INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

BEHAVIORAL OR CLASSICAL conditioning is an associative learning paradigm. Conditioning involves the pairing of a benign novel stimulus (conditioned stimulus, CS) with a stimulus that produces physiological changes (unconditioned stimulus, UCS). On re-presentation of the CS, the organism produces physiological alterations that are usually ascribed to the UCS. This paradigm has been implemented to produce conditioned alterations in immune functions (2). Commonly, animals are presented with a sweet taste (saccharin) in the drinking water and subsequently injected with a pharmacological agent that produces changes in immune status. At a later date, the saccharin solution is re-presented, at which time the animals avoid the stimulus (conditioned taste aversion) and experience concomitant alterations in immune function concordant with the effect of actual drug administration.

Conditioned effects have been demonstrated both in humoral and cellular immunity (2). Furthermore, a limited number of studies have attempted to examine the clinical relevance of conditioned changes in immune function. Specifically, the morbidity and mortality of animals with autoimmune disease are abated via conditioning with cyclophosphamide as the UCS (1). Furthermore, the survival of heterotopic heart allografts can be extended using a conditioning paradigm that pairs saccharin as the CS with cyclosporin A (CsA) as the UCS (8, 12). Despite such biologically relevant findings, a common criticism of the results from conditioning experiments is that the effects are relatively small in comparison to actual drug administration, and thus it is doubtful that conditioning has any clinical relevance as a stand-alone therapy. Nevertheless, the ability of a suboptimal, albeit therapeutic, dose of cyclophosphamide to inhibit the development of systemic lupus erythematosus in mice is enhanced by behavioral conditioning (1). Therefore, we extended these data by examining whether a combination of conditioning and subtherapeutic CsA treatment can prolong heterotopic heart allograft survival in rats.

Furthermore, the mechanisms of conditioned changes in immune function and disease progression are poorly understood. There is some evidence for the role of endocrine mediators such as opioids and catecholamines (16, 19); however, the results are inconclusive. One alternative hypothesis is that conditioning produces its effect via the autonomic innervation of lymphoid organs, where neurotransmitters and neuropeptides such as catecholamines (22) are released in close proximity to immunocompetent cells (10, 25). Catecholamines alter immune function via binding to functional adrenoceptors on lymphocytes (3, 11, 14). We have previously demonstrated that surgical denervation of sympathetic input to the spleen abrogated the conditioned inhibition of splenocyte proliferation and cytokine [interleukin (IL)-2 and interferon (IFN)-] production (8). Therefore, we examined whether the conditioned prolongation of heart allograft survival is mediated via sympathetic input to the spleen. As the present conditioning paradigm produces a reduction in IL-2 and IFN- secretion by proliferating splenocytes, we examined whether conditioning was also able to mimic the effect of CsA on intracellular IFN- production.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. Experimentally naive male Dark Agouti (DA) and Lewis rats (LEW; Harlan Laboratories, Borchen, Germany) weighing between 220 and 250 g were used. All rats were allowed to habituate for 3 wk before experimentation. Animals were individually housed in standard plastic-based laboratory cages (40 × 26 × 15 cm high) with a wire mesh lid. Cages were kept in an air-conditioned, soundproofed holding room at an ambient temperature of 24.0 ± 0.5°C. The animals had access to standard lab chow and tap water ad libitum except during the water deprivation phase of the experiment. A 12:12-h light cycle was maintained throughout the experiment, with lights off at 0700. This allowed stimuli presentation to be conducted during the dark (active) cycle of the animals. All conditioning procedures were completed under red light so as to avoid any interruption to the normal light-dark cycle of the rats.

