The role of type I interferon production by dendritic cells in host defense.

Type I interferons (IFN) and dendritic cells (DC) share an overlapping history, with rapidly accumulating evidence for vital roles for both production of type 1 IFN by DC and the interaction of this IFN both with DC and components of the innate and adaptive immune responses. Within the innate immune response, the plasmacytoid DC (pDC) are the "professional" IFN producing cells, expressing specialized toll-like receptors (TLR7 and -9) and high constitutive expression of IRF-7 that allow them to respond to viruses with rapid and extremely robust IFN production; following activation and production of IFN, the pDC subsequently mature into antigen presenting cells that help to shape the adaptive immune response. However, like most cells in the body, the myeloid or conventional DC (mDC or cDC) also produce type I IFNs, albeit typically at a lower level than that observed with pDC, and this IFN is also important in innate and adaptive immunity induced by these classic antigen presenting cells. These two major DC subsets and their IFN products interact both with each other as well as with NK cells, monocytes, T helper cells, T cytotoxic cells, T regulatory cells and B cells to orchestrate the early immune response. This review discusses some of the converging history of DC and IFN as well as mechanisms for IFN induction in DC and the effects of this IFN on the developing immune response.

Decreased interferon-alpha production in HIV-infected patients correlates with numerical and functional deficiencies in circulating type 2 dendritic cell precursors.

Peripheral blood mononuclear cells from patients with human immunodeficiency virus (HIV) infection exhibit a progressively marked decrease in the production of virus-induced interferon (IFN)-alpha, a finding that correlates with and is highly predictive of disease progression and opportunistic infections. The major IFN-alpha producing population has recently been defined as the precursor to type 2 dendritic cells (pDC2) or plasmacytoid DC (pDC). Using four-color flow cytometry, we have enumerated the pDC2 vs non-IFN-alpha producing myeloid DC1 in peripheral blood from HIV-infected patients and healthy controls and related these values to CD4 cell numbers, viral load, and functional activity. The patients had reductions in the numbers of both pDC2 (lin-/HLA-DR+/CD123(bright)) and DC1 (lin1-/HLA-DR+/CD123(dim)/CD11c+), both at an absolute level and as a percentage of cells. The decreases were most evident in patients with decreased CD4 levels. Viral load correlated with the functional frequency of the IFN producing cells but not with absolute pDC2 levels. Using intracellular flow cytometric analysis for IFN-alpha, the patients were demonstrated to have fewer pDC2, as well as a lower percentage of responding cells among those remaining. We conclude that deficient production of IFN-alpha by pDC2 from HIV-infected patients results from both selective loss of these cells and their qualitative dysfunction. Given the central role of DC, and in particular, DC2, in linking innate and adaptive immune responses, these qualitative and quantitative changes in pDC2 are likely to be key contributors to HIV pathogenesis.

Interferon-alpha generation and immune reconstitution during antiretroviral therapy for human immunodeficiency virus infection.

OBJECTIVES: To quantify the effect of HIV infection and HIV-suppressive therapy on interferon-alpha (IFN-alpha) production by human blood mononuclear cells; to compare, in parallel, effects on CD4+ T-cell numbers; and to ascertain the relationship of these interferon and CD4 parameters to resistance to opportunistic infections. DESIGN: Serial studies of 294 unselected patients with HIV infection during therapy, with outcomes analysis. METHODS: Determination of IFN generation by blood mononuclear cells via bioassay, and T-lymphocyte subset analysis via flow cytometry; serial studies of individual patients; linear regression and chi2 contingency table analysis. RESULTS: HIV burden is inversely related to interferon-alpha generation, much as it is to CD4+ T-cell counts. Both of these recover during HIV-suppressive therapy. Reconstitution of IFN-alpha generation to levels commensurate with protection against opportunistic infection occurs prior to similar restoration of CD4 counts. In the outcomes analyses, such immune reconstitution was associated with protection from recurrent or new opportunistic infection. Conversely, viral suppression without such immunologic recovery was not protective against opportunistic infection. CONCLUSIONS: Rapidly responding IFN-alpha generating cells appear to participate in resistance to opportunistic intracellular infection. Recovery of IFN-alpha generation may be an early marker of immune reconstitution in AIDS.

The nature of the principal type 1 interferon-producing cells in human blood.

