Modulation of alloimmune response by commensal gut microbiota and potential new avenues to influence the outcome of allogeneic transplantation by modification of the 'gut culture'.

Host defence response against microbial infections was the foundation for the Science of Immunology. Now, we know the mechanisms of such host defence which include innate immune responses that is generally nonspecific but effective in many cases and lead to more specific responses called adaptive immune response. The gene loci of class I, II and III of the major histocompatibility complex (MHC) play a major role in directing the adaptive immune responses by presenting processed antigens to T and B cells to induce appropriate antigen-specific cellular and or humoral immune responses. In humans, these are commonly referred to as human leucocyte antigens class I/II-HLA I/II). The class III region, the gamma region in the MHC complex, is mostly associated with regulation of immune responses along with genes associated with complement activation. The adaptive immune responses are orchestrated by T and B cells that are tuned to respond to antigens that are normally foreign to the body, because these cells are educated to avoid self-antigens by a process of thymic education and selection of the T cells that are mostly non-self-reactive which also helps the B cells in eliciting specific immune responses to non-self-antigens. A by-product of this is the ability of the T and B cells to elicit strong immune responses to foreign HLA/MHC (alloimmune response), which developed into the field of histocompatibility testing for allogeneic transplantation of stem cells and organs. Now, we are beginning to learn that such alloimmune responses can be influenced by the microbiota that symbiotically live in our body especially on the mucosal surfaces and on the skin. This review deals with new and emerging data on how the commensal mucosal and skin microbiota influence the immune homeostasis, and how manipulating the commensal microbiota of the mucosa and skin could influence the survival and long-term functions of the allografts. Also, alterations of the microbiota by the inevitable immunosuppression prior to and following allogeneic transplantation could contribute towards the outcome of the allografts by alloimmune responses generated due to microbial antigen vs HLA cross-reactivity.

Anti-vimentin Antibodies Present at the Time of Transplantation May Predict Early Development of Interstitial Fibrosis/Tubular Atrophy.

BACKGROUND: Anti-vimentin (a cytoskeletal protein) autoantibodies in renal transplant recipients have been correlated with interstitial fibrosis/tubular atrophy (IFTA). In this study, we examine the association between pretransplantation anti-vimentin antibodies and the subsequent development of IFTA. METHODS: Sera obtained before renal transplantation from 97 transplant recipients were analyzed for the presence of anti-vimentin antibodies via Luminex assays to determine the concentration of anti-vimentin antibodies. Results were correlated with findings of IFTA on biopsy as well as graft function and patient and graft survival. RESULTS: In our patient population, 56 of 97 patients were diagnosed by biopsy with IFTA 2.9 (±2.1) years after renal transplantation. Patients with IFTA on biopsy had higher mean concentration of anti-vimentin antibodies when compared to patients without IFTA (32.2 µg/mL [3.97-269.12 µg/mL] vs 14.57 µg/mL [4.71-87.81 µg/mL]). The risk of developing IFTA with a concentration of anti-vimentin antibody >15 µg/mL before transplantation was 1.96 (95% CI = 1.38-2.79, P = .011). Patients with elevated anti-vimentin antibody concentrations (>15 µg/mL) at the time of transplantation also had a higher risk of developing IFTA (81.4% vs 41.2%; P < .05). In addition, graft function was worse at 1, 3, and 5 years posttransplantation in patients with elevated concentrations of pretransplantation anti-vimentin antibody. Although there were more graft losses in the IFTA groups (49.12% vs 25.64%, P = .021) and the IFTA patients loss their grafts earlier (4.3 years vs 3.6 years), there was no statistical difference in graft loss rates. CONCLUSIONS: Pretransplantation anti-vimentin antibody concentrations >15 µg/mL may be a risk factor for IFTA.

16(th) IHIW: global distribution of extended HLA haplotypes.

