MHC Class II-deficient Mice Research Articles

MHC class II-deficient mice allow functional human CD4+ T-cell development.

Humanized mouse models have been developed to study cell-mediated immune responses to human pathogens in vivo. How immunocompetent human T cells are selected in a murine thymus in such humanized mice remains poorly explored. To gain insights into this mechanism, we investigated the differentiation of human immune compartments in mouse MHC class II-deficient immune-compromised mice (humanized Ab0 mice). We observed a strong reduction in human CD4+ T-cell development but despite this reduction Ab0 mice had no disadvantage during Epstein-Barr virus (EBV) infection. Viral loads were equally well controlled in humanized Ab0 mice compared to humanized NSG mice, and improved T-cell recognition of autologous EBV-transformed B cells was observed, especially with respect to cytotoxicity. MHC class II blocking experiments with CD4+ T cells from humanized Ab0 mice demonstrated MHC class II restriction of lymphoblastoid cell line recognition. These findings suggest that a small number of CD4+ T cells in humanized mice can be solely selected on human MHC class II molecules, presumably expressed by reconstituted human immune cells, leading to improved effector functions.

Correlation of cell-surface CD8 levels with function, phenotype and transcriptome of naive CD8 T cells.

We have previously demonstrated co-receptor level-associated functional heterogeneity in apparently homogeneous naive peripheral CD4 T cells, dependent on MHC-mediated tonic signals. Maturation pathways can differ between naive CD4 and naive CD8 cells, so we tested whether the latter showed similar co-receptor level-associated functional heterogeneity. We report that, when either polyclonal and T-cell receptor (TCR)-transgenic monoclonal peripheral naive CD8 T cells from young mice were separated into CD8hi and CD8lo subsets, CD8lo cells responded poorly, but CD8hi and CD8lo subsets of CD8 single-positive (SP) thymocytes responded similarly. CD8lo naive CD8 T cells were smaller and showed lower levels of some cell-surface molecules, but higher levels of the negative regulator CD5. In addition to the expected peripheral decline in CD8 levels on transferred naive CD8 T cells in wild-type (WT) but not in MHC class I-deficient recipient mice, short-duration naive T-cell-dendritic cell (DC) co-cultures invitro also caused co-receptor down-modulation in CD8 T cells but not in CD4 T cells. Constitutive pZAP70/pSyk and pERK levels exvivo were lower in CD8lo naive CD8 T cells and dual-specific phosphatase inhibition partially rescued their hypo-responsiveness. Bulk mRNA sequencing showed major differences in the transcriptional landscapes of CD8hi and CD8lo naive CD8 T cells. CD8hi naive CD8 T cells showed enrichment of genes involved in positive regulation of cell cycle and survival. Our data show that naive CD8 T cells show major differences in their signaling, transcriptional and functional landscapes associated with subtly altered CD8 levels, consistent with the possibility of peripheral cellular aging.

PCI-based vaccination: a novel method for targeting of MHC class I antigen presentation and stimulation of antigen-specific cytotoxic T cells

Abstract Cytotoxic CD8+ T cells are key players in fighting cancer and infectious diseases, hence, the design of therapeutic vaccines focuses on ways to prime strong cytotoxic immune responses. Yet, current vaccines efficacy is limited mainly due to the default processing of exogenous antigens via the MHC class II pathway and the activation of CD4+ and not CD8+ T cells. Photochemical internalization (PCI) is a novel intracellular delivery approach, able to circumvent the MHC class II processing. Briefly, after endocytosis of antigen and photosensitizer by antigen presenting cells, light activation of the photosensitizer causes disruption of endosomal membrane and antigen release into the cytosol, resulting in stimulation of MHC class I-restricted CD8+ T cells. Wild type and MHC class II-deficient mice were immunized intradermally with protein antigens and photosensitizer, followed by controlled light exposure for activation of the photosensitizer. The effectiveness of the PCI to stimulate CD8+ T cells was analyzed by measuring frequency, phenotype, and function of antigen-specific T cells in blood and spleens. PCI-treated mice showed a strong activation and proliferation of specific CD8+ T cells, with improved functions in terms of IFN-g production and cytotoxic capacity. The CD8+ T cell effector functions were not impaired in MHC class II-deficient or in CD4+ T cell-depleted mice, suggesting that the PCI-mediated antigen processing is independent of MHC class II and CD4+ T helper cells. Our data demonstrate that PCI–based vaccination can stimulation strong and functional cytotoxic CD8+ T cells, making PCI a potential method for use in therapeutic vaccines against cancer and infectious diseases.

