Highly proliferative and functional PD-1+ and TIM-3+ T cells are transiently increased in multiple myeloma following autologous hematopoietic stem cell transplantation.

The aim of our prospective study was to assess recovery dynamics and functional characteristics of PD-1+ and TIM-3+ T cells in multiple myeloma (MM) patients following high-dose chemotherapy (HDCT) with autologous hematopoietic stem cell transplantation (AHSCT). Peripheral blood, autograft and bone marrow samples were obtained from 46 MM patients before conditioning, at the engraftment, following six and 12 months post-transplant. Frequencies of CD4+ and CD8+ T cells expressing PD-1 and TIM-3 and intracellular expression of Ki-67 and Granzyme B were evaluated. Counts of PD-1+ and TIM-3+ T cells at the engraftment were significantly higher comparing with the levels before HDCT and 6-12 months following AHSCT. The post-transplant increase in the studied subsets was due to a temporary enhancement in proliferation activity. The cytotoxic potential of PD-1- and TIM-3-expressing CD8+ T cells was higher at the engraftment comparing with the pre-transplant and remained at the same level for at least 12 months. The increase in CD4+PD-1+ and CD8+TIM-3+ T cells at the engraftment was associated with higher absolute counts of their reinfused counterparts. Circulating PD-1+ CD8+ and TIM-3+ CD4+ T cells were increased in patients after post-transplant relapse comparing with the ones in remission. Homeostatic proliferation plays a key role in the upregulation of inhibitory checkpoint receptors on functional T cells under lymphopenic conditions. In this regard, it is difficult to predict both the efficacy and adverse reactions of therapy with checkpoint inhibitors on the course of MM after HDCT with AHSCT. Précis. Homeostatic proliferation plays apparently a key role in the upregulation of PD-1 and TIM-3 on functional T cells after AHSCT and appears to be a normal physiological process, contrary to relapse-associated increase in PD-1+ and TIM-3+ T cells.

Quantitative and functional characteristics of circulating and bone marrow PD-1- and TIM-3-positive T cells in treated multiple myeloma patients.

The aim of the present work was to evaluate counts and functional properties of PD-1+ and TIM-3+ T cells in peripheral blood (PB) and bone marrow (BM) of multiple myeloma (MM) patients following the induction therapy. Sixty patients were enrolled in the study, CD4+ and CD8+ T cells expressing PD-1 and TIM-3, intracellular production of IFNγ and intracellular expression of Granzyme B were assessed. Relative counts of the majority of circulating PD-1+, TIM-3+ and PD-1+TIM-3+ T cells were higher in MM patients with disease progression compared with individuals in remission. Frequencies of almost all evaluated PD-1+ and TIM-3+ T cell subsets were higher in BM samples compared with PB; circulating CD4+PD-1+, CD8+PD-1+, CD8+TIM-3+, CD8+PD-1+TIM-3+ T cells positively correlated with the same BM subsets. Circulating CD4+ T cells, expressing PD-1 and TIM-3 (including co-expressing subset), as well as CD8+PD-1+TIM-3+ T cells, and BM CD8+PD-1+ T cells correlated with serum B2-M levels. Sufficient frequencies of GrB+ and IFNγ+ subsets in PD-1-expressing T cells indicated their retained functional properties. TIM-3-expressing T cells and double positive PD-1+TIM-3+ populations showed diminished cytotoxic and cytokine-producing ability and therefore might be attributed to the exhausted compartment. To identify T cell exhaustion, it is necessary to evaluate T cells co-expressing PD-1, TIM-3 and other inhibitory signal molecules and to study their functional properties. Sustained functionality of PD-1-positive T cells may explain low efficacy and frequent immune-mediated adverse events during anti-PD-1 therapy in MM.

Increased circulating CD3+ T cells are associated with early relapse following autologous hematopoietic stem cell transplantation in patients with classical Hodgkin lymphoma.

