Virus-specific CD8+ T Lymphocytes Downregulate T Helper Cell Type 2 Cytokine Secretion and Pulmonary Eosinophilia during Experimental Murine Respiratory Syncytial Virus Infection

T lymphocytes play a pivotal role in the immune response during viral infections. In a murine model of experimental respiratory syncytial virus (RSV) infection, mice sensitized to either of the two major glycoproteins of RSV develop distinct patterns of cytokine secretion and lung inflammation upon subsequent RSV infection. Mice sensitized to RSV-G (attachment) glycoprotein exhibit a strong interleukin (IL)-4 and IL-5 response and develop pulmonary eosinophilia, whereas mice sensitized to RSV-F (fusion) glycoprotein develop a predominantly T helper cell (Th)1 response and pulmonary inflammation characterized by mononuclear cell infiltration. In this study, we examined the potential role of virus-specific CD8⁺ T cytolytic T cells on the differentiation and activation of functionally distinct CD4⁺ T cells specific to these viral glycoproteins. Mice primed with recombinant vaccinia virus expressing RSV-F glycoprotein mounted a strong RSV-specific, MHC class I–restricted cytolytic response, whereas priming with recombinant vaccinia virus expressing RSV-G glycoprotein failed to elicit any detectable cytolytic response. Priming for a RSV-specific CD8⁺ T cell response, either with a recombinant vaccinia virus expressing RSV-G glycoprotein in which a strong CD8⁺ T cell epitope from RSV-M2 (matrix) protein has been inserted or with a combination of vaccinia virus expressing the matrix protein and the RSV-G glycoprotein, suppressed the eosinophil recruitment into the lungs of these mice upon subsequent challenge with RSV. This reduction in pulmonary eosinophilia correlated with the suppression of Th2 type cytokine production. The importance of CD8⁺ T cells in this process was further supported by the results in CD8⁺ T cell deficient, β₂ microglobulin KO mice. In these mice, priming to RSV-F glycoprotein (which in normal mice primed for a strong cytolytic response and a pulmonary infiltrate consisting primarily of mononuclear cells on RSV challenge) resulted in the development of marked pulmonary eosinophilia that was not seen in mice with an intact CD8⁺ T cell compartment. These results indicate that CD8⁺ T cells may play an important role in the regulation of the differentiation and activation of Th2 CD4⁺ T cells as well as the recruitment of eosinophils into the lungs during RSV infection.Multiple arms of the immune system are activated in response to infection by foreign organisms. The outcomes of the infection, such as the clearance of the organisms and the generation of tissue injury, depend on these immune effector mechanisms that are mobilized against the pathogen. The role of T cells in the immune response to viral infections has been clearly established (1, 2). Mature T cells that express α/β T cell receptors can be divided into cells expressing either CD4 or CD8 surface antigen. CD8⁺ T cells recognize processed antigen in the context of MHC class I molecules, whereas CD4⁺ T cells recognize peptides in the context of MHC class II molecules (3, 4). The conventional view of CD8⁺ T cells has been primarily the killing of virally infected cells by direct cytolysis or by cytokines secreted by these T cells such as IFN-γ and TNF (1, 2). Functional subsets of CD4⁺ T cells have been described based on the cytokines produced by these cells, Th1 CD4⁺ T cells that secrete IL-2 and IFN-γ and Th2 T cells that secrete IL-4 and IL-5 (5, 6). The physiological relevance of these subsets of T cells with diverse functions have been shown in several models of viral infection including respiratory syncytial virus (RSV)¹.In the murine model of RSV infection, the roles of T cells and their products in the eradication of the virus as well as the pathogenesis of lung inflammation have been well documented (7–12). Recent studies have shown that mice sensitized to either of the two major glycoproteins of this virus developed distinct lung pathology when subsequently infected with RSV (12–15). Mice sensitized to the attachment glycoprotein (G) of RSV developed pulmonary eosinophilia that correlated with a strong induction of Th2 type response, whereas mice primed to the fusion (F) glycoprotein mounted a weak Th2 response and developed lung inflammation characterized by mononuclear cell infiltration. The underlying mechanisms that lead to these apparent distinct patterns of cytokine production and lung injury still remain unclear.The process by which CD4⁺ T cells mature and differentiate has been the subject of intense investigation (16). CD4⁺ T cells can be induced to differentiate into effector cells of either the Th1 or Th2 subsets, depending on the milieu in which these cells are activated. Cytokines such as IL-4 and IL-12 have been shown to play a crucial part in the regulation of CD4⁺ T cell differentiation (17–19). The presence of certain immune effector functions during the differentiation and expansion of CD4⁺ T cells may potentially be a key factor in determining the functional phenotypes acquired by these cells. In the murine model of experimental RSV infection, one important difference in the immune response to F and G glycoprotein of RSV is the lack of MHC class I–restricted cytolytic response to the G glycoprotein (13, 20). In the following studies, we began to investigate the impact of CD8⁺ T cells and MHC class I–restricted CTL responses on cytokine secretion and the subsequent development of pulmonary eosinophilia during experimental murine RSV infection. We have found that the induction of CD8⁺ T cell response to RSV proteins resulted in dramatically decreased levels of Th2 type cytokines and eosinophil recruitment into the lungs of RSV-infected mice previously sensitized to the RSV-G glycoprotein. The possible mechanisms underlying these observations and their potential significance are discussed.     

