H-2 class I mutants utilize novel restriction specificities in the trinitrophenyl-specific cytotoxic T-lymphocyte response

In antigen-specific cytotoxic T-lymphocyte (CTL) responses H-2 class I mutations usually result in a decreased recognition of the antigen in association with the mutant molecule by CTL from the strain of origin. However, the influence of class I mutations on the magnitude and specificity of CTL responses in the mutants has been studied in only a few instances, in which usually a partial or complete loss of responsiveness was found. We now report that class I mutants extensively use gained (novel) CTL restriction sites, generated by the mutations in the CTL response against the hapten trinitrophenyl (TNP), demonstrated both at the population level and in limiting dilution. TNP-specific CTL clones, restricted by mutant-specific determinants, were detected in all mutants. The percentages mutant-specific CTL clones in limiting dilution experiments were 43, 40, 35, and 13 in the Kb mutants bm1, bm8, bm3 and bm5, respectively, and 35 in the Db mutant bm 14. It is concluded that H-2 class I mutations led to changes in the TNP-specific CTL repertoire resulting in gain of CTLs uniquely restricted to the mutant molecule.     

Fine mapping of an H-2Kk restricted cytotoxic T lymphocyte epitope in SV40 T antigen by using in-frame deletion mutants and a synthetic peptide

The CTL response to SV40 in C3H/HeJ mice is directed against the tumor (T) Ag and is H-2Kk restricted. CTL specific for both the amino terminus (residues 1-271) and the carboxyl terminus (residues 512-708) of the T Ag molecule have been detected, and we have previously cloned CTL of both specificities. In this paper we show that the panel of 10 CTL clones specific for the C-terminal region includes clones specific for three different epitopes, termed C1, C2, and C3. Epitopes C1 and C2 are conserved in the T Ag of the related papova viruses BK and SA12, and only epitopes C2 and C3 are present on SV40 transformed targets bearing the Kk mutant Kkml. Epitopes C1 and C2 were mapped to residues 563-576 by using in-frame deletion mutants of SV40 T antigen, and all clones specific for these two epitopes can lyse Kk bearing target cells in the presence of a synthetic peptide comprising residues 559-576. Kk and Kkml differ at residue 152, which is located in the Ag-binding pocket. Because epitopes C1 and C2 can be formed by the same antigenic peptide, but epitope C1 is not present on SV40 transformed Kkml cells, epitopes C1 and C2 must differ in the contribution made by residue 152 of the MHC class I molecule. These data show that CTL epitopes on transformed cells can be made up of Ag fragments, and strengthen the idea that this is a general phenomenon for both class I and class II restricted T cell epitopes.     

Differential recognition of the serologically defined HLA-A2 antigen by allogeneic cytotoxic T cells

A comprehensive analysis of human alloimmune cytotoxic T lymphocytes (CTLs) specific for the HLA-A2 antigen identified 11% of HLA-A2 positive cells as outliers. In total, 11 unrelated serologically indistinguishable, but distinguishable by cell-mediated lympholysis (CML) HLA-A2 positive outlier cells were identified. The outlier cells could be subdivided in two subgroups according to reactivity patterns obtained with CTLs directed against the HLA-A2 antigen of outlier cells and their inhibitory capacity in specific competitive inhibition experiments. Thus, the serologically defined HLA-A2 specificity can be divided into at least three subtypes using CTLs specific for the HLA-A2 antigen. Moreover, CTLs specific for an HLA-A2 subtype could be induced when responder cells expressed a different HLA-A2 subtype antigen. On the basis of several family studies, we conclude that the subtype HLA-A2 antigens are inherited in a codominant way.     

Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants.

Five distinct cytotoxic T-lymphocyte (CTL) recognition sites were identified in the simian virus 40 (SV40) T antigen by using H-2b cells that express the truncated T antigen or antigens carrying internal deletions of various sizes. Four of the CTL recognition determinants, designated sites I, II, III, and V, are H-2Db restricted, while site IV is H-2Kb restricted. The boundaries of CTL recognition sites I, II, and III, clustered in the amino-terminal half of the T antigen, were further defined by use of overlapping synthetic peptides containing amino acid sequences previously determined to be required for recognition by T-antigen site-specific CTL clones by using SV40 deletion mutants. CTL clone Y-1, which recognizes epitope I and whose reactivity is affected by deletion of residues 193 to 211 of the T antigen, responded positively to B6/PY cells preincubated with a synthetic peptide corresponding to T-antigen amino acids 205 to 219. CTL clones Y-2 and Y-3 lysed B6/PY cells preincubated with large-T peptide LT220-233. To distinguish further between epitopes II and III, Y-2 and Y-3 CTL clones were reacted with SV40-transformed cells bearing mutations in the major histocompatibility complex class I antigen. Y-2 CTL clones lysed SV40-transformed H-2Dbm13 cells (bm13SV) which carry several amino acid substitutions in the putative antigen-binding site in the alpha 2 domain of the H-2Db antigen but not bm14SV cells, which contain a single amino acid substitution in the alpha 1 domain. Y-3 CTL clones lysed both mutant transformants. Y-1 and Y-5 CTL clones failed to lyse bm13SV and bm14SV cells; however, these cells could present synthetic peptide LT205-219 to CTL clone Y-1 and peptide SV26(489-503) to CTL clone Y-5, suggesting that the endogenously processed T antigen yields fragments of sizes or sequences different from those of synthetic peptides LT205-219 and SV26(489-503).     

