Cutaneous T cell lymphoma: translating immunobiology into therapeutic opportunities.

Cutaneous T cell lymphoma (CTCL) has always served as a proving ground where conceptual advances in immunology can be tested and the results translated into clinical practice. From the earliest studies that used sheep red blood cells to identify the malignant cell as a T lymphocyte to molecular demonstration of the clonalilty of the disease, basic science techniques have provided sign posts that allow us to understand the clinical features seen in the patients. We continue to apply this paradigm to develop new insights into the role of the immune system in CTCL with the goal of using this knowledge to enhance the therapeutic options available to the patient. This article will review the studies that have led to our current understanding of the immunobiology of CTCL and the new therapeutic approaches that are being tested in this disease.

Tryptophan 512 is sensitive to conformational changes in the rigid relay loop of smooth muscle myosin during the MgATPase cycle.

To examine the structural basis of the intrinsic fluorescence changes that occur during the MgATPase cycle of myosin, we generated three mutants of smooth muscle myosin motor domain essential light chain (MDE) containing a single conserved tryptophan residue located at Trp-441 (W441-MDE), Trp-512 (W512-MDE), or Trp-597 (W597-MDE). Although W441- and W597-MDE were insensitive to nucleotide binding, the fluorescence intensity of W512-MDE increased in the presence of MgADP-berellium fluoride (BeF(X)) (31%), MgADP-AlF(4)(-) (31%), MgATP (36%), and MgADP (30%) compared with the nucleotide-free environment (rigor), which was similar to the results of wild type-MDE. Thus, Trp-512 may be the sole ATP-sensitive tryptophan residue in myosin. In addition, acrylamide quenching indicated that Trp-512 was more protected from solvent in the presence of MgATP or MgADP-AlF(4)(-) than in the presence of MgADP-BeF(X), MgADP, or in rigor. Furthermore, the degree of energy transfer from Trp-512 to 2'(3')-O-(N-methylanthraniloyl)-labeled nucleotides was greater in the presence of MgADP-BeF(X), MgATP, or MgADP-AlF(4)(-) than MgADP. We conclude that the conformation of the rigid relay loop containing Trp-512 is altered upon MgATP hydrolysis and during the transition from weak to strong actin binding, establishing a communication pathway from the active site to the actin-binding and converter/lever arm regions of myosin during muscle contraction.

Smooth muscle myosin mutants containing a single tryptophan reveal molecular interactions at the actin-binding interface.

Elucidation of the molecular details of the cyclic actomyosin interaction requires the ability to examine structural changes at specific sites in the actin-binding interface of myosin. To study these changes dynamically, we have expressed two mutants of a truncated fragment of chicken gizzard smooth muscle myosin, which includes the motor domain and essential light chain (MDE). These mutants were engineered to contain a single tryptophan at (Trp-546) or near (Trp-625) the putative actin-binding interface. Both 546- and 625-MDE exhibited actin-activated ATPase and actin-binding activities similar to wild-type MDE. Fluorescence emission spectra and acrylamide quenching of 546- and 625-MDE suggest that Trp-546 is nearly fully exposed to solvent and Trp-625 is less than 50% exposed in the presence and absence of ATP, in good agreement with the available crystal structure data. The spectrum of 625-MDE bound to actin was quite similar to the unbound spectrum indicating that, although Trp-625 is located near the 50/20-kDa loop and the 50-kDa cleft of myosin, its conformation does not change upon actin binding. However, a 10-nm blue shift in the peak emission wavelength of 546-MDE observed in the presence of actin indicates that Trp-546, located in the A-site of the lower 50-kDa subdomain of myosin, exists in a more buried environment and may directly interact with actin in the rigor acto-S1 complex. This change in the spectrum of Trp-546 constitutes direct evidence for a specific molecular interaction between residues in the A-site of myosin and actin.

Photoactivated 8-methoxypsoralen treatment causes a peptide-dependent increase in antigen display by transformed lymphocytes.