Eliminating a Region of Respiratory Syncytial Virus Attachment Protein Allows Induction of Protective Immunity without Vaccine-enhanced Lung Eosinophilia

In a murine model of respiratory syncytial virus disease, prior sensitization to the attachment glycoprotein (G) leads to pulmonary eosinophilia and enhanced illness. Three different approaches were taken to dissect the region of G responsible for enhanced disease and protection against challenge. First, mutant viruses, containing frameshifts that altered the COOH terminus of the G protein, were used to challenge mice sensitized by scarification with recombinant vaccinia virus (rVV) expressing wild-type G. Second, cDNA expressing these mutated G proteins were expressed by rVV and used to vaccinate mice before challenge with wild-type respiratory syncytial virus (RSV). These studies identified residues 193–205 to be responsible for G-induced weight loss and lung eosinophilia and showed that this region was not was not necessary for induction of protective immunity. Third, mice were sensitized using an rVV that expressed only amino acids 124–203 of the G protein. Upon RSV challenge, mice sensitized with this rVV developed enhanced weight loss and eosinophilia. This is the first time that a region within RSV (amino acids 193–203) has been shown to be responsible for induction of lung eosinophilia and disease enhancement. Moreover, we now show that it is possible to induce protective immunity with an altered G protein without inducing a pathological response.     

CD4+CD25+Foxp3+ Tregs resolve experimental lung injury in mice and are present in humans with acute lung injury

Acute lung injury (ALI) is characterized by rapid alveolar injury, inflammation, cytokine induction, and neutrophil accumulation. Although early events in the pathogenesis of ALI have been defined, the mechanisms underlying resolution are unknown. As a model of ALI, we administered intratracheal (i.t.) LPS to mice and observed peak lung injury 4 days after the challenge, with resolution by day 10. Numbers of alveolar lymphocytes increased as injury resolved. To examine the role of lymphocytes in this response, lymphocyte-deficient Rag-1^–/– and C57BL/6 WT mice were exposed to i.t. LPS. The extent of injury was similar between the groups of mice through day 4, but recovery was markedly impaired in the Rag-1^–/– mice. Adoptive transfer studies revealed that infusion of CD4⁺CD25⁺Foxp3⁺ Tregs as late as 24 hours after i.t. LPS normalized resolution in Rag-1^–/– mice. Similarly, Treg depletion in WT mice delayed recovery. Treg transfer into i.t. LPS–exposed Rag-1^–/– mice also corrected the elevated levels of alveolar proinflammatory cytokines and increased the diminished levels of alveolar TGF-β and neutrophil apoptosis. Mechanistically, Treg-mediated resolution of lung injury was abrogated by TGF-β inhibition. Moreover, BAL of patients with ALI revealed dynamic changes in CD3⁺CD4⁺CD25^hiCD127^loFoxp3⁺ cells. These results indicate that Tregs modify innate immune responses during resolution of lung injury and suggest potential targets for treating ALI, for which there are no specific therapies currently available.     

