Autologous Macrophages Research Articles

Vaccination with photodynamic therapy-treated macrophages induces highly suppressive T-regulatory cells

The present study explores whether photodynamic therapy (PDT)-induced apoptosis can increase the number of tolerogenic regulatory T cells (Treg) and limit collateral tissue damage. BALB/c mice were vaccinated subcutaneously three times with PDT-induced apoptotic or thaw-frozen, necrotic non-infected autologous macrophages (MΦ). Two weeks after the last vaccination, mice were infected intradermally with 10(6) promastigotes of Leishmania major. Mice that received PDT-induced apoptotic MΦ had fewer parasites and higher numbers of Treg than mice vaccinated with thaw-frozen necrotic MΦ or phosphate-buffered saline (PBS). Interleukin (IL)-4 and IL-6 were significantly suppressed, while IL-10 was increased in mice that received the PDT-induced apoptotic MΦ. The role of Treg in this process was confirmed through Treg transfer from vaccinated to naïve mice. Mice receiving CD4(+) CD25(+) cells from mice vaccinated with PDT-induced apoptotic MΦ showed smaller lesions 3 weeks after infection and lower parasitic burdens than mice that received Tregs from mice of thaw-frozen necrotic MΦ or PBS groups. These changes were mediated by the depletion of CD3(+) CD8(+) and NKT cells and increased levels of IL-12p70 and interferon-γ, IL-10, and TGF-β in the cutaneous leishmaniasis lesions. Vaccination with apoptotic MΦ-induced tolerogenic Treg cells that limited collateral tissue damage and diminished parasitic burden.

Administration of LPS-Stimulated Autologous Macrophages Induces α-Synuclein Aggregation in Dopaminergic Neurons of Rat Brain

Administration of autologous macrophages isolated from the abdominal cavity and stimulated in vitro with bacterial LPS to rats increased blood level of antibodies against α-synuclein. Antibody titer reached its maximum during week 5 of the experiment and exceeded the levels of anti-α-synuclein antibodies appeared in response to transplantation of non-stimulated macrophages. Brain immunohistochemistry showed that additional administration of LPS (250 μg/kg) to animals during week 4 after injection of LPS-stimulated macrophages led to α-synuclein accumulation in 9.4±3.2% dopaminergic neurons of the substantia nigra. These findings attest to induction of humoral immune response to α-synuclein in rats after administration of autologous LPS-stimulated macrophages, which can affect α-synuclein metabolism in dopaminergic neurons of the brain.

Genetically modified macrophages expressing hypoxia regulated cytochrome P450 and P450 reductase for the treatment of cancer

This study describes a combined gene and cell therapy based on the genetic modification of primary human macrophages, as a treatment for cancer. Here, we have utilised the tumour-infiltrating properties of macrophages as vehicles to deliver a gene encoding a prodrug-activating enzyme such as human cytochrome P450 2B6 (CYP2B6) inside tumours followed by killing the tumour cells with the prodrug cyclophosphamide (CPA). Macrophages were transduced with an adenoviral vector that expresses human cytochrome CYP2B6 via a synthetic hypoxia responsive promoter (OBHRE) and with human P450 reductase (P450R), via the CMV promoter. In the presence of CPA, these genetically modified macrophages showed increased cytotoxicity against various tumour cell lines compared to untransduced macrophages or macrophages transduced with CYP2B6 alone. In human ovarian carcinoma xenograft models, the median survival of mice treated with genetically modified macrophages plus CPA increased up to two-fold compared to the survival of mice treated with untransduced macrophages and CPA. Genetically modified autologous macrophages may be a feasible therapeutic option for the treatment of some solid tumours, such as ovarian cancer.

