Abundance of unconventional CD8(+) natural killer T cells in the large intestine.

Natural killer T (NKT) cells are mainly present in the liver and thymus, and the majority of these T cells express either a CD4(+) or a double-negative (DN) CD4(-)8(-) phenotype. In the present study, we examined whether such NKT cells were present in the intestine. NKT cells were rare in all sites of the small intestine, including an intraepithelial site. However, a considerable number of NKT cells were found at an intraepithelial site in the large intestine. This result was confirmed by both immunofluorescence and immunohistochemistry. In contrast to conventional NKT cells, NKT cells in the large intestine were CD8(+) or DN CD4(-)8(-). In the case of conventional NKT cells, their existence is known to depend on non-classical MHC class I-like antigens (i. e. CD1d) but not on classical MHC class I antigens. However, the NKT cells in the large intestine were independent of the presence of both CD1d and classical MHC class I antigens. These results were obtained using knockout mice lacking the corresponding genes and molecules. NKT cells in the large intestine were mainly alpha betaTCR(+) (> 75 %) but did not use an invariant chain of Valpha14Jalpha281, which is preferentially used by conventional NKT cells. These NKT cells did not bias the TCR-Vbeta usage toward Vbeta8. These findings suggest that the large intestine is a site in which unconventional NKT cells carrying the CD8(+) phenotype (or DN CD4(-)8(-)) are abundant and that these cells are independent of MHC and MHC-like antigens.     

Systematic characterization of human CD8+ T cells with natural killer cell markers in comparison with natural killer cells and normal CD8+ T cells

We investigated the function of CD56+ CD8+ T cells (CD56+ T cells) and CD56- CD57+ CD8+ T cells (CD57+ T cells; natural killer (NK)-type T cells) and compared them with those of normal CD56- CD57- CD8+ T cells (CD8+ T cells) and CD56+ NK cells from healthy volunteers. After the stimulation with immobilized anti-CD3 antibodies, both NK-type T cells produced much larger amounts of interferon-gamma (IFN-gamma) than CD8+ T cells. Both NK-type T cells also acquired a more potent cytotoxicity against NK-sensitive K562 cells than CD8+ T cells while only CD56+ T cells showed a potent cytotoxicity against NK-resistant Raji cells. After the stimulation with a combination of interleukin (IL)-2, IL-12 and IL-15, the IFN-gamma amounts produced were NK cells > or = CD56+ T cells > or = CD57+ T cells > CD8+ T cells. The cytotoxicities against K562 cells were NK cells > CD56+ T cells > or = CD57+ T cells > CD8+ T cells while cytotoxicities against Raji cells were CD56+ T cells > CD57+ T cells > or = CD8+ T cells > or = NK cells. However, the CD3-stimulated proliferation of both NK-type T cells was smaller than that of CD8+ T cells partly because NK-type T cells were susceptible to apoptosis. In addition to NK cells, NK-type T cells but not CD8+ T cells stimulated with cytokines, expressed cytoplasmic perforin and granzyme B. Furthermore, CD3-stimulated IFN-gamma production from peripheral blood mononuclear cells (PBMC) correlated with the proportions of CD57+ T cells in PBMC from donors. Our findings suggest that NK-type T cells play an important role in the T helper 1 responses and the immunological changes associated with ageing.     

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.     

Human CD8(+) T cells expressing HLA-DR and CD28 show telomerase activity and are distinct from cytolytic effector T cells

Cycling lymphocytes may express the enzyme telomerase which is involved in maintenance of telomere length and cell proliferation potential. In CD8(+) T cells freshly isolated from peripheral blood, we found that in vivo cycling cells expressed HLA-DR. Furthermore, CD28-positive cells are known to have longer telomeres than CD28-negative T cells. Therefore we used HLA-DR- and CD28-specific antibodies to sort CD8(+) T cells and measure telomerase activity ex vivo. Relatively high levels of telomerase activity were found in HLA-DR/CD28 double-positive cells. In contrast, HLA-DR-negative and CD28-negative cells had almost no telomerase activity. In summary, HLA-DR expression correlates with proliferation, and CD28 expression with proliferative potential. We have previously identified that ex vivo cytolytic CD8(+) T cells are CD56 (NCAM) positive. Here we show that HLA-DR(+) cells were rarely CD56(+) and vice versa. This demonstrates that telomerase-expressing and cytolytic CD8(+) T cells can be separated on the basis of the cell surface markers HLA-DR and CD56. Thus, activated CD8(+) T cells specialize and exert distinct functions correlating with surface molecule expression.     