Liver Damage Preferentially Results from CD8+ T Cells Triggered by High Affinity Peptide Antigens

Little is understood of the anatomical fate of activated T lymphocytes and the consequences they have on the tissues into which they migrate. Previous work has suggested that damaged lymphocytes migrate to the liver. This study compares class I versus class II major histocompatibility complex (MHC)–restricted ovalbumin-specific T cell antigen receptor (TCR) transgenic mice to demonstrate that after in vivo activation with antigen the emergence of CD4⁻CD8⁻B220⁺ T cells occurs more frequently from a CD8⁺ precursor than from CD4⁺ T cells. Furthermore, this change in phenotype is conferred only by the high affinity native peptide antigen and not by lower affinity peptide variants. After activation of CD8⁺ cells with only the high affinity peptide, there is also a dramatically increased number of liver lymphocytes with accompanying extensive hepatocyte damage and elevation of serum aspartate transaminase. This was not observed in mice bearing a class II MHC–restricted TCR. The findings show that CD4⁻CD8⁻B220⁺ T cells preferentially derive from a CD8⁺ precursor after a high intensity TCR signal. After activation, T cells can migrate to the liver and induce hepatocyte damage, and thereby serve as a model of autoimmune hepatitis.     

An AXL/LRP-1/RANBP9 complex mediates DC efferocytosis and antigen cross-presentation in vivo

The phagocytosis of apoptotic cells (ACs), or efferocytosis, by DCs is critical for self-tolerance and host defense. Although many efferocytosis-associated receptors have been described in vitro, the functionality of these receptors in vivo has not been explored in depth. Using a spleen efferocytosis assay and targeted genetic deletion in mice, we identified a multiprotein complex — composed of the receptor tyrosine kinase AXL, LDL receptor–related protein–1 (LRP-1), and RAN-binding protein 9 (RANBP9) — that mediates DC efferocytosis and antigen cross-presentation. We found that AXL bound ACs, but required LRP-1 to trigger internalization, in murine CD8α⁺ DCs and human-derived DCs. AXL and LRP-1 did not interact directly, but relied on RANBP9, which bound both AXL and LRP-1, to form the complex. In a coculture model of antigen presentation, the AXL/LRP-1/RANBP9 complex was used by DCs to cross-present AC-associated antigens to T cells. Furthermore, in a murine model of herpes simplex virus–1 infection, mice lacking DC-specific LRP-1, AXL, or RANBP9 had increased AC accumulation, defective viral antigen-specific CD8⁺ T cell activation, enhanced viral load, and decreased survival. The discovery of this multiprotein complex that mediates functionally important DC efferocytosis in vivo may have implications for future studies related to host defense and DC-based vaccines.     

Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression

EAE is a mouse T cell–mediated autoimmune disease of the CNS used to model the human condition MS. The contributions of B cells to EAE initiation and progression are unclear. In this study, we have shown that EAE disease initiation and progression are differentially influenced by the depletion of B cells from mice with otherwise intact immune systems. CD20 antibody–mediated B cell depletion before EAE induction substantially exacerbated disease symptoms and increased encephalitogenic T cell influx into the CNS. Increased symptom severity resulted from the depletion of a rare IL-10–producing CD1d^hiCD5⁺ regulatory B cell subset (B10 cells), since the adoptive transfer of splenic B10 cells before EAE induction normalized EAE in B cell–depleted mice. While transfer of regulatory B10 cells was maximally effective during early EAE initiation, they had no obvious role during disease progression. Rather, B cell depletion during EAE disease progression dramatically suppressed symptoms. Specifically, B cells were required for the generation of CD4⁺ T cells specific for CNS autoantigen and the entry of encephalitogenic T cells into the CNS during disease progression. These results demonstrate reciprocal regulatory roles for B cells during EAE immunopathogenesis. The therapeutic effect of B cell depletion for the treatment of autoimmunity may therefore depend on the relative contributions and the timing of these opposing B cell activities during the course of disease initiation and pathogenesis.     

