Sex- and Age-based Comparison of Key Clinical Markers of Immunity After Heart and Kidney Transplantation

Abstract

Background.

The combined effects of age and sex impact important posttransplant outcomes. Despite key physiologic differences in metabolic and immune function, older women are often indiscriminately grouped with either young women or older men. We examined sex- and age-based differences in clinical markers of immunity in heart and kidney recipients, with specific attention to those of postmenopausal women.

Methods.

Blood was prospectively collected before transplantation, and at 1 and 6 mo posttransplantation, alongside 12 mo of clinical data. Patients were stratified by age, biological sex, and menopause status. Absolute lymphocyte count (ALC), CD4⁺ and CD8⁺ lymphocyte subsets, total IgG, 4 selected cytokines, estradiol and progesterone, and cumulative incidence of infection were quantified within groups. The relationship between menopause category (premenopausal women, postmenopausal women, men) and 6-mo ALC was tested by linear regression, controlling for multiple confounding variables.

Results.

The cohort included 40 heart, 23 kidney, and 3 heart-kidney recipients categorized as 10 women older than 50 y, 12 women 50 y and younger, 26 men older than 50 y, and 18 men 50 y and younger. At 6 mo posttransplant, mean ALC among older women (0.59 K/µL) fell to a far lower range of lymphopenia than in young women (0.9 K/µL), older men (0.85 K/µL), and younger men (0.82 K/µL). Postmenopausal women had significantly lower ALC compared with premenopausal women (P = 0.03) and men (P = 0.05). Women older than 50 y also had the greatest cumulative incidence of infection by 1 y compared with other groups.

Conclusions.

These findings support the concern for increased risk of infection in postmenopausal organ transplant recipients.

The combined effects of age and sex are associated with differences in important posttransplant outcomes. Although it is clear that older women have distinct physiology separating them from both younger female and similarly-aged male counterparts, there are very few data informing how these differences influence their unique immunologic profile in solid organ transplantation. Young female heart and kidney recipients experience a greater risk of graft failure and rejection compared with similarly aged men.^1-5 Among kidney recipients, women up to 45 y have a greater graft loss rate, particularly when the donor is a man^2-4,6; however, this difference recedes, and even reverses, in older age strata such that older women have significantly less graft failure than male counterparts.^2,4 In addition, female kidney transplant recipients older than 60 y have higher excess mortality compared with their male counterparts.⁷ Although the problem of graft failure and rejection in young women is extremely important, much less attention has been given to the fact that older women, with less robust immunity, may have increased vulnerability to infection.

Often when age and sex are examined in clinical studies, they are reported separately; however, the effect of sex on a given outcome may differ depending on the age of the comparator groups. Furthermore, female recipients are a minority of all heart (26% in 2021) and kidney (39% in 2021) recipients, with older female recipients comprising yet a smaller fraction. Thus, clinical research that reflect this underlying epidemiology often lacks the power to detect statistically significant differences in any given outcome in this specific population unless specifically designed to do so. Limited studies have looked at this particular subgroup in relation to specific infection outcomes or markers of infection risk.^8,9 As the number of transplants occurring in older patients with end-stage heart and kidney failure increases, and as concerted efforts to improve gender equity in transplantation are bolstered, our understanding of the unique needs of older female recipients becomes extremely important.^10-12

To understand variability in immunity within heart transplant (HT) and kidney transplant populations, we examined sex- and age-based differences in key clinical immunological markers including absolute lymphocyte count (ALC), T-cell subsets, total IgG, and 4 selected cytokines: interferon gamma (IFN-γ), interleukin-8 (IL-8), IL-2, and C-C motif chemokine ligand 8 (CCL-8), chosen based on a previous association with sex, transplant immunosuppressive medications and rejection outcomes, and/or effective control of cytomegalovirus (CMV) in transplant populations.^13-15 All markers were measured prospectively at multiple time points to capture the dynamic nature of the posttransplant period, and were collected alongside relevant clinical data, including some less commonly considered factors such as menopausal status, prior pregnancies, exogenous steroid hormone supplementation, serum concentrations of estradiol and progesterone, infection, and rejection.

MATERIALS AND METHODS

Study Population and Data Collection

A prospective cohort of adult HT and/or kidney transplant recipients was enrolled and transplanted between March 2021 and January 2023 at a single academic medical center with a large volume of both transplant types. Eligible patients were older than 18 y, awaiting HT and/or kidney transplant at Tufts Medical Center, and planned to have follow-up at this center for at least 6 mo after transplant. Patients were excluded if they had a prior solid organ transplant, allogeneic stem cell transplant, autologous stem cell transplant within 5 y, hematologic disorder, hemoglobin level consistently <7 mg/dL at the time of screening, or inability to provide informed consent. After enrollment, baseline clinical data were collected along with a baseline blood sample before transplantation. Patients were prospectively followed for 12 mo after transplantation with collection of blood specimens at 4–6 wk posttransplant and at 6 mo posttransplant (±3 wk). Clinical data were collected from the electronic medical record; if specific information on clinical variables was not found, this could be gathered in-person during clinical visits by research personnel as needed.

