Predicting and controlling the reactivity of immune cell populations against cancer.

Heterogeneous cell populations form an interconnected network that determine their collective output. One example of such a heterogeneous immune population is tumor-infiltrating lymphocytes (TILs), whose output can be measured in terms of its reactivity against tumors. While the degree of reactivity varies considerably between different TILs, ranging from null to a potent response, the underlying network that governs the reactivity is poorly understood. Here, we asked whether one can predict and even control this reactivity. To address this we measured the subpopulation compositions of 91 TILs surgically removed from 27 metastatic melanoma patients. Despite the large number of subpopulations compositions, we were able to computationally extract a simple set of subpopulation-based rules that accurately predict the degree of reactivity. This raised the conjecture of whether one could control reactivity of TILs by manipulating their subpopulation composition. Remarkably, by rationally enriching and depleting selected subsets of subpopulations, we were able to restore anti-tumor reactivity to nonreactive TILs. Altogether, this work describes a general framework for predicting and controlling the output of a cell mixture.

Minimally cultured or selected autologous tumor-infiltrating lymphocytes after a lympho-depleting chemotherapy regimen in metastatic melanoma patients.

Adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL) and high-dose interleukin-2 (IL-2), after nonmyeloablative chemotherapy, has been shown to result in tumor regression in half of refractory metastatic melanoma patients. In the present study, we describe 2 separate clinical protocols. Twelve patients were treated with "Selected"-TIL, as previously reported and 8 patients with the modified version of "Young"-TIL. Selected-TIL protocol required the establishment of multiple T-cell cultures from 1 patient and in vitro selection of cultures secreting interferon-gamma upon antigenic stimulation. In contrast, Young-TIL are minimally cultured T cells with superior in vitro features that do not require further selection. Two of 12 Selected-TIL patients experienced objective clinical responses (1 complete response, 1 partial response). Out of 8 treated Young-TIL patients, 1 experienced complete response, 2 partial response, and 4 patients had disease stabilization. Twenty-one of 33 enrolled Selected-TIL patients were excluded from the protocol, mainly as cultures failed the interferon-gamma selection criteria or due to clinical deterioration, compared with only 3 Young-TIL patients. Expected bone marrow suppression and high-dose IL-2 toxicity were transient. There was no treatment-related mortality. This study vindicates the feasibility and effectiveness of TIL technology and calls for further efforts to implement and enhance this modality. The use of minimally cultured, unselected Young-TIL enables the treatment of most enrolled patients. Although the cohort of Young-TIL patients treated so far is rather small and the follow-up short, the response rate is encouraging.

Collection of large-scale expanded lymphocyte cultures for adoptive immunotherapy using a COBE Spectra apheresis machine.

Adoptive cell transfer represents an important mode of cancer immunotherapy and posttransplant associated viral infections. This technique involves massive volume reduction, as the procedure starts with large-scale ex vivo expansion of lymphocyte cultures (volume up to 60 L), which are harvested at the end of the in vitro process and terminates in a small volume to be infused into the patient. Toward this end, we develop a novel efficient process based on the COBE Spectra apheresis machine usually used for apheresis process. As the COBE Spectra cell separator is easy to use and often available at medical centers, this novel technique is applicable to many transplant and cell processing centers. Our results show a high recovery yield (98%+/-15%) and viability (ranged 79% to 99%) of the large-scale expanded lymphocytes. It preserves sterility of the product and is therefore suitable for immunotherapy treatments.

Inhibition of AC-II activity following chronic agonist exposure is modulated by phosphorylation.

Chronic exposure to opiate agonists (followed by agonist withdrawal) leads to a large increase in the activity of adenylyl cyclase (AC) isozymes I, V, VI, and VIII, a phenomenon defined as AC superactivation (or supersensitization). On the other hand, AC isozymes belonging to the AC-II family (AC-II, AC-IV, and AC-VII) show decreased activity, referred to as superinhibition. Using COS-7 cells transiently transfected with mu-opioid receptor and AC-II, we show here that inhibition of PKC and tyrosine kinase activities synergistically reduced the level of AC-II superinhibition. Moreover, inhibitor of Raf-1 kinase also led to a decrease in AC-II superinhibition. These data suggest that Raf-1, activated by PKC and tyrosine kinase, has a role in the regulation of AC-II superinhibition.

Adenylyl cyclase type II activity is regulated by two different mechanisms: implications for acute and chronic opioid exposure.