MerTK inhibition in tumor leukocytes decreases tumor growth and metastasis

MerTK, a receptor tyrosine kinase (RTK) of the TYRO3/AXL/MerTK family, is expressed in myeloid lineage cells in which it acts to suppress proinflammatory cytokines following ingestion of apoptotic material. Using syngeneic mouse models of breast cancer, melanoma, and colon cancer, we found that tumors grew slowly and were poorly metastatic in MerTK^–/– mice. Transplantation of MerTK^–/– bone marrow, but not wild-type bone marrow, into lethally irradiated MMTV-PyVmT mice (a model of metastatic breast cancer) decreased tumor growth and altered cytokine production by tumor CD11b⁺ cells. Although MerTK expression was not required for tumor infiltration by leukocytes, MerTK^–/– leukocytes exhibited lower tumor cell–induced expression of wound healing cytokines, e.g., IL-10 and growth arrest-specific 6 (GAS6), and enhanced expression of acute inflammatory cytokines, e.g., IL-12 and IL-6. Intratumoral CD8⁺ T lymphocyte numbers were higher and lymphocyte proliferation was increased in tumor-bearing MerTK^–/– mice compared with tumor-bearing wild-type mice. Antibody-mediated CD8⁺ T lymphocyte depletion restored tumor growth in MerTK^–/– mice. These data demonstrate that MerTK signaling in tumor-associated CD11b⁺ leukocytes promotes tumor growth by dampening acute inflammatory cytokines while inducing wound healing cytokines. These results suggest that inhibition of MerTK in the tumor microenvironment may have clinical benefit, stimulating antitumor immune responses or enhancing immunotherapeutic strategies.     

Infiltration of CD4+ lymphocytes into the brain contributes to neurodegeneration in a mouse model of Parkinson disease

Parkinson disease (PD) is a neurodegenerative disorder characterized by a loss of dopamine-containing neurons. Mounting evidence suggests that dopaminergic cell death is influenced by the innate immune system. However, the pathogenic role of the adaptive immune system in PD remains enigmatic. Here we showed that CD8⁺ and CD4⁺ T cells but not B cells had invaded the brain in both postmortem human PD specimens and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD during the course of neuronal degeneration. We further demonstrated that MPTP-induced dopaminergic cell death was markedly attenuated in the absence of mature T lymphocytes in 2 different immunodeficient mouse strains (Rag1^–/– and Tcrb^–/– mice). Importantly, similar attenuation of MPTP-induced dopaminergic cell death was seen in mice lacking CD4 as well as in Rag1^–/– mice reconstituted with FasL-deficient splenocytes. However, mice lacking CD8 and Rag1^–/– mice reconstituted with IFN-γ–deficient splenocytes were not protected. These data indicate that T cell–mediated dopaminergic toxicity is almost exclusively arbitrated by CD4⁺ T cells and requires the expression of FasL but not IFNγ. Further, our data may provide a rationale for targeting the adaptive arm of the immune system as a therapeutic strategy in PD.     

A Novel Cancer Therapeutic Using Thrombospondin 1 in Dendritic Cells

Induction of thrombospondin 1 (TSP-1) is generally assumed to suppress tumor growth through inhibiting angiogenesis; however, it is less clear how TSP-1 in dendritic cells (DCs) influences tumor progression. We investigated tumor growth and immune mechanism by downregulation of TSP-1 in dendritic cells. Administration of TSP-1 small hairpin RNA (shRNA) through the skin produced anticancer therapeutic effects. Tumor-infiltrating CD4⁺ and CD8⁺ T cells were increased after the administration of TSP-1 shRNA. The expression of interleukin-12 and interferon-γ in the lymph nodes was enhanced by injection of TSP-1 shRNA. Lymphocytes from the mice injected with TSP-1 shRNA selectively killed the tumor cells, and the cytotoxicity of lymphocytes was abolished by depletion of CD8⁺ T cells. Injection of CD11c⁺ TSP-1–knockout (TSP-1-KO) bone marrow–derived DCs (BMDCs) delayed tumor growth in tumor-bearing mice. Similarly, antitumor activity induced by TSP-1-KO BMDCs was abrogated by depletion of CD8⁺ T cells. In contrast, the administration of shRNAs targeting TSP-2, another TSP family member, did not extend the survival of tumor-bearing mice. Finally, TSP-1 shRNA functioned as an immunotherapeutic adjuvant to augment the therapeutic efficacy of Neu DNA vaccination. Collectively, the downregulation of TSP-1 in DCs produces an effective antitumor response that is opposite to the protumor effects by silencing of TSP-1 within tumor cells.     