Interferons (IFNs) are the most important cytokines in antiviral immune responses. "Natural IFN-producing cells" (IPCs) in human blood express CD4 and major histocompatibility complex class II proteins, but have not been isolated and further characterized because of their rarity, rapid apoptosis, and lack of lineage markers. Purified IPCs are here shown to be the CD4(+)CD11c- type 2 dendritic cell precursors (pDC2s), which produce 200 to 1000 times more IFN than other blood cells after microbial challenge. pDC2s are thus an effector cell type of the immune system, critical for antiviral and antitumor immune responses.

The mannose receptor mediates induction of IFN-alpha in peripheral blood dendritic cells by enveloped RNA and DNA viruses.

Peripheral blood dendritic cells (DC) produce IFN-alpha in response to challenge by many enveloped viruses including herpes simplex virus (HSV) and HIV, whereas Sendai virus predominantly stimulates IFN-alpha production by monocytes. Glycosylated viral envelope proteins are known to be important for the induction of IFN-alpha. In this study we demonstrate that stimulation of IFN-alpha synthesis by HSV is inhibited by a number of monosaccharides, including fucose, N-acetylglucosamine, and N-acetylgalactosamine as well as the yeast polysaccharide mannan, supporting a role for lectin(s) in the IFN-alpha stimulation pathway. Furthermore, antiserum to the mannose receptor (MR) also inhibited HSV, vesicular stomatitis virus, and HIV-induced IFN-alpha production, but failed to inhibit the IFN-alpha induced by Sendai virus. We further demonstrated that freshly isolated blood DC and IFN-alpha-producing cells responding to HSV stimulation express the MR. This study therefore implicates the MR as an important receptor for the nonspecific recognition of enveloped viruses by DC and the subsequent stimulation of IFN-alpha production by these viruses. Thus, the MR probably serves as a critical link between innate and adaptive immunity to viruses, especially given the role of the MR in Ag capture by DC and the importance of IFN-alpha in shaping immunity.

Exogenous and endogenous IL-10 regulate IFN-alpha production by peripheral blood mononuclear cells in response to viral stimulation.

IL-10 is an important regulator of the production of proinflammatory cytokines. Its effect on IFN-alpha production, however, has not been reported. In this study, PBMC from healthy donors were stimulated with virus in the presence of IL-10. Human IL-10 (hIL-10) caused reductions in both the frequency of IFN-alpha-producing cells (IPC) and bulk IFN in response to herpes simplex virus type-1 (HSV-1), Sendai virus, Newcastle disease virus, and vesicular stomatitis virus. The inhibitory effect occurred when IL-10 was added 2 or 4 h before, or 2 h poststimulation with HSV or Sendai virus, but not when added 4 h postinduction. Unlike IL-10, IL-4 did not affect the IFN-alpha response to HSV. However, when PBMC were induced with Sendai virus, IFN-alpha production was also reduced by IL-4. IL-10 treatment of PBMC resulted in strong reductions in the steady state levels of both HSV- and Sendai virus-induced IFN-alpha1, -alpha2, and -beta mRNA as determined by RT-PCR. IFN-alpha production to Sendai virus occurs predominantly by monocytes, whereas most enveloped viruses stimulate low frequency "natural IFN-producing cells (NIPC)," which are thought to be dendritic cells. Peripheral blood dendritic cells were found to express the IL-10 receptor, suggesting that IL-10 may directly act on the dendritic IPC. Addition of monoclonal anti-IL-10 to PBMC resulted in a significant increase in both the frequency of IPC and the amount of secreted IFN-alpha in response to HSV but not Sendai virus. We conclude that human IL-10 can serve as both an endogenous and exogenous regulator of IFN-alpha production.

Role of tyrosine kinases, protein kinase C, and protein kinase A in the regulation of interferon-alpha production induced by herpes simplex virus type 1.

Herpes simplex virus 1 (HSV-1) is able to induce interferon-alpha production by natural IFN-alpha-producing cells. In this study, signal transduction in this process was examined. It was found that sequestering of calcium by EGTA abolished IFN-alpha induction by HSV-infected cells. Stimulation of human PBMC by HSV-1-infected fibroblasts resulted in the production of inositol triphosphate (InsP3) and tyrosine phosphorylation of cellular proteins. The protein kinase C inhibitor, H7, and the tyrosine kinase inhibitor, herbimycin A, were able to suppress IFN-alpha gene expression as determined by IFN bioassay and RT-PCR. An IFN-alpha-specific ELISpot assay revealed that herbimycin A and H7 remarkably decreased the number of IFN-alpha-producing cells. PMA or calcium ionophore A23187 alone did not increase IFN-alpha production. However, PMA in conjugation with ionophores increased IFN-alpha production as early as 2 h. HA1004 and 2',5'-dideoxyadenosine, which are potent inhibitors of PKA pathway, had no effect on IFN-alpha production. In contrast, BrcAMP, a specific PKA activator, inhibited the IFN-alpha secretion and number of IFN-alpha-producing cells and to a lesser extent reduced the level of IFN-alpha mRNA. Our results indicate that protein kinase C, tyrosine kinases, and protein kinase A are involved in the regulation of IFN-alpha production in response to HSV-1.

