CD3bright signals on γδ T cells identify IL-17A-producing Vγ6Vδ1+ T cells.

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine that has an important role at mucosal sites in a wide range of immune responses including infection, allergy and auto-immunity. γδ T cells are recognized as IL-17 producers, but based on the level of CD3 expression, we now define the remarkable ability of a CD3(bright) γδ T-cell subset with an effector memory phenotype to rapidly produce IL-17A, but not interferon-γ. CD3(bright) γδ T cells uniformly express the canonical germline encoded Vγ6/Vδ1(+) T-cell receptor. They are widely distributed with a preferential representation in the lungs and skin are negatively impacted in the absence of retinoic acid receptor-related orphan receptor gammat expression or endogenous flora. This population responded rapidly to various stimuli in a mechanism involving IL-23 and NOD-like receptor family, pyrin domain containing 3 (NLRP3)-inflammasome-dependent IL-1ß. Finally, we demonstrated that IL-17-producing CD3(bright) γδ T cells responded promptly and strongly to pneumococcal infection and during skin inflammation. Here, we propose a new way to specifically analyze IL-17-producing Vγ6/Vδ1(+) T cells based on the level of CD3 signals. Using this gating strategy, our data reinforce the crucial role of this γδ T-cell subset in respiratory and skin disorders.

A divergent transcriptional landscape underpins the development and functional branching of MAIT cells.

MR1-restricted mucosal-associated invariant T (MAIT) cells play a unique role in the immune system. These cells develop intrathymically through a three-stage process, but the events that regulate this are largely unknown. Here, using bulk and single-cell RNA sequencing-based transcriptomic analysis in mice and humans, we studied the changing transcriptional landscape that accompanies transition through each stage. Many transcripts were sharply modulated during MAIT cell development, including SLAM (signaling lymphocytic activation molecule) family members, chemokine receptors, and transcription factors. We also demonstrate that stage 3 "mature" MAIT cells comprise distinct subpopulations including newly arrived transitional stage 3 cells, interferon-γ-producing MAIT1 cells and interleukin-17-producing MAIT17 cells. Moreover, the validity and importance of several transcripts detected in this study are directly demonstrated using specific mutant mice. For example, MAIT cell intrathymic maturation was found to be halted in SLAM-associated protein (SAP)-deficient and CXCR6-deficient mouse models, providing clear evidence for their role in modulating MAIT cell development. These data underpin a model that maps the changing transcriptional landscape and identifies key factors that regulate the process of MAIT cell differentiation, with many parallels between mice and humans.

Innate lymphoid cells: models of plasticity for immune homeostasis and rapid responsiveness in protection.

Innate lymphoid cells (ILCs) have stormed onto the immune landscape as "newly discovered" cell types. These tissue-resident sentinels are enriched at mucosal surfaces and engage in complex cross talk with elements of the adaptive immune system and microenvironment to orchestrate immune homeostasis. Many parallels exist between innate cells and T cells leading to the initial partitioning of ILCs into rather rigid subsets that reflect their "adaptive-like" effector cytokines profiles. ILCs themselves, however, have unique attributes that are only just beginning to be elucidated. These features result in complementarity with, rather than complete duplication of, functions of the adaptive immune system. Key transcription factors determine the pathway of differentiation of progenitors towards an ILC1, ILC2, or ILC3 subset. Once formed, flexibility in the responses of these subsets to stimuli unexpectedly allows transdifferentation between the different subsets and the acquisition of altered phenotypes and function. This provides a mechanism for rapid innate immune responsiveness. Here, we discuss the models of differentiation for maintenance and activation of tissue-resident ILCs in maintaining immune homeostasis and protection.

Granzyme M has a critical role in providing innate immune protection in ulcerative colitis.