Ubiquitination of CD86 is a key mechanism in regulating antigen presentation by dendritic cells

Dendritic cells (DCs) require costimulatory molecules such as CD86 to efficiently activate T cells for the induction of adaptive immunity. DCs maintain minimal levels of CD86 expression at rest, but upregulate levels upon LPS stimulation. LPS-stimulated DCs produce the immune suppressive cytokine IL-10 that acts in an autocrine manner to regulate CD86 levels. Interestingly, the underlying molecular mechanism behind the tight control of CD86 is not completely understood. In this study, we report that CD86 is ubiquitinated in DCs via MARCH1 E3 ubiquitin ligase and that this ubiquitination plays a key role in CD86 regulation. Ubiquitination at lysine 267 played the most critical role for this regulation. CD86 is ubiquitinated in MARCH1-deficient DCs to a much lesser degree than in wild-type DCs, which also correlated with a significant increase in CD86 expression. Importantly, CD86 is continuously ubiquitinated in DCs following activation by LPS, and this was due to the autocrine IL-10 inhibition of MARCH1 downregulation. Accordingly, DCs lacking MARCH1 and DCs expressing ubiquitination-resistant mutant CD86 both failed to regulate CD86 in response to autocrine IL-10. DCs expressing ubiquitination-resistant mutant CD86 failed to control their T cell-activating abilities at rest as well as in response to autocrine IL-10. These studies suggest that ubiquitination serves as an important mechanism by which DCs control CD86 expression and regulate their Ag-presenting functions.     

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.     

H-2 antigen requirements in the in vitro induction of SV40-specific cytotoxic T lymphocytes

T cytotoxic cells generated to syngeneic SV40 virus transformants lyse only SV40 target cells that are syngeneic at the H-2 locus. In contrast, SV40-specific tumor transplantation immunity shows no requirements for syngeneic H-2. Inoculation of allogeneic or even xenogeneic transformants will confer immunity to a challenge of syngeneic SV40 tumor cells. The experiments described here represent an attempt to reconcile these apparently conflicting observations. In our hands, generation of SV40-specific T cytotoxic cells in vitro requires both in vivo priming and secondary in vitro sensitization. We have found that priming for a secondary syngeneic-restricted response requires only that the cell employed be SV40 transformed. That is, priming may be accomplished with syngeneic, allogeneic, or xenogeneic SV40 transformants. Thus, the apparent lack of H-2 restriction in vivo immunity does not eliminate a role for the H-2-restricted cytotoxic T cell in tumor transplantation immunity.     

An allosteric mechanism controls antigen presentation by the H-2K(b) complex.

The mechanism of assembly/dissociation of a recombinant water-soluble class I major histocompatibility complex (MHC) H-2Kb molecule was studied by a real-time fluorescence resonance energy transfer method. Like the H-2Kd ternary complex [Gakamsky et al. (1996) Biochemistry 35, 14841-14848], the interactions among the heavy chain, beta2-microglobulin (beta2m), and antigenic peptides were found to be controlled by an allosteric mechanism. Association of the heavy chain with beta2m increased peptide binding rate constants by more than 2 orders of magnitude and enhanced affinity of the heavy-chain molecule for peptides. Interaction of peptides with the heavy-chain binding site, in turn, increased markedly the affinity of the heavy chain for beta2m. Binding of peptide variants of the ovalbumin sequence (257-264) to the heavy chain/beta2m heterodimer was found to be a biphasic reaction. The fast phase was a second-order process with nearly the same rate constants as those of binding of peptides derived from the influenza virus nucleoprotein 147-155 to the H-2Kd heavy chain/beta2m heterodimer [(3.0 +/- 1.0) x 10(-6) M-1 s-1 at 37 degrees C]. The slow phase was a result of both the ternary complex assembly from the "free" heavy chain, beta2m, and peptide as well as an intramolecular conformational transition within the heavy chain/beta2m heterodimer to a peptide binding conformation. Biexponential kinetics of peptide or beta2m dissociation from the ternary complex were observed. They suggest that it can exist in two conformations. The rate constants of beta2m dissociation from the H-2Kb ternary complex were, in the limits of experimental accuracy, independent of the structure of the bound peptide, though their affinities differed by an order of magnitude. Dissociation of peptides from the Kb heavy chain was always faster than from the ternary complexes, yet the heavy chain/peptide complexes were considerably more stable compared with their Kd/nucleoprotein peptide counterparts.     