This report describes the project to identify the global distribution of extended HLA haplotypes, a component of 16th International HLA and Immunogenetics Workshop (IHIW), and summarizes the initial analyses of data collected. The project aims to investigate extended HLA haplotypes, compare their distribution among different populations, assess their frequency in hematopoietic stem cell unrelated donor registries and initiate an international family studies database and DNA repository to be made publicly available. HLA haplotypes compiled in immunogenetics laboratories during the evaluation of transplant candidates and related potential donors were analysed. Haplotypes were determined using the pedigree analysis tool publicly available from the National Marrow Donor Program (NMDP) website. Nineteen laboratories from 10 countries (11 laboratories from North America, five from Asia, two from Latin America and one from Australia) contributed data on a total of 1719 families comprised of 7474 individuals. We identified 10393 HLA haplotypes, of which 1682 haplotypes included high-resolution typing at HLA-A, B, C, DRB1 and DQB1 loci. We also present haplotypes containing MICA and other HLA loci and haplotypes containing rare alleles seen in these families. The project will be extended through the 17th IHIW, and investigators interested in joining the project may communicate with the first author.

Prevention and treatment of DNA vaccine encoding cockroach allergen Bla g 1 in a mouse model of allergic airway inflammation.

BACKGROUND: One-fourth of the US population is sensitized to the German cockroach. Primary German cockroach allergen Bla g 1 is detected in 63% of homes and 52% of childcare facilities in the United States. No effective treatment or vaccination strategies are yet available. OBJECTIVES: We evaluated the prophylactic and therapeutic efficacy of a plasmid DNA-mediated vaccination using the Bla g 1 gene in a mouse model of allergic inflammatory airway disease. METHODS: A plasmid DNA vector coding for the Bla g 1 allergen controlled by cytomegalovirus promoter was constructed. To estimate the protective efficacy, BALB/c mice were given three injections of plasmid DNA-Bla g 1 prior to sensitization with two priming doses of recombinant Bla g 1 (rBla g 1) antigens, followed by nebulized rBla g 1 challenge. In the therapeutic approach, sensitization was followed by administering Bla g 1 DNA vaccine. RESULTS: Bla g 1 vaccination significantly reduced allergen-induced airway inflammation, even after mice were presensitized and a Th2-dominant response was established. The Bla g 1 vaccination significantly reduced total inflammatory cell infiltrate, eosinophilia, secretion of Th2 cytokines IL-4 and IL-5 in bronchoalveolar lavage fluid, allergen-induced inflammatory infiltrates in the lungs, and Bla g 1-specific IgE in serum upon challenge with rBla g 1. Importantly, Bla g 1 DNA vaccination was able to induce IL-10-secreting regulatory T cells that could suppress the allergen-specific Th2 cells. CONCLUSION: DNA vaccination showed protective and therapeutic efficacy against a clinically relevant allergen Bla g 1.

Effects of methylprednisolone on intracellular bacterial growth.

Clinical studies have shown positive associations among sustained and intense inflammatory responses and the incidence of bacterial infections. Patients presenting with acute respiratory distress syndrome (ARDS) and high levels of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and IL-6, have increased risk for developing nosocomial infections attributable to organisms such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter spp., compared to those patients with lower levels. Our previous in vitro studies have demonstrated that these bacterial strains exhibit enhanced growth extracellularly when supplemented with high concentrations of pure recombinant TNF-alpha, IL-1 beta, or IL-6. In addition, we have shown that the intracellular milieu of phagocytic cells that are exposed to supraoptimal concentrations of TNF-alpha, IL-1 beta, and IL-6 or lipopolysaccharide (LPS) favors survival and replication of ingested bacteria. Therefore, we hypothesized that under conditions of intense inflammation the host's micromilieu favors bacterial infections by exposing phagocytic cells to protracted high levels of inflammatory cytokines. Our clinical studies have shown that methylprednisolone is capable of reducing the levels of TNF-alpha, IL-1 beta, and IL-6 in ARDS patients. Hence, we designed a series of in vitro experiments to test whether human monocytic cells (U937 cells) that are activated with high concentrations of LPS, which upregulate the release of proinflammatory cytokines from these phagocytic cells, would effectively kill or restrict bacterial survival and replication after exposure to methylprednisolone. Fresh isolates of S. aureus, P. aeruginosa, and Acinetobacter were used in our studies. Our results indicate that, compared with the control, stimulation of U937 cells with 100-ng/ml, 1.0-microg/ml, 5.0-microg/ml, or 10.0-microg/ml concentrations of LPS enhanced the intracellular survival and replication of all three species of bacteria significantly (for all, P = 0.0001). Stimulation with < or =10.0 ng of LPS generally resulted in efficient killing of the ingested bacteria. Interestingly, when exposed to graded concentrations of methylprednisolone, U937 cells that had been stimulated with 10.0 microg of LPS were able to suppress bacterial replication efficiently in a concentration-dependent manner. Significant reduction in numbers of CFU was observed at > or =150 microg of methylprednisolone per ml (P values were 0.032, 0.008, and 0.009 for S. aureus, P. aeruginosa, and Acinetobacter, respectively). We have also shown that steady-state mRNA levels of TNF-alpha, IL-1 beta, and IL-6 in LPS-activated cells were reduced by treatment of such cells with methylprednisolone, in a concentration-dependent manner. The effective dose of methylprednisolone was 175 mg, a value that appeared to be independent of priming level of LPS and type of mRNA. We therefore postulate that a U-shaped relationship exists between the level of expression of TNF-alpha, IL-1 beta, and IL-6 within the phagocytic cells and their abilities to suppress active survival and replication of phagocytized bacteria.