Inflammatory bowel disease (IBD) is an immunoregulatory disorder, associated with a chronic and inappropriate mucosal immune response to commensal bacteria, underlying disease states such as ulcerative colitis (UC) and Crohn's disease (CD) in humans. Granzyme M (GrzM) is a serine protease expressed by cytotoxic lymphocytes, in particular natural killer (NK) cells. Granzymes are thought to be involved in triggering cell death in eukaryotic target cells; however, some evidence supports their role in inflammation. The role of GrzM in the innate immune response to mucosal inflammation has never been examined. Here, we discover that patients with UC, unlike patients with CD, display high levels of GrzM mRNA expression in the inflamed colon. By taking advantage of well-established models of experimental UC, we revealed that GrzM-deficient mice have greater levels of inflammatory indicators during dextran sulfate sodium (DSS)-induced IBD, including increased weight loss, greater colon length reduction and more severe intestinal histopathology. The absence of GrzM expression also had effects on gut permeability, tissue cytokine/chemokine dynamics, and neutrophil infiltration during disease. These findings demonstrate, for the first time, that GrzM has a critical role during early stages of inflammation in UC, and that in its absence colonic inflammation is enhanced.

Intercellular and lymphatic pathways associated with tonsils of the soft palate in young pigs.

Tonsils of the soft palate of pigs are the main oropharyngeal lymphoid tissues that protect the body against antigens entering through the mouth. The aim of this work was to elucidate the intercellular and lymphatic pathways by which lymph and cells are transported through these tonsils. Tonsillar tissue from freshly-killed pigs was examined using light microscopy and electron microscopy, or was injected with Mercox for scanning electron microscopy of corrosion casts. Intercellular fluid passes between epithelial cells and is continuous with that of the subepithelium. Fluid from the subepithelium flows into sinuses that form a network around the apex of follicles. These sinuses are continuous with parafollicular sinuses that penetrate the parafollicular tissue between the follicles. Some parafollicular sinuses are traversed by a complex network of cell processes, whereas others appear to lack such processes. Some parafollicular sinuses are closely located (10 microm) to venules; others lie adjacent to the follicle capsule. No lymphatics enter or leave the follicles. All lymph from the tonsils must traverse parafollicular sinuses before entering septal vessels, and these are continuous with basal vessels. Basal vessels coalesce to form efferent vessels that transport lymph from the tonsil to the primary lymph nodes. Septal, basal and efferent lymphatic vessels contain prominent valves and many lymphocytes. Lymphatic sinuses appear to be a significant pathway for lymphocytes migrating from the tonsillar lymphoid tissue.

The epithelium of canine palatine tonsils.

The palatine tonsil is positioned to play a key role in protecting the body against ingested microorganisms. These microorganisms must traverse the tonsillar epithelium to initiate immune reactions, but no information is available from dogs on the structure of this epithelium. In this study, the morphology and ultrastructure of the epithelium of the palatine tonsil of dogs was examined using light and electron microscopy. The epithelium is of two types: reticular and non-reticular. Reticular epithelium, which is invaded by lymphoid cells, is located over the apices of nodules, separated indistinctly by islands of non-reticular squamous epithelium. The reticular epithelium contains M cells which are more abundant towards the periphery of lymphoid nodules. The apical membrane of the M cells forms folds from which microvilli extend into the lumen of the oropharynx. Abluminally, cytoplasmic processes enfold clusters of lymphocytes. Numerous desmosomes secure the marginal plasma membrane and lateral membranous interdigitations to adjacent epithelial cells, thereby maintaining the integrity of the tonsillar epithelium. Epithelial discontinuities, which are few, occupy similar positions to M cells and appear to contain migrating lymphocytes. A fenestrated basement membrane allows cell transport between the intraepithelial passageways of the reticular epithelium and the subepithelial lymphoid tissues.

An investigation of the use of chromium, platinum and gold coating for scanning electron microscopy of casts of lymphoid tissues.