Definition of target antigens for naturally occurring CD4(+) CD25(+) regulatory T cells

The antigenic targets recognized by naturally occurring CD4⁺ CD25⁺ regulatory T cells (T reg cells) have been elusive. We have serologically defined a series of broadly expressed self-antigens derived from chemically induced mouse sarcomas by serological identification of antigens by recombinant expression cloning (SEREX). CD4⁺ CD25⁺ T cells from mice immunized with SEREX-defined self-antigens had strong suppressive activity on peptide-specific proliferation of CD4⁺ CD25⁻ T cells and CD8⁺ T cells. The suppressive effect was observed without in vitro T cell stimulation. Foxp3 expression in these CD4⁺ CD25⁺ T cells from immunized mice was 5–10 times greater than CD4⁺ CD25⁺ T cells derived from naive mice. The suppressive effect required cellular contact and was blocked by anti-glucocorticoid–induced tumor necrosis factor receptor family–related gene antibody. In vitro suppressive activity essentially disappeared 8 wk after the last immunization. However, it was regained by in vitro restimulation with cognate self-antigen protein but not with control protein. We propose that SEREX-defined self-antigens such as those used in this study represent self-antigens that elicit naturally occurring CD4⁺ CD25⁺ T reg cells.     

Relationship of SARS-CoV-2–specific CD4 response to COVID-19 severity and impact of HIV-1 and tuberculosis coinfection

T cells are involved in control of coronavirus disease 2019 (COVID-19), but limited knowledge is available on the relationship between antigen-specific T cell response and disease severity. Here, we used flow cytometry to assess the magnitude, function, and phenotype of SARS coronavirus 2–specific (SARS-CoV-2–specific) CD4⁺ T cells in 95 hospitalized COVID-19 patients, 38 of them being HIV-1 and/or tuberculosis (TB) coinfected, and 38 non–COVID-19 patients. We showed that SARS-CoV-2–specific CD4⁺ T cell attributes, rather than magnitude, were associated with disease severity, with severe disease being characterized by poor polyfunctional potential, reduced proliferation capacity, and enhanced HLA-DR expression. Moreover, HIV-1 and TB coinfection skewed the SARS-CoV-2 T cell response. HIV-1–mediated CD4⁺ T cell depletion associated with suboptimal T cell and humoral immune responses to SARS-CoV-2, and a decrease in the polyfunctional capacity of SARS-CoV-2–specific CD4⁺ T cells was observed in COVID-19 patients with active TB. Our results also revealed that COVID-19 patients displayed reduced frequency of Mycobacterium tuberculosis–specific CD4⁺ T cells, with possible implications for TB disease progression. These results corroborate the important role of SARS-CoV-2–specific T cells in COVID-19 pathogenesis and support the concept of altered T cell functions in patients with severe disease.     

TLR4 signaling in effector CD4+ T cells regulates TCR activation and experimental colitis in mice