Non-malignant host immune cells are the main substrate in classical Hodgkin lymphoma (HL) microenvironment. Reconstitution of lymphocyte populations following the high-dose chemotherapy (HDC) with autologous hematopoietic stem cell transplantation (auto-HSCT) can support tumor growth in HL patients. We investigated recovery dynamics of circulating CD3+, CD4+, CD8+, CD16+/CD56+, CD19+, CD4+FOXP3+ lymphocytes following auto-HSCT in 79 HL patients and assessed relationship between these populations and the development of early relapse. Studied populations were not statistically significant between patients with high or standard/intermediate risk of relapse. CD3+ T cells at the time of engraftment were increased in patients with the early relapse of HL compared to non-relapsed patients (PU = 0.0028). Area under the curve was 0.76 (р = .0037). In logistic regression models, CD3+ T cell count was associated with early relapse/progression as a trend. These findings elucidate several interactions between early systemic T cell recovery and tumor progression following HDC with auto-HSCT.

Increased circulating CD4+FOXP3+ T cells associate with early relapse following autologous hematopoietic stem cell transplantation in multiple myeloma patients.

We investigated dynamics of CD4+FOXP3+ T cell recovery following the high-dose chemotherapy (HDC) with autologous hematopoietic stem cell transplantation (auto-HSCT) in multiple myeloma (MM) patients. Circulating CD4+FOXP3+ T cells of 79 MM patients were evaluated using flow cytometry before HDC with auto-HSCT, at the day of engraftment, and following 6 and 12 months. Percentage of CD4+FOXP3+ T cells restored rapidly following auto-HSCT, became higher than pre-transplant level at the day of engraftment and then subsequently decreased for a year. CD4+FOXP3+ T cells at the time of engraftment were increased in patients with the relapse or progression of MM during 12 months following auto-HSCT (n=10) compared to non-relapsed patients (n=50): 6.7% (5.3-8.9%) vs 4.9% (2.8-6.6%); PU = 0.026. Area under the curve was 0.72 (95% CI: 0.570-0.878; р=0.026). Circulating CD4+FOXP3+ T cell count was not associated with the percentage of myeloma plasma cells in a bone marrow but depended on its amount in autografts. CONCLUSIONS: Relative count of CD4+FOXP3+ T cells restored rapidly following auto-HSCT (at the day of engraftment), became higher than pre-transplant level and then subsequently decreased for a year. Their excess at the time of engraftment is associated with early relapse.

CD4+ memory T cells retain surface expression of CD31 independently of thymic function in patients with lymphoproliferative disorders following autologous hematopoietic stem-cell transplantation.

High-dose chemotherapy with autologous hematopoietic stem-cell transplantation (AHSCT) causes severe and long-lasting immunodeficiency in patients with lymphoproliferative disorders. The thymus begins to restore the T-cell repertoire approximately from the sixth month post-transplant. We assessed the dynamics of post-transplant recovery of CD4+CD45RA+CD31+ T cells, "recent thymic emigrants" (RTEs), and a poorly described subtype of CD4+CD45RA-CD31+ T cells in 90 patients with lymphoproliferative disorders following high-dose chemotherapy with AHSCT. Relative and absolute counts of CD4+CD31+ naïve and memory T cells were evaluated before AHSCT, at the day of engraftment, and 6- and 12-month post-transplant. The pre-transplant count of CD4+CD45RA+CD31+ T cells was lower than in healthy controls, and did not reach donors' values during the 12-month period. The pre-transplant number of CD4+CD45RA-CD31+ T cells was higher than in healthy controls and was restored rapidly following AHSCT. Post-transplant mediastinal radiotherapy reduced counts of RTEs and elongated recovery period. Non-thymic tissue irradiation did not reduce this subset. The obtained data indicate that homeostatic proliferation may decrease the significance of CD31 expression on CD4+CD45RA+ T cells as a marker of RTEs, and suggest that evaluation of RTEs recovery by flow cytometry requires an accurate gating strategy to exclude CD31+ memory T cells.

Gene expression profiling of tumor-initiating stem cells from mouse Krebs-2 carcinoma using a novel marker of poorly differentiated cells.

Using the ability of poorly differentiated cells to natively internalize fragments of extracellular double-stranded DNA as a marker, we isolated a tumorigenic subpopulation present in Krebs-2 ascites that demonstrated the features of tumor-inducing cancer stem cells. Having combined TAMRA-labeled DNA probe and the power of RNA-seq technology, we identified a set of 168 genes specifically expressed in TAMRA-positive cells (tumor-initiating stem cells), these genes remaining silent in TAMRA-negative cancer cells. TAMRA+ cells displayed gene expression signatures characteristic of both stem cells and cancer cells. The observed expression differences between TAMRA+ and TAMRA- cells were validated by Real Time PCR. The results obtained corroborated the biological data that TAMRA+ murine Krebs-2 tumor cells are tumor-initiating stem cells. The approach developed can be applied to profile any poorly differentiated cell types that are capable of immanent internalization of double-stranded DNA.