Functional deficiencies in two distinct interferon alpha-producing cell populations in peripheral blood mononuclear cells from human immunodeficiency virus seropositive patients.

Two populations of IFN-alpha producing cells (IPC) were examined to determine whether they are coordinately dysregulated in human immunodeficiency virus (HIV) disease. IFN-alpha produced in response to herpes simplex virus (HSV) and Sendai virus (SV) was measured and the frequencies of the IPC were obtained by ELISpot assay. IPC that respond to HSV (natural IFN-alpha producing cells) and those responding to SV (predominantly monocytes) were present, on average, at 7.6 and 138 per 10(4) PBMC in healthy controls, respectively. More patients had a reduced IFN-alpha response to HSV than to SV, and individual patients did not show a decreased response to SV without a decreased response to HSV. Neither IPC function was correlated with CD4+ cell levels. We conclude that the defects in IFN-alpha production in these two cell populations arise independently, possibly due to differences in susceptibility to HIV infection or molecular regulation.

Viral induction of low frequency interferon-alpha producing cells.

The human peripheral blood mononuclear cells responsible for IFN-alpha production in response to viral stimuli have been most often described as either monocytes (as typified by the response to Sendai virus) or as a light density, HLA-DR+ population which is negative for most cell surface markers characteristic of mature T cells, B cells, monocytes, or natural killer cells (as typified by the response to Herpes simplex virus (HSV)). The frequency of IFN-alpha-producing cells (IPC) responding to Sendai virus is typically 10-fold or more higher than those responding to HSV. In the current study, we have used ELISpot assays to determine the frequency of IPC responding to DNA and RNA viruses including HSV, Sendai, vesicular stomatitis virus, cytomegalovirus, adenovirus, SV40, influenza, measles, mumps, Newcastle disease virus (NDV) and human immunodeficiency virus (HIV). The enveloped viruses but not the nonenveloped viruses (adenovirus and SV40) elicited an IFN-alpha response. The frequency of IPC for each of the other viruses was more similar to the low frequency HSV-responding population than to the higher frequency Sendai virus response. These included several viruses in the same family as Sendai virus, namely the paramyxo viruses measles, mumps, and NDV. IPC were also tested for sensitivity to the lysosomotropic drug chloroquine, which diminishes IFN-alpha produced in response to HSV but not Sendai virus. With the exception of Sendai virus, chloroquine treatment abrogated the majority of IFN-alpha produced and IPC against each of the viruses. We conclude that low frequency, nonmonocytic NIPC account for the majority of IFN-alpha production in response to different viruses.

Impaired interferon alpha response in hairy cell leukemia is corrected by therapy with 2-chloro-2'-deoxyadenosine: implications for susceptibility to opportunistic infections.

Patients with hairy cell leukemia (HCL) are susceptible to opportunistic intracellular infections, suggesting defects in cellular immunity. Prior studies have indicated an association between failure of IFN-alpha generation by peripheral blood mononuclear cells (MNC) and susceptibility to such infections. We here present results on IFN-alpha generation in HCL patients pre- and post-therapy. Prior to treatment with 2-chloro-2'-deoxyadenosine (CdA), MNC from 24 HCL patients with active disease produced little or not IFN-alpha (geometric mean < 40 IU/ml) compared with controls (n = 140, geometric mean 1730 IU/ml, p < 0.0005). After treatment with CdA, IFN-alpha generation was studied in 16 patients, with a geometric mean value of 650 IU/ml (p < 0.0005 compared with pre-CdA levels). The severe depression of IFN-alpha generation improved progressively following CdA therapy-induced clinical remission. We propose that deficiency of IFN-alpha production may play a role in the susceptibility to intracellular infections of patients with active HCL.

Decreased frequency of functional natural interferon-producing cells in peripheral blood of patients with the acquired immune deficiency syndrome.