In addition to routine clinical laboratory collection, which routinely included a complete blood count with differential and a metabolic panel, research specimens were collected at all 3 time points for T-cell subsets, total IgG, IFN-γ, IL-8, IL-2, CCL-8, estradiol (E2), and progesterone (P4). All laboratory tests were run in the clinical laboratory at Tufts Medical Center, with the exception of the measurement of cytokines, which was conducted in collaboration with a research laboratory at Tufts University.

Clinical variables collected include demographic information of the recipient, medical comorbidities (diabetes mellitus, chronic kidney disease, and cause of end-stage organ failure), menopause status, last menstrual period, number of prior pregnancies, duration and type of current dialysis, immunosuppressive medications, hormonal medications, transplant type (heart, deceased donor kidney, living donor kidney, heart-kidney), donor sex and age, calculated panel-reactive antibodies closest to transplant, CMV serostatus of donor and recipient, perioperative complications, immunosuppressive regimen, antimicrobial prophylaxis, rejection episodes and treatment, occurrence of acute or chronic infections, and associated treatments.

The various immune markers measured in this study were each selected for specific reasons. Lymphocyte counts, T-cell subsets, and IgG were measured because of their well-established clinically significant associations with posttransplant outcomes, including CMV infection, non-CMV infection, and death.^16-21 The cytokines measured here have lesser-known associations and were chosen to explore distribution among different age and sex categories in the organ transplant population. IL-2 has a central role in T-cell expansion, is a primary target of key transplant immunosuppressive medications (basiliximab and others), and its synthesis differs by sex in aged (but not young) rats.²² Also along the T-cell pathway, IFN-γ, which plays a central role in response to viral pathogens, has known differences in expression by sex, mediated at least partly by estradiol.¹³ CCL-8 was of interest in this population as monocyte expression of this chemokine in CMV viremic organ recipients is associated with viral control¹⁴; CMV, one of the most common and problematic posttransplant infections, has shown age- and sex-related differences in clinical course.^9,23 Finally, IL-8, along with IL-2, has been found to be associated with early rejection in kidney transplant recipients,¹⁵ another transplant outcome with differences seen by age and sex.

This study was approved by the Tufts Health Sciences Institutional Review Board, and all patients provided consent pretransplantation before study participation (No. 00000934).

Immunosuppression and Rejection

Immunosuppressive strategies were different between HT and kidney transplant recipients. HT recipients did not routinely receive induction immunosuppression except in cases of concomitant kidney transplant or significant renal dysfunction (typically with a serum creatinine >2 mg/dL) in which case, basiliximab was preferred; or, in occasional highly sensitized recipients, in which case, antithymocyte globulin (ATG) was used. Heart recipients routinely received a triple drug regimen for maintenance immunosuppression, including tacrolimus (goal serum level 8–12 ng/mL), mycophenolate (1000 mg every 12 h), and steroid taper during 6 mo. Infrequently used alternatives to first-line medications were cyclosporine, azathioprine, and sirolimus.

Kidney recipients all received routine induction, primarily with ATG. Basiliximab was used in select recipients at low risk for rejection based on age (older than 65 y) and prior sensitization, or in people with a high risk of reaction to ATG. Patients received high-dose methylprednisolone, which was rapidly tapered unless they had a prior indication for steroids or evidence of donor-specific antibodies. Maintenance immunosuppression was continued with tacrolimus (goal 8–10 ng/mL in first 6 mo) and mycophenolate (1000 mg every 12 h). Less frequently used alternatives included cyclosporine, azathioprine, or belatacept (starting at least 3 mo posttransplantation). Among these alternatives, belatacept was preferred in patients who required high doses of calcineurin inhibitors, had difficulty with calcineurin inhibitor side effects, were younger (to limit prolonged use of calcineurin inhibitors), and/or had metabolic syndrome.

The rejection variable in this study represented treatment for rejection with at least high-dose steroids, regardless of biopsy result. Lower-grade rejection events treated only with adjustment of maintenance immunosuppression were not counted. By protocol, acute cellular rejection of cardiac graft grade III or higher (based on the International Society of Heart and Lung Transplantation grading system) was typically treated with IV methylprednisolone followed by steroid taper, and then ATG if severe, sustained, or recurrent. Similarly, acute cellular rejection in kidney recipients was treated with high-dose steroids if borderline or Banff grade 1, and with ATG if steroid-resistant or Banff grade ≥2. Antibody-mediated rejection of cardiac graft was treated with pulse corticosteroids, plasmapheresis with or without intravenous IgG, and rituximab. No cases of antibody-mediated rejection occurred in this group of kidney recipients.

