Human Innate Mycobacterium tuberculosis–Reactive αβTCR+ Thymocytes

The control of Mycobacterium tuberculosis (Mtb) infection is heavily dependent on the adaptive Th1 cellular immune response. Paradoxically, optimal priming of the Th1 response requires activation of priming dendritic cells with Th1 cytokine IFN-γ. At present, the innate cellular mechanisms required for the generation of an optimal Th1 T cell response remain poorly characterized. We hypothesized that innate Mtb-reactive T cells provide an early source of IFN-γ to fully activate Mtb-exposed dendritic cells. Here, we report the identification of a novel population of Mtb-reactive CD4⁻ αβTCR⁺ innate thymocytes. These cells are present at high frequencies, respond to Mtb-infected cells by producing IFN-γ directly ex vivo, and display characteristics of effector memory T cells. This novel innate population of Mtb-reactive T cells will drive further investigation into the role of these cells in the containment of Mtb following infectious exposure. Furthermore, this is the first demonstration of a human innate pathogen-specific αβTCR⁺ T cell and is likely to inspire further investigation into innate T cells recognizing other important human pathogens.     

Mycobacterium tuberculosis-reactive CD8+ T lymphocytes: the relative contribution of classical versus nonclassical HLA restriction

Previous studies in mice and humans models have suggested an important role for CD8+ T cells in host defense to Mycobacterium tuberculosis (Mtb). In humans, CD8+ Mtb-reactive T cells have been described that are HLA-A2-, B52-, as well as CD1-restricted. Recently, we have described Mtb-specific CD8+ T cells that are neither HLA-A-, B-, or C- nor group 1 CD1-restricted. At present, little is known about the relative contribution of each of these restriction specificities to the overall CD8+ response to Mtb. An IFN-gamma enzyme-linked immunospot assay was used to determine the frequency of Mtb-reactive CD8+ T cells directly from PBMC. The effector cell frequency among five healthy purified protein derivative-positive subjects was 1/7,600 +/- 4,300 compared with 1/16,000 +/- 7,000 in six purified protein derivative-negative controls. To determine the frequencies of classically, CD1-, and nonclassically restricted cells, a limiting dilution analysis was performed. In one purified protein derivative-positive subject, 192 clones were generated using Mtb-infected dendritic cells (DC). Clones were assessed for reactivity against control autologous DC, Mtb-infected autologous DC, and HLA-mismatched CD1+ targets (DC), as well as HLA-mismatched CD1- targets (macrophages). Of the 96 Mtb-reactive CD8+ T cell clones, four (4%) were classically restricted and 92 (96%) were nonclassically restricted. CD1-restricted cells were not detected. Of the classically restricted cells, two were HLA-B44 restricted and one was HLA-B14 restricted. These results suggest that while classically restricted CD8+ lymphocytes can be detected, they comprise a relatively small component of the overall CD8+ T cell response to Mtb. Further definition of the nonclassical response may aid development of an effective vaccine against tuberculosis.     

TGF-beta 1 uses distinct mechanisms to inhibit IFN-gamma expression in CD4+ T cells at priming and at recall: differential involvement of Stat4 and T-bet

TGF-beta1 plays a critical role in restraining pathogenic Th1 autoimmune responses in vivo, but the mechanisms that mediate TGF-beta1's suppressive effects on CD4(+) T cell expression of IFN-gamma expression remain incompletely understood. To evaluate mechanisms by which TGF-beta1 inhibits IFN-gamma expression in CD4(+) T cells, we primed naive wild-type murine BALB/c CD4(+) T cells in vitro under Th1 development conditions in the presence or the absence of added TGF-beta1. We found that the presence of TGF-beta1 during priming of CD4(+) T cells suppressed both IFN-gamma expression during priming as well as the development of Th1 effector cells expressing IFN-gamma at a recall stimulation. TGF-beta1 inhibited the development of IFN-gamma-expressing cells in a dose-dependent fashion and in the absence of APC, indicating that TGF-beta1 can inhibit Th1 development by acting directly on the CD4(+) T cell. During priming, TGF-beta1 strongly inhibited the expression of both T-bet (T box expressed in T cells) and Stat4. We evaluated the importance of these two molecules in the suppression of IFN-gamma expression at the two phases of Th1 responses. Enforced expression of T-bet by retrovirus prevented TGF-beta1's inhibition of Th1 development, but did not prevent TGF-beta1's inhibition of IFN-gamma expression at priming. Conversely, enforced expression of Stat4 partly prevented TGF-beta1's inhibition of IFN-gamma expression during priming, but did not prevent TGF-beta1's inhibition of Th1 development. These data show that TGF-beta1 uses distinct mechanisms to inhibit IFN-gamma expression in CD4(+) T cells at priming and at recall.     