Deficient in vitro production of interferon-alpha (IFN-alpha) in response to herpes simplex virus (HSV) occurs in patients infected with the human immunodeficiency virus (HIV), with the most deficient responses associated with opportunistic infections (OI). The peripheral blood mononuclear cells (PBMC) which produce IFN-alpha in response to HSV are light density, HLA-DR+ cells lacking any unique surface markers and have been termed "natural interferon-producing cells" (NIPC). In this study, IFN-alpha responses were measured and the ELISpot assay was utilized to determine the frequency of NIPC in response to HSV. As expected, HIV-infected patients had depressed IFN-alpha production. In the ELISpot assay, healthy controls had a mean frequency of 1:703 NIPC among PBMC; each NIPC made approximately 2 international units (IU) of IFN-alpha. HIV-infected patients on average had fourfold less NIPC than controls and produced 1 IU IFN-alpha/NIPC; the plaque size for patient samples was often smaller than that for controls. NIPC frequency and IFN-alpha production were lowest in patients with a history of OI. In conclusion, deficient IFN-alpha production by AIDS patients results from reductions in both the frequency and the activity of NIPC, probably reflecting a gradual turning off of IFN-alpha production.

Interferon-alpha generation in mice responding to challenge with UV-inactivated herpes simplex virus.

In humans with advanced human immunodeficiency virus (HIV) infection, an interferon-alpha (IFN-alpha) response by a specialized blood mononuclear cell to herpes simplex virus (HSV) in vitro is associated with resistance to opportunistic infections. A cell type of unknown lineage, designated the natural IFN-producing cell (NIPC), has been identified preliminarily as the source of these IFNs and may have a role in other host defense functions. Earlier studies suggested the existence of analogous HSV-responsive cell populations in mice. The role specifically of IFN-alpha in the murine system, however, has not been characterized. Using IFN bioassay and neutralization with antisera against Type I IFNs and IFN-beta, we have defined the types and sources of IFNs produced by mice in response to in vivo and in vitro challenge with UV-inactivated HSV. After intraperitoneal inoculation with HSV, BALB/c and C57Bl/6 strains produced characteristically different levels of serum IFNs that appeared principally to be IFN-alpha. The response of mononuclear cells from these mice differed from that of the intact mouse. Isolated cells from bone marrow and spleen released detectable IFNs much later than did whole animals, and the IFNs produced by marrow, spleen, and peritoneal cells were usually neutralized by the anti-IFN-beta. Only bone marrow cells produced detectable amounts of IFN-alpha. Both intact mice and their cells became refractory to restimulation with similar kinetics.

Human natural interferon-alpha producing cells.

Interferons (IFNs) are critical components of the host immune system, serving as antiviral agents, immunomodulators and inhibitors of cell growth. Among peripheral blood mononuclear cells, the primary IFN-alpha-producing cell is a light density, HLA-DR+ cell negative for cell surface markers typical for T cells, B cells, monocytes, natural-killer or progenitor cells and has been tentatively termed the 'natural IFN-producing cell' or NIPC. Although present in very low frequency (approximately 1:1000 among peripheral blood mononuclear cells), the NIPC are very potent, with an individual cell able to produce 1-2 IU of IFN. In this review, the characteristics, phenotype, regulation and relationship of NIPC to human disease are discussed.

Peripheral blood of AIDS patients contains cells capable of providing accessory function for the natural killer cell-mediated, lysis of herpes simplex virus-infected targets despite low interferon-alpha production.

We have previously demonstrated that in vitro production of interferon-alpha (IFN-alpha) in response to herpes simplex virus (HSV) by peripheral blood mononuclear cells PBMCs from patients infected with the human immunodeficiency virus (HIV-1) decreases dramatically with disease progression, with extremely low levels of IFN-alpha preceding and predictive of opportunistic infections. Natural killer (NK) lysis, however, was found to decay later in disease and often was within normal limits even when IFN-alpha production was severely compromised. The NK lysis of HSV-infected fibroblasts (HSV-FS) is dependent on an HLA-DR+ accessory cell (AC) population that shares the phenotype of the predominant IFN-alpha-producing cell (IPC) population. To determine whether there is a correlation between AC activity and IFN-alpha production in these patients, we tested the ability of PBMCs from AIDS patients to provide AC help to NK cells from heterologous donors. While NK cells were highly sensitive to gamma irradiation, AC activity was relatively radioresistant. Therefore, NK cells from healthy donors were depleted of HLA-DR+ ACs and added to irradiated PBMCs from either healthy or AIDS donors to test for the function of ACs in the irradiated populations. Irradiated cells from AIDS patients were found to provide normal AC activity despite decreased IFN-alpha production in the majority of the patients. We failed to observe NK augmenting activity in supernatants of irradiated PBMCs from IFN-deficient patients that had been stimulated with HSV-FS.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon-alpha-dependent and -independent participation of accessory cells in natural killer cell-mediated lysis of HSV-1-infected fibroblasts.