Conditioning paradigm: analysis of corticosterone, CsA, and intracellular IFN-. Male DA rats were placed on a water deprivation regimen for 5 days, allowing them 15 min of drinking at 0700 and again at 1700 each day (Fig. 1A). The present study implemented a three-learning (CS-UCS pairing)-trial paradigm. Each learning trial was separated by 72 h. On the fifth day animals received the first of three CS-UCS pairings. Conditioned animals received 0.2% saccharin solution (Sac) as the CS paired with 20 mg/kg ip CsA as the UCS on the training days (Fig. 1C). In the afternoon session they were administered water paired with intraperitoneal saline injection. Sham-conditioned rats were given water paired with CsA in the morning of the training days and Sac in combination with saline in the afternoon. Three days after the final pairing, the CS alone was presented during each drinking session. This was repeated for the subsequent 2 days. Two extra control groups were implemented (Fig. 1C). CsA-treated animals were treated similarly to sham-conditioned rats; however, these animals received an additional CsA injection (20 mg/kg) after each of the first three CS re-presentations. This allowed a comparison of the conditioned response with the actual drug effect. Additionally, an untreated group was used that was not manipulated during the entire conditioning procedure. One hour after the third CS re-presentation, animals were killed, and blood was drawn for analysis of corticosterone and CsA levels. Additionally, the spleen was removed for examination of intracellular IFN- in splenocytes.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   A: experimental design examining effect of conditioning on intracellular interferon (IFN)- production and concentration of corticosterone and cyclosporin A (CsA) in blood. Animals were habituated to experimental conditions for 3 wk and then placed on water deprivation (see MATERIALS AND METHODS). On 5th day animals received first of 3 conditioned stimulus (CS)-unconditioned stimulus (UCS) pairings. Three days after final pairing, CS alone was presented during drinking session. Animals were killed 1 h after third CS re-presentation, and spleen and blood were collected for assay. B: experimental design examining effect of combination conditioning and subtherapeutic CsA on heart allograft survival. Animals were conditioned with basic paradigm; however, a 2 mg/kg ip dose of CsA was injected after CS re-presentation. Heart allograft transplantation (Tx) was completed 1 h after second 2 mg/kg administration of CsA after third CS re-presentation. Subtherapeutic CsA administration was completed on 5 subsequent alternate days. CS was re-presented every day until rejection of the graft. C: experimental groups. Conditioned rats received a pairing of saccharin (Sac) and CsA on CS-UCS days, with saccharin alone presented on CS re-presentation days. Sham-conditioned rats were treated similarly to conditioned animals, with the modification that they received water (Wat) instead of saccharin. CsA-treated rats were conditioned in an identical manner to sham-conditioned animals. However, on each of first 3 CS re-presentation days (CS1-CS3) they were administered a further therapeutic dose of 20 mg/kg ip CsA. Untreated rats remained completely unhandled.

Conditioning paradigm with subtherapeutic CsA: heart allograft survival. The conditioning paradigm examining heart allograft survival was similar to the paradigm used for cytokine measurement (Fig. 1B). However, this paradigm differed in that instead of killing the animals 1 h after the third CS presentation, animals received a heterotopic heart allograft. The CS was subsequently re-presented every day until rejection of the graft. Additionally, on the first CS re-presentation day, conditioned, sham-conditioned, and CsA-treated groups were injected with a 2 mg/kg ip (subtherapeutic) dose of CsA. This procedure was repeated on six further CS re-presentation days, with each subtherapeutic CsA administration separated by 48 h. Thus these groups received a subtherapeutic regimen of seven 2-mg/kg CsA injections [subtherapeutic CsA given after CS3 (day 16), CS5 (day 18), CS7 (day 20), CS9 (day 22), CS11 (day 24), and CS13 (day 26)]. Untreated rats were neither conditioned nor administered subtherapeutic CsA. Similar to the basic conditioning paradigm, CsA-treated animals received an additional CsA injection (20 mg/kg) after each of the first three CS re-presentations.

Conditioning paradigm: role of sympathetic innervation in conditioned heart allograft survival. To examine the role of splenic innervation in the effects of combination subtherapeutic CsA plus conditioning, the subtherapeutic heart allograft conditioning regimen was again implemented (Fig. 1B). However, 2 wk before conditioning, the spleens of animals from all groups were denervated of sympathetic innervation. Additionally, a group of animals receiving the main experimental treatment (conditioned-subtherapeutic CsA) were sham denervated. Conditioning, heart allograft transplantation, subtherapeutic CsA administration, and monitoring of survival were conducted in an identical manner.