A strategy to eradicate well-developed Krebs-2 ascites in mice.

We describe the strategy, which allows curing experimental mice engrafted with Krebs-2 ascites. The strategy is based on the facts that i) Krebs-2 tumor-initiating stem cells (TISCs) are naturally capable of internalizing fragments of extracellular double-stranded DNA (dsDNA); ii) upon delivery into TISCs, these dsDNA fragments interfere with the on-going DNA repair process so that TISCs either die or lose their tumorigenic potential. The following 3-step regimen of therapeutic procedures leading to eradication of Krebs-2 ascites is considered. Firstly, three timed injections of cyclophosphamide (CP) exactly matching the interstrand cross-link (ICL) repair phases that lead to synchronization of ascites cells in late S/G2/M. Secondly, additional treatment of ascites 18 hours post each CP injection (at NER/HR transition timepoint) with a composite dsDNA-based preparation interfering with the NER and HR repair pathways, so that tumorigenic properties of ascites cells are compromised. Thirdly, final treatment of mice with a combination of CP and dsDNA injections as ascites cells undergo apoptotic destruction, and the surviving TAMRA+ TISCs arrested in late S/G2/M phases massively enter into G1/S, when they regain sensitivity to CP+dsDNA treatment. Thus, this regimen assures that no viable cells, particularly Krebs-2 TISCs, remain.

Intraocular cytokines in retinal vein occlusion and its relation to the efficiency of anti-vascular endothelial growth factor therapy.

PURPOSE: To analyze the change in the concentration of intraocular cytokines (ICs) in patients with retinal vein occlusion (RVO) before and after intravitreal ranibizumab therapy (IVR), and to find the correlations of IC with clinical activity of RVO and efficiency of treatment. MATERIALS AND METHODS: Forty-four patients aged 46-79 years old (mean age: 60.7 ± 7.5 years old) with RVO and macular edema (18 patients - with central RVO, 26 - with branch RVO) treated with IVR were included into the study. The concentrations of 27 cytokines were simultaneously measured in aqueous humor by flow fluorometry using Bio-Plex Pro Human Cytokine Panel, 27-Plex (Bio-Rad Laboratories, USA) at baseline and after the first IVR. Control group consisted of 20 age-matched patients. RESULTS: The levels of 11 cytokines (vascular endothelial growth factor [VEGF], receptor antagonist interleukin-1, interleukin-6 [IL-6], IL-8, IL-9, IL-10, IL-12r70, IL-13, IL-15, monocyte chemotactic protein-1 [MCP-1], regulated on activation, normal T expressed and secreted) were significantly (P < 0.05) different compared to control and significantly (P < 0.05) changed after IVR both in central and branch RVO. The patients were divided into two groups: the first -"effective" and the second - "partially effective" therapy. The second group characterized by the higher concentrations of VEGF, IL-8, IL-10, IL-17, and MCP-1 at baseline compared to the first group. CONCLUSION: The patients with RVO were characterized by the increased levels of VEGF and other pro- and anti-inflammatory cytokines and chemokines. Aqueous concentration of cytokines were different in patients with central and branch RVO and significantly changed after IVR. Insufficient response to IVR was associated with activation of immune-inflammatory processes.

Interferon alpha induces generation of semi-mature dendritic cells with high pro-inflammatory and cytotoxic potential.

