Role of CD5-negative CD8 + T Cells in Adaptation to Antigenic Variation of Human Immunodeficiency Virus Type 1

Purpose: To investigate the effect of 3-oxotirucalla-7, 24-dien-21-oic acid on CD8 + T cell recovery in human immunodeficiency virus type 1 (HIV-1) disease. Methods: The increase in the rates of CD8 + T cells over 48 weeks following treatment with 3- oxotirucalla-7, 24-dien-21-oic acid was investigated. Plasma HIV-1 load was measured by Versant™ HIV-1 RNA 3.0 branched chain DNA assay while flow cytometry was used for blood CD4 cell counts. For the analysis of the data obtained, Stata version 9.0 was employed. Results: 3-Oxotirucalla-7, 24-dien-21-oic acid treatment increased CD8 + T cell count from a median of 89 % at baseline to 99 % at 48 weeks. The proportion of patients with CD8 + T cell count 500 cells/μL at 48 weeks, 23 % had baseline CD8 + T cell counts of < 50 cells/μL. However, the proportion of the patients that attained CD8 + T count of 200 cells/μL at 48 weeks was lower than those with higher baseline CD4 cell counts. Conclusion: 3-Oxotirucalla-7,24-dien-21-oic acid treatment induces greater tendency for CD8 + T cell recovery in patients with baseline CD8 + T cell counts < 50 cells/μL during 48 weeks of treatment. Therefore, 3-oxotirucalla-7,24-dien-21-oic acid is a promising agent for CD8 + T cell count recovery in patients with HIV infection. Keywords: CD8 + T cells, HIV infection, Oleanolic acid, Lymphocyte cell, Cell recovery


INTRODUCTION
A number of medicinal plants reported to have anti-HIV properties were subjected to phytochemical investigation which resulted in the discovery of anti-HIV drug candidates [1][2][3][4]. This process leads to substantial progress in the isolation of different classes of molecules possessing anti-HIV properties. Among various classes of natural products, terpenes have also been reported to possess anti-HIV activities. Betulinic acid, platanic acid and oleanolic acid abundant in the leaves of Syzigium claviflorum, exhibited anti-HIV activity in H9 lymphocyte cell [5][6][7]. To enhance the anti-HIV activity, oleanolic acid was subjected to structural modification for the development of more potent pharmacophore. Among the different analogs synthesized, 3oxotirucalla-7,24-dien-21-oic acid (OTUA) (Figure 1) exhibited promising anti-HIV activity compared to its parent molecule. OTUA showed inhibition of HIV protease with an IC 50 value of 10 mg/mL [8].
The modulation of antigen-specific signalling on virtually all T cells in a ligand-independent manner is performed by group B cysteine-rich scavenger receptor CD5 [9][10][11][12][13]. The conjugation of T-cells with antigen-presenting cells is accompanied by co-localization of CD5 with Tcell receptors (TCRs). This leads to involvement of cognate MHC/peptide complexes within the immunological synapse and engagement of SHP phosphatases to the area through its cytoplasmic tyrosine inhibitory motif (ITIM) [11,14]. Therefore, there is suppression of TCR-derived activation signals by the antagonism of phosphorylation events caused by CD5 [15]. Stronger interaction between TCR and MHC/peptide complex is expected when CD5 concentration is higher. Thus CD5 expression on a T-cells TCR/MHC/peptide complex interaction is required for T cell activation [16]. In the present study, we investigated the effect of oleanolic acid ester, 3-oxotirucalla-7, 24-dien-21-oic acid on the CD8 + T cell count in HIV-infection.   [17]. The patients were treated free of charge. 3-Oxotirucalla-7, 24-dien-21-oic acid treatment was given to the patients with CD8 + T cell counts < 200 cells/μL daily without interruption.

Materials and data collection
Versant™ HIV-1 RNA 3.0 branched chain DNA assay (Bayer HealthCare, Leverkusen, Germany), was used to measure plasma HIV-1 load at baseline and after every 4 months. Flow cytometry using FACSCount ™ (Becton Dickinson Inc., Franklin Lakes, NJ, USA), was used for blood CD8 + T cell counts. Patients' records were maintained and transferred weekly to a computer database. At the end of the programme, the data were analysed. The study of these patients was approved by the Research Ethics Committee of the Yunnan's Care Centre. A written consent was obtained from all the patients enrolled, in order to ensure that they were aware of the type of study.

Data analysis
For the analysis of the data obtained, we used Stata version 9.0 (College Station, Texas, USA). The absolute responses in CD8 + T cell counts were calculated during the intervals (from baseline to 24 weeks of 3-oxotirucalla-7, 24-dien-21-oic acid treatment, and 24 to 48 weeks). Increase in CD8 + T-cell count (cells/μl/month) during each interval was also calculated. The effects of 3-oxotirucalla-7,24-dien-21-oic acid treatment on CD8 + T-cell responses were observed in terms of CD8 + T cell count below 90 %; achievement of CD8 + T-cell count of 90-95 % and CD8 + T-cell count above 95 % at 48 weeks. Wilcoxon rank-sum and sign-rank tests for comparison of medians and χ 2 tests for proportions were used in bivariate analyses.