Heterotopic heart transplantation. Transplantation was conducted using standard techniques (18). Briefly, the vena cava and the pulmonary veins of the donor rat (LEW rat; RT1^l) were ligated, and the pulmonary artery and aorta were transected 2-3 mm above their origins. The heart was perfused with Ringer solution and placed in a 4°C saline bath. The abdominal vessels were dissected free in the anesthetized recipient rat (DA; RT1^a) from the left renal vein to the bifurcation. The abdominal aorta and inferior vena cava were cross-clamped independently. The graft was placed into the abdominal cavity, and transplantation (donor aorta-recipient abdominal aorta; donor pulmonary artery-recipient inferior vena cava) was completed with end-to-side anastomoses. All grafts demonstrated good contractile function within 60 s of clamp removal. Grafts were palpated once daily to assess survival by an experimenter blind to the animals' treatment. Rejection was defined as the absence of a palpable heart beat.

Splenic denervation. Splenic denervation was conducted 2 wk before conditioning with standard techniques (20). Briefly, a midline incision opened the abdominal cavity, and the splenic nerve vascular package was exposed. The splenic nerve bundle was isolated from the splenic vasculature, and the neural bundle was cut before their bifurcation. The incision was then sutured, and the animal was allowed to recover. Sham denervation was completed by, again, isolating the splenic nerve bundle; however, the nerve was not sliced. Confirming previous data (8), splenic denervation reduced catecholamine content in the spleen to <20% of sham-denervated animals.

Corticosterone and CsA determination. One hour after the third CS re-presentation, blood was also collected for analysis of plasma corticosterone and CsA concentrations. Corticosterone concentrations were measured by radioimmunoassay as previously described (7). As previously detailed, concentrations of CsA and its metabolites (23) were assayed as double probes with commercial kits (Emit-test, Behring Diagnostic).

Intracellular IFN- determination in T cell subsets. DA rats were conditioned according to the basic protocol (Fig. 1A), and animals that did not receive subtherapeutic CsA were killed 1 h after the third CS re-presentation. The spleen was removed and, confirming previous data (8), showed comparable cell numbers and B and T cell subset composition among all groups (data not shown). Intracellular IFN- in splenic T cells was detected as described (17), with the following modifications. Briefly, 2 × 10⁶ splenocytes were incubated in 1 ng phorbol 12-myristate 13-acetate/ml and 250 ng Ionomycin (Sigma, Diesenhofen, Germany) for 2 h. Then 2 µl Brefeldin A (Golgiplug; Pharmingen, San Diego, CA) were added, and another 2-h incubation period followed. After being washed in ice-cold PBS, the cells were fixed in 2% formaldehyde for 20 min at room temperature. The cells were subsequently incubated for 5 min in PBS containing 0.5% saponin, 1% BSA, and 0.1% NaN₃. To detect intracellular IFN- the cells were incubated for 15 min at room temperature with a mouse anti-rat IFN- antibody (DB1; kindly provided by P. van der Meide, The Netherlands), which was dissolved in PBS containing 0.5% saponin, 1% BSA, and 0.1% NaN₃. After washing the cells were incubated with PBS containing saponin, 10% rat serum, and 0.1% NaN₃ for 10 min, and a phycoerythrin-conjugated anti-mouse antibody was used as a second step antibody (30 min; dissolved in PBS containing 0.5% saponin, 5% rat serum, and 0.1% NaN₃). Subsequently, CD4⁺ T cells (including their "naive" and "memory" subsets) were identified via appropriately conjugated antibodies against CD4 (W3/25) and CD45RC (Ox22) as previously described (28). Isotype-matched irrelevant antibodies served as controls. With the use of a FACScan and PC-LYSYS software (Becton Dickinson, Mountain View, CA), the percentage of IFN--positive cells was recorded in viable T cell subsets (2 × 10⁴).

Statistical analyses. Heart allograft survival was analyzed by Kaplan-Meier logrank analysis for survival curve comparison. Average differences in graft survival between groups were analyzed with the nonparametric Kruskall-Wallis rank sum test, because data from conditioned animals violated the assumption of a normal distribution required for parametric statistical analysis. One-way ANOVAs were used to examine statistical differences between the four groups in hormonal and immunologic data. Post hoc Fisher's least significant difference tests were implemented to examine specific differences between groups. Values are presented as means ± SE. Statistically significant differences are reported when P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Behavioral conditioning enhances subtherapeutic CsA treatment. To investigate the effect of combination therapy (conditioning-CsA), we combined behavioral conditioning with a subtherapeutic CsA regimen. This was completed by conditioning the rats with 20 mg/kg CsA as the UCS (Fig. 1B) and additionally administering seven injections of 2 mg/kg ip CsA (subtherapeutic dose), which were injected on alternate days and commenced 2 days before transplantation (Fig. 1B).