Ex vivo exposure of malignant human T cells to photoactivated 8-methoxypsoralen (8-MOPa), followed by their i.v. return, appears to vaccinate patients against tumor-associated antigens of cutaneous T cell lymphoma in a procedure termed photopheresis. The molecular basis of this Food and Drug Administration-approved therapy, administered in 100 centers worldwide, is unclear. Most of the attention to the mechanism of action of the drug has focused on its capacity to form covalent cross-links with pyrimidine bases of DNA, thereby inhibiting cellular proliferation. Because immunologic factors appear to be important in the clinical response and could potentially serve as a model for immunotherapy of other malignancies, we explored the possibility that 8-MOP-treated cells display increased quantities of antigenic peptides at their cell surface. In this work, human B-lymphoblastoid tissue culture lines were exposed to 8-MOPa and expression of cell surface class I major histocompatibility complex proteins assessed, since CD8 T cells recognize antigenic moieties in the context of class I molecules. A peak 200-300% increase in MHC class I expression in 8-MOPa-treated cells occurred at 20 hr. 8-MOPa was far more effective in inducing this increase in class I MHC than other modalities, including mitomycin C, gamma-irradiation, ultraviolet B or heat or cold shock. This increase in surface class I MHC molecules appears to be driven by the degradation of cytoplasmic proteins into small peptides, followed by the transport of these peptides to MHC class I molecules in the endoplasmic reticulum. The data suggest that 8-MOPa treatment may augment the immunogenicity of tumor and/or antigen-presenting cells by enhancing processing and transport of class I MHC antigenic peptides.

Juvenile friends, behavior, and immune responses to separation in bonnet macaque infants.

Individual differences in the response to maternal separation in nonhuman primate infants have been attributed to (among other variables) presence or absence of processes that may model social support in humans. Alternative attachments to other members of the social group buffer the infant against a depressive response to maternal separation. This hypothesis was tested in a group of bonnet macaques by manipulating the presence or absence of alternative juvenile attachment figures (friends) during separation. Infants who retained such attachments showed fewer behavioral evidences of depression when separated from their mothers. These infants without friends also showed changes in lymphocyte activation by mitogens or natural cytotoxicity that were not evident in the infants with juvenile friends. Across all separated infants, natural cytotoxicity was positively correlated with juvenile affiliative behavior directed toward the infants during the separation. These results support the hypothesis that social support, available from alternative attachments, can modulate the response to loss, and can account for some of the individual differences seen in these responses.

T-cell responses in photoimmune therapy.

The extracorporeal inactivation of a lymphocyte rich buffy coat suspension with ultraviolet A light and 8 methoxypsoralen can lead to dramatic clinical improvements following reinfusion of the damaged cells. This therapy is reviewed in the context of the disease it is most commonly used for: cutaneous lymphoma. Studies with cutaneous lymphoma patients have shown an active immune response against purified tumor cells. In addition a mouse model for an impact of therapy on a T-cell lymphoma has demonstrated results that parallel those from clinical studies in humans. The impact of photoimmune therapy on in vivo and in vitro T-cell responses to cutaneous lymphoma is discussed.

Antigen-specific tolerance induced by autoimmunization with photoinactivated syngeneic effector cells.

Development of a protocol that could invoke specific suppression of an undesired immune response, while sparing normal immune competence, would be of great clinical value. This report demonstrates that multiple infusions of splenocytes sensitized in vivo to sheep red blood cells (SRBC) and photoinactivated in vitro with 8-methoxypsoralen and ultraviolet A light can render a syngeneic recipient selectively unresponsive to subsequent challenge with this antigen. Mice treated in this fashion did not develop a T cell-mediated delayed type hypersensitivity (DTH) reaction to SRBC. In contrast, control mice exposed to nonimmune splenocytes pretreated in an identical manner developed a normal DTH response to SRBC, thereby demonstrating that drug and light in the absence of effector T cells were not suppressive. Inhibition of the DTH response was antigen specific, since animals rendered unresponsive to SRBC developed a normal DTH response to chicken red blood cells. Cell transfer experiments demonstrated that unprimed recipients of splenocytes from mice rendered unresponsive to SRBC could not mount a DTH reaction when challenged. Moreover, this procedure can also suppress established immunity to that antigen. The use of photoinactivated syngeneic antigen-reactive effector cells as immunosuppression agents suggests that this method may be clinically useful in inhibiting pathogenic antigen-specific immunologic reactions.