Analysis of the two-signal requirement for precursor cytolytic T lymphocyte activation using H-2Kk in liposomes

Activation of primed pre-cytolytic T lymphocytes (pCTL) requires two signals: recognition of antigen (signal 1) and interaction with a nonspecific helper factor (signal 2). The two signals necessary for generation of a secondary allogeneic CTL response have been analyzed using H-2Kk in liposomes as the stimulating antigen. Use of the liposomes allows the alloantigen to be separated from responder cells after a brief exposure. Thus, the requirements for effective delivery of each signal could be studied independently. A 12-hr exposure of pCTL to alloantigen was sufficient for optimum signal 1 delivery. pCTL recognition of the antigen occurs during this time, and no requirement for adherent cells could be demonstrated. The structure of the antigen-containing liposomes affects the efficiency of pCTL triggering. Factor(s) necessary for signal 2 could be provided by supernatants from mitogen-stimulated lymphocytes. Alternatively, it could be generated with alloantigen, providing that adherent cells were present. Optimum interaction of factor(s) with pCTL, i.e., optimum delivery of signal 2, occurred only if factor(s) was present at 12 to 24 hr after interaction of pCTL with alloantigen. The results suggest that alloantigen recognition triggers pCTL to synthesize and/or express receptors for the factor(s).     

T-lymphocyte heterogeneity in rat: role of adherent T-cell subpopulation in the regulation of cytotoxic T-cell response to alloantigens

Based on the property of adherence to glass bead columns, we have separated two populations of spleen cells from alloantigen-primed rats that interact synergistically in generation of secondary in vitro CML response. Further analysis of these populations reveals that the adherent cell fraction contains a radioresistant (2500 rad) T helper population (Th1) that enhances the CTL response to alloantigens, a macrophage-type suppressor cell (Møs), and a radio-sensitive suppressor component of yet undetermined nature. The nonadherent cell fraction contains precursors of CTLs and a helper T cell (Th2) that helps in the generation of a T-dependent antibody response to SRBC.     

Effect of HIV on antigen presentation by dendritic cells and macrophages

The antigen-presenting function of dendritic cells (DC) and macrophages (MO) following infection with HIV in vitro was examined. Using non-infected cells, DC, but not MO, stimulated primary proliferative responses in allogeneic lymphocytes in the mixed leukocyte reaction. Both DC and MO stimulated secondary responses to influenza virus and to tetanus toxoid in autologous T lymphocytes. After exposure of DC and MO to HIV1 in vitro for 2 days, 27 % of DC but < 1 % MO became infected as assessed by in situ hybridization. DC were blocked in their capacity to stimulate responses to alloantigens or to the recall antigens. By contrast, MO retained the ability to stimulate responses to the recall antigens. Similar effects during in vivo infection would allow activated T-cell clones to respond to antigens presented by MO early in infection. However, any loss of activated T cells might prove cumulative and damaging in the absence of an effective DC recruitment mechanism for resting T cells.     

Generation of cytotoxic T cells specific for minor histocompatibility antigens by cross challenge in vitro with H-2 disparate adherent cells

