Cross-presentation of tumor antigens by bone marrow-derived antigen-presenting cells is the dominant mechanism in the induction of T-cell tolerance during B-cell lymphoma progression.

Tumor antigen-specific T-cell tolerance may limit the efficacy of therapeutic cancer vaccines. Direct presentation of antigens by tumor cells incapable of providing adequate costimulation to tumor-specific T cells has been suggested as the basis for this unresponsiveness. Using parent-into-F1 bone marrow (BM) chimeras, this study unambiguously demonstrates that the induction of this tolerant state requires T-cell recognition of tumor antigen presented by BM-derived antigen-presenting cells (APCs), not tumor cells themselves. In the absence of host APC presentation, tumor-specific T cells remained functional, even in the setting of antigen expressed by B-cell lymphomas residing in secondary lymphoid tissues. The intrinsic APC capacity of tumor cells has therefore little influence over T-cell priming versus tolerance, a decision that is regulated at the level of host APCs. (Blood. 2001;98:1070-1077)

Enhanced antigen-specific antitumor immunity with altered peptide ligands that stabilize the MHC-peptide-TCR complex.

T cell responsiveness to an epitope is affected both by its affinity for the presenting MHC molecule and the affinity of the MHC-peptide complex for TCR. One limitation of cancer immunotherapy is that natural tumor antigens elicit relatively weak T cell responses, in part because high-affinity T cells are rendered tolerant to these antigens. We report here that amino acid substitutions in a natural MHC class I-restricted tumor antigen that increase the stability of the MHC-peptide-TCR complex are significantly more potent as tumor vaccines. The improved immunity results from enhanced in vivo expansion of T cells specific for the natural tumor epitope. These results indicate peptides that stabilize the MHC-peptide-TCR complex may provide superior antitumor immunity through enhanced stimulation of specific T cells.

Sustaining the graft-versus-tumor effect through posttransplant immunization with granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing tumor vaccines.

For many cancers, autologous bone marrow transplantation (BMT) achieves a minimal residual disease state, yet relapse rates remain high. Using a syngeneic murine bone marrow transplant model, we demonstrate that vaccination with irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing autologous tumor cells is effective in the post-BMT period and actually results in a greater tumor-free survival than vaccination in the nontransplant setting. Employing T cells specific for a model tumor-antigen, we find that transplantation of the tumor-bearing host results in a massive expansion and activation of tumor-specific T cells in the early posttransplant period, but this response rapidly declines in association with tumor progression. Immunization with irradiated GM-CSF tumor cells during the period of immune reconstitution results in the sustained amplification and activation of this response that closely correlates with freedom from relapse. These results demonstrate the feasibility of integrating GM-CSF vaccines in the postautologous BMT setting and suggest mechanisms that may contribute to the observed efficacy of immunization during the critical period of immune reconstitution.

Conversion of tumor-specific CD4+ T-cell tolerance to T-cell priming through in vivo ligation of CD40.

Tumor antigen-specific T-cell tolerance limits the efficacy of therapeutic cancer vaccines. Antigen-presenting cells mediate the induction of T-cell tolerance to self-antigens. We therefore assessed the fate of tumor-specific CD4+ T cells in tumor-bearing recipients after in vivo activation of antigen-presenting cells with antibodies against CD40. Such treatment not only preserved the responsiveness of this population, but resulted in their endogenous activation. Established tumors regressed in vaccinated mice treated with antibody against CD40 at a time when no response was achieved with vaccination alone. These results indicate that modulation of antigen-presenting cells may be a useful strategy for enhancing responsiveness to immunization.

Effects of ultraviolet B radiation on human Langerhans cells: functional alteration of CD86 upregulation and induction of apoptotic cell death.