Modulating cholesteryl ester transfer protein activity maintains efficient pre-β-HDL formation and increases reverse cholesterol transport

The mechanism by which cholesteryl ester transfer protein (CETP) activity affects HDL metabolism was investigated using agents that selectively target CETP (dalcetrapib, torcetrapib, anacetrapib). In contrast with torcetrapib and anacetrapib, dalcetrapib requires cysteine 13 to decrease CETP activity, measured as transfer of cholesteryl ester (CE) from HDL to LDL, and does not affect transfer of CE from HDL3 to HDL2. Only dalcetrapib induced a conformational change in CETP, when added to human plasma in vitro, also observed in vivo and correlated with CETP activity. CETP-induced pre-β-HDL formation in vitro in human plasma was unchanged by dalcetrapib ≤3 µM and increased at 10 µM. A dose-dependent inhibition of pre-β-HDL formation by torcetrapib and anacetrapib (0.1 to 10 µM) suggested that dalcetrapib modulates CETP activity. In hamsters injected with [3H]cholesterol-labeled autologous macrophages, and given dalcetrapib (100 mg twice daily), torcetrapib [30 mg once daily (QD)], or anacetrapib (30 mg QD), only dalcetrapib significantly increased fecal elimination of both [3H]neutral sterols and [3H]bile acids, whereas all compounds increased plasma HDL-[3H]cholesterol. These data suggest that modulation of CETP activity by dalcetrapib does not inhibit CETP-induced pre-β-HDL formation, which may be required to increase reverse cholesterol transport.

The interaction of human natural killer cells with either unpolarized or polarized macrophages results in different functional outcomes

The cross-talk among cells of the innate immunity can greatly affect both innate and adaptive responses. Here we analyzed the molecular interactions between human natural killer (NK) cells and autologous macrophages. Activated NK cells killed M0 and M2, whereas M1 macrophages were more resistant to lysis because of their higher expression of HLA class I molecules. Following exposure to LPS or bacillus Calmette-Guérin, M0 and M2, but not polarized (endotoxin tolerant) M1 macrophages, induced strong activation of resting NK cells. The expression of CD69 and CD25 activation markers and the acquisition of cytotoxicity against tumor cells and immature dendritic cells required soluble factors being mostly contact independent. On the contrary, IFN-γ production was contact dependent and required the interaction of DNAM-1 and 2B4 (on NK) with their ligands on macrophages as well as IL-18. IL-18 was involved also in the acquisition of CCR7 by NK cells. Interestingly, M0 and M2 cells expressed a membrane-bound form of IL-18, which was released in small amounts after LPS treatment. Our data indicate that, upon interaction with M0 macrophages exposed to microbial products, NK cells may amplify classical type 1 immune responses. In addition, M1-polarizing stimuli can rescue M2 macrophages from their immunomodulatory state and shape their functional behavior toward NK stimulatory capability.

Cytotoxic mechanisms may play a role in the local immune response in the central nervous system in neuroborreliosis

Aiming to investigate the role of cytotoxic mechanisms in neuroborreliosis (NB), the cytokines IL-2, IL-7, IL-10, IL-12p70, IL-15, GM-CSF and the Th17-cytokine IL-17 were analyzed in cerebrospinal fluid (CSF) and plasma from NB-patients. NB-patients showed increased levels in CSF compared to controls of all analyzed cytokines except IL-15 but not in plasma. Blood lymphocytes from three NB-patients showed functional cytotoxicity in response to autologous Borrelia-infected macrophages. The findings support a role for cytotoxic mechanisms in the local immune response in NB and in addition suggest an increase of IL-17.