Human natural killer (NK) cells require an HLA-DR+ accessory cell (AC) population to lyse herpes simplex virus-type 1 (HSV-1)-infected fibroblasts (HSV-Fs) but not K562 target cells. It has been postulated that ACs may function by producing interferon-alpha (IFN-alpha), which stimulates NK cells. Using a sequential enrichment protocol, ACs were found to coenrich with the interferon-producing cells (IPCs). Treatment of the ACs with a protein synthesis inhibitor, emetine, inhibited both their IFN production and AC function, results that support a central role for IFN in AC activity. In contrast, when the arginine analogue canavanine was added to NK assays, no IFN-alpha was produced and NK(HSV-Fs) activity was only partially inhibited. Consistent with IFN-independent AC function, treatment with either polyclonal sheep or bovine anti-IFN-alpha neutralized all the IFN-alpha produced during the NK assays but caused either no or partial reduction of NK(HSV-Fs) activity, respectively. However, when limiting numbers of ACs were used, the bovine antiserum neutralized both IFN-alpha and NK(HSV-Fs) activity. We further found that HLA-DR+ cells are required for cell clustering, suggesting a role for cell contact. Finally, fixation of activated ACs prevented the accessory function. Together, these results demonstrate that ACs can provide help to NK cells in both IFN-alpha-dependent and -independent manners.

Immediate-early gene expression is sufficient for induction of natural killer cell-mediated lysis of herpes simplex virus type 1-infected fibroblasts.

Herpes simplex virus type 1 (HSV-1)-infected human fibroblast (HSV-FS) targets are susceptible to lysis by natural killer (NK) cells, whereas uninfected FS are resistant to lysis. Studies were undertaken to determine the mechanism of this preferential susceptibility. HSV-FS were not intrinsically less stable than FS, as determined by a 51Cr release assay under hypotonic shock in the presence of rat granule cytolysin and by sensitivity to anti-human leukocyte antigen class I antibody plus complement. Single-cell assays in agarose demonstrated that although similar numbers of large granular lymphocytes bound to the HSV-FS and FS targets, the conjugates with HSV-FS were lysed at a much higher frequency than those with FS. These results suggested that both targets are bound by the NK cells but only the HSV-FS were able to trigger lysis. The requirement for active virus expression was demonstrated by failure of emetine-treated HSV-FS targets or targets infected with UV-inactivated HSV to be lysed by NK effectors. To evaluate the role of viral glycoproteins in conferring susceptibility to lysis, Fab were prepared from HSV-1-seropositive sera; these Fab were unable to block lysis of the HSV-FS. Furthermore, incubation in phosphonoacetic acid failed to reduce NK(HSV-FS) activity despite sharp reductions in viral glycoprotein synthesis. Finally, targets infected with tsLB2 at the nonpermissive temperature were lysed as well as or better than targets infected with wild-type virus, indicating that HSV immediate-early gene product expression is sufficient for conferring susceptibility to lysis. We conclude that expression of nonstructural viral proteins or virally induced cellular gene products early in the course of infection rather than structural glycoproteins is required for NK lysis of HSV-FS targets.

Natural killer-mediated lysis of some but not all HSV-1- or VSV-infected targets requires the participation of HLA-DR-positive accessory cells.