Leishmania: naive human T cells sensitized with promastigote antigen and IL-12 develop into potent Th1 and CD8(+) cytotoxic effectors.

Russo, D. M., Chakrabarti, P., and Higgins, A. Y. 1999. Leishmania: Naive human T cells sensitized with promastigote antigen and IL-12 develop into potent Th1 and CD8(+) cytotoxic effectors. Experimental Parasitology 93, 161-170. The differentiation of naive human T cells into Leishmania-specific Th1 or cytotoxic effector cells was examined by sensitizing T cells in vitro with dead Leishmania antigen in the presence or absence of IFN-gamma or IL-12. These Leishmania-specific T cell lines proliferated and produced cytokines in response to challenge with autologous Leishmania-infected macrophages. Sensitization in the presence of IL-12 or IFN-gamma induced Leishmania-specific human Th1 responses, with IL-12 inducing more potent Th1 responses. However, IL-12-induced Th1 responses were IFN-gamma dependent. T cell lines exhibited Th2 or Th0 phenotypes when primed in the absence of cytokines. Only T cell lines primed in the presence of IL-12 contained high percentages of CD8(+) cells. These cells lysed autologous Leishmania-infected but not uninfected macrophages in an MHC-dependent manner. Thus, this in vitro sensitization system can be used to delineate the conditions for optimally priming human Leishmania-specific effector cells.     

IL-4-dependent Th2 collateral priming to inhaled antigens independent of Toll-like receptor 4 and myeloid differentiation factor 88

Allergic asthma is an inflammatory lung disease thought to be initiated and directed by type 2 helper T cells responding to environmental Ags. The mechanisms by which allergens induce Th2-adaptive immune responses are not well understood, although it is now clear that innate immune signals are required to promote DC activation and Th2 sensitization to inhaled proteins. However, the effect of ongoing Th2 inflammation, as seen in chronic asthma, on naive lymphocyte activation has not been explored. It has been noted that patients with atopic disorders demonstrate an increased risk of developing sensitivities to new allergens. This suggests that signals from an adaptive immune response may facilitate sensitization to new Ags. We used a Th2-adoptive transfer murine model of asthma to identify a novel mechanism, termed "collateral priming," in which naive CD4(+) T cells are activated by adaptive rather than innate immune signals. Th2 priming to newly encountered Ags was dependent on the production of IL-4 by the transferred Th2 population but was independent of Toll-like receptor 4 signaling and the myeloid differentiation factor 88 Toll-like receptor signaling pathway. These results identify a novel mechanism of T cell priming in which an Ag-specific adaptive immune response initiates distinct Ag-specific T cell responses in the absence of classical innate immune system triggering signals.     

Dendritic cell-intrinsic expression of NF-kappa B1 is required to promote optimal Th2 cell differentiation