Dendritic cell-based vaccines are considered as a new and promising immunotherapeutic approach for cancer treatment. However, the choice of optimal protocol of dendritic cell generation in vitro represents the major challenge. Here, we compared phenotype and functional characteristics of human monocyte-derived dendritic cells (DCs) generated in the presence of IL-4/GM-CSF (IL4-DCs) and IFNα/GM-CSF (IFN-DCs). We showed that IFN-DCs displayed semi-mature phenotype and expressed higher level of CD123, TNF-related apoptosis-inducing ligand (TRAIL) and B7-H1 molecules in comparison with IL4-DCs. LPS-stimulated IFN-DCs were characterized by greater production of Th1/pro-inflammatory (IFN-γ, IL-2, IL-1ß, TNF-α, IL-17), Тh2/anti-inflammatory cytokines (IL-10, IL-5), hematopoietic growth factors (G-CSF) and chemokines (MCP-1). These data indicated more pronounced ability of IFN-DCs to induce cellular immune response as well as humoral immune response compared to IL4-DCs. LPS-stimulated IFN-DCs possessed higher direct cytotoxic activity against TRAIL-sensitive tumor cell line Jurkat and similar cytotoxicity against TRAIL-resistant tumor HEp-2 cells. Besides, IFN-DCs and IL4-DCs equally induced apoptosis of activated CD4(+) and CD8(+) T cells. These results suggest that IFN-DCs can be used as potent cell-based curative therapies for individuals with cancer.

Clinical experience with autologous M2 macrophages in children with severe cerebral palsy.

Stem cell-based therapy is considered to be a new approach for the treatment of cerebral palsy (CP). Given the potent anti-inflammatory activity and high regenerative potential of M2 macrophages, these cells may be an alternative source for cell transplantation. To evaluate the safety and efficacy of autologous M2 macrophages, we conducted a pilot clinical trial in 21 children with severe CP. The primary outcome measure was safety, which included assessment of mortality of any cause, immediate adverse reactions, and serious adverse effects and comorbidities during 5-year follow-up. The secondary outcome measure was functional improvement in Gross Motor Function Measure (66-item GMFM) test, Peabody Developmental Motor Scale-Fine Motor (PDMS-FM) test, Ashworth scale, MRC scale, and an easy-to-understand questionnaire for evaluation of cognitive functions in our modification. Intradural injection of M2 cells (in mean dose of 0.8 × 10(6)/kg) into the lumbar spinal area did not induce any serious adverse events. No cases of mortality, psychomotor worsening, exacerbation of seizures, and long-term comorbidities, including tumors, were observed during a 5-year follow-up. After 3 months, GMFM score increased from 13.7 ± 7.8 to 58.6 ± 14.6, PDMS-FM score improved from 0.76 ± 0.42 to 5.05 ± 0.97, and the Ashworth score decreased from 3.8 ± 0.21 to 3.3 ± 0.24. Along with gross and fine motor function enhancement, an improvement of cognitive activity (from 1.62 ± 0.41 to 4.05 ± 0.64, according to questionnaire assessment) and reduction of seizure syndrome were registered as well. The neurological improvements did not diminish during the 5-year follow-up period. The data obtained suggest that cell therapy based on M2 macrophages is safe, does not induce early adverse effects and long-term comorbidities, and is accompanied with a significant improvement of motor and cognitive activities in severe CP patients. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation.

Identification of cancer stem cells and a strategy for their elimination.

It has been established previously that up to 40% of mouse CD34(+) hematopoietic stem cells are capable of internalizing exogenous dsDNA fragments both in vivo and ex vivo. Importantly, when mice are treated with a combination of cyclophosphamide and dsDNA, the repair of interstrand crosslinks in hematopoietic progenitors is attenuated, and their pluripotency is altered. Here we show for the first time that among various actively proliferating mammalian cell populations there are subpopulations capable of internalizing dsDNA fragments. In the context of cancer, such dsDNA-internalizing cell subpopulations display cancer stem cell-like phenotype. Furthermore, using Krebs-2 ascites cells as a model, we found that upon combined treatment with cyclophosphamide and dsDNA, engrafted material loses its tumor-initiating properties which we attribute to the elimination of tumor-initiating stem cell subpopulation or loss of its tumorigenic potential.

Cytotoxic activity of ex-vivo generated IFNα-induced monocyte-derived dendritic cells in brain glioma patients.

Recent studies have revealed that besides the important role in triggering the adoptive antitumor immunity, dendritic cells (DCs) possess direct cytotoxic antitumor activity. Here, we investigated brain glioma patient monocyte-derived DCs generated in the presence of IFNα and GM-CSF (IFN-DCs). These DCs were characterized by reduced cytotoxic activity against TRAIL-resistant HEp-2 cells. The impairment of DC cytotoxic function was observed mainly in high-grade glioma patients and associated with poor survival. The dysfunction of patient DC cytotoxicity was partially restored under in vitro pretreatment of DCs with double-stranded human DNA as well as rIL-2. In contrast to healthy donors, IFN-DCs in a part of high-grade glioma patients also failed to lyse primary autologous or allogeneic glioma cells. Our findings point to possible contribution of DC impairment in tumor pathogenesis in brain glioma and justify the necessity to evaluate and correct DC cytotoxic function when exploring DCs as cancer vaccines in glioma.