Natural killer (NK)-mediated lysis of herpes simplex virus type 1-infected fibroblasts (HSV-FS) has been previously shown to require the co-operation of CD16-positive NK cells and an HLA-DR-positive accessory cell population. In contrast, lysis of K562 tumour cells requires the presence of only the Leu-11-positive cells. In the current study, targets of different morphologies, both virally infected and non-infected, were tested in an attempt to dissect out which target characteristics determine the need for accessory cell participation for NK-mediated lysis. Effector populations were obtained through antibody plus complement (C) depletions of subpopulations of human peripheral blood mononuclear cells using anti-HLA-DR+C (accessory cell depleted) or anti-CD16+C (NK depleted). The subpopulations were tested both alone and mixed together for their ability to mediate target lysis. Although NK-mediated lysis of most HSV-infected targets required the presence of HLA-DR-positive accessory cells, there was one set of exceptions. Lysis of the non-adherent Epstein-Barr virus (EBV)-transformed lymphoblastoid lines HSV-Raji, HSV-ARH and HSV-CCRF demonstrated only partial accessory cell dependence. All infected adherent cell lines were accessory cell dependent. In contrast, none of the adherent or non-adherent non-infected targets tested required the presence of DR-positive accessory cells for killing. Therefore, the presence of virus was an indicator of accessory cell dependence for NK-mediated kill except in the cases where HSV-infected EBV-transformed targets were used. Assay times of 4 hr versus 14 hr were conducted to determine if the kinetics of kill of various targets correlated with the requirement for accessory cells. A substantial percentage of the total lysis seen at 14 hr occurred within 4 hr for accessory cell independent lysis of the non-infected targets. In contrast, accessory cell-dependent kill of infected targets usually required longer incubation time for substantial lysis to occur, and correlated with interferon (IFN) production. NK-mediated lysis of vesicular stomatitis virus-infected fibroblasts required the presence of both the CD16- and HLA-DR-positive subpopulations, extending the role of DR-positive cells in NK-mediated killing beyond herpes virally infected targets.

Sequential enrichment and immunocytochemical visualization of human interferon-alpha-producing cells.

Human HLA-DR+ peripheral mononuclear cells (PBMC) produce interferon-alpha (IFN-alpha) in response to herpes simplex virus type 1 (HSV-1) or HSV-1-infected fibroblasts (HSV-FS). We have developed a protocol, based partly on a technique known to enrich for dendritic cells, that allows for a greater than 125-fold enrichment of these IFN-alpha-producing cells. Nylon wood nonadherent PBMC (NWNA) were fractionated on a 48% Percoll gradient into low-density (LD) and high-density (HD) populations. The LD cells were 10- to 30-fold enriched for the production of IFN-alpha compared to PBMC when stimulated with HSV-FS. LD cells were treated further to deplete any contaminating monocytes, CD3+ T cells and CD56+ natural killer (NK) cells. The resulting population (CD3/CD56-depleted) produced approximately 30,000 IU/ml IFN-alpha compared to 3,000-10,000 IU/ml for the corresponding LD cells and 30-300 IU/ml for PBMC. Immunocytochemistry to detect cytoplasmic IFN-alpha indicated that PBMC, NWNA, HD, LD, and CD3/CD56-depleted populations contained an average of less than 0.1%, 0.3%, less than 0.1%, 3%, and 12% IFN-alpha-producing cells, respectively. The cells responsible for IFN-alpha production in response to HSV-1 were of medium to large diameter and possessed eccentric nuclei that were often indented, with lightly staining perinuclear areas. The CD3/CD56-depleted populations were fivefold enriched for HLA-DR+ cells. This enrichment procedure partially overcomes the barrier of low frequency that has contributed to the elusive identification of these cells.

Positively selected Leu-11a (CD16+) cells require the presence of accessory cells or factors for the lysis of herpes simplex virus-infected fibroblasts but not herpes simplex virus-infected Raji.

Previous studies from our laboratory indicated that human NK activity against HSV-infected fibroblasts (HSV-Fs) but not K562 targets was sensitive to treatment with anti-HLA-DR plus C. In the current study, we have selected Leu-11a+ (CD-16) cells by fluorescence activated cell sorting and found that although Leu-11a enriched populations lysed K562 targets in 14-h 51Cr-release assays, they were unable to kill HSV-Fs targets unless a Leu-11a-depleted population was added back to the effectors or unless known activators of NK cells (IFN-alpha or IL-2) were added to the assays. In contrast, Leu-11a-enriched populations were able to mediate ADCC against HSV-Fs in the presence of sera from HSV-seropositive individuals without the requirement for accessory cells. We have begun preliminary characterization of the accessory cells which allow lysis of HSV-Fs by NK cells: they are HLA-DR+ cells which enrich in the light density fractions of Metrizamide density gradients. They need be present in very small numbers for lysis to take place and are not MHC restricted in that heterologous add-backs between anti-HLA-DR plus C and anti-Leu-11b plus C-treated populations are capable of target cell lysis at levels similar to those achieved with the autologous add-backs. Further, the levels of lysis in heterologous add-back experiments reflected the lytic potential of the effector rather than the accessory cell donor. Finally, although the requirement for accessory cells for NK lysis has been demonstrated for fibroblasts infected with HSV-1, CMV, and VZV, lysis of HSV-infected Raji lymphoblastoid cells is relatively accessory-cell independent, indicating that the requirement for accessory cells for lysis by NK cells is not a property of all herpesvirus-infected targets.