A number of receptors and signaling pathways can influence the ability of dendritic cells (DC) to promote CD4(+) Th type 1 (Th1) responses. In contrast, the regulatory pathways and signaling events that govern the ability of DC to instruct Th2 cell differentiation remain poorly defined. In this report, we demonstrate that NF-kappaB1 expression within DC is required to promote optimal Th2 responses following exposure to Schistosoma mansoni eggs, a potent and natural Th2-inducing stimulus. Although injection of S. mansoni eggs induced production of IL-4, IL-5, and IL-13 in the draining lymph node of wild-type (WT) mice, NF-kappaB1(-/-) hosts failed to express Th2 cytokines and developed a polarized Ag-specific IFN-gamma response. In an in vivo adoptive transfer model in which NF-kappaB-sufficient OVA-specific DO11.10 TCR transgenic T cells were injected into OVA-immunized WT or NF-kappaB1(-/-) hosts, NF-kappaB1(-/-) APCs efficiently promoted CD4(+) T cell proliferation and IFN-gamma responses, but failed to promote Ag-specific IL-4 production. Further, bone marrow-derived DC from NF-kappaB1(-/-) mice failed to promote OVA-specific Th2 cell differentiation in in vitro coculture studies. Last, S. mansoni egg Ag-pulsed NF-kappaB1(-/-) DC failed to prime for Th2 cytokine responses following injection into syngeneic WT hosts. Impaired Th2 priming by NF-kappaB1(-/-) DC was accompanied by a reduction in MAPK phosphorylation in Ag-pulsed DC. Taken together, these studies identify a novel requirement for DC-intrinsic expression of NF-kappaB1 in regulating the MAPK pathway and governing the competence of DC to instruct Th2 cell differentiation.     

Bidirectional negative regulation of human T and dendritic cells by CD47 and its cognate receptor signal-regulator protein-alpha: down-regulation of IL-12 responsiveness and inhibition of dendritic cell activation

Proinflammatory molecules, including IFN-gamma and IL-12, play a crucial role in the elimination of causative agents. To allow healing, potent anti-inflammatory processes are required to down-regulate the inflammatory response. In this study, we first show that CD47/integrin-associated protein, a ubiquitous multispan transmembrane protein highly expressed on T cells, interacts with signal-regulator protein (SIRP)-alpha, an immunoreceptor tyrosine-based inhibition motif-containing molecule selectively expressed on myelomonocytic cells, and next demonstrate that this pair of molecules negatively regulates human T and dendritic cell (DC) function. CD47 ligation by CD47 mAb or L-SIRP-alpha transfectants inhibits IL-12R expression and down-regulates IL-12 responsiveness of activated CD4(+) and CD8(+) adult T cells without affecting their response to IL-2. Human CD47-Fc fusion protein binds SIRP-alpha expressed on immature DC and mature DC. SIRP-alpha engagement by CD47-Fc prevents the phenotypic and functional maturation of immature DC and still inhibits cytokine production by mature DC. Finally, in allogeneic MLR between mDC and naive T cells, CD47-Fc decreases IFN-gamma production after priming and impairs the development of a Th1 response. Therefore, CD47 on T cells and its cognate receptor SIRP-alpha on DC define a novel regulatory pathway that may be involved in the maintenance of homeostasis by preventing the escalation of the inflammatory immune response.     

Protective antitumor immunity induced by a costimulatory thalidomide analog in conjunction with whole tumor cell vaccination is mediated by increased Th1-type immunity

Thalidomide and its novel T cell costimulatory analogs (immunomodulatory drugs) are currently being assessed in the treatment of patients with advanced cancer. However, neither tumor-specific T cell costimulation nor effective antitumor activity has been demonstrated in vivo. In this study, we assessed the ability of an immunomodulatory drug (CC-4047/ACTIMID) to prime a tumor-specific immune response following tumor cell vaccination. We found that the presence of CC-4047 during the priming phase strongly enhanced antitumor immunity in the vaccinated group, and this correlated with protection from subsequent live tumor challenge. Protection was associated with tumor-specific production of IFN-gamma and was still observed following a second challenge with live tumor cells 60 days later. Furthermore, CD8(+) and CD4(+) splenocyte fractions from treated groups secreted increased IFN-gamma and IL-2 in response to tumor cells in vitro. Coculture of naive splenocytes with anti-CD3 mAb in the presence of CC-4047 directly costimulated T cells and increased Th1-type cytokines. Our results are the first to demonstrate that a costimulatory thalidomide analog can prime protective, long-lasting, tumor-specific, Th1-type responses in vivo and further support their ongoing clinical development as novel anti-cancer agents.     