CD8 Cells of Patients with Diffuse Cutaneous Leishmaniasis Display Functional Exhaustion: The Latter Is Reversed, In Vitro, by TLR2 Agonists

Leishmania mexicana (Lm) causes localized (LCL) and diffuse (DCL) cutaneous leishmaniasis. DCL patients have a poor cellular immune response leading to chronicity. It has been proposed that CD8 T lymphocytes (CD8) play a crucial role in infection clearance, although the role of CD8 cytotoxicity in disease control has not been elucidated. Lesions of DCL patients have been shown to harbor low numbers of CD8, as compared to patients with LCL, and leishmanicidal treatment restores CD8 numbers. The marked response of CD8 towards Leishmania parasites led us to analyze possible functional differences between CD8 from patients with LCL and DCL. We compared IFNγ production, antigen-specific proliferation, and cytotoxicity of CD8 purified from PBMC against autologous macrophages (MO) infected with Leishmania mexicana (MOi). Additionally, we analyzed tissue biopsies from both groups of patients for evidence of cytotoxicity associated with apoptotic cells in the lesions. We found that CD8 cell of DCL patients exhibited low cytotoxicity, low antigen-specific proliferation and low IFNγ production when stimulated with MOi, as compared to LCL patients. Additionally, DCL patients had significantly less TUNEL+ cells in their lesions. These characteristics are similar to cellular “exhaustion” described in chronic infections. We intended to restore the functional capacity of CD8 cells of DCL patients by preincubating them with TLR2 agonists: Lm lipophosphoglycan (LPG) or Pam3Cys. Cytotoxicity against MOi, antigen-specific proliferation and IFNγ production were restored with both stimuli, whereas PD-1 (a molecule associated with cellular exhaustion) expression, was reduced. Our work suggests that CD8 response is associated with control of Lm infection in LCL patients and that chronic infection in DCL patients leads to a state of CD8 functional exhaustion, which could facilitate disease spread. This is the first report that shows the presence of functionally exhausted CD8 T lymphocytes in DCL patients and, additionally, that pre-stimulation with TLR2 ligands can restore the effector mechanisms of CD8 T lymphocytes from DCL patients against Leishmania mexicana-infected macrophages.

Interleukin‐10 Promotes NK Cell Killing of Autologous Macrophages by Stimulating Expression of NKG2D Ligands

Under inflammatory conditions, the pleiotropic cytokine interleukin-10 (IL-10) is released in many tissues. It mediates anti-inflammatory effects in particular by inhibiting the release of T helper type 1 (Th1) cytokines. In contrast, we show here that NK cell cytotoxicity against autologous macrophages is elevated if both cell types are cultured with IL-10. The expression of most activatory NK receptors is increased after culture in the presence of IL-10. On the other hand, macrophages cultured in the presence of IL-10 show elevated expression of the NKG2D ligands major histocompatibility complex (MHC) class 1-like molecules (MIC) - A and - B, as well as UL-16 binding proteins (ULBP) - ULBP-1, ULBP-2 and ULBP-3. By masking the interaction of NK cells with macrophages through interruption of the NKG2D receptor with its ligands, we could reverse the IL-10-induced lysis of macrophages. Our data therefore reveal that IL-10 may exert a novel immunomodulatory role by stimulating NKG2D ligand expression on macrophages, thereby rendering them susceptible to NK cell elimination. This suggests that NK cells would delete macrophages and potentially other immature antigen-presenting cells (APC) or their precursors under inflammatory conditions as a feedback mechanism to shut off uncontrolled immune responses.

P55 Syngenic bone marrow transfer stimulates hepatic progenitor cell expansion via TWEAK/Fn14 Signalling: implications for human autologous cell therapy

IntroductionAutologous bone marrow cell (BMC) therapy for liver disease has shown increased liver regeneration in animal studies and in phase 1 clinical studies. As yet no clear mechanism accounts for...