Dendritic cells as potential mediators of Mycobacterium tuberculosis infection of the central nervous system in a MHC class II-deficient mouse model.

Abstract Mycobacterium tuberculosis (Mtb) infection of the central nervous system (CNS) in a rare, but deadly form of TB affecting immune-compromised individuals. Due to the HIV-AIDS epidemic this population has recently increased, however the pathway by which Mtb gains access to the CNS is poorly understood. Our work seeks to understand the potential ability of dendritic cells to traffic Mtb to the CNS. Preliminarily, we established the importance of mycobacterium infected dendritic cells to the formation of nervous system infection in MHC class II-deficient mice. In this model we used bone marrow derived dendritic cells (BMDC) from a dendritic cell reporter mouse strain (CD11c-eYFP). These cells increased the dissemination of Mtb 6020 to the brain by one log compared to Mtb 6020 alone, though the systemic infection remained the same. When GM-CSF BMDCs were infected with tdTomato H37Rv Mtb and injected IV into MHC class II-deficient mice CFU was detected in the brain, cervical lymph nodes and lung with no Mtb observed in the liver and superficial lymph nodes. After infection with tdTomato Mtb, BMDCs matured using GM-CSF or FLT3 had increased expression of CD11c, eYFP, CD40 and CD86 as detected by flow cytometry. These immunocompromised mice, whose phenotype reflects aspects of immunodeficiency seen in human populations, have a predisposition to CNS infection when BMDCs are directly infected with mycobacterium, suggesting the ability of dendritic cells to facilitate the movement of Mtb across the BBB or choroid plexus to initiate infection. These data support the use of MHC class II-deficient mice as a model for future work.

Helper T cells are required for the development of IgM memory cells, but not IgM bone marrow plasmablasts

Abstract The processes and factors that regulate B cell fate decisions during IgM memory cell development and differentiation are unknown, and may differ from those required by canonical memory cells. In our experimental model of Ehrlichia muris infection, we have characterized two long-lived B cell populations: IgM memory cells, and IgM bone marrow (BM) antibody secreting cells (ASCs). The IgM+ memory B cells are required for recall IgG responses, while the BM ASCs produce antigen-specific IgM responsible for maintaining long-term immunity. Both populations are derived from an early AID-expressing CD4 T cell-independent splenic CD11c+ plasmablast population. Because abundant T follicular helper cells are generated in the spleen during early infection, we investigated whether T cell help regulates B cell fate. Infection of MHC class II-deficient mice revealed that the IgM+ memory B cells required CD4+ T cell help, while the IgM+ BM ASCs did not. Moreover, IgM memory cell transfer studies indicated that T cell help was required for the generation of IgM+ memory cells, but not for the generation of BM ASCs. On the basis of these observations, we propose that T cells drive B cell fate: developing spleen plasmablasts that receive T cell signals mature to become IgM memory cells, while cells that do not receive these signals follow a default pathway to become long-lived IgM BM ASCs.

Plasma‐derived MHC class II+ exosomes from tumor‐bearing mice suppress tumor antigen‐specific immune responses

Tumor-specific immunosuppression is frequently observed in tumor-bearing hosts. Exosomes are nano-sized, endosomal-derived membrane vesicles secreted by most tumor and hematopoietic cells and have been shown to actively participate in immune regulation. We previously demonstrated that antigen-specific immunosuppressive exosomes could be isolated from the blood plasma of antigen-immunized mice. Here, we demonstrate that plasma-derived exosomes isolated from mice bearing OVA-expressing tumors were able to suppress OVA-specific immune responses in a mouse delayed-type hypersensitivity model. Enrichment of tumor-derived exosomes in the plasma of mice bearing subcutaneous melanoma was not detected using an exosome-tagging approach. Instead, depletion of MHC class II(+) vesicles from plasma-derived exosomes or using plasma-derived exosomes isolated from MHC class II-deficient mice resulted in significant abrogation of the suppressive effect. These results demonstrate that circulating host-derived, MHC class II(+) exosomes in tumor-bearing hosts are able to suppress the immune response specific to tumor antigens.