Induction of IL-17+ T cell trafficking and development by IFN-gamma: mechanism and pathological relevance in psoriasis

Th1 and Th17 T cells are often colocalized in pathological environments, yet Th1-derived IFN-gamma inhibits Th17 cell development in vitro. We explored the physiologic basis of this paradox in humans. In this study, we demonstrate increased the number of CD4(+) and CD8(+) IL-17(+) T cells in skin lesions of psoriasis. Furthermore, we show that myeloid APCs potently support induction of IL-17(+) T cells, and that this activity is greatly increased in psoriasis. We tested stimuli that might account for this activity. Th1 cells and IFN-gamma are increased in psoriatic blood and lesional skin. We show that IFN-gamma programs myeloid APCs to induce human IL-17(+) T cells via IL-1 and IL-23. IFN-gamma also stimulates APC production of CCL20, supporting migration of IL-17(+) T cells, and synergizes with IL-17 in the production of human beta-defensin 2, an antimicrobial and chemotactic protein highly overexpressed by psoriatic keratinocytes. This study reveals a novel mechanistic interaction between Th1 and IL-17(+) T cells, challenges the view that Th1 cells suppress Th17 development through IFN-gamma, and suggests that Th1 and IL-17(+) T cells may collaboratively contribute to human autoimmune diseases.     

Cytokines regulate the capacity of CD8alpha(+) and CD8alpha(-) dendritic cells to prime Th1/Th2 cells in vivo

Prior studies have shown that subclasses of dendritic cells (DC) direct the development of distinct Th populations in rodents and in humans. In the mouse, we have recently shown that administration of Ag-pulsed CD8alpha(-) DC induces a Th2-type response, whereas injection of CD8alpha(+) DC leads to Th1 differentiation. To define the DC-derived factors involved in the polarization of Th responses, we injected either subset purified from mice genetically deficient for IFN-gamma, IL-4, IL-12, or IL-10 into wild-type animals. In this work, we report that DC-derived IL-12 and IFN-gamma are required for Th1 priming by CD8alpha(+) DC, whereas IL-10 is required for optimal development of Th2 cells by CD8alpha(-) DC. The level of IL-12 produced by the DC appears to determine the Th1/Th2 balance in vivo. We further show that the function of DC subsets displays some flexibility. Treatment of DC with IL-10 in vitro induces a selective decrease in the viability of CD8alpha(+) DC. Conversely, incubation with IFN-gamma down-regulates the Th2-promoting capacities of CD8alpha(-) DC and increases the Th1-skewing properties of both subsets.     

CD8alpha+ and CD11b+ dendritic cell-restricted MHC class II controls Th1 CD4+ T cell immunity

The activation, proliferation, differentiation, and trafficking of CD4 T cells is central to the development of type I immune responses. MHC class II (MHCII)-bearing dendritic cells (DCs) initiate CD4(+) T cell priming, but the relative contributions of other MHCII(+) APCs to the complete Th1 immune response is less clear. To address this question, we examined Th1 immunity in a mouse model in which I-A(beta)(b) expression was targeted specifically to the DCs of I-A(beta)b-/- mice. MHCII expression is reconstituted in CD11b(+) and CD8alpha(+) DCs, but other DC subtypes, macrophages, B cells, and parenchymal cells lack of expression of the I-A(beta)(b) chain. Presentation of both peptide and protein Ags by these DC subsets is sufficient for Th1 differentiation of Ag-specific CD4(+) T cells in vivo. Thus, Ag-specific CD4(+) T cells are primed to produce Th1 cytokines IL-2 and IFN-gamma. Additionally, proliferation, migration out of lymphoid organs, and the number of effector CD4(+) T cells are appropriately regulated. However, class II-negative B cells cannot receive help and Ag-specific IgG is not produced, confirming the critical MHCII requirement at this stage. These findings indicate that DCs are not only key initiators of the primary response, but provide all of the necessary cognate interactions to control CD4(+) T cell fate during the primary immune response.     