Monocyte Modulation of Aqueous Outflow and Recruitment to the Trabecular Meshwork Following Selective Laser Trabeculoplasty

To determine whether selective laser trabeculoplasty (SLT) induces monocyte recruitment to the trabecular meshwork (TM) in human and monkey eyes and whether monocytes increase both aqueous outflow in vivo and the conductivity of human Schlemm canal endothelial cells (SCEs) in vitro. Monocyte recruitment was examined morphometrically in control human and monkey eyes and compared with that following SLT applied 1 to 3 days earlier. Outflow facility was measured for up to 4 days after the intracameral infusion of autologous macrophages in rabbits. Schlemm canal endothelial cell conductivity was measured using flow meters after exposing cultured SCEs to monocytes and monocyte-secreted factors for 24 hours. Our estimates show that the TM in the human eye normally had an average of 15 003 monocytes, while in the monkey eye there were 3181 monocytes, and this number increased 4- to 5-fold following SLT. The intracameral infusion of autologous macrophages in rabbits increased outflow facility 2-fold in a rapid and sustained manner. Human monocytes and monocyte-secreted factors increased SCE conductivity 2-fold in vitro. The number of monocytes/macrophages in the TM increases substantially after SLT and monocytes augment both outflow facility and SCE conductivity. Clinical Relevance These findings indicate that the innate immune system in general and monocytes in particular play an important role in aqueous outflow homeostasis. The recruitment of monocytes in increased numbers after SLT likely plays a role in lowering the intraocular pressure after this procedure. The intracameral introduction of autologous monocytes harvested from a vein could have therapeutic potential as a cell-based individualized treatment of glaucoma.

Developing Capsid Inhibitor V-073 for Poliovirus

Autologous macrophage therapy represents a potentially significant therapeutic advance for the treatment of severe progressive liver cirrhosis. Administration of macrophages has been shown to reduce inflammation and drive fibrotic scar breakdown and tissue repair in relevant models. This therapeutic approach is being assessed for safety and feasibility in a first-in-human trial (MAcrophages Therapy for liver CirrHosis [MATCH] trial).We outline the development and validation phases of GMP production. This includes use of the CliniMACS Prodigy cell sorting system to isolate CD14+ cells; optimizing macrophage culture conditions, assessing cellular identity, product purity, functional capability and determining the stability of the final cell product.The GMP-compliant macrophage products have a high level of purity and viability, and have a consistent phenotypic profile, expressing high levels of mature macrophage markers 25F9 and CD206 and low levels of CCR2. The macrophages demonstrate effective phagocytic capacity, are constitutively oriented to an anti-inflammatory profile and remain responsive to cytokine and TLR stimulation. The process validation shows that the cell product in excipient is remarkably robust, consistently passing the viability and phenotypic release criteria up to 48 hours after harvest.This is the first report of validation of a large-scale, fully Good Manufacturing Practice–compliant, autologous macrophage cell therapy product for the potential treatment of cirrhosis. Phenotypic and functional assays confirm that these cells remain functionally viable for up to 48 h, allowing significant flexibility in administration to patients.

Mycobacterium chelonae sensitisation induces CD4+‐mediated cytotoxicity against BCG

Significant variability in efficacy of live Mycobacterium bovis BCG as a tuberculosis vaccine is observed globally. Effects of pre-vaccination sensitisation to non-tuberculous environmental mycobacteria (Env) are suspected to underlie this phenomenon, but the mechanisms remain unclear. We postulated that it could be due to Env-specific T cells exerting cytotoxicity against BCG-infected host cells. After murine sensitisation with heat-killed antigens of different Env species, splenocytes from M. chelonae (CHE)-sensitised mice exerted the strongest cytotoxicity against autologous BCG-infected macrophages. This cytotoxicity was correlated with reduced BCG viability. The cytotoxicity was reduced by the depletion of CD4(+), but not CD8(+) or CD56(+) cells, and CD4(+) cells showed higher percentage of cytotoxicity than CD4(-) cells, supporting a role for CD4(+) cells in CHE-induced, BCG-specific cytotoxicity. Additionally, this cytotoxicity was IFN-gamma, perforin and FasL dependent. After CHE-sensitisation and subsequent BCG intranasal infection, there was significant expansion of lung CD4(+) cells, the main cell type producing IFN-gamma. This was associated with 2- and 6-fold reductions in lung BCG counts 1 and 3 wk, respectively post- infection, relative to non-sensitised mice. This is the first report describing cytotoxicity against BCG-infected cells as a mechanism underlying the influence of Env sensitisation on subsequent BCG responses.

