In vivo administration of a JAK3 inhibitor to chronically siv infected rhesus macaques leads to NK cell depletion associated with transient modest increase in viral loads

Abstract

Innate immune responses are reasoned to play an important role during both acute and chronic SIV infection and play a deterministic role during the acute stages on the rate of infection and disease progression. NK cells are an integral part of the innate immune system but their role in influencing the course of SIV infection has been a subject of debate. As a means to delineate the effect of NK cells on SIV infection, use was made of a Janus kinase 3 (JAK3) inhibitor that has previously been shown to be effective in the depletion of NK cells in vivo in nonhuman primates (NHP). Extensive safety and in vitro/in vivo PK studies were conducted and an optimal dose that depletes NK cells and NK cell function in vivo identified. Six chronically SIV infected rhesus macaques, 3 with undetectable/low plasma viral loads and 3 with high plasma viral loads were administered a daily oral dose of 10 mg/kg for 35 days. Data obtained showed that, at the dose tested, the major cell lineage affected both in the blood and the GI tissues were the NK cells. Such depletion appeared to be associated with a transient increase in plasma and GI tissue viral loads. Whereas the number of NK cells returned to baseline values in the blood, the GI tissues remained depleted of NK cells for a prolonged period of time. Recent findings show that the JAK3 inhibitor utilized in the studies reported herein has a broader activity than previously reported with dose dependent effects on both JAK2 and JAK1 suggests that it is likely that multiple pathways are affected with the administration of this drug that needs to be taken into account. The findings reported herein are the first studies on the use of a JAK3 inhibitor in lentivirus infected NHP.

Figure 1. The effect of the JAK3 inhibitor on rhesus macaque NK cell function in vitro/ in vivo and on the frequencies of the major cell lineages in the blood.

A) PBMC from 9 normal rhesus macaques was assayed for NK activity in the presence and absence of varying concentrations of the JAK-3 inhibitor. The mean lytic units/10⁶ effector cells were calculated. The S.D. was always <10%. B) Two normal rhesus macaques were orally administered 10 mg/kg of the JAK3 inhibitor and PBMC samples obtained prior to and at 1, 3, 24, 48, 72 and 168 hrs. post JAK3 administration and assayed for NK activity. Results reflect Mean lytic units of NK activity/10⁶ effector cells performed in triplicated with the S.D. <10%. C) Three normal rhesus macaques were orally administered the JAK3 inhibitor at 10 mg/kg daily for 14 days and an aliquot of their PBMC obtained prior to and at day 7 and 14 subjected to polychromatic flow cytometric analysis for the frequencies of CD4⁺ T cells, CD8⁺ T cells, CD20⁺ B cells and CD3⁻/CD8⁺/NKG2a⁺ cells. An aliquot was utilized for CBC and the absolute numbers of each cell lineage calculated and illustrated. Note the major depletion of the NK cells D). Another aliquot of the PBMC from C) were assayed for NK cell function and the data shows the Mean lytic units/10⁶ effector cells. The S.D. was always <10%.

Figure 2. The effect of JAK3 oral administration on the frequencies of CD4⁺ and CD8⁺ T cells and their subsets in 6 SIV chronically infected rhesus macaques.

Each animal received an initial loading does of 20 mg/kg and thereafter a daily dose of 10 mg/kg for a total of 35 days. A) Aliquots of the PBMC obtained prior to (baseline) and at weeks 1, 3, 5, 6, 8 and 12 were analyzed for the frequencies of lymphoid cell subsets A) The mean % change from baseline values of CD4⁺ T cells is shown. B) The mean % change from baseline values of CD4⁺ T cells classified as naïve, central memory (CM) and effector memory (EM) is illustrated c) The mean % change from baseline of total CD8⁺ T cells is shown and D) The mean % change from baseline values of CD8⁺ T cells classified as naïve, central memory (CM) and effector memory (EM) is illustrated. Statistical differences are denote as asterisks with * (p<0.01), ** (p<0.05), *** (p<0.03) and **** (p<0.001).

Figure 3. Frequencies of CD3^-/CD8⁺/NKG2a⁺ (NK cells).

Aliquots of cells from the same 6 monkeys as described under Fig. 2 were assayed for the frequencies of CD3⁻/CD8⁺/NKG2a⁺ (NK cells) and the mean % change from baseline values illustrated for A) PBMC samples and B) Gastro-intestinal tissue samples. There was high statistical difference in the values obtained on both PBMC samples (p<0.001) and G.I. tissue samples (all p<0.0) that did not return to baseline values.

Figure 4. Frequencies of the 4 major subset of the CD3⁻/CD8⁺/NKG2a⁺ NK cells.

Aliquots of the same PBMC from the 6 SIV infected monkeys as used under Fig. 2 were also analyzed for the frequencies of the 4 major subset of the CD3⁻/CD8⁺/NKG2a⁺ NK cells. The data is once again expressed as the mean +/− S.D. of the baseline values from individual monkeys. A) The % change from baseline values for the CD16⁺/CD56⁻ (cytolytic subset) B) The % change from baseline for the CD16⁻/CD56⁺ (major cytokine synthesizing subset) C) The % change from baseline in the CD16-/CD56- subset and D) The % change from baseline of the CD16⁺/CD56⁺ subset. The statistical differences are denoted by asterisks and reflects * (p<0.01), ** (p<0.05), *** (p<0.03) and **** (p<0.0001).

Figure 5. Effect of JAK3 inhibitor on plasma and gastro-intestinal tissue viral loads.

Aliquots of the plasma samples obtained prior to and post administration of the JAK3 inhibitor from each of the 6 monkeys were assayed for levels of SIV (commercially performed). A) Reflects # of viral copies/ml of plasma obtained from each of the 6 monkeys at weekly intervals until week 6, bi-weekly until week 14 is illustrated using a log scale to highlight differences seen in monkeys with low viral loads B) Reflects # of viral copies/ml of plasma obtained from each of the 6 monkeys at weekly intervals until week 6, bi-weekly until week 14 is illustrated using an arithmetic scale to highlight differences seen in monkeys with high viral loads C) Reflects the viral copies/ng DNA isolated from a pool of colo-rectal biopsies from each of the 6 monkeys obtained prior to (baseline) and at weeks 1, 3, 6 and 12 post JAK3 inhibitor administration.