Enhanced extracellular growth of Staphylococcus aureus in the presence of selected linear peptide fragments of human interleukin (IL)-1beta and IL-1 receptor antagonist.

Replication of Staphylococcus aureus is significantly enhanced in the presence of recombinant interleukin (IL)-1beta. In this study, specific binding of IL-1beta to the surface of S. aureus significantly increased growth of S. aureus in the presence of IL-1beta and IL-1ra in a concentration-dependent manner. Although IL-1ra enhanced the growth of S. aureus, there was a significant reduction in IL-1beta-mediated growth enhancement of S. aureus when 25-fold excess amounts of IL-1ra (in comparison with the IL-1beta concentration) were present in the culture medium. Thus, IL-1beta may influence the growth of S. aureus through a receptor-mediated event. By using 5 linear peptides spanning limited regions of IL-1beta, the growth-promoting regions were localized to amino acid residues 118-147 and 208-240. These results build on the newly evolved concept of direct interactions between the soluble mediators of inflammation and infectious agents.

Cytokines IL-1beta, IL-6, and TNF-alpha enhance in vitro growth of bacteria.

We have previously reported that in acute respiratory distress syndrome (ARDS), nonsurvivors have persistent elevation in pulmonary and circulating proinflammatory cytokine levels over time and a high rate of nosocomial infections antemortem. In these patients, none of the proven or suspected nosocomial infections caused a transient or sustained increase in plasma proinflammatory cytokine levels above preinfection values. We hypothesized that cytokines secreted by the host during ARDS may favor the growth of bacteria. We conducted an in vitro study of the growth of three bacteria clinically relevant in nosocomial infections, evaluating their in vitro response to various concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6. We found that all three bacterial species showed concentration-dependent growth enhancement when incubated with one or more tested cytokines and that blockade by specific neutralizing cytokine MoAb significantly inhibited cytokine-induced growth. When compared with control, the 6-h growth response (cfu/ml) was maximal with IL-1beta at 1,000 pg for Staphylococcus aureus (36 +/- 16 versus 377 +/- 16; p = 0.0001) and Acinetobacter spp. (317 +/- 1,147 versus 1,124 +/- 147; p = 0.002) and with IL-6 at 1,000 pg for Pseudomonas aeruginosa (99 +/- 50 versus 509 +/- 50; p = 0.009). The effects of cytokines were seen only with fresh isolates and were lost with passage in vitro on bacteriologic medium without added cytokines. In this study we provide additional evidence for a newly described pathogenetic mechanism for bacterial proliferation in the presence of exaggerated and protracted inflammation.

Effects of cytokines and endotoxin on the intracellular growth of bacteria.

Patients with unresolving acute respiratory distress syndrome (ARDS) have persistently elevated levels of proinflammatory cytokines in the lungs and circulation and increased rates of bacterial infections. Phagocytic cells hyperactivated with lipopolysaccharide (LPS), which induces high levels of proinflammatory cytokines in monocytic cells, are inefficient in killing ingested bacteria despite having intact phagocytic activity. On the other hand, phagocytic cells that are activated with an analogue of LPS that does not induce the expression of proinflammatory cytokines effectively ingest and kill bacteria. We hypothesized that in the presence of high concentrations of proinflammatory cytokines, bacteria may adapt and utilize cytokines to their growth advantage. To test our hypothesis, we primed a human monocytic cell line (U937) with escalating concentrations of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-6 and with LPS. These cells were then exposed to fresh isolates of three common nosocomial pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, and an Acinetobacter sp. In human monocytes primed with lower concentrations of proinflammatory cytokines (10 to 250 pg) or LPS (1 and 10 ng), intracellular bacterial growth decreased. However, when human monocytes were primed with higher concentrations of proinflammatory cytokines (1 to 10 ng) or LPS (1 to 10 micrograms), intracellular growth of the tested bacteria increased significantly (P <0.0001). These results were reproduced with peripheral blood monocytes obtained from normal healthy volunteers. The specificity of the cytokine activity was demonstrated by neutralizing the cytokines with specific antibodies. Our findings provide a possible mechanism to explain the frequent development of bacterial infections in patients with an intense and protracted inflammatory response.