Cutting edge: a new approach to modeling early lung immunity in murine tuberculosis

In this study, we report a new approach that allows dissection of distinct pathways regulating induction of early adaptive immunity in response to Mycobacterium tuberculosis (Mtb). We used traceable murine dendritic cells (DCs) and macrophage populations to chart their migratory pattern in response to Mtb, and found that only DCs receiving inflammatory stimuli from Mtb up-regulated their expression of CCR7 and migrated specifically to the draining lymph nodes (LNs). Furthermore, these Mtb-modulated DCs initiated a Th1 response only in the draining LNs. Taken together, these results demonstrate that Mtb-induced modulation of DCs is critical for their migration to regional LNs and ensuing T cell priming.     

IL-1 beta enhances CD40 ligand-mediated cytokine secretion by human dendritic cells (DC): a mechanism for T cell-independent DC activation.

CD40 ligand (CD40L) is a membrane-bound molecule expressed by activated T cells. CD40L potently induces dendritic cell (DC) maturation and IL-12p70 secretion and plays a critical role during T cell priming in the lymph nodes. IFN-gamma and IL-4 are required for CD40L-mediated cytokine secretion, suggesting that T cells are required for optimal CD40L activity. Because CD40L is rapidly up-regulated by non-T cells during inflammation, CD40 stimulation may also be important at the primary infection site. However, a role for T cells at the earliest stages of infection is unclear. The present study demonstrates that the innate immune cell-derived cytokine, IL-1beta, can increase CD40L-induced cytokine secretion by monocyte-derived DC, CD34(+)-derived DC, and peripheral blood DC independently of T cell-derived cytokines. Furthermore, IL-1beta is constitutively produced by monocyte-derived DC and monocytes, and is increased in response to intact Escherichia coli or CD40L, whereas neither CD34(+)-derived DC nor peripheral blood DC produce IL-1beta. Finally, DC activated with CD40L and IL-1beta induce higher levels of IFN-gamma secretion by T cells compared with DC activated with CD40L alone. Therefore, IL-1beta is the first non-T cell-derived cytokine identified that enhances CD40L-mediated activation of DC. The synergy between CD40L and IL-1beta highlights a potent, T cell-independent mechanism for DC activation during the earliest stages of inflammatory responses.     

Mycobacterium leprae inhibits dendritic cell activation and maturation

Leprosy presents with a clinical spectrum of skin lesions that span from strong Th1-mediated cellular immunity and control of bacillary growth at one pole to poor Ag-specific T cell immunity with extensive bacillary load and Th2 cytokine-expressing lesions at the other. To understand how the immune response to Mycobacterium leprae is regulated, human dendritic cells (DC), potent inducers of adaptive immune responses, exposed to M. leprae, Mycobacterium tuberculosis (Mtb), and Mycobacterium bovis bacillus Calmette-Guerin (BCG) were studied for their ability to be activated and to prime T cell proliferation. In contrast with Mtb and BCG, M. leprae did not induce DC activation/maturation as measured by the expression of selected surface markers and proinflammatory cytokine production. In MLR, T cells did not proliferate in response to M. leprae-stimulated DC. Interestingly, M. leprae-exposed MLR cells secreted increased Th2 cytokines as well as similar Th1 cytokine levels as compared with Mtb- and BCG-exposed cells. Gene expression analysis revealed a reduction in levels of mRNA of DC activation and maturation markers following exposure to M. leprae. Our data suggest that M. leprae does not induce and probably suppresses in vitro DC maturation/activation, whereas Mtb and BCG are stimulatory.     