TLRs sense various microbial products. Their function has been best characterized in DCs and macrophages, where they act as important mediators of innate immunity. TLR4 is also expressed on CD4⁺ T cells, but its physiological function on these cells remains unknown. Here, we have shown that TLR4 triggering on CD4⁺ T cells affects their phenotype and their ability to provoke intestinal inflammation. In a model of spontaneous colitis, Il10^–/–Tlr4^–/– mice displayed accelerated development of disease, with signs of overt colitis as early as 8 weeks of age, when compared with Il10^–/– and Il10^–/–Tlr9^–/– mice, which did not develop colitis by 8 months. Similar results were obtained in a second model of colitis in which transfer of naive Il10^–/–Tlr4^–/– CD4⁺ T cells into Rag1^–/– recipients sufficient for both IL-10 and TLR4 induced more aggressive colitis than the transfer of naive Il10^–/– CD4⁺ T cells. Mechanistically, LPS stimulation of TLR4-bearing CD4⁺ T cells inhibited ERK1/2 activation upon subsequent TCR stimulation via the induction of MAPK phosphatase 3 (MKP-3). Our data therefore reveal a tonic inhibitory role for TLR4 signaling on subsequent TCR-dependent CD4⁺ T cell responses.     

Protein tyrosine phosphatase–σ regulates hematopoietic stem cell–repopulating capacity

Hematopoietic stem cell (HSC) function is regulated by activation of receptor tyrosine kinases (RTKs). Receptor protein tyrosine phosphatases (PTPs) counterbalance RTK signaling; however, the functions of receptor PTPs in HSCs remain incompletely understood. We found that a receptor PTP, PTPσ, was substantially overexpressed in mouse and human HSCs compared with more mature hematopoietic cells. Competitive transplantation of bone marrow cells from PTPσ-deficient mice revealed that the loss of PTPσ substantially increased long-term HSC-repopulating capacity compared with BM cells from control mice. While HSCs from PTPσ-deficient mice had no apparent alterations in cell-cycle status, apoptosis, or homing capacity, these HSCs exhibited increased levels of activated RAC1, a RhoGTPase that regulates HSC engraftment capacity. shRNA-mediated silencing of PTPσ also increased activated RAC1 levels in wild-type HSCs. Functionally, PTPσ-deficient BM cells displayed increased cobblestone area–forming cell (CAFC) capacity and augmented transendothelial migration capacity, which was abrogated by RAC inhibition. Specific selection of human cord blood CD34⁺CD38^–CD45RA^–lin^– PTPσ^– cells substantially increased the repopulating capacity of human HSCs compared with CD34⁺CD38^–CD45RA^–lin^– cells and CD34⁺CD38^–CD45RA^–lin^–PTPσ⁺ cells. Our results demonstrate that PTPσ regulates HSC functional capacity via RAC1 inhibition and suggest that selecting for PTPσ-negative human HSCs may be an effective strategy for enriching human HSCs for transplantation.     

Cytotoxic T Lymphocyte Antigen 4 Is Induced in the Thymus upon In Vivo Activation and Its Blockade Prevents Anti-CD3–mediated Depletion of ?Thymocytes

The development of a normal T cell repertoire in the thymus is dependent on the interplay between signals mediating cell survival (positive selection) and cell death (negative selection or death by neglect). Although the CD28 costimulatory molecule has been implicated in this process, it has been difficult to establish a role for the other major costimulatory molecule, cytotoxic T lymphocyte antigen (CTLA)-4. Here we report that in vivo stimulation through the T cell receptor (TCR)–CD3 complex induces expression of CTLA-4 in thymocytes and leads to the association of CTLA-4 with the SH2 domain–containing phosphatase (SHP)-2 tyrosine phosphatase. Moreover, intrathymic CTLA-4 blockade dramatically inhibits anti-CD3–mediated depletion of CD4⁺CD8⁺ double positive immature thymocytes. Similarly, anti-CD3–mediated depletion of CD4⁺CD8⁺ double positive cells in fetal thymic organ cultures could also be inhibited by anti–CTLA-4 antibodies. Thus, our data provide evidence for a role of CTLA-4 in thymic selection and suggest a novel mechanism contributing to the regulation of TCR-mediated selection of T cell repertoires.     