Herpes simplex virus replication-induced expression of chemokines and proinflammatory cytokines in the eye: implications in herpetic stromal keratitis.

On infection of the cornea with herpes simplex virus (HSV), an immunopathologic response termed herpetic stromal keratitis (HSK) ensues. This response is mediated primarily by CD4+ T cells and only occurs if mice are infected with replication-competent virus, although replication-defective mutants induce cellular immune responses following infection. To determine the consequences of HSV replication in the cornea, which is crucial for HSK manifestation, corneas infected with productive virus and replication-defective mutants were analyzed for chemokines and proinflammatory cytokine mRNA expression by RT-PCR at various times. While productive infection resulted in rapid upregulation and sustained expression of such chemokines as N51/KC, macrophage inflammatory protein-1beta (MIP-1beta), MIP-2, and monocyte chemotactic protein-1 (MCP-1) and such cytokines as interleukin-1 (IL-1), IL-6, IL-12, and tumor necrosis factor-alpha (TNF-alpha), expression of such inflammatory mediators was minimal and transient after unproductive infection. Expression of MIP-1alpha and lymphotactin along with a biphasic expression of IL-6 and MIP-2 were seen only with productive infection. Initial PMN recruitment into the cornea was approximately 50-fold greater with productive infection than with unproductive infection. These data suggest that a replication-induced proinflammatory milieu in the cornea is crucial for the subsequent progression of HSK possibly because of enhancement of the expression of corneal agonists that drive HSK manifestation.

Protection by minigenes: a novel approach of DNA vaccines.

To test the principle that genetically engineered epitopes in a plasmid DNA can efficiently induce specific immunity, a minigene cassette encoding cytotoxic T lymphocyte (CTL), helper T and B cell epitopes from herpes simplex virus (HSV) was constructed and placed in an expression vector named pcMini. Following immunizations with pcMini, mice developed epitope-specific CTLs comparable to the response induced by live HSV. Less effective but detectable antibody, lymphoproliferation, and T cell cytokine responses were also produced. In addition, pcMini-primed mice elicited a recall response upon restimulation with recombinant vaccinia virus expressing HSV antigen. The protection provided by minigene vaccination was significant, although not as efficient as live virus vaccine. The DNA minigene approach may prove useful to define and induce immune responses against minimal antigenic determinants.

Production of key molecules by ocular neutrophils early after herpetic infection of the cornea.

Herpes simplex virus infection of the eye can result in a blinding inflammatory lesion that is a T cell mediated immunopathological reaction. A prominent early event following HSV infection is neutrophil invasion of the corneal stroma. These cells may be involved in viral clearance and may influence the nature of the anti HSV T cell response which subsequently occurs. This article measures the expression of some key molecules which could participate in viral clearance and immune modulation. Using RT-PCR and in-situ hybridization, both corneal and peritoneal neutrophils were shown to be sources of iNOS and TNF alpha molecules which likely contribute to antiviral activity. Neutrophils also produce the cytokine IL-12, a key molecule which modulates the CD4+ T cell response to a type which mediates immunopathology. The present results indicate that neutrophils play an important role in the pathogenesis of herpetic ocular lesions.

Suppression of ongoing ocular inflammatory disease by topical administration of plasmid DNA encoding IL-10.