Antimicrobial Prophylaxis

All participants received CMV prophylaxis with duration according to international consensus guidelines.²⁴ Seronegative recipients of seropositive donors (high risk) routinely received 6 mo of valganciclovir or ganciclovir dosed for standard prophylaxis and adjusted for renal function.²⁴ Seropositive recipients (intermediate risk) received the same for 3 mo. There was a concomitant open-label clinical trial of letermovir primary prophylaxis among HTs during the study period. Several high and intermediate-risk patients were enrolled before their transplant and received letermovir (and famciclovir) in place of valganciclovir/ganciclovir. The participants in this concomitant trial were not enrolled on the basis of any criteria related to white blood cell counts. Seronegative recipients of seronegative donors (low risk) were given famciclovir, valacyclovir, or acyclovir for herpes simplex prophylaxis for 3 mo.

Heart recipients received trimethoprim-sulfamethoxazole (single strength daily, adjusted for kidney function) for Pneumocystis jirovecii prophylaxis for 1 y posttransplantation, dosed higher (double strength daily, adjusted for kidney function) for the additional prophylaxis of toxoplasmosis in settings where donor and/or recipient had prior toxoplasma exposure. Kidney recipients all received trimethoprim-sulfamethoxazole single strength daily, adjusted for kidney function, for 6 mo posttransplantation. In cases of sulfa allergy or persistent neutropenia, atovaquone was substituted for both organ transplant types. Nystatin oral solution was provided for HT recipients for the first year. Kidney recipients did not use prophylaxis for oropharyngeal candidiasis beyond the transplant hospitalization.

In heart recipients, routine prophylaxis was restarted for episodes of rejection treated with ATG or rituximab and was continued for 3 mo.

Laboratory Analysis

Blood samples for T-lymphocyte subset analysis, total serum IgG, E2, and P4 were collected in clinical settings and run in a Clinical Laboratory Improvement Amendments-certified lab according to clinical laboratory standards. Serum specimens were collected concomitantly with the others noted previously processed, aliquoted, and stored at –80 °C. All cytokine specimens were processed simultaneously. The concentrations of IFN-γ, IL-2, and IL-8 from each available serum sample were quantified using Quantikine ELISA (R&D Systems; Minneapolis, MN) following the manufacturer’s instructions. The concentration of CCL-8 (also known as monocyte chemoattractant protein 2) was quantified from all available samples using the DuoSet ELISA (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions. Samples were diluted with PBS 1:1 and run in a singlet.

Infection

To maximize the data available from a relatively small sample size, we broadly included any infection that was diagnosed clinically by the treating transplant and/or transplant Infectious Diseases specialist from the time of transplant surgery through 1 y posttransplantation. This included microbiologically proven infections and nonmicrobiologically proven infections (ie, skin and soft tissue infection, acute cholecystitis, diverticulitis, cutaneous herpes zoster infection, and other infections where standard care does not necessarily involve isolation of an organism).

Statistical Analysis

A 4-category variable was created according to biologic sex (male/female) and age (dichotomized at 50 y, which is roughly both the median age of the cohort and also the average age of menopause in the general US population).²⁵ Baseline characteristics were stratified according to these 4 groups and reported as frequencies and percentages for categorical variables, or as means and SDs for continuous variables. Distribution of ALC, CD4⁺ lymphocytes, CD8⁺ lymphocytes, IgG, IFN-γ, IL-8, IL-2, and CCL-8 at all 3 time points for the 4 categories was visualized and examined with means and medians, and plots stratified by age-sex categories, evaluating for relative differences and trends. Mean measures of estradiol and progesterone were examined both within age-sex categories and within a separate 3-level category of “menopause status,” a clinical variable reported by patients (premenopausal female, postmenopausal female, and male patients).

The relationship between menopause category and the primary outcome of 6-mo posttransplant ALC was tested by linear regression controlling for use of induction therapy, treatment for rejection within the first 6 mo, and use of valganciclovir at 6 mo. Model assumptions were checked using linear regression diagnostics.

Given the small sample size and 4 subgroups, the evaluation of infection incidence was primarily hypothesis-generating. The frequency of infection occurrence was tabulated within each age-sex group. The cumulative incidence of infections per person during the first 12 mo after transplant was calculated and compared between age-sex category groups by Kruskal-Wallis rank-sum test. Pathogen type and site of infection were characterized within each group to look for trends. Additional analyses using a narrower definition of infection and comparison of infection incidence between different organ transplant types are described in Supplemental Materials (SDC, https://links.lww.com/TXD/A782).

Because of important differences in underlying comorbidities and immunosuppression strategy between heart and kidney recipients, heart and kidney subgroups were also explored separately, although statistical comparisons were not performed because of limited power. ALC, CD4⁺ lymphocytes, CD8⁺ lymphocytes, IgG, IFN-γ, IL-8, IL-2, and CCL-8 were again assessed within age-sex groups, evaluating for trends in older versus younger women and in older women versus older men.

P values of <0.05 were considered statistically significant. R Studio version 1.3.1073 was used for statistical analysis.