Resin casts replicate the internal structure of organs and provide a three-dimensional representation of the arrangement of vessels and intercellular spaces. Casting media are insulators and must be coated with a conductor to prevent sample charging and to allow the adequate production of secondary electrons from the specimen to generate sufficient signal to form a clear image. Visualization of surface structures depends largely on the metal coating. The use of gold or platinum, deposited on Mercox casts of lymphoid tissues using plasma-magnetron sputtering, and of chromium coating of casts by Penning ion-beam coating, was investigated. Casts were examined using a field emission scanning electron microscope at 3-3.5 kV. Thick coatings of gold were necessary to reduce cast charging but they obscured fine structural information. Charging effects were less pronounced when casts were coated with platinum, but charge lines were present at slow scan rates. The dimensions of cast impressions for both platinum and chromium coatings were similar to those described in fixed tissues. Negligible charging and maximal cast thermal stability and structural information was obtained from casts which were tumbled during chromium coating.

IL-17-producing NKT cells depend exclusively on IL-7 for homeostasis and survival.

Natural killer T (NKT) cells are innate-like T cells that rapidly recognize pathogens and produce cytokines that shape the ensuing immune response. IL-17-producing NKT cells are enriched in barrier tissues, such as the lung, skin, and peripheral lymph nodes, and the factors that maintain this population in the periphery have not been elucidated. Here we show that NKT17 cells deviate from other NKT cells in their survival requirements. In contrast to conventional NKT cells that are maintained by IL-15, RORγt(+) NKT cells are IL-15 independent and instead rely completely on IL-7. IL-7 initiates a T-cell receptor-independent (TCR-independent) expansion of NKT17 cells, thus supporting their homeostasis. Without IL-7, survival is dramatically impaired, yet residual cells remain lineage committed with no downregulation of RORγt evident. Their preferential response to IL-7 does not reflect enhanced signaling through STAT proteins, but instead is modulated via the PI3K/AKT/mTOR signaling pathway. The ability to compete for IL-7 is dependent on high-density IL-7 receptor expression, which would promote uptake of low levels of IL-7 produced in the non-lymphoid sites of lung and skin. This dependence on IL-7 is also reported for RORγt(+) innate lymphoid cells and CD4(+) Th17 cells, and suggests common survival requirements for functionally similar cells.

An unusual structure of venules in tonsils of the soft palate of young pigs.

In tonsils of the soft palate of pigs high endothelial venules (HEV) occur throughout the parafollicular region. In some regions of the venules the endothelial lining is uniformly high; in other areas, mainly the nuclear region of the endothelial cell protrudes into the lumen and the cytoplasmic region is attenuated. In this portion of the venule endothelial cells may traverse the lumen of the vessel to form intravascular bridging processes. The endothelial cells are characterised by pinocytotic vesicles, many surface microvilli, cytoplasmic processes and local cell-cell contacts with adjacent endothelial cells. These specialised HEV may play a role in the unusual pattern of lymphocytes from tonsillar tissue into blood. In the tonsils of the soft palate of pigs, HEV may mediate both lymphocyte migration to and from the lymphoid tissue. Further investigation is required to determine the importance of these 2 migration pathways.

Lymph pathways of the medial retropharyngeal lymph node in dogs.

In dogs, lymph drains from tissues throughout the head, including the tonsils, along lymphatic vessels to the facial, parotid, lateral retropharyngeal and mandibular lymph nodes. From the mandibular lymph nodes, lymph may flow to the ipsilateral medial retropharyngeal lymph nodes, or along anastomotic connections to the contralateral node. Afferent lymphatics convey lymph from these nodes to defined areas in the medial retropharyngeal nodes. They divide over the surface of the node, and within trabeculae. Terminal afferent lymphatics are connected to the subcapsular and trabecular sinuses either through circular or oval holes in the vessel wall, or terminate at the sinus where the vessel contains a valve adjacent to the point of entry. The subcapsular sinus surrounds the entire node, and is continuous with an interconnecting network of trabecular and cortical sinuses which convey lymph through the cortex. Connective tissue septa extend through the sinuses and lymph flows freely between adjacent sinuses through holes in the septal walls. Initial efferent lymphatic vessels, which arise from the medullary sinuses between medullary cords, converge towards and unite within the network of medullary trabeculae. Other vessels, which contain valve-like flaps, drain lymph from the subcapsular sinus. Efferent vessels emerge along the hilus and coalesce to form the tracheal trunk. The tracheal trunk has several layers of smooth muscle cells, well developed elastic laminae and connective tissue, surrounding the lymphatic endothelium.