Ocular infection with herpes simplex virus leads to an inflammatory lesion in the cornea orchestrated by CD4+ Th1 lymphocytes. This immunopathologic disease, called herpetic stromal keratitis, is an important cause of impaired vision. In this study, we set out to determine whether established lesions of herpetic stromal keratitis could be controlled by topically administering naked plasmid DNA encoding cytokines to the corneal surface. A single topical administration of DNA encoding IL-10 was beneficial to the majority (75%) of treated animals, and 50% (vs 10% in controls) resolved their lesions completely over a 23-day observation period. Topical ocular application of DNA encoding foreign proteins was also shown to be an effective means of inducing systemic and mucosal immune responses. The direct application of DNA encoding cytokines may represent an additional therapeutic option for the management of immunoinflammatory disease.

On the essential involvement of neutrophils in the immunopathologic disease: herpetic stromal keratitis.

Corneal infection with herpes simplex virus-1 in immunocompetent mice induces an immunopathologic response termed herpetic stromal keratitis (HSK). The earliest sign of disease is neutrophil infiltration, which lasts for 48 to 72 h and then disappears. However, a secondary neutrophil infiltration, this time more massive, occurs, beginning 8 to 9 days postinfection, a time in which HSK becomes clinically evident. The role of neutrophils in HSK expression was investigated by eliminating such cells using a specific mAb (RB6-8C5). In neutrophil-depleted immunocompetent mice, virus replicated more abundantly, but no effects on HSK expression were observed, possibly because sustained neutropenia could not be maintained. However, using a severe combined immunodeficient mouse model, in which HSK does not occur unless given adoptive transfer of CD4+ T cells, the effects of neutrophil depletion were more pronounced. There were significantly less incidence and severity of HSK in CD4+ T cell-reconstituted severe combined immunodeficient mice that were depleted of neutrophils as compared with controls. Neutrophil-depleted mice displayed moderate to severe periocular skin lesions, progressively became cachetic, and developed signs of encephalitis. Virus was recovered at higher titers and for longer periods from eyes of neutrophil-depleted animals. Brain virus titers were also significantly higher on day 12 postinfection as compared with control animals. These results suggest that herpes simplex virus infection of the cornea rapidly invokes recruitment of neutrophils that may aid in viral clearance, and that neutrophils directly or indirectly serve as agonists in perpetuating a CD4+ T cell-mediated inflammatory reaction.

Enhancement of immune response to naked DNA vaccine by immunization with transfected dendritic cells.

Immunization with plasmid DNA encoding various proteins promises to be a valuable vaccine approach especially if its immunogenicity could be optimized. In this study we show that the intramuscular delivery in dendritic cells (DC) of naked plasmid DNA encoding two proteins of herpes simplex virus (HSV) leads to the induction of significantly enhanced levels of resistance to viral challenge. Whereas DC transfected in vitro with DNA induced enhanced immunity, similarly transfected macrophage (M phi) populations lacked immunogenicity even though plasmid expression occurred in vitro. The enhanced immunity induced by DC-delivered DNA appeared to be associated mainly with an increased Th1 CD4+ T cell response. Our results add evidence that DC are the essential antigen-presenting cell types involved in immune responses to intramuscularly administered DNA vaccines.

Cytokine expression in the gut associated lymphoid tissue after oral administration of attenuated Salmonella vaccine strains.

The use of attenuated Salmonella vaccine vectors as delivery vehicles for heterologous antigens has been extensive. The majority of work has analyzed the specific immune responses to the recombinant antigen in question. In addition, most analysis has been performed on animals maintained with sterile food, water, and bedding. This report defines the Salmonella specific cytokine responses in the gut associated lymphoid tissue after oral immunization with two highly publicized attenuated Salmonella carrier strains in animals maintained with nonsterile food, water, and bedding. Increases in expression of both IL-4 and IFN-gamma occur following immunization with either Salmonella construct. In addition, other cytokines (IL-6, IL-7, IL-12) increase at similar levels in either BRD509 or KR1 dosed animals. Proinflammatory cytokines IL-1 and TNF-alpha are also present but unchanged at early time points (6, 24, and 72 hours), increasing only after 7 days postimmunization. These data have implications in the generation of immunity to recombinant antigens since the response to Salmonella will dictate the direction of responses in terms of CD4 T cell activation.

The role of the innate immune system in the reconstituted SCID mouse model of herpetic stromal keratitis.

