Combination gene therapy with CD86 and the MHC class II transactivator in the control of lung tumor growth.

Early reports suggest that the costimulatory molecule CD86 (B7-2) has sporadic efficacy in tumor immunity, whereas changes in cancer immunity mediated by the MHC class II transactivator (CIITA) have not been extensively investigated. CIITA activates MHC class II expression in most cells; however, in the Line 1 lung carcinoma model system, CIITA activates MHC class I and well as class II. Here we show that CD86 is very effective in inducing a primary immune response against Line 1. Tumor cells expressing CD86 grew in only 50% of the mice injected with live cells, and those mice that developed tumors did so with significantly delayed kinetics. Furthermore, irradiated CD86-expressing Line 1 cells served as an effective tumor vaccine, demonstrating that CD86 is effective in inducing tumor immunity in the Line 1 system. These data suggest that if CIITA and CD86 cooperate, enhanced tumor immunity could be achieved. CIITA alone was mildly beneficial in slowing primary tumor growth but only when expressed at low levels. Clones expressing high levels of class II MHC grew as fast as or faster than parental tumor, and CIITA expression in a tumor vaccine assay lacked efficacy. When CIITA and CD86 were coexpressed, there was no cooperative immune protection from tumor growth. Cells that coexpress both genes also failed as a cancer vaccine, suggesting a negative role for CIITA in this lung carcinoma. These data suggest that human cancer vaccine trials utilizing CIITA gene therapy alone or in combination with CD86 should be approached with caution.     

Enhancement of DNA vaccine potency through coadministration of CIITA DNA with DNA vaccines via gene gun

Administration of DNA vaccines via gene gun has emerged as an important form of Ag-specific immunotherapy. The MHC CIITA is a master regulator of MHC class II expression and also induces expression of class I molecules. We reasoned that the gene gun administration of CIITA DNA with DNA vaccines employing different strategies to improve MHC I and II processing could enhance DNA vaccine potency. We observed that DC-1 cells transfected with CIITA DNA lead to higher expression of MHC I and II molecules, leading to enhanced Ag presentation through the MHC I/II pathways. Furthermore, our data suggested that coadministration of DNA-encoding calreticulin (CRT) linked to human papillomavirus (HPV) 16 E6 Ag (CRT/E6) with CIITA DNA leads to enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. In addition, coadministration of the combination of CRT/E6 DNA with CIITA DNA and DNA encoding the invariant chain (Ii) linked to the pan HLA-DR-reactive epitope (Ii-PADRE) further enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. Treatment with the combination vaccine was also shown to enhance the antitumor effects and to prolong survival in TC-1 tumor-bearing mice. Vaccination with the combination vaccine also led to enhanced E6-specific CD8(+) memory T cells and to long-term protection against TC-1 tumors and prolonged survival in vaccinated mice. Thus, our findings suggest that the combination of CIITA DNA with CRT/E6 and Ii-PADRE DNA vaccines represents a potentially effective means to combat tumors in the clinical setting.     

LILRA2 activation inhibits dendritic cell differentiation and antigen presentation to T cells

The differentiation of monocytes into dendritic cells (DC) is a key mechanism by which the innate immune system instructs the adaptive T cell response. In this study, we investigated whether leukocyte Ig-like receptor A2 (LILRA2) regulates DC differentiation by using leprosy as a model. LILRA2 protein expression was increased in the lesions of the progressive, lepromatous form vs the self-limited, tuberculoid form of leprosy. Double immunolabeling revealed LILRA2 expression on CD14+, CD68+ monocytes/macrophages. Activation of LILRA2 on peripheral blood monocytes impaired GM-CSF induced differentiation into immature DC, as evidenced by reduced expression of DC markers (MHC class II, CD1b, CD40, and CD206), but not macrophage markers (CD209 and CD14). Furthermore, LILRA2 activation abrogated Ag presentation to both CD1b- and MHC class II-restricted, Mycobacterium leprae-reactive T cells derived from leprosy patients, while cytokine profiles of LILRA2-activated monocytes demonstrated an increase in TNF-alpha, IL-6, IL-8, IL-12, and IL-10, but little effect on TGF-beta. Therefore, LILRA2 activation, by altering GM-CSF-induced monocyte differentiation into immature DC, provides a mechanism for down-regulating the ability of the innate immune system to activate the adaptive T cell response while promoting an inflammatory response.     