Cross-primed CD8(+) T cells mediate graft rejection via a distinct effector pathway

To prevent bystander destruction of healthy host tissues, cytotoxic CD8(+) T lymphocytes are fitted with specific receptors that direct their destructive forces specifically against chosen targets. We show here, however, that anti-H-Y monospecific, H-2(b-restricted MataHari CD8(+) T cells reject H-2(k) male skin grafts, with which they cannot directly interact. Such rejection is interferon-gamma-dependent and only occurs if the recipient endothelium expresses H-2(b). The findings suggest an alternate indirect effector pathway that requires processing and presentation of the donor H-Y antigen by recipient endothelium and have implications for both transplantation and autoimmune disease.     

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.     

In vivo acute depletion of CD8(+) T cells before murine cytomegalovirus infection upregulated innate antiviral activity of natural killer cells

In this study, we investigated the effect of depletion of CD8(+) T cells on the activity of natural killer (NK) cells at an early phase of murine cytomegalovirus (MCMV) infection. For CD8(+) T cell depletion, mice were intraperitoneally treated with anti-CD8 mAb, purified from 2.43 hybridoma, for 2 consecutive days before or after infection. Three days after infection, we found that an acute depletion of CD8(+) T cells before infection caused a significant decrease in the viral load in liver and spleen. This effect coincided with an increase in numbers of CD3(-) NK1.1(+) cells in spleen and their expression of the early activation molecule CD69. Although cytolytic activity of NK cells increased on day 3 of infection in CD8-depleted mice, the level of IFN-gamma decreased in serum and supernatant of cultured spleen cells. In contrast to the effect of acute depletion of CD8(+) T cells before infection, the depletion after infection had no effect on the viral load or number and cytolytic function of NK cells. Lack of effects of CD8(+) T cell depletion on the viral load and NK cytolytic activity is also observed in CD8(+) knockout mice. In conclusion, the results suggest that an acute depletion of CD8(+) T cells before MCMV infection effectively upregulated the antiviral activity of NK cells. This effect appears to be mediated through an increase in numbers, activation and cytolytic activity of NK cells.     

Molecular profiling reveals distinct functional attributes of CD1d-restricted natural killer (NK) T cell subsets

CD1d-restricted natural killer T (NKT) cells can have multiple effects on an immune response, including the activation, regulation and attraction of innate immune cells, and modulation of adaptive immunity. Recent studies reveal that there are distinct subsets of NKT cells which selectively perform some of the functions attributed to CD1d-restricted cells, but the mechanisms underlying these functional differences have not been resolved. Our aim in this study was to identify novel NKT cell associated traits that would provide important insight into NKT cell activation and function. To this end, we have performed gene expression profiling of two separate subsets of NKT cells, analyzing genes differentially expressed in these cells compared to conventional CD4(+)NK1.1(-) T cells. We identify different sets of genes over expressed in each of the two NKT cell types, as well as genes that are common to the two CD1d-restricted NKT cell populations analyzed. A large number of these genes are highly relevant for NKT cell development, activation and function. Each NKT subtype displayed a unique set of chemokine receptors, integrins and molecules related to effector function, supporting the notion that distinct NKT cells can be selectively engaged and have diverse functions in different types of immune reactions.     