Cytotoxic activity of dendritic cells as a possible mechanism of negative regulation of T lymphocytes in pulmonary tuberculosis.

The PD-1/B7-H1-mediated induction of T cell apoptosis/anergy as a possible mechanism of immune response failure was studied in 76 patients with pulmonary tuberculosis (TB) with normal and low-proliferative response to antigens of M. tuberculosis (purified protein derivative (PPD)). It was revealed that dendritic cells (DCs), generated in vitro from patient blood monocytes with GM-CSF + IFN-α, were characterized by increased B7-H1 expression, upproduction of IL-10, and reducing of allostimulatory activity in mixed lymphocyte culture (MLC). Moreover, DCs of patients with TB were able to enhance T cell apoptosis and to block T-cell division in MLC. It was shown that neutralizing anti-PD1 antibodies significantly decreased the proapoptogenic/tolerogenic effect of DCs. Correlation analysis revealed a direct relationship between IL-10 production and level of B7-H1 expression in the general group of investigated patients. It was demonstrated that generation of healthy donor DCs in the presence of IL-10 led to an increase in the number of DCs-expressed B7-H1 molecule, DC proapoptogenic activity, and a decrease in their allostimulatory activity. Obviously, the revealed phenomenon of the PD-1/B7-H1-mediated pro-apoptogenic activity of DCs is clinically significant since the cytotoxic/tolerogenic potential of DCs is more pronounced in patients with PPD anergy.

Peripheral T Cell Apoptosis and Its Role in Generalized Bacterial Infections: A Minireview.

In the present review we have attempted to analyze recent findings concerning apoptosis of mature peripheral T cells. The great attention is made to the factors underlying resistance or sensitivity of mature T lymphocytes to activation-induced cell death. The role of preactivation and altered costimulation is discussed in this regard. Besides, the possible role of cytokines in the modulation of T cell apoptosis is emphasized. Particular attention is paid to the studies of apoptosis disorders in the pathogenesis of generalized bacterial infections. In this connection some own results are summarized as well. To characterize T cell death and its role in the pathogenesis of bacterial infections an anti-CD3-mAb or Con A-induced apoptosis in patients with severe and generalized forms of surgical infections have been investigated. We have found a significant increase of activation-induced lymphocyte apoptosis and a high level of apoptosis in freshly isolated lymphocytes in patients with surgical infections. Alternatively, peripheral blood mononuclear cells from surgical patients without infectious complications did not exhibit a marked enhancement of activation-induced cell death. Activation-induced T cell death in surgical infections appeared to be Fas-dependent, involved reactive oxygen intermediates and was partly prevented by pro-inflammatory cytokines, among which IL-2 exhibited the most pronounced anti-apoptotic activity. Likewise, APACHE II score, as a marker of the infection severity, directly correlated with a rate of activation-induced T cell apoptosis. Accelerated T cell apoptosis at the early stage of infection was revealed in survivors and non-survivors, that appears to designate a common pathway for the restriction of systemic inflammation. At the late stage of infection altered T cell apoptosis could account for different outcomes, since the patients with lethal outcome showed 2-fold increase in activation-induced cell death compared to the opposite group. Immunotherapy with rIL-2 declined anti-CD3-induced apoptosis and promoted a reduction in the mortality rate from 29% to 13%.

T Cell Functional Disturbances in Patients with Pulmonary Tuberculosis.