Modulation of CD4 Th cell differentiation by ganglioside GD1a in vitro

Cell surface gangliosides are shed by tumors into their microenvironment. In this study they inhibit cellular immune responses, including APC development and function, which is critical for Th1 and Th2 cell development. Using human dendritic cells (DCs) and naive CD4(+) T cells, we separately evaluated Th1 and Th2 development under the selective differentiating pressures of DC1-inducing pertussis toxin (PT) and DC2-inducing cholera toxin (CT). High DC IL-12 production after PT exposure and high DC IL-10 production after CT exposure were observed, as expected. However, when DCs were first preincubated with highly purified G(D1a) ganglioside, up-regulation of costimulatory molecules was blunted, and PT-induced IL-12 production was reduced, whereas CT-induced IL-10 production was increased. The combination of these effects could contribute to a block in the Th1 response. In fact, when untreated naive T cells were coincubated with ganglioside-preincubated, Ag-exposed DCs, naive Th cell differentiation into Th effector cells was reduced. Both the subsequent DC1-induced T cell production of IFN-gamma (Th1 marker) and DC2-induced T cell IL-4 production (Th2) were inhibited. Thus, ganglioside exposure of DC impairs, by at least two distinct mechanisms, the ability to induce Th differentiation, which could adversely affect the development of an effective cellular antitumor immune response.     

Chemokine-guided CD4+ T cell help enhances generation of IL-6RalphahighIL-7Ralpha high prememory CD8+ T cells

CD4(+) T cells promote effective CD8(+) T cell-mediated immunity, but the timing and mechanistic details of such help remain controversial. Furthermore, the extent to which innate stimuli act independently of help in enhancing CD8(+) T cell responses is also unresolved. Using a noninfectious vaccine model in immunocompetent mice, we show that even in the presence of innate stimuli, CD4(+) T cell help early after priming is required for generating an optimal pool of functional memory CD8(+) T cells. CD4(+) T cell help increased the size of a previously unreported population of IL-6Ralpha(high)IL-7Ralpha(high) prememory CD8(+) T cells shortly after priming that showed a survival advantage in vivo and contributed to the majority of functional memory CD8(+) T cells after the contraction phase. In accord with our recent demonstration of chemokine-guided recruitment of naive CD8(+) T cells to sites of CD4(+) T cell-dendritic cell interactions, the generation of IL-6Ralpha(high)IL-7Ralpha(high) prememory as well as functional memory CD8(+) T cells depended on the early postvaccination action of the inflammatory chemokines CCL3 and CCL4. Together, these findings support a model of CD8(+) T cell memory cell differentiation involving the delivery of key signals early in the priming process based on chemokine-guided attraction of naive CD8(+) T cells to sites of Ag-driven interactions between TLR-activated dendritic cells and CD4(+) T cells. They also reveal that elevated IL-6Ralpha expression by a subset of CD8(+) T cells represents an early imprint of CD4(+) T cell helper function that actively contributes to the survival of activated CD8(+) T cells.     

Absence of allograft ICAM-1 attenuates alloantigen-specific T cell priming,but not primed T cell trafficking into the graft,to mediate acute rejection

The expression and function of ICAM-1 are critical components in the initiation and elicitation of many T cell-mediated responses. Whether ICAM-1 expression is required on the T cells or on the APC during T cell priming remains unclear. To address this issue in alloantigen-specific T cell activation, the priming and function of T cells in response to heart allografts from MHC-mismatched wild-type vs ICAM-1(-/-) donors were tested. Wild-type C57BL/6 (H-2(b)) heart allografts were rejected by A/J (H-2(a)) recipients on days 7-9, whereas B6.ICAM-1(-/-) allografts survived until days 18-23 post-transplant. On day 7 post-transplant, infiltrating macrophages and CD4(+) and CD8(+) T cells in the ICAM-1(-/-) allografts were 20-30% those observed in the wild-type allografts. ELISPOT analyses indicated that the number of alloantigen-specific T cells producing IFN-gamma from recipients of ICAM-1-deficient grafts was 60% lower than that from recipients of wild-type allografts. On day 16 post-transplant, these numbers did not markedly increase in ICAM-1-deficient allograft recipients. Consistent with the reduced priming of alloreactive T cells, isolated dendritic cells from ICAM-1(-/-) mice stimulated allogeneic T cell proliferation poorly compared with wild-type dendritic cells. When A/J mice were primed with wild-type dendritic cells and then received wild-type or ICAM-1-deficient heart allografts 3 days later, the primed recipients rejected the wild-type and ICAM-1(-/-) allografts on days 5-6 post-transplant. These results indicate that optimal priming of alloreactive T cells requires allograft expression of ICAM-1, but, once primed, recipient T cell infiltration into the allograft is independent of graft ICAM-1 expression.     