RESULTS

In total, 68 patients were enrolled and 66 proceeded to transplantation within the study period. The cohort included 40 HT recipients, 23 kidney recipients (6 living related, 16 living unrelated, and 1 deceased donor), and 3 simultaneous heart-kidney recipients. Three patients died during the study period (2 men younger than 50 y and 1 woman older than 50 y). One additional woman older than 50 y was lost to follow-up after the 6-mo visit. The data contributed from these 4 patients before the study exit date were included in the analysis.

The study cohort was one-third women (n = 22), reflecting the distribution of the underlying population of heart and kidney transplant recipients in the United States. The median age of the cohort was 51.9 y (54 y among men, 49.5 y among women). Baseline characteristics are displayed in Table 1. Groups included 12 women 50 y and younger, 10 women older than 50 y, 18 men 50 y and younger, and 26 men older than 50 y. It is notable that the majority within the young male group were kidney recipients, although not statistically different than other groups. Thirty percent of HT recipients received induction immunosuppression; all kidney recipients received induction. Baseline laboratory values before immunosuppression are also shown in Table 1. Notably, white blood cell count was very similar among groups; lymphocyte counts are lower among older men and women compared with younger men and women, but remain within the normal range.

TABLE 1.

Cohort characteristics stratified by age-sex category

Characteristics	Women ≤50 y (N = 12)	Women >50 y (N = 10)	Men ≤50 y (N = 18)	Men >50 y (N = 26)
Race, n (%)
Asian	0 (0.0)	0 (0.0)	2 (11.1)	2 (7.7)
Black	1 (8.3)	1 (10.0)	1 (5.6)	1 (3.8)
White	11 (91.7)	9 (90.0)	15 (83.3)	23 (88.5)
Age at transplant, mean (SD)	37.58 (10.32)	59.71 (7.00)	40.75 (6.91)	62.19 (6.33)
Organ transplant type, n (%)
Heart	8 (66.7)	6 (60.0)	7 (38.9)	19 (73.1)
Kidney	4 (33.3)	4 (40.0)	10 (55.6)	5 (19.2)
Deceased donor	0 (0.0)	0 (0.0)	1 (10.0)	0 (0.0)
Living unrelated donor	2 (50.0)	2 (50.0)	8 (80.0)	4 (80.0)
Living related donor	2 (50.0)	2 (50.0)	1 (10.0)	1 (20.0)
Heart-kidney	0 (0.0)	0 (0.0)	1 (5.6)	2 (7.7)
Pretransplant dialysis among kidney recipients, n (%)
None	2 (50.0)	2 (50.0)	4 (36.4)	5 (71.4)
Hemodialysis	1 (25.0)	2 (50.0)	6 (54.5)	1 (14.3)
Peritoneal dialysis	1 (25.0)	0 (0.0)	1 (9.1)	1 (14.3)
Duration of pretransplant dialysis among kidney recipients (%)
<1 y	1 (50.0)	1 (50.0)	4 (57.1)	1 (50.0)
1–2 y	1 (50.0)	0 (0.0)	0 (0.0)	1 (50.0)
>2 y	0 (0.0)	1 (50.0)	3 (42.9)	0 (0.0)
Female donor (%)	7 (58.3)	9 (90.0)	9 (50.0)	5 (19.2)
Cytomegalovirus sero-status risk group, n (%)
Low risk	6 (50.0)	3 (30.0)	10 (55.6)	6 (23.1)
Intermediate risk	5 (41.7)	6 (60.0)	3 (16.7)	9 (34.6)
High risk	1 (8.3)	1 (10.0)	5 (27.8)	11 (42.3)
BMI, mean (SD)	29.30 (6.24)	25.83 (4.82)	29.49 (6.16)	27.05 (4.94)
No. of prior pregnancies, mean (SD)	2.17 (1.85)	2.22 (1.72)	–	–
Postmenopausal,a n (%)	0 (0.0)	9 (90.0)	–	–
History of diabetes mellitus, n (%)	1 (8.3)	1 (10.0)	4 (22.2)	13 (50.0)
History of chronic kidney disease, n (%)	5 (41.7)	7 (70.0)	12 (66.7)	17 (65.4)
cPRA, mean (SD)	39.25 (43.05)	17.20 (24.88)	6.71 (17.08)	4.54 (9.49)
Baseline laboratory measurements, mean (SD)
WBC	8.80 (5.46)	8.55 (5.23)	8.49 (4.41)	9.21 (1.81)
ANC	5.86 (3.85)	6.42 (5.09)	5.59 (2.86)	6.79 (1.79)
ALC	1.79 (0.95)	1.24 (0.45)	1.74 (1.46)	1.06 (0.46)
CD4+	0.92 (0.49)	0.65 (0.28)	0.71 (0.26)	0.48 (0.18)
CD8+	0.43 (0.23)	0.29 (0.22)	0.38 (0.24)	0.25 (0.22)
CD4:CD8 ratio	2.16 (0.52)	2.81 (1.88)	2.30 (1.14)	2.98 (2.14)
CD3+	1.39 (0.74)	0.95 (0.44)	1.13 (0.49)	0.74 (0.35)
IgG	1035.25 (321.04)	985.90 (322.86)	1143.83 (340.97)	1114.92 (421.13)
eGFRb	58.33 (43.26)	37.70 (35.14)	35.78 (44.17)	52.15 (30.61)
Induction immunosuppression, n (%)	8 (66.7)	5 (50.0)	12 (66.7)	13 (50.0)
Type of induction, n (%)
None	4 (33.3)	5 (50.0)	6 (33.3)	13 (50.0)
Basiliximab	2 (16.7)	2 (20.0)	4 (22.2)	8 (30.8)
Antithymocyte globulin	6 (50.0)	3 (30.0)	8 (44.4)	5 (19.2)