Intercellular and lymphatic pathways of the canine palatine tonsils.

The palatine tonsils play a key role in initiating immune responses against antigenic material entering the mouth and their lymphatic pathways are important in disseminating immunological information to the lymph nodes and other mucosal surfaces. Scanning and transmission electron microscopy and Mercox casts were used to examine the intercellular and lymphatic pathways of the palatine tonsil in dogs. Intercellular fluid within the intraepithelial passageways of the reticular epithelium flows through pores in the basement membrane and intermingles with that of the subepithelial intercellular spaces. From there, tissue fluid enters initial lymphatics which form a plexus surrounding each follicle. No lymphatic vessels are seen entering or leaving the follicles but intercellular pathways of the follicles are continuous with those of the adjacent initial lymphatics. These pathways appear to provide the only route for lymphocytes leaving the follicles and directly entering the lymphatic pathway. Lymph then flows into sinuses adjacent to, and incompletely surrounding, the base of follicles; or it may enter a network of sinuses between and beneath the lymphoid follicles which convey lymph throughout the parafollicular tissue. Lymphoid cells enter the parafollicular sinuses which may be a major entry site of lymphocytes emigrating from the lymphoid parenchyma. Some lymph may bypass these sinuses by entering septal lymphatic vessels oriented perpendicular to the central connective tissue lamina of the palatine tonsil. All lymph is collected into basal lymphatic vessels where valves prevent retrograde flow. Basal vessels course within the central lamina and converge to form efferent lymphatic vessels which emerge at the caudal region of the palatine tonsil and convey lymph to the medial retropharyngeal lymph node.

Pathways of blood flow to and through superficial lymph nodes in the dog.

Wide variations occur in the arrangement of the blood vessels of the superficial lymph nodes in dogs. These vessels were studied using Microfil and Mercox casts, scanning and transmission electron microscopy, and the dye Alcian blue. Most nodes receive some arteries at a hilus, but this varies from a single indentation to one or more grooves and in some cases is a smooth and flattened area. Some nodes have no identifiable hilus and arteries enter at many different points. Most nodes that receive arteries at a hilus also receive additional vessels, some of which are derived from a network at the capsule; some of these enter by crossing the subcapsular sinus. In both cortex and medulla, some capillaries are within lymph sinuses, or adjacent to them. Within the cortex, networks of arterioles, capillaries and venules occur near subcapsular and trabecular sinuses, and around nodules, but the nodules themselves are relatively avascular. Some medullary capillaries are fenestrated, but all other capillaries have a complete endothelial lining, with basement membrane and pericytes. All venules, except those in the deep cortex, have a low endothelial lining. Although the distribution of vessels within the nodes follows a general pattern in dogs, the variation in the pattern of supply is such that it is not possible to ascribe any particular pattern to superficial nodes in general, or even to the nodes of a particular lymph centre. This contrasts with the commonly accepted description which implies that all blood vessels to all lymph nodes pass through a well defined hilus.

Lymphatic drainage from the tonsil of the soft palate in pigs.