Sand Fly Salivary Proteins Induce Strong Cellular Immunity in a Natural Reservoir of Visceral Leishmaniasis with Adverse Consequences for Leishmania

Immunity to a sand fly salivary protein protects against visceral leishmaniasis (VL) in hamsters. This protection was associated with the development of cellular immunity in the form of a delayed-type hypersensitivity response and the presence of IFN-γ at the site of sand fly bites. To date, there are no data available regarding the cellular immune response to sand fly saliva in dogs, the main reservoirs of VL in Latin America, and its role in protection from this fatal disease. Two of 35 salivary proteins from the vector sand fly Lutzomyia longipalpis, identified using a novel approach termed reverse antigen screening, elicited strong cellular immunity in dogs. Immunization with either molecule induced high IgG2 antibody levels and significant IFN-γ production following in vitro stimulation of PBMC with salivary gland homogenate (SGH). Upon challenge with uninfected or infected flies, immunized dogs developed a cellular response at the bite site characterized by lymphocytic infiltration and IFN-γ and IL-12 expression. Additionally, SGH-stimulated lymphocytes from immunized dogs efficiently killed Leishmania infantum chagasi within autologous macrophages. Certain sand fly salivary proteins are potent immunogens obligatorily co-deposited with Leishmania parasites during transmission. Their inclusion in an anti-Leishmania vaccine would exploit anti-saliva immunity following an infective sand fly bite and set the stage for a protective anti-Leishmania immune response.

Intrathecal transplantation of autologous macrophages genetically modified to secrete proenkephalin ameliorated hyperalgesia and allodynia following peripheral nerve injury in rats

To develop a novel genetic approach for the treatment of pain, we tested the transplantation of gene-transferred autologous macrophages by lumbar puncture. A rat neuropathic pain model was produced by chronic constriction of the sciatic nerve. Autologous macrophages were collected from the intraperitoneal space. Then human proenkephalin gene was transferred into the macrophages by electroporation. The gene-transferred macrophages were transplanted into the subarachnoid space by lumbar puncture. One week after transplantation, the heat hyperalgesia and allodynia induced by sciatic nerve constriction completely remitted. The analgesic action continued until at least 4 weeks after transplantation. The transplanted macrophages migrated into the spinal cord and expressed proenkephalin mRNA and Met-enkephalin protein. The method we tested in the present study may be a safe, simple and effective way to inhibit pain sensation after peripheral nerve injuries.

Degradation-resistant protein domains limit host cell processing and immune detection of mycobacteria

The Mycobacterium tuberculosis genome reveals a large family of glycine–alanine rich PE–PGRS proteins. Due to similarities with the glycine–alanine rich Epstein–Barr nuclear antigen 1, there has been interest in whether PE–PGRS proteins inhibit cellular processing and presentation via the major histocompatibility complex class I pathway. We investigated whether PE–PGRS proteins were resistant to ubiquitin–proteasome-dependent degradation and CD8+ T cell recognition. Upon transient expression of ubiquitin fusion constructs of either full-length Rv0978cPE–PGRS protein or its PE domain in HeLa cells, the former was markedly less susceptible to proteasomal degradation. When peptides of varying glycine and alanine content from different PE–PGRS proteins were fused to the N-terminus of SIINFEKL peptide, the alanine-rich fusions elicited lower interleukin-2 responses in SIINFEKL-specific CD8+ T cells, with corresponding decrease in lysis of cells presenting such peptides. When CD8+ T cells from Mycobacterium bovis BCG-immunized mice were stimulated with either full-length PE–PGRS protein Rv3812 or its PE domain, the former exhibited a lower level of cytotoxicity against BCG-infected autologous macrophages. These results suggest that mycobacterium PE–PGRS proteins have domains that confer resistance to ubiquitin–proteasome-dependent protein degradation, and the bacteria may have an abundance of such proteins to evade immune detection and killing of mycobacterium-infected cells.