The investigations of 38 patients with pulmonary tuberculosis (PT) revealed combined T cell and monocyte functional disturbances. Indeed, the percentages of CD4(+) and CD8(+) T lymphocytes, proliferative response and IL-2 production, as well as the percentages of HLA DR(+) monocytes and IL-1beta production were significantly decreased in PT patients as compared with normal individuals. Herewith the absolute T lymphocyte number did not undergo the pronounced changes. The decrease of T cell proliferative response was not mediated through immunosuppressive action of monocytes or T lymphocytes since removing of "adherent" cells from patient's peripheral blood mononuclear cells (PBMC) or pretreatment of PBMC with indomethacin and cyclophosphan failed to recover mitogenic reactivity in vitro. The patient's sera also did not significantly influence on PBMC proliferation. The decrease of IL-2 production and the stimulation of T cell proliferative response via TcR-CD3 complex, i.e. through the classic pathway of activation, indicated the anergy of T lymphocyte in tuberculosis patients. Furthermore, T lymphocytes were characterized by enhanced apoptosis. It should be noted, that patient's sera (especially in the patients with an initially high apoptosis) promoted significant anti-apoptotic activity. It is likely that this mechanism may be an explanation, why absolute T lymphopenia is absent during tuberculosis infection. Our findings suggest, that T lymphocyte dysfunctions in patients with PT are caused by impairments of T cell activation process, which lead to predominance of "negative" response (induction anergy, apoptosis) and to a lesser degree connected with direct suppressive mechanisms mediated by monocytes, T lymphocytes or serum factors.

Inflammatory Syndromes (SIRS, MARS, CARS) in Patients with Surgical Infection.

In the present study 37 patients with surgical infection were investigated and a new set of diagnostic tests for detection of major syndromes of systemic inflammation - systemic inflammatory response syndrome (SIRS), compensatory anti-inflammatory response syndrome (CARS) and mixed antagonist response syndrome (MARS) - was developed. In summary, we have demonstrated that patients with surgical infection were characterized by an immunodeficiency with significant reduction of mitogen-induced proliferation and IL-2/IL-4 production in vitro combined with decrease of HLA DR(+) monocytes. Furthermore, it was revealed that the patient's serum exhibited substantially enhanced suppressive and inflammatory activities as well as the level of C-reactive protein. We have defined the negative correlation between the serum inflammatory and suppressive activities (SIA and SSA) that was most prominent at the early stage of disease. Since the changes of serum bioactivity in the course of surgical infection were prominent and coherent, we supposed that tested activities might reflect the distinctive features of systemic inflammation. In according to this assumption, the patients were divided into 3 subgroups with predominance of SIRS, CARS and MARS by using the SIA and SSA expression. It has been shown that SIRS was more frequently detected at the early stage, whereas CARS - at the late stage of disease. Patients with SIRS, CARS or MARS significantly differed by the content of CD8(+) T and CD72(+) B lymphocytes, the concentration of IgG and IgA, the production of IL-2 and IL-4. Finally, the data obtained from patients, those were studied repeatedly, showed the possibility of transformation of the major systemic inflammatory syndromes during the disease course. Our findings suggest that measurement of serum inflammatory and suppressive activities may help to differentiate patients with SIRS, CARS or MARS and to select the appropriate strategy of immunotherapy.

IL-2-Activated Killer Cells and Native Cytokines in Treatment of Patients with Advanced Cancer.

We evaluated the efficiency/tolerability of and the immunological changes induced by the adoptive immunotherapy (AIT) with IL-2-activated killer cells, and preparation of native cytokines from swine spleen (PSS) in treatment of 20 patients with advanced cancer (10 patients with primary lung cancer; 3 with metastatic melanoma; 2 with advanced neuroblastoma; 2 with ovarian cancer; renal cancer; gastric adenocarcinoma; and colorectal cancer). The partial/minor response of duration period 2-10 months was observed in 20% of patients. 2/4 patients, who underwent partial surgical tumor resection and following AIT course, sustained the event-free survival for more than 24 months. The response to the therapy was revealed in 4/10 patients with lung cancer, 2/2 patients with neuroblastoma, of whom each had ovarian and colorectal cancers. The evaluation of a dose of infused LAKcells as well as combined i.v./local (endobronchial or endoperitoneal) LAK administration were necessary to assure positive response in patients. The cytokine and/or side effects were moderate and the combined LAK-PSS infusions were generally well tolerated by the patients. The treatment was followed by activation of the patient immune system that included: (i) rebound in amount of peripheral blood lymphocytes; (ii) gain in amount of CD3(+) T cells and those CD4(+) helper/inducer; (iii) enchantment of lymphocyte proliferation and cytokine production (IL-2, IL-1, TNF-alpha). Being injected to patients in combination with LAK cells, cytokines related to PSS action and/or those, either exogenous or secondary, and released by in vitro and in vivo, activated lymphocytes and could cause the therapeutic effects.