The tonsil of the soft palate, the predominant lymphoid tissue of the oropharynx in pigs, is important especially in initiating immune responses against antigenic material entering the mouth. The aim of this work was to describe the lymphatic pathways from the tonsils of the soft palate of pigs through lymph nodes of the head to the bloodstream. This was achieved by gross dissection, and by using Evans' Blue dye and Microfil casts. Efferent lymphatic vessels from the tonsil coalesce to form vessels which convey lymph to the primary nodes, the mandibular and medial retropharyngeal, and thence to the bloodstream, along two distinct pathways. In the superficial pathway, lymph flows through the mandibular lymph node, along lymphatic vessels closely associated with the linguofacial vein, to the ventral superficial cervical node (middle group) and the accessory mandibular node. Most efferent vessels from the accessory mandibular node enter the ventral superficial cervical node, but some may directly join the lymphatic vessels emanating from the ventral superficial cervical node. These vessels convey lymph to the dorsal superficial cervical node and thence, via the efferent lymphatics, to the circulatory system. In the deep pathway, lymph is conveyed directly to the medial retropharyngeal node and then to the tracheal trunk, as in other domestic animals. As the vessels from the tonsils course over the surface of the pharynx, the muscular movements of swallowing may help propel lymph towards the primary nodes and the bloodstream.

Tonsils of the soft palate of young pigs: crypt structure and lymphoepithelium.

BACKGROUND: Tonsils of the soft palate are especially important in pigs as the major pharyngeal mucosal-associated lymphoid tissue and as a portal of entry for microorganisms. They play a key role in initiating immune responses against antigens entering tonsillar crypts from the oropharynx. The aim of this study was to describe the architecture of the tonsillar crypst and the morphology of their epithelial surface. METHODS: Tissue taken from the tonsil of the soft palate of freshly-killed pigs was examined using light microscopy, electron microscopy, and three-dimensional reconstruction techniques. RESULTS: Tonsils of the soft palate in pigs are penetrated by numerous crypts which extend into, and branch extensively within, the lymphoid tissue. Stratified squamous non-keratinised epithelium covering the oropharyngeal surface is continuous with that lining the neck of crypts. Lymphoepithelium covers the tonsillar lymphoid tissue within the crypts. It consists of non-keratinised epithelial cells, M cells, goblet cells and many intraepithelial lymphoid cells. M cells have a variable surface morphology: some are covered by relatively regular and well-formed microvilli; others possess very long undulant microvilli emanating from broad membranous folds. CONCLUSIONS: Variations in M cell surface morphology occur and these may reflect alterations of the apical plasmalemma in response to antigenic stimuli. Further investigation will be required to determine molecular specializations of the apical membrane of M cells which may facilitate interactions with antigenic material.

Virus-specific and bystander CD8+ T-cell proliferation in the acute and persistent phases of a gammaherpesvirus infection.

The cycling characteristics of CD8+ T cells specific for two lytic-phase epitopes of murine gammaherpesvirus 68 (gammaHV68) have been analyzed for mice with high or low levels of virus persistence. The extent of cell division is generally reflective of the antigen load and suggests that gammaHV68 may be regularly reactivating from latency for some months after the resolution of the acute phase of the infectious process. Although gammaHV68 infection is also associated with massive proliferation of lymphocytes that are not obviously specific for the virus, the level of "bystander-induced" cycling in a population of influenza virus-specific CD8+ T cells was generally fourfold lower than the extent of cell division seen for the antigen-driven, gammaHV68-specific response. The overall conclusion is that turnover rates substantially in excess of 5 to 10% over 6 days for CD8+ "memory" T-cell populations are likely to be reflective of continued antigenic exposure.

Quantitative analysis of the CD8+ T-cell response to readily eliminated and persistent viruses.