^aOne woman who was amenorrheic before transplant and had categorized herself as postmenopausal was recategorized when regular menstruation returned after a kidney transplant.

^bCalculated according to the CKD-EPI equation based on serum creatinine.³⁹

ALC, absolute lymphocyte count; ANC, absolute neutrophil count; BMI, body mass index; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; cPRA, calculated panel-reactive antibody; eGFR, estimated glomerular filtration rate; WBC, white blood cell count.

Steroid hormone comparisons between groups are shown in Table 2. At baseline, a state of end-stage heart and/or kidney failure and hormonal dysregulation, estradiol among young women was not significantly different compared with all other groups.²⁶ However, at both posttransplant time points, estradiol among young women rose to more typical levels for premenopausal women, whereas among older women, it stayed the same or lower than that of both groups of men. Menopause category correlated well with age >50 y or <50 y (Table 1).

TABLE 2.

Mean serum estradiol and progesterone levels by age-sex category at 3 interval time points, including pretransplantation, 1 mo posttransplantation, and 6 mo posttransplantation

	Women ≤50 ya (N = 12)	Women >50 y (N = 10)	Men ≤50 y (N = 18)	Men >50 y (N = 26)
Estradiol, pg/mL, mean (SD)
Baseline	29.61 (14.76)	22.70 (21.80)	39.75 (10.76)	31.76 (16.07)
1 mo	75.88 (50.22)	24.64 (27.45)	26.51 (11.44)	24.57 (14.77)
6 mo	57.57 (35.51)	14.29 (5.44)	37.67 (14.83)	32.06 (11.28)
Progesterone, ng/mL, mean (SD)
Baseline	0.90 (1.09)	0.55 (0.38)	0.62 (0.40)	0.48 (0.42)
1 mo	2.18 (3.20)	0.22 (0.10)	0.22 (0.06)	0.22 (0.07)
6 mo	1.89 (2.90)	0.18 (0.06)	0.24 (0.09)	0.23 (0.10)

^aTwo premenopausal women were on systemic hormonal contraceptive medication (oral contraceptive pill or depot shot); others had copper or hormonal intrauterine devices, which have no hormone or far lower concentrations of progestin, respectively, compared with systemic options.

At 6 mo posttransplantation, the mean ALC for older women (0.59 K/µL) fell into a range of lymphopenia previously associated with increased risk of CMV and other posttransplant infection and was lowest relative to young women (0.9 K/µL), older men (0.85 K/µL), and younger men (0.82 K/µL), respectively.^16,17,19 ALC distribution stratified by age-sex category is shown in Figure 1A. Six-month ALC values were plotted against age individually for male and female recipients (Figure 2), clearly demonstrating down-trending ALC with age among women, and a flatter decline among men, crossing roughly around age 45 y. The same plot using pretransplant baseline lymphocyte count is provided for comparison (Figure 2).

FIGURE 1.

Distribution of ALC (A), and CD4⁺ lymphocyte count (B), at 6 mo posttransplant, stratified into 4 age-sex categories. Mean values are indicated in red and median values in blue. ALC, absolute lymphocyte count.

FIGURE 2.

Distribution of total lymphocyte count by age at baseline pretransplant measurement (A) and 6 mo posttransplant (B). Separate lines by sex. ALC, absolute lymphocyte count.

CD4⁺ lymphocyte counts at 6 mo (Figure 1B) show a similar relative pattern between groups as in ALC, although there is a larger range among young women. The lowest mean CD4⁺ lymphocyte count was among older women (0.22 K/µL). CD8⁺ lymphocyte counts and total IgG did not show similar trends between groups (Table 3).

TABLE 3.