Conditioned animals in the present paradigm avoid consumption of the saccharin stimulus after the first CS-UCS pairing (conditioned taste aversion) (8, 27). Such a response indicates acquisition of the association between the two stimuli, and was observed in the present series of experiments, in which conditioned animals typically drank <10% of the level of saccharin consumed by control animals. During assessment of graft survival, conditioned taste aversion extinguished between the 16th and 20th CS re-presentation (data not shown).

The mean graft survival time of heart allografts revealed that the subtherapeutic CsA regimen alone was not effective, inasmuch as no difference was observed between completely untreated animals and rats receiving a combination of subtherapeutic CsA treatment and sham conditioning (Fig. 2A). In contrast, animals that received subtherapeutic CsA and were behaviorally conditioned displayed a significant increase in survival time compared with rats that received subtherapeutic CsA and were only sham conditioned (P < 0.0001). Furthermore, even a short course of therapeutic CsA treatment (3 × 20 mg/kg) together with the subtherapeutic regimen (7 × 2 mg/kg CsA) produced an increase in the mean survival time that was significantly lower than that achieved by the combination of subtherapeutic CsA and behavioral conditioning (P < 0.001; Fig. 2C). The most striking effect observed by combining conditioning and subtherapeutic CsA was that 20% of these animals displayed long-term surviving grafts (>100 days).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Conditioning prolonged survival time of heterotopic heart allografts in rats treated with subtherapeutic doses of CsA. A: Kaplan-Meier survival curve revealed that subtherapeutic CsA did not extend survival time of heart allografts. However, rats that both received subtherapeutic CsA and were conditioned had significant prolongation of heart allograft survival compared with sham-conditioned animals receiving subtherapeutic CsA (P < 0.0001). Of conditioned animals, 20% displayed long-term graft survival (>100 days). B: second independent experiment confirmed that conditioning-subtherapeutic CsA prolonged heart allograft survival in sham-denervated rats, with 20% of animals displaying long-term graft survival. Conditioned increase of heart allograft survival in rats receiving subtherapeutic CsA was produced via innervation of spleen, inasmuch as splenic denervation completely abrogated this effect in conditioned animals (P < 0.001). C: mean graft survival time in animals pooled from 2 experiments that were either untreated, sham conditioned, CsA treated (3 × 20 mg/kg), conditioned, or conditioned after splenic denervation. Except first group, which was completely untreated, all other animals also received subtherapeutic CsA regimen (7 × 2 mg/kg). Subtherapeutic CsA protocol alone (sham conditioned-subtherapeutic CsA) was not effective in prolonging heart allograft survival, inasmuch as these animals showed no difference in survival time compared with untreated group. Therapeutic CsA treatment (therapeutic-subtherapeutic CsA) significantly increased survival time. However, combination of conditioning and subtherapeutic CsA (conditioned-subtherapeutic CsA) was able to produce a further increase in graft survival, which was abrogated by surgical denervation of spleen (sham denervation did not influence mean survival time; data analyzed with nonparametric Kruskall Wallis rank sum test; * P < 0.05, *** P < 0.001 compared with untreated animals).

Behavioral conditioning enhances subtherapeutic CsA treatment via sympathetic innervation of the spleen. We have previously shown that the present conditioning paradigm produces a significant reduction in splenocyte proliferation and cytokine (IL-2, IFN-) production that is mediated via nerve fibers innervating the spleen (8). Thus we examined whether autonomic innervation of the spleen influences the prolongation of heart allograft survival produced by the combination of subtherapeutic CsA and conditioning. A second independent experiment confirmed that conditioning plus subtherapeutic CsA prolonged heart allograft survival, revealed in sham-denervated rats. Furthermore, this replication again demonstrated that the combination therapy induced long-term graft survival in 20% of the animals. Moreover, splenic denervation completely abrogated the increased survival time induced by the combination of subtherapeutic CsA and behavioral conditioning (Fig. 2, B and C; P < 0.0001).

