Heterogeneity of CD4(+) and CD8(+) T cells

There is extensive plasticity in the T-cell response to antigen. Helper CD4(+) T cells, cytotoxic CD8(+) T cells, the progression from na?ve to effector and memory T cells, and differentiation into Th1, Tc1, Th2 and Tc2 subsets have long been recognized. More recently it has become apparent that T-cell populations display additional diversity in terms of phenotype, anatomical distribution and effector function.     

Functions of tetramer-stained HIV-specific CD4(+) and CD8(+) T cells

Significant insight has been gained into constraints on the sensitivity and specificity of staining with class I tetramers. The function of the populations that are defined varies with the clinical situation. Insight has also been gained into the determinants of the CD8(+) T cell response during primary and chronic HIV infection. Human class II tetramers have been synthesised but their role in defining CD4(+) T cell function in HIV infection remains to be determined.     

Oral and rectal immunization of adult female volunteers with a recombinant attenuated Salmonella typhi vaccine strain.

An attenuated strain of Salmonella typhi delta(cya) delta(crp-cdt) delta(asd) expressing a gene encoding a hepatitis B virus core-pre-S protein was tested in female adult volunteers for its ability to elicit a systemic and a mucosal immune response. Specifically, our purpose was to evaluate the potential of such a vaccine strain to induce specific secretory immunoglobulin A (sIgA) at genital and rectal surfaces. Oral and rectal routes of immunization were compared: oral immunization induced seroconversion against the bacterial lipopolysaccharide (LPS) in six out of seven volunteers, while after rectal immunization only one out of six volunteers seroconverted against LPS. To our disappointment, the latter volunteer was also the only one who seroconverted against the carried antigen (pre-S1), demonstrating the poor ability of this live vaccine to induce an immune response against the carried antigen. Anti-LPS sIgA was found in both the vaginal and cervical secretions of a volunteer who presented a strong seroconversion after oral immunization (16-fold increase in anti-LPS IgG). Smaller amounts of anti-LPS sIgA were found in the rectal secretions of one orally and one rectally immunized volunteer and in the saliva of three orally and one rectally immunized woman. Our data show for the first time that it is possible to induce specific sIgA in the genital and rectal tracts of women by using an S. typhi vaccine strain.     

A synthetic malaria vaccine elicits a potent CD8(+) and CD4(+) T lymphocyte immune response in humans. Implications for vaccination strategies.

We report the first synthetic peptide vaccine eliciting strong CD8(+) and CD4(+) T lymphocyte responses in humans. The vaccine, representing the C-terminal region of the circumsporozoite protein of Plasmodium falciparum (amino acids 282-383) was well tolerated and strong sporozoite-specific antibodies were elicited. In addition, robust lymphocyte proliferation responses were equally elicited with concomitant in vitro production of IFN-gamma, crucial in the elimination of the parasite. Most importantly, we also observed the development of CD8(+) T lymphocyte responses decisive in the immunity to malaria. The latter finding opens new, possibly safer, avenues for vaccination strategies when a CD8(+) T cell response is needed.     

H2-M3 major histocompatibility complex class Ib-restricted CD8 T cells induced by Salmonella enterica serovar Typhimurium infection recognize proteins released by Salmonella serovar Typhimurium

Salmonella enterica serovar Typhimurium causes a typhoid-like disease in mice which has been studied extensively as a model for typhoid fever in humans. CD8 T cells contribute to protection against S. enterica serovar Typhimurium in mice, but little is known about the specificity and major histocompatibility complex (MHC) restriction of the response. We report here that CD8 T-cell lines derived from S. enterica serovar Typhimurium-infected BALB/c mice lysed bone marrow macrophages infected with S. enterica serovar Typhimurium or pulsed with proteins from S. enterica serovar Typhimurium culture supernatants. Cytoxicity was beta-2-microglobulin dependent and largely TAP dependent, although not MHC class Ia restricted, as target cells of several different MHC haplotypes were lysed. The data suggested the participation of class Ib MHC molecules although no evidence for the presence of Qa1-restricted T cells could be found, unlike in previous reports. Instead, the T-cell lines lysed H2-M3-transfected fibroblasts infected with S. enterica serovar Typhimurium SL3261 or treated with Salmonella culture supernatants. Thus, this report increases the number of MHC class Ib antigen-presenting molecules known for Salmonella antigens to three: Qa-1, HLA-E, and now H2-M3. It also expands the range of pathogens that induce H2-M3-restricted CD8 T cells to include an example of gram-negative bacteria.     