Laboratory and clinical data from 1 and 6 mo posttransplant

	Women ≤50 y (N = 12)	Women >50 y (N = 10)	Men ≤50 y (N = 18)	Men >50 y (N = 26)
1-mo laboratory measurements, mean (SD)
WBC	8.46 (3.62)	9.08 (3.89)	6.95 (2.71)	8.03 (2.69)
ANC, K/µL	6.74 (2.95)	7.59 (3.58)	5.34 (2.15)	6.44 (2.13)
ALC, K/µL	0.99 (0.91)	0.79 (0.53)	0.86 (0.73)	0.81 (0.62)
CD4+, K/µL	0.38 (0.51)	0.34 (0.29)	0.39 (0.46)	0.35 (0.31)
CD8+, K/µL	0.23 (0.25)	0.23 (0.26)	0.19 (0.18)	0.18 (0.17)
CD3+, KµL	0.62 (0.78)	0.57 (0.49)	0.59 (0.61)	0.54 (0.45)
CD4+/CD8+ ratio	1.12 (0.89)	2.10 (2.24)	1.78 (1.83)	2.65 (2.34)
Total IgG, mg/dL	679.4 (225.5)	576.3 (319.1)	770.2 (325.2)	597.8 (224.9)
eGFRa	60.08 (36.86)	63.30 (20.96)	64.94 (35.14)	58.27 (31.51)
Tacrolimus level	9.55 (2.20)	7.87 (2.45)	7.82 (2.84)	7.85 (3.11)
Valganciclovir/ganciclovir at time of 1-mo laboratory values, n (%)	4 (33.3)	6 (60.0)	7 (41.2)	13 (50.0)
Reduction or discontinuation of mycophenolate at time of 1 mo laboratory values, n (%)	1 (8.3)	0 (0)	1 (5.5)	4 (15)
TMP-SMX at time of 1-mo laboratory values, n (%)	12 (100.0)	9 (90.0)	17 (100.0)	24 (92.3)
6-mo laboratory measurements, mean (SD)
WBC	5.79 (2.53)	4.68 (1.81)	5.88 (2.75)	5.70 (2.38)
ANC, K/µL	4.03 (2.13)	3.44 (1.48)	4.18 (2.17)	4.04 (1.85)
ALC, K/µL	0.90 (0.55)	0.59 (0.34)	0.82 (0.41)	0.85 (0.52)
CD4+, K/µL	0.31 (0.32)	0.22 (0.15)	0.30 (0.20)	0.34 (0.23)
CD8+, K/µL	0.23 (0.15)	0.23 (0.18)	0.19 (0.11)	0.22 (0.18)
CD3+, K/µL	0.56 (0.47)	0.44 (0.26)	0.51 (0.29)	0.57 (0.32)
CD4+/CD8+ ratio	1.08 (0.70)	1.39 (1.12)	1.65 (1.27)	2.47 (2.66)
Total IgG, mg/dL	655.2 (207.0)	663.0 (292.2)	839.9 (293.5)	585.0 (171.8)
eGFRa	59.17 (26.90)	41.00 (17.36)	61.88 (19.71)	48.19 (20.82)
Tacrolimus level	7.65 (3.31)	7.19 (1.46)	6.81 (2.83)	8.10 (2.19)
Valganciclovir/ganciclovir at time of 6-mo laboratory values, n (%)	3 (25.0)	1 (10.0)	2 (12.5)	3 (11.5)
TMP-SMX at time of 6-mo laboratory values, n (%)	11 (91.7)	9 (90.0)	14 (87.5)	18 (69.2)
Reduction or discontinuation of mycophenolate at time of 6-mo laboratory values, n (%)	5 (42)	8 (80)	4 (25)	18 (69)
Belatacept use at time of 6-mo laboratory values, n (%)	3 (25.0)	0 (0.0)	7 (43.8)	1 (4.0)
Treatment for rejection in first 6 mo, n (%)	4 (33.3)	5 (50.0)	3 (16.7)	6 (23.1)
Treatment included ATG	1 (8.3)	0 (0.0)	2 (11.1)	1 (3.8)
Treatment included rituximab	1 (8.3)	0 (0.0)	0 (0.0)	0 (0.0)

^aCalculated according to the CKD-EPI equation based on serum creatinine.

ALC, absolute lymphocyte count; ANC, absolute neutrophil count; ATG, antithymocyte globulin; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; eGFR, estimated glomerular filtration rate; TMP-SMX, trimethoprim-sulfamethoxazole; WBC, white blood cell count.

IFN-γ levels measured over time demonstrated similar patterns to ALC and CD4⁺ lymphocyte trends, with the lowest mean and median measures found among older women compared with all other groups. A large number of unavailable samples for cytokine analysis from the 6-mo time point (>50% in all groups) limited definitive conclusions. However, at the 1-mo time point, where more complete data were available (87% of all participants), mean IFN-γ was lowest among older women (13.1 pg/mL), compared with young women (59.5 pg/mL), young men (92.8 pg/mL), and older men (106.7 pg/mL; Table S1 and Figure S1, SDC, https://links.lww.com/TXD/A782). IL-8, IL-2, and CCL-8 were largely undetectable in all groups (Table S1, SDC, https://links.lww.com/TXD/A782).

In the multivariable regression model adjusted for induction, rejection treatment, and valganciclovir/ganciclovir use at 6 mo, postmenopausal women had significantly lower ALC compared with premenopausal women (P = 0.03) and men (P = 0.05). Postmenopausal women had an average expected ALC that was 0.438 K/µL below (95% confidence interval, 0.096-0.858) that of premenopausal women, and 0.331 K/µL below (95% confidence interval, 0.009-0.651) that of men.