Behavioral conditioning does not induce corticosterone secretion or alter CsA metabolism. Psychological stress activates the hypothalamus-pituitary-adrenal axis, resulting in the release of glucocorticoids, which may potentially produce the current immunosuppression (29). Thus we examined whether conditioning may enhance allograft survival via inducing the production of adrenal steroids. We investigated this in nontransplanted, nonsubtherapeutic CsA-administered animals that were conditioned with the basic paradigm (Fig. 1A). One hour after the third conditioned stimulus re-presentation, blood was collected to investigate glucocorticoid levels. Because no differences were observed between the different experimental groups (Fig. 3A), it is unlikely that conditioning-induced changes in glucocorticoid levels are responsible for the prolonged allograft survival. To ensure that conditioning did not produce immunosuppression via altering CsA metabolism (9), we examined CsA levels at the same time. Only in CsA-treated animals (3 × 20 mg · kg¹ · day¹) was a detectable level of CsA (Fig. 3B) and CsA metabolites (Fig. 3C) revealed, indicating that conditioning did not effect graft prolongation via altering CsA metabolism.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Analysis of plasma corticosterone and CsA concentrations in rats that underwent basic conditioning paradigm. Samples were taken from animals that were exsanguinated 1 h after 3rd CS re-presentation. These animals were not treated with subtherapeutic CsA and did not receive heterotopic heart transplantation. A: plasma corticosterone (means ± SE) did not differ between untreated, sham- conditioned, CsA-treated (3 × 20 mg/kg), and conditioned animals (n = 10 for each group), which were conditioned without the addition of subtherapeutic CsA. CsA (B) and CsA metabolite (C) serum concentrations (means ± SE) were only detectable in CsA-treated group, in which levels were under detection limit of assay revealed in other animals (n = 10 for each group; *** P < 0.001).

Behavioral conditioning increases, and CsA decreases, intracellular IFN- in splenocytes. The present conditioning paradigm has been shown to significantly reduce mitogen-induced splenocyte proliferation and IL-2 and IFN- levels in the supernatant (8), the degree of reduction comparable to that achieved by therapeutic CsA treatment (3 × 20 mg/kg). Because it is likely that CsA treatment and behavioral conditioning prolong heart allograft survival via different mechanisms, we further characterized the effect of conditioning by examining the number of splenocytes positive for intracellular IFN-. We investigated this in nontransplanted, nonsubtherapeutic CsA-administered animals that were conditioned with the basic paradigm (Fig. 1A). Despite similar reductions in extracellular cytokine levels in CsA-treated and conditioned rats (8), only CsA-treated rats showed the expected reduction in the number of IFN--positive CD4⁺ splenocytes (Fig. 4A). In contrast, in conditioned animals a significant increase of IFN--positive CD4⁺ splenocytes was observed (Fig. 4A). This effect was more pronounced in "memory" than in "naive" CD4⁺ T cells (Fig. 4, B and C).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   A: Intracellular IFN- expression (means ± SE) in splenic CD4+ T cell subsets in untreated, sham-conditioned, CsA-treated (3 × 20 mg/kg), and conditioned rats (n = 10 for each group), which were conditioned with basic protocol. These animals did not undergo heart allograft transplantation and were not administered subtherapeutic CsA. In agreement with reduction of secreted IFN- (8), CsA-treated animals showed significant decline of IFN--positive CD4+ splenocytes. However, in contrast to reduction of secreted IFN- (8), conditioned animals showed increased numbers of IFN--positive CD4+ T cells compared with untreated and sham-conditioned rats (*** P < 0.001). This effect was seen in both "naive" (CD45RC+; B) and "memory" (CD45RC+; C) CD4+ T cells; however, it was more pronounced in latter (** P < 0.01; *** P < 0.001).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our results demonstrate that behavioral conditioning can supplement subtherapeutic doses of CsA to prolong heart allograft survival in the rat. This is achieved by the central nervous system via autonomic innervation of the spleen. In two independent experiments, subtherapeutic doses of CsA combined with behavioral conditioning exceeded the level of graft prolongation achieved by the present conditioning paradigm in isolation (8), and 20% of the animals retained a fully functional allograft 100 days posttransplantation. Therefore, the present data indicate that behavioral conditioning produces a clinically meaningful alteration of heart allograft survival when combined with subtherapeutic CsA treatment.