Total glucosides of paeony induces regulatory CD4(+)CD25(+) T cells by increasing Foxp3 demethylation in lupus CD4(+) T cells

Total glucosides of paeony (TGP), an active compound extracted from Paeony root, has been used in therapy for autoimmune diseases. However the molecular mechanism of TGP in the prevention of autoimmune response remains unclear. In this study, we found that TGP treatment significantly increased the percentage and number of Treg cells in lupus CD4(+) T cells. Further investigation revealed that treatment with TGP increased the expression of Foxp3 in lupus CD4(+) T cells by down-regulating Foxp3 promoter methylation levels. However, we couldn't observe similar results in healthy control CD4(+) T cells treated by TGP. Moreover, our results also showed that IFN-γ and IL-2 expression was enhanced in TGP-treated lupus CD4(+) T cells. These findings indicate that TGP inhibits autoimmunity in SLE patients possibly by inducing Treg cell differentiation, which may in turn be due to its ability to regulate the methylation status of the Foxp3 promoter and activate IFN-γ and IL-2 signaling.     

Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity

Several studies have indicated that CD8(+) T cells require CD4(+) T cell help for memory formation. Evidence suggests that such help can be antigen independent, challenging whether the 'licensing' of dendritic cells (DCs) by CD4(+) T cells is ever required for cytotoxic T lymphocyte (CTL) responses. We show here that help is essential for the generation of CTL immunity to herpes simplex virus 1 and that CD4(+) T cells mediate help in a cognate, antigen-specific way. We provide direct in vivo evidence for DC licensing by helper T cells and show that licensing is rapid and essential for the formation of effector and memory CTLs. In situations in which DCs are poorly licensed by pathogen-derived signals, our findings suggest that CTL immunity may be heavily dependent on cognate DC licensing.     

Oral immunization with ATP-dependent protease-deficient mutants protects mice against subsequent oral challenge with virulent Salmonella enterica serovar typhimurium

We evaluated the efficacy of mutants with a deletion of the stress response protease gene as candidates for live oral vaccine strains against Salmonella infection through infection studies with mice by using a Salmonella enterica serovar Typhimurium mutant with a disruption of the ClpXP or Lon protease. In vitro, the ClpXP protease regulates flagellum synthesis and the ClpXP-deficient mutant strain exhibits hyperflagellated bacterial cells (T. Tomoyasu et al., J. Bacteriol. 184:645-653, 2002). On the other hand, the Lon protease negatively regulates the efficacy of invading epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). When 5-week-old BALB/c mice were orally administered 5 x 10(8) CFU of the ClpXP- or Lon-deficient strain, bacteria were detected with 10(3) to 10(4) CFU in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum 1 week after inoculation and the bacteria then decreased gradually in each tissue. Significant increases of lipopolysaccharide-specific immunoglobulin G (IgG) and secretory IgA were detected at week 4 and maintained until at least week 12 after inoculation in serum and bile, respectively. Immunization with the ClpXP- or Lon-deficient strain protected mice against oral challenge with the serovar Typhimurium virulent strain. Both the challenged virulent and immunized avirulent salmonellae were completely cleared from the spleen, mesenteric lymph nodes, Peyer's patches, and even cecum 5 days after the challenge. These data indicate that Salmonella with a disruption of the ATP-dependent protease ClpXP or Lon can be useful in developing a live vaccine strain.     

Peripheral blood extrathymic CD4(+)CD8(+) T cells with high cytotoxic activity are from the same lineage as CD4(+)CD8(-) T cells in cynomolgus monkeys