Patterns of constitutive and IFN-γ inducible expression of HLA class II molecules in human melanoma cell lines

Major histocompatibility complex (MHC) class II proteins (HLA-DR, HLA-DP and HLA-DQ) play a fundamental role in the regulation of the immune response. The level of expression of human leukocyte antigen (HLA) class II antigens is regulated by interferon-gamma (IFN-gamma) and depends on the status of class II trans-activator protein (CIITA), a co-activator of the MHC class II gene promoter. In this study, we measured levels of constitutive and IFN-gamma-induced expression of MHC class II molecules, analysed the expression of CIITA and investigated the association between MHC class II transactivator polymorphism and expression of different MHC class II molecules in a large panel of melanoma cell lines obtained from the European Searchable Tumour Cell Line Database. Many cell lines showed no constitutive expression of HLA-DP, HLA-DQ and HLA-DR and no IFN-gamma-induced increase in HLA class II surface expression. However, in some cases, IFN-gamma treatment led to enhanced surface expression of HLA-DP and HLA-DR. HLA-DQ was less frequently expressed under basal conditions and was less frequently induced by IFN-gamma. In these melanoma cell lines, constitutive surface expression of HLA-DR and HLA-DP was higher than that of HLA-DQ. In addition, high constitutive level of cell surface expression of HLA-DR was correlated with lower inducibility of this expression by IFN-gamma. Finally, substitution A-->G in the 5' flanking region of CIITA promoter type III was associated with higher expression of constitutive HLA-DR (p<0.005). This study yielded a panel of melanoma cell lines with different patterns of constitutive and IFN-gamma-induced expression of HLA class II that can be used in future studies of the mechanisms of regulation of HLA class II expression.     

MHC II类分子表达调控的研究进展

MHCII类分子提呈经过加工的抗原给CD4 T淋巴细胞 ,在诱发免疫反应中起重要作用。MHCII类分子不正常表达会引起严重的免疫缺陷疾病 ,如裸淋巴细胞综合征 (BLS)等。目前已识别出四种不同的MHCII调控基因。这些基因分别编码RFXANK、RFX5、RFXAP和CIITA。其中 ,前三个是RFX复合物的亚基 ,RFX是一种结合于所有MHCII类基因启动子上的泛式表达的因子。CIITA是MHCII类分子表达的主要调控因子 ,其严密调控的表达模式决定了MHCII类分子表达的细胞特异性 ,及能否被诱导且在何种水平上表达。本文着重介绍近年来国内外对MHCII类分子表达及其调控研究的新进展     

An MHC class Ib-restricted CD8+ T cell response to lymphocytic choriomeningitis virus

Conventional MHC class Ia-restricted CD8(+) T cells play a dominant role in the host response to virus infections, but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, K(b-/-)D(b-/-)CIITA(-/-) mice lacking expression of MHC class Ia and class II molecules were infected with lymphocytic choriomeningitis virus (LCMV). These animals have a large class Ib-selected CD8(+) T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared with K(b-/-)D(b-/-)β(2)-microglobulin(-/-) mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8(+) T cells and induction of granzyme B and IFN-γ effector molecules in CD8(+) T cells. Partial virus clearance was dependent on CD8(+) cells. In vitro T cell restimulation assays demonstrated induction of a population of β(2)-microglobulin-dependent, MHC class Ib-restricted CD8(+) T cells with specificity for viral Ags and yet to be defined nonclassical MHC molecules. MHC class Ib-restricted CD8(+) T cell responses were also observed after infection of K(b-/-)D(b-/-)mice despite the low number of CD8(+) T cells in these animals. Long-term infection studies demonstrated chronic infection and gradual depletion of CD8(+) T cells in K(b-/-)D(b-/-)CIITA(-/-) mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8(+) T cells have the potential to participate in the host immune response to LCMV.     