Trends in organ subgroups were examined without statistical testing due to the small sample size. Older female heart recipients had the lowest mean ALC, CD4⁺ lymphocytes, and IgG relative to other groups at both times. At 6 mo, these counts fell below thresholds that are historically associated with increased infection risk (means: ALC 0.60 K/µL, CD4⁺ lymphocytes 0.18 K/µL, and IgG 384 mg/dL). Table 4 and Figure 3 display these data.

TABLE 4.

Cohort restricted to heart recipients only

	Women ≤50 y (N = 8)	Women >50 y (N = 6)	Men ≤50 y (N = 7)	Men >50 y (N = 19)
1-mo measures, mean (SD)
ALC, K/µL	1.16 (1.08)	0.72 (0.36)	1.52 (0.91)	0.97 (0.67)
CD4+, K/µL	0.50 (0.59)	0.34 (0.24)	0.84 (0.51)	0.44 (0.31)
CD8+, K/µL	0.27 (0.30)	0.12 (0.07)	0.31 (0.26)	0.22 (0.19)
CD3+, K/µL	0.78 (0.90)	0.47 (0.27)	1.16 (0.72)	0.67 (0.47)
CD4+/CD8+ ratio	1.38 (0.93)	2.99 (2.57)	3.41 (2.13)	3.11 (2.51)
Total IgG, mg/dL	592.14 (196.99)	378.33 (128.40)	514.17 (262.25)	617.63 (257.29)
6-mo measures, mean (SD)
ALC, K/µL	0.99 (0.65)	0.60 (0.35)	1.21 (0.41)	0.92 (0.54)
CD4+, K/µL	0.39 (0.37)	0.18 (0.15)	0.57 (0.05)	0.38 (0.24)
CD8+, K/µL	0.25 (0.16)	0.15 (0.12)	0.26 (0.16)	0.19 (0.12)
CD3+, K/µL	0.65 (0.55)	0.33 (0.12)	0.86 (0.15)	0.59 (0.31)
CD4+/CD8+ ratio	1.24 (0.81)	1.76 (1.43)	2.94 (1.89)	2.70 (2.93)
Total IgG, mg/dL	543.00 (220.45)	488.00 (238.52)	650.33 (397.61)	587.83 (181.11)

Mean values of ALC, lymphocyte subsets, and total IgG, stratified by age-sex category.

ALC, absolute lymphocyte count.

FIGURE 3.

Cohort restricted to heart recipients only. Distribution of 6-mo ALC (A), CD4⁺ lymphocyte count (B), CD8⁺ lymphocyte count (C), and total Ig G (D) stratified by age-sex categories. ALC, absolute lymphocyte count.

Infection events are summarized in Table S2 (SDC, https://links.lww.com/TXD/A782). Overall, the data indicate that older women experienced the greatest number of infection events per person cumulatively in the first year (mean 3.4) when compared with the other 3 groups (P = 0.01); young men had the fewest (mean 1.28). Results of additional exploratory analyses are shown in Tables S3 and S4 (SDC, https://links.lww.com/TXD/A782) and described in Supplemental Results (SDC, https://links.lww.com/TXD/A782).

DISCUSSION

Our study demonstrates the importance of considering sex in conjunction with age when evaluating immunologic profile and risk after HT or kidney transplantation. We identified a less robust lymphocyte count among older, postmenopausal female heart and kidney recipients compared with young, premenopausal women, and with men in both age categories. Furthermore, the mean ALC at 6 mo posttransplant among this group was 0.59 K/µL (median 0.54 K/µL, 25th–75th percentile 0.41–0.72 K/µL), a level associated with a greater risk of CMV and non-CMV infection as shown in multiple organ transplant studies.^16-19 Definitions of lymphopenia associated with infection have often ranged from 0.6 to 0.75 K/µL, in prior studies; in our cohort, 75% of older women had lymphopenia below the cut-point of 0.75 K/µL.^16,17,19,27 Trends in markers were most pronounced among heart recipients (Table 4 restricted to heart recipients only), where older women had a mean ALC of <0.75 K/µL, a mean CD4⁺ lymphocyte count of <0.200 K/µL, and a mean total IgG of <500 mg/dL at both 1 and 6 mo. These thresholds are all surrogates, based on prior studies, for increased infection risk.^20,21,28,29

Of the women in this cohort treated for rejection, a greater proportion was older than 50 y compared with 50 y and younger (Table 3); however, only 1 (a woman 50 y and younger) received lymphocyte-depleting ATG. Regardless, rejection treatment was a prespecified confounding variable adjusted for in the multivariable model. It is also noteworthy that 80% of women older than 50 y had reduction or discontinuation of mycophenolate at the time of their 6 mo laboratory measurement (Table 3). This might indicate not only that these women would have had a lower ALC if still on standard immunosuppression with a mycophenolate product but also that this medication change could have been a response to leukopenia. This observation warrants further consideration and investigation.