The recent development of techniques for the direct staining of peptide-specific CD8+ T cells has revolutionized the analysis of cell-mediated immunity (CMI) in virus infections. This approach has been used to quantify the acute and long-term consequences of infecting laboratory mice with the readily eliminated influenza A viruses (fluA) and a persistent gammaherpesvirus (gammaHV). It is now, for the first time, possible to work with real numbers in the analysis of CD8+ T CMI, and to define various characteristics of the responding lymphocytes both by direct flow cytometric analysis and by sorting for further in vitro manipulation. Relatively little has yet been done from the latter aspect, though we are rapidly accumulating a mass of numerical data. The acute, antigen-driven phases of the fluA and gammaHV-specific response look rather similar, but CD8+ T-cell numbers are maintained in the long term at a higher 'set point' in the persistent infection. Similarly, these 'memory' T cells continue to divide at a much greater rate in the gammaHV-infected mice. New insights have also been generated on the nature of the recall response following secondary challenge in both experimental systems, and the extent of protection conferred by large numbers of virus-specific CD8+ T cells has been determined. However, there are still many parameters that have received little attention, partly because they are difficult to measure. These include the rate of antigen-specific CD8+ T-cell loss, the extent of the lymphocyte 'diaspora' to other tissues, and the diversity of functional characteristics, turnover rates, clonal life spans and recirculation profiles. The basic question for immunologists remains how we reconcile the extraordinary plasticity of the immune system with the mechanisms that maintain a stable milieu interieur. This new capacity to quantify CD8+ T-cell responses in readily manipulated mouse models has obvious potential for illuminating homeostatic control, particularly if the experimental approaches to the problem are designed in the context of appropriate predictive models.

Contemporary analysis of MHC-related immunodominance hierarchies in the CD8+ T cell response to influenza A viruses.

Early studies of influenza virus-specific CD8+ T cell-mediated immunity indicated that the level of CTL activity associated with H2Db is greatly diminished in mice that also express H2Kk. Such MHC-related immunodominance hierarchies are of some interest, as they could lead to variable outcomes for peptide-based vaccination protocols in human populations. The influence of H2Kk on the H2Db-restricted response profile has thus been looked at again using a contemporary, quantitative, IFN-gamma-based flow cytometric assay. The depressive effect of H2Kk was very apparent for the influenza DbPA224 epitope and was also reproduced when CTL activity was measured for H2Db-expressing targets pulsed with the immunodominant NP366 peptide. The secondary CD8+IFN-gamma+ DbNP366-specific response was much greater in parental H2b than in H2kxbF1 mice, but the sizes of the CD8+ sets specific for KkNP50 and DbNP366 were essentially equivalent in the F1 animals. Thus, although the immunodominance profile associated with DbNP366 is lost when H2Kk is also present, the response is still substantial. A further, MHC-related effect was also identified for the KkNS1152 epitope, which was consistently associated with a greater CD8+IFN-gamma+ response in H2KkDb recombinant than in (H2KkDk x H2KbDb)F1 mice. The diminished DbPA224 response in H2kxbF1 mice was characterized by loss of a prominent Vbeta7 TCR responder phenotype, supporting the idea that TCR deletion during ontogeny shapes the available repertoire. The overall conclusion is that these MHC-related immunodominance hierarchies are more subtle than the early CTL assays suggested and, although inherently unpredictable, are unlikely to cause a problem for peptide-based vaccine strategies.

Characteristics of virus-specific CD8(+) T cells in the liver during the control and resolution phases of influenza pneumonia.

Dissection of the primary and secondary response to an influenza A virus established that the liver contains a substantial population of CD8(+) T cells specific for the immunodominant epitope formed by H-2Db and the influenza virus nucleoprotein peptide fragment NP366-374 (DbNP366). The numbers of CD8(+) DbNP366(+) cells in the liver reflected the magnitude of the inflammatory process in the pneumonic lung, though replication of this influenza virus is limited to the respiratory tract. Analysis of surface phenotypes indicated that the liver CD8(+) DbNP366(+) cells tended to be more "activated" than the set recovered from lymphoid tissue but generally less so than those from the lung. The distinguishing characteristic of the lymphocytes from the liver was that the prevalence of the CD8(+) DbNP366(+) set was always much higher than the percentage of CD8(+) T cells that could be induced to synthesize interferon gamma after short-term, in vitro stimulation with the NP366-374 peptide, whereas these values were generally comparable for virus-specific CD8(+) T cells recovered from other tissue sites. Also, the numbers of apoptotic CD8(+) T cells were higher in the liver. The results overall are consistent with the idea that antigen-specific CD8(+) T cells are destroyed in the liver during the control and resolution phases of this viral infection, though this destruction is not necessarily an immediate process.