Raising allo-restricted cytotoxic T lymphocytes by co-culture of murine splenocytes with autologous macrophage bearing the peptide/allo–major histococompatibility complex

Generation and adoptive transfusion of alloreactive cytotoxic T lymphocytes (CTLs) specific for tumor are expected to circumvent tumor tolerance. Here we describe a novel protocol to raise allo-restricted, peptide-specific CTLs by co-culture of murine splenocytes and autologous macrophage bearing an allogeneic H-2K molecule associated with its restricted peptide (peptide/allo-MHC). The extracellular domains of H-2K d were fused with constant domains of murine IgG2a heavy chain to generate a fusion protein (peptide/H-2K d/IgG2aFc, the dimer) consisting of divalent TCR-ligands and an IgG Fc receptor type I (FcγRI)-reactive moiety. The dimer is able to bind to macrophage (Mϕ) of H-2K k via the interaction of the Fc part with FcγRI, and cause the H-2K k Mϕ to be coated with the peptide/H-2K d complex. The results show that proliferation of CD8+ cells is enhanced and that the specific-tetramer stained CD8+ cells appear more frequently by co-culture of H-2K k splenocytes with the autologous Mϕ loaded with the dimer. Furthermore, the CD8+ T cells from the co-cultural bulk exhibit an elevated cytotoxicity against a specific target (H-2K d-restricted, peptide-specific cytotoxicity), compared with that against the irrelevant targets. This study provides a strategy for preparation of allo-restricted, peptide-specific CTLs, which may add to our arsenal for adoptive immunotherapy to eliminate chronic virally infected or tumor cells.

ORIGINAL ARTICLE: Human Uterine NK Cells Interact with Uterine Macrophages via NKG2D upon Stimulation with PAMPs

Problem The initiation of an immune response often involves the cooperation of various innate immune cells. In the human endometrium, uterine natural killer (uNK) cells and uterine macrophages are present in significant numbers and in close proximity, yet how they cooperatively respond to infectious challenge is poorly understood.Method of study Primary autologous uNK cells and macrophages were co‐cultured to determine functional interactions after stimulation with pathogen‐associated molecular patterns.Results After stimulation by polyI:C, human uNK cells interact with autologous uterine macrophages and produce interferon‐γ in an NKG2D‐dependent manner. Stimulated primary uterine macrophages up‐regulated the expression of MHC Class I chain‐related protein A (MICA), but expression of the cognate receptor NKG2D remained unchanged on uNK cells, even in the presence of cytokines.Conclusion This study demonstrates that the NKG2D‐MICA interaction is an important molecular mechanism that is involved in the innate immune response to microbial signals in the human uterine endometrium.

Activated autologous macrophage implantation in a large-animal model of spinal cord injury

Axonal regeneration may be hindered following spinal cord injury (SCI) by a limited immune response and insufficient macrophage recruitment. This limitation has been partially surmounted in small-mammal models of SCI by implanting activated autologous macrophages (AAMs). The authors sought to replicate these results in a canine model of partial SCI. Six dogs underwent left T-13 spinal cord hemisection. The AAMs were implanted at both ends of the lesion in 4 dogs, and 2 other dogs received sham implantations of cell media. Cortical motor evoked potentials (MEPs) were used to assess electrophysiological recovery. Functional motor recovery was assessed with a modified Tarlov Scale. After 9 months, animals were injected with wheat germ agglutinin-horseradish peroxidase at L-2 and killed for histological assessment. Three of the 4 dogs that received AAM implants and 1 of the 2 negative control dogs showed clear recovery of MEP response. Behavioral assessment showed no difference in motor function between the AAM-treated and control groups. Histological investigation with an axonal retrograde tracer showed neither local fiber crossing nor significant uptake in the contralateral red nucleus in both implanted and negative control groups. In a large-animal model of partial SCI treated with implanted AAMs, the authors saw no morphological or histological evidence of axonal regeneration. Although they observed partial electrophysiological and functional motor recovery in all dogs, this recovery was not enhanced in animals treated with implanted AAMs. Furthermore, there was no morphological or histological evidence of axonal regeneration in animals with implants that accounted for the observed recovery. The explanation for this finding is probably multifactorial, but the authors believe that the AAM implantation does not produce axonal regeneration, and therefore is a technology that requires further investigation before it can be clinically relied on to ameliorate SCI.