An Essential Role for Interleukin 10 in the Function of Regulatory T Cells That Inhibit Intestinal Inflammation

A T helper cell type 1–mediated colitis develops in severe combined immunodeficient mice after transfer of CD45RB^high CD4⁺ T cells and can be prevented by cotransfer of the CD45RB^low subset. The immune-suppressive activities of the CD45RB^low T cell population can be reversed in vivo by administration of an anti-transforming growth factor β antibody. Here we show that interleukin (IL)-10 is an essential mediator of the regulatory functions of the CD45RB^low population. This population isolated from IL-10–deficient (IL-10^−/−) mice was unable to protect from colitis and when transferred alone to immune-deficient recipients induced colitis. Treatment with an anti–murine IL-10 receptor monoclonal antibody abrogated inhibition of colitis mediated by wild-type (WT) CD45RB^low CD4⁺ cells, suggesting that IL-10 was necessary for the effector function of the regulatory T cell population. Inhibition of colitis by WT regulatory T cells was not dependent on IL-10 production by progeny of the CD45RB^high CD4⁺ cells, as CD45RB^low CD4⁺ cells from WT mice were able to inhibit colitis induced by IL-10^−/− CD45RB^high CD4⁺ cells. These findings provide the first clear evidence that IL-10 plays a nonredundant role in the functioning of regulatory T cells that control inflammatory responses towards intestinal antigens.     

Induction and Exhaustion of Lymphocytic Choriomeningitis Virus–specific Cytotoxic T Lymphocytes Visualized Using Soluble Tetrameric Major Histocompatibility Complex Class I–Peptide Complexes

This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2D^b, chemically biotinylated β2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33–41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8⁺ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2D^b in association with peptide GP33–41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8⁺ T cells were GP33 tetramer⁺ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8⁺ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.The ability to clear infections with noncytopathic viruses is predominantly attributed to CD8⁺ CTLs. CTLs recognize infected cells via an interaction between TCRs and their corresponding ligands, class I MHC molecules (1). MHC class I molecules are expressed on the cell surface in association with self or pathogen-derived peptides that are generated intracellularly by proteolytic degradation of the parent proteins (2). After recognition of an infected cell, naive CTLs become activated, proliferate, and attain not only the ability to lyse infected cells, but also the ability to produce IFN-γ (3). Although IFN-γ may have a direct antiviral effect (4, 5), it has also been shown to improve the efficiency of antigen presentation by class I molecules (6–9) thereby promoting the induction of a CTL response and improving the efficiency with which CTL can recognize their infected targets.CTLs have been shown to be essential for the recovery of mice from the acute phase of infection with the noncytopathic lymphocytic choriomeningitis virus (LCMV; reference 10). So far, it has not been possible to follow the kinetics of appearance and disappearance of antigen-specific effector CTLs during the acute phase of both low- and high-dose LCMV infections in non-TCR-transgenic mice. A recent study by Altman et al. (11) described a method, using tetrameric soluble MHC class I–peptide complexes, for the identification of antigen-specific CD8⁺ cells in the PBMCs of HIV-infected humans. This study describes an adaptation of this method for the identification of antigen-specific CD8⁺ cells in B6 (H-2^b) mice infected with LCMV. In this case, soluble peptide–MHC complexes were generated using the mouse class I heavy chain D^b, chemically biotinylated human β2 microglobulin (β₂M) and the LCMV peptide epitope glycoprotein (GP)33–41 (GP33-KAVYNFATC). Fluorescence-labeled tetrameric complexes were subsequently produced by mixing the biotinylated complexes with phycoerythrin-labeled neutravidin. Peptide GP33–41 (GP33) was used for the purposes of this study since, after LCMV-WE infection of C57BL/6 mice, most CTL activity (∼50–60%) is directed towards this epitope. Two other epitopes, defined by residues 276– 286 of the viral glycoprotein (GP276) and residues 396– 404 of the viral nucleoprotein (NP396), represent 10–20 and 20–30% of the total CTL activity, respectively (12–17).The tetrameric class I–peptide complexes, which stained CTLs specifically, were used to follow the fate of GP33-specific CD8⁺ T cells in mice during the acute phase of LCMV infection. This study demonstrates the accummulation of stained virus-specific CTLs in the CSF and spleens of mice after intracranial infection with a substrain of LCMV-WE called LCMV-DOCILE, and in the spleens of mice infected intravenously with the same virus. In both cases, the accummulation of GP33-specific CTLs, after both low- and high-dose infection with LCMV-DOCILE, was monitored in relation to the capacity of the cells to produce IFN-γ; to mediate cytotoxic activity, and to mediate virus clearance.     