Because splenic denervation completely abrogates the conditioned prolongation of heart allograft survival, the spleen is important for both receiving signals from the central nervous system during CS re-presentation and mediating the subsequent immunosuppression. Sympathetic nerves are in close contact with lymphocytes in the spleen (10, 25), and they release catecholamines that influence splenic IL-6 production (25) via functional adrenoceptors (3, 11, 14). In addition, it is known that splenic T cell proliferation and synthesis of both IL-2 and IFN- can be reduced by sympathetic nervous system input (15, 21). Furthermore, in the current conditioning paradigm, splenocyte proliferation and the secretion of IL-2 and IFN- from these cells is reduced, although the cell number of lymphocyte subpopulations in the spleen is unaltered. Because the release of cytokines (e.g., IL-2) from CD4⁺ T cells activates CD8⁺ T cells, which are responsible for mediating allograft rejection in this model (13), it is likely that conditioning contributes to graft prolongation via suppression of the functional capacity of T cells within the spleen.

The current data demonstrate that conditioning does not increase the effectiveness of subtherapeutic CsA via inducing corticosteroid release or altering CsA metabolism. Behavioral paradigms potentially increase corticosteroid secretion as a result of stress. Corticosteroids then potentiate the suppression of immune functions and may synergize with CsA to prolong graft survival (5, 26). However, it is unlikely that stress-induced steroids are responsible for the present effect of subtherapeutic CsA, inasmuch as no differences in plasma corticosterone were observed between groups. It was also possible that conditioning may have been effective by altering CsA metabolism. Certain drugs increase the effectiveness of CsA via increasing its concentration in the blood (9, 24). However, detectable levels of CsA or its metabolites were only observed in CsA-treated rats, who received therapeutic CsA on the three CS re-presentation days.

Taken together, this evidence indicates that splenic innervation is responsible for producing the conditioned alterations in immune function, whereas the cellular mechanism is still unknown. However, a possible clue may be provided by the finding that although behavioral conditioning reduces IL-2 and IFN- in the supernatant of proliferating splenocytes without altering lymphocyte subpopulation cell numbers in the spleen (8), this paradigm actually increases the number of CD4⁺ T cells that are positive for intracellular IFN-. This contrasts with CsA treatment, which leads to the expected lower number of IFN--positive splenocytes. Thus these data indicate that behavioral conditioning promotes a different cellular action than CsA treatment. That is, conditioning acts to inhibit cellular cytokine release, whereas CsA induces its effect via arresting cytokine production. This may explain the synergistic effect of combination subtherapeutic CsA and behavioral conditioning in prolonging heart allograft survival. Similarly, subtherapeutic doses of other immunosuppressants potentiate subtherapeutic CsA, producing significant prolongation of graft survival that is not possible with either drug dose administered in isolation (4, 6, 24).

Although the current data showed that conditioning prolongs heart allograft survival, it cannot rule out the possibility that this effect was produced by the CS-UCS acquisition trials and not by CS re-presentation. However, this is unlikely, inasmuch as both conditioned and sham-conditioned animals received the same stimuli, albeit in different combinations, on CS-UCS acquisition days. Nevertheless, future research should account for this possibility by incorporating a conditioned group that does not receive the CS on re-presentation trials (1, 16, 19).

In summary, the current data show that behavioral conditioning and a subtherapeutic CsA regimen synergize to prolong heart allograft survival. Conditioning prolongs graft survival via a neural mechanism, inasmuch as removal of sympathetic innervation of the spleen abrogates the conditioned effect. The synergistic combination of conditioning and subtherapeutic CsA may result from the distinct alterations of cytokine production. That is, although CsA blocks IL-2 and IFN- production, conditioning may supplement this immunosuppression by limiting extracellular cytokine release.

Perspectives