Diversity of epitope and cytokine profiles for primary and secondary influenza a virus-specific CD8+ T cell responses.

Screening with the flow cytometric IFN-gamma assay has led to the identification of a new immunogenic peptide (SSYRRPVGI) [corrected] from the influenza PB1 polymerase (PB1(703--711)) and a mimotope (ISPLMVAYM) from the PB2 polymerase (PB2(198--206)). CD8(+) T cells specific for K(b)PB1(703) make both IFN-gamma and TNF-alpha following stimulation with both peptides. The CD8(+) K(b)PB1(703)(+) population kills PB2(198)-pulsed targets, but cell lines stimulated with PB2(198) neither bind the K(b)PB1(703) tetramer nor become CTL. This CD8(+)K(b)PB1(703)(+) population is prominent in the primary response to an H3N2 virus, although it is much less obvious following secondary challenge of H1N1-primed mice. Even so, we can now account for >40% of the CD8(+) T cells in a primary influenza pneumonia and >85% of those present after H3N2 --> H1N1 challenge. Profiles of IFN-gamma and TNF-alpha staining following in vitro stimulation have been traced for the four most prominent influenza peptides through primary and secondary responses into long-term memory. The D(b)NP(366) epitope that is immunodominant after the H3N2 --> H1N1 challenge shows the lowest frequencies of CD8(+) IFN-gamma(+)TNF-alpha(+) cells for >6 wk, and the intensity of IFN-gamma staining is also low for the first 3 wk. By 11 wk, however, the IFN-gamma/TNF-alpha profiles look to be similar for all four epitopes. At least by the criterion of cytokine production, there is considerable epitope-related functional diversity in the influenza virus-specific CD8(+) T cell response. The results for the K(b)PB1(703) epitope and the PB2(198) mimotope also provide a cautionary tale for those using the cytokine staining approach to identity antigenic peptides.

A previously unrecognized H-2D(b)-restricted peptide prominent in the primary influenza A virus-specific CD8(+) T-cell response is much less apparent following secondary challenge.

Respiratory challenge of H-2(b) mice with an H3N2 influenza A virus causes an acute, transient pneumonitis characterized by the massive infiltration of CD8(+) T lymphocytes. The inflammatory process monitored by quantitative analysis of lymphocyte populations recovered by bronchoalveolar lavage is greatly enhanced by prior exposure to an H1N1 virus, with the recall of cross-reactive CD8(+)-T-cell memory leading to more rapid clearance of the infection from the lungs. The predominant epitope recognized by the influenza virus-specific CD8(+) set has long been thought to be a nucleoprotein (NP(366-374)) presented by H-2D(b) (D(b)NP(366)). This continues to be true for the secondary H3N2-->H1N1 challenge but can no longer be considered the case for the primary response to either virus. Quantitative analysis based on intracellular staining for gamma interferon has shown that the polymerase 2 protein (PA(224-233)) provides a previously undetected epitope (D(b)PA(224)) that is at least as prominent as D(b)NP(366) during the first 10 days following primary exposure to either the H3N2 or H1N1 virus. The response to D(b)NP(366) seems to continue for longer, even when infectious virus can no longer be detected, but there is no obvious difference in the prevalence of memory T cells specific for D(b)NP(366) and D(b)PA(224). The generalization that the magnitude of the functional memory T-cell pool is a direct consequence of the clonal burst size during the primary response may no longer be useful. Previous CD8(+)-T-cell immunodominance heirarchies defined largely by cytotoxic T-lymphocyte assays may need to be revised.