We have previously reported that CD4/CD8 double-positive (DP) T cells with the resting memory phenotype are present in the periphery of healthy cynomolgus monkeys. In the present study, we performed functional studies on the T cells. The expression of CD4 and CD8 on DP, CD4 single-positive (SP) or CD8 SP T cells was stable in cultures with either mitogen or anti-CD3 antibody stimulation. In spite of lacking CD28 expression, DP T cells showed similar proliferative ability and apoptosis sensitivity to CD4 SP and CD8 SP T cells. DP T cells showed both helper and cytotoxic activities. Although the helper activity of DP T cells was lower than that of CD4 SP T cells, cytotoxic activity was comparable to that of CD8 SP T cells. Fresh DP T cells killed target cells mainly by the perforin-granzyme pathway. In addition, fresh DP T cells expressed a high level of mRNA for IFN-gamma and produced a high level of IFN-gamma when they were activated by anti-CD3 antibody ligation. On the other hand, several expanded DP T cell clones shared TCR V(beta) with expanded CD4 SP T cell clones, strongly suggesting that those two corresponding clones with DP and CD4 SP phenotypes might be derived from the same ancestor T cell. These results showed that the DP T cells are a novel T cell subset with functions overlapping with those of CD4 SP and CD8 SP T cells, and that they might play protective and regulatory roles in secondary immune response in cynomolgus monkeys.     

Ciprofloxacin-resistant Salmonella enterica serovar typhi from Kuwait with novel mutations in gyrA and parC genes

Blood isolates of Salmonella enterica serovar Typhi from two recently returned Bangladeshi patients in Kuwait were ciprofloxacin resistant, with ciprofloxacin MICs of 12 mg/liter for both isolates. Both isolates had three novel gyrA mutations (55-Leu→Trp, 87-Asp→Ala, and 106-Gln→Arg) and three novel parC mutations (84-Glu→Lys, 106-Trp→Gly, and 128-Tyr→Asp).     

Naive CD8(+) but not CD4(+) T cells induce maturation of dendritic cells

We have shown previously that the generation of tumor-reactive CD8(+) cytotoxic T lymphocytes require qualitatively different signals from CD4(+) and CD8(+) T cells that most likely are provided to dendritic cells (DCs). This raises the question of whether the two T cell subsets are equally able to deliver the initial activation signal to DCs. Using ovalbumin as a model antigen we show that naive CD4(+) T cells cannot activate immature DCs and do not become activated, even though they recognize antigen on immature DCs. In contrast, naive CD8(+) T cells rapidly activate DCs and subsequently start to proliferate. This suggests that CD8(+) T cells contribute to DC activation prior to CD4(+) T cells and implies that CD8(+) T cells can provide help to CD4(+) T cells.     

Immunization of volunteers with Salmonella enterica serovar Typhi strain Ty21a elicits the oligoclonal expansion of CD8+ T cells with predominant Vbeta repertoires

CD8(+) T cells are likely to play an important role in host defense against Salmonella enterica serovar Typhi by several effector mechanisms, including lysis of infected cells (cytotoxicity) and gamma interferon (IFN-gamma) secretion. In an effort to better understand these responses, we studied the T-cell receptor (TCR) repertoire of serovar Typhi-specific CD8(+) T cells in humans. To this end, we determined the TCR beta chain (Vbeta) usage of CD8(+) T cells from three volunteers orally immunized with Ty21a typhoid vaccine by flow cytometry using a panel of monoclonal antibodies. Although TCR Vbeta usage varied among volunteers, we identified oligoclonal Vbeta subset expansions in individual volunteers (Vbeta 2, 5.1, 8, 17, and 22 in volunteer 1; Vbeta 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and Vbeta 3, 8, 14, and 16 in volunteer 3). These subsets were antigen specific, as shown by cytotoxicity and IFN-gamma secretion assays on Vbeta sorted cells and on T-cell clones derived from these volunteers. Moreover, eight-color flow cytometric analysis showed that these clones exhibited a T effector memory phenotype (i.e., CCR7(-) CD27(-) CD45RO(+) CD62L(-)) and coexpressed gut homing molecules (e.g., high levels of integrin alpha4beta7, intermediate levels of CCR9, and low levels of CD103). In conclusion, our results show that long-term T-cell responses to serovar Typhi in Ty21a vaccinees are oligoclonal, involving multiple TCR Vbeta families. Moreover, these serovar Typhi-specific CD8(+) T cells bearing defined Vbeta specificities are phenotypically and functionally consistent with T effector memory cells with preferential gut homing potential.     