Inhibition of antiskin allograft immunity induced by infusions with photoinactivated effector T lymphocytes (PET cells). Is in vivo cell transferrable?

We previously reported producing donor-specific tolerance to alloantigens by intravenous exposure to pretreated antidonor T cells. The current study extends that work by adoptively transferring the donor-specific tolerance into naive syngeneic recipients. Eight days after BALB/c mice received histoincompatible CBA/j skin grafts, their splenocytes which included an expanded population of cells mediating rejection were treated with 100 ng/ml 8-methoxypsoralen (8-MOP) photoactivated by 1 Joule/cm2 of ultraviolet A (UVA) light prior to infusion into naive BALB/c recipients. Whereas 8-MOP itself is biologically inert, photoactivated 8-MOP crosslinks DNA by covalently binding to pyrimidine bases. Recipient BALB/c mice which had been previously demonstrated to be hyporesponsive to CBA/j alloantigens in mixed leukocyte culture (MLC), cytotoxicity (CTL) and in vivo delayed type hypersensitivity (DTH) assays were the donors of spleen cells for the adoptive transfer experiments. Fifty to one hundred million viable spleen cells from these pretreated BALB/c mice were transferred into naive syngeneic recipients which then were tested for DTH response and allograft survival to the relevant and irrelevant antigens. The radiosensitivity of this transferrable suppression was evaluated by exposing the adoptively transferred cell population to 3200 rads of C-irradiation prior to cell transfer. The phenotype of the cells transferring this suppressive response was performed by depleting specific populations of cells with monoclonal antibodies prior to cell transfer. In vivo the DTH response of the pretreated BALB/c mice was specifically suppressed to the relevant alloantigen, correlating with retention of CBA/j skin grafts for up to 42 days post engraftment without visual evidence of rejection, in comparison to control mice complete rejection of the skin graft in less than 8 days. In vitro, splenocytes from BALB/c recipients of pretreated syngeneic splenocytes containing large numbers of BALB/c anti-CBA/j T cells proliferated less in MLC and generated lower cytotoxic T cell responses to CBA/j alloantigens than did controls and suppressed the naive and sensitized BALB/c MLC and CTL responses to CBA/j alloantigen. This specific suppressive response to alloantigen was optimally transferred into syngeneic naive recipients when the adoptive transfer was performed on the sixth day after the last infusion received by the spleen cell donor mice. The adoptive transfer of this suppressive response was abrogated by the prior X-irradiation of the donor spleen cells and significantly abolished by the depletion of Thy-1+, Lyt-2+, L3T4- T lymphocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Autoregulation of the immune response in autoimmune disease and cardiac transplantation by photoinactivated autologous lymphocytes.

These studies demonstrate that photochemotherapy can be successfully evaluated in animal models. The therapy mediates specific suppression of immune responses and appears to operate at the level of the effector T cells. Future studies will focus on isolation and characterization of the host response to photochemotherapy. The extention of this form of therapy to conditions mediated by dysfunctional regulation of effector T cells is already in progress in clinical trials of cardiac allograft transplantation and autoimmune disease. The results of these trials will provide more evidence on the role of this form of therapy in autoregulation of the immune response.

Inhibition of autoimmune disease in a murine model of systemic lupus erythematosus induced by exposure to syngeneic photoinactivated lymphocytes.

MRL/l mice develop progressive, virulent autoimmune disease that has many of the features of systemic lupus erythematosus. Prophylactic treatment of MRL/l mice with syngeneic photoinactivated autoimmune splenocytes improves survival and inhibits the fulminant hyperproliferation of abnormal T cells and the production of high titer anti-DNA antibody invariably found in untreated mice. The proliferation of Thy 1+ splenic T cells was significantly decreased, and prolonged retention of the response to T-cell mitogen was found in treated mice. Treatment with unmodified cells induced a partial inhibition of disease features which did not prolong survival rates. These results suggest that phototherapy potentiates a normal immunoregulatory process which enables suppression of the development of abnormal cell populations in young MRL/l mice with relatively intact immune systems.