Nasopharyngeal colonization by Streptococcus pneumoniae elicits long-lasting immune responses that confer protection from invasive lung infections. (49.23)

Abstract Streptococcus pneumoniae can cause life-threatening pulmonary and systemic infection, but more commonly resides harmlessly in the nasopharynx. We demonstrated that nasopharyngeal (n.p.) colonization by S. pneumoniae in mice generates an antibody response that provides at least 10-week protection against lethal lung challenge. B cells were required for this protection, and transfer of sera from n.p.-colonized mice or antibodies against phosphorylcholine, a common S. pneumoniae surface antigen, protected naïve mice. Whereas T-independent anti-capsular antibodies were previously shown to be sufficient for protection against bloodstream infection , we found that T cells were required to generate a fully protective response following n.p. colonization. Depletion of CD4+ T cells at the time of pulmonary challenge or colonization of MHC class II-deficient mice did not compromise protection, suggesting a limited role for CD4+ T cells in our model. Sub-lethal irradiation of mice, which inactivates memory B cells but not long-lived plasma cells, did not significantly diminish protection, and transfer of bone marrow cells enriched for long-lived plasma cells from n.p.-colonized mice to B cell-deficient recipients increased pneumococcal-specific antibody titers and afforded significant protection. Our results suggest that n.p. colonization is capable of eliciting extended protection against invasive pneumococcal disease by generating long-lived antibody-secreting cells.

Vaccination against Lymphocytic Choriomeningitis Virus Infection in MHC Class II-Deficient Mice

The impact of prophylactic vaccination against acute and chronic infection in a Th-deficient host has not been adequately addressed because of difficulties in generating protective immunity in the absence of CD4(+) T cell help. In this study, we demonstrated that a broad CD8(+) T cell immune response could be elicited in MHC class II-deficient mice by vaccination with adenovirus encoding lymphocytic choriomeningitis virus (LCMV) glycoprotein tethered to MHC class II-associated invariant chain. Moreover, the response induced conferred significant cytolytic CD8(+) T cell-mediated protection against challenge with a high dose of the invasive clone 13 strain of LCMV. In contrast, vaccination with adenovirus encoding unlinked LCMV glycoprotein induced weak virus control in the absence of CD4(+) T cells, and mice may die of increased immunopathology associated with incomplete protection. Acute mortality was not observed in any vaccinated mice following infection with the less-invasive Traub strain. However, LCMV Traub infection caused accelerated late mortality in unvaccinated MHC class II-deficient mice; in this case, we observed a strong trend toward delayed mortality in vaccinated mice, irrespective of the nature of the vaccine. These results indicated that optimized vaccination may lead to efficient protection against acute viral infection, even in Th-deficient individuals, but that the duration of such immunity is limited. Nevertheless, for select immunodeficiencies in which CD4(+) T cell deficiency is incomplete or transient, these results are very encouraging.

Costimulation blockade inhibits the indirect pathway of allorecognition in nerve allograft rejection

Nerve allografts provide a temporary scaffold for host nerve regeneration. The need for systemic immunosuppression limits clinical application. Characterization of the immunological mechanisms that induce immune hyporesponsiveness may provide a basis for optimizing immunomodulating regimens. We utilized wild-type and MHC class II-deficient mice, as both recipients and donors. Host treatment consisted of triple costimulatory blockade. Quantitative assessment was made at 3 weeks using nerve histomorphometry, and muscle testing was performed on a subset of animals at 7 weeks. Nerve allograft rejection occurred as long as either the direct or indirect pathways were functional. Indirect antigen presentation appeared to be more important. Nerve allograft rejection occurs in the absence of a normal direct or indirect immune response but may be more dependent on indirect allorecognition. The indirect pathway is required to induce costimulatory blockade immune hyporesponsiveness.