A new mechanism for inhalational priming: IL-4 bypasses innate immune signals

Signaling via innate immune mechanisms is considered pivotal for T cell-mediated responses to inhaled Ags. Furthermore, Th2 cells specific for one inhaled Ag can facilitate priming of naive T cells to unrelated new inhaled Ags, a process we call "Th2 collateral priming". Interestingly, our previous studies showed that collateral priming is independent of signals via the innate immune system but depends on IL-4 secretion by CD4(+) T cells. We thus hypothesized that IL-4 can bypass the need for signals via the innate immune system, considered essential for pulmonary priming. Indeed, we were able to show that IL-4 bypasses the requirement for TLR4- and MyD88-mediated signaling for responses to new allergens. Furthermore, we characterized the mechanisms by which IL-4 primes for new inhaled allergens: "IL-4-dependent pulmonary priming" relies on IL-4 receptor expression on hematopoietic cells and structural cells. Transfer experiments indicate that within the hematopoietic compartment both T cells and dendritic cells need to express the IL-4 receptor. Finally, we were able to show that IL-4 induces recruitment and maturation of myeloid dendritic cells in vivo and increases T cell recruitment to the draining lymph nodes. Our findings bring new mechanistic knowledge to the phenomenon of polysensitization and primary sensitization in asthma.     

Initial characterization of a lymphokine pathway for the immunologic induction of tumor necrosis factor-alpha release from human peripheral blood mononuclear cells

Under endotoxin-free conditions, unstimulated human PBMC do not release TNF-alpha, as measured in a sensitive assay with 51Cr release in 6 h from actinomycin D-treated WEHI 164 cells. IFN-gamma alone at less than or equal to 10,000 U/ml is insufficient to elicit TNF-alpha release. Similarly, the lymphokine-rich supernatant of PBMC stimulated by allogeneic cells is also insufficient to induce TNF-alpha release in a short term assay. However, when PBMC are first primed with IFN-gamma for 48 h and then exposed to lymphokine supernatant for 6 h, effector cells within the PBMC population are triggered to express TNF-alpha-mediated cytotoxicity. All of the measured cytotoxicity is attributable to TNF-alpha because it could be abolished by a specific anti-TNF-alpha neutralizing mAb. Although IFN-gamma serves to prime PBMC in this assay system, it fails to trigger the release of TNF-alpha. Instead, a second lymphokine (provisionally termed "cytotoxicity triggering factor" (CTF) is required to induce TNF-alpha release from IFN-gamma-primed human PBMC. In kinetic studies, IFN-gamma priming was optimal when PBMC were exposed to IFN-gamma (150 U/ml) for 48 h. In contrast to the prolonged interval for priming, CTF need be present for 6 h or less for maximal induction of TNF-alpha-mediated cytotoxicity. In dose-response studies, IFN-gamma priming (48 h) required at least 4 U/ml and was complete with 20 to 100 U/ml. By using fully primed PBMC, the response to CTF followed a sigmoidal dose-response curve, which allowed the quantitation of CTF in half-maximal units. Activated Th lymphocytes constitute one cellular source for CTF. CTF is produced by cloned allorective T3+T4+T8-M1- Th cells after alloantigen stimulation, and also by nylon wool-purified T cells after stimulation with PMA and A23187 calcium ionophore. Unstimulated T cells do not release CTF. In physicochemical studies, CTF activity elutes from Sephadex G-100 as a major discrete peak of Mr 55 kDa and minor peaks of 14 kDa and greater than 150 kDa. On the basis of multiple criteria, CTF is distinguishable from several other cytokines: IFN-gamma, IL-1, IL-2, GM-CSF, MIF, CSF-1, TNF-alpha, and lymphotoxin (TNF-beta). We conclude that, by acting together, IFN-gamma and CTF provide a lymphokine pathway whereby Ag-responsive human Th cells induce the immunologic release of TNF-alpha from effector cells present in PBMC.