Although it is well known that an H-2-restricted cytotoxic T cell response to minor histocompatibility antigens (MIHA) can be primed in vivo with H-2 disparate spleen cells, it has not been previously possible to induce cytotoxic T lymphocyte (CTL) precursors (CTLp) in vitro by this type of challenge. In this work, we demonstrate that the inability to cross challenge in vitro is due to the existence of inhibitory effects that can be obviated by cell fractionation, and to insufficient priming in vivo. BALB/c CTLp (H-2d) that have been repeatedly primed in vivo with B10.D2 can be challenged in vitro with C57BL10/J (H-2b) or B10.BR (H-2k)-adherent cells to generate CTL able to lyse B10.D2 (H-2d) target cells. The H-2 restriction properties of the cross-challenged CTL specific for MIHA were analyzed by using the technique of cold target competition. Within the limits of detection in bulk cultures, the entire response appeared to be H-2 unrestricted, whether the cross challenge was with intact C57BL10/J-adherent cells, or with membrane fragments of C57BL10/J presented by BALB/c adherent cells. The frequency of CTLp responsive to cross challenge was analyzed by limiting dilution, with cold target competition at each cell number to establish the restriction properties of the MIHA-specific CTL induced. We were able to detect two subsets of H-2-unrestricted CTLp responsive to intact C57BL10/J-adherent cells; one present at high frequency (1/250 T cells) and subject to suppressive effects at high cell number, and a second present at lower frequency (1/9800 T cells). There appeared to be a relatively infrequent subset of H-2-restricted CTLp as well (1/52,500 T cells). The frequency of CTLp responsive to cross challenge is of comparable magnitude to the frequency of H-2-restricted CTLp responsive to H-2-matched cells bearing MIHA. These observations are discussed in relationship to immunodominance and clonal dominance effects in the response to MIHA.     

Possible mechanisms by which the H-2Kbm3 mutation may decrease cytotoxic T-lymphocyte recognition of vesicular stomatitis virus nucleoprotein antigen.

Spleen cells from C57BL/6 (B6) mice generate a strong in vitro cytotoxic T-lymphocyte (CTL) response specific for vesicular stomatitis virus (VSV). Spleen cells from VSV-primed B6-H-2bm3 (bm3) mice, which have a mutation in H-2Kb, require approximately 10-fold more UV-inactivated VSV to generate in vitro secondary anti-VSV CTL, compared with spleen cells from primed B6 mice. Anti-VSV CTL elicited in both bm3 and B6 mice are primarily specific for the viral nucleocapsid protein (N protein), as demonstrated by using recombinant vaccinia viruses that express the VSV N protein. bm3 CTL were found to exhibit only a very low level of lytic activity when tested against autologous VSV-infected concanavalin A spleen cell blasts as well as several H-2b tumor cell lines. The weak anti-VSV response of bm3 CTL was found to be the result of a combination of inefficient recognition of VSV-infected target cells and decreased elicitation of secondary effector cells. VSV-infected bm3 target cells were not killed as well as B6 targets by either bm3 or B6 effectors. This is because of the inefficient recognition of targets, as demonstrated by the fact that VSV-infected bm3 cells were unable to competitively inhibit the lysis of VSV-infected B6 target cells by either bm3 or B6 effectors. By using cells from recombinant mice, it was shown that the CTL response restricted by H-2Kb was low in the bm3 mice, compared with that of the B6 mice. However, the H-2Db-restricted CTL activity was similarly low in both the B6 and bm3 mice. The possibility that the low response to VSV-infected bm3 cells is caused by differences between the bm3 and B6 cells in expression of either viral antigens or H-2K was investigated by radiolabeling and immunoprecipitation. VSV-infected B6 and bm3 cells were found to express equivalent levels of both viral antigens and H-2K. These results indicate that the bm3 mutation alters a functional site on the H-2Kb molecule that is involved in the recognition of VSV-infected cells. The observation that elicitation of bm3 CTL can occur at high antigen doses further suggests that the bm3 mutation results in a lower affinity of H-2K either for viral antigen or for receptor sites on the CTL.     

Frequency of allogeneic helper T cells responding to whole H-2 differences and to an H-2K difference alone.

Limiting dilution analysis was used to determine the frequency of splenic T cells that are stimulated by alloantigen to give help in a primary antibody response to SRBC. Several haplotype combinations were tested. A semilogarithmic plot of the fraction of nonresponding culture as a function of the number of T cells added to excess B cells gave a straight line intercepting with the origin. Thus a single cell-type was limiting, which was required to help B cells respond to SRBC. The frequency of syngeneic precursors of T helper cells specific for SRBC ranged from 1/10,000 to 1/55,000 with a mean of about 1/20,000. Allohelpers generated by whole H-2 differences gave precursor frequencies that ranged from 1/1000 to 1/7000 with a mean of about 1/2500. Thus allohelpers to whole H-2 differences were approximately 8-fold more frequent than SRBC-specific helpers. When the stimulation was limited to the H-2K difference between the mutant B6.C-H-2ba and wild-type B6, frequencies of from 1/2600 to 1/7900 allohelpers were found with a mean of about 1/5000, approximately half the frequency of allohelpers to whole H-2 differences. Thus some, but probably not all, of the magnitude of allogeneic halp can be attributed to the high frequency of helper T cells that respond to a given alloantigen.     

