Among the 47 subjects with available samples for analysis here, the study subject median age was 47 years; 79% were male; 53% white, 32% black and 15% Hispanic. The median CD4 cell count at entry was 444 cells/mm3, 77% were on antiretroviral treatment and 53% had undetectable HIV-1 RNA (<50 copies/mL).
As recently described [13
], there was a modest trend towards a higher proportion of vaccine responders (HBsAb≥10 mIU/mL) in the GM-CSF arm at week 4 (26% vs. 9%, p=0.24), but by week 8 this trend was lost (week 8: 26% vs. 35%, p=0.75; week 16: 52% vs. 65%, p=0.54; week 36: 55% vs. 64%, p=0.76; week 60: 35% vs. 45%, p=0.54).
To identify whether GM-CSF receptor expressing myeloid populations could account for variability in vaccine response we measured peripheral blood hematopoietic progenitor cell (CD34+), monocyte (CD14+), and MDSC. MDSCs
are a heterogeneously defined population of cells that have been described as CD34+, CD33+/lineage marker (including CD14-HLADR-) and CD11b+CD14-CD33+[18
]. Additionally, another sub-population of MDSCs has been described as CD14+HLADR- [18
]. We focused here on CD14+HLADR-CD11b+, CD34+CD14-HLADR-CD11b+, and CD34+CD14-HLADR- cell frequencies at baseline (). Comparing week 16 vaccine responders to nonresponders, irrespective of study arm, baseline CD34+ progenitor frequency was higher in subjects with a week 16 antibody response (n=26; median [Q1, Q3]: 0.94% [0.62%, 1.28%]) than those without (n=18; 0.57% [0.47%, 0.68%]; p=0.005) (). When stratified by study arm, this remained statistically significant (p=0.01). Additionally, this relationship held as a positive correlation between CD34+ progenitor cell frequency and week 16 antibody titer (r=0.34, p=0.03, ). A subset of CD34+ progenitors with phenotypic markers of MDSCs (CD34+CD14-HLADR-) were also higher in frequency at baseline in week 16 vaccine responders than in nonresponders (0.14% [0.08%, 0.42%] vs. 0.08% [0.03%, 0.19%], p=0.02; p=0.04 when stratified by study arm). In contrast, baseline CD14+ monocyte frequency was lower in week 16 antibody responders than in nonresponders (6.87% [4.15%, 8.29%] vs. 10.17% [7.97%, 12.48%], p=0.02; p=0.06 when stratified by study arm, ). This was consistent with an observed negative correlation between baseline CD14+ cell frequency and week 16 antibody titer (r= -0.38, p=0.01, ).
Baseline CD34+ progenitor and monocyte frequencies are associated with week 16 vaccine response
We next asked if administration of GM-CSF affected the frequencies of these cell populations. At week 4 we observed a decline in CD34+CD14-HLADR- and CD34+CD14-HLADR-CD11b+ cell frequencies in the vaccine arm that did not receive GMCSF (-0.01% [-0.02%, 0%], p=0.02; and 0% [-0.02%, 0%], p=0.01 respectively, and ) while there were no such changes observed in the GMCSF arm (p=0.007 and p=0.03 for the comparison of study arms). Notably, 2 subjects in the arm that did not receive GM-CSF had greater frequencies of these cells at baseline (potentially driving this difference). Also, there were more subjects without such measurable cells at baseline in the GM-CSF arm (5 in the GM-CSF arm vs. 4 in the Vaccine only arm and 12 vs. 9 for CD34+CD14-HLADR- and CD34+CD14-HLADR-CD11b+, respectively) who could have only had increased frequency subsequently. However, though overall there were no statistically significant differences in these cell frequencies at baseline. When we evaluated change in cell frequency in relation to week 16 antibody response, vaccine responders had a minimal week 4 decline in CD34+CD14-HLADR- cell frequency (0% [-0.02%, 0%], p=0.02) while non-responders showed no decline (0.01% [-0.01%, 0.01%], p=0.03 for the comparison of responders and nonresponders; p=0.08 when stratified by study arm). In other words, GM-CSF administration may have preserved CD34+CD14-HLADR-(CD11b+/-) cells to a modest extent, and preservation of these cells in circulation was associated with a failure to respond to the vaccine.
GMCSF administration is associated with preservation of CD34+ CD14-HLADR-and CD34+ CD14-HLADR- CD11b+ cells
On the other hand, a decline in the frequency of MDSCs with the CD34+CD14-HLADR-CD11b+ phenotype at week 4 was associated with a week 4 nonresponse to vaccine (p=0.03; p=0.16 when stratified by study arm). While there were few week 4 vaccine responders (8 responders total [13
]), it appears that the relation between flux in cell subset frequency and vaccine response is complex and differs depending on time point of response analysis, with a potential delay in effect. Nonetheless, it appears that GM-CSF administration selectively alters peripheral myeloid subset frequencies.
Evaluating frequencies of a different phenotype of MDSCs, CD14+HLADR-CD11b+, we found no statistically significant difference between the study arms, nor between vaccine responders and nonresponders. We also measured GM-CSF receptor (CD116) expression on monocytes and myeloid suppressor cells at baseline and week 4 of the HBV vaccine protocol. Similarly, there was no change in GM-CSF receptor expression from baseline to week 4 in either study arm ( for Monocytes: p=0.9 in the Vaccine only arm, p=0.8 in the GM-CSF arm; p=0.9 for the comparison between the study arms), or between week 4 vaccine responders and nonresponders (p=0.9 in both responders and nonresponders; p=0.7 for the comparison between responders and nonresponders, p=0.6 when stratified by study arm; and p>0.1 for all CD14+HLADR-CD11b+ comparisons, not shown).