Our findings have an important place in a growing body of literature looking at sex- and age-based differences in transplant outcomes. Two recent meta-analyses combining data from the largest transplant registries worldwide showed that female heart and kidney recipients in all age categories carried greater excess mortality than men (modified by donor sex).^7,30 In the younger age categories, this aligns well with greater rates of graft failure in women due to robust alloimmune response.^2,4,5 In contrast, excess mortality in the older, postmenopausal female population is not well explained by graft failure.³¹ One analysis actually showed that female kidney recipients older than 55 y have lower graft loss rates than men.² Therefore, among these older female recipients, we must consider alternative factors such as infection or other complications of excessive immune suppression, including medication toxicity or malignancy (the latter not examined here). Although these large registries are incredibly important in establishing that differences in excess mortality exist, they do not reliably provide data on specific causes of mortality, nor on infection- or immunosuppression-related morbidity, which both may differ by sex and age. Our study provides a more granular view of these subpopulations and suggests a greater degree of immunosuppression and possibly a greater risk of infection in older women.

This is the first study, to our knowledge, to prospectively analyze the relationship of steroid hormones (measurement of concomitant serum levels, documentation of exogenous sources, and menopause status) with immune function in solid organ transplant recipients. Generally, healthy postmenopausal women have decreased levels of CD4⁺ T and B lymphocytes, cytotoxic activity of natural killer cells, and even circulating IFN-γ.^32-35 This becomes even more relevant in the transplant population, where immunosuppressive medications primarily target cell-mediated immunity, creating heightened susceptibility to opportunistic infection. We previously showed higher ALC among all women compared with all men in a large cohort at 1 mo post-HT.³⁶ Importantly, however, in a post hoc analysis with age dichotomization at 50 y, ALC was far lower among older (mean 0.92, median 0.60 K/µL) compared with younger (mean 1.64, median 1.45 K/µL) women (P < 0.001; unpublished data).

Sex steroids, including estrogens, progesterone, and androgens, have far-reaching effects on immune cells, many of which express sex steroid receptors and involve a multitude of response cascades ultimately affecting the proliferation, differentiation, and function of the cell.¹³ At physiologic levels, estradiol generally promotes proinflammatory functions; for example, the enhancement of toll-like receptor 7-induced type I IFN signaling, and the increase of IFN-γ secretion by T cells.¹³ These particular pathways are central to human responses to viral pathogens. Progesterone typically promotes a tolerogenic state.¹³ Both of these hormones vary by menstrual cycle, age, and sex. In addition, end-stage kidney and heart failure commonly induce hormonal dysregulation, often resulting in amenorrhea in young women. The normalization of steroid hormones has been studied in the kidney transplant population, where the typical hormonal milieu is restored by 6 mo posttransplantation.²⁶ For these reasons, the direct measurement of serum concentrations of estradiol and progesterone taken concomitantly with measures of lymphocyte counts, IgG, and cytokines was essential to accurately characterize the dynamic relationship between aging and sex simultaneously. These measurements, as well as reported experience of menopause and/or exogenous sources of estrogen and progesterone, are a strength of this analysis. In future study, measurement of testosterone, which in many pathways acts as an immune inhibitory, may elucidate additional parallel sex hormone-related patterns critical for understanding immunity and risk of infection posttransplantation.¹³

The effect of aging on the immune profile has been investigated in kidney transplant recipients; older recipients experience increased T-cell immune senescence and accelerated maturation phenotypes, which are associated with a greater risk of infection.³⁷ However, there has been little comparison of the magnitude and rate of immune aging between male and female organ recipients and only a limited study of the variable effects of immune suppression between sexes with age.³⁸ Notably, the metabolism of routinely used immunosuppressive drugs (dosed uniformly for recipients of a given organ type) varies by sex, with lower clearance of both glucocorticoids and mycophenolic acid among women compared with men, and lower clearance of glucocorticoids among postmenopausal women compared with premenopausal women.³⁸

This analysis adds an important dimension to our understanding of the many patient-specific factors that affect global immune function after transplantation and highlights older, postmenopausal women as a group with potentially greater vulnerability to posttransplant infection. Important limitations to this study include a small sample size, heterogeneity introduced by different organ transplant types, and a broadly defined infection outcome that included various pathogens and syndromes without uniform screening. Despite this, the trends seen in these data provide a strong basis for future adequately powered and more targeted studies of the effect of sex and age on the solid organ transplant host responses to infection. Based on our findings related to lymphocytes, larger future studies of single organ types should systematically evaluate the incidence and severity-specific viral infections (CMV and others) defined by commonly used research criteria. They should also ideally examine 3 age categories rather than 2, to account for a perimenopausal age range, which may elucidate greater contrast between pre- and postmenopausal groups.

Supplementary Material

txd-11-e1846-s001.pdf ^{(685KB, pdf)}