Regulation of antigen presentation machinery in human dendritic cells by recombinant adenovirus

Recombinant adenoviral vectors (AdV) are potent vehicles for antigen engineering of dendritic cells (DC). DC engineered with AdV to express full length tumor antigens are capable stimulators of antigen-specific polyclonal CD8+ and CD4+ T cells. To determine the impact of AdV on the HLA class I antigen presentation pathway, we investigated the effects of AdV transduction on antigen processing machinery (APM) components in human DC. Interactions among AdV transduction, maturation, APM regulation and T cell activation were investigated. The phenotype and cytokine profile of DC transduced with AdV was intermediate, between immature (iDC) and matured DC (mDC). Statistically significant increases in expression were observed for peptide transporters TAP-1 and TAP-2, and HLA class I peptide-loading chaperone ERp57, as well as co-stimulatory surface molecule CD86 due to AdV transduction. AdV transduction enhanced the expression of APM components and surface markers on mDC, and these changes were further modulated by the timing of DC maturation. Engineering of matured DC to express a tumor-associated antigen stimulated a broader repertoire of CD8+ T cells, capable of recognizing immunodominant and subdominant epitopes. These data identify molecular changes in AdV-transduced DC (AdV/DC) that could influence T cell priming and should be considered in design of cancer vaccines.     

Salmonella infection of bone marrow-derived macrophages and dendritic cells: influence on antigen presentation and initiating an immune response

Traditionally macrophages (MPhi) have been considered to be the key type of antigen presenting cells (APC) to combat bacterial infections by phagocytosing and destroying bacteria and presenting bacteria-derived antigens to T cells. However, data in recent years have demonstrated that dendritic cells (DC), at their immature stage of differentiation, are capable of phagocytosing particulate antigens including bacteria. Thus, DC may also be important APC for initiating an immune response to bacterial infections. Our studies focus on studying how DC and MPhi process antigens derived from bacteria with no known mechanism of phagosomal escape (i.e. Salmonella typhimurium) for T cell stimulation as well as what role these APC types have in Salmonella infection in vivo. Using an in vitro antigen processing and presentation assay with bone marrow-derived (BM) APC showed that, in addition to peritoneal elicited MPhi and BMMPhi, BMDC can phagocytose and process Escherichia coli and S. typhimurium for peptide presentation on major histocompatibility complex (MHC) class I (MHC-I) and class II MHC-II. These studies showed that both elicited peritoneal MPhi and BMMPhi use an alternate MHC-I presentation pathway that does not require the transporter associated with antigen processing (TAP) or the proteasome and involves peptide loading onto a preformed pool of post-Golgi MHC-I molecules. In contrast, DC process E. coli and S. typhimurium for peptide presentation on MHC-I using the cytosolic MHC-I presentation pathway that requires TAP, the proteasome and uses newly synthesized MHC-I molecules. We further investigated the interaction of Salmonella with BMDC and BMMPhi by analyzing surface molecule expression and cytokine secretion following S. typhimurium infection of BMDC and BMMPhi. These data reveal that Salmonella co-incubation with BMDC as well as BMMPhi results in upregulation of MHC-I and MHC-II as well as several co-stimulatory molecules including CD80 and CD86. Salmonella infection of BMDC or BMMPhi also results in secretion of cytokines including IL-6 and IL-12. Finally, injecting mice with BMDC that have been loaded in vitro with S. typhimurium primes na?ve CD4(+) and CD8(+) T cells to Salmonella-encoded antigens. Taken together, our data suggest that DC may be an important type of APC that contributes to the immune response to Salmonella.     

Microbial and T cell-derived stimuli regulate antigen presentation by dendritic cells in vivo

B cells and dendritic cells (DC) internalize and degrade exogenous Ags and present them as peptides bound to MHC class II molecules for scrutiny by CD4(+) T cells. Here we use an Ab specific for a processed form of the model Ag, hen egg lysozyme (HEL), to demonstrate that this protein is not efficiently presented by lymph node DC following s.c. immunization. HEL presentation by the DC can be dramatically enhanced upon coinjection of a microbial adjuvant, which appears to act by enhancing peptide loading onto MHC class II. CD40 cross-linking or the presence of a high frequency of T cells specific for HEL can similarly improve presentation by DC in vivo. For any of these activating stimuli, CD8alpha(+) DC consistently display the highest proportion of HEL-loaded MHC class II molecules. These data indicate that exogenous Ags can be displayed to T cells in lymphoid tissues by a large cohort of resident DC whose presentation is regulated by innate and adaptive stimuli. Our data further reveal the existence of a feedback mechanism that augments Ag presentation during cognate APC-T cell interactions.