Salmonella enterica serovar Typhi live vector vaccines delivered intranasally elicit regional and systemic specific CD8+ major histocompatibility class I-restricted cytotoxic T lymphocytes

We investigated the ability of live attenuated Salmonella enterica serovar Typhi strains delivered to mice intranasally to induce specific cytotoxic T-lymphocyte (CTL) responses at regional and systemic levels. Mice immunized with two doses (28 days apart) of Salmonella serovar Typhi strain Ty21a, the licensed oral typhoid vaccine, and genetically attenuated mutants CVD 908 (DeltaaroC DeltaaroD), CVD 915 (DeltaguaBA), and CVD 908-htrA (DeltaaroC DeltaaroD DeltahtrA) induced CTL specific for Salmonella serovar Typhi-infected cells in spleens and cervical lymph nodes. CTL were detected in effector T cells that had been expanded in vitro for 7 days in the presence of Salmonella-infected syngeneic splenocytes. A second round of stimulation further enhanced the levels of specific cytotoxicity. CTL activity was observed in sorted alphabeta+ CD8+ T cells, which were remarkably increased after expansion, but not in CD4+ T cells. CTL from both cervical lymph nodes and spleens failed to recognize Salmonella-infected major histocompatibility complex (MHC)-mismatched cells, indicating that the responses were MHC restricted. Studies in which MHC blocking antibodies were used showed that H-2L(d) was the restriction element. This is the first demonstration that Salmonella serovar Typhi vaccines delivered intranasally elicit CD8+ MHC class I-restricted CTL. The results further support the usefulness of the murine intranasal model for evaluating the immunogenicity of typhoid vaccine candidates at the preclinical level.     

Induction of cellular immune response and anti-Salmonella enterica serovar typhi bactericidal antibodies in healthy volunteers by immunization with a vaccine candidate against typhoid fever

Typhoid fever remains a serious public health problem. We have developed a vaccine from Salmonella enterica serovar typhi (S. typhi) outer-membrane proteins (OMPs) known as porins. A single subcutaneous dose of 10 microg of porins induced a five-fold (P = 0.05) seroconversion index consisting of IgM and IgG at 7 and 15 days after vaccination as well as the production of IgG1 and IgG2 isotypes. The porins-based vaccine induced a two-fold increase (P = 0.05) in bactericidal titres in volunteers, whom also developed a T-cell response characterized by the production of interferon-gamma (INF-gamma). Side effects after vaccination were mild and transient. The data showed that our S. typhi porins-based candidate vaccine is safe and immunogenic in healthy humans.     

IL-2 induces in vivo suppression by CD4(+)CD25(+)Foxp3(+) regulatory T cells

Interleukin-2 (IL-2) treatment is currently used to enhance T cell-mediated immune responses against tumors or in viral infections. At the same time, IL-2 is essential for the peripheral homeostasis of CD4(+)CD25(+)Foxp3(+ )regulatory T cells (Treg). In our study, we show that IL-2 is also an important activator of Treg suppressive activity in vivo. IL-2 treatment induces Treg expansion as well as IL-10 production and increases their suppressive potential in vitro. Importantly, in vivo application of IL-2 via gene-gun vaccination using IL-2 encoding DNA plasmids (pIL-2) inhibited naive antigen-specific T cell proliferation as well as a Th1-induced delayed type hypersensitivity response. The suppressive effect can be transferred onto naive animals by Treg from IL-2-treated mice and the suppression depends on the synergistic action of IL-10 and TGF-beta. These data highlight that during therapeutic treatment with IL-2 the concomitant activation of Treg may indeed counteract the intended activation of cellular immunity.     

Differential effect of CD8(+) and CD8(-) dendritic cells in the stimulation of secondary CD4(+) T cells

Dendritic cells (DC), in their role in initiation of the adaptive immune response, have been extensively studied for their capacity to interact and stimulate naive T cells. Subsets of mature murine DC isolated directly from the spleen have been shown to differ in their ability to induce proliferative responses in both primary CD4(+) and primary CD8(+) T cells; the myeloid-related CD8alpha(-) DC induce a more intense or prolonged proliferation of naive T cells than do the lymphoid-related DC bearing CD8alpha despite similar expression of MHC and co-stimulatory molecules. Here we examine the interaction of these DC subpopulations with T cells already in the activated or memory state which are known to have greater sensitivity to antigen stimulation and bear receptors with increased capacity for signal transduction. We show that influenza virus-specific CD4(+) T cell clones and splenic T cells from peptide-primed animals proliferated in response to antigen presented by separated splenic CD8(-) DC. In contrast, these T cells showed only weak, if any, proliferation in response to CD8(+) DC despite observable cluster formation in the cultures. The differential between the two DC types in inducing proliferation was even more pronounced than previously seen with primary T cells and did not reflect differential longevity of the DC in culture, altered response kinetics or deviation from IL-2 to IL-4 induction with CD8(+) DC, but was related to the levels of IL-2 induced. The deficiency in the CD8(+) DC was not overcome by using infectious virus rather than synthetic peptide as the antigen source. These results show that lymphoid-related CD8(+) splenic DC, despite their mature phenotype, fail to provide appropriate signals to secondary CD4(+) T cells to sustain their proliferation.     