We have recently reported that in vitro low dose of ultraviolet B radiation (UVB, 100-200 J per m2) directly impaired the antigen-presenting function of human Langerhans cells. In this study, we analyzed the effect of UVB irradiation on the Langerhans cells expression of several accessory molecules, namely CD54, CD80, and CD86. Langerhans cells phenotype was determined either immediately after UVB exposure (100 J per m2) or after a 2 d culture. No modification in cell surface antigen levels was observed immediately after irradiation. Prior UVB exposure did not modify the levels of CD80 at the Langerhans cells surface after a 2 d culture. In contrast, CD54 and, above all, CD86 expression were significantly decreased. Addition of exogenous anti-CD28 monoclonal antibodies partly restored the allostimulatory property of irradiated Langerhans cells in mixed epidermal cell-lymphocyte reaction, demonstrating that impairment of CD86 upregulation contributes to the UVB-induced immunosuppressive effect. Furthermore, we found that UVB irradiation at 200 J per m2 significantly reduced the number of viable Langerhans cells after 2 d of culture. UVB-induced cytotoxicity was due to apoptotic cell death, as demonstrated by typical morphologic alterations and by DNA fragmentation yielding a classical ladder pattern on gel electrophoresis. Interestingly, interaction of Langerhans cells with CD40-ligand transfected L cells improved the viability of irradiated Langerhans cells, counteracted the inhibition of CD86 expression, and efficiently reduced the number of apoptotic cells after a 2 d culture. Collectively, these results demonstrate that in vitro UVB exposure affects Langerhans cells via at least two distinct pathways: (i) decreased CD86 costimulatory molecule upregulation; and (ii) induction of Langerhans cells apoptosis, a phenomenon partly prevented by CD40 triggering.

Effects of ultraviolet B radiation on in vitro hapten T-cell sensitization by human langerhans cells.

The deleterious effects of ultraviolet B radiation (UVB) on the antigen-presenting function of human epidermal Langerhans cells (LC) were studied by using the in vitro primary and secondary T-cell proliferative responses to the trinitrophenyl hapten (TNP) modified autologous LC. Increasing doses of UVB radiation (100-200 J/m(2)) induced a dose dependent inhibition of the primary and secondary TNP-specific T cell response. However, this decreased T-cell proliferative response after UVB radiation, was strongly enhanced when freshly isolated LC, as compared with cultured LC, were used as antigen-presenting cells (APC), suggesting an impaired development of LC accessory function. Moreover, the exogenous addition of IL1beta, TNFalpha, IL10 or their specific monoclonal antibodies neither modified nor reversed the immunosuppressive effect of UVB radiation. Even if the low doses of UVB radiation (100 and 200 J/m(2)) seemed to slightly affect HLA-DR synthesis, the antigen-presenting function of human LC cannot be related to the decreased expression of these molecules but might be associated with an impaired development of accessory molecules such as a downregulation of B7-2 antigen.

Expression and function of B7-1 (CD80) and B7-2 (CD86) on human epidermal Langerhans cells.

In addition to T cell receptor triggering, activation of T cells requires costimulatory signals that have been shown to be mainly initiated through CD28. We analyzed the expression and function of the two ligands for CD28, B7-1 (CD80) and B7-2 (CD86), on human Langerhans cells (LC), the antigen-presenting cells from epidermis. Human LC freshly isolated from epidermis (fLC) expressed significant level of B7-2, which was increased upon a short culture in vitro. In contrast, B7-1 was undetectable on fLC but appeared at the cell surface after a 3-day culture in vitro. Pre-incubation of 18-h cultured LC with anti-B7-2 monoclonal antibodies (mAB) was sufficient to abrogate the binding of CTLA4-Ig fusion protein, while a combination of both mAB against B7-1 and B7-2 was necessary to obtain a complete inhibition of CTLA4-Ig binding on 3-day cultured LC, showing the absence of a third CTLA4 ligand. The function of B7-1 and B7-2 on human LC has been analyzed by adding mAb at the beginning of mixed epidermal cell lymphocyte reactions. Anti-B7-2 mAb and CTLA4-Ig, but not anti-B7-1 mAb, strongly inhibited allogenic. as well as recall antigen-induced T cell proliferation supported by fLC or 3-day cultured LC. Collectively, these results demonstrate that B7-2 is the major ligand for CD28/CTLA4 at the LC surface and that it plays a crucial role in human LC co-stimulatory function with little, if any, dependence of B7-1 expression.

Functional expression of CD40 antigen on human epidermal Langerhans cells.

It is now well established that interactions of CD40 on the B cells, along with its ligand (CD40-L) on the T cells, regulate B cell proliferation and differentiation. However, the functional significance of CD40 expression on cells known for most efficient Ag-presenting function, i.e., dendritic cells, is not so clear. In this study, we demonstrate that CD40 is expressed on human dendritic Langerhans cells (LC) freshly isolated from epidermis. Using CD40-L transfected cells, CD40 triggering was found to enhance LC viability when cultured and to result in phenotypic alterations. Thus, a 2-day CD40 activation induced up-regulation of CD54 and CD86 at the LC surface, while it did not significantly affect the levels of HLA-DR, CD1a, CD58, and CD80 expression. These phenotypic changes correlate with enhanced LC allostimulatory property, as shown by the use of paraformaldehyde-fixed LC. Furthermore, mAbs against CD40, as well as CD40-L, strongly inhibit the primary T cell response to allogeneic LC. Collectively, these data support a role for CD40/CD40-L pair in the development of normal T cell functions.

cis-urocanic acid failed to affect in vitro human Langerhans cell allostimulatory function.