Herpetic stromal keratitis (HSK) has an immunopathological basis, thought primarily to involve a CD4+ T cell-mediated immune response to viral antigen. Other cell types, however, particularly those involved in nonspecific immunity, such as natural killer (NK) cells or neutrophils, may also contribute to tissue destruction in the cornea. The reconstituted SCID mouse model of HSK provides a powerful system in which to study the interactions of the innate and adaptive immune responses to herpes simplex virus type 1 corneal infection. In the present study, reconstituted SCID mice depleted of NK cells had a reduced incidence and severity of clinical and histopathological HSK. The levels of T cell cytokine protein and message in restimulated splenocytes and cytokine message in corneas did not differ between experimental groups. However, significantly fewer neutrophils were seen within the inflamed corneas of NK-depleted SCID mice. Therefore, endogenous NK cells may indirectly influence the severity of HSK in reconstituted SCID mice by affecting neutrophil migration into the cornea.

HSV-1-mediated modulation of cytokine gene expression in a permissive cell line: selective upregulation of IL-6 gene expression.

Pathological effects of herpes simplex virus (HSV) can result due to a combination of direct viropathic effects and immunological reactions to viral antigens. The immunological reactions are orchestrated by a variety of cytokines and chemokines released by the host cells. Therefore, the cytokine gene expression in response to HSV-1 infection in a permissive murine cell line was investigated. The data demonstrate that HSV-1 induced a selective activation of IL-6 gene expression at the mRNA and protein levels, in the permissive cell line. The cell line used was capable of expressing IL-1, IL-7, and IL-10 in addition to IL-6, upon lipopolysaccharide stimulation. UV or heat-inactivated viruses were unable to upregulate IL-6 expression. However, mutant HSV-1 strains lacking fully functional ICP0, ICP4, ICP8, or ICP27 genes, thereby rendering them replication incompetent or impaired in in vitro cell growth (ICP0), enhanced IL-6 expression selectively. Considering the role of IL-6 in inflammation, immune response, and its known association with increased levels of MyD116 and GADD 34 mRNAs (genes involved in the prevention of apoptotic death of cells), the present data may have relevance to HSV-1-mediated diseases as well as to the prevalence of HSV-1 in the host.

Herpes simplex virus type 1-mediated up-regulation of IL-12 (p40) mRNA expression. Implications in immunopathogenesis and protection.

In this report we investigated whether induction of IL-12 occurs in response to herpes simplex virus (HSV) infection of the mouse eye, which may serve to regulate the nature of the subsequent immune response. The data show early induction and continued maintenance of IL-12 (p40) mRNA in the cornea and draining lymph node upon ocular infection with HSV. Using a very sensitive radioimmunoassay technique, IL-12 (p40) protein also was detected in the cornea upon ocular HSV infection. Unfractionated splenocytes and enriched populations of dendritic cells, macrophages, and neutrophils all responded to HSV infection in vitro by up-regulating the expression of IL-12 (p40) mRNA. However, cultured corneal cells failed to generate IL-12 (p40) upon exposure to HSV. The data indicate that inflammatory cells that infiltrate the cornea in response to HSV-1 infection are the main source of IL-12 in the eye. Our results are consistent with the hypothesis that IL-12 induction in the eye acts as a triggering event that biases HSV-specific immunity to a type 1 T cell response, which, in the environment of the eye, culminates in an immunopathologic disease.

Viral replication is required for induction of ocular immunopathology by herpes simplex virus.

Corneal infection of BALB/c mice with herpes simplex virus type 1 results in a chronic inflammatory response in the stroma termed herpetic stromal keratitis (HSK). This disease is considered to be immunopathological and mediated primarily by CD4+ T cells of the type 1 cytokine profile. However, the nature of the antigens, virus or host derived, which drive the inflammatory response remains in doubt. In this study, the relevance of infection with replicating virus for the subsequent development of HSK was evaluated with immunocompetent mice as well as with SCID mice reconstituted with herpes simplex virus-immune CD4+ T cells. In the corneas of immunocompetent mice, infectious virus, viral antigen, and mRNA expression were detectable for only a brief period of time (< or = 7 days postinfection), and all were undetectable by the time clinical lesions were evident (10 to 15 days). Viral replication, however, was necessary for the development of HSK in both models, since infection with UV-inactivated virus or with mutant viruses which were incapable of multiple rounds of replication in vivo failed to induce HSK. The inactivated and mutant viral preparations did, however, stimulate T-cell immune responses in immunocompetent mice. The results are discussed in terms of possible involvement of host antigens exposed in response to transient progeny virion replication in the immune-privileged cornea.