Inhibition of antiskin allograft immunity induced by infusions with photoinactivated effector T lymphocytes (PET cells).

Induction of tolerance for skin allotransplantation requires selective suppression of the host response to foreign histocompatibility antigens. This report describes a new approach which employs pre-treatment with 8-methoxypsoralen (8-MOP) and ultraviolet A light (UVA) to render the effector cells of graft rejection immunogenic for the syngeneic recipient. Eight days after BALB/c mice received CBA/j skin grafts, their splenocytes were treated with 100 ng/ml 8-MOP and 1 J/cm2 UVA prior to reinfusion into naive BALB/c recipients. Recipient mice were tested for tolerance to alloantigens in mixed leukocyte culture (MLC), cytotoxicity (CTL), delayed-type hypersensitivity assays (DTH), and challenge with a fresh CBA/j graft. Splenocytes from BALB/c recipients of photoinactivated splenocytes containing the effector cells of CBA/j alloantigen rejection proliferated poorly in MLC and generated lower cytotoxic T-cell responses to CBA/j alloantigens in comparison with sensitized and naive controls and suppressed the MLC and CTL response to alloantigen from sensitized and naive BALB/c mice. In vivo, the DTH response was specifically suppressed to the relevant alloantigen in comparison with controls. BALB/c mice treated in this fashion retained a CBA/j skin graft for up to 42 days post-transplantation without visual evidence of rejection. These results showed that reinfusion of photoinactivated effector cells resulted in an immunosuppressive host response which specifically inhibited in vitro and in vivo responses that correlate with allograft rejection and permitted prolonged retention of histoincompatible skin grafts.

Experimental murine and primate models for dissection of the immunosuppressive potential of photochemotherapy in autoimmune disease and transplantation.

This paper reviews the results achieved in murine and primate models of autoimmune disease and transplantation. These studies have attempted to clarify the nature and specificity of the response induced by reinfusion of phototreated immunoactive lymphocytes. Results obtained in murine lupus have demonstrated that some of the disease features related to the abnormal proliferation of inducer T cells can be inhibited both prophylactically and therapeutically by exposure to photoinactivated autoimmune splenocytes. Radiolabeling studies performed in normal syngeneic mice have shown that, if immunoactive cells are phototreated and injected, their recirculation pattern is altered, and increased sequestration in the spleen, bone marrow, and kidney is noted. These studies suggest that reinfused, phototreated, antigen-activated lymphocytes may localize in sites where they are available for induction of immune responses. Primate cardiac xenotransplantation models have demonstrated that reinfusion of phototreated autologous leukocytes, administered with cyclosporine A and steroids, mediates enhanced specific suppression of both the cellular and humoral host response to foreign tissue. Taken as a whole, the experimental models suggest that photopheresis may provide a means of inducing specific suppression of immunoactive T cells. This form of therapy may have a role as an immunosuppressive agent in both autoimmune disease and transplantation.

Effect of monochromatic UVA light and 8-methoxypsoralen on human lymphocyte response to mitogen.

The ultraviolet-A (UVA) action spectrum for inhibition of DNA synthesis by lymphocytes stimulated with phytohemagglutinin and sensitized with 8-methoxypsoralen (8-MOP) (100 ng/ml) was determined by exposing cells to 0.33 and 1.0 J/cm2 of sub-bands of UVA light at 322, 334, 346, 358 and 370 nm. For lymphocytes irradiated with 0.33 J/cm2, the wavelength for optimal inhibition of DNA synthesis was 334 nm (P less than 0.001). Relatively high doses of monochromatic UVA light (6J/cm2) significantly inhibited lymphocyte proliferation at all wavelengths tested: 64% at 370 nm, 90% at 358 nm, 78% at 346 nm and greater than 99% at both 334 and 322 nm (P less than 0.001 at each wavelength). The optimal wavelength for selective 8-MOP enhanced inhibition of DNA synthesis by lymphocytes in the 334-346 nm range is shown to support the usual assumption that crosslinks represent a lethal cellular event.