Molecular follow-up of Salmonella enterica subsp. enterica serovar Agona infection in cattle and humans

Salmonella enterica subsp. enterica serovar Agona was not frequently encountered in Finland until an increase in rates of isolation among animal and feed was seen in 1994. A small outbreak among cattle farms in the regions of Oulu and Vaasa in northwestern Finland in 1994-1995 included eight farms. After the outbreak, an increase in the number of serovar Agona infections in humans was seen in 1999: the number of annual microbiologically confirmed cases in humans increased from about 10 from 1990 to 1998 to 84 in 1999, including an outbreak in which more than 50 people were infected. To gather epidemiological data on serovar Agona and to trace the origin of the human infections, 110 serovar Agona isolates isolated from animal, feed, and other sources as well as from humans with cases of salmonellosis of domestic and foreign origin, which were recovered from 1984 to 1999, were analyzed for their pulsed-field gel electrophoresis (PFGE), plasmid, and IS200 profiles and antibiograms. Of these typing methods, PFGE with restriction endonucleases XbaI, BlnI, NotI, and SpeI was the most useful. The PFGE profile of the strain causing an outbreak among cattle in Finland in 1994-1995 was not seen previously. The strain with this profile was later only sporadically found in human infections. The profile of the strain causing the human outbreak in 1999 was not found among isolates from cattle or any other sources. Molecular typing was valuable in showing that although the outbreaks in cattle and humans seemed to be related regionally, they were not related otherwise.     

Functional subsets within clonally expanded CD8(+) memory T cells in elderly humans

With advancing age, healthy humans frequently demonstrate large clonal expansions of CD8(+) T cells in the peripheral blood, which persist for long periods of time and appear to be maintained as a population of memory cells. We studied nine large T cell clones in five elderly individuals. We noted that in most cases the expanded clones were dominated by cells that did not express CD28, a pivotal molecule in T cell activation, and these clones proliferated poorly in culture. However, nearly all of the clonal expansions had CD28(+) fractions and some of these cells appeared to lose CD28 gene expression with stimulation in culture. CD28(+) cells demonstrated greater proliferation in both bulk and limiting dilution cultures compared to CD28(-) cells bearing the same TCR, whereas CD28(-) cells showed increased perforin expression. Together, these data suggest that loss of CD28 expression marks functional differentiation to cytotoxic memory cells within these clonal expansions and likely within CD8(+) memory populations in general.     

Peptide immunization in humans: a combined CD8+/CD4+ T cell-targeted vaccine restimulates the memory CD4 T cell response but fails to induce cytotoxic T lymphocytes (CTL)

Immunization with short antigenic peptides represents a potential strategy to induce peptide-specific CTL in vivo. In this study, a synthetic vaccine consisting of an HIV-derived, HLA-A2.1-binding CTL epitope and a tetanus toxin-derived T helper epitope was evaluated for its capacity to induce peptide-specific CTL in humans. Thirteen volunteers were immunized and boosted twice with 100 μg of the CTL epitope plus 300 μg of the T helper peptide (p30). Peripheral blood mononuclear cells (PBMC) were regularly analysed for cytotoxic and proliferative responses before, between and after the immunizations, and the serum was tested for anti-peptide antibodies. No unequivocal induction of HIV peptide-specific CTL in any of the volunteers was observed. However, a wide pattern of mild and transient side reactions was observed, ranging from local redness at the injection site to generalized exanthema, myalgias, arthralgias and fever. The side-effects were related to the T helper epitope, as they were similar to the side-effects experienced after tetanus immunization, correlated to the magnitude of the p30-specific in vitro proliferative response, and occurred only if p30 was co-injected. No antibodies against the HIV-derived peptides nor against p30 were detectable in the serum after repeated immunizations. The data suggest that the CTL peptide, at the concentration used in this study, failed to induce a cytotoxic immune response in vivo, although the T helper peptide seems to be capable of restimulating the specific memory T cells.