Homeostatic Turnover of Virus-Specific Memory CD8 T Cells Occurs Stochastically and Is Independent of CD4 T Cell Help

Memory CD8 T cells persist by Ag-independent homeostatic proliferation. To examine the dynamics of this cell turnover, we transferred lymphocytic choriomeningitis virus specific memory CD8 T cells into naive mice and analyzed their in vivo division kinetics longitudinally in individual recipients.Using mathematical modeling, we determined that proliferation of this stably maintained memory CD8 T cell population was homogeneous and stochastic with a small fraction of cells completing division at any given time with an intermitotic interval of 50 d. This homeostatic turnover was comparable between memory CD8 T cells of different viral epitope specificities and also the total memory phenotype (CD44(high)) CD8 T cells. It is well established that CD4 T cell help is critical for maintenance of CD8 T cells during chronic infections, but recent studies have suggested that CD4 T cell help is also required for maintenance of memory CD8 T cells following acute infections. Hence, we assessed the role of CD4 T cells in Ag-independent maintenance of memory CD8 T cells. Consistent with previous reports, we found that memory CD8 T cells declined when transferred into MHC class II-deficient mice. However, their numbers were maintained stably when transferred into CD4 T cell-deficient mice. Interestingly, their homeostatic proliferation, ability to make recall responses, and phenotype were independent of CD4 T cell help because none of these qualities were affected when memory CD8 T cells were transferred and maintained in either MHC class II- or CD4-deficient recipients.

APCs Expressing High Levels of Programmed Death Ligand 2 Sustain the Development of CD4 T Cell Memory

The role APCs play in the transition of T cells from effector to memory remains largely undefined. This is likely due to the low frequency at which long-lived T cells arise, which hinders analysis of the events involved in memory development. In this study, we used TCR transgenic T cells to increase the frequency of long-lived T cells and developed a transfer model suitable for defining the contribution of APCs to the development of CD4 T cell memory. Accordingly, naive TCR transgenic T cells were stimulated in vitro with Ag presented by different types of APCs and transferred into MHC class II-deficient mice for parking, and the hosts were later analyzed for long-lived T cell frequency or challenged with suboptimal dose of Ag, and the long-lived cells-driven memory responses were measured. The findings indicate that B cells and CD8alpha(+) dendritic cells sustained elevated frequencies of long-lived T cells that yielded rapid and robust memory responses upon rechallenge with suboptimal dose of Ag. Furthermore, both types of APCs had significant programmed death (PD) ligand 2 expression prior to Ag stimulation, which was maintained at a high level during presentation of Ag to T cells. Blockade of PD ligand 2 interaction with its receptor PD-1 nullified the development of memory responses. These previously unrecognized findings suggest that targeting specific APCs for Ag presentation during vaccination could prove effective against microbial infections.

CD4+ T Cells and Lactobacillus casei Control Relapsing Colitis Mediated by CD8+ T Cells

Evidence that CD4(+) regulatory T cells can control Ag-specific CD8(+) T cell-mediated colitis in immunocompetent mice is poorly documented. To examine the potential of CD4(+) T cells to control colitis, we used our model of CD8(+) T cell-mediated colitis induced by intracolonic sensitization followed by challenge with the hapten 2,4-dinitrobenzene sulfonic acid. The defect of CD4(+) T cells in MHC class II-deficient (Abeta(degrees/degrees)) mice allowed priming of 2,4-dinitrobenzene sulfonic acid-specific IFN-gamma-producing CD8 colitogenic effectors and development of colitis in the otherwise resistant C57BL/6 strain. Cotransfer experiments in RAG2(degrees/degrees) mice and ex vivo studies showed that CD4(+)CD25(+) T cells completely prevented CD8(+) T cell-mediated colitis and controlled CD8(+) T cell activation, respectively. In the susceptible BALB/c strain, Ab depletion revealed that lack of CD4(+) regulatory T cells resulted in 1) acute colitis elicited by a suboptimal dose of hapten challenge and 2) more severe relapsing episodes of colitis induced by effector/memory CD8(+) T cell-mediated colitis at an optimal dose of hapten challenge, even when CD4 depletion was performed just before the second challenge. Oral administration of the probiotic strain Lactobacillus casei DN-114 001 alleviated colitis and increased the suppressive function of Foxp3(+)CD4(+) regulatory T cells of colon lamina propria. These data demonstrate that CD4(+) regulatory T cells exert a protective effect on colitis by controlling colitogenic effector/memory CD8(+) T cells during the effector (symptomatic) phase of acute and relapsing colitis, respectively. Probiotics with natural adjuvant effects on mucosal regulatory T cells may represent a valuable approach to alleviate the colitogenic effect of Tc1-type CD8(+) effectors.