IFN-γ expression is up-regulated by peripheral blood mononuclear cells (PBMC) from non-exposed dogs upon Leishmania chagasi promastigote stimulation in vitro

While the response to Leishmania spp. is well characterized in mice and humans, much less is known concerning the canine immune response, particularly soon after exposure to the parasite. Early events are considered to be a determinant of infection outcome. To investigate the dog's early immune response to L. chagasi, an in vitro priming system (PIV) using dog naïve PBMC was established. Until now, dog PIV immune response to L. chagasi has not been assessed. We co-cultivated PBMC primarily stimulated with L. chagasi in vitro with autologous infected macrophages and found that IFN-γ mRNA is up-regulated in these cells compared to control unstimulated cells. IL-4 and IL-10 mRNA expression by L. chagasi-stimulated PBMC was similar to control unstimulated PBMC when incubated with infected macrophages. Surprisingly, correlation studies showed that a lower IFN-γ/IL-4 expression ratio correlated with a lower percentage of infection. We propose that the direct correlation between IFN-γ/IL-4 ratio and parasite load is dependent on the higher correlation of both IFN-γ and IL-4 expression with lower parasite infection. This PIV system was shown to be useful in evaluating the dog immune response to L. chagasi, and results indicate that a balance between IFN-γ and IL-4 is associated with control of parasite infection in vitro.

Specific lysis of mycobacterial antigen-bearing macrophages by class II MHC-restricted polyclonal T cell lines in healthy donors or patients with tuberculosis

The cytolytic capacity of mycobacterial antigen-stimulated peripheral blood mononuclear cells, from healthy Mantoux-positive volunteers and from patients with tuberculosis was investigated. Polyclonal T cell lines induced by 7 days of stimulation in vitro with PPD or a sonicate of Mycobacterium tuberculosis lysed both autologous macrophages and Epstein-Barr virus (EBV) transformed B cell lines which had been pulsed with mycobacterial antigens, to a greater extent than unpulsed target cells or target cells pulsed with an irrelevant antigen (streptokinase/streptodornase). The killing of mycobacterial antigen-pulsed macrophages and EBV-transformed B cell line targets was inhibited by monoclonal antibodies to MHC class II antigens but not by antibodies directed against MHC class I antigens. PPD-pulsed EBV-transformed lymphoblastoid cell lines (LCL) competitively inhibited the killing of mycobacterial antigen-pulsed macrophages, whereas natural killer (NK) sensitive K562 cells (with or without antigen pulsing) did not inhibit mycobacterial antigen-dependent cytolysis of macrophages. Patients with tuberculosis showed a spectrum of mycobacterial antigen-induced cytolytic capacity. Those with extensive tissue necrosis (e.g. cavitatory pulmonary tuberculosis or caseous, extrathoracic tuberculosis) had high levels while patients with disseminated (miliary) tuberculosis or disease refractory to treatment showed little evidence of mycobacterial antigen induced cytotoxicity. The ability of mycobacterial antigen-stimulated lymphoblasts to kill specific antigen-pulsed autologous macrophages was not significantly different between healthy donors and patients with tuberculosis. However, the 'mycobacterial antigen-specific' component of this cytolysis was significantly deficient (P less than 0.01) in patients. We conclude that mycobacterial antigen-specific cytotoxic T cell responses may play a significant part in the immune response to mycobacterial infection.