Thalidomide and its analogues have distinct and opposing effects on TNF-alpha and TNFR2 during co-stimulation of both CD4(+) and CD8(+) T cells

Thalidomide (Thd) is clinically useful in a number of conditions where its efficacy is probably related to its anti-TNF-alpha activity. More recently, Thd has also been shown to co-stimulate T cells and second generation co-stimulatory (IMiD trade mark ) analogues are currently being assessed in the treatment of cancer patients. However, in contrast to their known suppressive effects during inflammatory stimuli, the effects of Thd/IMiDs on TNF-alpha and TNF receptors (TNFRs) during T cell co-stimulation are not known. We sought to determine the effect of Thd, two clinically relevant IMiDs (CC-4047, ACTIMID trade mark and CC-5013, REVIMID trade mark ) and a non-stimulatory SelCID analogue (CC-3052) on TNF-alpha production and on the expression and shedding of TNFRs during co-stimulation. We found that co-stimulation of PBMC with Thd/IMiDs, but not CC-3052, prevented alphaCD3-induced T cell surface expression of TNFR2 and thereby reduced soluble TNFR2 (sTNFR2) levels. However, there was no effect on total (surface/intracellular) TNFR2 protein expression, suggesting inhibition of trafficking to the cell membrane. The extent of co-stimulation by Thd/IMiDs (assessed by CD69/CD25 expression and IL-2/sIL-2Ralpha production) was similar for CD4+ and CD8+ T lymphocytes and correlated with TNFR2 inhibition. Co-stimulation, but not the early inhibitory effect on TNFR2, was IL-2-dependent and led to increased TNF-alpha production by both CD4+ and CD8+ T lymphocytes. The clinical relevance of this observation was confirmed by the elevation of serum TNF-alpha during REVIMID trade mark treatment of patients with advanced cancer. Together, these results suggest a possible role for TNF-mediated events during co-stimulation and contrast with the TNF inhibitory effects of Thd and its analogues during inflammatory stimuli.     

Functional impairment of CD8(+) T cells by regulatory T cells during persistent retroviral infection

The establishment of viral persistence generally requires evasion of the host CD8(+) T cell response. Here we describe a form of evasion wherein the CD8(+) T cells are fully capable of recognizing their cognate antigen but their effector functions are suppressed by regulatory T cells. Virus-specific CD8(+) T cells adoptively transferred into mice persistently infected with Friend virus proliferated and appeared activated, but failed to produce IFNgamma or reduce virus loads. Cotransfer experiments revealed that a subpopulation of CD4(+) T cells from persistently infected mice suppressed IFNgamma production by the CD8(+) T cells. Treatment of persistently infected mice with anti-GITR antibody to ameliorate suppression by regulatory T cells significantly improved IFNgamma production by transferred CD8(+) T cells and allowed a significant reduction in viral loads. The results indicate that CD4(+) regulatory T cells contribute to viral persistence and demonstrate an immunotherapy for treating chronic retroviral infections.     

Lipid-antigen presentation by CD1d(+) B cells is essential for the maintenance of invariant natural killer T cells

B cells perform many immunological functions, including presenting lipid antigen to CD1d-restricted invariant natural killer T (iNKT) cells, known to contribute to maintaining tolerance in autoimmunity. Patients with systemic lupus erythematous (SLE) display dysregulated B cell responses and reduced peripheral iNKT cell frequencies. The significance of these defects and how they relate to SLE pathogenesis remain elusive. We report that B cells are essential for iNKT cell expansion and activation in healthy donors but fail to exert a similar effect in SLE patients. Defective B cell-mediated stimulation of iNKT cells in SLE patients was associated with altered CD1d recycling, a defect recapitulated in B cells from healthy donors after stimulation with interferon-α (IFN-α) and anti-immunoglobulin (Ig). iNKT cell number and function were restored in SLE patients responding to anti-CD20 treatment upon normalization of CD1d expression exclusively in repopulated immature B cells. We propose that healthy B cells are pivotal for iNKT cell homeostasis.     