Cutting Edge: Viral Infection Breaks NK Cell Tolerance to “Missing Self”

NK cells attack cells lacking MHC class I, yet MHC class I-deficient mice have normal numbers of NK cells with intact, albeit diminished, functions. Moreover, wild-type NK cells are tolerant of MHC class I-deficient cells in mixed bone marrow chimeras. In this study, we investigated how the absence of MHC class I affects NK cells. NK cells from beta(2)-microglobulin-deficient (B2m(-/-)) and wild-type mice exhibit similar phenotypic and functional characteristics. Both B2m(-/-) and wild-type Ly49H(+) NK cells proliferated robustly and produced IFN-gamma after infection with mouse CMV. NK cells in mixed wild-type:B2m(-/-) chimeric mice were initially tolerant of MHC class I-deficient host cells. However, this tolerance was gradually lost over time and after mouse CMV infection was rapidly broken, with a pronounced rejection of host B2m(-/-) hematopoietic cells. Thus, although NK cells can be held in check against "missing self," acute inflammation driven by infection can rapidly break established self-tolerance.

CD11c Expression Identifies a Population of Extrafollicular Antigen-Specific Splenic Plasmablasts Responsible for CD4 T-Independent Antibody Responses during Intracellular Bacterial Infection

Although T-independent immunity is known to be generated against bacterial capsular and cell wall polysaccharides expressed by a number of bacterial pathogens, it has not been studied in depth during intracellular bacterial infections. Our previous study demonstrated that Ehrlichia muris, an obligate intracellular tick-borne pathogen, generates protective classical TI responses in CD4 T cell-deficient C57BL/6 mice. We found that E. muris T-independent immunity is accompanied by the expansion of a very large extrafollicular spleen population of CD11c(low)-expressing plasmablasts that exhibit characteristics of both B-1 and marginal zone B cells. The plasmablasts comprised up to 15% of the total spleen lymphocytes and approximately 70% of total spleen IgM(high)IgD(low) cells during peak infection in both wild-type and MHC class II-deficient mice. The CD11c(low) cells exhibited low surface expression of B220, CD19, and CD1d, high expression of CD11b, CD43, but did not express CD5. Approximately 50% of the CD11c(low) cells also expressed CD138. In addition to CD11b and CD11c, the plasmablasts expressed the beta(1) (CD29) and alpha4 (CD49d) integrins, as well as the chemokine receptor CXCR4, molecules which may play roles in localizing the B cells extrafollicular region of the spleen. During peak infection, the CD11c(low) cells accounted for the majority of the IgM-producing splenic B cells and nearly all of the E. muris outer membrane protein-specific IgM-secreting cells. Thus, during this intracellular bacterial infection, CD11c expression identifies a population of Ag-specific spleen plasmablasts responsible for T-independent Ab production.

T cell differentiation and generation of the antigen-specific T cell repertoire in man: observations in MHC class II deficiency.

The circulating T cell pool of an MHC class II-deficient patient was shown to lack the MHC class II-specific T cell functions. This was demonstrated by the absence of MHC class II-specific alloreactive T cells and a substantially decreased number of circulating CD4+ lymphocytes. The patient's T cells did respond to an allostimulus, although the restriction pattern of this reaction remains speculative. The function and distribution of peripheral T cell subsets from the patient resemble findings in MHC class II-deficient mice, which also lack interaction of T cell precursors with MHC class II-bearing accessory cells during thymic differentiation. Our data support the concept that T cell differentiation in humans is similar, and that the human MHC-restricted T cell repertoire depends on prior interaction of T cell precursors with self MHC.

Circulating intercellular adhesion molecule-1 (ICAM-1) as an early and sensitive marker for virus-induced T cell activation.

The effect of systemic virus infection on the level of circulating ICAM-1 (cICAM-1) in serum, and the role of virus-activated T cells in this context, were studied using the murine lymphocytic choriomeningitis virus infection as primary model system. A marked virus-induced elevation in cICAM-1 in serum was revealed, the presence of which coincided with the phase of virus-induced T cell activation. However, high levels of cICAM-1 in serum were observed well before maximal T cell activation could be demonstrated. No increase in cICAM-1 was observed in the serum of infected T cell-deficient nude mice, clearly demonstrating that T cells were mandatory. Analysis of MHC class I and MHC class II-deficient mice revealed that either CD4+ or CD8+ T cells alone are sufficient, despite a markedly reduced inflammatory exudate in the former animals. These results indicate that virus-activated T cells induce shedding of ICAM-1 into the circulation, and this parameter may be used as an early and sensitive marker for immune activation.