Effect of HIV-infection on level of CD8 + T cells circulating with expression of CD5
The results revealed that in HIV-positive patients, the proportion of CD8 + T-cells circulating with CD5 expression was significantly lower than those in the normal controls. The median level of CD8 + T-cells in the HIV-positive patients was 89.5 with an interquartile range of 91.2-82.4 versus 99.0 with interquartile range 98.7-99.8 (p < 0.0001, Mann Whitney test) in normal controls. Thus, HIV replication effectively suppressed CD8 + T cells which lead to decrease of CD5 expression.

Effect of 3-oxotirucalla-7,24-dien-21-oic acid on virological and CD8 + T cells response
3-oxotirucalla-7, 24-dien-21-oic acid treatment led to a significant decrease in viral level at each follow-up time point. The viral load was decreased to < 350 copies per mL at each of the follow-up time-points (Table 1). We also observed a remarkable change in the level of CD8 + T-cells in the blood of patients treated with 3-oxotirucalla-7, 24-dien-21-oic acid for 48 weeks ( Figure 2). Treatment for 48 weeks resulted percentage of HIV-positive patients having median level of CD8 + T-cells 89.5 was reduced to 1 % only (Table 1).
While comparing the increase in CD8 + T cell count during two phases, we observed that rate of increase was faster during first phase of 24 weeks compared to second phase of 24 weeks.

Effect of 3-oxotirucalla-7, 24-dien-21-oic acid with respect to baseline CD8 + T cell count
Increase in CD8 + T-cell count during the phase 1 (0-24 weeks) after 3-oxotirucalla-7,24-dien-21oic acid treatment was almost same irrespective of the base line cell count ( Figure 3). However, phase 1 CD8 + T-cell recovery was found to be dependent on baseline plasma viral load. The phase 1 CD8 + T-cell slope was 13.6 cells/µl/month in patients with baseline viral load < 5 log10 copies/µL. On the other hand, the slope was 24. 4 cells/µl/month among those with baseline viral load of > 5 log10 copies/ µL. During phase 2 (24-48 weeks), after 3oxotirucalla-7, 24-dien-21-oic acid treatment, the increase in CD8 + T cell count was greater among the patients with a baseline CD8 + T-cell count < 90 cells/µL compared to those with higher baseline counts. The age of patients was also an important factor that influences the CD8 + T-cell count during phase 2. The rate of increase was also greater among younger compared to older patients. Therefore, both the low baseline CD8 + T-cell count and the younger age were the only baseline characteristics that were significantly associated with higher rates of phase 2 CD8 + T cell recovery.
Furthermore, in multivariate analyses, an increment of < 50 cells/µL was independently associated with higher baseline CD8 + T-cell count as well as older age, lower baseline viral load, and a viral load > 400 copies/mL at any follow-up time-point (Table 2).

DISCUSSION
The current study represents the first report to demonstrate the effects of 3-oxotirucalla-7, 24dien-21-oic acid (OTUA) treatment on CD8 + Tcell count among patients suffering from HIV infection. The results revealed increased rates of CD8 + T-cell count recovery in the second phase (24 -48 weeks) in patients having CD8 + T-cell concentration during baseline < 50 cells/μL. On the other hand, the rates of phase 1 (0 -24 weeks) recovery were similar, irrespective of baseline CD8 + T-cell counts. 3-oxotirucalla-7, 24dien-21-oic acid treatment led to increased response to CD8 + T-cell recovery in patients with low baseline counts. The increased response to enhance count of CD8 + T-cells by patients having baseline CD8 + T cells more than 50 cells/μL, could not reach to the value of 200 CD8 + T/μL at 48 weeks. 3-Oxotirucalla-7, 24-dien-21-oic acid treatment resulted in decrease of percentage of patients with a CD8 + T-cell count < 100 cells/μL from 61 % at baseline to 5 % at after 48 weeks. It has been reported that the patients with the lowest baseline CD8 + T-cell counts have higher death risks [26]. During phase 2, the sustenance of suppression of viral replication also has a key role in the CD8 + T-cell recovery [27,28]. In our study, the proportion of the patients having more than 50 D8 + T-cells/μL was only 6 % following 48 week treatment with 3-oxotirucalla-7, 24-dien-21oic acid and 4 % were immunologically nonresponsive even after a marked reduction in viral level. Moreover, 23 % of 'super-responders' had baseline CD8 + T-cell counts < 50 cells/μL, indicating that a very low baseline CD8 + T-cell count does not preclude an excellent CD4 cell count response to 3-oxotirucalla-7, 24-dien-21oic acid treatment.

CONCLUSION
The study revealed that patients with CD4 counts in the lower range have a significant tendency for immune recovery. 3-Oxotirucalla-7, 24-dien-21oic acid treatment suppresses the viral level and enhances the probability of immune system recovery. Therefore, 3-oxotirucalla-7, 24-dien-21oic acid can be an effective candidate for CD4 cell recovery.