Low-dose cyclophosphamide combined with IL-2 inhibits tumor growth by decreasing regulatory T cells and increasing CD8+ T cells and natural killer cells in mice

Interleukin-2 (IL-2) benefits some cancer patients by promoting the proliferation of cytotoxic effector T cells, but this process is limited by the expansion of regulatory T cells (Tregs). Low-dose cyclophosphamide (CTX) can inhibit the number and function of Tregs. We treated carcinoma-bearing mice with Vehicle, CTX, IL-2 and CTX + IL-2 to investigate the effects of low-dose CTX combined with IL-2 in antitumor treatment. In comparison to monotherapy, CTX + IL-2 significantly limited tumor growth, via tumor cell proliferation inhibition and increased apoptosis. The infiltration of CD8⁺ T cells in tumor tissues was significantly increased in the CTX + IL-2 group. CTX + IL-2 safely increased CD8⁺ T and natural killer cells in the spleen, lymph nodes and peripheral blood, and CTX attenuated the increase in Tregs induced by IL-2 in the spleen.     

Role of new population of peripheral CD11c(+)CD8(+) T cells and CD4(+)CD25(+) regulatory T cells during acute and remission stages in rheumatoid arthritis patients.

BACKGROUND AND PURPOSE: Rheumatoid arthritis (RA) is a CD4(+)-dependent chronic systemic inflammatory disease with autoimmune features. Autoreactive CD4(+) T-cell activation can result in autoimmune diseases. One of the key regulators is the CD4(+)CD25(high) regulatory T (Treg) cell. In an animal arthritis model, CD11c(+)CD8(+) T cells were found to be elevated, and could suppress pathogenic CD4(+) T cells after cross-linking with CD137. The purpose of this study was to compare the expression of CD137, CD4(+)CD25(high) Treg cells, and CD11c(+)CD8(+) in the peripheral blood T lymphocytes of RA patients during active and remissive states, and evaluate the correlation with disease activity. METHODS: Thirty nine RA patients treated at the rheumatology outpatient clinic at the Changhua Christian Hospital were assessed clinically for disease activity and classified as either highly active or remissive by the Disease Activity Score 28. Peripheral blood mononuclear cells were isolated from these patients and compared against normal controls. RESULTS: The presence of CD11c(+)CD8(+) T cells or the expression of CD137 molecules in peripheral blood cells was not related to disease activity. In contrast, CD4(+)CD25(high) Treg cell levels were increased significantly in patients with active RA compared with patients with remissive RA or controls (p<0.05). These lymphocytes were intact, without evidence of apoptosis. CONCLUSIONS: Our results indicate that CD4(+)CD25(high) Treg cells play an important role in modulating RA disease activity and can serve as a parameter of disease activity.     

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.     

Proliferative arrest and cell cycle regulation in CD8(+)CD28(-) versus CD8(+)CD28(+) T cells

CD8(+)CD28(-) T cells have been characterized by oligoclonal expansions, impaired proliferative responses, but preserved cytotoxicity and reduced telomeres. To examine this subset further and define the underlying mechanisms of proliferation arrest, we investigated several features of this cell type compared with CD8(+)CD28(+) controls. We analyzed expression of various activation markers, thymidine incorporation upon activation, T-cell receptor (TCR) zeta-chain phosphorylation, cell cycle characteristics, and cell cycle related gene expression. Flow cytometry revealed higher expression of CD11b, CD29, CD57, and CD94, and lower expression of CD25 in CD8(+)CD28(-) compared with CD8(+)CD28(+) T cells. Sorted CD8(+)CD16(-)CD28(-) cells exhibited decreased phosphorylation of the TCR zeta-chain in three of four probands. Proliferation of these T cells was impaired, even when activated with mitogens that bypass TCR signaling. Cell cycle profiles demonstrated a lower percentage of cycling cells and significantly higher levels of cyclin dependent kinase inhibitor p16(INK4a) in the CD28(-) subset compared with the CD28(+) control. These observations suggest that expanded CD8(+)CD28(-) T cells in normal elderly individuals have reduced proliferation concomitant with increased p16(INK4a) expression. Defects in TCR signaling were associated with altered TCR zeta-chain phosphorylation.     

Memory CD8(+) T cells undergo peripheral tolerance

Memory T cells differ from naive T cells in that they respond more rapidly and in greater numbers. In addition, memory T cells are generally believed to be less susceptible to tolerance induction than naive T cells. In this study, we show that this is not the case. Using two different methods of tolerance induction, peptide-induced tolerance and crosstolerance, we present evidence that memory CD8(+) T cells are as susceptible to tolerance as naive cells. These results have a direct impact on manipulating T cell responses to self-antigens in order to improve immunotherapy of cancer and autoimmune diseases.     