Urocanic acid (UCA) represents the major ultraviolet B (UVB, 290-320 nm)-absorbing component of the skin. Trans-UCA is naturally produced in the stratum corneum and converts to the cis isomer upon UVB irradiation. In this study, we examined the effect of purified cis-UCA (about 99% of cis isomer) on the human Langerhans cell (LC) allostimulatory function by using the mixed epidermal cell-lymphocyte reaction (MELR). We found that addition of increasing amounts (6.5-400 micrograms/mL) of purified cis-UCA or trans-UCA did not modify the T-cell response supported by enriched LC (eLC: 8-25% LC) as well as purified LC (pLC: 70-90% LC) suspensions. Because cis-UCA had no effect on the allostimulatory function of untreated LC, we investigated whether this compound could modify T-cell proliferation induced by UVB-irradiated LC. The UVB exposure of eLC or pLC to 100 J/m2 significantly inhibited the capacity of both suspensions to mount a T-cell response. However, addition of cis-UCA did not potentiate this UVB-induced immunosuppression. The eLC or pLC were then incubated with cis-UCA for 18 h at 37 degrees C and washed before adding to allogeneic T cells. The obtained proliferative response was similar to that induced by control LC incubated in medium alone, demonstrating that pretreatment with cis-UCA did not alter human LC function. In conclusion, these results strongly suggest that cis-UCA has no direct effect on human LC antigen-presenting function.

In vitro effects of ultraviolet B radiation on human Langerhans cell antigen-presenting function.

The effects of ultraviolet B radiation (UBV) on the immune function of human epidermal Langerhans cells (LC) were studied by using the mixed epidermal cell-lymphocyte reaction (MELR). Exposure of both enriched LC suspensions (eLC, 8-20% LC) and purified LC suspensions (pLC, 70-90% LC) to increasing doses of UVB radiation (25 to 200 J/m2) decreased the proliferative T cell response in a very similar dose-dependent way, suggesting that keratinocytes did not play a major role in the UVB-induced inhibition of MELR. Supernatants from irradiated cultured eLC or pLC failed to inhibit T cell proliferation induced by untreated pLC. Furthermore, addition of irradiated eLC to untreated pLC did not affec the allogeneic T cell response. Taken together, these results provide evidence that in vitro UVB-induced immunosuppression was not mediated by inhibitory soluble factors that could affect either LC allostimulatory property or T cell proliferative response. UVB irradiation of human LC inhibited the capacity of these cells to induce CD4+ as well as CD8+ T cell proliferation. UVB-irradiated LC also induced a decreased T cell response to recall antigen or mitogen. Moreover, addition of exogeneous cytokines such as IL-1 beta, IL-1 alpha, or IL-2 did not reverse the defective function of UVB-irradiated LC in MELR. The inhibitory effect of UVB radiation on human LC was not related to a decreased HLA-DR expression. Because cultured LC appeared to be less sensitive than freshly isolated LC to UVB-induced suppressive effects, the deleterious effects of UVB radiation on human LC allostimulatory properties may be associated with an impaired development of LC accessory function.

Topical application of isotretinoin did not modify human Langerhans cell allostimulatory function.

The beneficial effect of isotretinoin on the repair of photodamaged skin is well documented. Little is known, however, on the action of this compound on immunological functions of epidermis. In a double-blind study, we analyzed the effect of topical applications of isotretinoin on human Langerhans cell (LC) function by using the mixed epidermal cell lymphocyte reaction (MELR). Isotretinoin cream (0.1%) was applied daily for 4 months on the back of one hand of 5 healthy volunteers, 50-60 years of age. The back of the other hand received vehicle alone and was used as control. Skin biopsy specimens were taken at the end of treatment. Epidermal cell (EC) suspensions were obtained by the use of trypsin and allogeneic T cells were purified from the peripheral blood of allogeneic donors. MELR was performed in microtiter plates and T cell proliferation was assessed by 3H-thymidine incorporation during the last 18 h of culture. Results did not show any differences between allogeneic T cell responses to EC from isotretinoin-treated or nontreated skin. These results therefore suggest that isotretinoin, applied topically onto photodamaged skin, did not alter human LC antigen-presenting function.