Acute and chronic activation of opioid receptors differentially regulate the activity of the various adenylyl cyclase (AC) isoforms. In several AC isoforms (I, V, VI and VIII) acute opioid activation (by agonists such as morphine) leads to AC inhibition, while prolonged opioid activation leads to increase in AC activity, a phenomenon known as AC sensitization or superactivation. In several other AC isoforms (II, IV and VII), acute opioid activation leads to AC stimulation, while chronic opioid exposure inhibits AC activity, in a process, which in analogy to the term "superactivation" is referred to as "superinhibition". AC-II is highly regulated by multiple and independent biochemical stimuli, including Gbetagamma, Galphas and PKC activation. We investigated the regulation of AC-II by Galphas and by PKC under conditions of acute and chronic exposure to opioid agonists in COS-7 transfected cells. We found that acute opioid exposure led to an increase in AC-II activity by either Galphas or PKC stimulation. This effect seems to be regulated by Gbetagamma subunits, in both activation pathways, as the increase in AC-II activity was abolished by pertussis toxin treatment and by Gbetagamma scavengers. On the other hand, while chronic opioid exposure led to a decrease in AC-II activity ("superinhibition") upon stimulation of the Galphas pathway, this superinhibition was not observed when the opioid treated cells were stimulated via PKC activation.

Adenylyl cyclase type-VIII activity is regulated by G(betagamma) subunits.

The Ca2+-activated adenylyl cyclase type VIII (AC-VIII) has been implicated in several forms of neural plasticity, including drug addiction and learning and memory. It has not been clear whether Gi/o proteins and G-protein coupled receptors regulate the activity of AC-VIII. Here we show in intact mammalian cell system that AC-VIII is inhibited by mu-opioid receptor activation and that this inhibition is pertussis toxin sensitive. Moreover, we show that G(betagamma) subunits inhibit AC-VIII activity, while constitutively active alphai/o subunits do not. Different Gbeta isoforms varied in their efficacies, with Gbeta1gamma2 or Gbeta2gamma2 being more efficient than Gbeta3gamma2 and Gbeta4gamma2, while Gbeta5 (transfected with gamma2) had no effect. As for the Ggamma subunits, Gbeta1 inhibited AC-VIII activity in the presence of all gamma subunits tested except for gamma5 that had only a marginal activity. Moreover, cotransfection with proteins known to serve as scavengers of Gbetagamma dimers, or to reduce Gbetagamma plasma membrane anchorage, markedly attenuated the mu-opioid receptor-induced inhibition of AC-VIII. These results demonstrate that Gbetagamma (originating from agonist activation of these receptors) and probably not Galphai/o subunits are involved in the agonist inhibition of AC-VIII.

Inhibition and superactivation of the calcium-stimulated isoforms of adenylyl cyclase: role of Gbetagamma dimers.

It was shown previously that chronic exposure to opiate agonists increases adenylyl cyclase (AC) activity, a phenomenon termed AC superactivation (or supersensitization). More recently, we showed that acute Gi/o- coupled receptor activation inhibits the activity of several AC isozymes, including Ca2+/calmodulin-stimulated AC-I and -VIII, whereas chronic receptor activation induces their superactivation. Here, we report that both acute Mu-opioid receptor-induced inhibition and chronic induced superactivation of AC-I and -VIII are pertussis toxin sensitive. In addition, we show that proteins that interfere with the activity of Gbetagamma subunits (Gbetagamma scavengers) strongly attenuate the acute inhibition of AC-I and -VIII and the superactivation of AC-I, and abolish the superactivation of AC-VIII. Based on these results, we suggest that Gbetagamma is involved in the acute inhibition and chronic agonist-induced superactivation of AC types I and VIII.

Regulation of adenylate cyclase type VIII splice variants by acute and chronic Gi/o-coupled receptor activation.

We previously reported that acute agonist activation of G(i/o)-coupled receptors inhibits adenylate cyclase (AC) type VIII activity, whereas agonist withdrawal following chronic activation of these receptors induces AC-VIII superactivation. Three splice variants of AC-VIII have been identified, which are called AC-VIII-A, -B and -C (with AC-VIII-B missing the glycosylation domain and AC-VIII-C lacking most of the C1b area). We report here that AC-VIII-A and -B, but not -C, are inhibited by acute mu-opioid and dopaminergic type D2 receptor activation, indicating that the C1b area of AC-VIII has an important role in AC inhibition by G(i/o)-coupled receptor activation. On the other hand the glycosylation sites in AC-VIII did not play a role in AC-VIII regulation. Although AC-VIII-A and -C differed in their capacity to be inhibited by acute agonist exposure, agonist withdrawal after prolonged treatment led to a similar superactivation of all three splice variants, with no significant change in AC-VIII expression. AC-VIII superactivation was not affected by pre-incubation with a cell permeable cAMP analogue, indicating that the superactivation does not depend on the agonist-induced reduction in cAMP levels. The superactivated AC-VIII-A, -B and -C were similarly re-inhibited by re-application of agonist (morphine or quinpirole), returning the activity to control levels. These results demonstrate marked differences in the agonist inhibition of the AC-VIII splice variants before, but not after, superactivation.