Emerging concepts in CD8(+) T regulatory cells

CD8(+) T regulatory cells (T regs) are elicited by unique antigen presenting cells during viral infections, by manipulation of co-stimulatory molecules, or in the development of tumors. CD8(+) T regs display antigen-specificity, which is most exquisitely manifested by the HLA-E-restricted cytolytic CD8(+) T regs in MS. There is evidence that some CD8(+) T regs also express organ specificity. In many cases, IFN-gamma is required for either the induction or expression of CD8(+) T regs. CD8(+) T regs can produce suppression directly by killing immune cells or indirectly by co-opting other cells to elaborate end-stage suppressive molecules such as TGF-beta, IL-10, and indoleamine dioxygenase (IDO).     

Frequency and function of HIV-specific CD8(+) T cells

The virus-specific CD8(+) T cell responses of 27 HIV-infected patients were studied, including a unique cohort of long term nonprogressors (LTNP) with normal CD4(+) T cell counts, low levels of plasma viral RNA, strong proliferative responses to HIV antigens and an over-representation of the HLA B*5701 class I allele. The frequencies of CD8(+) T cells specific to the majority of HIV gene products were measured by flow cytometric detection of intracellular interferon-gamma (IFN-gamma) in response to HIV-vaccinia recombinant infected autologous B cells. Very high frequencies (1.4-22%) of circulating CD8(+) T cells were found to be HIV-specific and were not only found in LTNP with reduced plasma virus. No correlation was evident between the frequency of HIV-specific CD8(+) T cells and levels of plasma viremia. In each case, the vast majority of cells (up to 17.2%) responded to Gag-Pol gene products. Although similar frequencies of Gag peptide-specific CD8(+) T cells were found in LTNP and progressors by either intracellular IFN-gamma or MHC class I tetramer staining, the breadth of these responses was greater in patients with progressive HIV infection compared with the LTNP group. The frequency of CD8(+) T cells specific for a single peptide was not representative of an individual patient's total HIV-specific CD8(+) T cell response. These data demonstrate that high numbers of HIV-specific CD8(+) T cells exist even in patients with high level viremia and progressive disease. Further, they suggest that other qualitative parameters of the CD8(+) T cell response may differentiate some patients with very low levels of plasma virus and nonprogressive infection.     

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.     

Compartmentalized cytotoxic immune response leads to distinct pathogenic roles of natural killer and senescent CD8+ T cells in human cutaneous leishmaniasis

Cytotoxic activity mediated by CD8⁺ T cells is the main signature of the immunopathogenesis of cutaneous leishmaniasis (CL). Here, we performed a broad evaluation of natural killer (NK) cell phenotypic and functional features during cutaneous leishmaniasis. We demonstrate for the first time that CL patients present the accumulation of circulating NK cells with multiple features of replicative senescence including low proliferative capacity and shorter telomeres, elevated expression of CD57, KLRG1 but diminished CD27 stimulatory receptor expression. Moreover, they exhibited higher cytotoxic and inflammatory potential than age-matched controls. The accumulation of circulating senescent NK cells (CD56^dim CD57^bright) correlated positively with skin lesion size in the same patients, suggesting that they, like circulating senescent CD8⁺ T cells, may contribute to the immunopathology of CL. However, this senescent population had lower cutaneous lymphocyte antigen expression and so had diminished skin-homing potential compared with total or senescent CD8⁺ T cells. This was confirmed in CL skin lesions where we found a predominance of CD8⁺ T cells (both senescent and non-senescent) that correlated with the severity of the disease. Although there was also a correlation between the proportions of senescent NK cells (CD56⁺ CD57⁺) in the skin and lesion size, this was less evident. Collectively our results demonstrate first-hand that senescent cytotoxic cells may mediate skin pathology during human cutaneous leishmaniasis. However, as senescent cytotoxic CD8⁺ T cells predominate in the skin lesions, they may have a greater role than NK cells in mediating the non-specific skin damage in CL.