Host Plasmacytoid or Conventional Dendritic Cells Alone Are Sufficient To Initiate Graft-Versus-Host Disease.

Graft-versus-host disease (GVHD) is a major complication after allogeneic hematopoietic stem cell transplantation. Alloantigen expression on host dendritic cells (DCs) is critical to initiate GVHD. DCs can be divided into two main subpopulations; conventional DCs (cDCs) and plasmacytoid DCs (pDCs), however, the contribution of each DC subset to elicit GVHD remains unclear. We examined the ability of cDCs and pDCs to initiate GVHD. pDCs, cDCs and B cells were isolated from C57BL/6 (B6: H–2b) mice treated with Flt3 ligand in order to expand DCs. pDCs were enriched from bone marrow by depleting CD3+, CD19+, CD11b+, and CD49b+ cells, followed by a FACS sorting of CD11cint B220+ cells. cDCs and B cells were sorted from splenocytes as CD11chi B220− cells and CD11c− B220+ cells, respectively. Isolated pDCs showed plasmacytoid morphology, produced IFN-α in response to CpG oligonucleotide. Although pDCs stimulated allogeneic T cells far less potently than cDCs, stimulation with CpG enhanced their allostimulatory capacity as potent as cDCs. We compared the ability of each DC subset to initiate GVHD by an add-back study of MHC class II-expressing DCs into MHC class II-deficient (II−/−) mice that were resistant to CD4-dependent GVHD. Lethally irradiated II−/− B6 mice were injected with 2 × 106 pDCs, cDCs or B cells from wild-type (II+/+) B6 mice on day -1 and injected with 2 × 106 CD4+ T cell from BALB/c (H–2d) mice on day 0. A flow cytometric analysis of the mesenteric lymph nodes on day +5 demonstrated significantly greater expansion of donor CD4+ T cells in recipients of pDCs or cDCs than those of B cells (Table). While injection of B cells did not cause any sign of GVHD, injection of pDCs or cDCs alone was sufficient to produce clinical and pathological GVHD (Table), thus breaking GVHD resistance of II−/− mice. We next examined the ability of pDCs to induce CD8-dependent GVHD in MHC-matched transplant using mice deficient in functional MHC class I expression (β2m−/−). Again, injection of pDCs or cDCs alone was sufficient to cause expansion of donor CD8+ T cells (p<0.05). We next asked whether signaling through Toll-like receptors (TLRs) could be required for pDCs to initiate GVHD. However, injection of pDCs isolated from MyD88/TRIF-double deficient mice was able to initiate GVHD as potent as wild-type pDCs, thus demonstrating that pDCs initiate GVHD in a TLR signaling independent manner. These results provide important information for developing strategies aimed at inactivating host DCs to prevent GVHD.Impact of each APC subpopulation on GVHDAPCDonor CD4 expansion (×103±SE)Clinical GVHD score (mean±SE)Pathological GVHD score (mean±SE)B cell0.1 ± 0.02.1 ± 0.22.1 ± 0.2pDC5.3 ± 2.4*4.3 ± 0.3*7.4 ± 0.5*cDC9.7 ± 3.8 *3.8 ± 0.5 *7.2 ± 0.7**p<0.05 compared with B cells

