naive T cells. Moreover, among the memory T cells, CCR7- effector cells were more sensitive to TLR ligands than CCR7+ central memory cells. These data demonstrate for the first time a direct effect of TLR5 and TLR7/8 ligands on human T cells, and highlight an innate arm in T cell functions. They also suggest that some components from invading microorganisms may directly stimulate effector memory T cells located in tissues by up-regulating cytokine and chemokine production.     

The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands

The programmed death (PD)-1 molecule and its ligands (PD-L1 and PD-L2), negative regulatory members of the B7 family, play an important role in peripheral tolerance. Previous studies have demonstrated that PD-1 is up-regulated on T cells following TCR-mediated activation; however, little is known regarding PD-1 and Ag-independent, cytokine-induced T cell activation. The common gamma-chain (gamma c) cytokines IL-2, IL-7, IL-15, and IL-21, which play an important role in peripheral T cell expansion and survival, were found to up-regulate PD-1 and, with the exception of IL-21, PD-L1 on purified T cells in vitro. This effect was most prominent on memory T cells. Furthermore, these cytokines induced, indirectly, the expression of PD-L1 and PD-L2 on monocytes/macrophages in PBMC. The in vivo correlate of these observations was confirmed on PBMC isolated from HIV-infected individuals receiving IL-2 immunotherapy. Exposure of gamma c cytokine pretreated T cells to PD-1 ligand-IgG had no effect on STAT5 activation, T cell proliferation, or survival driven by gamma c cytokines. However, PD-1 ligand-IgG dramatically inhibited anti-CD3/CD28-driven proliferation and Lck activation. Furthermore, following restimulation with anti-CD3/CD28, cytokine secretion by both gamma c cytokine and anti-CD3/CD28 pretreated T cells was suppressed. These data suggest that gamma c cytokine-induced PD-1 does not interfere with cytokine-driven peripheral T cell expansion/survival, but may act to suppress certain effector functions of cytokine-stimulated cells upon TCR engagement, thereby minimizing immune-mediated damage to the host.     

Production of tumor necrosis factor-alpha by naive or memory T lymphocytes activated via CD28.

While it is well established that activated T cells can produce tumor necrosis factor alpha (TNF-alpha), it is less clear whether this function is confined to a given subset, e.g., memory cells. To approach this question, we investigated the production of TNF-alpha by human peripheral blood T lymphocytes activated with anti-CD28 mAb since this activation pathway is known to potentiate cytokine production. Under the culture conditions used, the amount of TNF-alpha produced was markedly enhanced compared to that obtained after activation with immobilized anti-CD3 mAb. The enhancement of TNF-alpha production was already apparent after incubation of T cells for 6 hr. Up to 5 ng/ml of TNF-alpha was measured on Day 2 in supernatants of cultures of 10(4) T lymphocytes. To determine the source of the cells producing high amounts of TNF-alpha, T lymphocytes were separated into two subpopulations, namely naive cells (expressing the CD45RA isoform) and memory cells (expressing the CD45RO isoform). While both subpopulations proliferated equally well after stimulation with anti-CD28 mAb, up to 90% of the TNF-alpha produced under these conditions originated from memory T cells. These results thus document that T cell activation via CD28 results in a marked increase in TNF-alpha production without affecting the functional disparity that exists between naive and memory T cells.     

NKT cell ligand recognition logic: molecular basis for a synaptic duet and transmission of inflammatory effectors

NKT cells that express the semi-invariant TCR are innate-like lymphocytes whose functions are regulated by self and foreign glycolipid ligands presented by the Ag-presenting, MHC class I-like molecule CD1d. Activation of NKT cells in vivo results in rapid release of copious amounts of effector cytokines and chemokines with which they regulate innate and adaptive immune responses to pathogens, certain types of cancers, and self-antigens. The nature of CD1d-restricted ligands, the manner in which they are recognized, and the unique effector functions of NKT cells suggest an immunoregulatory role for this T cell subset. Their ability to respond fast and our ability to steer NKT cell cytokine response to altered lipid ligands make them an important target for vaccine design and immunotherapies against autoimmune diseases. This review summarizes our current understanding of CD1d-restricted ligand recognition by NKT cells and how these innate-like lymphocytes regulate inflammation.     

Preservation of functional virus-specific memory CD8+ T lymphocytes in vaccinated, simian human immunodeficiency virus-infected rhesus monkeys

Functional impairment of virus-specific memory CD8(+) T lymphocytes has been associated with clinical disease progression following HIV, SIV, and simian human immunodeficiency virus infection. These lymphocytes have a reduced capacity to produce antiviral cytokines and mediators involved in the lysis of virally infected cells. In the present study, we used polychromatic flow cytometry to assess the frequency and functional capacity of central memory (CD28(+)CD95(+)) and effector memory (CD28(-)CD95(+)) subpopulations of Gag-specific CD8(+) T cells in SIV/simian human immunodeficiency virus-infected rhesus monkeys. The aim of this study was to determine whether Ag-specific, memory CD8(+) T cell function could be preserved in infected monkeys that had been immunized before infection with a vaccine regimen consisting of a plasmid DNA prime followed by a recombinant viral vector boost. We observed that vaccination was associated with the preservation of Gag-specific central memory CD8(+) T cells that were functionally capable of producing IFN-gamma, and effector memory CD8(+) T cells that were capable of producing granzyme B following viral Ag exposure.     

TGF-beta 1 attenuates the acquisition and expression of effector function by tumor antigen-specific human memory CD8 T cells

TGF-beta1 is a potent immunoregulatory cytokine. However, its impact on the generation and effector function of Ag-specific human effector memory CD8 T cells had not been evaluated. Using Ag-specific CD8 T cells derived from melanoma patients immunized with the gp100 melanoma Ag, we demonstrate that the addition of TGF-beta1 to the initial Ag activation cultures attenuated the gain of effector function by Ag-specific memory CD8 T cells while the phenotypic changes associated with activation and differentiation into effector memory were comparable to control cultures. These activated memory CD8 T cells consistently expressed lower mRNA levels for T-bet, suggesting a mechanism for TGF-beta1-mediated suppression of gain of effector function in memory T cells. Moreover, TGF-beta1 induced a modest expression of CCR7 on Ag-activated memory CD8 T cells. TGF-beta1 also suppressed cytokine secretion by Ag-specific effector memory CD8 T cells, as well as melanoma-reactive tumor-infiltrating lymphocytes and CD8 T cell clones. These results demonstrate that TGF-beta1 suppresses not only the acquisition but also expression of effector function on human memory CD8 T cells and tumor-infiltrating lymphocytes reactive against melanoma, suggesting that TGF-beta1-mediated suppression can hinder the therapeutic benefits of vaccination, as well as immunotherapy in cancer patients.     

Memory B cells and CD8⁺ lymphocytes do not control seasonal influenza A virus replication after homologous re-challenge of rhesus macaques

This study sought to define the role of memory lymphocytes in the protection from homologous influenza A virus re-challenge in rhesus macaques. Depleting monoclonal antibodies (mAb) were administered to the animals prior to their second experimental inoculation with a human seasonal influenza A virus strain. Treatment with either anti-CD8α or anti-CD20 mAbs prior to re-challenge had minimal effect on influenza A virus replication. Thus, in non-human primates with pre-existing anti-influenza A antibodies, memory B cells and CD8α⁺ T cells do not contribute to the control of virus replication after re-challenge with a homologous strain of influenza A virus.