Butyrate inhibits functional differentiation of human monocyte-derived dendritic cells

Dendritic cells (DCs) play a key role in immune function through antigen presentation by MHC and CD1, as well as cytokine production that shapes the immune response. Here we report that butyrate, a histone deacetylase inhibitor, inhibits the functional differentiation of human monocyte-derived DCs. Mature DCs were generated from monocytes in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2 day LPS stimulation. Butyrate treatment throughout the culture period inhibited the expression of CD1 molecules, but not on CD83, CD86, and MHC molecules. The suppression was exerted at protein and mRNA levels. Butyrate-treated immature DCs also showed decreased expression of CD1 molecules. Moreover the butyrate-treated immature DCs showed lower production of IL-12 p40 and IL-6 in response to lipopolysaccharides and induced less Th1 cells in allogenic mixed lymphocyte reactions. Our results imply that histone acetylation is involved in regulating immune responses through regulating functional differentiation of DC. Thus HDAC may be one of the targets for controlling the immune response.     

Aberrant expression of Fas ligand in mice deficient for the MHC class II transactivator

The MHC class II transactivator (CIITA) is a critical regulator of MHC class II genes and other genes involved in the Ag presentation pathway. CIITA-deficient mice lack MHC class II expression on almost all APCs. In this study, we show that these mice also have aberrant Fas ligand expression on both CD4 T cells and B cells. We found that Fas ligand expression was greatly increased on CIITA-deficient CD4 T cells during the Th1 differentiation process. However, both CIITA-deficient and control Th1 effector cells up-regulated Fas ligand to similar levels if cells were reactivated. The introduction of CIITA into primary CD4 T cells via retroviral infection resulted in a reduction in the level of Fas ligand and delay in apoptosis after activation. Interestingly, activated B cells from the CIITA-deficient mice also showed increased levels of Fas ligand that could be to some degree inhibited by the introduction of IL-4.     

Infection of APC by human cytomegalovirus controlled through recognition of endogenous nuclear immediate early protein 1 by specific CD4(+) T lymphocytes

Infections by human CMV are controlled by cellular immune responses. Professional APC such as monocytes and macrophages can be infected in vivo and are considered as a reservoir of virus. However, CMV-specific CD4(+) responses against infected APC have not been reported. To develop a model of CD4-infected APC interaction, we have transfected the U373MG astrocytoma cell line with the class II transactivator (CIITA). Confocal microscopy experiments showed that U373MG-CIITA cells expressed markers characteristic of APC. Functional assays demonstrated that infected U373MG-CIITA APC processed and presented both exogenous and endogenously neosynthesized nuclear immediate early (IE) protein 1 through the MHC class II pathway. More importantly, endogenous presentation of IE1 by infected APC lead to efficient control of CMV infection as revealed by decreased viral titer. Thus, these results describe the endogenous presentation of a nuclear viral protein by the MHC class II pathway and suggest that IE1-specific CD4(+) T cells may play an important role in CMV infection by directly acting against infected APC.     

Functional and phenotypic analysis of thymic CD34+CD1a- progenitor-derived dendritic cells: predominance of CD1a+ differentiation pathway

Whether thymic dendritic cells (DC) are phenotypically and functionally distinct from the monocyte lineage DC is an important question. Human thymic progenitors differentiate into T, NK, and DC. The latter induce clonal deletion of autoreactive thymocytes and therefore might be different from their monocyte-derived counterparts. The cytokines needed for the differentiation of DC from thymic progenitors were also questioned, particularly the need for GM-CSF. We show that various cytokine combinations with or without GM-CSF generated DC from CD34+CD1a- but not from CD34+CD1a+ thymocytes. CD34+ thymic cells generated far fewer DC than their counterparts from the cord blood. The requirement for IL-7 was strict whereas GM-CSF was dispensable but nonetheless improved the yield of DC. CD14+ monocytic intermediates were not detected in these cultures unless macrophage-CSF (M-CSF) was added. Cultures in M-CSF generated CD14-CD1a+ DC precursors but also CD14+CD1a- cells. When sorted and recultured in GM-CSF, CD14+ cells down-regulated CD14 and up-regulated CD1a. TNF-alpha accelerated the differentiation of progenitors into DC and augmented MHC class II transport to the membrane, resulting in improved capacity to induce MLR. The trafficking of MHC class II molecules was studied by metabolic labeling and immunoprecipitation. MHC class II molecules were transported to the membrane in association with invariant chain isoforms in CD14+ (monocyte)-derived and in CD1a+ thymic-derived DC but not in monocytes. Thus, thymic progenitors can differentiate into DC along a preferential CD1a+ pathway but have conserved a CD14+ maturation capacity under M-CSF. Finally, CD1a+-derived thymic DC and monocyte-derived DC share very close Ag-processing machinery.