Xenogeneic β2-Microglobulin Substitution Alters NK Cell Function

Recently, it has been shown that human beta(2)-microglobulin (h-beta(2)m) blocks the association between the NK cell inhibitory receptor Ly49C and H-2K(b). Given this finding, we therefore sought to assess the immunobiology of NK cells derived from C57BL/6 (H-2(b)) mice expressing exclusively h-beta(2)m. Initial analysis revealed that the Ly49C expression profile of NK cells from h-beta(2)m(+) mice was modified, despite the fact that H-2K(b) expression was normal in these mice. Moreover, the NK cells were not anergic in that IL-2 treatment of h-beta(2)m(+) NK cells in vitro enabled efficient lysis of prototypic tumor cell lines as well as of syngeneic h-beta(2)m(+) lymphoblasts. This loss of self-tolerance appeared to correlate with the activation status of h-beta(2)m(+) NK cells because quiescent h-beta(2)m(+) transplant recipients maintained h-beta(2)m(+) grafts but polyinosine:polycytidylic acid-treated recipients acutely rejected h-beta(2)m(+) grafts. NK cell reactivity toward h-beta(2)m(+) targets was attributed to defective Ly49C interactions with h-beta(2)m:H-2K(b) molecules. With regard to NK cell regulatory mechanisms, we observed that h-beta(2)m:H-2K(b) complexes in the cis-configuration were inefficient at regulating Ly49C and, furthermore, that receptor-mediated uptake of h-beta(2)m:H-2K(b) by Ly49C was impaired compared with uptake of mouse beta(2)m:H-2K(b). Thus, we conclude that transgenic expression of h-beta(2)m alters self-MHC class I in such a way that it modulates the NK cell phenotype and interferes with regulatory mechanisms, which in turn causes in vitro-expanded and polyinosine:polycytidylic acid-activated NK cells to be partially self-reactive similar to what is seen with NK cells derived from MHC class I-deficient mice.

The Antiviral CD8+ T Cell Response Is Differentially Dependent on CD4+ T Cell Help Over the Course of Persistent Infection

Although many studies have investigated the requirement for CD4(+) T cell help for CD8(+) T cell responses to acute viral infections that are fully resolved, less is known about the role of CD4(+) T cells in maintaining ongoing CD8(+) T cell responses to persistently infecting viruses. Using mouse polyoma virus (PyV), we asked whether CD4(+) T cell help is required to maintain antiviral CD8(+) T cell and humoral responses during acute and persistent phases of infection. Though fully intact during acute infection, the PyV-specific CD8(+) T cell response declined numerically during persistent infection in MHC class II-deficient mice, leaving a small antiviral CD8(+) T cell population that was maintained long term. These unhelped PyV-specific CD8(+) T cells were functionally unimpaired; they retained the potential for robust expansion and cytokine production in response to Ag rechallenge. In addition, although a strong antiviral IgG response was initially elicited by MHC class II-deficient mice, these Ab titers fell, and long-lived PyV-specific Ab-secreting cells were not detected in the bone marrow. Finally, using a minimally myeloablative mixed bone marrow chimerism approach, we demonstrate that recruitment and/or maintenance of new virus-specific CD8(+) T cells during persistent infection is impaired in the absence of MHC class II-restricted T cells. In summary, these studies show that CD4(+) T cells differentially affect CD8(+) T cell responses over the course of a persistent virus infection.

Agonistic Anti-CD40 Antibody Profoundly Suppresses the Immune Response to Infection with Lymphocytic Choriomeningitis Virus

Previous work has shown that agonistic Abs to CD40 (anti-CD40) can boost weak CD8 T cell responses as well as substitute for CD4 T cell function during chronic gammaherpes virus infection. Agonistic anti-CD40 treatment has, therefore, been suggested as a potential therapeutic strategy in immunocompromised patients. In this study, we investigated whether agonistic anti-CD40 could substitute for CD4 T cell help in generating a sustained CD8 T cell response and prevent viral recrudescence following infection with lymphocytic choriomeningitis virus (LCMV). Contrary to expectations, we found that anti-CD40 treatment of MHC class II-deficient mice infected with a moderate dose of LCMV resulted in severe suppression of the antiviral CD8 T cell response and uncontrolled virus spread, rather than improved CD8 T cell immune surveillance. In Ab-treated wild-type mice, the antiviral CD8 T cell response also collapsed prematurely, and virus clearance was delayed. Additional analysis revealed that, following anti-CD40 treatment, the virus-specific CD8 T cells initially proliferated normally, but an increased cell loss compared with that in untreated mice was observed. The anti-CD40-induced abortion of virus-specific CD8 T cells during LCMV infection was IL-12 independent, but depended partly on Fas expression. Notably, similar anti-CD40 treatment of vesicular stomatitis virus-infected mice resulted in an improved antiviral CD8 T cell response, demonstrating that the effect of anti-CD40 treatment varies with the virus infection studied. For this reason, we recommend further evaluation of the safety of this regimen before being applied to human patients.