Regulatory CD4+ T Cells Expressing Endogenous T Cell Receptor Chains Protect Myelin Basic Protein–specific Transgenic Mice from Spontaneous Autoimmune Encephalomyelitis

The development of T cell–mediated autoimmune diseases hinges on the balance between effector and regulatory mechanisms. Using two transgenic mouse lines expressing identical myelin basic protein (MBP)–specific T cell receptor (TCR) genes, we have previously shown that mice bearing exclusively MBP-specific T cells (designated T/R⁻) spontaneously develop experimental autoimmune encephalomyelitis (EAE), whereas mice bearing MBP-specific T cells as well as other lymphocytes (designated T/R⁺) did not. Here we demonstrate that T/R⁻ mice can be protected from EAE by the early transfer of total splenocytes or purified CD4⁺ T cells from normal donors. Moreover, whereas T/R⁺ mice crossed with B cell–deficient, γ/δ T cell–deficient, or major histocompatibility complex class I–deficient mice did not develop EAE spontaneously, T/R⁺ mice crossed with TCR-α and -β knockout mice developed EAE with the same incidence and severity as T/R⁻ mice. In addition, MBP-specific transgenic mice that lack only endogenous TCR-α chains developed EAE with high incidence but reduced severity. Surprisingly, two-thirds of MBP-specific transgenic mice lacking only endogenous TCR-β chains also developed EAE, suggesting that in T/R⁺ mice, cells with high protective activity escape TCR-β chain allelic exclusion. Our study identifies CD4⁺ T cells bearing endogenous α and β TCR chains as the lymphocytes that prevent spontaneous EAE in T/R⁺ mice.     

Aplastic Anemia Rescued by Exhaustion of Cytokine-secreting CD8+ T Cells in Persistent Infection with Lymphocytic Choriomeningitis Virus

Aplastic anemia may be associated with persistent viral infections that result from failure of the immune system to control virus. To evaluate the effects on hematopoiesis exerted by sustained viral replication in the presence of activated T cells, blood values and bone marrow (BM) function were analyzed in chronic infection with lymphocytic choriomeningitis virus (LCMV) in perforin-deficient (P^0/0) mice. These mice exhibit a vigorous T cell response, but are unable to eliminate the virus. Within 14 d after infection, a progressive pancytopenia developed that eventually was lethal due to agranulocytosis and thrombocytopenia correlating with an increasing loss of morphologically differentiated, pluripotent, and committed progenitors in the BM. This hematopoietic disease caused by a noncytopathic chronic virus infection was prevented by depletion of CD8⁺, but not of CD4⁺, T cells and accelerated by increasing the frequency of LCMV-specific CD8⁺ T cells in T cell receptor (TCR) transgenic (tg) mice. LCMV and CD8⁺ T cells were found only transiently in the BM of infected wild-type mice. In contrast, increased numbers of CD8⁺ T cells and LCMV persisted at high levels in antigen-presenting cells of infected P^0/0 and P^0/0 × TCR tg mice. No cognate interaction between the TCR and hematopoietic progenitors presenting either LCMV-derived or self-antigens on the major histocompatibility complex was found, but damage to hematopoiesis was due to excessive secretion and action of tumor necrosis factor (TNF)/lymphotoxin (LT)-α and interferon (IFN)-γ produced by CD8⁺ T cells. This was studied in double-knockout mice that were genetically deficient in perforin and TNF receptor type 1. Compared with P^0/0 mice, these mice had identical T cell compartments and T cell responses to LCMV, yet they survived LCMV infection and became life-long virus carriers. The numbers of hematopoietic precursors in the BM were increased compared with P^0/0 mice after LCMV infection, although transient blood disease was still noticed. This residual disease activity was found to depend on IFN-γ–producing LCMV-specific T cells and the time point of hematopoietic recovery paralleled disappearance of these virus-specific, IFN-γ–producing CD8⁺ T cells. Thus, in the absence of IFN-γ and/or TNF/LT-α, exhaustion of virus-specific T cells was not hampered.